Download BRAF - Circulating Tumor DNA (ctDNA)

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
general
visit
treatment
PHYSICIAN
Result
sample
Pathological studies
PATHOLOGIST
Lab
Future paradigm
PATIENT
Personalised
visit
treatment
PHYSICIAN
PHARMA
Result
sample
Molecular testing
Pathologist
LAB
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 :
Colon cancer :
Prostate cancer :
Lung cancer :
• Melanoma :
10 %
5-10 %
low
very low
10 %
Germline mutations in MM
Protein
Function
AD
p16 (INK4)
p16 : CDK inhibitor
20 -40 %
p14 (ARF)
p14 : binds MDM2p53
Gene/Locus
CDKN2A
(cyclin-dependent
kinase inhibitor 2)
CDK4
(cyclin-dependent
kinase 4)
MC1R
XRCC3
AD
<10 fam
melanocortin-1 receptor
Risk factor
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
POT1
Risk factor
ACD
Risk factor
TERF2IP
BAP1
Risk factor
AD
Telomerase integrity
POT1-interacting protein 1
TERF2-interacting protein
Breast cancer associated prtotein
Telomerase integrity
Telomerase integrity
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
23
34
48
16
< 10
19
35
5
Few
1-4
8-15
Few
TP53
10
KRAS
Few
NRAS
13-25
BRAF
10-50
PIK3CA
Few
26
4
22
2
EGFR
Few
< 10
34
< 10
4
MLL3
Few
7
10
12
5
< 10
< 10
< 10
4
CTNNB1 2-3
Somatic mutations in MM
% Mutations
Gene
Targeted therapy
BRAF
Activating point mutations
10-50
Dabrafenib,
vemurafenib
NRAS
Activating point mutations
13-25
MEK162
KIT
Activating point mutations
2-6
MEK1
Activating point mutations
6
CTNNB1
Activating point mutations
2-3
CDKN2A
Deletions
50
CDK4
Activating point mutations
10
GNA11
Activating point mutations
2
PTEN
Deletions
p53
Activating point mutations
10
GNAQ
Activating point mutations
1
PIC3CA
Activating point mutations
5
Overall
20-40
60-70
Dasatinib, imatinib
Trametinib, MEK162
Cyclin D1 inhibitor
LY2835219
Somatic mutations in MM
Gene
% Mutations
BRAF
+
Skin
Normal
Sun
50-60
NRAS
+
20
10-15
5-15
10-15
<1
KIT
+
<1
2
20
15
<1
CDK4
CCND1
CDKN2A
+
Low
Low
High
High
Low
_
Low
Low
High
High
Low
MANY
MANY
CNV
Other
Skin
Much
sun
10
Mucosa
Acra
Eye
5-10
15-25
<1
BAP1
GNAQ
GNA11
Somatic mutations in uvual MM
Gene
% Mutations
in MM
% Mutations
in 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
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
Gene
% Mutations
Targeted therapy
Respons
Resistance
Relaps
BRAF
10-50
Dabrafenib,
vemurafenib
50 %
Most
NRAS
13-25
MEK162
Resistance to BRAF inhibitors
with reactivation opf MAPK pathway
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
BRAF
MEK
Dabrafenib
Trametinib
Vemurafenib
Cobimetinib
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
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
Mechanism
Re-Activation
MAPK pathway
Activating
PI3K/AKT pathway
Therapy
Inhibition
ERK inhibitors
distal MAPK pathway
Inhibition
PI3K/AKT pathway
PI3K inhibitors
AKT inhibitors
mTOR 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 :
•
•
•
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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
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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