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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
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
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
• Breast Cancer : 10 %
• Colon cancer : 3-5%
• Prostate cancer : low
• Lung cancer : very low
Majority of cancers are caused by genetic anomalies in the tumor
(somatic mutations)
Minority of cancers is inherited (germline 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)
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 genesBRCA1/2 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
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
Breast Lung Colon Prostate
EGFR < 10 34 20-80 4
KRAS < 10 19 36-40 5
NRAS 1-6
BRAF Few 1-4 8-15 Few
PIK3CA 26 4 10-30 2
TP53 23 34 48 16
MLL3 7 10 12 5
CTNNB1 < 10 < 10 < 10 4
Somatic mutations in CRC
P
Gene Mechanism % Mutations Targeted therapy
EGFR Activating point mutations Gene Amplification Overexpression ligands Overexpression nuclear EGFR
20-80 %
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
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
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
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
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 :– 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
P
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)
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
• No tissue biopsy needed
• No FFPE fixation
• Profiling the overall genotype of cancer
• primary cancer
• circulating cells
• metastases
• Better evaluation of :
• reaction to therapy
• development of resistance
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 CRC
1. DESCRIPTION : ct DNA testing on liquid biopsies :
• 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