1. Molecular prognosis of colon cancer David J Kerr University of Oxford President ESMO

4. Irinotecan 5-FU / FA Oxaliplatin Capecitabine ? ? UFT? Anti-VEGF? Cetuximab Tomudex? Anti-EGFR?

5. Individualising CRC therapy Increasing diversity of drugs, combinations and schedules Maximise efficacy, minimise toxicity Individualisation based on: Clinical factors Pharmacokinetics (AUC, etc) Molecular markers (TS, VEGF, etc) Pharmacogenomics (genetic changes, microarrays)

6. Pharmacogenomics Using inter individual differences in genetic pre-disposition to toxicity and response to optimise drug therapy

7. Genetic polymorphisms Single nucleotide changes which occur in >1% of population Occur in drug metabolising enzymes Are one of the most important reason for inter patient variability in toxicity and response Eg, a metabolic polymorphism that reduces drug clearance can lead to life-threatening toxicity

8. UGT Uridine diphosphate glucuronosyltransferase Involved in catabolism of SN-38 to an inactive species Polymorphisms in promoter region of gene (eg, (TA)6TAA  (TA)7TAA) associated with reduced enzyme levels and so reduced irinotecan clearance Patients with TA7 develop severe diarrhoea Innocenti et al 2001

9. ‘Uncertain indication’ randomisation Adjuvant fluorouracil & folinic acid based chemotherapy Observation only (with chemotherapy considered on recurrence)

10. Survival – Dukes stage B

11. Died despite adjuvant chemotherapy 80% Cured by adjuvant chemotherapy and surgery 16% Cured by surgery 4% alone But Should All Stage II patients Receive Adjuvant Therapy?

12. Technical Feasibility Gene Discovery & Refinement Analytical Validation Clinical Validation Oncotype DX® Colon Cancer AssayDevelopment Overview

13. Development and Validation of a Multi-Gene RT-PCR Colon Cancer Assay Colon Cancer Technical Feasibility Development Studies Surgery Alone NSABP C-01/C-02 (n=270) Cleveland Clinic (n = 765) Development Studies Surgery + 5FU/LV NSABP C-04 (n=308) NSABP C-06 (n=508) Selection of Final Gene List & Algorithm Standardization and Validation of Analytical Methods Clinical Validation Study – Stage II Colon Cancer QUASAR (n=1,436) Test Prognosis and Treatment Benefit Kerr et al., ASCO® 2009, #4000

14. Quencher Reporter Forward Primer Probe Q R Polymerization Reverse Primer R Q Strand Displacement and Cleavage of Probe Q R Polymerization Completed RT-PCR for RNA Quantification from Fixed Paraffin-Embedded Tumor Tissue Clark-Langone, BMC Genomics: 2007; 8:279. Cronin et al. Am J Pathol. 2004;164:35-42

15. Oncotype DX® Colon Cancer Technical Feasibility Studies RNA yield and RNA quality after extraction from FPET tissues Gene expression differences and similarities between whole section and enriched colon tumor tissue sections Established need for manual microdissection of colon tumor specimens Reference gene selection Compensates for known variability of pre-analytical factors (e.g. Delay to fixation, duration of fixation, fixative)

17. Reference normalization compensates for these differences in sample processing and sample ageReference Normalized Expression (CT) Hollandes Reference Normalized Expression (CT) Formalin Genomic Health, data on file

18. Technical Feasibility Gene Discovery & Refinement Analytical Validation Clinical Validation Oncotype DX® Colon Cancer AssayDevelopment Overview

19. Gene Discovery and Gene Refinement Studies: Oncotype DX® Colon Correlation between gene expression and recurrence-free interval (RFI) across four independent studies. Total of 1851 patients. O’Connell et al. 2010 JCO 28:3937

20. Assessment of 761 Candidate Genes in 1,851 Patients in the Development Studies to Yield Final Pre-specified Assay for Validation in QUASAR 48 Recurrence and 66 Treatment Benefit Genes Significant Across Development Studies Modeling and Analytical Performance FINAL ASSAY 7 Recurrence Genes 6 Treatment Benefit Genes 5 Reference Genes RECURRENCE SCORE (0-100) TREATMENT SCORE (0-100) O’Connell et al. 2010 JCO 28:3937 Kerr et al., ASCO® 2009, #4000

21. The 12-Gene Oncotype DX® Colon Cancer Recurrence Score® Reference Genes Recurrence Score ATP5E GPX1 PGK1 UBB VDAC2 STROMAL FAP INHBA BGN CELL CYCLE Ki-67 C-MYC MYBL2 GADD45B RS = 0.15 x Stromal Group - 0.30 x Cell Cycle Group + 0.15 x GADD45B O’Connell et al. 2010 JCO 28:3937Kerr et al., ASCO 2009, #4000

22. A quantitative multi-gene RT-PCR assay for prediction of recurrence in stage II colon cancer: Selection of the genes in 4 large studies and results of the independent, prospectively-designed QUASAR validation study David Kerr1, Richard Gray2, Philip Quirke3, Drew Watson4, Greg Yothers5, Ian Lavery6, Mark Lee4, Michael O'Connell5, Steven Shak4, Norman Wolmark5 and the Genomic Health & QUASAR Colon Teams 1. University of Oxford, Oxford, UK & SIDRA, Qatar; 2. Birmingham Clinical Trials Unit, Birmingham, UK; 3. Leeds Institute of Molecular Medicine, Leeds, UK; 4. Genomic Health, Inc., Redwood City, CA; 5. National Surgical Adjuvant Breast and Bowel Project, Pittsburgh, PA; 6. Cleveland Clinic, Cleveland, OH

23. Stage II Colon Cancer: Overall Goal To develop and validate a multi-gene expression assay which improves treatment decisions for patients with stage II colon cancer, providing: Individualized assessment of recurrence risk following surgery Identification of patients with differential 5FU/LV benefit Independent clinical value in the context of other measures such as T-stage and MMR Optimized for fixed, paraffin-embedded colon tumor tissue Kerr et al., ASCO® 2009, #4000

25. Parent study demonstrated 3-4% absolute benefit of adjuvant 5FU/LV for stage II disease (approximate 20% relative risk reduction)* Lancet 2007 370:2020-9

26. QUASAR: Demographics of 1,436 Evaluable Patients Two Arms are Balanced Kerr et al., ASCO® 2009, #4000

27. RECURRENCE SCORE Calculated from Tumor Gene Expression STROMAL FAP INHBA BGN CELL CYCLE Ki-67 C-MYC MYBL2 GADD45B REFERENCE ATP5E GPX1 PGK1 UBB VDAC2 QUASAR: Pre-Specified Primary Endpoint: Recurrence Risk Is there a significant relationship between the risk of recurrence and the pre-specified continuous Recurrence Score® in stage II colon cancer patients randomized to surgery alone? Kerr et al., ASCO® 2009, #4000

28. 35% 30% 25% 20% Risk of Recurrence at 3 years 15% 10% 5% | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | 0% 0 10 20 30 40 50 60 70 Recurrence Score QUASAR Results: Colon Cancer Recurrence Score® Predicts Recurrence Following Surgery Prospectively-Defined Primary Analysis in Stage II Colon Cancer (n=711) p=0.004 Kerr et al., ASCO® 2009, #4000

29. QUASAR Results: Recurrence Risk in Pre-specified Recurrence Risk Groups Comparison of High vs. Low Recurrence Risk Groups using Cox Model: HR = 1.47 (p=0.046) n=711 Kerr et al., ASCO® 2009, #4000

30. HR Variable Categories HR 95% CI P value Mismatch Repair (MMR) 13% Deficient vs. 87 % Proficient 0.32 (0.15,0.69) <.001 T Stage 15% T4 vs. 85% T3 1.83 (1.23,2.75) 0.005 Tumor Grade 29% High vs. 71 % Low 0.62 (0.40,0.96) 0.026 Number of Nodes Examined 62% <12 vs. 38 % >12 1.47 (1.01,2.14) 0.040 Lympho - Vascular Invasion 13% Present vs. 87% Absent 1.40 (0.88,2.23) 0.175 Recurrence Score® continuous per 25 units 1.61 (1.13,2.29) 0.008 QUASAR Results: Clinical/Pathological Covariates and Recurrence Pre-specified Multivariate Analysis, Surgery Alone Patients (n=605) Kerr et al., ASCO® 2009, #4000

31. Prognostic AND predictive Risk Score Prognostic AND predictive Risk Score Relationship of 5FU/LV Benefit to Recurrence Score® : QUASAR Results Secondary Analysis in QUASAR Examination of Recurrence Score in surgery alone and 5FU/LV-treated patients: RS by Treatment interaction p=0.76 Prognostic, NOT predictive Risk Score Surgery Alone Surgery + Chemo

33. With similar relative risk reduction across the range of RS, patients at high RS would be expected to derive larger absolute benefit than patients at low RS

35. Summary: QUASAR Validation Study The Recurrence Score® is a validated multi-gene RT-PCR clinical assay which independently and quantitatively predicts individual recurrence risk and provides additional clinical value beyond other available measures These results strongly support a new paradigm for quantitative assessment of recurrence risk in stage II colon cancer, emphasizing the role of three measures, Recurrence Score, MMR/MSI, and T stage The continuous RS will have the greatest clinical utility for T3, MMR-proficient patients, who constitute the majority of stage II colon cancer (~70% of pts)

36. Acknowledgements QUASAR Study Team Laura Magill Kelly Handley Zoe Gray Claire Beaumont Rachel Midgley NSABP Study Team Joe Costantino Soon Paik Cleveland Clinic Study Team Genomic Health Colon Team Kim Langone Rick Baehner Joffre Baker Margarita Lopatin Carl Yoshizawa Wayne Cowens Lauren Intagliata Claire Pomeroy MRC, UK and CRUK for funding QUASAR trial Patients and investigators who participated in the NSABP, Cleveland Clinic, and QUASAR studies