DNA diagnosis in malignant melanoma Patrick Willems GENDIA

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DNA diagnosis in malignant melanoma Patrick Willems GENDIA Antwerp, Belgium

DNA diagnosis in malignant melanoma Patrick Willems GENDIA Antwerp, Belgium

Personalized cancer treatment • Immunotherapy to stimulate immune response to cancer PD-1 inhibitors PD-L

Personalized cancer treatment • Immunotherapy to stimulate immune response to cancer PD-1 inhibitors PD-L 1 inhibitors CTLA-4 inhibitors • Targeted therapy with designer drugs that target the genetic cause of the tumor m. AB: Herceptin TKI: Gleevec

Treatment of Malignant melanoma • • surgery radiation Chemotherapy Targeted treatment – BRAF inhibitor

Treatment of Malignant melanoma • • surgery radiation Chemotherapy Targeted treatment – BRAF inhibitor (Vemurafenib) – MEK inhibitor • Immunotherapy – Interferon (IFN) alfa-2 b, IL 2 (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

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

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)

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

 Market for tumor biomarkers in Liquid biopsies TARGETS DRUGS SEQUENCING Liquid biopsy market

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 general visit treatment PHYSICIAN Result sample Pathological studies PATHOLOGIST Lab

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

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

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

Cancer Morbidity and Mortality Melanoma : 1 -8 %

Cancer Morbidity and Mortality Melanoma : 1 -8 %

New cancers per year in Belgium • Lung : 7. 100 • Colon :

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 •

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

 Skin cancer • Basal cell carcinoma : 75 % • Spinocellular epithelioma: 5%

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

 Malignant melanoma • Melanoma is a malignant tumor of melanocytes. • Fifth most

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

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 •

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

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 : Colon cancer : Prostate cancer : Lung cancer : • Melanoma : 10 % 5 -10 % low very low 10 % :

Germline mutations in MM Protein Function AD p 16 (INK 4) p 16 :

Germline mutations in MM Protein Function AD p 16 (INK 4) p 16 : CDK inhibitor 20 -40 % p 14 (ARF) p 14 : binds MDM 2 p 53 Gene/Locus CDKN 2 A (cyclin-dependent kinase inhibitor 2) CDK 4 (cyclin-dependent kinase 4) MC 1 R XRCC 3 AD <10 fam melanocortin-1 receptor Risk factor MITF TERT control of cell proliferation X-ray repair cross-complementing protein 3 microphthalmia-associated transcription factor Risk factor DNA repair protein transcription factor Telomerase integrity telomerase reverse transcriptase POT 1 Risk factor ACD Risk factor TERF 2 IP BAP 1 Risk factor AD Telomerase integrity POT 1 -interacting protein 1 TERF 2 -interacting protein Breast cancer associated prtotein Telomerase integrity

Inheritance of MM 10 % germline mutations MANY somatic mutations

Inheritance of MM 10 % germline mutations MANY somatic mutations

Cancer genes and mutations • 140 driver genes • 60 % TSG • 40

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

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 23 34 48 16 <

Somatic mutations in cancer Melanoma Breast Lung Colon Prostate 23 34 48 16 < 10 19 35 5 Few 1 -4 8 -15 Few TP 53 10 KRAS Few NRAS 13 -25 BRAF 10 -50 PIK 3 CA Few 26 4 22 2 EGFR Few < 10 34 < 10 4 MLL 3 Few 7 10 12 5 < 10 4 CTNNB 1 2 -3

Somatic mutations in MM % Mutations Gene Targeted therapy BRAF Activating point mutations 10

Somatic mutations in MM % Mutations Gene Targeted therapy BRAF Activating point mutations 10 -50 Dabrafenib, vemurafenib NRAS Activating point mutations 13 -25 MEK 162 KIT Activating point mutations MEK 1 Activating point mutations CTNNB 1 Activating point mutations 2 -3 CDKN 2 A Deletions 50 CDK 4 Activating point mutations 10 GNA 11 Activating point mutations 2 PTEN Deletions p 53 Activating point mutations 10 GNAQ Activating point mutations 1 PIC 3 CA Activating point mutations Overall 2 -6 6 20 -40 5 60 -70 Dasatinib, imatinib Trametinib, MEK 162 Cyclin D 1 inhibitor LY 2835219

Somatic mutations in MM Gene % Mutations Skin Normal Much Sun sun 50 -60

Somatic mutations in MM Gene % Mutations Skin Normal Much Sun sun 50 -60 10 Mucosa Acra Eye 5 -10 15 -25 < 1 BRAF + NRAS + 20 10 -15 5 -15 10 -15 <1 KIT + < 1 2 20 15 <1 CDK 4 CCND 1 CDKN 2 A + Low High Low _ Low High Low MANY CNV Other BAP 1 GNAQ GNA 11

Somatic mutations in uvual MM Gene % Mutations in MM % Mutations in uveal

Somatic mutations in uvual MM Gene % Mutations in MM % Mutations in uveal MM BRAF 50 % < 1 % NRAS 13 -25 % < 1 % MEK 1 6 % < 1 % KIT 2 -6 % < 1 % CTNNB 1 2 -3 % < 1 % GNA 11 2 % 32 % GNAQ 1 % 50 % BAP 1 < 1 %

Cell growth and survival pathway

Cell growth and survival pathway

Cell growth pathway • Ligands • Receptors : KIT (EGFR, HER 2, MET) •

Cell growth pathway • Ligands • Receptors : KIT (EGFR, HER 2, MET) • Secondary messengers : 2 pathways : 1. MAPK pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK 4/6 2. PI 3 K / AKT pathway : PI 3 K, PTEN, AKT, m. TOR

Designer molecules

Designer molecules

DNA testing to orient personalised treatment Gene % Mutations Targeted therapy BRAF 10 -50

DNA testing to orient personalised treatment Gene % Mutations Targeted therapy BRAF 10 -50 Dabrafenib, vemurafenib NRAS 13 -25 MEK 162 MEK 1 6 Trametinib, MEK 162 KIT 2 -6 Dasatinib, imatinib CTNNB 1 2 -3 Cyclin D 1 inhibitor CDK 4 10 LY 2835219

DNA testing to follow treatment and detect metastasis and resistance Gene % Mutations Targeted

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 MEK 162

Resistance to BRAF inhibitors with reactivation opf MAPK pathway Gene Mechanism BRAF Amplification Splice

Resistance to BRAF inhibitors with reactivation opf MAPK pathway Gene Mechanism BRAF Amplification Splice variants NRAS Activating point mutation MEK 1 Activating point mutation MEK 2 Activating point mutation PTEN loss Activating PI 3 K/AKT pathway PI 3 CA Activating PI 3 K/AKT pathway

Cell growth and survival pathway

Cell growth and survival pathway

Combination therapy BRAF en MEK inhibitors BRAF MEK Dabrafenib Trametinib Vemurafenib Cobimetinib

Combination therapy BRAF en MEK inhibitors BRAF MEK Dabrafenib Trametinib Vemurafenib Cobimetinib

Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK pathway or PI Gene Mutation

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 Activation MAPK pathway MEK 1 Activating point mutation Activation MAPK pathway MEK 2 Activation MAPK pathway PTEN Activating point mutation loss Activating PI 3 K/AKT pathway PI 3 CA Activating PI 3 K/AKT pathway

Cell growth and survival pathway

Cell growth and survival pathway

Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK or PI 3 K pathway

Resistance to BRAF-MEK inhibitors combi with reactivation of MAPK or PI 3 K pathway Mechanism Re-Activation MAPK pathway Activating PI 3 K/AKT pathway Therapy Inhibition ERK inhibitors distal MAPK pathway Inhibition PI 3 K/AKT pathway PI 3 K inhibitors AKT inhibitors m. TOR inhibitors

Why perform genetic studies on tumor DNA ? • Initial diagnosis and prognosis •

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

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 : CDKN 2 A - CDK 4 (melanoma) BAP 1 (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

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 (ct. DNA)

Ct DNA cell-free DNA (cf. DNA) is released from healthy, inflamed or cancerous tissue

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

cell-free DNA (cf. DNA) • Cell-free DNA (cf. DNA) in plasma of healthy individuals

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

Advantages of liquid biopsies vs FFPE • No biopsy needed • Better representation of

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

ct. DNA circulating tumor DNA testing in blood for detection of cancer www. circulatingtumor.

ct. DNA circulating tumor DNA testing in blood for detection of cancer www. circulatingtumor. DNA. net

Technology to detect mutations in ct. DNA Next gen sequencing (NGS) + specific technology

Technology to detect mutations in ct. DNA 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 ct. DNA • Pangaea Biotech • • • • Cynvenio BGI

Companies focusing on ct. DNA • 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

ct DNA testing on liquid biopsy for malignant melanoma 1. DESCRIPTION : ct DNA testing on liquid biopsies : • BRAF: 10 -50 % – V 600 E : 80– 90% – V 600 K : 5 -12% – V 600 R or V 600 D : 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 ct. DNA testing to your patients ? 1. Refer to our consultation

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

www. circulatingtumor. DNA. net

www. circulatingtumor. DNA. net