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J Lung Cancer 2012;11(2):59-65 http://dx.doi.org/10.6058/jlc.2012.11.2.59 Mechanisms of Acquired Resistance to Epidermal Growth Factor Receptor Inhibitors and Overcoming Strategies in Lung Cancer Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR- TKIs) such as gefitinib and erlotinib show good response and survival benefit in EGFR-mutant lung cancer, acquired resistance inevitably develops which limits the median response duration to around 1 year. Secondary T790M gatekeeper mutation is the most common mechanism representing approximately 50% of resistance. The suggested strategies for overcoming T790M including irreversible EGFR-TKIs, mutant-selective EGFR-TKIs, EGFR dual targeting and HSP90 inhibitors should be further investigated for clinical application. Bypass signals through MET or AXL also contribute to resistance, which lead to development of MET or AXL inhibitors. Other mechanisms such as small cell transformation, epithelial-to-mesenchymal transition, PI3KCA mutation, ERK/ HER2 amplification and mirnas are other suggested candidates awaiting validation. As many resistant mechanisms have been recognized, it is important to obtain tissue samples after resistance to provide appropriate treatment. In this review, recent advances in the understanding of resistance and novel ways of overcoming it are discussed. (J Lung Cancer 2012;11(2):59 65) Key Words: Lung neoplasms, EGFR tyrosine kinase inhibitor, Drug resistance Chang-Min Choi, M.D. 1,2 and Jae Cheol Lee, M.D., Ph.D. 1 Departments of 1 Oncology and 2 Pulmonology and Critical Care Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea Received: October 29, 2012 Accepted: November 7, 2012 Address for correspondence Jae Cheol Lee, M.D., Ph.D. Department of Oncology, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea Tel: 82-2-3010-3208 Fax: 82-2-3010-6961 E-mail: jclee@amc.seoul.kr 서론폐암은전세계적으로암발생률과사망률에서모두 1위를차지하고있고 5년생존율이 15% 내외에불과할정도로예후가좋지않은데이는대부분의환자들이수술이어려운상태로진단되고이때사용하는항암제의효과가좋지않았기때문이다. 기존에사용하던세포독성항암제는독성또한심하여많은환자들이치료에따른부작용으로고통을받았던것도사실이다. 하지만 2000년대에들어서면서폐암에서 epidermal growth factor receptor (EGFR) 를타겟으로하는표적치료제가등장하면서이러한양상이많이달라지고있는데 gefitinib과 erlotinib이라는 EGFR tyrosine kinase를억제하는 small molecule inhibitor (EGFR tyrosine kinase inhibitors, EGFR-TKIs) 는경구로복용이가능하여치료를받는데에따른불편함이많이감소하였고피부발진, 설사등의일부부작용을보이긴하지만기존의약제에비해다른부작용들이거의없어치료에대한순응도또한 많이향상되었다. EGFR의 tyrosine kinase를 coding하는부위인 exon 18에서 exon 21에걸쳐돌연변이를가지고있는환자에게서매우뛰어난효과를보여주는것이확인되었고, 기존항암제와효과를비교한 Iressa Pan-Asia Study (IPASS), European Tarceva (erlotinib) versus Chemotherapy (EURTAC) study 등을통해생존율향상을입증함으로써현재 EGFR 돌연변이가있는폐암환자에게서 1차요법제로널리쓰이고있다 (1,2). EGFR 돌연변이는지금까지 30개가넘는종류가보고되어왔는데대부분이 exon 19 deletion과 exon 21 L858R로서이두가지가 90% 이상을차지한다 (3). 이외에도 G719A, L861Q와같이반응과의관련성이잘알려진돌연변이도있지만나머지대부분은빈도가낮아그의미가불확실한상태이다 (Fig. 1). 하지만 EGFR-TKI에대한초기반응이좋더라도결국은약제내성이생기는것이가장큰문제인데평균반응지속기간은 1년정도로생각되고있다. 현재까지가장널리알려진내성기전은 T790M 이차돌연변이에의한것과 MET 59

60 J Lung Cancer 2012;11(2):59-65 Fig. 1. Epidermal growth factor receptor (EGFR) tyrosine kinase mutations (reprinted from Chan SK, et al. Eur J Cancer 2006;42: 17-23 (3), with permission from Elsevier). 내성연구결과를비교하고평가하는데도움이될것으로생각된다. T790M Fig. 2. The frequency of observed drug resistance mechanisms (reprinted from Sequist LV, et al. Sci Transl Med 2011; 3:75ra26 (6), with permission from American Association for the Advancement of Science). EGFR: epidermal growth factor receptor, PIK3CA: phosphatidylinositol 3 kinase catalytic subunit, SCLC: small cell lung cancer. amplification을통한우회신호전달체계의활성화가있다 (Fig. 2) (4-6). 2010년 Jackman 등 (7) 은비소세포폐암에서 EGFR-TKI에대한획득내성을다음과같이정의하였는데 (Table 1) 이는내성을일정한기준에의해진단함으로써내성의치료및 내성을획득한환자의약 50% 는 T790M 이차돌연변이에의한것이다 (8). 이는 790번째아미노산인 threonine이크기가큰 methionine으로치환되어약제결합력을떨어뜨림으로써내성을가져오는것으로이해되고있지만 T790M에의해본래존재하던 EGFR sensitizing mutation의 ATP에대한결합력이증가하는것이더중요한기전이라는설명도있다 (9). Threonine이 kinase 활성부위근처에위치하기때문에 gatekeeper mutation 이라고하는데이런현상은이미가장먼저등장한표적치료제인 imatinib에대한내성이만성백혈병의원인인 BCR-ABL에발생한 T315I 돌연변이등과유사하다고볼수있다. 또한폐암의새로운표적치료제인 crizotinib도결국내성이발생하는데 L1196M과같은이차돌연변이에의한것이가장중요한것으로 (30 40%) 밝혀지고있어 (10) 모든표적치료제에나타나는공통된현상임을알수있다. T790M의경우내성이증가함에따라유전자증폭을통해 T790M을포함한 allele이늘어나기때문에내성의초기에는그양이많지않아민감도가낮은검사법을쓰게되면 T790M이없는 allele에희석이되면서발견하지못하는경

Resistance to EGFR-TKI 61 Table 1. Proposed Definition of Acquired Resistance to EGFR-TKI Criteria Definition 1 Previously received treatment with single-agent EGFR-TKI (e.g., gefitinib or erlotinib) 2 Either of the following: (A) A tumor that harbors an EGFR mutation known to be associated with drug sensitivity (B) Objective clinical benefit from treatment with EGFR-TKI as defined by either documented partial or complete response (RECIST or WHO), or significant and durable (>6 months) clinical benefit (stable disease as defined by RECIST or WHO) after inititation of gefitinib or erlotinib 3 Systemic progression of disease (RECIST or WHO) while on continuous treatment with gefitinib or erlotinib within the last 30 days 4 No intervening systemic therapy between cessation of gefitinib or erlotinib and initiation of new therapy EGFR-TKI: epidermal growth factor receptor tyrosine kinase inhibitors, RECIST: Response Evaluation Criteria in Solid Tumors, WHO: World Health Organization. Table 2. Sensitivity of EGFR Mutation Tests Technique Sensitivity (mutant DNA, %) Direct sequencing 25 PCR-SSCP 10 TaqMan PCR 10 Loop-hybrid mobility shift assay 7.5 Cycleave PCR 5 PCR-RFLP and length analysis 5 MALDI-TOF MS-based genotyping 5 PNA-LNA PCR clamp 1 Scorpions ARMS 1 dhplc 1 Single-molecule sequencing 0.2 Mutant-enriched PCR 0.2 SMAP 0.1 EGFR: epidermal growth factor receptor, PCR-SSCP: polymerase chain reaction-single-stranded conformation polymorphism, PCR-RFLP: polymerase chain reaction-restriction fragment length polymorphism, MALDI-TOF MS: matrix-assisted laser desorption/ionization-time of flight mass spectrometry, PNA-LNA: peptide nucleic acid-locked nucleic acid, ARMS: amplified refractory mutation system, dhplc: denaturing highperformance liquid chromatography, SMAP: smart amplification process. 우가있다. Arcila 등 (11) 의보고에따르면처음에 direct sequencing에서음성으로나왔던 30명의환자중 peptide nucleic acid test를다시했더니 11명에서추가로 T790M을발견할수있었다. 따라서민감도가낮은 direct sequencing 이현재가장대표적으로사용되는검사법임을감안할때 T790M은지금알려진빈도보다더많을것으로추정된다 (Table 2). 흥미로운사실중하나는 T790M 이차돌연변이가생긴환자는다른기전에의해내성이발생한환자에비해더예후가좋다는점이다. 이는 T790M을지닌세포의성장이 Fig. 3. Slow, indolent growth of lung cancer cells harboring EGFR T790M (reprinted from Oxnard GR, et al. Clin Cancer Res 2011; 17:5530-5537 (8), with permission from American Association for Cancer Research). 느리고전이도덜일어나는성질을지니는것에기인하는것으로생각되고있다 (Fig. 3) (12). T790M 극복방안 1) Irreversible EGFR-TKIs Gefitinib, erlotinib과같이현재사용중인 EGFR-TKI는 reversible inhibitor이지만 PF-002999804 (dacomitinib), BIBW2992 (afatinib) 와같은 2세대 EGFR-TKI들은 Michael-acceptor group을함유하고있어 EGFR ATP 결합부위에공유결합하는 irreversible inhibitor이고 EGFR, HER2, HER4 등을같이억제하는 pan-her inhibitor로작용하기때문에효과가훨씬뛰어난것으로알려졌다 (13). 초기실험결과에서는이러한 irreversible inhibior가 T790M 내성을극복할수있다는것을보여주었지만 PF-002999804의경우사용중 T790M 의증폭이더늘어나면서효과가없어지고 (14), BIBW2992

62 J Lung Cancer 2012;11(2):59-65 역시가장약제효과가높다고알려져있음에도내성환자에서생존율향상을입증하는데결국실패하였다 (15). 2) Mutant-selective inhibitors 기존의 EGFR inhibitor가 quinazoline 기본구조를가지고있어정상 EGFR에도 ATP와경쟁적결합함으로써부작용을초래하는데최근 T790M EGFR에특이적으로반응하는 pyrimidine EGFR inhibitor가개발되어이를활용하는연구가진행중이다. 이런약제는기존의 EGFR inhibitor에비해 T790M EGFR에 30 100배정도더강력한반면정상 EGFR에는오히려 100배정도덜작용함으로써효과는더좋고부작용이감소되는결과를보여주고있어앞으로의전망이밝다 (Fig. 4) (16). 3) Heat shock protein 90 (HSP90) inhibitors HSP90는단백질의 conformational maturation, stability에관여하는 chaperone으로기능을하고있는데정상단백질보다암단백질의 HSP90에대한의존성이훨씬높아 HSP90 을억제하여암을치료하려는시도가이루어져왔다 (17). EGFR의경우에도 T790M을포함한 mutant EGFR이정상 EGFR보다 HSP90 억제에더민감하게반응하기때문에 T790M 내성을극복하는데효과가있다는연구결과들이보고되었다 (18). 하지만 HSP90 inhibitor의하나인 IPI-504로시행한임상시험에서는과거 EGFR-TKI 치료후내성이생긴환자 28명중단 1명에서만반응을보인실망스런결과를얻었다 (19). 그럼에도 IPI-504와같은기존의 geldanamycin 계열의약물과는다른종류의 HSP90 inhibitor인 AUY922나 STA-9090의경우에는현재진행중인임상시험에서좋은반응을나타내고있는것으로알려지고있어그결과가기대되고있다. 4) Dual targeting T790M transgenic mouse를이용한동물실험을통해 EGFR blocking antibody인 cetuximab과 BIBW2992의병합이 T790M 내성극복에효과적임을보여주는보고가있었다 (20). HER2+ breast cancer에서도 trastuzumab+lapatinib을사용하여 HER2의 extracellular and intracellular domain을모두차단하는소위 vertical inhibition 을통해치료효과를향상시킨결과와유사한것이다 (21). 이연구결과를바탕으로 phase I 연구가진행되고있는데반응평가가가능하고 T790M이확인된 13명의환자중 4명에서 partial response를보여주었다 (22). 이는실제내성환자에서시행된연구중가장좋은성적으로, 앞으로 update 될결과에많은기대가모아지고있다. OTHER SECONDARY MUTATION Exon 21의 T854A나 exon 19의 L747S, D761Y 등과같은이차돌연변이도내성환자에서보고되었지만매우드물다. 이들도나름의방식으로약제의결합부위에영향을주게되고이로인해내성이발생하는것으로설명되고있다 (23). MET BYPASS 내성의다른중요한기전중하나가 MET 유전자증폭을통한 MET receptor 우회신호전달체계의활성인데초기에약 20% 의내성환자에서 MET 유전자증폭이관찰되었다고보고되었으나 (5) 이후에는그보다는빈도가좀낮은것으로알려져한조사에따르면약 5% 를차지하였다 (6). MET 의 ligand인 hepatocyte growth factor가 gefitinib 내성을유도하는실험적모델이발표되기도하였다 (24). 현재 MET receptor blocking monoclonal antibody인 MetMAb과 small molecule inhibitor인 ARQ197 (tivantinib) 등의 MET inhibitor 에대한임상시험들이진행중이지만아직내성과관련된뚜렷한효과를입증한연구결과는나와있지않다. AXL BYPASS Fig. 4. Novel mutant-selective EGFR inhibitors (Zhou W, et al. Nature 2009;462;1070-1074 (16), with permission from Nature Publishing Group). AXL도세포표면수용체의하나로 GIST의 imatinib 내성, 유방암의 lapatinib 내성에관여하는것으로알려져있다. 최근 ligand인 GAS6의 upregulation과함께 AXL의과발현이 EGFR-TKI 내성의기전으로작용한다는보고가나오고다

Resistance to EGFR-TKI 63 양한 AXL inhibitor들이개발되면서주목을받고있다 (25). 내성을획득한환자중약 20% (7/35) 에서 AXL의발현이증가하였는데그중 2명은 T790M을같이가지고있었다. AXL inhibitor의병합투여를통해내성이극복될수있을지, 정확한빈도가어떻게되는지등에대한추가적인연구가필요할것으로보인다. EPTHELIAL-TO-MESENCHYMAL TRANSITION (EMT) EMT는 epithelial polarized phenotype이 mesenchymal fibroblastoid phenotype으로변하는현상으로전이및다양한항암제내성과의관련성은이미알려져왔는데 2009년 EGFR- TKI 내성과관련되어있다는실험결과가나오고 2010년첫증례가발표되었다 (26). Sequist 등 (6) 도 3명의내성환자에서 EMT가관찰되었다고보고한바있어빈도가높지않아도 EGFR-TKI 내성의한기전임을알수있다. SMALL CELL TRANSFORMATION Massachusetts General Hospital 연구자들은 37명의내성환자조직을분석했을때그중 5명 (14%) 에서형태학적인변화와함께 synaptophysin, chromogranin, CD56과같은소세포폐암의마커들의발현이증가되었고소세포폐암에사용되는 etoposide+cisplatin 항암제에반응이좋았다고보고하였다 (6). 이전에도비슷한증례보고가있어왔고 (27,28) 소세포폐암에서도드물게 EGFR 돌연변이가발견된다는 (29) 연구결과들을종합해볼때이또한가능한내성기전의하나로생각된다. PI3KCA MUTATION 빈도는아주낮지만내성환자의조직에서 PIK3CA 돌연변이가발견되었고 (6) EGFR 돌연변이가있는폐암세포주에 mutant PIK3CA의발현을유도할경우내성이발생하였다는보고가있다 (30). 현재 BKM120, GDC-0941, MK-2206 등다양한 inhibitor 들이개발되어임상시험이진행중인상태이다. ERK/HER2 AMPLIFICATION 다른내성기전이없는환자에서 ERK (5%), HER2 (12%) amplification이관찰되었고이러한변화가 in vitro model에서내성을유도하는것을확인한연구결과가최근보고된바있어 (31,32) 내성과의관련성에대한추가연구가필요할것으로보인다 (Fig. 5) (33). mirna mirna는약 19 25개의 nucleotide로구성된 RNA로 target mrna와결합함으로써유전자의발현을억제하는역할을하는데인체유전자의 30% 이상이영향을받는것으 Fig. 5. Schematic of pathways to EGFR inhibitor acquired resistance and pharmacologic approaches (Blakely CM and Bivoma TG. Cancer Discov 2012;2:872-875 (33), with permission from American Association for Cancer Research). EGFR: epidermal growth factor receptor, EMT: epithelial mesenchymal transition, ERK: extracellular signal-regulated kinase, NF-κB: nuclear factor κb, PIK3CA: phosphatidylinositol 3 kinase catalytic subunit.

64 J Lung Cancer 2012;11(2):59-65 Fig. 6. mirnas associated with gefitinib resistance and EMT (Garofalo M, et al. Nat Med 2011;18:74-82 (34), with permission from Nature Publishing Group). APAF-1: apoptosis proteinase activating factor-1, EGFR: epidermal growth factor receptor, EMT: epithelial mesenchemal transition, PKC-ε: protein kinase Cε, PTEN: phosphatase and tensin homologue. 로알려지고있다. 특히 mir-16, Let-7i 등의 mirna는항암제반응과관련이되어있고 mir-521, Let-7g 등은방사선감수성에영향을주는것으로보고되었다. Garofalo 등 (34) 은 2011년 gefitinib 투여후암세포에 apoptosis가오는과정에관여하고있는 BIM, APAF-1 등이 mir-30b/c, mir-221/ 222에의해조절된다는사실을증명하여이를치료에응용할수있음을시사한바있다 (Fig. 6). 결 EGFR-TKI 치료를받고있는환자의대부분에서내성이발생하기때문에내성의기전을이해하고그기전에따라극복방안을마련하는것이 EGFR-TKI 치료에있어매우중요한일이다. 따라서앞으로는내성이생기면조직검사를다시시행하고알려진내성기전에대한검사를의뢰하는방향으로변하게될것으로예상된다. MET나 AXL과같은우회신호전달을통하는내성과 small cell transformation 과같은경우에는치료약제를찾는것이어렵지않지만가장많은내성을차지하고있는 T790M의경우에는현재제시되고있는치료방안들의효과에대한추가적인연구가더진행되어야할것으로보인다. 론 REFERENCES 1. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-957. 2. Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol 2012;13:239-246. 3. Chan SK, Gullick WJ, Hill ME. Mutations of the epidermal growth factor receptor in non-small cell lung cancer: search and destroy. Eur J Cancer 2006;42:17-23. 4. 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 Med 2005;2:e73. 5. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 2007;316:1039-1043. 6. Sequist LV, Waltman BA, Dias-Santagata D, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med 2011;3:75ra26. 7. Jackman D, Pao W, Riely GJ, et al. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J Clin Oncol 2010;28:357-360. 8. Oxnard GR, Arcila ME, Chmielecki J, Ladanyi M, Miller VA, Pao W. New strategies in overcoming acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer. Clin Cancer Res 2011;17:5530-5537. 9. Yun CH, Mengwasser KE, Toms AV, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci U S A 2008;105: 2070-2075. 10. Doebele RC, Pilling AB, Aisner DL, et al. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res 2012;18:1472-1482. 11. Arcila ME, Oxnard GR, Nafa K, et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clin Cancer Res 2011;17:1169-1180. 12. Oxnard GR, Arcila ME, Sima CS, et al. Acquired resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant lung cancer: distinct natural history of patients with tumors harboring the T790M mutation. Clin Cancer Res 2011;17:1616-1622. 13. Kwak EL, Sordella R, Bell DW, et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci U S A 2005;102:7665-7670. 14. Ercan D, Zejnullahu K, Yonesaka K, et al. Amplification of EGFR T790M causes resistance to an irreversible EGFR inhibitor. Oncogene 2010;29:2346-2356. 15. Miller VA, Hirsh V, Cadranel J, et al. Afatinib versus placebo for patients with advanced, metastatic non-small-cell lung cancer after failure of erlotinib, gefitinib, or both, and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial. Lancet Oncol 2012;13:528-538.

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