Promising Future Biomarkers For Colorectal Cancer: Focus on Targeted Therapies

1. Promising Future Biomarkers For Colorectal Cancer:Focus on Targeted Therapies Lee M. Ellis, MD Depts of Surgical Oncology and Cancer Biology UT MD Anderson Cancer Center Houston, Texas USA

2. A Few DefinitionsA “marker” is not a “marker” is not a “marker” • Prognostic marker • associated with clinical outcome, independent of specific treatment • Predictive marker • identifies groups receiving different degrees of benefitfrom a therapy • specific for a given treatment (i.e., Ras for EGFR MoABs in CRC, HER2 expression for trastuzumab) • Surrogate / activity marker • modulated by treatment • may be on target tissue (tumor) or surrogate tissue (i.e., lymphocytes, skin) • might or might not correlate with clinical activity

3. CRYSAL TrialWe Need to Do Better!! Van Cutsem et al. NEJM 2009

4. Biomarkers for Targeted Therapies in CRC I apologize in advance for some mixing and matching as some studies report more than one marker. • EGFR • Downstream signaling pathways • RAS (Lenz) • Raf • PTEN/PI3K • AACR Presentations (including EGFR amplifications) • VEGF • ?

5. EGF Receptor: Its Role in CRC Therapy Meyerhardt & Mayer, N Engl J Med 2005 Venook, Oncologist 2005 pY PI3K pY pY pY PTEN STAT AKT Ligand Antibodies to EGFRcetuximab, panitumumab RAS RAF EGFR-TK MEK MAPK Gene transcription Cell-cycle progression Proliferation Survival (anti-apoptosis) Chemotherapy /radiotherapy resistance Invasion and metastasis Angiogenesis

6. Clin Ca Res, Jan 2005 “Bypass” Pathways and EGFR Resistance

7. Now that Mutated K-Ras is an Established Marker of Resistance, the Next Advance will be in the identification of resistance markers in K-Ras Wild-type tumors CRC K-Ras WT K-Ras MT B-Raf PI3K PTEN EGFR copy # ? EGFR MoAB

8. Bypass Pathways and EGFR Resistance Camp et al Clin Ca Res 2005

9. B-Raf and Resistance to EGFR MoABs • Mutated in 3-15% CRC • Ras and Raf mutations exclusive of each other • Gain-of-function mutations • Inability to convert the active form to inactive confirmation Schubbert et al. Nat Rev Ca, 2007

10. All pts had progressed on at least one line of therapy ~50% received monotherapy with EGFR MoAB ~50 MoAB with chemotherapy In patients with tumors with Mut B-raf, there were NO objective responses “We hypothesized that in K-ras wild-type patients, B-raf mutations could have prognostic/predictive value.” Nicolantonio et al. JCO 2008 Wild-Type BRAF is Required for Response to..(EGFR MoABS)

11. Ras and PFS B-Raf Predicts for Benefit of Anti-EGFR Therapy in Patients with WT Ras and the Entire Cohort PFS OS Wild-type K-ras All Patients

12. Bypass Pathways and EGFR Resistance Camp et al Clin Ca Res 2005

13. Classic PTEN Pathway: Inhibition of the Activation of AKT • PTEN is a phosphatase that blocks activation of Akt / survival pathway • Loss of function of PTEN associated with an increase in cell survival signaling Planchon et. al., J Cell Sci, 2008

14. PTEN Pathway – Functions in the Cytoplasm and Nucleus Nuclear PTEN plays a role in chromosome stability, DNA repair, cell cycle arrest and cellular stability. Planchon et. al., J Cell Sci, 2008

15. 44th ASCO Annual Meeting, 2008 “PTEN …. Intensity was scored according to a four-tier system: 0, no staining; 1, weak; 2, moderate; and 3, strong. We attributed one, two, or three additional points if the percentage of positive cells was less than 25%, 25% to 50%, or greater than 50%, respectively. Specimens were defined as positive if the score was 4 or greater.” Loupakis et al. JCO 2009

16. PTEN Expression and PFS • Only PTEN in the metastasis was predictive of efficacy. • PTEN in the primary tumor was NOT predictive of efficacy. • PTEN in the primary tumor and liver metastasis was concordant in only 60% of cases • pAKT was NOT predictive of efficacy. Logrank Test: p=0.005 HR = 0,49 95% CI: 0.20-0.75 PTEN+ PTEN- Loupakis et al. JCO 2009

17. Challenges with PTEN • Expression in primary tumors does not reflect expression in metastases • Although it is not lost or mutated in CRC, its expression can be regulated by methylation or miRNA • It will be hard to standardize IHC in different labs Supplemental Figure 1: Representative examples of PTEN positive (A, B) and negative (C, D) cases. The cases reported in A and C panels were evaluated at Ospedale Niguarda Ca’ Granda (Milan, Italy) whereas those in B and D at the Institute of Pathology in Locarno (Switzerland). Sartore-Bianchi et al. Cancer Res 2009.

18. Bypass Pathways and EGFR Resistance Camp et al Clin Ca Res 2005

19. Point Mutations in PIK3CA Observed in Human Tumors CRC Exon 9 Hotspots Exon 20 Bader et al., Nat Rev Cancer 2005

20. Fig. 1 Prenen, H. et al. Clin Cancer Res 2009

21. PFS and PIK3CA Mutational Status in mCRC Patients Treated With Panitumumab and Cetuximab 110 pts > 85% received at least 1 prior Rx Sartore-Bianchi, A. et al. Cancer Res 2009

22. Biomarkers for Targeted Therapies in CRC • EGFR • Downstream signaling pathways • RAS (Lenz) • Raf • PTEN/PI3K • AACR Presentations (including EGFR amplifications) • VEGF • ?

23. CAIRO -2 STUDYCAPOX + Bevacizumab +/- Cetuximab755 Pts Tissue in 545 Loss PTEN 42% no difference in PFS EGFR copy number 7% no difference in PFS among arms EGFR amplification and PTEN did NOT predict for response to chemo + cetuximab therapy Tol et al. Proc AACR, 2009 Abstract 691

24. 173 Pts BRAF & EGFR Amplification in Metastatic CRC with Wild-type K-Ras Cetuximab based therapy Ras Mut (57) Ras Wt (116) Raf Mt PTEN EGFR Amplification WT Kras/ WT BRAF/ EGFR Amp = 80% RR Negative Positive Laurent-Puig et al, Proc AACR 2009, A1897

25. Do Mutations / Aberrations in the Primary Tumor Reflect the Metastasis?

26. Overall, mutations do not change between the primary and the metastasis. But….expression levels or gene amplification may change.

27. The Role of PREDICTIVE Markers for Efficacy of EGFR MoABs • Excluding patients from EGFR MoAB Rx by use of multiple predictive factors will greatly increase the efficacy of EGFR MoABS • It is imperative to PROSPECTIVELY include biomarkers and tissue procurement in clinical trials • When possible, biomarkers in primary tumors and liver metastasis should be compared • Mutational status gives you a “black and white” answer and is more likely to be reproducible relative to other biomarkers (IHC, etc)

28. Biomarkers for Targeted Therapies in CRC • EGFR • Downstream signaling pathways • RAS (Lenz) • Raf • PTEN/PI3K • AACR Presentations (including EGFR amplifications) • VEGF • ?

29. What Holds Promise in Anti-VEGF Therapy? None currently identified in CRC!

30. Kaplan-Meier estimates for progression-free survival (PFS) of patients with low and high baseline volume transfer constant of contrast agent (Ktrans) and blood plasma volume fraction (Vp) Hahn, O. M. et al. J Clin Oncol; 2008

31. VEGF Polymorphisms and Predictive Value in ECOG-2100 (Pac +/- Bev Metastatic Breast Cancer) Kaplan-Meier curve for overall survival (OS) in experimental arm by genotype; (A) vascular endothelial growth factor (VEGF)-2578 C/A; (B) VEGF-1154 G/A • Caveats: • Predictive for OS, but not PFS • Small numbers • Did not include Pac Rx alone group Schneider, et al. J Clin Oncol; 2008

32. A Few DefinitionsA “marker” is not a “marker” is not a “marker” • Prognostic marker • associated with clinical outcome, independent of specific treatment • Predictive marker • identifies groups receiving different degrees of benefitfrom a therapy • specific for a given treatment (i.e., Ras for EGFR MoABs in CRC, HER2 expression for trastuzumab) • Surrogate / activity marker • modulated by treatment • may be on target tissue (tumor) or surrogate tissue (i.e., lymphocytes, skin) • might or might not correlate with clinical activity

33. The Harvest of a Decade of Biomarker Studies for Anti-angiogenic Therapy George Sledge

34. Grade 3/4 Hypertension Is Associated With Improved Median OS in E2100 Median OS: 25.3 mo vs. 38.7 mo p=0.002 Schneider et al; J Clin Oncol, 2008

35. Relationship Between HTN and the Efficacy of Anti-VEGF Rx Mir et al. Ann Onc 2009

36. Numerous Studies Testing Dose Escalation of VEGF Inhibitors A Dose Escalation Study of Sorafenib (BAY 43-9006, NSC 724772) in Normotensive Patients with Advanced Malignancies-Michael Maitland, Univ of Chicago

37. Angiogenic Cytokines Are Increased Prior to Disease Progression In Metastatic Colorectal Cancer Patients Treated With Bevacizumab Scott Kopetz, Paulo M. Hoff, Cathy Eng, Michael Overman, Katrina Y. Glover, David Z. Chang, Robert A. Wolff, James L. Abbruzzese, Lee M. Ellis, John V. Heymach The University of Texas, M.D. Anderson Cancer Center, Houston, Texas Centro de Oncologia, Hospital Sírio Libanês, Sao Paulo, Brazil

38. Study Methods • Sample Collection Study Aim 1: • Subsequent samples were obtained every two weeks • n=40 with evaluable samples at the selected time points Cycle 1 Day 1 Cycle 2 Day 15 Cycle 3 Day 29 Cycle 4+ Day 43 Bevacizumab Bevacizumab Bevacizumab FOLFIRI FOLFIRI Baseline After Bevacizumab After FOLFIRI + Bev Every 2 weeks

39. Cytokines Increased Prior to Progression

40. Conclusions: Future PREDICTIVE Biomarkers for Targeted Therapies in mCRC • EGFR MoABs • K-Ras ---- current and validated! • B-Raf • Not ready for prime time • PI3K mutations? • PTEN? • EGFR amplifications? • Gene arrays (under study)? • VEGF MoAB • Wide open • Can we learn from other disease sites? • VEGF polymorphisms? • I predict that imaging will be the answer

41. ASCO 2009…Tons of Colorectal CancerPredictive Biomarker Abstracts Type: General Poster Session Time: Sunday May 31, 8:00 AM to 12:00 PM Location: Level 2, West Hall C Type: Poster Discussion Time: Monday June 1, 8:00 AM to 12:00 PM Location: Level 2, W240A Discussion: Monday June 1, 11:00 AM to 12:00 PM Location: Level 2, West Hall E1