*University Hospital Gasthuisberg, Leuven, Belgium Cetuximab plus FOLFIRI: Final data from the CRYSTAL study on the association of KRAS and BRAF biomarker status with treatment outcome Eric Van Cutsem*, I. Lang, G. Folprecht, M. Nowacki, C. Barone, I. Shchepotin, J. Maurel, D. Cunningham, I. Celik, C-H. Köhne. *University Hospital Gasthuisberg, Leuven, Belgium (Presenting author)

Background (1) In the CRYSTAL study,1 patients with KRAS wild-type tumors (KRAS wt) treated 1st-line with FOLFIRI plus cetuximab compared with FOLFIRI alone experienced: A significantly reduced risk of disease progression (hazard ratio [HR], 0.68, p=0.02) An increased chance of tumor response (odds ratio, 1.91) This confirmed earlier findings that cetuximab efficacy was confined to patients with KRAS wt metastatic colorectal cancer (mCRC)2,3,4 1Van Cutsem E, et al. N Engl J Med 2009;360:1408-17 2Bokemeyer C, et al. J Clin Oncol 2009;27:663-71 3De Roock W, et al. Ann Oncol 2008;19:508-15 4Lievre A, et al. J Clin Oncol 2008;26:374-9

Background (2) Serine-threonine kinase BRAF is a direct downstream effector of KRAS BRAF gene mutations have been detected in 8% of colon cancers (stage II/III – PETACC-3)1 BRAF mutation status has been suggested to be predictive of cetuximab efficacy in pretreated patients with mCRC2,3 1Roth A, et al. J Clin Oncol 2010; 28:466-74 2Di Nicolantonio F, et al. J Clin Oncol 2008;26:5705-12 3Tejpar S et al on behalf of EU consortium, ECCO/ESMO, 2009

Background (3) An updated analysis of the CRYSTAL study primary analysis population showed: Significantly longer survival in the FOLFIRI plus cetuximab vs FOLFIRI alone arm (HR 0.88, p=0.04)1 In an updated retrospective analysis, the impact of BRAF mutation status on cetuximab efficacy in patients with KRAS wt tumors was investigated 1Lang I, et al. Eur J Cancer Supplements 2009; 7: S345; P-607

CRYSTAL study endpoints Primary endpoint Progression-free survival (PFS) Secondary endpoints Overall survival (OS), best overall response (OR), safety Retrospective analysis The effect of KRAS and BRAF tumor mutation status on PFS time, OR and OS time

Stratification factor: The CRYSTAL study FOLFIRI EGFR - expressing R mCRC Stratification factor: FOLFIRI + cetuximab ECOG PS 0-1, 2 FOLFIRI (q2w) Cetuximab Irinotecan 180 mg/m2, day 1 400 mg/m2 initial dose then 5-FU 400 mg/m2 bolus then 250 mg/m2 weekly 600 mg/m2 infusion, day 1 and 2 LV 200 mg/m2, day 1 Treatment until progression, symptomatic deterioration or unacceptable toxicity ECOG PS, Eastern Cooperative Oncology Group performance status; 5-FU, 5-fluorouracil; LV, leucovorin

KRAS/BRAF tumor mutation analysis Sample numbers for KRAS and BRAF mutation status were increased by using DNA extracted from formalin fixed paraffin embedded slide mounted tumor sections prepared to evaluate tumor EGFR expression KRAS mutations at codons 12/13 and BRAF (V600E) mutations were detected using a polymerase chain reaction clamping and melting curve technique1 1Van Cutsem E, et al. N Engl J Med 2009;360:1408-17

CRYSTAL study data cut-offs PFS and overall response by independent review committee 27 July, 2006 Overall survival 31 May, 2009

Patient characteristics (1) Of 1198 patients in the primary analysis population 1063 (89%) were evaluable for KRAS mutation status 1000 (83%) were evaluable for BRAF mutation status BRAF mutations were detected in 60/1000 (6%) evaluable samples 1 tumor was both KRAS mt and BRAF mutant (BRAF mt) 666/1063 (63%) patients had KRAS wt tumors

Patient characteristics (2) 625 KRAS wt tumors were evaluable for BRAF mutation analysis 566 (91%) were BRAF wt 59 (9%) were BRAF mt

Patient characteristics (3) Baseline characteristics were generally balanced across populations and by treatment group1 Noteworthy differences were in patients with KRAS wt/BRAF mt tumors receiving FOLFIRI plus cetuximab vs FOLFIRI: ≥65 years old (50% vs 33%) Liver metastases only (35% vs 12%) ECOG PS 2 (0 vs 9%) 1Van Cutsem E, et al. ASCO Gastrointestinal Cancer Symposium Proceedings 2010: Abstract 281

Efficacy data in patients with KRAS wt tumors KRAS wt/BRAF wt (n=566) KRAS wt/BRAF mt (n=59) FOLFIRI n=350 FOLFIRI + cetuximab n=316 n=289 n=277 n=33 n=26 Overall survival Median, mo [95% CI] 20.0 [17.4‒21.7] 23.5 [21.2‒26.3] 21.6 [20.0‒24.9] 25.1 [22.5‒28.7] 10.3 [8.4‒14.9] 14.1 [8.5‒18.5] Hazard ratio p-value 0.796 [0.670‒0.946] 0.0093 0.830 [0.687‒1.004] 0.0547 0.908 [0.507‒1.624] 0.7435 PFS 8.4 [7.4‒9.2] 9.9 [9.0‒11.3] 8.8 [7.6‒9.4] 10.9 [9.4‒11.8] 5.6 [3.8‒8.1] 8.0 [3.6‒9.1] P-value 0.696 [0.558‒0.867] 0.0012 0.673 [0.528‒0.858] 0.0013 0.934 [0.425‒2.056] 0.8656 Tumor response OR rate (%) 39.7 [34.6‒45.1] 57.3 [51.6‒62.8] 42.6 [36.8‒48.5] 61.0 [55.0‒66.8] 15.2 [5.1‒31.9] 19.2 [6.6‒39.4] Odds ratio 2.069 [1.515‒2.826] <0.0001 2.175 [1.551‒3.051] 1.084 [0.264‒4.446] 0.9136 CI, confidence interval; mt, mutant; PFS, progression-free survival; OR, best overall response rate; OS, overall survival; wt, wild-type

Tumor regression according to treatment status in patients with KRAS wt tumors -100 -80 -60 -40 -20 20 40 60 80 100 FOLFIRI, n=350* FOLFIRI + cetuximab, n=316** % change in lesion (SOPD) *Data for 21 patients were missing; **Data for 16 patients were missing wt, wild-type; SOPD, sum of the product diameters In patients with KRAS wt tumors the addition of cetuximab to FOLFIRI compared with FOLFIRI alone, led to a mean difference in the best % change in sum of the product diameters of 13.9

PFS by subgroups in KRAS wt patients Subgroup (number of patients in Group A vs B) HR [95% CI] All KRAS wt (316 vs 350) 0.70 [0.56‒0.87] Age <65 years (200 vs 234) 0.66 [0.50‒0.87] ≥65 years (116 vs 116) 0.79 [0.54‒1.15] Gender Male (196 vs 211) 0.60 [0.44‒0.80] Female (120 vs 139) 0.83 [0.59‒1.17] ECOG PS 0 - 1 (303 vs 336) 0.68 [0.54‒0.85] 2 (13 vs 14) 1.03 [0.44‒2.43] Number of metastatic sites ≤2 (277 vs 295) [0.55‒0.89] >2 (33 vs 49) 0.78 [0.43‒1.42] Liver metastases only Yes (68 vs 72) 0.56 [0.32‒0.97] No (248 vs 278) 0.74 [0.58‒0.94] Leukocytes ≤10000/mm3 (258 vs 284) [0.55‒0.90] >10000/mm3 (48 vs 58) 0.73 [0.43‒1.26] LDH at baseline > upper normal range (138 vs 150) 0.75 [0.54‒1.05] ≤ upper normal range (144 vs 161) 0.69 [0.50‒0.97] Alkaline phosphatase at baseline ≥300 U/L (30 vs 42) 0.77 [0.39‒1.52] <300 U/L (272 vs 295) [0.53‒0.86] Prior adjuvant chemotherapy Yes (80 vs 73) [0.49‒1.21] No (236 vs 277) 0.67 [0.52‒0.87] 0.3 0.4 0.5 1 2 3 4 HR and 95% CI Benefit under cetuximab No benefit under cetuximab Group A, FOLFIRI + cetuximab; Group B, FOLFIRI CI, confidence interval; ECOG PS, Eastern Cooperative Group performance status; HR, hazard ratio; LDH, lactate dehydrogenase; PFS, progression-free survival; wt, wild-type

PFS in patients with KRAS wt tumors 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 FOLFIRI (n=350) FOLFIRI + cetuximab (n=316) No of events 189 146 Median PFS 8.4 months 9.9 months [95% CI] [7.4‒9.2] [9.0‒11.3] HR [95% Cl] p-value 0.696 [0.558‒0.867] 0.0012 Probability of PFS FOLFIRI + cetuximab FOLFIRI 12 4 8 16 20 Time (months) Number of patients FOLFIRI 350 237 111 22 4 FOLFIRI + cetuximab 316 227 128 40 8 1 CI, confidence interval; HR, hazard ratio; PFS, progression-free survival; wt, wild-type

OS in patients with KRAS wt tumors 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 FOLFIRI* (n=350) FOLFIRI + cetuximab** (n=316) No of events 288 242 Median OS 20.0 months 23.5 months [95% CI] [17.4‒21.7] [21.2‒26.3] HR [95% Cl] p-value 0.796 [0.670‒0.946] 0.0093 Median follow up was *46 .9 months and **46.2 months. Probability of overall survival FOLFIRI + cetuximab FOLFIRI 18 6 12 24 54 30 36 42 48 Time (months) Number of patients FOLFIRI 350 311 246 179 132 92 64 48 18 2 FOLFIRI + cetuximab 316 281 237 198 144 108 82 65 21 4 CI, confidence interval; HR, hazard ratio; OS, overall survival; wt, wild-type

Treatment interactions Significant interactions between treatment outcome and KRAS tumor mutation status were observed for:1 Tumor response (p=0.0005) PFS (p=0.003) OS (p=0.046) No significant interactions between treatment outcomes and BRAF tumor mutation status were observed for: Tumor response (p=0.87) PFS (p=0.56) OS (p=0.25) 1Lang I, et al. Eur J Cancer Supplements 2009; 7: S345. P-607

Conclusions: KRAS This final analysis shows for the first time in a randomized study that patients with KRAS wt mCRC treated with FOLFIRI plus a targeted agent (cetuximab) in the 1st-line setting had significantly prolonged OS compared with FOLFIRI alone For all efficacy endpoints including survival, this analysis confirms the value of KRAS mutational status as a predictor of treatment outcome in patients with mCRC receiving FOLFIRI plus cetuximab 1st-line

Conclusions: BRAF The analysis suggests that a mutation in BRAF is an indicator of poor prognosis in patients receiving 1st-line treatment for mCRC The role of BRAF mutation as a predictive biomarker for the efficacy of cetuximab added to FOLFIRI 1st-line in patients with mCRC is not proven in this study

Acknowledgments The authors would like to thank patients, investigators, co-investigators and the study teams at each of the participating centers and at Merck KGaA, Darmstadt, Germany