Capitolo 2. Il DNA tumorale circolante (ctDNA)
3.5 Conclusioni
Nell’ultimo decennio, un numero crescente di evidenze scientifiche ha suggerito l’impiego della biopsia liquida, e in particolare dell’analisi mutazionale del DNA tumorale circolante (ctDNA), come approccio alternativo alla caratterizzazione molecolare eseguita sul tessuto tumorale dei pazienti affetti da tumore polmonare.
Dati i sostanziali vantaggi della biopsia liquida in termini di minima invasività e facile accessibilità, questo lavoro si è proposto di indagare il ruolo che tale approccio possa ricoprire nella gestione clinica routinaria dei pazienti affetti da adenocarcinoma polmonare, sia dal punto di vista diagnostico che del monitoraggio terapeutico.
I risultati del nostro studio suggeriscono delle differenze in termini di concordanza per quanto riguarda la determinazione delle mutazioni di EGFR e KRAS. I risultati della ricerca delle mutazioni del gene EGFR sul ctDNA mediante Real-Time PCR hanno mostrato una concordanza dell’84.2%, con una sensibilità del 62.5% e una specificità del 100%. Sebbene la determinazione delle mutazioni di EGFR sul ctDNA non abbia la stessa accuratezza diagnostica di quella sul tessuto e pertanto non possa essere considerata un’alternativa equi-efficace, la biopsia liquida può rappresentare uno strumento utile in quei casi in cui non sia possibile reperire tessuto idoneo per l’analisi molecolare, al fine di garantire al paziente EGFR mutato un trattamento di prima linea con inibitori tirosin chinasici, con benefici significativi in termini di PFS e OS rispetto alla chemioterapia. Ulteriori sforzi devono essere invece compiuti per migliorare la sensibilità nell’individuazione delle mutazioni di KRAS su ctDNA, in vista della futura introduzione nella pratica clinica di inibitori diretti della proteina da esso codificata. Nel nostro studio abbiamo infatti riportato una sensibilità del 52.8% e una specificità del 100%, con una concordanza del 75.3%.
Inoltre la biopsia liquida, essendo rappresentativa della massa tumorale globale nel paziente, potrebbe costituire uno strumento semiquantitativo per stimare la probabilità di risposta al trattamento con TKI-EGFR. I dati a nostra disposizione oggi non sono sufficienti per sostenere tale ipotesi, tuttavia sono opportuni ulteriori studi al fine di poter trarre conclusioni definitive.
Infine, i casi clinici descritti suggeriscono alcune riflessioni circa l’utilità del ctDNA nel monitoraggio della risposta alla terapia, in particolare per quanto riguarda la possibilità di individuare precocemente, mediante periodiche rivalutazioni, l’insorgenza di mutazioni acquisite di resistenza al trattamento con inibitori tirosin chinasici di EGFR, alcuni mesi prima della progressione radiologica della malattia.
Questo aspetto è rilevante per l’attuale disponibilità di TKI di terza generazione in grado di superare la resistenza acquisita determinata dalla mutazione T790M. In quest’ottica la biopsia liquida rappresenta uno strumento importante, data la scarsa invasività e facile ripetibilità rispetto alle rebiopsie tissutali.
Tuttavia, a causa dell’eterogeneità dei meccanismi di resistenza acquisita al momento della progressione, come evidenziato dai casi discussi, l’analisi dello stato mutazionale del ctDNA può attualmente rappresentare solo uno strumento complementare alla rebiopsia nella valutazione della resistenza al trattamento.
Le prospettive delineate da questo lavoro consistono dunque nella possibilità di aumentare ulteriormente la sensibilità dell’analisi mutazionale condotta sul ctDNA mediante l’impiego di nuove metodiche, come la NGS e la ddPCR, in particolare per quanto riguarda l’identificazione delle mutazioni di KRAS, e di progettare uno studio prospettico che abbia l’obiettivo primario di monitorare la dinamica delle alterazioni tumorali mediante ripetute analisi del ctDNA in corso di trattamento con TKI-EGFR.
Bibliografia
1. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 http://globocan.iarc.fr; 2013. Accessed 03.2017.
2. SEER Cancer Statistics Review, 1975-2014. 2017;
https://seer.cancer.gov/csr/1975_2014/. Accessed 03.2017.
3. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA: a cancer journal for
clinicians. 2017;67(1):7-30.
4. Society AC. Cancer Facts & Figures 2017. Atlanta: American Cancer Society;2017. 5. AIOM-AIRTUM. I numeri del cancro in Italia 2016. 2016.
6. Lortet-Tieulent J, Renteria E, Sharp L, et al. Convergence of decreasing male and increasing female incidence rates in major tobacco-related cancers in Europe in 1988-2010. European journal of cancer (Oxford, England : 1990). 2015;51(9):1144-1163.
7. North CM, Christiani DC. Women and lung cancer: what is new? Seminars in
thoracic and cardiovascular surgery. 2013;25(2):87-94.
8. Egleston BL, Meireles SI, Flieder DB, Clapper ML. Population-based trends in lung cancer incidence in women. Seminars in oncology. 2009;36(6):506-515.
9. Jemal A, Simard EP, Dorell C, et al. Annual Report to the Nation on the Status of Cancer, 1975-2009, featuring the burden and trends in human papillomavirus(HPV)-associated cancers and HPV vaccination coverage levels.
Journal of the National Cancer Institute. 2013;105(3):175-201.
10. Bosetti C, Malvezzi M, Rosso T, et al. Lung cancer mortality in European women: trends and predictions. Lung cancer (Amsterdam, Netherlands). 2012;78(3):171-178.
11. Torre LA, Islami F, Siegel RL, Ward EM, Jemal A. Global cancer in women: burden and trends. Cancer epidemiology, biomarkers & prevention : a publication of the
American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2017.
12. Torre LA, Siegel RL, Ward EM, Jemal A. International variation in lung cancer mortality rates and trends among women. Cancer epidemiology, biomarkers &
prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2014;23(6):1025-1036.
13. Marshall AL, Christiani DC. Genetic susceptibility to lung cancer--light at the end of the tunnel? Carcinogenesis. 2013;34(3):487-502.
14. Howlader N NA, Krapcho M, Miller D, Bishop K, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review. Bethesda: National Cancer Institute; 1975-2014. 15. White C. Research on smoking and lung cancer: a landmark in the history of
chronic disease epidemiology. The Yale journal of biology and medicine. 1990;63(1):29-46.
16. Dubey AK, Gupta U, Jain S. Epidemiology of lung cancer and approaches for its prediction: a systematic review and analysis. Chinese journal of cancer. 2016;35(1):71.
17. Alberg AJ, Brock MV, Ford JG, Samet JM, Spivack SD. Epidemiology of lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest. 2013;143(5 Suppl):e1S-29S.
18. Cancer IAfRo. Tobacco Smoke and Involuntary Smoking. Lion: World Health Organization; 2004.
19. Alavanja MC. Biologic damage resulting from exposure to tobacco smoke and from radon: implication for preventive interventions. Oncogene.
2002;21(48):7365-7375.
20. The Health Consequences of Smoking-50 Years of Progress: A Report of the Surgeon General. Atlanta (GA)2014.
21. Hecht SS. Biochemistry, biology, and carcinogenicity of tobacco-specific N- nitrosamines. Chemical research in toxicology. 1998;11(6):559-603.
22. Sopori M. Effects of cigarette smoke on the immune system. Nature reviews.
Immunology. 2002;2(5):372-377.
23. Yang P, Cerhan JR, Vierkant RA, et al. Adenocarcinoma of the lung is strongly associated with cigarette smoking: further evidence from a prospective study of women. American journal of epidemiology. 2002;156(12):1114-1122.
24. Boffetta P, Pershagen G, Jockel KH, et al. Cigar and pipe smoking and lung cancer risk: a multicenter study from Europe. Journal of the National Cancer
25. The Health Benefits of Smoking Cessation. A report of the Surgeon General. Washington,
DC: US Department of Health and Human Services;1990. 90-8416.
26. Oberg M, Jaakkola MS, Woodward A, Peruga A, Pruss-Ustun A. Worldwide burden of disease from exposure to second-hand smoke: a retrospective analysis of data from 192 countries. Lancet (London, England). 2011;377(9760):139-146. 27. Sun S, Schiller JH, Gazdar AF. Lung cancer in never smokers--a different
disease. Nature reviews. Cancer. 2007;7(10):778-790.
28. Hackshaw AK. Lung cancer and passive smoking. Statistical methods in medical
research. 1998;7(2):119-136.
29. Catelinois O, Rogel A, Laurier D, et al. Lung cancer attributable to indoor radon exposure in france: impact of the risk models and uncertainty analysis.
Environmental health perspectives. 2006;114(9):1361-1366.
30. Lubin JH, Boice JD, Jr., Edling C, et al. Lung cancer in radon-exposed miners and estimation of risk from indoor exposure. Journal of the National Cancer
Institute. 1995;87(11):817-827.
31. Health Effects of Exposure to Radon (BEIR VI). Washington, DC: Committee on
Health Risks of Exposure to Radon;1999.
32. Saracci R. Asbestos and lung cancer: an analysis of the epidemiological evidence on the asbestos-smoking interaction. International journal of cancer. 1977;20(3):323- 331.
33. Heintz NH, Janssen-Heininger YMW, Mossman BT. Asbestos, Lung Cancers, and Mesotheliomas: From Molecular Approaches to Targeting Tumor Survival Pathways. American Journal of Respiratory Cell and Molecular Biology. 2010;42(2):133- 139.
34. Nishimura Y, Miura Y, Maeda M, et al. Impairment in cytotoxicity and expression of NK cell- activating receptors on human NK cells following exposure to asbestos fibers. International journal of immunopathology and pharmacology. 2009;22(3):579-590.
35. O'Reilly KM, McLaughlin AM, Beckett WS, Sime PJ. Asbestos-related lung disease. American family physician. 2007;75(5):683-688.
36. Saracci R. The interactions of tobacco smoking and other agents in cancer etiology. Epidemiologic reviews. 1987;9:175-193.
37. Cancer IAfRo. Ionizing radiation. part 1: x- and Gamma radiations and neutrons. Lion: World Health Organization; 2000.
38. Shimizu Y, Kato H, Schull WJ. Studies of the mortality of A-bomb survivors. 9. Mortality, 1950-1985: Part 2. Cancer mortality based on the recently revised doses (DS86). Radiation research. 1990;121(2):120-141.
39. Darby SC, McGale P, Taylor CW, Peto R. Long-term mortality from heart disease and lung cancer after radiotherapy for early breast cancer: prospective cohort study of about 300,000 women in US SEER cancer registries. The Lancet.
Oncology. 2005;6(8):557-565.
40. Alberg A, Yung R, Strickland PT, et al. Respiratory cancer and exposure to arsenic, chromium, nickel and polycyclic aromatic hydrocarbons. Clin Occup
Environ Med. 2002;2(4):779-801.
41. Raaschou-Nielsen O, Beelen R, Wang M, et al. Particulate matter air pollution components and risk for lung cancer. Environment international. 2016;87:66-73. 42. Cancer IAfRo. Outdoor pollution. Lion: World Health Organization; 2013.
43. Barone-Adesi F, Chapman RS, Silverman DT, et al. Risk of lung cancer associated with domestic use of coal in Xuanwei, China: retrospective cohort study. Bmj. 2012;345:e5414.
44. Mu L, Liu L, Niu R, et al. Indoor air pollution and risk of lung cancer among Chinese female non-smokers. Cancer causes & control : CCC. 2013;24(3):439-450. 45. Kurmi OP, Arya PH, Lam KB, Sorahan T, Ayres JG. Lung cancer risk and solid
fuel smoke exposure: a systematic review and meta-analysis. The European
respiratory journal. 2012;40(5):1228-1237.
46. Gordon SB, Bruce NG, Grigg J, et al. Respiratory risks from household air pollution in low and middle income countries. The Lancet. Respiratory medicine. 2014;2(10):823-860.
47. Wu CY, Hu HY, Pu CY, et al. Pulmonary tuberculosis increases the risk of lung cancer: a population-based cohort study. Cancer. 2011;117(3):618-624.
48. Liang HY, Li XL, Yu XS, et al. Facts and fiction of the relationship between preexisting tuberculosis and lung cancer risk: a systematic review. International
journal of cancer. 2009;125(12):2936-2944.
50. Ballaz S, Mulshine JL. The potential contributions of chronic inflammation to lung carcinogenesis. Clinical lung cancer. 2003;5(1):46-62.
51. Auerbach O, Garfinkel L, Parks VR. Scar cancer of the lung: increase over a 21 year period. Cancer. 1979;43(2):636-642.
52. Skillrud DM, Offord KP, Miller RD. Higher risk of lung cancer in chronic obstructive pulmonary disease. A prospective, matched, controlled study. Annals
of internal medicine. 1986;105(4):503-507.
53. O'Callaghan DS, O'Donnell D, O'Connell F, O'Byrne KJ. The role of inflammation in the pathogenesis of non-small cell lung cancer. Journal of thoracic
oncology : official publication of the International Association for the Study of Lung Cancer.
2010;5(12):2024-2036.
54. Yang SR, Chida AS, Bauter MR, et al. Cigarette smoke induces proinflammatory cytokine release by activation of NF-kappaB and posttranslational modifications of histone deacetylase in macrophages. American journal of physiology. Lung cellular
and molecular physiology. 2006;291(1):L46-57.
55. Yang P, Schwartz AG, McAllister AE, Aston CE, Swanson GM. Genetic analysis of families with nonsmoking lung cancer probands. Genetic epidemiology. 1997;14(2):181-197.
56. Antoniou KM, Tomassetti S, Tsitoura E, Vancheri C. Idiopathic pulmonary fibrosis and lung cancer: a clinical and pathogenesis update. Current opinion in
pulmonary medicine. 2015;21(6):626-633.
57. Hessel PA, Gamble JF, Nicolich M. Relationship between silicosis and smoking.
Scandinavian journal of work, environment & health. 2003;29(5):329-336.
58. Spigno F, Mortara V, Vitto V, Biagioli M, Traversa F. Lung cancer in subjects suffering from silicosis in the Province of Genoa from 1979 to 2004. Giornale
italiano di medicina del lavoro ed ergonomia. 2007;29(4):898-902.
59. Gonzalez FJ, Crespi CL, Gelboin HV. DNA-expressed human cytochrome P450s: a new age of molecular toxicology and human risk assessment. Mutation
research. 1991;247(1):113-127.
60. How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking- Attributable Disease: A Report of the Surgeon General. Atlanta (GA)2010.
61. Yokota J, Shiraishi K, Kohno T. Genetic basis for susceptibility to lung cancer: Recent progress and future directions. Advances in cancer research. 2010;109:51-72.
62. Landi MT, Chatterjee N, Yu K, et al. A genome-wide association study of lung cancer identifies a region of chromosome 5p15 associated with risk for adenocarcinoma. American journal of human genetics. 2009;85(5):679-691.
63. Hung RJ, McKay JD, Gaborieau V, et al. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25. Nature. 2008;452(7187):633-637.
64. Wang Y, Broderick P, Webb E, et al. Common 5p15.33 and 6p21.33 variants influence lung cancer risk. Nature genetics. 2008;40(12):1407-1409.
65. Amos CI, Wu X, Broderick P, et al. Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1. Nature genetics. 2008;40(5):616-622.
66. McKay JD, Hung RJ, Gaborieau V, et al. Lung cancer susceptibility locus at 5p15.33. Nature genetics. 2008;40(12):1404-1406.
67. Bailey-Wilson JE, Amos CI, Pinney SM, et al. A major lung cancer susceptibility locus maps to chromosome 6q23-25. American journal of human genetics. 2004;75(3):460-474.
68. Matakidou A, Eisen T, Houlston RS. Systematic review of the relationship between family history and lung cancer risk. British journal of cancer. 2005;93(7):825-833.
69. Torok S, Hegedus B, Laszlo V, et al. Lung cancer in never smokers. Future
oncology (London, England). 2011;7(10):1195-1211.
70. Sato M, Shames DS, Girard L, Gazdar AF, Minna JD. Chapter 30 - Molecular Basis of Lung Cancer. The Molecular Basis of Cancer (Third Edition). Philadelphia: W.B. Saunders; 2008:397-407.
71. Herbst RS, Heymach JV, Lippman SM. Lung cancer. The New England journal of
medicine. 2008;359(13):1367-1380.
72. Sekido Y, Fong KM, Minna JD. Molecular genetics of lung cancer. Annual review
of medicine. 2003;54:73-87.
73. Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P. Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking- associated cancers. Oncogene. 2002;21(48):7435-7451.
75. Moriya Y, Niki T, Yamada T, Matsuno Y, Kondo H, Hirohashi S. Increased expression of laminin-5 and its prognostic significance in lung adenocarcinomas of small size. An immunohistochemical analysis of 102 cases. Cancer. 2001;91(6):1129-1141.
76. Bredin CG, Sundqvist KG, Hauzenberger D, Klominek J. Integrin dependent migration of lung cancer cells to extracellular matrix components. The European
respiratory journal. 1998;11(2):400-407.
77. Shibue T, Weinberg RA. Integrin beta1-focal adhesion kinase signaling directs the proliferation of metastatic cancer cells disseminated in the lungs. Proceedings of
the National Academy of Sciences of the United States of America. 2009;106(25):10290-
10295.
78. Quint LE, Tummala S, Brisson LJ, et al. Distribution of distant metastases from newly diagnosed non-small cell lung cancer. The Annals of thoracic surgery. 1996;62(1):246-250.
79. Hiyama K, Hiyama E, Ishioka S, et al. Telomerase activity in small-cell and non- small-cell lung cancers. Journal of the National Cancer Institute. 1995;87(12):895-902. 80. Travis WD, Brambilla E, Nicholson AG, et al. The 2015 World Health
Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. Journal of thoracic oncology :
official publication of the International Association for the Study of Lung Cancer.
2015;10(9):1243-1260.
81. Parkin DM, Whelan SL, Ferlay J, Teppo L, Thomas DB. Cancer Incidence in Five
Continents. Lyon: International Agency for Research on Cancer; 2002.
82. Dela Cruz CS et.al. Lung cancer: epidemiology, etiology, and prevention. Clinics
in chest medicine. 2011(32(4)):605–644
83. Meza R et al. Lung cancer incidence trends by gender, race and histology in the United States, 1973–2010. 2015;PLoS ONE(10(3)).
84. Moran CA. Chapter 3 - Non–Small Cell Carcinomas of the Lung. In: Suster S, ed. Tumors and Tumor-Like Conditions of the Lung and Pleura. New York: W.B. Saunders; 2010:51-110.
85. Ding L, Getz G, Wheeler DA, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature. 2008;455(7216):1069-1075.
86. Larsen JE, Minna JD. Molecular biology of lung cancer: clinical implications.
Clinics in chest medicine. 2011;32(4):703-740.
87. Krawczyk K W-K. Screening of Gene Mutations in Lung Cancer for Qualification to Molecularly Targeted Therapies. 2012.
88. Rosell R, Moran T, Queralt C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. The New England journal of medicine. 2009;361(10):958-967.
89. Pao W, Wang TY, Riely GJ, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS medicine. 2005;2(1):e17. 90. Eberhard DA, Johnson BE, Amler LC, et al. Mutations in the epidermal growth
factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. Journal of clinical oncology : official journal of the American
Society of Clinical Oncology. 2005;23(25):5900-5909.
91. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Journal
of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2013;8(7):823-859.
92. Wang L, Hu H, Pan Y, et al. PIK3CA mutations frequently coexist with EGFR/KRAS mutations in non-small cell lung cancer and suggest poor prognosis in EGFR/KRAS wildtype subgroup. PloS one. 2014;9(2):e88291. 93. AIOM AIdOM. Neoplasie del polmone. AIOM; 2016.
94. Zhang H, Berezov A, Wang Q, et al. ErbB receptors: from oncogenes to targeted cancer therapies. The Journal of clinical investigation. 2007;117(8):2051-2058. 95. Lemmon MA, Schlessinger J, Ferguson KM. The EGFR family: not so prototypical receptor tyrosine kinases. Cold Spring Harbor perspectives in biology. 2014;6(4):a020768.
96. Schneider MR, Wolf E. The epidermal growth factor receptor ligands at a glance. Journal of cellular physiology. 2009;218(3):460-466.
98. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000;103(2):211- 225.
99. Prigent SA, Lemoine NR. The type 1 (EGFR-related) family of growth factor receptors and their ligands. Progress in growth factor research. 1992;4(1):1-24.
100. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nature reviews. Cancer. 2007;7(3):169-181.
101. Fujimoto J, Wistuba, II. Current concepts on the molecular pathology of non- small cell lung carcinoma. Seminars in diagnostic pathology. 2014;31(4):306-313. 102. Eck MJ, Yun CH. Structural and mechanistic underpinnings of the differential
drug sensitivity of EGFR mutations in non-small cell lung cancer. Biochimica et
biophysica acta. 2010;1804(3):559-566.
103. Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science. 2004;305(5687):1163-1167.
104. Ono M, Hirata A, Kometani T, et al. Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signal-regulated kinase 1/2 and EGF receptor/Akt pathway for proliferation. Molecular cancer therapeutics. 2004;3(4):465-472.
105. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304(5676):1497-1500. 106. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common
in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proceedings of the National Academy of Sciences of the
United States of America. 2004;101(36):13306-13311.
107. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. The New England journal of medicine. 2004;350(21):2129-2139.
108. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS medicine. 2005;2(3):e73.
109. Yu HA, Arcila ME, Rekhtman N, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clinical cancer research : an official journal of the American Association for
Cancer Research. 2013;19(8):2240-2247.
110. Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. The New England journal of medicine. 2005;352(8):786-792.
111. Kwak EL, Sordella R, Bell DW, et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proceedings of the National Academy
of Sciences of the United States of America. 2005;102(21):7665-7670.
112. Yun CH, Mengwasser KE, Toms AV, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proceedings of the
National Academy of Sciences of the United States of America. 2008;105(6):2070-2075.
113. Chmielecki J, Foo J, Oxnard GR, et al. Optimization of dosing for EGFR- mutant non-small cell lung cancer with evolutionary cancer modeling. Science
translational medicine. 2011;3(90):90ra59.
114. Li H, Hu H, Wang R, et al. Primary concomitant EGFR T790M mutation