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-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)

PHYSICIAN

Current paradigm

sampleResult

Pathological studies

PATIENT

PATHOLOGIST

general

treatmentvisit

Lab

PHYSICIAN

Future paradigm

sampleResult

Molecular testing

PHARMA

PATIENT

LAB

Personalised

treatmentvisit

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

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

• Melanoma : 10 %

Majority of cancers are caused by genetic anomalies in the tumor

(somatic mutations)

Minority of cancers is inherited (germline mutations) :

Germline mutations in MM

P

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)

AD

<10 fam

control of cell proliferation

MC1R melanocortin-1 receptorXRCC3 Risk factor

X-ray repair cross-complementing protein 3

DNA repair protein

MITF Risk factor microphthalmia-associated

transcription factortranscription factor

TERT Risk factor

telomerase reverse transcriptase

Telomerase integrity

POT1 Risk factor Telomerase integrity

ACD Risk factor POT1-interacting protein 1 Telomerase integrity

TERF2IPRisk factor

TERF2-interacting protein Telomerase integrity

BAP1 AD Breast cancer associated prtotein

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

Tumors with high mutation load

due to Mutagens or genomic instability

form many neoantigens

and are candidates for immunotherapy

TUMOR MUTATIONS EXPLANATION

HNPCC 1782 Genomic instability

Lung 150 Mutagen (smoke)

Melanoma 80 Mutagen (sun)

Somatic mutations in cancer

P

Melanoma Breast Lung Colon Prostate

TP53 10 23 34 48 16

KRAS Few < 10 19 35 5

NRAS 13-25

BRAF 10-50 Few 1-4 8-15 Few

PIK3CA Few 26 4 22 2

EGFR Few < 10 34 < 10 4

MLL3 Few 7 10 12 5

CTNNB1 2-3 < 10 < 10 < 10 4

Somatic mutations in MM

P

Gene % Mutations Targeted therapy

BRAF Activating point mutations 10-50 Dabrafenib, vemurafenib

NRAS Activating point mutations 13-25 MEK162

KIT Activating point mutations 2-6 Dasatinib, imatinib

MEK1 Activating point mutations 6 Trametinib, MEK162

CTNNB1 Activating point mutations 2-3 Cyclin D1 inhibitor

CDKN2A Deletions 50

CDK4 Activating point mutations 10 LY2835219

GNA11 Activating point mutations 2

PTEN Deletions 20-40

p53 Activating point mutations 10

GNAQ Activating point mutations 1

PIC3CA Activating point mutations 5

Overall 60-70

Somatic mutations in MM

P

Gene % Mutations

SkinNormal

Sun

SkinMuch sun

Mucosa Acra Eye

BRAF + 50-60 10 5-10 15-25 < 1

NRAS + 20 10-15 5-15 10-15 <1

KIT + < 1 2 20 15 <1

CDK4CCND1

+ Low Low High High Low

CDKN2A _ Low Low High High Low

CNV MANY MANY

Other BAP1GNAQGNA11

Somatic mutations in uvual MM

P

Gene % Mutationsin MM

% Mutationsin uveal MM

BRAF 50 % < 1 %

NRAS 13-25 % < 1 %

MEK1 6 % < 1 %

KIT 2-6 % < 1 %

CTNNB1 2-3 % < 1 %

GNA11 2 % 32 %

GNAQ 1 % 50 %

BAP1 < 1 %

Cell growth and survival pathway

Cell growth pathway

• Ligands

• Receptors : KIT (EGFR, HER2, MET)

• Secondary messengers : 2 pathways :

1. MAPK pathway : RAS, BRAF, MEK, ERK, Cyclins, CDK4/6

2. PI3K / AKT pathway : PI3K, PTEN, AKT, mTOR

Designer molecules

DNA testing to orient personalised treatment

P

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

DNA testing to follow treatment and detect metastasis and resistance

P

Gene

% Mutations Targeted therapy Respons ResistanceRelaps

BRAF 10-50 Dabrafenib, vemurafenib

50 % Most

NRAS 13-25 MEK162

Resistance to BRAF inhibitors with reactivation opf MAPK pathway

P

Gene Mechanism

BRAF Amplification Splice variants

NRAS Activating point mutation

MEK1 Activating point mutation

MEK2 Activating point mutation

PTEN loss Activating PI3K/AKT pathway

PI3CA Activating PI3K/AKT pathway

Cell growth and survival pathway

Combination therapy BRAF en MEK inhibitors

P

BRAF MEK

Dabrafenib Trametinib

Vemurafenib Cobimetinib

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

P

Gene Mutation Mechanism

BRAF Amplification Splice variants

Activation MAPK pathway

NRAS Activating point mutation Activation MAPK pathway

MEK1 Activating point mutation

Activation MAPK pathway

MEK2 Activating point mutation

Activation MAPK pathway

PTEN loss Activating PI3K/AKT pathway

PI3CA Activating PI3K/AKT pathway Activating PI3K/AKT pathway

Cell growth and survival pathway

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

P

Mechanism Therapy

Re-Activation MAPK pathway

Inhibition distal MAPK pathway

ERK inhibitors

Activating PI3K/AKT pathway

Inhibition PI3K/AKT pathway

PI3K inhibitorsAKT inhibitorsmTOR inhibitors

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

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 ?

1. Refer to our consultation :

Email [email protected] to ask for an appointment

2. Take blood yourself :

Email [email protected] to ask for kits

www.circulatingtumorDNA.net

www.circulatingtumorDNA.net