DNA diagnosis in malignant melanoma Patrick Willems GENDIA Antwerp, Belgium

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

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)

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)

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

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

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

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

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

Cancer Morbidity and Mortality Melanoma : 1-8 %

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

Incidence MM Higher in sunny countries Higher in light skin people Increasing everywhere

Skin cancer Basal cell carcinoma :75 % Spinocellular epithelioma: 5% Melanoma : 10 % Other : 10 %

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%

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)

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

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 %

Germline mutations in MM Gene/Locus Protein Function CDKN2A (cyclin-dependent kinase inhibitor 2) AD 20 -40 % p16 (INK4) p14 (ARF) p16 : CDK inhibitor p14 : binds MDM2- p53 CDK4 (cyclin-dependent kinase 4) <10 fam control of cell proliferation MC1R melanocortin-1 receptor XRCC3 Risk factor X-ray repair cross-complementing protein 3 DNA repair protein MITF microphthalmia-associated transcription factor transcription factor TERT telomerase reverse transcriptase Telomerase integrity POT1 ACD POT1-interacting protein 1 TERF2IP TERF2-interacting protein BAP1 Breast cancer associated prtotein P

Inheritance of MM 10 % germline mutations MANY somatic mutations

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)

Driver and passenger gene mutations TUMOR MUTATIONS EXPLANATION HNPCC 1782 Genomic instability Lung 150 Mutagen (smoke) Melanoma 80 Mutagen (sun) Tumors with high mutation load due to Mutagens or genomic instability form many neoantigens and are candidates for immunotherapy

Somatic mutations in cancer Melanoma Breast Lung Colon Prostate TP53 10 23 34 48 16 KRAS Few < 10 19 35 5 NRAS 13-25 BRAF 10-50 1-4 8-15 PIK3CA 26 4 22 2 EGFR MLL3 7 12 CTNNB1 2-3 P

Somatic mutations in MM Gene % Mutations Targeted therapy BRAF Activating point mutations 10-50 Dabrafenib, vemurafenib NRAS 13-25 MEK162 KIT 2-6 Dasatinib, imatinib MEK1 6 Trametinib, MEK162 CTNNB1 2-3 Cyclin D1 inhibitor CDKN2A Deletions 50 CDK4 10 LY2835219 GNA11 2 PTEN 20-40 p53 GNAQ 1 PIC3CA 5 Overall 60-70 P

Somatic mutations in MM Gene % Mutations Skin Normal Sun Much sun Mucosa Acra Eye BRAF + 50-60 10 5-10 15-25 < 1 NRAS 20 10-15 5-15 <1 KIT 2 15 CDK4 CCND1 Low High CDKN2A _ CNV MANY Other BAP1 GNAQ GNA11 P

Somatic mutations in uvual MM Gene % Mutations in MM in uveal MM BRAF 50 % < 1 % NRAS 13-25 % MEK1 6 % KIT 2-6 % CTNNB1 2-3 % GNA11 2 % 32 % GNAQ 1 % 50 % BAP1 < 1 % P

Cell growth and survival pathway

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

Designer molecules

DNA testing to orient personalised treatment Gene % Mutations Targeted therapy BRAF 10-50 Dabrafenib, vemurafenib NRAS 13-25 MEK162 MEK1 6 Trametinib, MEK162 KIT 2-6 Dasatinib, imatinib CTNNB1 2-3 Cyclin D1 inhibitor CDK4 10 LY2835219 P

DNA testing to follow treatment and detect metastasis and resistance Gene % Mutations Targeted therapy Respons Resistance Relaps BRAF 10-50 Dabrafenib, vemurafenib 50 % Most NRAS 13-25 MEK162 P

Resistance to BRAF inhibitors with reactivation opf MAPK pathway Gene Mechanism BRAF Amplification Splice variants NRAS Activating point mutation MEK1 MEK2 PTEN loss Activating PI3K/AKT pathway PI3CA P

Cell growth and survival pathway

Combination therapy BRAF en MEK inhibitors Dabrafenib Trametinib Vemurafenib Cobimetinib P

Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK pathway or PI Gene Mutation Mechanism BRAF Amplification Splice variants Activation MAPK pathway NRAS Activating point mutation MEK1 MEK2 PTEN loss Activating PI3K/AKT pathway PI3CA P

Cell growth and survival pathway

Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK or PI3K pathway Mechanism Therapy Re-Activation MAPK pathway Inhibition distal MAPK pathway ERK inhibitors Activating PI3K/AKT pathway PI3K inhibitors AKT inhibitors mTOR inhibitors P

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

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 .

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)

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

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

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

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

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

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

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 weeks 4. PRICE : < 1000 Euro

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

www.circulatingtumorDNA.net