Targeting the MAPK pathway Josep Tabernero, MD PhD Medical Oncology Department Vall d’Hebron University Hospital & Vall d’Hebron Institute of Oncology Barcelona Great Debates & Updates in GI Malignancies NY, March 28th, 2014

DISCLOSURES Speaker’s Bureau Amgen Merck Kga Novartis Sanofi Roche

Advances in the treatment of mCRC 5-FU Oral fluoropyrimidines Irinotecan Oxaliplatin 2000 More options RR ≈ 50% mTTP 8−9 months mOS 17-21 months Optimal regimens Sequencing Treatment duration 1995 One option RR 50%+ mTTP 9+ months mOS 22+ months Cure 5-FU Oral fluoropyrimidines Irinotecan Oxaliplatin Cetuximab, Panitumumab Bevacizumab, Aflibercept Regorafenib 2014 Many options Optimal regimens Correct sequencing & dosing Cost Biomarkers 5-FU First-line efficacy RR 20% mTTP 5−6 months mOS 10−12 months Issues Low efficacy

Targeting the EGFR pathway in CRC TGF-α Amphiregulin Epiregulin Anti-EGFR MoAbs (RAS inh.) RAF inh. MEK inh. ERK inh. Inhibitors EGFR expression 27–95% KRAS mutation 45–50% NRAS mutation 5-8% KRAS EGFR Sos Grb2 BRAF BRAF mutation 5-10% EGFR mutation 1-2% MEK MAPK TGF = transforming growth factor Adapted from Roberts and Der. Oncogene 2007 4

CO.17: phase III trial Failed or intolerant to all recommended therapies REGISTER RANDOMI ZE Cetuximab* + BSC EGFR testing by IHC Disease progression or Unacceptable toxicity BSC alone 1:1 Stratification: Centre ECOG PS (0 or 1 vs. 2) Jonker et al, NEJM 2007;357:2040-8

NCIC CTG CO.17: mCRC Cetuximab vs BSC CO.17: phase III trial NCIC CTG CO.17: mCRC Cetuximab vs BSC HR OS: ITT 0.7  KRAS wt 0.55 1Jonker, DJ et al. NEJM;357:2040-8,2007 2Karapetis, CS. et al. NEJM;359:1757-65,2008

CRYSTAL: Study design Cetuximab + FOLFIRI R FOLFIRI Cetuximab IV 400 mg/m2 on day 1, then 250 mg/m2 weekly + irinotecan (180 mg/m2) + 5-FU (400 mg/m2 bolus + 2400 mg/m2 as 46-hr continuous infusion) + FA every 2 weeks EGFR-expressing mCRC R Stratification by: Regions ECOG PS Populations: Randomized patients (n=1217) Safety population (n=1202) ITT population (n=1198) FOLFIRI Irinotecan (180 mg/m2) + 5-FU 400 mg/m2 bolus + 2400 mg/m2 as 46-h continuous infusion) + FA every 2 weeks Van Cutsem E, et al. NEJM 2009

CRYSTAL study – final results ITT KRAS wild-type KRAS mutant FOLFIRI FOLFIRI + cetuximab (n=599) (n=350) (n=316) (n=87) (n=105) RR (%) 39 47 40 57 36 31 p=0.0038a p<0.001a p=0.35a mPFS (months) 8.0 8.9 8.4 9.9 7.7 7.4 Hazard ratio 0.85 0.7 1.17 p=0.048 p=0.0012 p=0.26 mOS (months) 18.6 19.9 20.0 23.5 16.7 16.2 0.93 0.8 1.04 p=0.30 p=0.0093 p=0.75 aCochran–Mantel–Haenszel test Van Cutsem E, et al. J Clin Oncol 2011; 29:2011-9 8

CRYSTAL study – final results by KRAS/BRAF status KRAS/BRAF wild-type KRAS wild-type / BRAF mutant FOLFIRI CETUXIMAB + FOLFIRI Cetuximab + FOLFIRI (n=289) (n=277) (n=33) (n=26) RR (%) 43 61 15 8.0 p<0.001a p=1.08a mPFS (months) 8.8 10.9 5.6 7.4 Hazard ratio 0.67 1.17 p=0.0013 p=0.87 mOS (months) 21.6 25.1 10.3 14.1 0.83 0.91 p=0.0547 p=0.74 aCochran–Mantel–Haenszel test Van Cutsem E, et al. J Clin Oncol 2011; 29:2011-9 9

PRIME trial FOLFOX4  panitumumab in 1st-line treatment of mCRC Design amended to focus on prospective hypothesis testing in the KRAS WT† stratum Study endpoints: PFS (1º); OS, ORR, safety, HRQoL L o n g t e r m f o l l o w u p E n d o f t r e a t m e n t Panitumumab 6 mg/kg (Q2W) + FOLFOX4 (Q2W) mCRC (n = 1183) R 1:1 Disease assessment every 8 weeks †WT in codons 12 and 13. HRQoL, Health-related quality of life; mCRC, metastatic colorectal cancer; OS, overall survival; PFS, progression-free survival; ORR, objective response rate. Douillard JY, et al. J Clin Oncol 2010;28:4697–705; www.amgentrials.com; protocol ID: 20050203; ClinicalTrials.gov identifier: NCT00364013.

KRAS, NRAS and BRAF mutation hotspots in the PRIME study EXON 2 EXON 3 EXON 4 EXON 1 12 13 61 117 146 BRAF EXON 15 EXON 16… EXON 1… 600 40% 4% 6% 3% 1% 59 8% All RAS mutation testing might identify an additional 15–20% mutants

PRIME RAS/RAF OS analysis* Overall survival Original WT KRAS exon 2 testing WT RAS 2 4 6 8 10 12 14 16 18 24 20 22 36 26 28 30 32 34 Proportion alive (%) 100 90 70 60 80 50 40 HR = 0.83 (95% CI, 0.67–1.02) P = 0.072 Months 2 4 6 8 10 12 14 16 18 24 20 22 36 26 28 30 32 34 Proportion alive (%) 100 90 70 60 80 50 40 Months HR = 0.78 (95% CI, 0.62–0.99) P = 0.043 Events n (%) Median (95% CI) months Panitumumab + FOLFOX4 (n = 325) 165 (51) 23.9 (20.3–28.3) FOLFOX4 (n = 331) 190 (57) 19.7 (17.6–22.6) Events n (%) Median (95% CI) months Panitumumab + FOLFOX4 (n = 259) 128 (49) 26.0 (21.7–30.4) FOLFOX4 (n = 253) 148 (58) 20.2 (17.7–23.1) *Predefined retrospective analysis; 7 patients harbouring Codon 59 mutations were not excluded from this analysis. 1. Douillard JY, et al. J Clin Oncol 2010;28:4697–705; 2. Douillard JY et al New Engl J Medicine Sept 12 2013.

Potential biomarkers in mCRC for EGFR inhibitors On/Off marker Threshold marker KRAS mut BRAF mut NRAS mut PIK3CA mut TP53 mut PTEN mut EGFR mut EGF & EGFR polymorphisms COX-2 PTEN EGF Epiregulin Amphiregulin EGFR expression DUSPs Mitogen-activated protein kinase phosphatase-1 (MKP-1) Signatures Genomic/Proteomic

BRAF mutant CRC Small population: 8-10% early stage 4-5% late stage BRAF V600E mutations as a biomarker? very poor prognosis in late stage (mCRC) no clear prognostic effect in early stage predictive: negative predictive effect for anti-EGFR MoAbs in some studies: Cetuximab: refractory (European cons.)1,2 & first-line setting (CRYSTAL study)3 Panitumumab: 2nd line setting (PICCOLO study)4 No change in the label by any regulatory authority 1 Di Nicolantonio F, J Clin Oncol 2018; 2 De Roock et al. Lancet Oncol 2010; 3Van Cutsem et al, J Clin Oncol 2011; 4Seymour MT et al, Proc ASCO 2011

Vemurafenib in BRAF V600E mutant mCRC (RECIST cutoff for PR, 30%) n=19 Evaluable Patients 1 PR and 4 MRs (≥10% shrinkage) Interim - 12/31/09 Outstanding activity of vemurafenib in metastatic melanoma with BRAF V600E mutations Preclinical activity shown in limited CRC BRAF V600 mutated cells1 Marginal activity in metastatic CRC with BRAF V600E mutations2 1Yang H et al. Clin Cancer Res 2011; 2Kopetz S et al, Proc ASCO 2010

BRAF inhibitors + EGFR inhibitors have in vivo activity in BRAFV600E mutant CRC xenografts Corcoran RB et al, Cancer Discov 2012;2:227-35 Prahallad A et al, Nature 2012;483:100-3

New studies in the BRAFV600E mutant CRC population As examples of clinical trials evaluating the combination of BRAFV600E inhibitors plus anti-EGFR inhibitors in the BRAF mutant population in CRC: NCT01524978: Vemurafenib + Cetuximab (BASKET) – Phase Ib NCT01750918: BRAF/MEK Inhibitors (dabrafenib + trametinib) + Panitumumab – Phase Ib RP2 NCT01719380: LGX818 and Cetuximab or LGX818, BYL719, and Cetuximab – Phase IbRP2

Connolly K et al. Curr Oncol 2014

RAS mutant CRC Big population: 50% KRAS mutations 5-8% NRAS mutations RAS mutations as a biomarker? no clear prognostic effect in early/late stage predictive: negative predictive effect for anti-EGFR MoAbs: KRAS for Cetuximab & Panitumumab: 1st, 2nd and late stage NRAS for Panitumumab: 1st & 2nd line NRAS for Cetuximab: 1st line 1 Di Nicolantonio F, J Clin Oncol 2018; 2 De Roock et al. Lancet Oncol 2010; 3Van Cutsem et al, J Clin Oncol 2011; 4Seymour MT et al, Proc ASCO 2011 4Douillard JY et al, N Engl J Med 2013; 5Stintzing S et al. Proc ECCO 2013

MEK inhibition leads to PI3K/AKT activation by relieving a negative feedback on ERBB receptors 1Turke A et al. Cancer Research 2012

New studies in the RAS mutant CRC population MEK inhibitors + EGFR inhibitors in RAS mutant CRC: CMEK162X2116: A phase Ib/II study of MEK162 in combination with panitumumab in patients with metastatic KRAS-mutant or -WT CRC MEK inhibitors + EGFR/HER3 inhibitors in RAS mutant CRC: GO29030: A phase Ib study of MEHD7945A in combination with cobimetinib (GDC-0973) in patients with metastatic KRAS-mutant cancers MEK inhibitors + IGF1R inhibitors in RAS mutant CRC: CMEK162X2111: A phase Ib study of AMG479 (anti IGF-1R) in combination with MEK162 in patients with solid tumors

Unsupervised molecular subtyping of CRC - Microarray

PIs: Justin Guinney Rodrigo Dienstmann

Summary Target discovery has resulted in numerous novel drugs in clinical development Different CRC subtypes: genomic signatures Tumor heterogeneity and clonal selection/evolution Signal transduction inhibition does not guarantee tumor response (BRAF V600 E mutant): Target presence and dependence Redundancy Cross-talk Need for molecular profile and selection Combinations: mechanistic interactions, rationale-based Need to sequentially evaluate tumor cells (tumor tissue, CTCs, cfDNA, …)