1. Mace L. Rothenberg, M.D. Professor of Medicine Ingram Professor of Cancer Research Biomarkers in Colorectal Cancer Management: KRAS Mutations and EGFR Inhibitors

2. Conflict of Interest Disclosure Consultant or Advisory Role AntigenicsOSI Array BioPharmaPfizer Bristol Myers-SquibbRoche Genentechsanofi-aventis IderaSynta ImCloneTakeda Johnson & JohnsonZymogenetics Novacea Stock Ownership SyntaTargeted Therapeutics

3. Biomarkers in CRC Management A specific, measurable, physical trait that can be used as a surrogate for a process of interest The trait can be a physical finding, a drug level, activation status of a molecule, or imaging characteristic The process that it reflects should be clinically meaningful: tumor presence or absence, response to therapy, development of toxicity, etc. What is a biomarker?

4. Prognostic Factor A measurement or characteristic present at the time of diagnosis that correlates with clinical outcome regardless of treatment Predictive Factor A measurement or characteristic present at the time of diagnosis or initiation of treatment that is associated with likelihood of response to therapy Definitions Biomarkers in CRC Management

5. Relationship of efficacy with KRAS status in patients with irinotecan-refractory mCRC treated with irinotecan and escalating doses of cetuximab – the EVEREST experience Biomarkers in CRC Management S Tejpar, M Peeters, Y Humblet, JB Vermorken, G De Hertogh, W. De Roock, J. Nippgen, A. von Heydebreck, C. Stroh, E. Van Cutsem

6. KRAS status and Outcome in CRC Patients Treated with Irinotecan and Standard or Escalating-Doses of Cetuximab: EVEREST Key Findings Escalating cetuximab dose until Grade 2+ skin toxicity occurs results in higher RR (30% vs 16%) – but not PFS (median 4.8 vs 3.9 months) or OS (median 8.6 vs 10.0 months) than with standard doses S Tejpar et al: ASCO 2007, Abst. 4037 Association of skin rash with PFS was present in KRAS wt and mutant subsets (but stronger in KRAS wt) There was a non-significant trend towards higher RR in the cetuximab dose escalation arm only in KRAS wt patients (42% vs 30%). No responses were seen in KRAS mutant patients, regardless of cetuximab dose. In fact, the rate of SD patients in the dose escalation arm was lower than the standard arm (33% vs 45%)

7. Conclusions Patients with KRAS wt tumors benefited from irinotecan + cetuximab treatment Agree In the dose escalation arm, a trend towards increased responses was observed in patients with KRAS wild-type tumors Yes, but the 39% relative improvement in RR was not matched by the 14% improvement in PFS KRAS status and Outcome in CRC Patients Treated with Irinotecan and Standard or Escalating-Doses of Cetuximab: EVEREST Dose escalation did not improve the efficacy in KRAS mutant tumors Agree

8. Conclusions Skin toxicity and KRAS status are independent predictors of outcome Yes, but KRAS was the much stronger of the two Predictive markers that act independently of KRAS were identified But these will only help us if we understand their biological significance KRAS status and Outcome in CRC Patients Treated with Irinotecan and Standard or Escalating-Doses of Cetuximab: EVEREST

9. C. Bokemeyer, I. Bondarenko, J. T. Hartmann, F. G. De Braud, C. Volovat, J. Nippgen, C. Stroh, I. Celik, P. Koralewski K-Ras status and efficacy of 1 st -line treatment of patients with mCRC with FOLFOX ± cetuximab: OPUS experience Biomarkers in CRC Management Abstract #4000

10. KRAS status and 1 st -line FOLFOX ± cetuximab: OPUS Key Findings In unselected patients, the addition of cetuximab to FOLFOX improves RR but not PFS

11. 37 61 0 10 20 30 40 50 60 70 Response rate (%) Cetuximab + FOLFOXFOLFOX KRAS wt 24% absolute  65% relative  FOLFOX ± Cetuximab RR in KRAS wild-type

12. Kaplan-Meier Estimate 0.5 1.0 0.4 0.3 0.2 0.1 0.0 0.6 0.7 0.8 0.9 8 02 4 610 12 Progression-free time (months) FOLFOX ± Cetuximab FOLFOX Cetuximab + FOLFOX PFS in KRAS wild-type Progression HR = 0.57 for FOLFOX + cetuximab

13. 49 33 0 10 20 30 40 50 60 Response rate (%) FOLFOXCetuximab + FOLFOX KRAS mt 16% absolute  33% relative  FOLFOX ± Cetuximab RR in KRAS mutant

14. Kaplan-Meier Estimate 0.5 1.0 0.4 0.3 0.2 0.1 0.0 0.6 0.7 0.8 0.9 8 02 4 610 12 Progression-free time (months) FOLFOX Cetuximab + FOLFOX FOLFOX ± Cetuximab PFS in KRAS mutant Progression HR = 1.83 for FOLFOX + cetuximab

15. 37 61 0 10 20 30 40 50 60 70 Response rate (%) Cetuximab + FOLFOXFOLFOX 49 33 0 10 20 30 40 50 60 Response rate (%) FOLFOXCetuximab + FOLFOX KRAS mt KRAS wt KRAS status and 1 st -line FOLFOX ± cetuximab: OPUS 12% absolute  32% relative 

16. 37 61 0 10 20 30 40 50 60 70 Response rate (%) Cetuximab + FOLFOXFOLFOX 49 33 0 10 20 30 40 50 60 Response rate (%) FOLFOXCetuximab + FOLFOX KRAS mt KRAS wt KRAS status and 1 st -line FOLFOX ± cetuximab: OPUS 28% absolute  46% relative 

17. KRAS status and 1 st -line FOLFOX ± cetuximab: OPUS Toxicity and Tolerability Patients treated with FOLFOX + cetuximab received roughly the same chemotherapy doses and dose intensity as those treated with FOLFOX alone But … There was a difference in patterns of toxicity based on KRAS status: Patients with KRAS wt tumors treated with FOLFOX + cetuximab tended to have a higher rate of Grade 3/4 hematological and GI toxicities than those treated with FOLFOX alone Patients with KRAS mutant tumors treated with FOLFOX + cetuximab had lower rates of these toxicities

18. KRAS status and 1 st -line FOLFOX ± cetuximab: OPUS Conclusions Addition of cetuximab to 1 st -line FOLFOX  RR and PFS in patients with KRAS wt tumors Agree Patients with KRAS mutant tumors do not profit from the addition of cetuximab Agree but … Is it possible that patients with KRAS mutant tumors are harmed by the addition of cetuximab to FOLFOX? Trend towards lower RR and shorter PFS when compared to those treated with FOLFOX alone is of concern.

19. KRAS status and 1 st -line FOLFOX ± cetuximab: OPUS Questions and Concerns - 1 Is this effect limited to FOLFOX or is it seen with other regimens like FOLFIRI See Van Cutsem - CRYSTAL presentation (Abst #2) – Sunday, June 1 and Cervantes poster (Abst #4129), Monday, June 2 Is this effect limited to cetuximab or is it also observed with panitumumb? See Cohn PRECEPT poster (Abst #4127) – Monday, June 2

20. KRAS status and 1 st -line FOLFOX ± cetuximab: OPUS Questions and Concerns - 2 Is this effect limited to 1 st -line therapies or is it seen in all lines of therapy? See Di Fiore poster discussion (Abst #4035) – Sunday, June 1 Is this effect seen with EGFR mAbs when used as a single agent? Apparently not (Amado – J Clin Oncol – 2008) These findings will determine whether KRAS status should be established prior to the use of EGFR mAbs in patients with mCRC

21. Possible Mechanisms for Resistance of KRAS Mutated Tumors to EGFR Inhibitors Ras-induced up-regulation of VEGF Zachary & Gliki: Cardiovasc Res 49:568-581, 2001 Activation of Ras   terminal differentiation and  tumor stem cell population KM Haigis et al: Nature Genetics 40:600-608, 2008 K-Ras mutation   DNA methylation   expression of tumor suppressor and apoptotic genes SK Patra: Exp Cell Res 314:1193-1201, 2008 KRAS mutation   expression or activity of DNA repair genes Pure speculation