Theradiagnostics for cancer Patrick Willems GENDIA Antwerp, Belgium

1. Theradiagnostics • for cancer • Patrick Willems • GENDIA • Antwerp, Belgium

2. Treatment of cancer • Surgery • Radiation • Chemotherapy • Personalised treatment

4. Personalized cancer treatment Immunotherapy to modulate immune response : • Interferon (IFN) alfa-2b, IL2 (interleukin 2) • CTLA-4 inhibitors • PD-1 inhibitors • PD-L1 inhibitors Targetedtherapywith designer drugs to target the geneticcause of the tumor • EGFR inhibitors • BRAF inhibitor • MEK inhibitor

5. Bottlenecks 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. Bottlenecks in personalized cancer treatment The very high cost of personalised treatment makes companion diagnostics (cancer biomarkers) necessary These are referred to as Theradiagnostics

8. Theradiagnostics Tumor DNA (FFPE - biopsy) Circulating tumor DNA (ctDNA in liquid biopsy)

9. Market for theradiagnostics TARGETS DRUGS SEQUENCING Theradiagnostics market : 40 Billion USD per year

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 Belgium11 million inhabitants • Lung : 7.100 • Colon : 6.500 • Prostate : 8.800 • Breast : 9.700 TOTAAL : 65.000

15. Treatment of cancer • Surgery • Radiation • Chemotherapy • Personalised treatment : • Immunotherapy • Targeted therapy with designer drugs

16. Immunotherapy for cancer • 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

17. Inhibition immune checkpoints

18. Biomarkers for immunotherapy for Colorectal cancer 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)

19. MSI as biomarker for immunotherapy MMR deficiency Genomic instability Large mutation load in CRC Many mutant proteins – neo antigens Immune response with immunotherapy

20. MSI as biomarker for immunotherapy in CRC MMR deficiency Genomic instability Large mutation load in CRC Many mutant proteins – neo antigens Immune response with immunotherapy

21. Treatment of cancer • Surgery • Radiation • Chemotherapy • Personalised treatment : • Immunotherapy • Targeted therapy with designer drugs

22. Targeted treatment for cancer Personalised targeted treatment inhibits specific mutations that cause cancer These mutations are patient-specific Mutations can be detected by molecular studies of : . tumor material (biopsy) : FFPE, fresh or frozen . blood (liquid biopsy) Therapy is dependent upon the specific mutation Personalised medicine

23. Which genetic anomalies cause cancer ?

24. Genetics 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. Cancer gene mutations

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

27. Two step cancer theory (Knudson) Inheritedcancer : • Germlinemutation in all cells • Somaticmutation in cancercell Sporadiccancer : • No germlinemutation • Somaticmutations in the 2 gene copies in cancercell

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

29. Cancer genes and mutations • 140 driver genes • 60 % Tumor suppressor genes • 40 % Oncogenes • > 1000 driver gene mutations (Most tumors 2-10 driver gene mutations) • Millions of passenger gene mutations (Most tumors 10-100 passenger gene mutations)

30. 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

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

32. Somatic mutations P

33. Inactivating somatic mutations in cancer P

34. Activating somatic mutations in cancer P

35. Cell growth and survival pathway

36. Cell growth pathway • Ligands • Receptors : EGFR • Secondarymessengers : 2 pathways : • 1. MAPK / RAS pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6 • 2. mTOR / AKT pathway : PIK3CA, PTEN, AKT, mTOR

37. Classicaltreatment in coloncancer • Surgery • Chemotherapy • In case of EGFR mutation or overexpression Start anti EGFR therapy : • mAB : cetuximab, panitumumab • TKI : erlotinib, gefitinib, afatinib

38. EGFR mutations • Lung Ca : activatingmutations in TK domain • Glioblastoma: activatingmutations in Extracellular domain • Colorectal ca : unclear : Overexpressionmembrane EGFR (mEGFR) Overexpressionnuclear EGFR (nEGFR) Gene Amplification Overexpressionligands Activating point mutations

39. EGFR status

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

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

42. EGFR resistance : KRAS and BRAF mutations TREATMENT RELAPSE

43. EGFR resistance in CRC: KRAS and BRAF mutations 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

44. EGFR resistance in CRC : PIK3CA mutation Resistance against EGFR therapy PIK3CA mutation : 10-30 % PTEN loss Addition of mTOR inhibitor

45. PIK3CA • PIK3CA encodes p110 subunit of Phosphatidylinositol 3-kinase PIK3 phosphorylates PI PI is central in AKT/mTOR pathway • PIK3CA driver mutations in : • Breast cancer (26 %) • Endometrium (23 %) • Colon (22 %) • Non-tumor : somatic overgrowth syndromes (Cowden and Clove syndrome) • Therapy : PIK3, AKT, mTOR inhibitors

46. Why genetic studies on tumor DNA ? • Initial diagnosis and prognosis • Monitoring recurrence – metastasis

47. Genetic studies in cancer • Blood DNA If CRC occurs in different family members : Genetic studies on DNA frombloodtoidentify a germlinemutation (BRCA) • Tumor • MSI : in order todeterminesensitivityforimmunotherapy • Mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA todeterminesensitivityfortargetedtherapy • Liquid biopsy • Initialtheradiagnosticsif tumor material is unavailable • Follow up duringcancer treatment • Screening of high risk patients (HNPCC carriers, BRCA carriers)

48. Genetic studies of somatic mutations • DNA studies on tumor material Analysis of DNA from tumor (FFPE, fresh, frozen) • Circulating tumor DNA (ctDNA) in Liquid biopsy Analysis of circulatingtumor DNA (ctDNA) in blood

49. Circulating tumor DNA (ctDNA)

50. ctDNA from tumor tissue is released through secretion, necrosis and apoptosis, but mainly through apoptosis. ctDNA