Download Dia 1 - Circulating Tumor DNA (ctDNA)

DNA diagnosis for colorectal carcinoma
Patrick Willems
GENDIA
Antwerp, Belgium
Treatment of Colorectal carcinoma
• surgery
• radiation
• Chemotherapy
• Targeted treatment
• Immunotherapy
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
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)
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
The changing face of cancer diagnosis
Cancer Morbidity and Mortality
New cancers per year in Belgium
• Lung :
7.100
• Colon :
6.500
• Prostate :
8.800
• Breast :
9.700
• Melanoma :
1.500
TOTAAL :
65.000
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
Treatment of CRC
•
•
•
•
surgery
radiation
Chemotherapy
Targeted treatment
– BRAF inhibitor
– MEK inhibitor
• Immunotherapy
– CTLA-4 inhibitors
– PD-1 inhibitors
– PD-L1 inhibitors
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
Inhibition immune checkpoints
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)
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
Microsatellite instability (MSI)
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)
Why liquid biopsies for CRC ?
• 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 :
3-5%
• Prostate cancer :
low
• Lung cancer :
very low
Inheritance of CRC
3-5 % germline mutations
MANY somatic mutations
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
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)
Two step cancer theory
(Knudson)
Retinoblastoma (RB1 gene)
Mesothelioma
Uveal melanoma (BAP1 gene)
Multistep cancer theory
(Vogelstein)
Vogelstein et al, Science Aug 22, 2013
Colon cancer
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)
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
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
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
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
Breast
Lung
Colon
Prostate
EGFR
< 10
34
20-80
4
KRAS
< 10
19
36-40
5
NRAS
BRAF
1-6
Few
1-4
8-15
Few
PIK3CA
26
4
10-30
2
TP53
23
34
48
16
MLL3
7
10
12
5
< 10
< 10
< 10
4
CTNNB1
Somatic mutations in CRC
Gene
Mechanism
% Mutations
Targeted therapy
EGFR
Activating point mutations
Gene Amplification
Overexpression ligands
Overexpression nuclear EGFR
KRAS
Activating point mutations
36-40
Tipifarnib, lonafarnib
BRAF
Activating point mutations
8-15
Dabrafenib,
vemurafenib,
sorafenib
NRAS
Activating point mutations
1-6
MEK162
PIC3CA
Activating point mutations
10-30
mTOR
20-80 %
Cell growth and survival pathway
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
Driver mutations in CRC
1. MAPK pathway : KRAS, BRAF, NRAS
2. PI3K / AKT pathway : PIK3CA
Classical treatment in colon cancer
• Surgery
• Chemotherapy
• If pathology shows EGFR overexpression
Start anti EGFR therapy :
– mAB : Cetuximab, panitumumab
– TKI : erlotinib, gefitinib, afatinib
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
EGFR overexpression
Overexpression membrane EGFR (mEGFR)
Overexpression nuclear EGFR (nEGFR)
Gene Amplification
Overexpression ligands
Activating point mutations
EGFR status
Anti-EGFR therapy
mAB : cetuximab, panitumumab
TKI : erlotinib, gefitinib, afatinib
EGFR Resistance : T790M mutation
Inhibitors of EGFR with the T790M mutation :
AZD9291
CO-1831
EGFR resistance : KRAS and BRAF mutations
TREATMENT
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RELAPSE
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
BRAF en MEK inhibitors
BRAF
MEK
Dabrafenib
Trametinib
Vemurafenib
Cobimetinib
EGFR resistance treatment in CRC
Resistance against EGFR therapy
PIC3CA mutation : 10-30 %
PTEN loss
Addition of mTOR inhibitor
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 :
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–
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Breast cancer (25 -40 %)
Endometrium (23 %)
Ovarium
Colon
Non-tumor : somatic overgrowth syndromes
(Cowden and Clove syndrome)
• Therapy : PIK3, AKT, mTOR inhibitors
Resistance to BRAF-MEK inhibitors combi
with reactivation of MAPK pathway or PI
Gene
Mutation
Mechanism
BRAF
Amplification
Splice variants
Activation MAPK pathway
KRAS
Activating point mutation
Activation MAPK pathway
MEK1
Activating point mutation
Activation MAPK pathway
PTEN
loss
Activating PI3K/AKT pathway
PI3CA
Activating PI3K/AKT pathway
Activating PI3K/AKT pathway
Why perform genetic studies on tumor DNA ?
• Initial diagnosis and prognosis
• Monitoring recurrence – metastasis
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
.
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)
Circulating tumor DNA (ctDNA)
ctDNA
ctDNA from tumor tissue is released
through secretion, necrosis and apoptosis,
but mainly through apoptosis.
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) 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
Advantages liquid biopsies
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No tissue biopsy needed
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No FFPE fixation
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Profiling the overall genotype of cancer
• primary cancer
• circulating cells
• metastases
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Better evaluation of :
• reaction to therapy
• development of resistance
Tissue biopsy
TISSUE BIOPSY
EGFR TREATMENT
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Liquid biopsy
LIQUID BIOPSY
TREATMENT
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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 CRC
1. DESCRIPTION : ct DNA testing on liquid biopsies :
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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
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