Transcript Papel del ADN tumoral circulante en el manejo del paciente con cáncer colorrectal metastásico J.

Papel del ADN tumoral circulante
en el manejo del paciente con
cáncer colorrectal metastásico
J. Tabernero
Servicio de Oncología Médica
Hospital Universitario Vall d’Hebron
Vall d’Hebron Instituto de Oncología (VHIO)
Barcelona
Diagnosis evolution in cancer
Currently
Clinical diagnosis
Clinical
approach
Pathology diagnosis
Pathology
approach
Molecular diagnosis
Personalized
Oncology
Molecular
approach
Genomics & Personalized Medicine
First-generation sequencing platforms, Sanger technique
First complete human genome sequencing
Second-generation sequencing platforms
Need for Non-Invasive Molecular Testing
● Tissue availability for molecular testing varies from 90% (CRC) to 10% (HCC)
 25% - 35% inadequate tissue in NSCLC
 Long lead times for 2nd opinions from hospital to hospital
● Complication rate with biopsies
 2% major complication rate with lung biopsy
 15% minor complication rate
● Increased need for rapid turn-around time for treatment decisions
 Early integration of molecular targeted therapies
● Tumor Heterogeneity (CRC)
0%
13%
19%
0/19
1/8
20/108
Sites with Low Discordance
32%
33%
10/31
11/33
Sites with High Discordance
50%
9/18
Overman et al, J Clin Oncol 2013,
Kopetz et al, ASCO 2014, A3509
Characteristics of cell-free tumor DNA
● Cell-free tumor DNA is cell-free DNA
released from a solid tumor
● cfDNA ≠ CTCs
● Total cfDNA elevated for various
reasons:
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Inflammation
Wound healing
Malignant lesions
Menstruation
Sport
● Healthy individual:
3,000 – 5,000 Genomic Equivalents/ml
● Patient with a tumor:
average of 10,000 Genomic Equivalents/ml
Cell-Free
Tumor DNA
Circulating
Tumor Cell
Cell-Free
Normal DNA
Advantages of cell-free tumor DNA testing
Patient / Oncologist
High compliance
Minimally invasive, few risks
Fresh DNA
Physician Specialist
(i.e. Surgeon)
Archival tissue can be degraded & have different
mutation profile
Accessible
Tissue not always accessible; e.g. NSCLC
Tissue
Testing
Direct
Plasma
Testing
Pathology
No selection bias
Assess primary tumor and metastases with one sample
Tumor intra/inter-heterogeneity not a problem
Monitoring possible
Allows monitoring for drug response and resistance
Clinical Laboratory
Cell-free tumor DNA
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•
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An alternative source of DNA
DNA can be shed as both single- and double-stranded DNA (in complexes with
histones)
Genomic, epigenomic, mitochondrial and viral DNA
cfDNA is fragmented to an average length of 140 to 170 base pairs
Not cancer specific
– Healthy individuals: ~1 ng DNA/ml
– Tumor 100g: 3x1010 tumor cells, 3.3% tumor DNA enter circulation every day
– Cancer patients: 4-fold greater levels
cfDNA is present in a few thousand amplifiable copies/milliliter blood
Circulating mutant DNA: 0.01% of total cfDNA
Stroun et al, 1987; Rumore and Steinman, 1990;
Lee et al, 2001; Schwarzenbach et al, 2011
Cell-free tumor DNA
 Sources of cfDNA release:

Types of DNA alterations
found in cfDNA
The tumour burden and rate of tumor cell
proliferation have a substantial role
Schwarzenbach et al, Nat Reviews Cancer 2011
Cell-free tumor DNA in stage IV
(n = 177)
Bettegowda et al, Sci Tran Med Feb 2014
Cell-free tumor DNA in stage IV
Localized vs Metastatic Disease (n = 223)
Bettegowda et al, Sci Tran Med Feb 2014
Principles of BEAMing
Beads / Emulsification / Amplification / Magnetics
BEAMing
PreAmplification
Emulsion PCR
Hybridization
Flow
Cytometry
Flow cytometry analysis
Wild-type DNA
Wild-type signal
Mutant &
Wild-type DNA
Detection Capability
(mutant DNA/ total DNA)
100%
10%
Mutant
DNA
1%
0.1%
Mutant signal
0.01%
Sanger Sequencing
Pyrosequencing
Real-Time PCR
BEAMing
Concordance tissue vs cftDNA
Tissue
Analysis
Plasma
Analysis
Concordance
78
78
SOC
SOC
BEAMing
BEAMing
99%
92%
IV
38
SOC
BEAMing
93%
93%
PIK3CA
IV
34
BEAMing
BEAMing
100%
BC
PIK3CA
IV
50
BEAMing
BEAMing
100%
Vogelstein et al,
Nat Med 2008
CRC
KRAS
IV
10
Sanger
Sequencing
BEAMing
100%
METRIC study,
J Clin Oncol 2013
Melanoma
BRAF
V600E
V600K
IV
305
PCR
BEAMing
96%
Citation
Cancer
Marker
Stage
Nexus study,
AACR 2013
NSCLC
EGFR
KRAS
IV
Astra Zeneca
studies,
ASCO 2014
Cross platform
comparison of
Cobas EGFR,
Therascreen,
BioRad and
BEAMing
NSCLC
L858R
Exon19 del
Higgins et al,
CCR 2012
BC
Agenedt et al,
JCO 2010
Patient
Number
Cell-free tumor DNA utilities
Screening
&
Diagnosis
Prognosis
Targeted
Therapy
Response
Prediction
Monitoring
Resistance/
Recurrence
cftDNA in CRC
Applications in CRC:
– Monitoring tumor burden
– Monitoring response
– Monitoring resistance
– Molecular characterization and tailoring targeted
treatment
cftDNA in CRC
Applications in CRC:
– Monitoring tumor burden
– Monitoring response
– Monitoring resistance
– Molecular characterization and tailoring targeted
treatment
cftDNA: Monitoring of tumor burden
Over a 56 day period of time, a total of 162
plasma samples were analyzed from 18
patients. APC was used as the mutation
marker.
Diehl et at. Nat Med. 2008
Normal signal
cftDNA: Monitoring of tumor burden
Before Surgery
After Surgery
After Surgery
Day of Surgery
Day 3
Day 244
Residual
Mutant cfDNA
Mutant signal
Diehl et at. Nat Med. 2008
cftDNA and monitoring
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cftDNA in plasma: reliable tool to monitor
mCRC dynamics
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Case study :
– CRC pt: PIK3CA mutation in tissue and
plasma
– Sigmoid adenocarcinoma and solitary mets
in both hepatic lobes.
– Surgery 1: sigmoidectomy with hepatic
metastectomy.
– A right-sided liver metastasis was left in
place while the he was treated with
systemic chemotherapy (Chemo 1).
– Surgery 2: right hepatectomy. After surgery,
the subject was treated for 4 months with
Chemo 2.
– Progression day 400
Diehl et at. Nat Med. 2008
cftDNA: Monitoring of tumor burden
Cell-free tumor DNA
No detectable mutations in plasma
(n = 4)
Detectable mutations in plasma
(n = 14)
CEA
No detectable CEA (normal range boundary)
(n = 10)
Detectable CEA
(n = 8)
Diehl et at. Nat Med. 2008
cftDNA in CRC
Applications in CRC:
– Monitoring tumor burden
– Monitoring response
– Monitoring resistance
– Molecular characterization and tailoring targeted
treatment
cftDNA: monitoring response
Baseline Imaging
8 weeks later
Courtesy of: Jeanne Tie, Peter Gibbs
cftDNA in CRC
Applications in CRC:
– Monitoring tumor burden
– Monitoring response
– Monitoring resistance
– Molecular characterization and tailoring targeted
treatment
Clonal selection vs evolution: Detection of KRAS mt
cftDNA in patients treated with anti-EGFR MoAbs
Misale S, et al. Nature 2012
Diaz E, et al. Nature 2012
Clonal selection vs evolution: Detection of KRAS mt
cftDNA in patients treated with anti-EGFR MoAbs
Initial response to
cetuximab followed by PD
Quantitative analysis of
KRAS(Q61H) mutant DNA in
plasma, as assessed
by BEAMing
Misale S, et al. Nature 2012
Diaz E, et al. Nature 2012
Vilar E & Tabernero J, Nature 2012
“Under pressure” clonal selection vs evolution
Vilar E & Tabernero J, Nature 2012
cftDNA: monitoring resistance
● 24 cases of metastatic CRC that
responded to EGFR blockade and
then progressed
● Examined known resistance
mutations in KRAS, NRAS, BRAF,
EGFR and PIK3CA
● 23 of 24 cases had a detectable
resistance mutation in the plasma
● 69 resistant mutations detected
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49% in KRAS codon 12/13
23% in NRAS codon 61
22% in KRAS codon 61
15 of 24 (63%) had codon 61
Bettegowda et al, Sci Tran Med Feb 2014
cftDNA: monitoring resistance
● Eligibility: progressive mCRC patients previously treated with FOLFOX or FOLFIRI ±
anti-VEGF and ± anti-EGFR if KRAS wild-type; CORRECT study.
● Plasma of 503 patients evaluated by BEAMing for KRAS mutations. Archival tissue of
239 patients evaluated by BEAMing.
● Correlation between mutational status in patient´s matched fresh plasma: 76%
● Mismatches of fresh plasma and archived tissue: mainly WT KRAS in tissue and mutant
in plasma, due to acquired KRAS mutations following anti-EGFR therapy.
● High frequency of Q61H mutations (1% in archival tissue & 12% in plasma)
● Mutational analysis of fresh plasma may more accurately reflect a patients current
mutational status than analysis of archival tumor tissue.
Jeffers, ASCO GI 2013 A381
Multiple mechanisms of acquired resistance to EGFR
inhibition in mCRC
Hobor S et al, Clin Cancer Res 2014; Salazar R & Tabernero J, Clin Cancer Res 2014
cftDNA in CRC
Applications in CRC:
– Monitoring tumor burden
– Monitoring response
– Monitoring resistance
– Molecular characterization and tailoring targeted
treatment
All RAS mutations
KRAS exon 2 wild-type
(2008)
Extended RAS wild-type
(2014)
NRAS Exon 3
mutant
NRAS Exon 2
mutant
KRAS
codon 13
mutant
NRAS
Exon 4
mutant
KRAS Exon 4
mutant
KRAS Exon 3
mutant
KRAS
codon 12
mutant
KRAS
wild-type
RAS wild-type
KRAS codon
13 mutant
KRAS codon
12 mutant
All RAS mutations
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9 RCTs (N= 5,948) evaluated for both established and new RAS mutations
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7 studies evaluated the addition of an anti-EGFR mAb to therapy (FOLFOX, FOLFIRI, irinotecan,
oxaliplatin and fluoropyrimidine chemotherapy or best supportive care). 2 studies compared the addition of
anti-EGFR mAb or bevacizumab to cytotoxic therapy (FOLFOX or FOLFIRI)
Sorich et al, Ann Oncol 2014
Sysmex-Inostics panel of RAS mutations
in cftDNA in CRC (Sysmex Inostics OncoBEAM™)
Gene
Exon
Mutation
COSMIC ID
KRAS
2
2
2
2
2
2
2
3
3
3
3
3
4
4
4
4
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
4
4
4
G12S
G12R
G12C
G12D
G12A
G12V
G13D
A59T
Q61L
Q61H
Q61H
Q61R
K117N
K117N
A146T
A146V
G12S
G12R
G12C
G12D
G12A
G12V
G13R
G13D
G13V
A59T
Q61K
Q61R
Q61L
Q61H
Q61H
K117N
K117N
A146T
COSM517
COSM518
COSM516
COSM521
COSM522
COSM520
COSM532
COSM546
COSM553
COSM554
COSM555
COSM552
COSM19940
COSM28519
COSM19404
COSM19900
COSM563
COSM561
COSM562
COSM564
COSM565
COSM566
COSM569
COSM573
COSM574
COSM578
COSM580
COSM584
COSM583
COSM585
COSM586
TBD
TBD
COSM27174
NRAS
● Final mutation profile is under discussion
● BRAF mutation (RUO) under consideration for certain countries (e.g., France)
Cell-free tumor DNA in CRC - Summary
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Cell-free tumor DNA testing can play a key role in realizing Precision Medicine
 Directing Therapy
 Monitoring Response to Therapy
 Monitoring Disease Recurrence
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For solid tumor cancers, cell-free tumor DNA shows strong correlation
between tissue and plasma in metastatic settings
•
BEAMing is highly sensitive in monitoring disease, with the potential of earlier
treatment intervention compared to current diagnostic methods (CEA and
imaging)
•
Emergence of resistance mutations is common in mCRC patients and can be
detected earlier than imaging with BEAMing
•
Sysmex Inostics OncoBEAM™ assays target clinically actionable mutations in
mCRC.
Gracias