DNA diagnosis of lung cancer Patrick Willems GENDIA Antwerp, Belgium

1. DNA diagnosis of lung cancer Patrick Willems GENDIA • Antwerp, Belgium

2. Treatment of Lung Cancer • Small Cell Lung Cancer (SCLC) • chemotherapy • radiation • Non-Small Cell Lung Cancer (NSCLC) • 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 mAB: Herceptin 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. Mortality UK, 2009-2011

13. Cancer Morbidity and Mortality Canada, 2007

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

15. 14 % of all cancer • 80 % is non–small cell lung cancer (NSCLC) • Belgium : 7.100 new cases per year • Worldwide : 10 million new cases per year • Worldwide : 8 million fatalities per year • The main cause (20-30%) of cancer-related death in both men and women : • More women die of lung ca than breast, cervical and uterine ca combined. • More men die of lung ca than prostate and colorectal ca combined. Lung cancer

16. Treatment of Lung Cancer • Small Cell Lung Cancer (SCLC) • chemotherapy • Radiation • Non-Small Cell Lung Cancer (NSCLC) • surgery • radiation • chemotherapy • immunotherapy • personalised targeted treatment

17. Immunotherapy for NSCLC • CTLA-4 (cytotoxic T-lymphocyte–associated antigen 4) : ipilimumab • PD-1 (programmed death-1) : nivolumab, pembrolizumab • PD-L1 (programmed death-1 ligand) BMS-935559, MPDL3280A

18. Inhibition immune checkpoints

19. Biomarkers for immunotherapy for Lung Ca Few biomarkers for immunotherapy First real biomarker : Tumor load (amount of mutations-driver and passenger) Response to pembrolizumab (PD-1 inhibitor) better if high mutation load Science, April 3, 2015 (Rizvi et al)

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

21. Immunotherapy for NSCLC • Extremely expensive (100-300.000 Euro/year) • No biomarkers to select patients

22. Targetedtherapywith designer drugs • Receptor antibodies (---- ab) • HER2 : Trastuzumab (Herceptin) • EGFR : Cetuximab, Pertuzumab • MET : AMG102 • VEGF : Befacizumab • Tyrosine Kinase Inhibitors : TKI (---- ib) • BRC-ABL : Imatinib (Gleevec) • KRAS : Tipifarnib • BRAF : Sorafenib • MEK • ERK • mTOR : Everolimus

23. inhibit receptor kinases by interfering with ligand-receptor binding Preventing intracellularsignaling Receptor antibodies

24. Herceptin (Trastuzumab) Inhibits HER2 dimerisation / activation and the downstream signaling pathways MAPK and AKT/mTOR Active whenthere is HER2 overexpression • Breast ca (25 %) • Gastric ca (20 %) Herceptin

25. Tyrosine kinase inhibitors (TKI) TKI inhibits a Tyrosine kinase by binding to its kinase domain Preventing phosphorylation (activation) of target

26. Gleevec Gleevec (Imatinib) inhibitsTyrosinekinases by binding to its kinase domain Thereby preventing phosphorylation (activation) of targets : • BCR-ABL (CML) • cKIT (GIST, Mastocytosis) • PDGFR (GIST)

27. Targeted treatment • Non-Small Cell Lung Cancer (NSCLC) • surgery • radiation • chemotherapy • personalised targeted treatment • immunotherapy • Small Cell Lung Cancer (SCLC) • chemotherapy • radiation

28. Targeted treatment of NSCLC Expensive, but many biomarkers to select patient Personalised targeted treatment targets specific somatic mutations that cause NSCLC These mutations are patient-specific These mutations can be detected by molecular studies of : tumor (biopsy) blood (liquid biopsy)

29. Progress in lung ca treatment

30. Problems in targeted cancer treatment The very high cost of personalised treatment makes companion diagnostics (cancer biomarkers) necessary The mutations leading to lung ca are the biomarkers to guide targeted therapy

31. 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 : 5-10 % • Prostate cancer : low • Lung cancer : very low

32. Inheritance of lung cancer • NO germline mutations • MANY somatic mutations

33. Driver and passenger gene mutations Vogelstein et al, Science Aug 22, 2013 NEJM May 30, 2015

34. Somatic mutations in cancer P

35. Somatic mutations in adeno ca NSCLC TP53 : 34 % EGFR : 10-30 % KRAS : 15-25 % MLL3 : 10 % STK11 : 9 % CDKN2A : 8 % ALK fusions : 5 % HER 2 : 2% BRAF : 1-2 % Ros fusions : 2 % PTEN : 25 % (loss) P

36. Somatic mutations in adeno ca NSCLC

37. Cell growth and survival pathway

38. Genetic testing for lung cancer • EGFR: deletions in exon 19 L858R mutation in exon 21 T790M mutation in exon 20 • KRAS: mutations of codons 12 and 13 • BRAF: V600E, G469A and D594G mutations • ALK-EML4 fusion

39. EGFR Mutations in lung cancer EGFR mutations : 10 % (Europe) 30 % (Asia) women, non-smokers, adenocarcinoma (NSCLC) 90% of EGFR mutations : L858R in exon 21 (Sensitivity to TKIs) Small deletions in exon 19 (Sensitivity to TKIs) T790M in exon 20 (Resistance to TKIs) First-generation EGFR tyrosine-kinase inhibitors : Erlotinib (Tarceva) Gefitinib (Iressa) Second-generation EGFR tyrosine-kinase inhibitors : Dacomitinib Afatinib (Gilotrif) P

40. EGFR mutations

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. KRAS Mutations in lung cancer KRAS mutations : 15-25 % in NSCLC smokers 90% of KRAS mutations : codon 12 (90 %) codon 13 (5-10 %) KRAS Mutations are contraindications for EGFR TKI

44. BRAF Mutations in lung cancer BRAF mutations : 1-4 % in NSCLC 55 % of KRAS mutations : V600E BRAF Mutations are contraindications for EGFR TKI

45. ALK Mutations in lung cancer • ALK mutations : 5 % in NSCLC • ALK activation is caused by EML4-ALK fusion generated by inv(2)(p21p23) • ALK mutations are sensitive to ALK inhibitors : Crizotinib (Xalkori) Ceritinib (Zykadia)

46. ROS1 Mutations in lung cancer ROS1 mutations : 2 % ROS1 activation is caused by ROS1 fusion to different partners ROS1 mutations are sensitive to Crizotinib (Xalkori) P

47. Why perform genetic studies on tumor DNA ? • Initial diagnosis and prognosis Initial therapy • Monitoring recurrence – metastasis Secundary therapy

48. Prognosis according to EGFR mutations

49. Prognosis according to BRAF mutations

50. Why perform genetic studies on tumor DNA ? • Initial diagnosis and prognosis Initial therapy • Monitoring recurrence – metastasis Secundary therapy