1. A.O.U.P. "P. Giaccone" University Hospital DEPARTMENT OF ONCOLOGY MEDICAL ONCOLOGY UNIT (Dir.: Prof. Antonio Russo) Sergio Rizzo EGFR inhibitors for overcoming resistance

2. Efficacy of EGFR-TKIs in EBFR mut. NSCLC Studio di fase IIITKI N. Pts (TKI arm) ORR (%) mPFS (mesi) IPASS Mok, 2009 Gefitinib609715,7 WJTOG3405 Mitsudomi, 2010 Gefitinib88629,2 NEJ002 Maemondo, 2010 Gefitinib1147410,4 First-SIGNAL Han, 2012 Gefitinib159858,0 TORCH Gridelli,2012 Erlotinib380426,4 OPTIMAL Zhou, 2011 Erlotinib828313,1 EURTAC Rosell, 2011 Erlotinib86649,7 LUX-Lung 3 Sequist, 2012 Afatinib2306010,1 LUX-Lung 6 Wu, 2013 Afatinib2425611,1 SUMMARY TOT: 1990 40-856-13 Review of literature 2016 Acquired resistance remains a major clinical problem in EGFR-mutant lung cancer, usually occurring within a year of starting treatment

3. Criteria for Acquired Resistance to EGFR TKIs Jackman, D. et al. J. Clin. Oncol. 28, 357–360 (2009) 1 Patient has received prior therapy with an EGFR TKI (monotherapy). 2 Tumor genotyping confirms the presence of a typical EGFR mutation that is associated with sensitivity to EGFR TKIs (i.e. exon 19 deletions, L858R, and G719X). 3 Patient achieves either a documented CR or PR or prolonged SD (≥ 6 months) based on RECIST or WHO criteria. 4 Disease progression occurs despite uninterrupted exposure to an EGFR TKI within 30 days. 5 Patient has not received additional systemic therapy between the cessation of EGFR TKIs and the initiation of new therapy.

4. Causes of Acquired Resistance to EGFR TKIs Genomic alterationsPhenotypic alterations Epithelial-to-Mesenchymal Transition Second site mutations By-pass signaling (Gene Amplifications) By-pass signaling (Gene Mutations) Gainor & Shaw JCO 2013 Sequist, et al. Sci Trasl Med 2011 NSCLC  SCLC

5. Genetic alterations in EGFR T790M mutations50% D761Y, T854A, L747S mutations<5% Bypass signaling tracks MET amplification5-22% HER2 amplification12% PIK3CA mutations5% BRAF mutations1% CRKL amplification9% HGF over-expressionRare cases Phenotypic alterations Transformation to SCLC3-14% Adapted from Gainor et al. J Clin Oncol, 2013 Percentage of Acquired-Resistance Mechanisms

6. Del Re M, et al Expert Rev Mol Diagn. 2014 EGFR T790M (~50%) MET ampl. (5-15%) HER2 ampl. (12%) PIK3CA mut. (5%) BRAF mut. (1%) SCLC trans. (3-14%) EMT (?%) Tumours from approximately 40% of pts with acquired resistance do not harbour a second-site mutation or MET amplification. Multiple investigations to identify resistance mechanisms are ongoing.

7. 57213161718-2122-2428 EGF binding TMTyrosine kinaseAutophosphorilatyon EXON 18192021 Nucleotide-binding loopActivation loop Chromosome 7 D761Y T790M Acquired-Resistance secondary mutations Second-site EGFR genetic alterations L747S V759L T854A EGFR gene The T790M accounts for ~90% of secondary mutations observed in EGFR Del Re M, et al Expert Rev Mol Diagn. 2014ins770-771

8. EGFR secondary somatic mutation in exon 20: T790M T790M: mutation that interfere with drug binding in the EGFR ATP pocket Michalczyk A et al. Bioorg Med Chem. 2008 Apr 1;16(7):3482-8. Threonine (~116 A) - Methionine (~163 A) STERIC HINDRANCE

9. Dead cells Progenitor cell Oncogenic activation Proliferation Additional Mutation Resistant tumor cells TKIs Primary mutation L858R (exon 21) Secondary mutation T790M (exon 20) Russo A. et Al Cancer Treatment Reviews. 2010 Clonal selection hypothesis How to explain the development of acquired resistance to TKIs

10. mod. da Alvaro L. J Thorac Oncol 2013 T790M mutation occurs exclusively in cis with the primary activating mutations in EGFR Same allele (in cis) Opposite allele (in trans) Different clone Fast growing Unpredictable growth behavior More indolent tumor growth

11. Retrospective study on 173 pts harboring sensitive EGFR mut. Lee Y. et al, Cancer 2014 Prognostic role of BASELINE T790M mutation TTP OS Pts with a high preexisting T790M % had worse clinical outcomes to EGFR-TKIs than patients with a low T790M % 25% of MT pts

12. Li W. et al, Lung Cancer 2014 Prognostic role of T790M-related ACQUIRED resistance PFSOS T790M+ T790M- T790M+ T790M- Prospective study on 369 pts harboring sensitive EGFR mut. progressed after TKI and treated with CT Lower rates of disease progression in T790M+ pts

13. Rationale of TKI-continuation or Re-challenge Following the discontinuation of TKI therapy disease flares may occur Re-responses to EGFR TKIs following a TKI-free interval (case report) Hata A, et al. J Thorac Oncol 2013 Pz ♂ (66 aa.) NSCLC Adenocarcinoma, EGFR mut a proportion of cells in a resistant tumour cell population remain sensitive to EGFR inhibition

14. Re-challenge of EGFR-TKI after a TKI-free interval Tumor Volume Time PR PD PR PD Start TKI Stop TKI IMAGING TKI-sensitive clone TKI-resistant clone Restart TKI TKI withdrawal

15. Advanced NSCLC EGFR+ TKI PD* Symptomatic Asymptomatic Brain Systemic Isolated lesion Multiple lesions Continue TKI First-line CT Local therapy +TKI Local therapy +TKI WBRT +TKI NCCN Clinical Recommendations 2015 Advanced NSCLC guidelines: sensitizing EGFRmut+ *according to RECIST criteria Isolated lesion Multiple lesions

16. TKI PD Prior to changing therapy, a biopsy is reasonable to determine mechanism of acquired resistance Tissue biopsy Liquid biopsy Advanced NSCLC EGFR+ Advanced NSCLC guidelines: sensitizing EGFRmut+ NCCN Clinical Recommendations 2016

17. mod. da Li et al, Oncogene, 2008; Ranson et al, WCLC, 2013 Afatinib Dacomitinib OsimerinibGefitinib Erlotinib EGFRm T790M Wt 1x 10x 100x Relative IC50 2 nd and 3 rd generation of TKIs to overcome T790M-mediated resistance

18. Li D et al. Oncogene. 2008 Aug 7;27(34):4702-11. % CONTROL VIABLE CELLS (72 HRS) DRUG CONCENTRATION (  M) Growth inhibition of lung cancer cell lines harboring T790M by AFATINIB and ERLOTINIB Erlotinib Afatinib 2 nd generation EGFR TKIs for overcoming resistance: AFATINIB

19. Reckamp KL, et al. Cancer 2014 Phase II study with Dacomitinib 66 pre-treated (CT and erlotinib) advanced NSCLC pts T790M SD = 3 pts PD = 3 pts 2 nd generation EGFR TKIs for overcoming resistance: DACOMITINIB (panHER inhibitor)

20. Janne PA, et al.. NEJM 2015 PFS AZD9291 in NSCLC EGFR mut. pts pre-treated with TKI (N=253) Overall population T790M+ T790M- ORR 3 rd generation EGFR TKIs for overcoming resistance: OSIMERTINIB (AZD9291)

21. Yu Z, et al.. Cancer Res 2007 Structural analysis of the EGFR kinase domain mutations Acquired Resistance to 3 rd generation EGFR TKI: C797S

22. Emergence of a Third Mutation in the EGFR Tyrosine Kinase Domain (case report) Pt ♀ (60 yr) NSCLC Adenocarcinoma, EGFR mut (del ex19) Yu HA, et al. JAMA Onc. 2015 Acquired Resistance to 3 rd generation EGFR TKI: C797S TKI Local therapy CT±TKI AZD9291 ?

23. Emergence of a Third Mutation in the EGFR Tyrosine Kinase Domain (case report) Mutation Analysis of Exon 20 of EGFR in Tumor Samples Yu HA, et al. JAMA Onc. 2015 Acquired Resistance to 3 rd generation EGFR TKI: C797S Erlotinib AZD9291 ?

24. The Allelic Context of T790M and C797S Acquired Mutations Niederst MJ, et al. Clin Cancer Res 2015 ?

25. Tumor Volume Time PR PD SD PD Start 1-2°g-TKI IMAGING TKI-sensitive EGFR resistance mutations in response to TKI treatment and clonal selection 3°g-TKI-resistant Stop 1-2°g-TKI Start 3°g-TKI

26. Acquired Resistance to EGFR TKIs: MET Amplification Turke AB, et al. Cancer Cell 2010 Engelman JA, et al. Science 2007 MET Amplification in EGFR TKI resistant cells (FISH) of MET/EGFR/CEP7 probe set Pre-treatment Post-treatment High-throughput FISH Pre-treatment Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC

27. Z. Wang et al. PLOSone 2014 Higher rate of c-MET amplification Decreased Increased Decreased OS PFS Survival analysis on EGFR-TKI according to the dynamic change of T790M Pts with decreasing trend of T790M from pre- to post-TKI Better prognosis Increased T790M in ctDNA during TKI Acquired Resistance to EGFR TKIs: MET Amplification

28. Combined mechanisms of resistance: T790M and MET amplif. Scheffler M, et al. J Thorac Oncol 2015 Pt ♂ (55 yr), NSCLC Adenocarcinoma, EGFR mut (L858R) Jun-2013 Lung -EGFR L858R -TP53 C277* -TP53 R110C Oct-2013 Muscle -EGFR L858R -TP53 C277* -  MET amp Jan-2014 Pleural eff. -EGFR L858R -EGFR T790M -TP53 R110C -  MET amp Apr-2014 Pleural eff. -EGFR L858R -EGFR T790M -TP53 R110C TKI  CTCTTKI Crizotinib + Erlotinib TKI Crizotinib + Erlotinib 3a g TKI

29. Baseline H&E SF Adenocarcinoma CT Cis+Eto Sequist, et al. Sci Trasl Med 2011 PD SCLC PR Pt ♀ (40 yr), NSCLC Adenocarcinoma, EGFR mut (ex19 del) TKI Erlotinib 36 mths Re-biopsy Other mechanisms of resistance: NSCLC  SCLC RE-BIOPSY TO CLARIFY THE MECHANISM OF ACQUIRED RESISTANCE BEFORE CHANGING THERAPY

30. EGFR inhibitors for overcoming resistance CONCLUSIONS  What are the mechanisms of resistance to EGFR TKIs? There are a several hypothesized mechanisms. The most studied are the T790 mutation.  Could the identification of the mechanisms of resistance be useful in clinical management? Yes. Many drugs targeting specifical mechanisms of resistance are now available. A sequential strategy should be used.