Cetuximab plus FOLFIRI in the treatment of metastatic colorectal cancer: the influence of KRAS and BRAF biomarkers on outcome: updated data from the CRYSTAL trial Eric Van Cutsem*, I. Lang, G. Folprecht, M. Nowacki, S. Cascinu, I. Shchepotin, J. Maurel, D. Cunningham, P. Rougier, I. Celik, C.H. Köhne *University Hospital Gasthuisberg, Leuven, Belgium

Background: The CRYSTAL trial KRAS wild-type mCRC patients treated 1 st -line with cetuximab plus FOLFIRI compared with FOLFIRI alone experienced –Reduced risk of disease progression (Hazard Ratio, 0.68) 1 –Increased chance of tumor response (59% vs 43%, p=0.0025) 2 In this final analysis we report –Increased follow-up time –Increased population of evaluable tumors for KRAS and BRAF mutations –The impact of BRAF mutation status on cetuximab efficacy in patients with KRAS wild-type tumors 1 Van Cutsem E, et al. N Engl J Med 2009;360: ; 2 Van Cutsem E, et al. J Clin Oncol 2008;26: (suppl; abstr 2)

The CRYSTAL study ECOG PS, Eastern Cooperative Oncology Group performance status; 5-FU, 5 fluorouracil; LV, leucovorin Endpoints: Primary- PFS time Secondary- OS time, best overall response (OR) rate and safety Retrospective subgroup analyses: OS, PFS and OR by KRAS mutation status Treatment until progression, symptomatic deterioration or unacceptable toxicity

Background: BRAF 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 mCRC 2,3 1 Roth A, et al. J Clin Oncol 2010; 28:466-74; 2 Di Nicolantonio F, et al. J Clin Oncol 2008;26: ; 3 Tejpar S et al on behalf of EU consortium, ECCO/ESMO, 2009

KRAS/BRAF evaluable population 666 (63%) KRAS wild-type 540/1198 subjects (45% of ITT) 1 : KRAS evaluable population 1063 (89%) subjects: updated KRAS evaluable population 625 (59%) KRAS wild-type/BRAF evaluable KRAS wild-type/BRAF wild-type 566/625 (91%) KRAS wild-type/BRAF mutant 59/625 (9%) Sample numbers for KRAS and BRAF mutation status were increased using DNA extracted from tumor from paraffin blocks or formalin fixed paraffin embedded slide mounted sections prepared to evaluate tumor EGFR expression KRAS (codons 12/13) and BRAF (V600E) mutations were detected using a PCR clamping and melting curve technique BRAF mutations were detected in a total of 60/1000 (6%) evaluable samples,1 patient was also KRAS mutant 1 Van Cutsem E, et al. N Engl J Med 2009;360:

Baseline characteristics according to tumor KRAS/BRAF mutation status KRAS wt (n=666) KRAS wt/ BRAF wt (n=566) KRAS wt/ BRAF mt (n=59) ParameterFOLFIRI (n= 350) Cetuximab + FOLFIRI (n= 316) FOLFIRI (n= 289) Cetuximab +FOLFIRI (n= 277) FOLFIRI (n=33) Cetuximab +FOLFIRI (n=26) Gender, male % Median age, yrs (range) 59 (19–84) 61 (24–79) 59 (19–84) 60 (24–79) 58 (25–75) 65 (34–79) <65 yrs, % ECOG PS 0-1, % Liver-limited disease, % Involved disease sites ≤2, % Prior adjuvant chemotherapy, % ECOG PS, Eastern Cooperative Group performance status; mt, mutant; wt, wild-type

PFS in patients with KRAS wild-type tumors FOLFIRI Number of patients Cetuximab + FOLFIRI CI, confidence interval; HR, hazard ratio; PFS, progression-free survival FOLFIRICetuximab + FOLFIRI Median PFS8.4 months9.9 months [95% CI][ ][ ] HR [95% Cl] p-value [ ] (log-rank) Probability of PFS Time (months) Cetuximab + FOLFIRI FOLFIRI

Subgroup (number of patients in Group A vs B)HR [95% CI][95% CI] All ITT Subjects (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 (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)0.70[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] Leucocytes ≤10000/mm 3 (258 vs 284)0.70[0.55, 0.90] > 10000/mm 3 (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)0.68[0.53, 0.86] Prior adjuvant chemotherapy Yes (80 vs 73) 0.77[0.49, 1.21] No (236 vs 277)0.67[0.52, 0.87] PFS by subgroups in KRAS wild-type patients HR and 95% CI Group A, cetuximab + FOLFIRI; Group B, FOLFIRI Benefit under cetuximab No benefit under cetuximab CI, confidence interval; ECOG PS, Eastern Cooperative Group performance status; HR, hazard ratio; LDH, lactate dehydrogenase; PFS, progression-free survival

OS in patients with KRAS wild-type tumors FOLFIRI Number of patients Cetuximab + FOLFIRI CI, confidence interval; HR, hazard ratio; OS, overall survival FOLFIRICetuximab + FOLFIRI Median OS20.0 months23.5 months [95% CI][ ][ ] HR [95% Cl] p-value [ ] (log-rank) Probability of overall survival Time (months) Cetuximab + FOLFIRI FOLFIRI Median follow up was 46 months

Subgroup (number of patients in Group A vs B)HR [95% CI][95% CI] All ITT Subjects (316 vs 350)0.80[0.67, 0.95] Age <65 years (200 vs 234)0.71[0.57, 0.89] ≥65 years (116 vs 116)0.98[0.73, 1.30] Gender Male (196 vs 211)0.82[0.66, 1.03] Female (120 vs 139)0.72[0.55, 0.95] ECOG PS (303 vs 336)0.78[0.65, 0.93] 2 (13 vs 14)1.31[0.57, 3.03] Number of metastatic sites ≤2 (277 vs 295)0.81[0.67, 0.98] >2 ( 33 vs 49)0.69[0.41, 1.14] Liver metastases only Yes ( 68 vs 72)0.85[0.57, 1.28] No (248 vs 278)0.79[0.65, 0.95] Leucocytes ≤10000/mm 3 (258 vs 284)0.78[0.64, 0.94] >10000/mm 3 (48 vs 58)0.94[0.61, 1.44] LDH at baseline > upper normal range (138 vs 150)0.72[0.56, 0.93] ≤ upper normal range (144 vs 161)0.94[0.72, 1.22] Alkaline Phosphatase at baseline ≥300 U/L (30 vs 42)0.74[0.43, 1.28] < 300 U/L (272 vs 295)0.81[0.67, 0.98] Prior adjuvant chemotherapy Yes (80 vs 73)0.83[0.58, 1.21] No (236 vs 277)0.79[0.65, 0.96] OS by subgroups in KRAS wild-type patients HR and 95% CI Benefit under cetuximab No benefit under cetuximab Group A, cetuximab + FOLFIRI; Group B, FOLFIRI CI, confidence interval; ECOG PS, Eastern Cooperative Group performance status; HR, hazard ratio; LDH, lactate dehydrogenase; OS, overall survival

Response in patients with KRAS wild-type tumors FOLFIRI (n= 350) Cetuximab + FOLFIRI (n= 316) OR rate (%) [95% CI] 39.7 [34.6–45.1] 57.3 [51.6–62.8] Odds ratio [95% CI] p-value a [1.5154–2.8258] < a Cochran-Mantel-Haenszel test CI, confidence interval; OR, best overall response

Tumor regression according to treatment in patients with KRAS wild-type tumors For patients receiving cetuximab + FOLFIRI compared with FOLFIRI alone, the mean difference in the best % change of the lesion (based on WHO criteria) was 13.9% * Data for 16 patients were missing; ** Data for 21 patients were missing Cetuximab + FOLFIRI, n=316* FOLFIRI, n=350** Change in lesion (%)

Clinical efficacy in KRAS wild-type tumors by BRAF mutation status 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 [0.687–1.004] [0.507–1.624] 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 [0.533–0.864] [0.425–2.056] 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 < 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

Conclusions: KRAS in the CRYSTAL trial This final analysis shows for the first time in a randomized study that the addition of a targeted agent (cetuximab) to FOLFIRI in the 1 st -line treatment of patients with metastatic colorectal cancer with KRAS wild-type tumors significantly improved OS compared to FOLFIRI alone This final analysis confirms KRAS tumor mutation status to be a predictive factor across all efficacy endpoints examined for cetuximab in combination with FOLFIRI

Conclusions: BRAF in the CRYSTAL trial The analysis suggests BRAF tumor mutation to be a poor prognostic factor in 1 st -line mCRC BRAF mutation status does not appear to be a strong predictive biomarker for the addition of cetuximab to FOLFIRI in 1 st -line treatment of mCRC

Acknowledgements 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