Download Lung cancer - Circulating Tumor DNA (ctDNA)

DNA diagnosis of lung cancer
Patrick Willems
GENDIA
•Antwerp, Belgium
Treatment of Lung Cancer
– Small Cell Lung Cancer (SCLC)
• chemotherapy
• radiation
– Non-Small Cell Lung Cancer (NSCLC)
• 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
mAB: Herceptin
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
Mortality
UK, 2009-2011
Cancer Morbidity and Mortality
Canada, 2007
New cancers per year in Belgium
• Lung :
7.100
• Colon :
6.500
• Prostate :
8.800
• Breast :
9.700
TOTAAL :
65.000
Lung cancer
•
14 % of all cancer
•
80 % is non–small cell lung cancer (NSCLC)
• Belgium : 7.100 new cases per year
• Worldwide : 10 million new cases per year
•
Worldwide : 8 million fatalities per year
•
The main cause (20-30%) of cancer-related death
in both men and women :
• More women die of lung ca than breast, cervical and uterine ca combined.
• More men die of lung ca than prostate and colorectal ca combined.
Treatment of Lung Cancer
– Small Cell Lung Cancer (SCLC)
• chemotherapy
• Radiation
– Non-Small Cell Lung Cancer (NSCLC)
• surgery
• radiation
• chemotherapy
• immunotherapy
• personalised targeted treatment
Immunotherapy for NSCLC
• CTLA-4 (cytotoxic T-lymphocyte–associated antigen 4) :
ipilimumab
• PD-1 (programmed death-1) :
nivolumab, pembrolizumab
• PD-L1 (programmed death-1 ligand)
BMS-935559, MPDL3280A
Inhibition immune checkpoints
Biomarkers for immunotherapy for Lung Ca
Few biomarkers for immunotherapy
First real biomarker : Tumor load
(amount of mutations-driver and passenger)
Response to pembrolizumab (PD-1 inhibitor)
better if high mutation load
Science, April 3, 2015 (Rizvi et al)
MSI as Biomarker for immunotherapy
MMR deficiency
Genomic instability
Large mutation load in tumor (driver and passenger)
Many mutant proteins - neoantgens
Immune response
Immunotherapy for NSCLC
– Extremely expensive
(100-300.000 Euro/year)
– No biomarkers to select patients
Targeted therapy with designer drugs
•
Receptor antibodies (---- ab)
•
•
•
•
•
HER2 : Trastuzumab (Herceptin)
EGFR : Cetuximab, Pertuzumab
MET : AMG102
VEGF : Befacizumab
Tyrosine Kinase Inhibitors : TKI (---- ib)
•
•
•
•
•
•
BRC-ABL : Imatinib (Gleevec)
KRAS : Tipifarnib
BRAF : Sorafenib
MEK
ERK
mTOR : Everolimus
Receptor antibodies
inhibit receptor kinases
by interfering with ligand-receptor binding
Preventing intracellular signaling
Herceptin
Herceptin (Trastuzumab) Inhibits HER2 dimerisation / activation
and the downstream signaling pathways MAPK and AKT/mTOR
Active when there is HER2 overexpression
•
Breast ca (25 %)
•
Gastric ca (20 %)
Tyrosine kinase inhibitors (TKI)
TKI inhibits a Tyrosine kinase
by binding to its kinase domain
Preventing phosphorylation (activation) of target
Gleevec
Gleevec (Imatinib) inhibits Tyrosine kinases
by binding to its kinase domain
Thereby preventing phosphorylation (activation) of targets :
•
BCR-ABL (CML)
•
cKIT (GIST, Mastocytosis)
•
PDGFR (GIST)
Targeted treatment
– Non-Small Cell Lung Cancer (NSCLC)
• surgery
• radiation
• chemotherapy
• personalised targeted treatment
• immunotherapy
– Small Cell Lung Cancer (SCLC)
• chemotherapy
• radiation
Targeted treatment of NSCLC
Expensive, but many biomarkers to select patient
Personalised targeted treatment
targets specific somatic mutations
that cause NSCLC
These mutations are patient-specific
These mutations can be detected
by molecular studies of :
tumor (biopsy)
blood (liquid biopsy)
Progress in lung ca treatment
Problems in targeted cancer treatment
The very high cost of personalised treatment makes
companion diagnostics (cancer biomarkers) necessary
The mutations leading to lung ca
are the biomarkers to guide targeted therapy
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 :
5-10 %
• Prostate cancer :
low
• Lung cancer :
very low
Inheritance of lung cancer
• NO germline mutations
• MANY somatic mutations
Driver and passenger gene mutations
TUMOR
MUTATIONS
EXPLANATION
HNPCC
1782
Genomic instability
Lung
150
Mutagen (smoke)
Melanoma
80
Mutagen (sun)
Colon with MSS
73
Breast
60
Prostate
40
Leukemia
10
Fast (acute) tumor
Pediatric tumors
10
Young age
Vogelstein et al, Science Aug 22, 2013
NEJM May 30, 2015
Somatic mutations in cancer
Breast
NSCLC
Colon
Prostate
TP53
23
34
48
16
KRAS
< 10
15-25
35
5
26
4
22
2
EGFR
< 10
10-30
< 10
4
MLL3
7
10
12
5
< 10
< 10
< 10
4
PIK3CA
CTNNB1
Somatic mutations in adeno ca NSCLC
TP53 :
34 %
EGFR :
10-30 %
KRAS :
15-25 %
MLL3 :
10 %
STK11 :
9%
CDKN2A :
8%
ALK fusions : 5 %
HER 2 :
2%
BRAF :
1-2 %
Ros fusions : 2 %
Somatic mutations in adeno ca NSCLC
Cell growth and survival pathway
Genetic testing for lung cancer
– EGFR: deletions in exon 19
L858R mutation in exon 21
T790M mutation in exon 20
– KRAS: mutations of codons 12 and 13
– BRAF: V600E, G469A and D594G mutations
– ALK-EML4 fusion
EGFR Mutations in lung cancer
EGFR mutations :
10 % (Europe)
30 % (Asia)
women, non-smokers, adenocarcinoma (NSCLC)
90% of EGFR mutations :
L858R in exon 21 (Sensitivity to TKIs)
Small deletions in exon 19 (Sensitivity to TKIs)
T790M in exon 20 (Resistance to TKIs)
First-generation EGFR tyrosine-kinase inhibitors :
Erlotinib (Tarceva)
Gefitinib (Iressa)
Second-generation EGFR tyrosine-kinase inhibitors :
Dacomitinib
Afatinib (Gilotrif)
EGFR mutations
EGFR Resistance : T790M mutation
Inhibitors of EGFR with the T790M mutation :
AZD9291
CO-1831
EGFR resistance : KRAS and BRAF mutations
TREATMENT
E
G
F
R
K
R
A
S
W
I
L
D
RELAPSE
KRAS Mutations in lung cancer
KRAS mutations : 15-25 % in NSCLC
smokers
90% of KRAS mutations : codon 12 (90 %)
codon 13 (5-10 %)
KRAS Mutations are contraindications for EGFR TKI
BRAF Mutations in lung cancer
BRAF mutations : 1-4 % in NSCLC
55 % of KRAS mutations : V600E
BRAF Mutations are contraindications for EGFR TKI
ALK Mutations in lung cancer
•
ALK mutations : 5 % in NSCLC
•
ALK activation is caused by EML4-ALK fusion generated by
inv(2)(p21p23)
•
ALK mutations are sensitive to ALK inhibitors :
Crizotinib (Xalkori)
Ceritinib (Zykadia)
ROS1 Mutations in lung cancer
ROS1 mutations : 2 %
ROS1 activation is caused by ROS1 fusion to different partners
ROS1 mutations are sensitive to Crizotinib (Xalkori)
Why perform genetic studies on tumor DNA ?
• Initial diagnosis and prognosis
Initial therapy
• Monitoring recurrence – metastasis
Secundary therapy
Prognosis according to EGFR mutations
Prognosis according to BRAF mutations
Why perform genetic studies on tumor DNA ?
• Initial diagnosis and prognosis
Initial therapy
• Monitoring recurrence – metastasis
Secundary therapy
Monitoring recurrence - metastasis
• Clinical : imaging
• Tumor markers : CEA
• Circulating tumor cells (CTC)
• Circulating tumor DNA (ctDNA)
Diagnostic tests on tumor DNA
• Solid tumor biopsy
FFPE
Frozen
Fresh
• Liquid biopsy : Circulating tumor DNA (ctDNA)
Blood plasma/serum
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
Why liquid biopsies for Lung cancer ?
• The main cause (20-30%) of cancer-related death
• High percentage of driver oncogenic mutations
• Druggable targets
• Lung biopsy difficult – liquid biopsy easy
• Relatively cheap test
• Association with expert group of Rafael Rosell
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
a small fraction of cfDNA is circulating tumor (ctDNA)
very sensitive technology is needed
to detect mutations in ct DNA
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) for Down syndrome:
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
Tissue biopsy
•
•
E
G
F
R
K
R
A
S
W
I
L
D
TISSUE BIOPSY
EGFR TREATMENT
•
RELAPSE
Liquid biopsy
•
•
E
G
F
R
K
R
A
S
B
R
A
F
W
I
L
D
LIQUID BIOPSY
TREATMENT
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
Companies focusing on ctDNA
Most companies have an expensive test (5000 USD)
based upon NGS (Next Generation sequencing)
detecting many mutations in many cancer genes
of which the majority are currently “nondruggable”
Sensitivity to detect mutant sequence
• Sanger sequencing : 10 %
• Next gen sequencing (NGS) : 1 %
• NGS + specific technology : 0.1 %
Pangaea biotech
• Simple, relatively cheap test for lung cancer
• Spin off company from IOR (Instituto oncologico Rosell)
• CEO : Rafael Rosell
• Focusing on Lung cancer
• Technology : Mutant Allele - specific PCR using PNAs
Mutant Allele - specific PCR
PCR amplification of wild type allele is blocked
by PNA probe, while mutant is allele is PCR-amplified
ctDNA testing for lung ca
1. DESCRIPTION : ct DNA testing on liquid biopsies :
• EGFR: deletions in exon 19, L858R mutation in exon 2, T790M mutation in
exon 20
• KRAS: codon 12 and 13 mutations
• BRAF: V600E, G469A and D594G mutations
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. Send blood :
Email [email protected] to ask for tubes
www.circulatingtumorDNA.net