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

11. PHYSICIAN Future paradigm sample Result Molecular testing PHARMA PATIENT LAB Personalised treatment visit 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. Microsatellite instability (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 Breast Cancer : 10 % Colon cancer : 3-5% Prostate cancer : low Lung cancer : very low Majority of cancers are caused by genetic anomalies in the tumor (somatic mutations) Minority of cancers is inherited (germline mutations) :

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

26. Germline mutations in Colon cancer 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 TUMORMUTATIONSEXPLANATION HNPCC1782Genomic instability Lung150Mutagen (smoke) Melanoma80Mutagen (sun)

36. Somatic mutations in cancer P BreastLungColonProstate EGFR< 103420-804 KRAS< 101936-405 NRAS1-6 BRAFFew1-48-15Few PIK3CA26410-302 TP5323344816 MLL3710125 CTNNB1< 10 4

37. Somatic mutations in CRC P GeneMechanism % MutationsTargeted therapy EGFR Activating point mutations Gene Amplification Overexpression ligands Overexpression nuclear EGFR 20-80 % KRAS Activating point mutations36-40Tipifarnib, lonafarnib BRAF Activating point mutations8-15Dabrafenib, vemurafenib, sorafenib NRAS Activating point mutations1-6MEK162 PIC3CA Activating point mutations 10-30 mTOR

38. Cell growth and survival pathway

39. Cell growth pathway Ligands Receptors : EGFR Secondary messengers : 2 pathways : 1.MAPK pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6 2.PI3K / AKT pathway : PI3K, PTEN, AKT, mTOR

40. 1.MAPK pathway : KRAS, BRAF, NRAS 2.PI3K / AKT pathway : PIK3CA Driver mutations in CRC

41. Classical treatment in colon cancer 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 : activating mutations TK domain of EGFR In Glioblastoma : activating mutations Extracellular domain of EGFR In CRC : unclear : Overexpression membrane EGFR (mEGFR) Overexpression nuclear EGFR (nEGFR) Gene Amplification Overexpression ligands Activating point mutations

43. EGFR overexpression Overexpression membrane EGFR (mEGFR) Overexpression nuclear EGFR (nEGFR) Gene Amplification Overexpression ligands 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 EGFREGFR KRASKRAS WILDWILD TREATMENTRELAPSE

49. EGFR resistance in CRC Resistance against EGFR therapy –KRAS mutation : 40 % –BRAF mutation : 8-15 % –NRAS mutation : 1-6 % Mostly pre-existent – selection due to anti-EGFR treatment Also new due to ongoing mutagenesis ? Addition of BRAF or MEK inhibitor

50. BRAF en MEK inhibitors P BRAFMEK DabrafenibTrametinib VemurafenibCobimetinib

51. EGFR resistance treatment in CRC Resistance against EGFR therapy PIC3CA mutation : 10-30 % PTEN loss Addition of mTOR inhibitor

52. PIK3CA Driver gene PIK3CA encodes p110 subunit of Phosphatidylinositol 3-kinase PIK3 phosphorylates PI PI is central in AKT/mTOR pathway PIK3CA driver mutations in : –Breast cancer (25 -40 %) –Endometrium (23 %) –Ovarium –Colon –Non-tumor : somatic overgrowth syndromes (Cowden and Clove syndrome) Therapy : PIK3, AKT, mTOR inhibitors

53. Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK pathway or PI P GeneMutationMechanism BRAFAmplification Splice variants Activation MAPK pathway KRASActivating point mutationActivation MAPK pathway MEK1Activating point mutation Activation MAPK pathway PTENlossActivating PI3K/AKT pathway PI3CAActivating PI3K/AKT pathway

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

55. On which tissue should genetic studies be performed ? If CRC occurs in different family members : Genetic studies on DNA from blood to identify a germline mutation : Polyposis coli : APC, MUTYH HNPCC : MLH1, MESH2, MSH6, PMS1 If CRC is sporadic : Genetic studies on Tumor or liquid biopsy to identify a somatic mutation : EGFR KRAS BRAF.

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

57. Circulating tumor DNA (ctDNA)

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

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

60. cell-free DNA (cfDNA) testing 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

61. Advantages liquid biopsies No tissue biopsy needed No FFPE fixation Profiling the overall genotype of cancer primary cancer circulating cells metastases Better evaluation of : reaction to therapy development of resistance

62. Tissue biopsy EGFREGFR KRASKRAS WILDWILD EGFR TREATMENTRELAPSE TISSUE BIOPSY

63. Liquid biopsy EGFREGFR KRASKRAS BRAFBRAF WILDWILD TREATMENT LIQUID BIOPSY

64. 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

65. 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

66. ct DNA testing on liquid biopsy for CRC 1. DESCRIPTION : ct DNA testing on liquid biopsies : EGFR KRAS BRAF PIK3CA 2. SAMPLE : blood in specific test kits with Streck tubes provided by GENDIA 3. TURNAROUND TIME : 3 weeks 4. PRICE : < 1000 Euro

67. How offer ctDNA testing to your patients ? 1.Refer to our consultation : Email ctDNA@GENDIA.net to ask for an appointment@GENDIA.net 2.Take blood yourself : Email ctDNA@GENDIA.net to ask for kits@GENDIA.net www.circulatingtumorDNA.net