DNA diagnosis in malignant melanoma Patrick Willems GENDIA Antwerp, Belgium

1. DNA diagnosis in malignant melanoma • Patrick Willems • GENDIA • Antwerp, Belgium

3. 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

4. Treatment of Malignant melanoma • surgery • radiation • Chemotherapy • Targeted treatment • BRAF inhibitor (Vemurafenib) • MEK inhibitor • Immunotherapy • Interferon (IFN) alfa-2b, IL2 (interleukin 2) • CTLA-4 inhibitors (Ipilimumab) • PD-1 inhibitors (Pembrolizumab and nivolumab)

5. Problems in personalized cancer treatment • Immunotherapy Very Expensive (100-300.000 Euro/year Few biomarkers (companion diagnostics) • Designer drugs 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. Cancer Morbidity and Mortality Melanoma : 1-8 %

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

14. Incidence MM • Higher in sunny countries • Higher in light skin people • Increasing everywhere

15. Skin cancer • Basalcellcarcinoma :75 % • Spinocellularepithelioma: 5% • Melanoma : 10 % • Other : 10 %

16. Malignant melanoma • Melanoma is a malignant tumor of melanocytes. • Fifth most common cancer in men and the seventh in women • 76.100 new cases in 2014 in the US • 9.710 deaths in 2014 in the US • Five-year survival rates for patients with metastatic disease < 10%

17. Personalised targeted treatment of MM 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) blood (liquid biopsy)

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

19. 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 • Melanoma : 10 %

20. Germline mutations in MM P

21. Inheritance of MM 10 % germline mutations MANY somatic mutations

22. 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)

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

24. Somatic mutations in cancer P

25. Somatic mutations in MM P

26. Somatic mutations in MM P

27. Somatic mutations in uvual MM P

29. Cell growth and survival pathway

30. Cell growth pathway • Ligands • Receptors : KIT (EGFR, HER2, MET) • Secondarymessengers : 2 pathways : • MAPK pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6 • PI3K / AKT pathway : PI3K, PTEN, AKT, mTOR

31. Designer molecules

32. DNA testing to orient personalised treatment P

33. DNA testing to follow treatment and detect metastasis and resistance P

34. Resistance to BRAF inhibitors with reactivation opf MAPK pathway P

35. Cell growth and survival pathway

36. Combination therapy BRAF en MEK inhibitors P

37. Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK pathway or PI P

38. Cell growth and survival pathway

39. Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK or PI3K pathway P

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

41. On which tissue should genetic studies be performed ? • If melanoma occurs in different family members : Genetic studies on DNA from blood to identify a germline mutation : CDKN2A - CDK4 (melanoma) BAP1 (uveal melanoma, mesothelioma) • If melanoma is sporadic : Genetic studies on Tumor DNA or liquid biopsy to identify a somatic mutation BRAF NRAS KIT .

42. Genetic studies to identify somatic mutations • FFPE material of the tumor Analysis of DNA from Formaldehyde Fixed-Paraffin Embedded Melanoma tissue • Liquid biopsy Analysis of DNA from circulating tumor cells in blood (ctDNA)

43. Ct DNAcell-free DNA (cfDNA) is released from healthy, inflamed or cancerous tissue undergoing apoptosis or necrosiscirculating tumor (ctDNA) is only a small fraction of cfDNA in blood

44. cell-free DNA (cfDNA) • Cell-free DNA (cfDNA) in plasma of healthy individuals : Mandel and Métais (1948) • A proportion of cfDNA in pregnant women is fetus-derived (cffDNA) : Lo et al. (1997) • Non-Invasive Prenatal testing (NIPT) : 2012 : start 2015 : > 1 million tests Market : 4 billion USD • Increased concentrations of cfDNA in the circulation of cancer patients : Leon et al. (1977) • A proportion of cfDNA is tumor-derived : Stroun et al. (1987) • Circulating tumor DNA (ctDNA) testing (liquid biopsy) : 2015 : start Market : 40 billion USD

45. Advantages of liquid biopsies vs FFPE • No biopsy needed • Better representation of : • Total mutation load • Mutations in metastatic cells • Reaction to therapy • Development of resistance

46. ctDNA circulating tumor DNA testing in blood for detection of cancer www.circulatingtumorDNA.net

47. Technology to detect mutations in ctDNA Next gen sequencing (NGS) + specific technology • Digital PCR (dilution over many wells) • Epcam selection for epithelial tumors • Selection of mutant sequence Mutant Allele - specific PCR

48. Companies focusing on ctDNA • Pangaea Biotech • Cynvenio • BGI • Agena Bioscience • Boreal Genomics • Chronix Biomedical • Genomic Health • Guardant Health • Inivata • Molecular MD • Myriad Genetics • Natera • Personal Genome Diagnostics • Sysmex Inostics • Trovagene Liquid biopsy market for tumor biomarkers: 40 Billion USD per year

49. ct DNA testing on liquid biopsy for malignant melanoma 1. DESCRIPTION :ct DNA testing on liquid biopsies : • BRAF: 10-50 % • V600E : 80–90% • V600K : 5-12% • V600R or V600D : 5% • NRAS : 13-25 % • positions 12, 13, or 61 2. SAMPLE :blood in specific test kits with Streck tubes provided by GENDIA 3. TURNAROUND TIME :3 weeks4. PRICE :< 1000 Euro

50. How offer ctDNA testing to your patients ? • Refer to ourconsultation : Email ctDNA@GENDIA.net to askforanappointment • Takebloodyourself : Email ctDNA@GENDIA.net to askfor kits www.circulatingtumorDNA.net