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Alberto Bardelli Institute for Cancer Research and Treatment

Molecular mechanisms of resistance to anti EGFR based therapies in colorectal cancer. Alberto Bardelli Institute for Cancer Research and Treatment University of Torino - Medical School. DISCLOSURES Founder: Horizon Discovery (Cambridge, UK) Consultant: Merck-Serono, Amgen.

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Alberto Bardelli Institute for Cancer Research and Treatment

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  1. Molecular mechanisms of resistance to anti EGFR based therapies in colorectal cancer Alberto Bardelli Institute for Cancer Research and Treatment University of Torino - Medical School

  2. DISCLOSURES Founder: Horizon Discovery (Cambridge, UK) Consultant: Merck-Serono, Amgen

  3. Mutations and the cancer genome Mutations and resistance to therapies in CRCs Parallel clinical trials in cells, mice and patients

  4. “Cancer is, in essence, a genetic disease. Although cancer is complex, and environmental and other nongenetic factors clearly play a role in many stages of the neoplastic process, the tremendous progress made in understanding tumorigenesis in large part is owing to the discovery of the genes, that when mutated, lead to cancer.” Bert Vogelstein (1988) NEJM 1988; 319:525-532.

  5. Cancer: a genetic disease

  6. DNA IS DIGITAL Tumour Normal Mutation

  7. Tyrosine kinome mutations Residue is evolutionarily conserved Mutations of equivalent residues in other kinases are pathogenic Bardelli et. Al., Science: 300;949 (2003)

  8. Mutational lansdscapes of cancer genomes TP53 PIK3CA PIK3CA TP53 APC KRAS Wood et al., Science : 318 (2007)

  9. The genetic bases of response and resistance to EGFR therapies BRAF PIK3CA

  10. Mutation X Drug Y Parallel clinical trials in cells, mice and patients

  11. EGFR-targeted therapies in CRCs TK Inhibitors 3 Anti-ligand- blocking Antibodies 2 Ligand– toxin Conjugates 4 Antibody– toxin Conjugates 5 Anti-HER1/EGFR-blocking antibodies 1 Noonberg SB, Benz CC. Drugs 2000;59:753–67

  12. Who will benefit from treatment with antibodies targeting EGFR in mCRCs ? Responders (15-20%) Non-Responders Bardelli and Siena, J Clin Oncol 2010

  13. EGFR Mutations EGFR Protein expression (IHC) EGFR Gene Copy Number Ras Raf PI3K MEK p85 MAPK PDK GSK AKT S6K Cetuximab Panitumumab EGFR Ras Ras Ras Ras Ras Ras Ras Ras Ras Ras Ras SOS Raf Raf Raf Raf Raf Raf Raf Raf Raf Raf Raf Grb2 PI3K PI3K PI3K PI3K PI3K PI3K PI3K PI3K Shc MEK MEK MEK MEK MEK MEK MEK MEK MEK MEK p85 p85 p85 p85 p85 p85 p85 PTEN MAPK MAPK MAPK MAPK MAPK MAPK MAPK MAPK MAPK DUSPs PDK PDK PDK PDK PDK GSK GSK AKT AKT AKT AKT S6K S6K S6K Moroni et al Lancet Oncology 2005

  14. mCRC patients treated with panitumumab or cetuximab, N=114 BRAF mutational status on Wild-Type KRAS tumors (N=79) Benvenuti et al.,Cancer Research. 2007 Di Nicolantonio et al.,J Clin Oncol. 2008

  15. Responder (15%) KRAS/PIK3CA mutated KRAS-NRAS mutated (35-45%) BRAF/PIK3CA mutated BRAF mutated (8%) 20-25% ??? PIK3CA mutated and/or PTEN loss (15-20%) Bardelli and Siena, J Clin Oncol 2010

  16. Sartore-Bianchi A et al., PLOS One 21010

  17. Siena; Di Nicolantonio and Bardelli JNCI 2009

  18. KRAS, NRAS, or BRAF mutations are non overlapping, while PIK3CA mutations may occur concomitantly with any of the above Janakiraman M et al.,Cancer Res; 70(14) July 15, 2010

  19. From gene targeted therapies to mutant targeted therapies • Example 1: PIK3CA mutations • Example 2: KRAS mutations

  20. PIK3CA mutations and resistance to anti EGFR MoAbs ? • Sartore-Bianchi A et al., Cancer Res 2009 YES • Prenen et al., ClinCancer Res 2009 NO

  21. Different role for individual PIK3CA mutations on the response to EGFR MoAbs in mCRCs Zhao and Vogt PNAS 2008

  22. Effects of KRAS, BRAF, NRAS and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal Sample characteristics Total number of samples successfully assessed 969/1000 (97%) Type of tissue sample Primary tumor 790/969 (81.5%) Metastasis 118/969 (12.2%) Missing 61/969 (6.3%) Total number of chemotherapy-refractory tumors 717/969 (74%) Treatment type in chemotherapy-refractory tumors Panitumumab monotherapy 16/717 (2.2%) Cetuximab monotherapy 43/717 (6%) Cetuximab + chemotherapy 658/717 (91.8%) De Roock et al., EU Consortium Lancet Oncology, 2010

  23. Multivariate Cox regression analysis of overall survival in the unselected and KRAS wild-type population Unselected population KRAS wild-type population Genotype Adjusted hazard ratioOS (95% CI) LRT p-value Adjusted hazard ratio OS (95% CI) LRT p-value KRAS (mutant vs. wild-type) 1.87 (1.51-2.31) <0.0001 NC NC PIK3CAexon 9 (mutant vs. wild-type) 1.08 (0.77-1.51) 0.67 1.27 (0.75-2.14) 0.39 PIK3CAexon 20 (mutant vs. wild-type) 1.57 (0.90-2.76) 0.14 3.69 (1.69-8.02) 0.0055 BRAF (mutant vs. wild-type) 2.68 (1.70-4.22) 0.00016 2.97 (1.88-4.70) <0.0001 NRAS (mutant vs. wild-type) 1.81 (1.00-3.26) 0.069 1.96 (1.08-3.55) 0.042 De Roock et al., EU Consortium Lancet Oncology, 2010

  24. From gene targeted therapies to mutant targeted therapies • Example 1: PIK3CA mutations • Example 2: KRAS mutations

  25. mCRC patients N=114 Cancer Res 2007;67(6):2643–8 & J Clin Oncol. 2008; 26:5705-5712.

  26. KRAS mutations: clinical results from cetuximab treated mCRC Moroni Lancet Oncol 2005 n=31 Lièvre Clin Cancer Res 2006 n=30 Di Fiore Br J Cancer 2007 n=59 Frattini Br J Cancer 2007 n=27 Benvenuti Cancer Res 2007 n=48 Khambata-Ford J Clin Oncol 2007 n=80 De Roock ASCO Proc 2007 n=37 Finocchiaro ASCO Proc 2007 n=81 Response rate: analysis of 8 studies available in PubMed or from ASCO RAS mutated (7.0%) RAS mutated (43.9%) wt (93.0%) wt (56.1%) Responders (n=82) Non-Responders (n=312)

  27. KRAS mutations Smith G, et al., British Journal of Cancer (2010), 1 –11 GEP GDI GDP GTP GDP GTP RAS (inactive) RAS (active) Effectors: RAF/MAPK/ERK PI3K/AKT GAP Pi Farnesyl Geranylgeranyl

  28. Meta-analysis of 3 Chemotherapy Refractory Datasets NCIC CTG dataset from CO.17 trial Leuven dataset from clinical trials: EVEREST, BOND, SALVAGE, BABEL Italian dataset: from clinical trials mentioned above from non-trial patients with advanced, irinotecan-refractory CRC considered suitable to receive an EGFR MAb

  29. KRAS Mutation Status and Therapy by Dataset

  30. Baseline Patient Characteristics by Tumour KRAS status * between biomarker positive and negative groups from chi-square test for categorical variables and t-test for continuous variables.

  31. KRAS G13D Mutation status as a prognostic factor for OS in patients not treated with Cetuximab or Panitumumab? 100 80 60 Proportion alive 40 20 0 0.0 5.0 10.0 15.0 Time from randomization (months) De Roock et al JAMA 2010

  32. OS Predictive Analysis by KRAS status: EGFR MabMonotherapyvs no EGFR Mab 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 0 0 0.0 5.0 10.0 15.0 20.0 0.0 5.0 10.0 15.0 20.0 0.0 5.0 10.0 15.0 KRAS G13D Mutation Other KRAS Mutation KRAS Wild-type HR 0.56 (0.42 to 0.73) p<0.0001 HR 0.98 (0.70 to 1.38) p=0.91 Proportion alive Proportion alive Proportion alive HR 0.23 (0.09 to 0.61) p=0.002 Time from randomization (months) Time from randomization (months) Time from randomization (months) Monotherapy with cetuximab or panitumumab No Treatment with cetuximab or panitumumab De Roock et al JAMA 2010

  33. Molecular bases of G12V versus G13D mediated resistance to cetuximab in cellular and animal models

  34. Mutation X Drug Y Parallel clinical trials in cells, mice and patients

  35. Isogenic models of tumour progression Knock-in of oncogenic mutations Knock-out of cancer genes wt p53 -/- Ras / Raf PI3K EGFR Homologous recombination A B A Isogenic cells carrying cancer mutations B

  36. Mutation-specific pharmacogenomic profiles + Parental cell line Knock-in cell line Drug screening Incubate cells with drugs Mutated genotype selective drug Drug with no selectivity Wild genotype selective drug Di Nicolantonio et al., J Clin Invest, 2010 Di Nicolantonio; Arena et al., PNAS 2008

  37. Experimental design Cellularmodel Gene targeting (Knock-in approach) KRAS: G12D, G12V, G12C, G12A, G12S, G12R, G13D BRAF: V600E, PIK3CA: E545K (exon 9), H1047R (exon 20) Biochemical validation (pathway activation) Measure drug response

  38. G12V (G35>G/T) NotI NotI P Neo LoxP LoxP AAV-KRas-12V ITR ITR ITR ITR A NotI NotI G13D (G38>G/A) P Neo LoxP LoxP LoxP AAV-KRas-13D Knock-in G12V (or G12D / G12C) B Homologous recombination KRAS WT CRC cells C Knock-in G13D SW48 KRAS WT SW48 KRAS G12V SW48 KRAS G13D

  39. KRAS G12V or G13D and chemotherapy in cellular models De Roock et al JAMA 2010

  40. KRAS G12V and G13D and ceruximab in cellular models De Roock et al JAMA 2010

  41. Cetuximab delays growth of SW48 tumor xenografts De Roock et al JAMA 2010

  42. Cetuximab does not affect growth of G12V tumors, but inhibits the growth of G13D tumor xenografts SW48 KRAS G12V SW48 KRAS G13D Start of treatment Start of treatment De Roock et al JAMA 2010

  43. Secondary resistance to targeted therapies Responders (15-20%) Non-Responders 2007

  44. Secondary resistance to targeted therapies Responders (15-20%) Non-Responders 2010

  45. Mutation X Drug Y Parallel clinical trials in cells, mice and patients

  46. DNA, RNA and protein extraction, FFPE blocks stored by the pathologist DNA, RNA and protein extraction, FFPE blocks stored by the pathologist Liver Met implanted s.c. in NOD SCID mice Marker A Drug X Patient undergoing liver metastasectomy of CRC Using this approach 112 samples were succesfully engrafted since Oct 2008 A. Bertotti & L. Trusolino, Molecular Oncology, IRCC Marker B Drug Y Expansion

  47. Xenopatients from the pathologist NUMBER OF SAMPLES FFPE blocks DMSO RNA later 148 SURGERY p0 engraftment (2 mice) >90% DMSO RNA later Archive RNA extraction Genomic DNA extraction p1 expansion (6 mice) DMSO RNA later Snap Frozen DMSO RNA later Snap Frozen FFPE blocks p2 treatment (24 mice) 44 A. Bertotti & L. Trusolino, Molecular Oncology, IRCC

  48. In vivo – M016

  49. Understanding secondary resistance to cetuximab Time 0: Molecular analysis using multiple omics’ technologies (WP3) Time x: Molecular analysis using multiple omics’ technologies (WP3) control cetuximab secondary resistance Chronic treatment with cetuximab (0.5 mg/injection/2x/week)

  50. Xenopatient M026: development of resistance

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