The Influence of Molecular Biology in the Treatment of Colorectal Cancer Chongqing, June 2009

1. The Influence of Molecular Biology in the Treatment of Colorectal CancerChongqing, June 2009 Cynthia Gail Leichman, M.D. Aptium Oncology Desert Regional Medical Center Palm Springs, CA

2. Thank You for this Invitation I am honored to address you today. I have been treating patients with colorectal cancer for nearly 30 years When I started, it was a one drug disease We now have several chemotherapies and targeted agents Future progress depends not only on developing new treatment agents, but understanding what drives the disease for designing more specific and effective agents

8. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Focus of this talk: Current US standard adjuvant therapies for colon and rectal cancer (CRC) Evidence for the impact of specific gene expression, mutation and molecular pathways on the natural history of CRC The impact of molecular biology on current clinical trials

9. Currently Available Therapeutic Options in Colorectal Cancer (CRC) Fluoropyrimidines Irinotecan Oxaliplatin Cetuximab Panitumumab Bevacizumab Combinations

10. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Standard US colon cancer treatment ADJUVANT THERAPY: Stage II: Treat as stage III or clinical trial Molecular risk assessment Stage III: Adjuvant therapy with 6 months of: FOLFOX or FLOX or Capecitabine or Clinical trial

11. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Standard rectal cancer therapy in the U.S.: Neoadjuvant 5-FU infusion with radiation; post-op 5-FU/leucovorin or Postoperative radiation with 5-FU/leucovorin or Clinical Trial ** Even though not yet confirmed by randomized clinical trials, many clinicians are substituting capecitabine for infusion 5-FU and/or adding oxaliplatin

12. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Standard US colon cancer therapy Stage IV: 1st Line: FOLFOX + bevacizumab 2nd Line: Irinotecan (single agent or FOLFIRI)+/-cetuximab*or clinical trial 3rd Line: Varying combinations of the above or clinical trial

13. The Influence of Molecular Biology in the Treatment of Colorectal Cancer A Primer of US �Trialspeak� National Cancer Institute: US government research, treatment and funding agency for cancer Cooperative Group: An organization of university and clinical cancer centers and private oncology groups designing and conducting clinical trials SWOG, ECOG, NSABP, CALGB, RTOG, NCCTG, ACOSOG Intergroup: An NCI sponsored organization of disease site representatives from the cooperative groups that designs, oversees and endorses phase III randomized controlled trials

14. The Influence of Molecular Biology in the Treatment of Colorectal Cancer A Primer of US �Trialspeak� Example: N0417 is an adjuvant colon trial led by the NCI sponsored group NCCTG, endorsed by CALGB, ECOG, SWOG and NSABP

15. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Current or Recent Adjuvant Trials INT E5202: Testing FOLFOX +/- bevacizumab in high-risk stage II colon cancer (18q LOH) INT N0147: Testing the question of addition of cetuximab to FOLFOX (PETACC-8 in Europe asking the same question)

16. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Current or Recent Adjuvant Trials NSABP C-08: Tested addition of bevacizumab to FOLFOX (negative � ASCO 2009) NSABP R-04: Neo-adjuvant rectal trial testing external beam radiation and: a) capecitabine vs infusion 5-FU b) addition of bevacizumab

17. The Influence of Molecular Biology in the Treatment of Colorectal Cancer How is trial eligibility decided? Is knowing the cancer stage enough?

19. Colorectal Cancer Tumorigenesis Vogelstein, et. al. NEJM, 1988

20. Colorectal Cancer Tumorigenesis (Now)

21. Molecular Assessment of Response in Disseminated Disease

22. Microsatellite Instability Loss of mismatch repair - independent developmental pathway for CRC ~ 15% CRC exhibit MSI Can MMR be exploited for therapeutic decisions?

23. Microsatellite Instability�then Issues: Incidence too low for prediction? MSI in liver metastases not predictive for 5-FU response N=56 (1.8% MSI-H), Rosty, IntJ Can 2001 MSI may influence other response markers : Generally diploid cell populations Decreased VEGF in MSI-H CRC N=120, Wynter J Pathol 1999

24. Microsatellite Instability � and Now Microsatellite Instability Predicts Improved Response to Adjuvant Therapy With Irinotecan, Fluorouracil, and Leucovorin in Stage III Colon Cancer: Cancer and Leukemia Group B Protocol 89803 Bertagnolli, et al JCO 2009 1264 stage III colon cancer pts Randomized to 5FU + leucovorin vs Irinotecan + 5FU+LV Prospective tumor block collection for MSI analysis and outcome correlation

25. Microsatellite Instability

26. Microsatellite Instability

27. p53 and CRC Probably late event in tumorigenesis p53 overexpression/LOH 50% colon cancers right sided: 30% left sided: 60% p53 status and fluoropyrimidine therapy: p53 mutated cells less sensitive p53 wt more sensitive

28. Evidence: +/- for prognostic, predictive impact Many small analyses suggest trend toward increased radio-, chemo-sensitivity Relation to ploidy, microvessel density, other genes (p21, Bax, Bcl-2,TS) is defining p53 and CRC

29. Evidence: P53 has not been an independent therapeutic target Now related to other target pathways May exist in balance with other tumor suppressor genes Associated with TS and prognostic of recurrence Over-expressors benefit more from 5FU-based adjuvant therapy p53 and CRC

30. Bcl-2 Family of intracellular membrane proteins Evolutionarily conserved Affects apoptosis regulation mitochondrial cytochrome-c release Some members of family block apoptosis: Bcl-2 Some members of family promote apoptosis: Bax

31. Bcl-2 Evidence: Favorable Independent Survival Predictor? N=154 stg II, Sinicrope, CCR 1995; N=93, Bukholm,Virch Arch 2000 No impact on chemoradiotherapy - Rectal N=24(preop), Scott, EurJCan, 1998 No impact on chemotherapy - CRC N=231, Schneider, BrJCan 1997; N=84, Paradiso, BrJCan 2001 Survival Impact with p53 Status N=160(rectal), Schwander, EurJCan 2000

32. Bcl-2 Evidence: 188 stage III colon cancer PTS treated with 5FU based adjuvant therapy Tumors analyzed for p53, MSI, BAX and Bcl2 High BAX + better DFS High BAX/-p53 better than high BAX/+p53 Nehls et al; British J Cancer 2007

33. Vascular Endothelial  Growth Factor Regulates pathologic angiogenesis via: ?endothelial cell mitogenesis ?migration and remodeling of extracellular matrix may associate with thymidine phosphorylase (TP) VEGF may be p53 independent mechanism for aggressive biology especially in MSI CRC Antibodies/scoring technique complicate data

34. Vascular Endothelial  Growth Factor Evidence: Correlates with microvessel count, PD-EGF, survival N=136, Amaya, CanLett 1997 Correlates with stage, invasion, metastases N=111, Takeda, OncRept 2000 Serum levels correlate with response N=41(15CRC), Dirix, BrJCan 1997

35. Vascular Endothelial Growth Factor and Treatment Anti-VEGF Moab increases response to 5-FU, metastatic CRC N= 104, Bergsland, ASCO, 2000

36. Vascular Endothelial Growth Factor and Treatment 813 metastatic CRC PTS randomized to IFL* + bevacizumab vs IFL + placebo Overall survival 20.3 vs 15.6 mo (p=0.001)

37. Vascular Endothelial Growth Factor and Treatment

38. Vascular Endothelial Growth Factor and Treatment Association of k-ras, b-raf, and p53 Status With the Treatment Effect of Bevacizumab Ince et al, JNCI 2005 295 (of 813) tumor samples from Hurwitz trial; (274 primary tumor, 71 metastases) Analyzed for mutation KRAS, BRAF and p53 ( or nuclear p53 expression by IHC) No statistically significant impact on benefit of bevacizumab treatment by mutation in any of these genes

39. Forest plots illustrating hazard ratios for risk of death according to biomarker status and treatment subgroup

40. Vascular Endothelial  Growth Factor

41. Vascular Endothelial  Growth Factor

42.   Thymidylate Synthase (TS)  

43. Thymidylate Synthase Evidence: Prospective: PCR TS gene expression analysis correlates with response to PI 5-FU N=42, Leichman, JCO 1997 Prospective: IHC TS expression correlates with response to bolus, IA 5-FU N=41, Cascinu, CCR 1999; N=36, Davies, CCR 1999 Retrospective: IHC TS expression correlates with response to MTX/5-FU, PI N=48, Aschele, JCO 1999

44. Thymidylate Synthase Evidence: Retrospective: IHC TS expression in primary may not predict 5-FU response in metastases N=134, Findlay BrJCan 1997 Prospective: PCR TS expression differs by metastatic site (Liver < lung, nodes, peritoneum) N=19, Gorlick, JCO 1998; N=90, Ichikawa, ASCO 2001 Issues: Methodology PCR? IHC? Microarray? Genotype (Polymorphisms)?

45. Dihydropyrimidine Dehydrogenase  Initial and rate-limiting step in FP catabolism ? expression in tumors vs normal tissue ? levels in CRC non-responders vs responders Interaction with other molecular parameters of fluoropyrimidine metabolic pathway

46. Thymidine Phosphorylase Catalyzes the reversible phosphorylation of thymidine to thymine activates pyrimidine antimetabolites Identical to platelet-derived endothelial cell growth factor (PD-ECGF) promotes angiogenesis and cell motility ? expression in tumor than in normal tissue

47. Thymidine Phosphorylase ? levels in tumor cells = sensitivity to FPs ? TP associated with advanced stage, node positivity, microvessel count Optimum expression for activity not known May exist in balance with catalytic enzymes, e.g. DPD

48. Molecular Assessment of Response in Disseminated DiseaseTS:QUANTITATION IN DISSEMINATED COLORECTAL CANCER

49. *p-value based on Fisher's Exact test (2-tail) Molecular Assessment of Response in Disseminated DiseaseResponse to CI 5-FU in colon cancer: Effect of TS, TP, and DPD

50. TP/DPD Ratio and Response Prediction Higher TP/DPD ratios in tumor vs normal tissue colon primary and metastases N= 25, Collie-Duguid et al, Int J Cancer 2001 Predictive of response to doxifluridine in gastric cancer N=93, Terashima et al, Eur J Cancer 2002 N=22, Nishina et al, Eur J Cancer 2004

51. ERCC-1 and repair in Colorectal Cancer

53. ERCC-1 and repair in Colorectal Cancer

54. Cetuximab and Mechanism of Action Cetuximab is a chimeric antibody directed at the EGFR receptor on the cell surface to block signal transduction cascade EGFR receptor presumed necessary for activity and it�s presence required by IHC analysis for many trials Chung, et al, identified 16 chmotherapy refractory patients treated with cetuximab =/- irinotecan 4 (25%) responses seen

55. K-RAS mutation and Resistance KRAS operates downstream of the cell-surface location of the epidermal growth factor in the signal transduction pathway Mutations at KRAS codons 12 and 13 cause constitutive KRAS activation Mutations at these cites associated with anti-EGFR antibody resistance Mutations found in 30-50% of colon cancers

56. EGFR-KRAS Signaling Pathway

57. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Large clinical trials have now produced objective data for association of KRAS mutation with resistance to anti-EGFR antibodies in treatment of metastatic colon cancer This is the first body of data conclusive enough to form a selection criterion for on-going and future clinical trial in CRC

58. Panitumumab in the Treatment of Colorectal Cancer

59. Panitumumab in the Treatment of Colorectal Cancer

60. Cetuximab and Chemotherapy as Initial Treatment for Metastatic Colorectal Cancer Metastatic colorectal cancer with EGFR expression 599 PTS received FOLFIRI; 599 received FOLFIRI + cetuximab Retrospective analysis of KRAS mutation

61. Cetuximab and Chemotherapy as Initial Treatment for Metastatic Colorectal Cancer

62. Impact of other mutations in the KRAS signal transduction pathway PIK3CA Mutation Is Associated With Poor Prognosis Among Patients With Curatively Resected Colon Cancer Ogino, et al, JCO 2009 450 resected colon cancer PTS stages I-III 18% had mutated PIK3CA Worse colon CA specific survival mutated vs wild-type PIK3CA Worse survival with mutated PIK3CA seen in KRAS wild-type vs mutated

63. Impact of other mutations in the KRAS signal transduction pathway

64. Impact of other mutations in the KRAS signal transduction pathway Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer Di Nicolantonio et al, JCO 2008 113 CRC PTS treated with cetuximab or panitumumab 70% KRAS; 11/79 of these had BRAF V600E mutation None of BRAF mutated PTS responded; none of responders carried BRAF mutation

65. Impact of other mutations in the KRAS signal transduction pathway

66. Integration of Molecular Markers:Methodology Issues Immunohistochemistry Ease, Cost, Specimen Availability Interpretation, Reagent, Quantitation Real-Time PCR Quantitative, Reproducible, Small Sample Tissue Requirement, Tumor Heterogeneity

67. Integration of Molecular Markers:Impact on Trial Design Select Populations by Molecular Expression: Smaller Trial, Shorter Time, Greater Benefit Multiple Correspondence Analysis (Buglioni, et al) Graphs multiple frequency table variables Categories plotting closely - statistically related Enrichment Approach (McKay, et al) Examine multiple factors; �Degree of Difference� Group factors with trends to clinical significance

68. Integration of Molecular Markers in the Treatment of CRC Future phase II and III trials should include molecular parameters Select Population by Developmental Mechanism: MIN vs CIN Select Population by �First Tier� (e.g. TS) Stratify by associated factors Add to first tier of subsequent trials

69. RECTAL CANCER: A Model for Translational Trials Direction of treatment and trials is neo-adjuvant therapy with clinical goal of organ (sphincter) sparing Tissue is fairly easily available for biopsy to study genetic markers A reasonable body of molecular-genetic data exists to build on for assessing new treatments and further defining critical cellular pathways

70. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Patients With Curative Resection of cT3-4 Rectal Cancer After Preoperative Radiotherapy or Radiochemotherapy: Does Anybody Benefit From Adjuvant Fluorouracil-Based Chemotherapy? Collette et al, JCO 2007 1011 PTS withT3 or T4 rectal cancer Randomize: pre-op RT; pre-op chemoRT; pre-op RT+post-op chemo; pre-op chemoRT + post-op chemo No survival difference; chemo increased local control

71. RECTAL CANCER: Selection Trial How are we proceeding with molecular selection in clinical trials? A US example: SWOG is conducting a phase II clinical neoadjuvant trial for rectal cancer utilizing: Molecular selection pCR as clinical endpoint Molecular analysis to define a molecular profile correlating with pCR

72. RECTAL CANCER: Selection trial SWOG 7013 Primary Objective: To determine the complete pathologic response rate for the combination of oxaliplatin, capecitabine and cetuximab alone and concomintantly with external beam radiation (EBRT) pre-operatively for patients with adenocarcinoma of the rectum, stages II and III.

73. SWOG 7013 SCHEMA Cycle 1:D1 D8 D15 D22 D29 D36 D42 D49 OXP X X X X X Erb X X X X X XEL X X X X Biopsy X Cycle 2: OXP X X X X X Erb X X X X X XEL X X X X XRT X X Biopsy X*

74. RECTAL CANCER: Selection Trial SWOG 7013 Secondary Objectives: To assess the value of the intratumoral expressions of : TS, TP, DPD (predictive markers for 5-FU) ERCC1, GST-P1, XPD (predictive for oxalipaltin) EGFR, VEGF (predictive for cetuximab?) IL-6, COX-2 (? from baseline prognostic for pCR) in determining patients who will have pathologic complete responses at surgery.

75. RECTAL CANCER: Selection Trial SWOG 0713 ELIGIBILITY: Full colonoscopy c Biopsy = adenocarcinoma Paraffin block available Wild-type K-RAS by mutational analysis Clinical stage I-III by CT C-A-P; optional EUS >18 yrs old; no other CA x 5 yrs; PS < 2; No prior rectal CA Tx Standard good practice lab values; CEA No unbypassed bowel obstruction; Non-pregnant No peripheral neuropathy Able to give informed consent

76. RECTAL CANCER: Selection Trial Questions that remain: Do we need to give more therapy to those with pCR? 10% recurrence with pCR in breast cancer; 30% recurrence with pCR in esophagus cancer European data suggests this is the cohort benefitting from post-op adjuvant therapy Do we give more of what worked, or something different assuming anything not detectable is resistant? Will the other molecular parameters in the tumor profile direct this decision?

77. RECTAL CANCER: Is Less Better? Is less better in the neo-adjuvant therapy of rectal cancer than prolonged post-operative adjuvant therapy? It could be if: A pre-treatment molecular profile predicts pCR with the therapy being employed A pre-treatment molecular profile suggests a different treatment needs to be used at the outset A pre-treatment molecular profile suggests lack of response to currently available agents and surgery first will be the most useful intervention

78. COLORECTAL CANCER: New Potential Targets IL6 Overexpression associated with tumor adhesion/invasion Overexpression asociated with chemoresistance COX 2 Over-expression associated with tumor growth and progression Enhances invasion Increased levels associated with K-RAS mutation IGFR Separate pathway from EGFR Activation is upstream of EGFR

79. COLORECTAL CANCER: New Potential Targets ERK1-2 Functions in anchorage independent cell growth Target of MEK inhibitor therapy MEK inhibitor active in KRAS/BRAF mutated but not wild-type CRC cells mTOR Serine/threonine kinase; regulates cell proliferation, survival angiogenesis mTOR inhibition synergistic with irinotecan throug the HIF1a pathway mTOR inhibition synergistic with tyrosine kinases through the EGFR pathway

80. The Influence of Molecular Biology in the Treatment of Colorectal Cancer

81. Molecular Assessment of Response in Disseminated Disease

82. The Influence of Molecular Biology in the Treatment of Colorectal Cancer Intracellular pathways driving cancer growth, progression and metastases are both complex and redundant Therapies targeting single genes are unlikely to be adequate Proof of concept for multi-agent, mult-target therapies is a daunting challenge for future clinical trials We aren�t there yet, but�

83. The Influence of Molecular Biology in the Treatment of Colorectal Cancer IN THE APPROACHING AGE OF PERSONALIZED MOLECULAR MEDICINE, THE CANCER WILL INFORM US OF ITS APPROPRIATE THERAPY

84. Thank you !