1. Progress towards an individualized approach to therapy: colorectal cancer Alan P. Venook, M.D. University of California, SF

2. Breast Cancer Oncologist Envy Alan P. Venook, M.D. University of California, SF

3. Personalized decision-making: predicting risk:benefit

7. Meropol, et al; JCO, 2007 Escalating cost of cancer care

8. “Predictive” vs. “Prognostic” Predictive: response to treatment Prognostic: independent of treatment Variables: host (germline) tumor

9. Towards Personalized Therapy of Colon Cancer Tumor genetic profiling Stage II colon cancer Stage IV colorectal cancer Pharmacogenetics Oxaliplatin efficacy Irinotecan - UGT1A1 Therapeutic Drug Monitoring

10. CRC Stage at Diagnosis 13.7% Stage I 27.9% Stage II 37.2% Stage III 21.2% Stage IV Treatment Algorithms: Colorectal Cancer 5th Edition. Datamonitor 2003.

11. Colorectal Cancer: TNM Staging System Extent of invasion through bowel wall (T) Extent of LN metastases (N) Presence of distant metastases (M)

12. Renouf et al, Clin Col Can, 2008

13. QASAR, Lancet, 2007

15. Colorectal Cancer, 2007

16. 18Q deletion & outcome in untreated colorectal cancer 18Q + 18Q - Jen, et al. NEJM, 1994

17. mFOLFOX6 v. mFOLFOX6 + Bevacizumab qow Accrual Goal: 3,125 Tumor risk assessed based on biology (18q/MSI) High-risk (MSS and 18q LOH) Low-risk (MSI + or no loss 18q) OBSERVATION Surgery E5202 – Stage II Colon Cancer

18. Khambata-Ford, JCO, 2007

19. Towards Personalized Therapy: Stage II Colon Cancer MSI and del 18q Microarrays Composite of prognostic and predictive genes Nodal analysis

20. Towards Personalized Therapy: Tumor Genetics EGF-R antibodies: yes or no? KRAS BRAF PTEN VEGF antibody: yes or no?

21. Personalized Therapy: Tumor Genetics EGF-R antibodies KRAS BRAF PTEN

22. The Ras Oncogene 100% of mice developed tumors in weeks Rapid development of sarcomas and erythroleukemias Kirsten and Harvey: 1964 Identification of a virus that produced tumors in mice HaMSV KiMSV Harvey (1964) Nature 204:1104; Somers and Kirsten Science 1967;40:1053

23. Ras Family of Proteins Monomeric G proteins (H-Ras, K-Ras and N-Ras) Respond to activation of membrane-associated receptors for cell growth and survival Cycle between GDP bound “off” state and GTP bound “on” state Act as “molecular switches” linking extracellular signals through membrane receptors to intracellular signals

24. Copyright ©2008 American Association for Cancer Research Sebolt-Leopold, J. S. Clin Cancer Res 2008;14:3651-3656 Ras: Downstream Signaling

25. Ras mutations: 30% of Human Cancers Pancreatic carcinoma72-90% Cholangiocarcinoma 55% Colon adenocarcinoma 32-57% Thyroid carcinoma 30% Seminoma 40% Embryonal rhabdomyosarcoma 35% Acute myelogenous leukemia 35% Myeloblastic syndromes 30% Lung carcinoma 15-50%

26. CRC: Adenoma-Carcinoma Sequence 32-57% K-Ras mutant

27. Detecting tumor K-Ras mutations DNA extracted from tumor (FFPE, cell-free DNA) Mutational analysis by sequencing (various methods) or mutant allele specific amplification Detection threshhold: 1% of mutant DNA in a background of wild-type genomic DNA Gly 12 Asp Gly 12 Ala Gly 12 Val Gly 12 Ser Single base substitutions that render GTPase domain insensitive to inactivation by GAP Gly 12 Arg Gly 12 Cys Gly 13 Asp

28. Prognostic implications: K-Ras Probably NO Smakman et al; Biochem Biophys Acta 2005; 1756:103 Stage I-IV CRC; 24 studies with >100 patients (range 100-3439) Mutation rate 24-69% Association between K-Ras status and stage: Yes: 7; No: 13; N/A: 4 Association between K-Ras status and DFS: Yes: 3; No: 3; N/A: 18

29. Retrospective studies supporting K-ras and lack of anti- EGFR response

30. Advanced CRC treated with cetuximab: case series N=30 Lievre, A. et al. Cancer Res 2006;66:3992-3995 Most with prior irinotecan exposure 97% irinotecan + cetuximab

31. Single agent cetuximab: N=80 Khambata-Ford et al J Clin Oncol, 2007; 25: 3230-3237

32. Single agent panitumumab: N=208 K-Ras Mutation Wild-Type K-Ras Amado RG, et al. J Clin Oncol. 2008;26:1626-1634. Panitumumab registration trial

33. CRYSTAL RANDOMIZERANDOMIZE FOLFIRI FOLFIRI + Cetuximab n = 540 Van Cutsem ASCO 2008; JCO 28: May 20 suppl; abstr 2. FOLFIRI FOLFIRI + Cetuximab P value K-ras wildtypeK-ras mutant HR 0.68HR 1.07 (0.051-0.934) 0.017 (0.71-1.61) 0.75

34. CALGB/SWOG 80405 Study Design Untreated advanced or mCRC N = 2600 Bevacizumab followed by FOLFOX or FOLFIRI q 2 wks Cetuximab followed by FOLFOX or FOLFIRI q 2 wks Cetuximab followed by Bevacizumab followed by FOLFOX or FOLFIRI q 2 wks One cycle=8 weeks mCRC=metastatic colorectal cancer Open-label Phase III Study Screen for eligibility Send tumor tissue block to SWOG PCO Randomize Patients w/ Wild type K-ras tumor Register Patient

35. Copyright ©2008 American Association for Cancer Research Sebolt-Leopold, J. S. Clin Cancer Res 2008;14:3651-3656 Ras: Downstream Signaling

36. DiNicolantonio et al, JCO, 2008

38. Copyright ©2008 American Association for Cancer Research Sebolt-Leopold, J. S. Clin Cancer Res 2008;14:3651-3656 Ras: Downstream Signaling

39. 44th ASCO Annual Meeting May 30-June 3, 2008 McCormick Place, Chicago, Illinois Evaluation of PTEN expression in colorectal cancer (CRC) metastases (mets) and in primary tumors as predictors of activity of cetuximab plus irinotecan treatment F. Loupakis 1,6, L. Pollina 2, I. Stasi 1, G. Masi 1, N. Funel 2, M. Scartozzi 3, I. Petrini 4, D. Santini 5, S. Cascinu 3, A. Falcone 1,6. 1 Department of Oncology, Azienda USL 6 - Istituto Toscano Tumori Livorno, Italy, 2 Division of Pathology, AOUP, Pisa, Italy, 3 Division of Medical Oncology, Azienda Ospedaliera Ospedali Riuniti, Università Politecnica delle Marche, Ancona, Italy, 4 Division of Medical Oncology, AOUP, Pisa, Italy, 5 Division of Medical Oncology, Campus Biomedico University, Rome, Italy, 6 Department of Oncology, Transplants and New Technologies in Medicine, University of Pisa, Italy

40. PTEN PIP3 Ligands AKT Nucleus EGF receptor mTOR PIP2 PTEN (phoshatase and tensin homologue deleted on chromosome 10) gene encodes a phosphatase, whose major substrate is PIP-3 Loss of PTEN (mono or bi-allelic inactivation, but also epigenetic silencing) results in increased PIP-3 concentration Increase of PIP-3 leads to AKT hyperactivation PROTECTION FROM APOPTOSIS PTEN PI3K

41. CONCLUSIONS Primaries and related mets from CRC differed in terms of PTEN immunoreactivity in 40% of cases. KRAS mutations found on primaries are almost always (95% of cases) confirmed on mets. Such analysis may be ruled out on any available tumor sample. Loss of PTEN tested on mets predicted lack of activity of cetuximab plus irinotecan combination treatment in metastatic CRC pts. KRAS is confirmed to be a predictor of resistance to cetuximab plus irinotecan combination treatment in metastatic CRC pts. The combination of PTEN IHC performed on mets and KRAS mutational analysis identified a subgroup of patients with higher chances of benefiting from cetuximab plus irinotecan treatment.

42. PTEN: remaining challenges Reproducible assay Immunohistochemistry Gene copy number Sequence Proving predictive / prognostic value

43. Wong et al, JCO, 2008

44. Towards Personalized Therapy of Colon Cancer Tumor genetic profiling Stage II colon cancer Stage IV colorectal cancer Pharmacogenetics Oxaliplatin efficacy Irinotecan - UGT1A1 Therapeutic Drug Monitoring

45. Genetics and Pharmacokinetics / Pharmacodynamics Pharmacokinetics -Absorption -Distribution -Metabolism -Excretion Pharmacodynamics Tumor Host ResponseToxicity Dose Compliance Pharmacogenetics

46. H. L. McLeod, K. Owzar, D. Kroetz, F. Innocenti, S. Das, P. Friedman, K. Giacomini, R. Goldberg, A. Venook, M. J. Ratain Univ of North Carolina-Chapel Hill, Chapel Hill, NC; Duke, Durham, NC; UCSF, San Francisco, CA; University of Chicago, Chicago, IL; CALGB, Chicago, IL Cellular transporter pharmacogenetics in metastatic colorectal cancer: initial analysis of C80203

47. Predicting oxaliplatin efficacy? Genomic DNA from 180/238 patients on C80203 (FOLFOX vs. FOLFIRI +/- cetuximab) Genotype transporter genes involved in irinotecan and oxaliplatin clearance: ABCC2, ABCC4, ABCG2, SLCO1B1, SLC22A1, SLC22A2 Association of genotype with response and toxicity Result: ABCG2 34 G>A associated with response to FOLFOX, resistance to FOLFIRI but not to toxicity

48. Irinotecan pathway CPT-11 cell membrane CPT-11 SN-38 TOP1 Cell Death APC SN-38G ABCB1 CYP3A4 CYP3A5 CES1 CES2 UGT1A1 CES1 CES2 ABCC2 ABCG2 ABCC1 ADPRT TDP1 CDC45L XRCC1 NFKB1 NPC ABCB1

49. UGT1A1: promoter polymorphism and toxicity UGT1A1 gene structure Iyer et al 2002

50. UGT1A1 TA repeat: irinotecan neutropenia/activity 35.7 16.3 8.6 0 5 10 15 20 25 30 35 40 45 50 6/66/77/7 P=0.007 UGT1A1 genotype % grade 4/5 neutropenia McLeod et al, ASCO 2003 N=524 41.9 33.8 14.3 0 5 10 15 20 25 30 35 40 45 6/66/77/7 UGT1A1 genotype Objective response (%) P=0.045

51. Camptosar package insert, May 2005

52. Towards Personalized Therapy of Colon Cancer: Pharmacogenetics Oxaliplatin efficacy Rare polymorphism Small sample size Irinotecan toxicity Regimen depednent Not all or none VEGF antibody efficacy Polymorphisms? Cetuximab efficacy FC  R polymorphisms?

53. Towards Personalized Therapy of Colon Cancer Tumor genetic profiling Stage II colon cancer Stage IV colorectal cancer Pharmacogenetics Oxaliplatin efficacy Irinotecan - UGT1A1 Therapeutic Drug Monitoring

58. Biochemical Pathways of 5-FU Metabolism

60. Au et al.,1982 5D CVI, CRC LeucopeniaCpss > 1.5 µM Trump et al., 1991 3D CVI, CRC LeucopeniaCpss Yoshida et al.,1990 7D CVI, CRC LeucopeniaAUC > 33 mg/h.L Thyss et al.,1986 5D CVI, HNC LeucopeniaAUC  30 mg/h.L Vokes et al., 1996 3D CVI, HNC LeucopeniaCpss Reference Schedule, Tumor Toxicity PK variable, threshold AUC: Area under the curve; CCR: Colorectal cancer; Cpss: Steady state plasma concentration; CVI: Continuous venous infusion; HNC: Head and Neck Cancer IVB: Intravenous bolus

61. Hillcoat et al., 1978 5D CVI, CRC 36.1 19.2 Yoshida et al.,1990 7D CVI, CRC27.422.5 Milano et al.,1994 5D CVI, HNC 29.727.2 Vokes et al., 1996 5D CVI, HNC 27.5 21.0 Reference Schedule, Tumor AUC AUC (mg/h.L) (mg/h.L) (Responder) (Non-Responder) AUC: Area under the curve; CCR: Colorectal cancer; CVI: Continuous venous infusion; HNC: Head and Neck Cancer; LV: Leucovorin Studies Correlating Systemic Exposure with Tumor Response for 5-FU Infusional Regimen

62. Relationship between Systemic Exposure Relationship between Systemic Exposure and survival Milano et al., J Clinical Oncology 1994; 12: 1291

63. Remaining questions include: What is the target therapeutic range? For toxicity For efficacy With oxaliplatin When to sample? Would clinicans do this? Is this important?

64. Opportunities for TDM Most oral chemotherapy drugs Imatinib Sorafenib Sunitinib Taxanes Biologics

65. GI Stromal Tumor Oncologist Envy

66. Joensuu et al. N Engl J Med. 2001;344:1052. Copyright  2001 Massachusetts Medical Society. GIST: PET change after 4 weeks imatinib Multiple liver and upper abdominal 18 FDG-accumulating metastases A marked decrease in 18 FDG uptake 4 weeks after starting imatinib

67. Overall Survival by Genotype (Kaplan-Meier Estimate) Joensuu H et al. Eur J Cancer. 2007;5(suppl):404. Abstract 7506. over all KIT mutation groups Von Mehren, ASCO, 2008

70. Towards Personalized Therapy of Colon Cancer Tumor genetic profiling Nearing clinical reality for stage II patients KRAS a standard; BRAF coming; others pending Pharmacogenetics Complex Therapeutic Drug Monitoring Is there enough benefit to justify the effort? Can we change the paradigm for clinical trials?