Personalized Cancer Treatment and Biomarkers in Colorectal Carcinoma

1. DNA diagnosis for colorectal carcinoma • Patrick Willems • GENDIA • Antwerp, Belgium

3. Treatment of Colorectal carcinoma • surgery • radiation • Chemotherapy • Targeted treatment • Immunotherapy

4. Personalized cancer treatment • Immunotherapy to stimulate immune response to cancer PD-1 inhibitors PD-L1 inhibitors CTLA-4 inhibitors • Targeted therapy with designer drugs that target the genetic cause of the tumor Monoclonal antibodies (mAB): Herceptin Tyrosine kinase inhibitors (TKI): Gleevec

5. Problems in personalized cancer treatment • Immunotherapy Extremely expensive (100-300.000 Euro/year) Few biomarkers (companion diagnostics) • Targeted therapy with designer drugs Very expensive (50-100.000 Euro/year) Biomarkers (companion diagnostics)

6. Problems in personalized cancer treatment The very high cost of personalised treatment makes companion diagnostics (cancer biomarkers) necessary

8. Cancer biomarkers tumor material (biopsy) blood (liquid biopsy)

9. Market for tumor biomarkers in Liquid biopsies TARGETS DRUGS SEQUENCING Liquid biopsy market for tumor biomarkers: 40 Billion USD per year (Illumina estimate)

10. Current paradigm • PATIENT • general • treatment • visit • PHYSICIAN • Result • Pathological studies • sample • PATHOLOGIST • Lab

11. Future paradigm • PATIENT • Personalised • treatment • visit • PHYSICIAN • PHARMA • Result • Molecular testing • sample • LAB • Pathologist

12. The changing face of cancer diagnosis

13. Cancer Morbidity and Mortality

14. New cancers per year in Belgium • Lung : 7.100 • Colon : 6.500 • Prostate : 8.800 • Breast : 9.700 • Melanoma : 1.500 TOTAAL : 65.000

15. Colorecal carcinoma (CRC) • second leading cause of cancer related mortality (12.2 %) • 132.700 new cases anticipated in 2015 in the US • 49.700 deaths in 2015 in the US • Five-year survival rates for patients with metastatic disease still low

16. Treatment of CRC • surgery • radiation • Chemotherapy • Targeted treatment • BRAF inhibitor • MEK inhibitor • Immunotherapy • CTLA-4 inhibitors • PD-1 inhibitors • PD-L1 inhibitors

17. Immunotherapy for CRC • CTLA-4 (cytotoxic T-lymphocyte–associated antigen 4) : ipilimumab, tremelimumab • PD-1 (programmed death-1) : nivolumab, pembrolizumab, Lambrolizumab, pidilizumab • PD-L1 (programmed death-1 ligand) : BMS-935559, MEDI4736, MPDL3280A and MSB0010718C • Other checkpoints : TIM3, LAG3, VISTA, KIR, OX40, CD40, CD137

18. Inhibition immune checkpoints

19. Biomarkers for immunotherapy for CRC Few biomarkers for immunotherapy First real biomarker : MicroSatellite Instability (MSI) Response to pembrolizumab (PD-1 inhibitor) in CRC MMR-proficient : 0 % MMR-deficient : 40 % NEJM : May 30, 2015 (Vogelstein group)

20. MSI as Biomarker for immunotherapy in CRC MMR deficiency Genomic instability Large mutation load in CRC (driver and passenger) Many mutant proteins - neoantgens Immune response

21. Microsatelliteinstability (MSI)

22. Targeted treatment for CRC Personalised targeted treatment inhibits specific somatic mutations that cause MM These mutations are patient-specific These mutations can be detected by molecular studies of : tumor material (biopsy) : FFPE, fresh or frozen blood (liquid biopsy)

23. Why liquid biopsies for CRC ? • Common cancer • High mortality • High load of driver oncogenic mutations • Druggable targets

24. Inheritance of cancer Majority of cancers are caused by genetic anomalies in the tumor (somatic mutations) Minority of cancers is inherited (germline mutations) : • Breast Cancer : 10 % • Colon cancer : 3-5% • Prostate cancer : low • Lung cancer : very low

25. Inheritance of CRC 3-5 % germline mutations MANY somatic mutations

26. Germline mutations in Coloncancer Polyposis coli: APC gene (Autosomal dominant) MUTYH (Autosomal recessive) Hereditary Non Poliposis Coli (HNPCC) : Autosomal dominant mutations in : MLH1, MSH2, MSH6, PMS1

27. HNPCC Autosomal dominant germline mutation : 1. MLH1, MSH2, MSH6, PMS2 : majority 2. Constitutional (germline) epimutation in MLH1 3. Germline deletion EPCAM gene leading to epigenetic change (methylation-downsilencing of MSH2)

28. Two step cancer theory (Knudson) Retinoblastoma (RB1 gene) Mesothelioma Uveal melanoma (BAP1 gene)

29. Multistep cancer theory (Vogelstein) Vogelstein et al, Science Aug 22, 2013

30. Colon cancer

31. Cancer genes and mutations • 140 driver genes • 60 % TSG • 40 % oncogenes • > 1000 driver gene mutations (Most tumors 2-10 driver gene mutations) • Millions (?) passenger gene mutations (Most tumors 10-100 passenger gene mutations)

32. Mutations in cancer • Gate keeper mutations : transforms normal cell into tumor cell Rb in retinoblastoma APC in colon cancer • Driver mutations : confers growth advantage to tumor cell HER2 in breast cancer KRAS in colon cancer • Passenger mutations : accidental mutation not conferring growth advantage to tumor cell Any gene Also driver gene

33. Mutations in cancer • Inactivation of tumor suppressor genes TP53 in breast cancer APC in colon cancer • Activation of oncogenes HER2 in breast cancer KRAS in colon cancer • Inactivation of DNA repair genes BRCA1/2 in breast cancer MLH1, MSH2, MSH6 in colon cancer

34. Mutations in cancer • Inactivation of tumor suppressor gene or DNA repair gene : • Intragenic inactivating mutation • Promotor Methylation • Gene Loss • Activation of oncogenes : _ Intragenic activating mutation • Gene amplification

35. Driver and passenger gene mutations Tumors with high mutation load due to Mutagens or genomic instability form many neoantigens and are candidates for immunotherapy

36. Somatic mutations in cancer P

37. Somatic mutations in CRC P

38. Cell growth and survival pathway

39. Cell growth pathway • Ligands • Receptors : EGFR • Secondarymessengers : 2 pathways : • MAPK pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6 • PI3K / AKT pathway : PI3K, PTEN, AKT, mTOR

40. Driver mutations in CRC • MAPK pathway : KRAS, BRAF, NRAS • PI3K / AKT pathway : PIK3CA

41. Classicaltreatment in coloncancer • Surgery • Chemotherapy • If pathology shows EGFR overexpression Start anti EGFR therapy : • mAB : Cetuximab, panitumumab • TKI : erlotinib, gefitinib, afatinib

42. EGFR overexpression in CRC • In Lung Ca : activatingmutations TK domain of EGFR • In Glioblastoma: activatingmutationsExtracellular domain of EGFR • In CRC : unclear : Overexpressionmembrane EGFR (mEGFR) Overexpressionnuclear EGFR (nEGFR) Gene Amplification Overexpressionligands Activating point mutations

43. EGFR overexpression Overexpressionmembrane EGFR (mEGFR) Overexpressionnuclear EGFR (nEGFR) Gene Amplification Overexpressionligands Activating point mutations

45. EGFR status

46. Anti-EGFR therapy • mAB : cetuximab, panitumumab • TKI : erlotinib, gefitinib, afatinib

47. EGFR Resistance : T790M mutation Inhibitors of EGFR with the T790M mutation : AZD9291 CO-1831

48. EGFR resistance : KRAS and BRAF mutations TREATMENT RELAPSE

49. EGFR resistance in CRC Resistanceagainst EGFR therapy • KRAS mutation : 40 % • BRAF mutation : 8-15 % • NRAS mutation : 1-6 % • Mostly pre-existent – selectiondue to anti-EGFRtreatment • Alsonewdue to ongoingmutagenesis ? Addition of BRAF or MEK inhibitor

50. BRAF en MEK inhibitors P