Download Mutationer og blod

Mutationer og blod
Peter Meldgaard
Aarhus Universitetshospital
Evolution of Lung Cancer
Evolution of Lung Cancer
Evolution of Lung Cancer
Evolution of Lung Cancer
Genomiske Ændringer ved Lunge Adenocarcinomer
No known
genotype
1984 - 2003
2009
No known
genotype
2004
2013
EGFR mutants in lung adenocarcinoma
•
Somatic EGFR mutations found in
~30 to 60% of East Asian lung
adenocarcinomas, ~10-15% of U.S.
and European patients
•
Clustered in four areas
– Nucleotide binding loop (P loop),
G719
– Exon 19 deletions (ELREA)
– Exon 20 insertions (C-terminal to
alpha helix)
– Activation loop mutations (L858)
N lobe
C lobe
Associated with response to
erlotinib/gefitinib/afatinib (except for
exon 20 insertion mutants)
Promising results for
AZD9291/rocelitinib for T790M
resistance mutations
•
EGFR mutations (M+) are amongst the commonest druggable alterations in NSCLC
Waterfall plot depicting best response to erlotinib (EURTAC)1
PFS: 10-14 months
RR: 50-80%
Primary
Resistance
Acquired
Resistance
1Rosell
et al. Lancet Oncology 2012
Personalized medicine aims at selecting the right therapy
Cancer
Patient
Tumour
type
Oncologist
selects
therapy
based on
experience,
histology and
tumour site
Therapy
Wrong
match
The right drug for
the tumour type
Wrong
match
Adapted from D Weaver
Targeted
Therapies
Structural DNA/RNA
changes
Chemosensitivity
Immune contexture
PDL1 staining
Mutational load
IHC panels +/- FISH
13
Lung Cancer Patient in the near future
Lung nodule/metastases
Tumor Biopsy/ blood
GLOBAL MOLECULAR PORTRAIT
Diagnostic
Prognosis
Targeted therapy Immunotherapy
sensitivity
sensitivity
PD1/PDL1
ERCC1
OX40
RRM1
Other
Pathways
…
Which treatment and when ?
Chemotherapy
sensitivity
Cancer? Yes/No
Need for treatment ?
Genome comparison
adenocarcinoma vs. squamous cell carcinoma
High mutation rates in both lung adenocarcinoma and
lung squamous cell carcinoma
Lawrence et al., Nature, 2013
Different genes are mutated in lung adenocarcinoma
and lung squamous cell carcinoma
RTK/Ras/Raf pathway in
lung adenocarcinoma:
KRAS, EGFR, NF1,
BRAF, MET, ERBB2,
RIT1, RAF1
Common squamous cell
cancer genes including
NFE2L2, FAT1,
NOTCH1 in lung SqCC
Campbell et al., submitted
Common and different genes are amplified in lung
adenocarcinoma and lung squamous cell carcinoma
Lineage oncogenes:
SOX2 in squamous;
NKX2-1 in
adenocarcinoma
RTK amplifications
mostly in
adenocarcinoma but
EGFR, PDGFRA,
FGFR1 are common
TERT and MYC in both
Campbell et al., submitted
Lung adenocarcinoma driver mutations:
current status
Multiple known RTK/Ras/Raf drivers known
EGFR and ALK already actionable
BRAF, ERBB2, MET should be actionable soon
New alterations still being discovered: RIT1, ARAF, RAF1,
SOS1, RASA1, MAPK1
Many more yet to be discovered—problem of statistical
power and functional understanding
18 SPECTA-lung participating sites
Ireland
Stephen Finn - St James Institute
Belgium
Thierry Berghmans - Institute Jules Bordet
Johan Vansteenkiste - Institute KU Leuven
Jan Van Meerbeeck – UZA Antwerp
UK
Sanjay Popat – Royal Marsden
Netherlands
Egbert Smit - VU University Medical Center
France
Benjamin Besse – Gustave Roussy
Julien Mazieres - Hôpital Larrey
Denmark
P. Meldgaard - Aarhus University Hospital
Spain
Enriqueta Felip – Vall d'Hebron
University Hospital
Luis Paz-Ares – Hospital Universitario
Doce de Octubre
Germany
Martin Reck - Center of Pneumology and
Thoracic Surgery of Grosshansdorf
Poland
Rafal Dziadziuszko - University of Gdansk
Italy
Silvia Novello – University of Turin
Andrea Ardizzoni – S. Orsola-Malpighi
Switzerland
Rolf Stahel – University Hospital of Zurich
Solange Peters – University of Lausanne
Slovenia
Tanja Cufer – University Clinic Golnik
Central Biobank
Gustave Roussy
Cancer Campus
Next Generation Sequencing (NGS) by 14 MG
Resistens
Til målrettet behandling
Kinase
Target
Kinase
Inhibitor
Tumor Response
Kinase
Target
Target Reactivation
Activating “downstream”
effectors
• Secondary genetic alterations
• Activation of upstream effectors
Pathway Reactivation
Histologic Transformation
Failure to undergo apoptosis
Disease Progression
Garraway and Jänne Cancer Discovery 2012
BYPASS of (onco)protein
effector
Mechanisms of drug resistance to EGFR
and ALK targeted therapies
EGFR mutant
Junko Tanizaki
ALK rearranged
Efficacy of mutant selective EGFR inhibitors in
EGFR inhibitor resistant EGFR T790M NSCLC
Jänne et al. NEJM 2015; Sequist et al. NEJM 2015
Serial ddPCR profiling of cfDNA reveals 3 molecular
subtypes of acquired resistance to AZD9291
EGFR Activating Mutation
EGFR T790M
EGFR C797S
Thress et al, Nature Medicine, 2015
EGFR Activating Mutation
EGFR T790M
EGFR Activating Mutation
Loss of T790M
Evolution of resistance mechanisms in EGFR mutant lung
cancer following EGFR TKI therapy
T790M+
AZD9291
T790M+ plus
C797S
Erlotinib
T790M+ plus
unknown
resistance
T790M+
EGFR
activating
mutation
T790M-
No response
T790M -
Increased
Heterogeneity
Liquid biopsies
Også kaldet blodprøver
Tumor diagnostik
• Timing
• Metastisk sygdom
• Beskadigelse af DNA
Plasma DNA for tumor diagnostic
Fleischhacker & Schmidt
Nature Medicine 2008
Blodprøve --- Tumor DNA Bibliotek.
Schwarzenbach H, Hoon DS, Pantel K.; Nat Rev Cancer. 2011,Jun;11(6):426-37
What is cell-free DNA?
•
•
•
•
166 bp average length
Due to apoptotic/necrotic/lytic cell turnover
Mostly of hematopoietic origin (disease-free)
<2hr half-life
Courtesy A Bardelli
Cell-free DNA Sequencing as a “Summary”
of Inter-Tumor Heterogeneity
Plasma DNA---kræft behandling
Plasma DNA forudsiger response på erlotinib.
Step 1
Step 2
Step 3
Step 4
Blod prøve
Oprensning
PCR Setup
Resultat
cobas® 4800 v2.0
2 mL Plasma
P<0.05
EGFR M+
EGFR M-
Weber et al. 2014,
BMC Cancer
10000
Mutated DNA (Copy/ml)
Mutated DNA (Copy/ml)
Erlotinib --- T790M resistens
5000
100
100
50
0
0
100
200
time (days)
Blå = EGFR del19
Rød = EGFR T790M
300
400
10000
5000
100
100
50
0
0
100
200
time (days)
300
400
250
250
00
0
0
0
100
200
300 opsporing
400
50 100 150 => ny behandling
Tidligere
af0 resistens
time (days)
time (days)
D.
300
400400 600 800 10001200
!
Blood based monitoring of
erlotinib in stage 4
EGFRM+ NSCLC
Eva Boysen Hansen
Peter Meldgaard
Boe Sandahl Soerensen
Mut ated DNA (Copy / ml )
Background
1
0
Pat ient 3
5
0
1
0
1
0
5
0
0
0
1
0
2
0
3
0
ti me (days)
4
0
A.
Mutated DNA (Copy/ml)
Mutated DNA (Copy/ml)
800
10000
5000
500
500
250
Patient 2
600
400
200
0
0
0
100
200
300
400
100
200
300
500
150
200
50000
1000
1000
500
250
50
50
400
1000
1000
500
200
300
400
500
Patient 9
10000
5000
500
500
250
0
0
600
100
time (days)
Mutated DNA (Copy/ml)
Mutated DNA (Copy/ml)
Mutated DNA (Copy/ml)
100
0
500000
0
100
time (days)
B.
Patient 6
150
150
1000000
0
200
100
Patient 8
1500000
500
400
200
time (days)
1000
300
0
0
Patient 7
0
200
250
100000
time (days)
1500
100
time (days)
0
100
50
0
Mutated DNA (Copy/ml)
1000
1000
50
100
100
400
Patient 5
150000
Patient 4
10000
0
Patient 3
5000
time (days)
Mutated DNA (Copy/ml)
Mutated DNA (Copy/ml)
20000
10000
0
0
time (days)

Mutated DNA (Copy/ml)
Patient 1
15000
200
300
400
0
500
50
100
150
time (days)
time (days)
D.
C.
500
20000
400
10000
2000
2000
Mutated DNA (Copy/ml)
Tissue samples
Mutated DNA (Copy/ml)
300
200
100
50
50
40
30
20
300
300
200
100
10
0
0
0
10
20
time (days)
 Blood samples
1000
30
0
20
40
60
80
100 100
200
300
400400 600 800 10001200
time (days)
Soerensen BS et al. ”Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing
!
and resistance mutations in the plasma DNA of patients with advanced
non-small cell lung cancer during
treatment with erlotinib” Cancer. 2014 Aug.
Methods
•
Multicenter: 4 oncology centres in DK (Aalborg, Herning, Odense, Aarhus)
•
200 patients in 2 years: Stage 4, EGFRM+, NSCLC (app 80 recruited)
•
Standard treatment supplemented with blood based monitoring
Methods
•
Examining resistance mechanisms
•
Ca. 50% are expected to be T790M driven
•
The rest: ?  Exome sequencing of tissue samples at time of diagnosis vs. PD
Aims
1. Closer monitoring of erlotinib
treatment  identification and
treatment of oligoprogression.
2. Applicability of blood sample
monitoring in a clinically
meaningful setting.
3. Identification of new resistance
drivers
Urinprøver
Dynamic Changes in EGFR
Mutation Circulating Tumor DNA in
Urine on Anti-EGFR Therapy
Hatim Husain MD
Center of Personalized Cancer Therapy
Liquid Biopsy Program
University of California, San Diego Moores Cancer Center
September 9, 2015
IASLC World Conference on Lung Cancer
44
Clinical Questions
1. Can EGFR T790M resistance be
Detected by ctDNA in urine to
inform decisions by about tumor
progression in EGFR mutant lung cancer?
Yu et al Clin Can Res 2013
2. Can early daily dynamics of ctDNA in urine within the first
week on second line anti-EGFR tyrosine kinase therapy
provide insight to tumor biology and inform response?
Response within 3 weeks of treatment initiation
45
Blocking Wild-Type DNA Amplification Enriches
for Mutant Sequences in Urine
Daily collection
Mutants
Wild Type
Blocker
10x DNA yield in
urine vs plasma
due to higher urine
volumes collected
Blocking
technology
facilitates PCR
enrichment
followed by NGS
detection (MiSeq)
46
HRPP 130794 Project Schema
Specific Aim 1: Test the hypothesis that T790M could be identified prior to radiographic progression
• 34 patients receiving treatment with erlotinib monitored longitudinally for acquisition of T790M mutation
• Collection frequency q3-6 weeks
Specific Aim 2: Test the hypothesis that T790M mutational load in ctDNA within the first week may predict
radiographic response to therapy with second line anti-EGFR tyrosine kinase inhibitors
• 14 patients positive for T790M by tissue biopsy received treatment with second line anti-EGFR TKIs
• Urine specimens were collected from patients prior to treatment, daily for 1 week, then weekly for 3
weeks, then monthly
47
Results:
Correlation Data Between Urine and Tissue Biopsy for EGFR T790M
T790M Tissue
Total Patients (N=65)
Positive
T790M Tissue
Negative
T790M Tissue
not yet
tested
Total
T790M Urine Positive
14
3
12
29
T790M Urine
Negative
1
1
7
9
Total
15
4
19
38
48
Results:
Urinary T790M Detection Compared with Date of Radiographic Progression
PD
300
T790M
detected
200
100
0
Early T790M Detection
(Days to Radiographic Progression)
T790M
detected
PD
Patient 1
111
Patient 3
52
Patient 10
56
Patient 20
29
28-10-2014
0
400
08-10-2014
10
Patient 3
49
Urine T790M Copies/100K GE
20
500
29-08-2014
18-09-2014
09-08-2014
30-06-2014
21-05-2014
0
30
PD
08-10-2014
20
T790M
detected
29-08-2014
40
40
20-07-2014
60
PD
Patient 20
50
10-06-2014
T790M
detected
Urine T790M Copies/100K GE
80
11-04-2014
Urine T790M Copies/100K GE
Patient 10
18-09-2014
Patient 1
100
Can the early dynamics of ctDNA within the first week on second line antiEGFR TKI therapy provide insight to tumor biology and inform response?
50
51
Week 1 on drug
Week 1 on drug
Week 5
Week 4
Week 3
Week 2
Day 7
Day 6
Day 5
0
Day 4
50
20000
Day 3
Ex19 del
Day 2
150
Day 1
T790M
4 hr on Drug
200
Urine EGFR Copies/100K GE
250
10/13/2014
Day 7
Day 6
Day 5
Day 4
100
Day 3
Day 2
Day 1
Day 1 (4 hrs)
Baseline
Urine EGFR Copies/100K GE
Results:
Daily Urinary ctDNA Detection within the First Week of Therapy
25000
L858R
15000
10000
T790M
5000
0
52
Week 1 on drug
6 wks CT scan
0
0
12 wks CT scan
Week 1 on drug
6 wks CT scan
1/19/2015
1/12/2015
1/5/2015
50
12/29/2014
5000
12/22/2014
100
12/15/2014
T790M
100
12/8/2014
10000
150
150
12/1/2014
200
11/24/2014
Size on CT scan
11/17/2014
250
11/10/2014
20000
11/3/2014
300
Urine EGFR Copies/100K GE
250
200
70
Size on CT scan
60
T790M
50
40
Ex19 del
30
50
20
10
0
0
12 wks CT scan
Sum of Longest Diameters (mm)
350
10/27/2014
L858R
Sum of Longest Diameters (mm)
25000
10/20/2014
1/14/2015
1/7/2015
12/31/2014
12/24/2014
12/17/2014
12/10/2014
12/3/2014
11/26/2014
11/19/2014
11/12/2014
11/5/2014
15000
10/29/2014
10/22/2014
10/15/2014
10/8/2014
Urine EGFR Copies/100K GE
Results:
Urinary ctDNA Kinetics Compared to Radiographic Change
80
Results:
Re-treatment Kinetics in ctDNA EGFR Ex19del and T790M
3.9x3.7
3.7x3.1
3.2x3.0
Change in the Target
Lesion on CT (cm)
2.1x1.9
1.7x1.5
200
1st gen TKI
150
3rd gen
TKI
100
Held
for
Toxicity
3rd gen TKI
(dose change)
50
T790M
25
E x19del
7 /12/2 015
6 /22/2 015
6 /8 /2 0 1 5
4 /23/2 015
4 /3 /2 0 1 5
0
3 /14/2 015
53
250
2 /22/2 015
U r in e E G F R C o p ie s / 1 0 0 K G E
300
Working Hypothesis:
Early Pharmacodynamics of Urine ctDNA as a Putative
Biomarker of Response
54
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