1. Exploring Biomarkers and Surrogates in Optimizing Patient Outcomes Eric Van Cutsem, MD, PhD Professor of Medicine University of Leuven Head, Digestive Oncology Unit University Hospital Gasthuisberg Leuven, Belgium This activity is supported by an educational donation provided by

2. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer About These Slides Disclaimer The materials published on the Clinical Care Options Web site reflect the views of the authors, not those of Clinical Care Options, LLC, the CME providers, or the companies providing educational grants. The materials may discuss uses and dosages for therapeutic products that have not been approved by the United States Food and Drug Administration. A qualified healthcare professional should be consulted before using any therapeutic product discussed. Readers should verify all information and data before treating patients or using any therapies described in these materials.  Users are encouraged to include these slides in their own presentations, but we ask that content and attribution not be changed. Users are asked to honor this intent.  These slides may not be published or posted online or used for any other commercial purpose without written permission from Clinical Care Options.  We are grateful to Eric Van Cutsem, MD, PhD, University Hospital Gasthuisberg, Leuven, Belgium, who aided in the content creation of these slides.

3. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Proliferation Metastasis Angiogenesis Apoptosis Resistance Shc PI3-K Raf MEKK-1 MEK MKK-7 JNK ERK Ras mTOR Grb2 AKT Sos-1 The Epidermal Growth Factor Receptor Pathway

4. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Predicting the Effect of EGFR Inhibitors  Skin rash  Measuring EGFR status –EGFR—IHC –FISH expression –Mutations –Gene levels  Measuring EGFR activation –KRAS –EGFR ligands –EGFR phosphorylation

5. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer 1 2 3 4 5 6 7 8 9 EGFR Inhibitor–Induced Skin Reactions Description of severe cases Post inflammatory effectsAcne-like rash Paronychia Dry skin Topical antiacne creams (drying effect) ± tetracyclines ± antihistamines Pruritus Pulse dye laser EmollientsHydrocolloid dressing or propylene glycol ± acetylsalicyl Antiseptic soaks, silver nitrate (pyogenic granuloma) Fissura Segaert S, Van Cutsem E, et al. Ann Oncol. 2005;16:1425-1433. Therapy Suggestions

6. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Correlation of Rash and Survival After Treatment With Cetuximab 1. Saltz L, et al. Proc ASCO. 2001. 2. Saltz L, et al. J Clin Oncol. 2004;22:1201- 1208. 3. Cunningham D, et al. N Engl J Med. 2004;351:337-345. 4. Van Cutsem E, et al. EORTC/NCI Geneva. 2004. 5. Xiong H, et al. J Clin Oncol. 2004;22:2610- 2616. 6. Kies MS, et al. Proc ASCO. 2002. 0 No reactionGrade 2 Grade 1 Grade 3 Survival (Months) 16 12 8 4 CRC 9923 Saltz (2001) [1] CRC 0141 Saltz (2004) [2] CRC BOND Cunningham [3] CRC Van Cutsem (2004) [4] Pancreatic Xiong (2004) [5] SCCHN Kies (2002) [6]

7. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Pooled Analysis: Predictive Value of Skin Toxicity With Panitumumab *Berlin study is interim. † Patients who developed progressive disease while receiving best supportive care in the phase III study were allowed to cross over to this extension study to receive panitumumab until disease progression or drug intolerability. Michelini T, et al. ASHP 2007. Abstract P141D. Van Cutsem E, et al. J Clin Oncol. 2007;25:1658-1664. Berlin J, et al. ASCO 2006. Abstract 3548. Mitchell EP, et al. ASCO 2007. Abstract 4082. Hecht JR, et al. Cancer. 2007;110:980-988. Van Cutsem, et al. 2007 Berlin, et al. 2006* Mitchell, et al. 2007 Hecht, et al. 2007 Phase III Panitumumab Crossover Phase IIIIII (ES † )II Patients enrolled, n23117793160150 Patients included in current analysis, n21816652145146 Dose schedule6 mg/kg q2w 2.5 mg/kg qw Response assessment RECIST central review RECIST local review WHO central review RECIST central review Assessment schedule Wks 8-48 and q3m after until PD q8w~ q8w for Wks 8-48 and q3m after until PD q9w

8. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Severity of Skin Toxicity Throughout Treatment Is Associated With PFS Michelini T, et al. ASHP 2007. Abstract P141D. 2.6 8.4 1.8 5.4 0 2 4 6 8 10 12 PFSOS Median Survival (months) Grade 2-4 Grade 0-1 Hazard ratio (grade 2-4:0-1): 0.66 (95% Cl: 0.56-0.78); P <.0001 Hazard ratio (grade 2-4:0-1): 0.62 (95% Cl: 0.5-0.74); P <.0001 Skin Toxicity

9. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer *There were no grade 4 skin reactions. Van Cutsem E, et al. ASCO 2007. Abstract 4000. Skin reaction grade 0 or 1 (n = 244) 0.02.55.07.510.012.515.017.520.0 PFS Time (Months) 1.00 0.75 0.50 0.25 0.00 PFS Estimate Skin reaction grade 2 (n = 243) Skin reaction grade 3* (n = 112) 11.3 months5.4 months 9.4 months CRYSTAL: PFS by On-Study Skin Reactions: Cetuximab + FOLFIRI

10. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer EVEREST: Study Design Day 22 Not eligible for randomization All patients continued to receive irinotecan Treatment until disease progression, unacceptable toxicity or withdrawal of consent. Skin and tumor biopsy at baseline, Week 3 and, in Arm B, at maximum cetuximab dose. Van Cutsem E, et al. WCGIC 2007. Abstract 0-034. Arm C Standard Cetuximab regimen (250 mg/m 2 /wk) Cetuximab 400 mg/m 2 initial dose then 250 mg/m 2 /wk + Irinotecan (180 mg/m 2 q2w) Arm A Standard cetuximab regimen (250 mg/m 2 /wk) Arm B Cetuximab dose escalation (dose increases of 50 mg/m 2 q2w up to maximum 500 mg/m 2 /wk) Screening

11. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer EVEREST: Most Common Treatment-Related Grade 3/4 AEs *Safety population includes 9 additional patients in Arm C who were not randomized because they discontinued study medication before day 22. † All grade 2 and 3 skin reactions in Arms A and B started after randomization. Van Cutsem E, et al. WCGIC 2007. Abstract 0-034. Adverse Event, % RandomizedNonrandomized Arm A standard (n = 45) Arm B escalation (n = 44) Arm C (n = 77*) Diarrhea1123 Fatigue795 Hypomagnesemia293 Neutropenia16221 Leucopenia228 Skin reaction  Any  Grade 1 (at any time)  Grade 1 (onset after randomization)  Grade 2 †  Grade 3 † 91 56 38 36 -- 98 41 34 45 11 91 23 -- 53 14

12. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer EVEREST: Efficacy Outcomes Van Cutsem E, et al. WCGIC 2007. Abstract 0-034. RandomizedNonrandomized Response OutcomesArm A standard (n = 45) Arm B escalation (n = 44) Arm C (n = 68) Best confirmed response, %  PR  SD  PD  Not evaluable 16 42 29 13 30 41 27 2 25 37 34 4 Confirmed response rate, % (95% CI) 16 (7-30) 30 (17-45) 25 (15-37) Disease control rate, % (95% CI) 58 (42-72) 71 (55-83) 62 (49-73) Median PFS, months (95% CI) 3.9 (2.8-5.3) 4.8 (3.8-6.7) 4.7 (2.8-6.2) Median OS, months (95% CI) 10.0 (6.0-12.1) 8.6 (7.3-12.5) 8.7 (6.4-12.6)

13. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Predicting the Effect of EGFR Inhibitors  Skin rash  Measuring EGFR status –EGFR—IHC –FISH expression –Mutations –Gene levels  Measuring EGFR activation –KRAS –EGFR ligands –EGFR phosphorylation

14. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer 0 10 20 30 40 50 22.9 20.0 22.2 24.2 20.8 24.7 22.7 7.1 31.3 0 9.4 4.8 12.7 11.8  10 > 10 -  20 > 35FaintWeak/ moderate Strong Irinotecan/cetuximab Cetuximab > 20 -  35 0 10 20 30 40 50 Percentage EGFR-expressing Cells (%)EGFR-staining Intensity Clinical Response Rate Correlation of Response Rate and EGFR Expression (IHC): BOND Data Cunningham D…Van Cutsem D, et al. N Eng J Med. 2004;351:337-345. P for trend =.87P for trend =.64

15. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer OS for Panitumumab by EGFR Tumor Membrane Staining Categories (IHC) 0.0 0.2 0.4 0.6 0.8 1.0 Months From Randomization 024681012141618202224 Survival Probability Van Cutsem E, et al. WCGIC 2005. Abstract O-027. EGFR Tumor Membrane Staining Category (IHC) > 35% (n = 76) 10% - 35% (n = 63) 1% - < 10% (n = 43)

16. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Correlation Between EGFR Gene Copy Number and Clinical Response  Metastatic colorectal cancer patients treated with cetuximab or panitumumab (N = 31) screened for EGFR copy number and mutation profile –Objective response (n = 10) –Stable or progressive disease (n = 21) Moroni M, et al. Lancet Oncol. 2005;6:279-286. 89.9 4.8 0 20 40 60 80 100 Objective responders Nonresponders Increased EGFR Copy Number by FISH (%) P <.0001

17. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer EGFR Inhibitors: EGFR Expression by FISH  Retrospective analyses suggest a correlation between anti-EGFR therapy and EGFR gene copy numbers by FISH [2,3]  Methodology issues for translation into clinical practice [4] Cetuximab Treatment of mCRC (n = 85) [1] 1. Cappuzzo F, et al. Ann Oncol. 2007;Epub. 2. Moroni M, et al. Lancet 2005;6:279-286. 3. Sartore-Bianchi A, et al. J Clin Oncol. 2007;25:3238-3245. 4. Personeni N, et al. J Clin Oncol. 2007;25:18S. Abstract 10569. 6.6 11.3 3.5 8.5 0 4 8 12 16 TTPOS Months EGFR FISH+ EGFR FISH- P =.02 P =.8

18. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer 100 70 30 0 20 40 60 80 100 < 2.47≥ 2.47 CR + PR PD + SD 100 32 0 20 40 60 80 100 < 43%≥ 43% 68 P =.0009 P =.0007 Patients (%) EGFR gene copy number Chromosome 7 polysomy or amplification EGFR Gene Copy Number and Outcome After Panitumumab Sartore-Bianchi A, et al. J Clin Oncol. 2007;25:3238-3245.  Survival, response outcomes on panitumumab associated with EGFR gene copy number

19. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer  In contrast to a subset of NSCLC patients, somatic EGFR gene mutations appear to be rare in CRC patients  However, EGFR inhibitors seem to be effective in cancer cells regardless of wild-type or mutated EGFR gene status Khambata-Ford S, et al. J Clin Oncol. 2007;25:3230-3237. Tsuchihashi Z, et al. N Engl J Med. 2005;353:208-209. EGFR Mutations

20. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer  Measuring EGFR phosphorylation by immunohistochemistry may predict higher response rates  Major methodological issues for translation into clinical practice pEGFR ≥ 7 (n = 7) pEGFR < 7 (n = 13) 0 20 40 60 80 100 Disease Control Rate (%) 100 54 P =.05 Level of EGFR Phosphorylation Personeni N, et al. Semin Oncol. 2005;32:S59-S62. EGFR Phosphorylation

21. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer EGFR Pathway and KRAS  EGFR pathway is the entry way to multiple intracellular signaling cascades  When KRAS gene is mutated, KRAS protein (p21 ras) is active regardless of EGFR activation  KRAS gene mutations are an early event and are found in 40% to 45% of CRC patients Mendelsohn J, et al. Oncogene. 2000;19:6550-6565. Khambata-Ford S, et al. J Clin Oncol. 2007;25:3230-3237.

22. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer OS With Cetuximab Is Significantly Reduced With Mutant KRAS  30 mCRC patients treated with cetuximab –43% with KRAS mutation  KRAS mutation observed in –0% of 11 responders –68% of 19 nonresponders –P =.0003  OS in patients without KRAS mutation was significantly better than patients with mutation Lievre A, et al. Cancer Res. 2006;66:3992-3995. 6.3 16.9 0 5 10 15 20 Median Survival (months) Mutated KRASWild type KRAS P =.016

23. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Predictive Role of KRAS in CRC Patients Treated With Cetuximab Khambata-Ford S, et al. J Clin Oncol. 2007;25:3230-3237. P =.003 11 51 89 46 0 20 40 60 80 100 Disease Control Group Patients (%) Mutant at KRAS codon 12 or 13 Wild-type KRAS Nonresponders

24. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Treatment (Panitumumab or Cetuximab) No. of Patients (WT:MT) Objective Response n (%) MTWT Liévre, et al (AACR Proceedings 2007) Cmab ± CT76 (49:27)0 (0)24 (49) Benvenuti, et al (Cancer Res, 2007) Pmab or cmab or cmab + CT 48 (32:16)1 (6)10 (31) De Roock, et al (Ann Oncol. 2007;Epub) Cmab or cmab + irinotecan 113 (67:46)0 (0)27 (40) Capuzzo, et al (Ann Oncol. 2007;Epub) Cmab ± CT81 (49:32)2 (6)13 (26) Di Fiore, et al (Br J Cancer. 2007) Cmab + CT59 (43:16)0 (0)12 (28) Khambata-Ford, et al (J Clin Oncol. 2007) Cmab80 (50:30)0 (0)5 (10) Single-Arm Studies: KRAS as a Biomarker for EGFR Inhibitors

25. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Lièvre A, et al. J Clin Oncol. 2008;26:374-379. Outcome & KRAS Status in Iri- Refractory mCRC Treated With Cmab KRAS status Median PFS (95% CI), n = 88 Median OS (95% CI), n = 88 KRAS mutation 10.1 weeks (8-16)10.1 months (5.1-13) Wild type 31.4 weeks (19.4-36)14.3 months (9.4-20) P Value.0001.026

26. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Survival & Skin Toxicity in Iri- Refractory CRC Treated With Cmab N = 113 Lièvre A, et al. J Clin Oncol. 2008;26:374-379. Skin toxicity 2-3 Skin toxicity 0-1 P =.029 13.9 8.2 0 5 10 15 20 Median Survival (months) Median OS

27. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer OS in Iri-Refractory CRC Treated With Cmab: KRAS and Skin Toxicity Lièvre A, et al. J Clin Oncol. 2008;26:374-379. P =.0008 5.6 10.7 15.6 0 5 10 15 20 Median OS Median Survival (months) 0 good prognostic factors (KRAS mutation and grade 0-1 skin toxicity) 1 good prognostic factors (wild type or grade 2-3 skin toxicity) 2 good prognostic factors (wild type and grade 2-3 skin toxicity)

28. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer KRAS in Irinotecan-Refractory mCRC Treated With Cetuximab and Irinotecan P =.020; HR: 0.620 (95% CI: 0.41-0.92) De Roock W…Van Cutsem E, et al. Ann Oncol. 2007;Epub. 43.0 10.7 0 10 20 30 40 50 Median Survival (weeks) KRAS wild type KRAS mutant 17 26 42 74 39 2 0 20 40 60 80 100 Wild type KRASMutant KRAS CR PR SD PD Best Response (%)

29. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Randomized by ECOG score 0-1 vs 2 and geographic region (Western EU vs Central and Eastern EU vs rest of world) Panitumumab vs Best Supportive Care in Colorectal Cancer: KRAS Analysis Van Cutsem E, et al. J Clin Onc. 2007;25:1658-1664. Panitumumab 6.0 mg/kg q2w + BSC (n = 231) PD Follow-up BSC (n = 232) Optional Panitumumab crossover study PD Follow-up  Hypothesis: clinical benefit of panitumumab would be confined to colorectal cancer patients with wild-type KRAS

30. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Progression-Free Survival: Final Analysis KRAS Evaluable Group PFSPmab + BSCBSC AloneHR (95% CI) Events/N (%)191/208 (92)209/219 (95) 0.59 (0.48-0.72) Median, wks8.07.3 Mean, wks15.49.6 Wild-Type KRAS Group PFSPmab + BSCBSC AloneHR (95% CI) Events/N (%)115/124 (93)114/119 (96) 0.45 (0.34-0.59); P <.0001 Median, wks12.37.3 Mean, wks19.09.3 Mutant KRAS Group PFSPmab + BSCBSC AloneHR (95% CI) Events/N (%)76/84 (90)95/100 (95) 0.99 (0.73-1.36) Median, wks7.47.3 Mean, wks9.910.2 Amado R...Van Cutsem E, et al. ECCO 2007. Abstract 0007.

31. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Objective Tumor Response and Progression-Free Survival Amado R...Van Cutsem E, et al. ECCO 2007. Abstract 0007. KRAS All EvaluableMutantWild Type Response, n (%) Pmab + BSC (n = 208) BSC (n = 219) Pmab + BSC (n = 84) BSC (n = 100) Pmab + BSC (n = 124) BSC (n = 119) CR0 (0) PR21 (10)0 (0) 21 (17)0 (0) SD52 (25)22 (10)10 (12)8 (8)42 (34)14 (12) PD104 (50)149 (68)59 (70)60 (60)45 (36)89 (75) CR, PR, SD73 (35)22 (10)10 (12)8 (8)63 (51)14 (12)

32. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer BRAF and KRAS Mutations in CRC  BRAF mutations in colorectal cancer present only in tumors that do not carry KRAS mutations [1] –BRAF linked to the ability of tumors to repair mismatched DNA bases in DNA –Results suggest that BRAF and KRAS mutations exert equivalent effects in tumorigenesis  Time to progression [2] –Shorter TTP with mutated KRAS vs wild type (P =.0443) –Equivalent TTP for mutated vs wild type BRAF (P =.5369) –Shorter TTP with KRAS or BRAF mutation vs wild type of both (P =.0259) 1. Rajagopalan H, et al. Nature. 2002;418:934. 2. Benvenuti S, et al. Cancer Res. 2007;67:2643-2648.

33. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer EGFR Ligand Expression: A Predictor for Increased PFS? EGFR Ligand Expression HighLow 0 20 40 60 80 100 120 140 Median PFS (Days) 103.5 days 115.5 days 57 days EREG (P =.0002) AREG (P =.0002) Single study data, further validation required in prospective studies n = 110, cetuximab monotherapy. Khambata-Ford S, et al. J Clin Oncol 2007;25:3230-3237.

34. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Association of PFS and OS with the baseline expression level of epiregulin (EREG) Van Cutsem E, et al. WCGIC 2007. Abstract 0–034. EVEREST Study: Analysis of Epiregulin Expression PFS Survival High Low 1.0 0.8 0.6 0.4 0.2 0.0 0 100 200 300 400 5000 200 400 600 800 High Low Refseq NM_001432 (EREG), 205767_at P =.013 Refseq NM_001432 (EREG), 205767_at P =.00033 Time (Days) Proof of PFS Proof of OS 1.0 0.8 0.6 0.4 0.2 0.0

35. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Polymorphism of the FC Receptors  Positive association between FC  2A H151H and FC  3A F158F genotype in 39 patients  These polymorphisms may indicate a role of ADCC Zhang W, Lenz H, et al. J Clin Oncol. 2007;25:3712-3718. Log-rank P =.004 FCGR2A H/H or H/R and FCGR3A F/F or F/V (n = 22) FCGR2A R/R or FCGR3A V/V (n = 13) PFS for patients with metastatic colorectal cancer receiving cetuximab by fragment c  receptor (FCGR) polymorphisms. 3.7 1.1 0 2 4 6 8 10 Median PFS Median PFS (months)

36. clinicaloptions.com/oncology Advances in Metastatic Colorectal Cancer Potential Predictors of Clinical Activity of Cetuximab, Panitumumab in mCRC  Skin reactions (“rash”) correlate with outcome  No role of IHC selection in CRC  No role of somatic EGFR gene mutations in CRC  Increased EGFR gene copy numbers assessed by FISH correlate with higher RR and PFS –Retrospective evidence from different studies, mainly in chemotherapy refractory patients; major methodology issues for clinical practice  High evidence for a role of KRAS gene mutations and lack of activity –Strong evidence from different studies, in chemotherapy-refractory patients treated with cetuximab (± irinotecan) and panitumumab  Other markers with emerging role: ligands  Need for data from first-line therapy randomized trials

37. Go Online for Virtual Presentations and Downloadable Slides! Download the PowerPoint slides from the symposium and listen and watch the CME-certified Virtual Presentations from: Edward Chu, MD Cathy Eng, MD J. Randolph Hecht, MD Eric Van Cutsem, MD, PhD clinicaloptions.com/CRCsatellite2007