James R. Rigas Comprehensive Thoracic Oncology Program Molecular Basis of Lung Cancer Therapy James R. Rigas Comprehensive Thoracic Oncology Program

Traditional View of Lung Cancer Adenoca Small Cell Large Cell Squamous

Presentation EGFR mutations ALK gene rearrangements BRAF mutations RADIANT adjuvant results in EGFR mutations Uncommon EGFR mutations Resistance in EGFR mutations (+) patients ALK gene rearrangements Resistance in ALK gene rearrangement (+) patients BRAF mutations Other mutations, fusions and amplifications

Locations and Types of the 134 EGFR Gene Mutations Detected in Lung Cancers Del 19 T790M Locations and types of the 134 epidermal growth factor receptor (EGFR) gene mutations detected in lung cancers. The structure of the EGFR gene is shown at left, and the locations and types of the mutations in the tyrosine kinase (TK) domain are shown at right. All mutations were located within exons 18–21, which encode the N lobe and part of the C lobe of EGFR (shaded area of the gene on the left, which is presented in magnified form on the right). Three major types of mutations (shown in bold) formed 94% of the 134 mutations detected and consisted of deletions in exon 19 immediately 5′ of the αC-helix (11 types, labeled Δ1–Δ11), duplications and/or insertions in exon 20 immediately 3′ of the αC-helix (eight types labeled D1–D8), and a single-point mutation, L858R (labeled M1), in the A-loop. The remaining 6% of mutations consisted of missense mutations in the P-loop in exon 18 (six types labeled M2–M7), in the αC-helix in exon 20 (a single type labeled M8), or in the A-loop in exon 21 (a single type labeled M9). L858R Shigematsu H et al. JNCI J Natl Cancer Inst 2005;97:339-346 Journal of the National Cancer Institute, Vol. 97, No. 5, © Oxford University Press 2005, all rights reserved.

Platinum-based doublet chemotherapy q3wks x 4 cycles* EURTAC Study Design Stage IIIB/IV NSCLC EGFR exon 19 deletion or exon 21 L858R mutation (DNA sequencing/Genescan and Taqman) Chemonaive ECOG PS 0–2 Measurable or evaluable disease Erlotinib 150 mg/day PD Stratification Mutation type ECOG PS (0 vs 1 vs 2) R Platinum-based doublet chemotherapy q3wks x 4 cycles* PD Secondary endpoints Objective response rate Overall survival (OS) Location of progression Safety EGFR mutation analysis in serum Quality of life Primary endpoint Progression-free survival (PFS) US FDA approval May 14, 2013 Cobas® EGFR Mutation Test 41 mutations in Exons 18, 19, 20 and 21 ECOG = Eastern Cooperative Oncology Group; PS = performance status; PD = progressive disease *Cisplatin 75mg/m2 d1 / docetaxel 75mg/m2 d1; cisplatin 75mg/m2 d1 / gemcitabine 1250mg/m2 d1,8; carboplatin AUC6 d1 / docetaxel 75mg/m2 d1; carboplatin AUC5 d1 / gemcitabine 1000mg/m2 d1,8 5 5

Primary endpoint: PFS in ITT population (updated analysis 26 Jan 2011) 1.0 0.8 0.6 0.4 0.2 Erlotinib (n=86) Chemotherapy (n=87) HR=0.37 (0.25–0.54) Log-rank p<0.0001 PFS probability 5.2 9.7 0 3 6 9 12 15 18 21 24 27 30 33 Time (months) Patients at risk Erlotinib 86 63 54 32 21 17 9 7 4 2 2 0 Chemo 87 49 20 8 5 4 3 1 0 0 0 0 Data cut-off: 26 Jan 2011 US FDA approval May 14, 2013

Study design US FDA approval July 12, 2013 Stage IIIB (wet)/IV lung adenocarcinoma (AJCC version 6) EGFR mutation in tumor (central lab testing; Therascreen EGFR29* RGQ PCR) Randomization 2:1 Stratified by: EGFR mutation (Del19/L858R/other) Race (Asian/non-Asian) Afatinib 40 mg/day† Cisplatin + Pemetrexed 75 mg/m2 + 500 mg/m2 i.v. q21 days, up to 6 cycles Primary endpoint: PFS (RECIST 1.1, independent review)‡ Secondary endpoints: ORR, DCR, DoR, tumor shrinkage, OS, PRO§, safety, PK *EGFR29:19 deletions in exon 19, 3 insertions in exon 20, L858R, L861Q, T790M, G719S, G719A and G719C (or G719X), S768I. †Dose escalated to 50 mg if limited AE observed in cycle 1. Dose reduced by 10 mg decrements in case of related G3 or prolonged G2 AE. ‡Tumor assessments: q6 weeks until Week 48 and q12 weeks thereafter until progression/start of new therapy. §Patient-reported outcomes: Q-5D, EORTC QLQ-C30 and QLQ-LC13 at randomization and q3 weeks until progression or new anti-cancer therapy. US FDA approval July 12, 2013 Yang JC, et al.

EGFR Gene Mutations in Adenocarcinoma Lung Cancer Therascreen ® EGFR (29) RGQ PCR Kit

PFS: Common mutations (Del19/L858R) Independent review – patients with Del19/L858R (n=308) 1.0 Afatinib n=204 Cis/pem n=104 PFS event, n (%) 130 (64) 61 (59) Median PFS (months) 13.6 6.9 Hazard ratio (95% confidence interval) 0.47 (0.34–0.65) p<0.0001 0.8 0.6 Progression-free survival (probability) 51% 0.4 0.2 21% 0.0 0 3 6 9 12 15 18 21 24 27 Progression-free survival (months) Number at risk Afatinib 204 169 143 115 75 49 30 10 3 0 Cis/Pem 104 62 35 17 9 6 2 2 0 0 US FDA approval July 12, 2013 Yang JC, et al.

RADIANT Study Schematic

RADIANT Study Results Hierarchical testing rendered all secondary endpoints non-significant.

LUX-Lung clinical trials and eligibility Treatment Line of treatment Mutation test LUX-Lung 2 Phase II N=129 Afatinib First- and second-line (after chemo) Direct sequencing (central) LUX-Lung 3 Phase III N=345 Afatinib vs. Pemetrexed/ cisplatin First-line EGFR29* (central) LUX-Lung 6 N=364 Afatinib vs. Gemcitabine/ cisplatin *EGFR mutations detected by TheraScreen EGFR29 test: Common: 19 deletions in exon 19 and L858R in exon 21 Uncommon: 3 insertions in exon 20, L861Q, T790M, G719S, G719A and G719C, S768I

EGFR mutation-positive patients in LUX-Lung trials Del19 n=408 L858R n=330 Uncommon n=100 LUX-Lung 2 Phase II N=129 n=52 n=54 n=23 LUX-Lung 3 Phase III N=345 n=170 n=138 n=37 LUX-Lung 6 N=364 n=186 n=40 Patients with uncommon mutations treated with afatinib Uncommon n=75 n=23 n=26 n=26

Subgroups of patients with uncommon mutations Categories De novo T790M Exon 20 insertions Other (exon 18, 19, 20, 21) n= 14 23 38 Mutations (n) T790M alone (3) T790M+Del19 (3) T790M+L858R (6) T790M+G719X (1) T790M+L858R+G719X (1) n/a L861Q alone (12) G719X alone (8) G719X+S768I (5) G719X+L861Q (3) E709G or V+L858R (2) S768I+L858R (2) S768I alone (1) L861P alone (1) P848L alone (1) R776H+L858R (1) L861Q+Del19 (1) K739_1744dup6 (1)

Tumour shrinkage in patients with uncommon mutations Independent review (n=67†) * De novo T790M (n=14): T790M alone(*), T790M+Del19, T790M+L858R, T790M+G719X, T790M+L858R+G719X Exon 20 insertions (n=20) Other (n=33): L861Q, G719X, G719X+S768I, G719X+L861Q, E709G or V+L858R, S768I+L858R, S768I, L861P, P848L, R776H+L858R, L861Q+Del19, K739_1744dup6 * * †8 patients were not included due to insufficient data

EGFR TKI resistance mechanisms T790M+ EGFR- TKI failures 2nd/3rd line NSCLC An area of high unmet medical need There are no current proven treatment options for these patients. Chemotherapy or EGFR-TKIs are used, but their benefit isn’t proven EGFR TKI resistance mechanisms AZD9291 or CO1686 “Patients with EGFR-mutant lung adenocarcinoma develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) after a median of 10-16 months. In half of these cases, a second EGFR mutation, T790M, underlies acquired resistance” Oxnard, et al. Clin Cancer Res Dec 6, 2010

Clinical activity AZD9291 in T790M+ Fast Track status granted by FDA in October 2013 Breakthrough Therapy Designation submission early February 2014 Best % change from baseline in target lesions in central testing T790M+ T790M+ Response Rate* = 64% (95% CI 53%-74%) For the 89 T790M+ evaluable patients there are 57 PRs (35 confirmed), 28 SDs, 4 PDs Population: T790M+ patients with observed or imputed target lesion data (n=89) *Confirmed+ unconfirmed response Imputations and exclusions Two patients with a BOR of PD are imputed as +20% increase as no follow up assessments for target lesions (E6004502, E6004504, both died prior to first scan). No patients are excluded Response ongoing for 34/35 T790M+ patients with confirmed PR D Discontinued treatment * Imputed T790M status is assigned by central testing of a recent tumour sample Independent review of scans is underway as per FDA request 41/43 confirmed so far Preliminary data, data cut off 16th January 2014

CO1686

FISH Assay for ALK Rearrangement* Telomere 2p23 region Centromere p25.2 p25.2 ALK 29.3 p24.3 p24.3 t(2;5) ALK gene breakpoint region p24.1 p24.1 p23.2 p23.2 p22.3 p22.3 p22.1 p22.1 EML4 42.3 p16.3 p16.3 p16.1 p16.1 3’ 5’ p14 p14 p13.2 p13.2 p12 p12 ~250 kb ~300 kb q12.1 q12.1 q12.3 q12.3 Break-apart FISH assay for ALK-fusion genes1 q14.1 q14.1 q14.3 q14.3 q21.2 q21.2 q22.1 q22.1 q23.2 q22.2 q22.2 q23.2 q24.1 q24.1 q24.3 q24.3 q31.3 q31.3 q32.1 q32.3 q33.2 q32.1 q32.3 q33.2 Non-split signal q34 q34 Split signal q36.1 q36.3 q37.2 q36.1 q36.3 q37.2 ALK break-apart FISH assay [Courtesy John Iafrate, Massachusetts General Hospital] *Assay is positive if rearrangements can be detected in ≥15% of cells FISH = fluorescence in situ hybridization 1Shaw AT et al. J Clin Oncol 2009;27:4247–4253

Crizotinib: First-in-human/Patient Trial Cohort 5 (n=6) 300 mg BID 2 DLTs: grade 3 fatigue Part 1: Dose escalation Cohort 6 (n=9) 250 mg BID MTD/RP2D Cohort 4 (n=7) 200 mg BID Cohort 3 (n=8) 200 mg QD 1 DLT: grade 3 ALT elevation Part 2: Molecularly enriched cohorts (ALK and c-MET) Cohort 2 (n=4) 100 mg QD Enrolling patients with ALK-positive NSCLC after preliminary observation of impressive activity in a few patients Data from database April 7, 2010 Data presented for 82 patients, study ongoing Cohort 1 (n=3) 50 mg QD ALT = alanine aminotransferase

Tumor Responses to Crizotinib for Patients with ALK-positive NSCLC 60 40 20 –20 –40 –60 –80 –100 Progressive disease Stable disease Confirmed partial response Confirmed complete response Maximum change in tumor size (%) –30% US FDA accelerated approval Aug 26, 2011 Full approval Nov 21, 2013 * *Partial response patients with 100% change have non-target disease present

Study Design – LDK378 (Ceritinib) Dose-Escalation Phase Advanced ALK-rearranged malignancies NCT01283516 Completed n=59 Dose escalation started at 50 mg/day Escalate to MTD (750 mg/day) Primary objective: determination of MTD Secondary objectives: characterize safety, PK, and antitumor activity Patients received treatment until disease progression, unacceptable toxicity, or withdrawal of consent Continuous oral dosing 21-day cycles Expansion Phase ALK-rearranged NSCLC Other ALK-activated tumors Additional n=71 enrolled Crizotinib naive Crizotinib pretreated Shaw AT et al. N Engl J Med. 2014;370:1189-1197.

Best Change (%) in Tumor Response from Baseline in NSCLC Patients Treated with Ceritinib 100 Crizotinib Pretreated Crizotinib Naive 80 PFS Event 60 40 20 Best Change (%) from Baseline -20 -40 -60 -80 US FDA accelerated approval Apr 30, 2014 -100 Based on investigator assessment of response. PFS, progression-free survival. Shaw AT et al. N Engl J Med. 2014;370:1189-1197. From N Engl J Med, Shaw AT, Kim DW, Mehra R, et al, Ceritinib in ALK-Rearranged Non–Small-Cell Lung Cancer, Vol 370, Page 1194. Copyright © 2014 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

BRAF-Mutations in NSCLC harbor BRAF V600E-mutations W. Pao, N. Girard Lancet Oncology, February 2011 A. Marchetti et al., J. Clin. Oncol. 29, 1, 2011 1,046 NSCLC samples Frequency of BRAF-mutations 3.5% BRAF-mut. in 4.9% adeno- and 0.3% SCC 57% BRAFV600E-mutations 43 % non-activating BRAF mutations 697 NSCLC-adeno. ca. Samples Frequency of BRAF-mutations 3% 50% BRAFV600E-mutations 50% non-activating BRAF mutations P. Paik et al., J. Clin. Oncol. 29, 2046, 2011 1.5-2.5 % of NSCLC harbor BRAF V600E-mutations

BRF113928: Initial Study Design Single arm, Phase II, open label Green-Dahlberg 2-stage: H(0): ORR ≤ 10% versus H(1): ORR ≥30% NSCLC (Adenocarcinoama) BRAFV600E-mutation ≥ 2nd Line dabrafenib 150 mg twice daily N = 40 Primary objective: Investigator-assessed ORR Secondary objectives: PFS, duration of response, OS, safety and tolerability, population PK

Interim Analysis (ASCO 2013) US FDA granted Breakthrough Therapy Jan 13, 2014 Investigator based response assessments demonstrated: 7PRs (5 confirmed—duration 29 and 49 weeks for 2 pts and remaining 3 patient 6+ to 24+ weeks) 1 stable disease 4 progressive disease Planchard et. al. 2013 ASCO Annual Meeting Proceedings

Rationale for the Combination Sustained target inhibition to observe more prolonged and durable anti-tumor effect Delay and potentially prevent the development of resistance Prevent/delay hyperproliferative lesions and secondary malignancies (Cu SCC) RAS BRAF V600 BRAF WT mutBRAF BRAFi + MEKi MEK pERK Proliferation, survival Invasion , Metastasis

Molecular Subsets Adenocarcinoma Lung Cancer MAP2K1 NRAS ROS1 fusions KIF5B-RET AKT1 PIK3CA BRAF HER2 ALK Fusions EGFR KRAS Unknown 1. Mascaux C et al. Br J Cancer 2005;92:131–9; 2. Winton et al. N Engl J Med 2005;352:2589–97; 3. Eberhard et al JCO 2005;23:5900–9; 4. Pao et al. PLOS Medicine 2005 2(1): e17; 5. Pao et al. Nat Med 2012;18:349–51; 6. Kris et al. ASCO 2011

Copy number alterations Point mutations & indels Gene expression Structural variants Translocations Fusions Inversion Copy number alterations Amplifications Deletions LOH Point mutations & indels Missense Nonsense Splice site Frameshift Gene expression Outlier expression Isoform usage Pathways & signatures Wild type AGTGA Mutant AGAGA Adapted from: Roychowdhury et al. Sci Transl Med; 20122

Conclusion Future Directions Molecular selection of patients improves therapy outcomes for patients with advanced adenocarcinoma of the lung Future Directions Extent patient selection and targeted therapies to squamous cell and earlier stages of NSCLC (i.e. RADIANT) Next generation sequencing needed Molecular characterization of resistance Development of a structure to conduct studies in uncommon molecular selected populations (BRAF, HER2, RET, etc) Clinical integration of tumor genomic and proteomic testing to better direct therapy for NSCLC (Gerber)