1. A Report from ASCO 2007 Adjuvant Colorectal Cancer John L. Marshall, MD Chief, Division of Hematology/Oncology Associate Director of Clinical Research Director, Developmental Therapeutics and GI Oncology Lombardi Comprehensive Cancer Center Georgetown University Medical Center Washington, DC

2. Abstract 4007 Oxaliplatin/5-FU/LV in Adjuvant Colon Cancer: Updated Efficacy Results of the Mosaic Trial, Including Survival, with a Median Follow-up of 6-Years Aimery de Gramont, Corrado Boni, Matilde Navarro, Josep Tabernero, Tamas Hickish, Clare Topham, Andrea Bonetti, Philip Clingan, Christelle Lorenzato, Thierry André, and MOSAIC investigators

3. MOSAIC: Study Design Primary end-point: disease-free survival Secondary end-points: safety, overall survival N = 2246 Enrollment: Oct 1998–Jan 2001 (146 centres; 20 countries) Completely resected colon cancer Stage II, 40%; Stage III, 60% Age 18–75 years KPS ≥60 No prior chemotherapy R LV5FU2 FOLFOX4 (LV5FU2 + oxaliplatin 85 mg/m²) (N = 1,123) LV5FU2: Leucovorin 200 mg/m 2 iv over 2 hours followed by 5-fluorouracil 400 mg/m 2 bolus and 5-fluorouracil 600 mg/m 2 iv over 22 hours on Days 1 and 2, every 14 days FOLFOX4: LV5FU2 + oxaliplatin 85 mg/m 2 iv over 2 hours on Day 1

4. MOSAIC: Cut-off Dates for Efficacy Analyses 2003 3-year DFS: primary endpoint 2006 5-year DFS: final update (No further updates on relapses) 2007 Overall Survival: 6-year, final analysis André, et al. N Engl J Med 2004;350:2343–2351.

5. Primary End-Point: Disease-Free Survival DFS allows for a quicker determination regarding the efficacy of a new treatment Clinical trials can be completed more quickly Drug development time can be shortened Better therapy can be made available to patients more quickly DFS can be considered as an endpoint of its own merit in decreasing the high cost, quality-of life impact, and debilitating consequence of recurrent disease Sargent, et al. J Clin Oncol 2005;23:8664–8670.

6. 3-Year DFS vs. 5-Year OS Sargent, et al. J Clin Oncol 2005;23:8664–8670. 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.50.550.60.650.70.750.8 r = 0.88 3-Year DFS 5-Year OS

7. 3-years (April 2003) 5-years (June 2006) FOLFOX4LV5FU2FOLFOX4LV5FU2 Median follow-up, mos.37.937.873.573.4 Events (%)21.126.127.132.1 DFS (%)78.272.973.367.4 HR [95% CI] 0.77 [0.65 – 0.91] 0.80 [0.68 – 0.93] P-value0.0020.003 Andre, et al. N Engl J Med 2004;350:2343–2351. MOSAIC: Disease-Free Survival Events = Relapse + Second Primary Colon Cancer + Death by any cause

8. MOSIAC: Disease-Free Survival (ITT) Data cut-off: June 2006 Disease-Free Survival (months) Probability 1.0 0.8 0.6 0.4 0.2 0 0.9 0.7 0.5 0.3 0.1 06121824603036424854 Events FOLFOX4 304/1123 (27.1%) LV5FU2 360/1123 (32.1%) HR [95% CI]: 0.80 [0.68–0.93] 5.9% P = 0.003

9. MOSIAC: Disease-Free Survival Stage II and Stage III Patients Data cut-off: June 2006 Months HR [95% CI] P-value Stage II 0.84 [0.62–1.14] 0.258 Stage III 0.78 [0.65–0.93] 0.005 FOLFOX4 stage II LV5FU2 stage II FOLFOX4 stage III LV5FU2 stage III Probability 1.0 0.8 0.6 0.4 0.2 0 0.9 0.7 0.5 0.3 0.1 061218246030364248546672 3.8% 7.5% P = 0.258 P = 0.005

10. MOSIAC: Disease-Free Survival High-Risk Stage II Patients Disease-Free Survival (months) FOLFOX4 (N = 286) LV5FU2 (N = 290) Probability 1.0 0.8 0.6 0.4 0.2 0 0.9 0.7 0.5 0.3 0.1 061218246030364248546672 3-year 5-year FOLFOX4 85.4% 82.1% LV5FU2 80.4% 74.9% HR [95% CI]: 0.74 [0.52–1.06] High-Risk Stage II – defined as at least one of the following: T4 Tumor perforation Bowel obstruction Poorly differentiated tumor Venous invasion <10 lymph nodes examined 7.2% Exploratory analysis Data cut-off: June 2006

11. MOSIAC: Long-Term Safety FOLFOX 5.3% LV5FU2 5.7% Data cut-off: January 2007 Second CancerPeripheral Sensory Neuropathy Evaluable Patients (N = 811) Grade 084.3% Grade 112.0% Grade 22.8% Grade 30.7% De Gramont A, et al. ASCO 2007. Abstract #4007.

12. MOSIAC: Overall Survival (ITT) Data cut-off: January 2007 Overall Survival (months) Probability 1.0 0.8 0.6 0.4 0.2 0 0.9 0.7 0.5 0.3 0.1 06121824603036424854669672788490 Events FOLFOX4 243/1123 (21.6%) LV5FU2 279/1123 (24.8%) HR [95% CI]: 0.85 [0.72–1.01] 2.6% P = 0.057 De Gramont A, et al. ASCO 2007. Abstract #4007.

13. MOSIAC: Overall Survival Stage II and Stage III Data cut-off: January 2007 FOLFOX4 stage II LV5FU2 stage II FOLFOX4 stage III LV5FU2 stage III Overall Survival (months) Probability 1.0 0.8 0.6 0.4 0.2 0 0.9 0.7 0.5 0.3 0.1 06121824603036424854669672788490 HR [95% CI] Stage II 1.00 [0.71–1.42] Stage III 0.80 [0.66–0.98] 0.1% 4.4% P = 0.996 P = 0.029 De Gramont A, et al. ASCO 2007. Abstract #4007.

14. MOSIAC: Conclusions For FOLFOX4 vs. LV5FU2: The DFS benefit at 3-years was maintained at 5-years Trend showing improved DFS in “high-risk” stage II patients Significant OS benefit in stage III patients No increase in the rate of secondary cancers Continued recovery from sensory neuropathy De Gramont A, et al. ASCO 2007. Abstract #4007.

15. Abstract 4022 Tissue Biomarkers (BIOM) in Colon Cancer (COC): The Translational Study on the Randomized Phase III Trial Comparing Infused Irinotecan/5-fluorouracil (5- FU)/Folinic Acid (FA) to 5-FU/FA in Stage II-III COC Patients (Pts) (PETACC 3 - EORTC 40993 -SAKK 60-00) A.D. Roth 1, S. Tejpar 2, P. Yan 3, R. Fiocca 4, D. Dietrich 5, G. Bodoky 6, R. Labianca 7, D. Cunningham 8, E. Van Cutsem 2, F. Bosman 3 1 Oncosurgery, Geneva University Hospital, Geneva, Switzerland, 2 Digestive Oncology Unit, University Hospital Gasthuisberg, Leuven, Belgium, 3 Dpt of Pathology, Lausanne University, Lausanne, Switzerland, 4 Dpt of Surgical and Morphological Sciences, University of Genova, Genova, Italy, 5 Swiss Group of Clinical Cancer Research, Bern, Switzerland, 6 Oncology, St Lazlo Hospital, Budapest, Hungary, 7 Unit of Medical Oncology, Ospedali Riuniti, Bergamo, Italy, 8 Medical Oncology, The Royal Marsden Hospital, Sutton, United Kingdom.

16. Methods (1) FFPE tissue blocks prospectively collected and cut in 5- 20µ sections Immunohistochemistry (IHC) –P53: mouse mAb clone D07, ABC Basic DAB Detection (Ventana medical system) –SMAD4: mouse mAb clone B8 (IgG1, Santa Cruz Biotechnology). Novocastra polymer detection kit –Thymidylate Synthetase (TS): Monoclonal antibody TS 106/4H4B1 (IgG1, Zymed). DAKO EnVision detection system –Telomerase (HTERT): Monoclonal antibody NCL-hTERT (IgG2, Novocastra). DAKO EnVision detection system Roth AD, et al. ASCO 2007. Abstract #4022.

17. Methods (2) DNA was extracted with phenol/chloroform from normal (Nor) and tumoral (Tu) tissues after microdissection of FFPE sections Molecular analysis: –Microsatellite Instability (MSI): assessed on 10 markers (BAT-25, BAT-26, D5S346, D2S123, D17S250, BAT-40, TGF-ß RII, D18S58, D18S69, D17S787) –18q and 8p LOH: multiple SNPs typing by pyrosequencing on Nor/Tu DNA –KRAS exon 2 and BRAF exon 15: Allele specific real time PCR on Tu DNA –UGT1A1 7/7 genotype: PCR and fragment sizing on Nor DNA Roth AD, et al. ASCO 2007. Abstract #4022.

18. Analysis Success Rate 1,530 patient slides analyzed by IHC DNA successfully extracted from 1,201 patient slides (91.2%) Roth AD, et al. ASCO 2007. Abstract #4022. Number of Samples with Results MarkerSamples Success Rate (%) Telomerase82654% TS1,26983% SMAD41,44394% P531,44795% MSI1,32794% KRAS1,37998% BRAF1,38699% LOH in 18q - at least one snip OK 1,22087% UGT1A11,33595%

19. Biomarker Alteration Observed (Mutation, Expression, or Deletion) Alterations Rate Observed Alteration Rate Reported (literature) P53 overexpression*37%25-76% SMAD4 loss**15%13-63% TS***48%No consistent data available HTERT48%No data available MSI15%10-17% 18q65%70% KRAS37%32-40% BRAF8%10% UGT1A1 (7/7 genotype)12%10-15% * Intense expression, More than 45% cells positive ** Any loss *** Positive = more than 25% cell positive Roth AD, et al. ASCO 2007. Abstract #4022.

20. SMAD4: Preliminary Results (Stage III) At Risk:14595572 81766344938 0500 1000 1500 0.4 0.5 0.6 0.7 0.8 0.9 1.0 // /////////// //// //////////////////////////////////// //////////// / / / / / / / / / / / / / //// / // /////// / /// / /////// // / /// ///// //////////////// /////// //////// /// /// //////////////// ////////////// ////////////// //// ////////////////////////////////////////// //// ////////////// ////////////////////////////// ///// //////////////////////////// //// /////// ///// ////// //////////////////////////////////////////////////// Day Proportion Disease-Free No Expression Expression Present Roth AD, et al. ASCO 2007. Abstract #4022. Cutpoint % Patients ≤ Cutpoint Estimated 3-Year DFS Log-Rank P-value ≤ Cutpoint> Cutpoint 015%53%68%< 0.001

21. MSI: Preliminary Results (Stage III) At Risk: 75359539633 10489613 05001000 1500 0.5 0.6 0.7 0.8 0.9 1.0 / / / / / / // / / /// ////////// //// ////// //////// /// ///////////////////// ////////// // ////////////// ///////////////////////////////////////////////// Day Proportion Disease-Free Stable/Low High % Patients High Estimated 3-Year DFS Log-Rank P-value Stable/LowHigh 12%65%74%< 0.04 Roth AD, et al. ASCO 2007. Abstract #4022.

22. Abstract 4008 Time-dependent Patterns of Failure and Treatment Benefit from Adjuvant Therapy for Resectable Colon Cancer: Lessons from the 20,800 Patient ACCENT Dataset D Sargent, for the Adjuvant Colon Cancer Endpoints (ACCENT) Group

23. Total: 18 trials; 20,898 pts 3,517 QUASAR867 GIVIO 905 GERCOR718 NSABP C02 3,547 INT 0089773 NSABP C01 1,078 SWOG 9415259 FFCD 878 N914653359 NCIC 915 N894651239 Siena 2,176 NSABP C05408 N874651 2,151 NSABP C04926 INT 0035 1,081 NSABP C03247 N784852 N TrialN Active ControlNo Treatment Control ACCENT Dataset Trials Included Sargent D, et al. ASCO 2007. Abstract #4008.

24. Three Questions Facing Adjuvant Colon Cancer Trialists Nature and duration of treatment benefit on overall survival (OS), disease-free survival (DFS) Long-term recurrence rates Adequacy of statistical assumptions, using DFS endpoint Sargent D, et al. ASCO 2007. Abstract #4008.

25. Hazard Rates for OS and DFS Disease-Free Survival Follow-up Time (Years) Hazard Rate 02468 0.0 0.0002 0.0004 0.0006 Surgery Alone Arms 5-FU Based Rx Arms Overall Survival Follow-up Time (Years) Hazard Rate 02468 0.0 0.0002 0.0004 0.0006 Surgery Alone Arms 5-FU Based Rx Arms Sargent D, et al. ASCO 2007. Abstract #4008.

26. After 5 years, recurrence rates < 1.5% / year After 8 years, recurrence rates < 0.5% / year Recurrence Rate by Time from Randomization (All Patients) Sargent D, et al. ASCO 2007. Abstract #4008.

27. Adequacy of Statistical Models Conclusions Similar exercises demonstrated < 2% power loss due to non-constant risk of event Real world impact of DFS endpoint on trial design, for range of treatment effects observed in ACCENT, is minimal Continued use of standard approaches for sample size determination remains appropriate Sargent D, et al. ASCO 2007. Abstract #4008.

28. Abstract 4009 Survival Following Recurrence in Patients with Adjuvant Colon Cancer: Findings from the ACCENT Dataset MJ O’Connell, for the ACCENT Collaborative Group

29. Total: 17 trials; 17,381 pts 3,517 QUASAR867 GIVIO 905 GERCOR718 NSABP C02 3,547 INT 0089773 NSABP C01 1,078 SWOG 9415259 FFCD 878 N914653359 NCIC 915 N894651239 Siena 2,176 NSABP C05408 N874651 2,151 NSABP C04926 INT 0035 1,081 NSABP C03247 N784852 N TrialN Active ControlNo Treatment Control ACCENT Dataset Trials Included O’Connell M, et al. ASCO 2007. Abstract #4009.

30. Prognostic Factors Examined Time from randomization on surgical adjuvant protocol to tumor recurrence ( 4 years) Initial stage of colon cancer (II, III) 5-FU-based adjuvant therapy vs. surgery alone Era patient entered onto surgical adjuvant protocol (1978-1985, 1986-1992, 1993-1999) O’Connell M, et al. ASCO 2007. Abstract #4009.

31. Time from Recurrence to Death by Year of Recurrence 100 Time (Years) Year 0- 1 (N = 1,846) Year 1-2 (N = 1,854) Year 2-3 (N = 924) Year 3-4 (N = 516) Year 4+ (N = 582) Total (N = 5,722) 0 20 40 60 80 012345678 % Alive P < 0.0001

32. O’Connell M, et al. ASCO 2007. Abstract #4009. Time from Recurrence to Death by Year of Recurrence for Stage II Patients

33. O’Connell M, et al. ASCO 2007. Abstract #4009. Time from Recurrence to Death by Year of Recurrence for Stage III Patients

34. O’Connell M, et al. ASCO 2007. Abstract #4009. Time from Recurrence to Death by Stage

35. O’Connell M, et al. ASCO 2007. Abstract #4009. Time from Recurrence to Death by Era

36. O’Connell M, et al. ASCO 2007. Abstract #4009. Time from Recurrence to Death by Adjuvant Treatment vs. Surgery Alone

37. Conclusions Time from initial surgery and stage of the primary colon cancer were important prognostic variables in patients with recurrent colon cancer Patients who have recurrent tumor following 5-FU-based adjuvant therapy had worse prognosis than those without adjuvant chemotherapy Survival following recurrence improved from 1978-1999 O’Connell M, et al. ASCO 2007. Abstract #4009.

38. Abstract 4019 The Impact of Dietary Patterns on Cancer Recurrence and Survival in Patients with Stage III Colon Cancer: Findings from CALGB 89803 Jeffrey A. Meyerhardt 1, Donna Niedzwiecki 2, Donna Hollis 2, Leonard B. Saltz 3, Walter Willett 4, Robert J. Mayer 1, Charles S. Fuchs 1 1 Dana-Farber Cancer Institute, Boston, MA; 2 CALGB Statistical Center, Durham, NC; 3 Memorial Sloan-Kettering Cancer Center, New York, NY; 4 Harvard School of Public Health, Boston, MA

39. Pearson Correlation Coefficients for the Relationship Between Food Intake and Factors Representing Dietary Patterns *values < 0.15 are not shown (---). † Vegetables other than yellow, cruciferous, or leafy-green vegetables. ‡ Potato, corn chips, crackers, or popcorn. Meyerhardt J, et al. ASCO 2007. Abstract #4019.

40. Impact of Western Pattern Diet on Colon Cancer Recurrence and Mortality Meyerhardt J, et al. ASCO 2007. Abstract #4019. Quintile of Western Pattern Diet 12345P-value Cancer recurrence or death-any cause (DFS) # of events / # at risk71/20157/20273/20268/20283/202 Multivariate adjusted hazard ratio Ref1.22.032.163.91<0.0001 (0.76-1.89)(1.30-3.16)(1.32-3.52)(2.21-6.89) Cancer recurrence (Recurrence-Free Survival) # of events / # at risk68/20151/20268/20261/20276/202 Multivariate adjusted hazard ratio Ref1.071.841.773.14<0.0001 (0.66-1.73)(1.16-2.90)(1.06-2.95)(1.73-5.69) Overall Mortality # of events / # at risk57/20135/20251/20253/20255/202 Multivariate adjusted hazard ratio Ref0.962.092.843.75<0.0001 (0.54-1.71)(1.22-3.57)(1.56-5.05)(1.90-7.41) Adjusted for gender, age, depth of invasion through bowel wall (T1-2 vs. T3-4), number of positive lymph noses (1-3 vs. 4 or more), presence of clinical perforation at time of surgery, presence of bowel obstruction at time of surgery, baseline performance status (0 vs. 1-2), treatment arm, weight change between 1 st and 2 nd questionnaire, time-varying body mass index, time-varying physical activity level, and time-varying total calories.

41. Impact of Prudent Pattern Diet on Colon Cancer Recurrence and Mortality Meyerhardt J, et al. ASCO 2007. Abstract #4019. Quintile of Prudent Pattern Diet 12345P-value Cancer recurrence or death-any cause (DFS) # of events / # at risk79/20179/20271/20253/20270/202 Multivariate adjusted hazard ratio Ref1.130.960.71.260.79 (0.71-1.67)(0.63-1.46)(0.44-1.11)(0.80-1.97) Cancer recurrence (Recurrence-Free Survival) # of events / # at risk73/20168/20267/20252/20264/202 Multivariate adjusted hazard ratio Ref1.050.960.761.20.76 (0.70-1.60)(0.62-1.49)(0.47-1.22)(0.75-1.94) Overall Mortality # of events / # at risk63/20158/20244/20234/20252/202 Multivariate adjusted hazard ratio Ref1.140.750.591.140.75 (0.73-1.78)(0.44-1.29)(0.33-1.65)(0.81-2.45) Adjusted for gender, age, depth of invasion through bowel wall (T1-2 vs. T3-4), number of positive lymph noses (1-3 vs. 4 or more), presence of clinical perforation at time of surgery, presence of bowel obstruction at time of surgery, baseline performance status (0 vs. 1-2), treatment arm, weight change between 1 st and 2 nd questionnaire, time-varying body mass index, time-varying physical activity level, and time-varying total calories.

42. Adjuvant Colorectal Cancer Closing Comments John L. Marshall, MD