Download Multiplexed ICE COLD-PCR (MX-ICP): For

Multiplexed ICE COLD-PCR (MX-ICP):
For Detection
of
Low-Level Mutations
in
Liquid Biopsies
Katherine Richardson
Vice President,
Research & Development
Transgenomic®
Advancing Personalized Medicine
1
Transgenomic
Background & Introduction
Biopsy
Learn more
Ultra-sensitive mutation detection in circulating free DNA (cfDNA)
Transgenomic®
Advancing Personalized Medicine
2
Transgenomic Technologies that Enable Biomarker
Discovery & Development of Molecular Diagnostics
Transgenomic Technology Development Focus:
Holds rights to IP on ICE COLD-PCR developed by Dr. Mike Makrigiorgos at the Dana
Farber Cancer Institute
Develops enrichment assays to enable sensitive mutation detection
Supports oncology and immuno-oncology biomarker development
Collaborates in biomarker discovery for colorectal, lung, melanoma and other cancers
Holds additional IP for several targets including PIK3CA and FC gamma receptor
1997
WAVE® System
WAVEMAKER™
Software –
nucleic acid
variation
analysis
2002
2004
Navigator™
SURVEYOR®
Software
Nuclease Kits
Automated
Mutation
Calling on the
WAVE System
2010
REVEAL®
ICE COLD-PCR
Kits
2011
RAScan™
& RASseq™
CRC Somatic
Mutation
Detection Kits
2014
Multiplex
ICE COLDPCR
Coupled
with NGS
2015
ICEme
Mutation
Enrichment
Kit
3
Discovery
Discovery (Novel
Targets)
• Identification of any and all
mutations
• Proof-of-concept
• Assay Development
Services
Kits
Services
Kits (Off-the-shelf)
• Targeted Sequencing
• RNA Sequencing
• microRNA Panels
• Gene Expression
• Methylation
• SNP Genotyping
• DNA Quantitation
• Mutation Enrichment
• Gene Panels
• ICE COLD-PCR
• Choice of exons/reactions
• Technical Support hotline
• RUO / 510K / CE-IVD
• MX-PCR/ICP
Multi-Disciplinary Team:
Expert Knowledge and Expertise
ICEme Kits
Knowledgeable and Experienced Scientists.
Discovery
Services
Expertise in ISO and QSR, CDx and CLIA/CAP.
Medical device development expertise.
Clinical, pharmaceutical and molecular genomics
expertise.
Kits
Preclinical Studies, extensive expertise in AAALAC
Regulatory Consultants with extensive expertise in
CDx development.
4
Liquid Biopsies
Traditional Tissue Biopsies are:
Costly
Invasive
May Require lengthy workflows
Tumor heterogeneity can be an issue
Biopsy
Ultra-sensitive mutation
detection in circulating cfDNA
ICE COLD-PCR is a technology exquisitely made for
mutational analysis of Liquid Biopsy
Circulating cell-free DNA (cfDNA) is a potential
surrogate for the entire tumor genome
Liquid Biopsy enables Precision Medicine (diagnosis,
prognosis, and monitoring of cancer, or as predictive
markers for the patient’s response to a targeted
therapy)
Transgenomic®
Advancing Personalized Medicine
5
ICE COLD-PCR Overview
Biopsy
Learn more
Ultra-sensitive mutation detection in circulating free DNA (cfDNA)
Transgenomic®
Advancing Personalized Medicine
6
ICE COLD-PCR Advantages
Non-Allele Specific – Enriches ANY & ALL Variants
Improves clinical outcome by use
of non-invasive liquid biopsies
for treatment & monitoring
Works on any sample – tissue, plasma,
cells, exosomes, FFPE, CTC’s
Increased sensitivity of detection on any platform:
Simple integration into existing workflow on NGS,
ddPCR, Sanger
Significantly improves sensitivity – 100 to 500 fold increase. Therefore
enables Sanger confirmation of low level NGS mutations
Exclusive license from Dana
Farber Cancer Institute
7
ICE COLD-PCR : A Unique Technology for
Preferential Enrichment of Mutations
Liquid
Biopsy
Pre-amplification
Multiplex-PCR
(Traditional PCR)
Mutant
0
5
10
15
20
# Cycles
25
30
Mutant
# PCR Products
DNA
Extraction &
Amplification
# PCR Products
Wild-Type
ICEme Kit
0
35
MX-ICP
(Specialized
Wild-Type
Nested-PCR)
5
10
15
20
# Cycles
25
30
35
Analyze ICP
Product on any
Sequence
Analysis Platform
The Pre-amplification step increases the amount of starting material
A small portion of the MX-PCR is used in the ICE COLD-PCR reaction to enrich for any and
all mutations present
After the ICE COLD PCR reaction, the samples can be analyzed on any downstream platform
8
ICE COLD-PCR Assay
Input Sample
from MX-PCR
5’
5’
5’
5’
5’
5’
PO4
PO4
PO4
5’
Pol.
Buffer, dNTP’s
Step 1:
Denature
5’
PO4
5’
5’
PO4
5’
XXXXX
5’
5’
XXXXX
PO4
PO4
5’
5’
5’
5’
PO4
5’
5’
Step 5:
Polymerase
Extension
5’
5’
5’
5’
Wild-Type Sequence
Mutated Sequence
PO4
Exp. Amp
Analysis on
platform of
choice
for mutation
detection
5’
Linear Amp
5’
5’
5’
5’
PO4
5’
Tc
5’
Step 4:
PCR Primer
Hybridize
PCR Primer 1
5’
5’
Step 3:
Denature at
Critical
Temperature (Tc)
Repeat
5’
5’
5’
Step 2:
RS-oligo
Hybridize
PO4
PO4
5’
5’
5’
5’
5’
5’
PO4
5’
PO4
PCR Primer 2
RS-oligo
PCR Process that Enriches for ANY Mutation
in the Region Interrogated by the RS-oligo.
9
MX-ICP Flexible Workflow
Sanger Sequencing of Individual
MX-ICP Assays
Detects ANY and ALL of the possible
mutations present
Sample DNA
MX-PCR to
increase the
amount of DNA
present for
multiple gene
regions
MX-ICP
Enrichment of ANY
and ALL mutations
present in the
gene regions
amplified in the
MX-PCR.
NGS: Pool all MX-ICP
Reactions/Sample then Analyze
Detects ANY and ALL of the possible
mutations present
ddPCR
Sufficient mutation-enriched DNA for
detection of ANY and ALL possible
mutations. The number of different
mutations which can be detected is
limited by the number of available
ddPCR assays.
10
Detection of ALL sequence variations
Selection of Exons Available
# of COSMIC Alterations
Detectable in MX-ICP Region
Single Multiplex PCR amplification
Detection of ALL sequence variations
including point mutations and small
insertions/deletions in:
KRAS Exons 2, 3, and 4
Including codons 12, 13, 59, 61, 117 & 146
NRAS Exons 2, 3, and 4
Including codons 12, 13, 59, 61, 117 & 146
BRAF Exons 11 and 15
Including codons 469 & 600
PIK3CA Exons 9 and 20
Including codons 542, 545 & 1047
EGFR Exons 12, 18, 19, 20, and 21
Including codons 492, 719, All Exon 19
dels/complex mutations, 790, 797, 858 & 861
*No mutations listed in COSMIC but observed in Colorectal Cancer
COSMIC
Insertions/
Deletions
COSMIC
Mutations
BRAF Exon 11
0
35
BRAF Exon15
20
53
EGFR Exon 12
0
3
EGFR Exon 18
5
44
EGFR Exon 19
>87
45
EGFR Exon 20
1
21
EGFR Exon 21
1
34
KRAS Exon 2
8
69
KRAS Exon 3
5
29
KRAS Exon 4A
0
4
KRAS Exon 4B
0
6
NRAS Exon 2
9
30
NRAS Exon 3
0
28
NRAS Exon 4A
0
0*
NRAS Exon 4B
0
1
PIK3CA Exon 9
0
41
PIK3CA Exon 20
0
51
Genes/Exons
Available
11
Genotyping Platforms
ICP increases sensitivity on all platforms
Platform
Sensitivity
Sanger
ICP + Sanger
Pyrosequencing
ICP +
Pyrosequencing
NGS
ICP + NGS
SNaPshot
BEAMing
ddPCR
10 -20%
≥0.01%
5%
Mutation
Quantitative
type
ALL
Yes
Mut, Indel
Yes
Site
Yes
≥0.05%
Site
Yes
3-5%
≥0.01%
5%
≥0.01%
≥0.01%
ALL
Mut, Indel
Site
Hotspot
Hotspot
Yes
Yes
Yes
Yes
Yes
Fold
Increase
>1000
>100
>500
12
Validation Data Generated using
ICE COLD-PCR
Biopsy
Learn more
Ultra-sensitive mutation detection in circulating free DNA (cfDNA)
Transgenomic®
Advancing Personalized Medicine
13
Initial Mutation (%)
1%
0.10%
0.01%
0.001%
100
10
1
0
45
5
0
200
100
10
1
400
152
600
303
455
800
152
1,000
30
3
0
1,200
500
330
150
100
50
33
10
15
2
0
1,000
1,400
15
2
Mutations in
Starting Material
1,600
DNA (ng)
# Genomic
Equivalents
151,515
100,000
45,455
30,303
15,152
10,000
3,030
30
3
0
0
1,515
Number of Genomic Copies and Mutations
Present in Starting Material
0
500 ng
330 ng
150 ng
100 ng
50 ng
33 ng
10 ng
Amount of Starting DNA (ng)
33 ng: Theoretical Limit of Detection for 0.01%
150 ng: Robust detection of 0.01% mutation using MX-ICP
330 ng: Robust detection of 0.01% mutation using ddPCR
14
MX-ICP plus Sanger Enhances Mutation Detection
of EGFR Exon 19 Deletions
Input
Mut
Output
Input
Mut
WT
1%
del
100%
del
0.1%
del
WT
0.1%
del
95%
del
0.01%
del
WT
0.01%
del
60%
del
WT
WT
WT
1%
del
Standard PCR, M13-Based
Sequencing
ICE COLD-PCR w/Sanger
Output
WT
15
MX ICP Enhances Mutation Detection by NGS:
Resulting in Increased Sensitivity of NGS
EGFR is one of the most common mutations present in up to 15-20% of all lung cancer patients*
% MT (sensitivity) refers to the concentration of mutant DNA in the total DNA sample
Next Gen Without MX-ICP
% MT
% MT
0%
0%
20%
5%
1%
0.5%
0.1%
0.05%
0.01%
0.005%
Next Gen With MX-ICP
✔
✔
No del
No del
No del
No del
No del
No del
20%
✔
✔
✔
✔
✔
✔
✔
5%
1%
0.5%
0.1%
0.05%
0.01%
0.005%
No del
EGFR * Present is 14-20% of lung cancer in Caucasians & 40-50% in Asian: Journal of Clinical Oncology
David a Eberhard et al Vol 23. Number 25 Sept 2015
16
Droplet Digital PCR and MX-ICP of EGFR T790M:
Bio-Rad QX200
150 ng of starting DNA containing 0.1 or 0.01% EGFR Exon 20 T790M Mutation
Enriched using MX-ICP prior to ddPCR
Amount of
Mutation in
Starting
Sample
Dilution of Starting MX-ICP product Necessary
1 / 100,000
1 / 1 Million
1/ 10 Million
AS = Droplets
containing
Mutations
0.1%
T790M
22% Mutation
25% Mutation
22% Mutation
WT = Wild-Type
Droplets
WT + AS =
Dual Occupancy
Droplets
0.01%
T790M
3% Mutation
5% Mutation
7% Mutation
Less DNA Needed = Less Plasma = Less Blood
17
Monitoring Mutations in cfDNA from a Patient:
Example of Effective and Improved Treatment
Detects actionable Mutations in DNA from plasma of patients prior to tumor growth
Supports determination of when to start treatment and monitoring of response
BRAF-Targeted
Therapy
1
0.8
0.6
V600E
decreasing
in cfDNA
0.4
0.2
1.4
1.2
1
0.8
0.6
0.4
0.2
0
9/5/13
8/5/13
7/5/13
6/5/13
5/5/13
4/5/13
3/5/13
2/5/13
1/5/13
12/5/12
11/5/12
9/5/12
10/5/12
0
9/1/13
8/1/13
7/1/13
6/1/13
5/1/13
4/1/13
3/1/13
2/1/13
1/1/13
12/1/12
11/1/12
10/1/12
Tumor
burden
decreasing
9/1/12
Tumor Burden %
cfDNA BRAF V600E %
No Therapy
18
Improvement of Mutation Detection using MX-ICP:
One Patient; Multiple Mutations, Pre- and Post- Treatment
Monitoring
100
90
90
Amount of Mutation Detected (%)
90
80
70
SANGER without ICP (No Treatment)
SANGER with ICP (No Treatment)
SANGER with ICP (BRAF-Inhibitor)
60
50
50
40
40
35
30
25
23.7
20
10
0
10.8
10
00
0
Wild-Type
Sequence
V600
Q61
G12
E542
19
Patient Monitoring using ICP – Plasma from Stage IV CRC
(Tumor Analysis not Performed at Transgenomic)
Tumor
Status/FFPE
G12C
G12D
G12D
NVD
Without ICE
COLD-PCR
With
ICE COLD-PCR
G12C
G12C
NVD
G12D
NVD
NVD
G12D
NVD
Input DNA (ng);
Gen Equiv (G.E.);
Calc Starting
Mut % from ICP
Outcome
Amt: 102 ng
G.E. 31,400
Mut % calc: 9.5%
Stable
Disease
Enriches for ANY
& ALL Mutations
from Low
Amounts of
Starting DNA
Amt: 67 ng
G.E.: 20,800
Mut % calc: 0.8%
Stable
Disease
Detects Mutations
from Liquid
Biopsy Samples
Amt: 18 ng
G.E.: 5,700
Mut % calc: 0.3%
Partial
Response
Amt: 22 ng
G.E.: 6,800
Mut % calc: 0%
Partial
Response
Helps Predict
Therapeutic
Options
Improves Clinical
Outcomes
20
MX-ICP Enables Quantification of Amount
of Mutation in Starting Sample:
LoD for EGFR S492R (3 reps)
10
Average LOD
Amount of Alteration
Prior to MX-ICP
ICP (%)
y=
0.0199e0.0557x
Digitally verified Sample
4.1% before MX-ICP
90% after MX-ICP
R² = 0.986
1
Sample Dilution (Rep 4)
0.04 before MX-ICP
15% after MX-ICP
CRC: Sample
?? before MX-ICP
40% after MX-ICP
Calculated 0.2% Before MX-ICP
0.1
0.01
0
20
40
60
80
100
CRC: Sample
?? before MX-ICP
10% after MX-ICP
Calculated 0.03% Before MX-ICP
Amount of Alteration After MX-ICP (%)
21
New MX-ICP Assay for C797S Mutation in EGFR
Exon 20 – no additional MX-PCR
EGFR Exon 20 C797S – G to C
20%→80%
5→65%
1→50%
0.5→35%
0.25→20%
0.1→10%
0.5→20%
0.25→10%
0.1→5%
EGFR Exon 20 C797S – T to A
20%→95%
5→55%
1→35%
1 MX-PCR including EGFR Exon 20
ICP assays for T790M and C797S
1 RS-oligo for T790M Region
1 RS-oligo for C797S Region
2 different mutations for C797S
22
Questions/Challenges
What levels of mutations are relevant for the patient?
In Tissues - tumor heterogeneity?
In FFPE what does it mean for the Pathologist/Oncologist/Patient?
In Liquid Biopsies – amounts of sample needed?
Clinical Trials have limited amounts
IRBs allow only so much blood drawn from a patient
Patients not on Clinical Trials can have more blood drawn
Are these levels the same for all mutations?
Will different mutations have different cut points?
Will cut points be both mutation and patient dependent?
Are these levels the same for all cancers?
Suspect different cancers will have same mutation but different cut point
Maybe cancer and patient dependent
23
Solutions?
Inclusion of ICP in research projects will help provide answers!
Use of ICP for mutational analysis of nucleic acids isolated from
Liquid Biopsies will help provide answers!
Inclusion of ICP in Clinical Trial Protocols will help provide
answers!
For all of the above, Correlation to Patient Treatment Outcomes
will be required!
24
Summary and Products
Biopsy
Learn more
Ultra-sensitive mutation detection in circulating free DNA (cfDNA)
Transgenomic®
Advancing Personalized Medicine
25
Summary of the Advantages of MX ICP
Pharma/biotech
“Plug-and Play” into
existing workflow for
in-house use or
preferred service
provider
Detection of low level
mutations that
correlate to response
Patient Stratification:
drug sensitivity,
resistance markers
Clinical Diagnostic
Development of
companion
diagnostic
Ease of technology
transfer with
Pharma/Biotech
Partner
Real-time
monitoring of
patient condition,
improving clinical
outcome
Clinical Oncologists
Real-time monitoring
of patient condition
and to improve patient
care.
Rapid identification of
all relevant DNA
alterations using
currently validated
platforms.
Implementation of
plasma/serum
oncology assays
26
Products Released
Biomarker Services: Launched new MX-ICP
services at AACR
CLIA Testing: First Test launched at ASCO EGFR
T790M Mutation, 6 more targeted tests/panels this
year NSCLC, CRC & Melanoma
Kits Research Use Only (ICEme): multiplex and
customizable kits launched globally
Licensing: Commercial arm of University of
Melbourne, others
27
Thank You
28