1. Colon cancer: Combining molecular diagnostics and the art of medicine in the long term management of advanced colon cancer J. Randolph Hecht, MD Professor of Clinical Medicine Director, UCLA GI Oncology Program David Geffen School of Medicine at UCLA

2. Median Overall Survival in First-Line MCRC: The Golden Age BSC 06121824 Median OS (mo) ~4-6 mo 12-14 mo ~ 15-16 mo 20.3 mo ? 19-20 mo 5-FU/LV FOLFOX4 or CAPEOX IFL + bevacizumab IFL or FOLFIRI 21.5 mo FOLFOX6 FOLFIRI + bevacizumab BSC = best supportive care. *FOLFOX6 followed by FOLFIRI. 24 mo

3. BSC5-FUIFL FOLFOX/IRI

4. BSC5-FUIFLFOLFOX/IRI

5. Modern Chemotherapy for Unresectable Metastatic Disease: 50 Years of Work Infusional 5-FU is better than bolus (? Capecitabine) Irinotecan and Oxaliplatin: ? Equivalent 3 drug hypothesis (Grothey) Almost all the improvement in mCRC survival is due to better cytotoxic therapies, not biologic therapies

6. 1st Line Metastatic Colorectal Cancer Irinotecan IFL > 5-FU Oxaliplatin FOLFOX > 5-FU FOLFOX > IFL FOLFOX = FOLFIRI

7. Treatment: Chemotherapy 1st line therapy: CPT-11+5FU/LV (Saltz) –Median survival 14.8 vs. 12.6 mo. –Similar to Douillard, Lancet 2000 NEJM, 2000

8. 1st Line Metastatic Colorectal Cancer Irinotecan IFL > 5-FU Oxaliplatin FOLFOX > 5-FU FOLFOX > IFL FOLFOX = FOLFIRI

9. 0 10 20 30 40 50 60 70 80 90 100 012 Years % Alive IFL (med 15.0 mo) FOLFOX4 (med 19.5 mo) IROX (med 17.4 mo) FOLFOX4 vs IFL (P=.0001; HR=0.66) IROX vs IFL (P=.04) N9741: Overall Survival Goldberg et al. J Clin Oncol. 2004;22:23.

10. 1st Line Metastatic Colorectal Cancer Irinotecan IFL > 5-FU Oxaliplatin FOLFOX > 5-FU FOLFOX > IFL FOLFOX = FOLFIRI

11. Colucci, G. et al., J Clin Oncol; 23:4866-4875 2005. FOLFOX n = 172, 34% RR FOLFIRI n = 164, 31% RR P = 0.60 FOLFOX vs. FOLFIRI in First-Line Metastatic CRC: Overall Survival

12. Access to Chemotherapy Improves Survival Grothey J Clin Oncol 2005;23:9441–9442

13. Capecitabine Converted to systemic 5-FU Single agent trials comparable to 5-FU CapeOx = FOLFOX (Cassidy) ? CapeIRI –Some trials with excessive toxicity –? Reduced efficacy BICC-C (Fuchs)

14. Modern Synthesis of Chemotherapy for mCRC 2012 Combination chemotherapy if possible “Continuum of Care” Chose for toxicity, not efficacy 2nd Line Chemotherapy Whatever they didn’t get 1st line

15. Molecular Markers Colorectal cancers are molecularly heterogeneous –MSI, KRAS, CIMP –We already do this in other cancers –Cytotoxics have targets too! –And yet we treat them all the same! –Still no validated markers for efficacy or toxicity (sorry Response Genetics!) Eng at al. BMC Cancer 2010

16. Advanced Disease: “Standard” Biological Therapies Angiogenesis inhibitors –Cancers need new vasculature to grow beyond a certain size (Folkman) –Multiple angiogenic factors –Hypoxia Epidermal growth factor receptor (EGFR) antagonists –Member of HER (ErbB) family of tyrosine kinases –Multiple ligands: TGF- , EGF, amphiregulin, betacellulin, HB-EGF, and epiregulin –Undergoes hetero and homodimerization –Activation of multiple downstream pathways including Ras, ERK1/2, PI3K/AKT, and STAT pathways –Leads to proliferation, survival, angiogenesis, and metastasis

17. The VEGF and VEGF-Receptor Family VEGF regulates angiogenesis via interaction with receptor tyrosine kinases –VEGFR-2/KDR and VEGFR-1/Flt-1 VEGFR-1 (Flt-1) VEGF-A Receptor isoforms Ligand isoforms VEGFR-2 (KDR) VEGF-B VEGFR-1s Angiogenesis VEGF-E VEGF-C VEGF-D VEGFR-3 (Flt-4) Lymph angiogenesis tumor metastases Extracellular Intracellular VEGF-A 165 NRP-1 PlGF

18. Agents Targeting the Vascular Endothelial Growth Factor (VEGF) Pathway VEGFR-2 VEGFR-1 P P P P P P P P Endothelial cell Small-molecule VEGFR inhibitors (regorafenib, PTK787, sunitinib) Anti-VEGFR antibodies (IMC-1121b) Soluble VEGF receptors (aflibercept) VEGF Anti-VEGF antibodies (bevacizumab)

19. First-Line Irinotecan/5-FU/LV + Bevacizumab Hurwitz H et al. N Engl J Med. 2004;350:2335-2342. HR = 0.66, P <.001 Percent Surviving Duration of Survival (months) 1.0 0.8 0.6 0.4 0.2 0.0 010203040 IFL/bevacizumab IFL/placebo 20.315.6 10.6 100 80 60 40 20 0 0102030 Progression-free Survival (%) Progression-Free Survival (months) 6.2 (n = 402) (n = 411) HR = 0.54, P <.001

20. How was this information processed by the oncology community ? FOLFOX/bevacizumab became the standard of care –FOLFOX > IFL –IFL+bev > IFL –Ergo, FOLFOX+bev must be > FOLFOX alone, right? What was the FOLFOX/ bevacizumab data? –Giantonio E3200 second line –TREE phase II trials –NO16966

21. PFS chemotherapy + bevacizumab superiority: primary objective met 0510152025 Months PFS estimate HR = 0.83 [97.5% CI 0.72–0.95] (ITT) p = 0.0023 9.48.0 1.0 0.8 0.6 0.4 0.2 0 FOLFOX+placebo/XELOX+placeboN=701; 547 events FOLFOX+bevacizumab/XELOX+bevacizumab N=699; 513 events

22. Second Line Bevacizumab: E3200 FOLFOX4 + Bevacizumab (10 mg/kg, q 2 weeks) FOLFOX4 Bevacizumab (10mg/kg, q 2 wks) Previously treated metastatic CRC PD Stratification factors: –ECOG PS: 0 vs 1, 2 –Prior XRT Giantonio PASCO 2005

23. E3200: Overall Survival P r o b a b i l i t y 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 OS (months) 0369121518212427303336 ALIVEDEADMEDIANTOTAL A:FOLFOX4 + bevacizumab 2892464312.9 B:FOLFOX42902573310.8 C:bevacizumab2432162710.2 HR = 0.76 A vs B: p = 0.0018 B vs C: p = 0.95 Giantonio BJ, et al. ASCO 2005

24. Bevacizumab Beyond Progression Bevacizumab Approved “in combination with intravenous 5-fluorouracil– based chemotherapy, is indicated for first- OR second-line treatment of patients with metastatic carcinoma of the colon or rectum.” (PI) What is the data supporting second-line bevacizumab in bevacizumab failures? BRITE Registry: Nonrandomized! ML 18147/AIO Trial FOLFIRI +bev > FOLFIRI OS PR 1/26/12

25. Aflibercept (VEGF-TRAP) Fully human fusion protein and soluble recombinant decoy VEGF receptor composed of Domain 2 of VEGFR1 and Domain 3 of VEGFR2 fused to the Fc of IgG1 Higher affinity for VEGF-A than bevacizumab and also blocks PlGF; T 1/2 17 days Where has it been?

26. EFC10262: VELOUR Phase III Trial 2 nd Line FOLFIRI +/- VEGF-TRAP (Aflibercept) Stratification factors: Prior bevacizumab (Y/N) ECOG PS (0 vs 1 vs 2) 1:1 mCRC after failure of an oxaliplatin based regimen R 600 pts Aflibercept 4 mg/kg IV + FOLFIRI q 2 weeks 600 pts Placebo + FOLFIRI q 2 weeks 26 30% of patients had prior BEV PI: Allegra N=1226

27. VELOUR: Results Van Cutsem, et al. WCGC 2011

28. VELOUR Discussion 30% had received bev, reportedly similar results How does this compare to bev 2nd line? What about EGFR Ab in KRAS WT: SPIRITT? Does bev treatment change the tumor?

29. Regorafenib: What A Difference a F Makes!

30. Regorafenib: Small molecule inhibitor of VEGFR and FGFR-1 CORRECT Trial Grothey et al. 760 pts 2:1 Chemorefractory mCRC vs BSC, interim analysis PFS: 1.9 v 1.7m (HR=0.493) p<0.000001 OS: 6.4 v 5.0m (HR=0.773) p=0.0051 Statistically positive but is it clinically significant?

31. Overall survival (primary endpoint) Primary endpoint met prespecified stopping criteria at interim analysis (1-sided p<0.009279 at approximately 74% of events required for final analysis) 1.00 0.50 0.25 0 0.75 200100500150300250400350450 Days from randomization Survival distribution function Placebo N=255 Regorafenib N=505 Median 6.4 mos 5.0 mos 95% CI 5.9–7.3 4.4–5.8 Hazard ratio: 0.77 (95% CI: 0.64–0.94) 1-sided p-value: 0.0052 RegorafenibPlacebo

32. Why these results? Possibly benefit from long term anti-VEGF inhibition (BRITE) Can anti-VEGF therapy worsen post-therapy outcome? (Bevacizumab Addiction) –Bevacizumab only leads to modest improvement in OS –VEGF inhibition may up-regulate other parts of pathway and other pathways –Preclinical models of increased metastasis with VEGFR-2 inhibition (Rip- TAG Paez-Ribes, 2009 and sunitinib conditioning Ebos, 2009) –Differences between PFS and OS with PTK/ZK (Hecht ECCO 2007)

33. Yarden & Sliwkowski Nat Rev Mol Cell Biol 2001

34. BOND Trial: Randomized Phase II Study With Cetuximab and Irinotecan % RR for cetuximab alone (n) % RR for cetuximab + irinotecan (n)P-value All patients11 (111)23 (218)0.007 Irinotecan- oxaliplatin failure 11 (44)24 (80)0.09 Irinotecan refractory 15 (69)26 (1320.07 Cetuximab PI. February 2004

35. What About Earlier in Treatment? 1st Line –High RR in phase II trials –CRYSTAL: FOLFIRI +/- cetuximab 2nd Line –EPIC: irinotecan +/- cetuximab PFS not OS But –Clearly, only some helped –Significant toxicities, particularly with long-term use in most

36. Why Do We Try to Predict Response? Modestly effective drugs that help a subset Toxicity Expense

37. Predictors of Response

38. NSCLC Cancer EGFR mutation, K-ras wt correlate with response CRC Mutations rare EGFR staining not helpful (Hecht, 2010) EGFR copy # (Moroni, 2005) Ligands: amphiregulin, epiregulin (Ford 2007) K-ras (Lievre, 2006; DiFiore 2007)

40. Extracellular Intracellular Ligand EGFR PI3K Akt Ras Raf MEK MAPK Cell motility Metastasis Angiogenesis Proliferation Cell survival DNA PTEN

42. KRAS as a Biomarker for Panitumumab Response in Metastatic CRC PFS log HR significantly different depending on KRAS status (p <.0001) Percentage decrease in target lesion greater in patients with wild-type KRAS receiving panitumumab Approved in EU in KRAS WT Patients With Mutant KRAS Mean in Wks Stratified log rank test: P <.0001 115/124 (93) Patients With Wild-Type KRAS 1.0 0.9 Proportion With PFS 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 024 6810 Events/N (%) Median in Wks Pmab + BSC BSC alone 114/119 (96) 12.3 7.3 19.0 9.3 HR: 0.45 (95% CI: 0.34–0.59) 12141618 20 2224262830 32 343638 40 42 44 46485052 Weeks Proportion With PFS 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 24 681012141618 20 2224262830 32 343638 40 42 44 464850 Weeks Pmab + BSC BSC alone Mean in Wks 76/84 (90) Events/N (%) Median in Wks 95/100 (95) 7.4 7.3 9.9 10.2 HR: 0.99 (95% CI: 0.73–1.36) 52 Amado et al., JCO 2008.

43. EGFR Abs Earlier in Treatment in the Era of KRAS CRYSTAL positive, but others (COIN, NORDIC VII) negative Second line trials (EPIC, 181) negative for OS SPIRITT: FOLFIRI+bev vs FOLFIRI+Pmab 2012 Role in second line remains unclear

44. Other Biomarkers For Anti- EGFR Abs? BRAF KRAS G13D

45. BRAF Background BRAF is a serine/threonine kinase downstream of KRAS V600E mutation is found in 5-10% of CRCs Mutation mutually exclusive with KRAS mut Correlatated with poor prognosis –Ogino, Gut, 2009 –Tol, NEJM, 2009 (letter) Retrospective studies correlated with lack of response –Di Nicolantonio, JCO, 2008

46. Does BRAF Mutation Identify Nonresponders? Extracellular Intracellular Ligand EGFR PI3K Akt Ras Raf MEK MAPK Cell motility Metastasis Angiogenesis Proliferation Cell survival DNA PTEN

47. Clinical efficacy in KRAS wild-type tumors by BRAF mutation status: CRYSTAL KRAS wt/BRAF wt (n=566) KRAS wt/BRAF mt (n=59) FOLFIRI (n= 289) Cetuximab +FOLFIRI (n= 277) FOLFIRI (n=33) Cetuximab +FOLFIRI (n=26) Median OS mo [95% CI] 21.6 [20.0–24.9] 25.1 [22.5–28.7] 10.3 [8.4–14.9] 14.1 [8.5–18.5] HR [95% CI] p-value a 0.830 [0.687–1.004] 0.0549 0.908 [0.507–1.624] 0.7440 Median PFS mo [95% CI] 8.8 [7.6–9.4] 10.9 [9.4–11.8] 5.6 [3.5–8.1] 8.0 [3.6–9.1] HR [95% CI] p-value a 0.679 [0.533–0.864] 0.0016 0.934 [0.425–2.056] 0.8656 OR rate (%) [95% CI] 42.6 [36.8–48.5] 61.0 [55.0–66.8] 15.2 [5.1–31.9] 19.2 [6.6–39.4] p-value b <0.00010.9136 CI, confidence interval; OR, best overall response; OS, overall survival; PFS, progression-free survival; mo, months; mt, mutant; wt, wild-type a Stratified log-rank test; b Cochran-Mantel-Haenszel test Van Cutsem GI ASCO 2010

48. KRAS G13D mutations: Effect on outcome in pts with mCRC treated with First Line Chemotherapy +/- cetuximab Tejpar et al. Abs 3511 ASCO 2011.

49. Once Again Larger Numbers Needed: Peeters GI ASCO 2012 Analysis of PRIME, 181, 408 data sets: 2606 pts! 40-45% KRAS codon 12 or 13 mutations No allele prognostic for PFS or OS G13D worse prognosis in PRIME Would not treat G13D pts with an anti-EGFR Ab

50. What is the treatment of mCRC in 2012? 1st Line –FP/Ox +bevacizumab (timing of reintroduction?) –FOLFIRI+ bevacizumab 2nd Line –If FP/OX+bevacizumab 1st line FOLFIRI+bevacizumab ? FOLFIRI+aflibercept (FP)+IRI+panitumumab or cetuximab (KRAS WT) –IF FOLFIRI+bevacizumab 1st line FP/Ox+bevacizumab (FP)+IRI+panitumumab or cetuximab (KRAS WT) 3rd Line (KRAS WT) –Whatever wasn’t used Salvage –? Regorafenib