Download ICE COLD-PCR - Transgenomic

The 'Perfect Pairing’:
ICE COLD-PCR with
cfDNA for Highly
Sensitive Mutation
Detection
Transgenomic develops and commercializes biomarkers and personalized
g
with the g
goal of improving
p
g medical diagnoses
g
and p
patient outcomes.
diagnostics
Personalized Oncology Solutions for the Global Community
©Transgenomic‐2013
Agenda

History of Transgenomic

Technology and History ICE COLD-PCR

Project 1 (Transgenomic)

Project 2 (MD Anderson, Dr.Filip Janku)

Conclusions

Next Phase (Transgenomic)

Future Direction and Advances

L
Long
T
Term Vision
Vi i
©Transgenomic‐2013
Transgenomic
Leverage Proprietary Technology and Molecular Genetics Expertise
to Provide a Fully Integrated Molecular Diagnostics Solution
Biomarker
Identification
Genetic Assays and
Platforms
Identifies new genetic
biomarkers for pharma sector
and develops assays for
patient testing
Provides genetic assays,
testing platforms and
equipment to labs worldwide
©Transgenomic‐2013
Patient Testing
Provides g
genetic testing
g
services
Oncology
Cardiology
Neurology
ICE COLD-PCR Methodology
Selectively Amplifies Mutant Sequences = Enriches for All Mutations
Denature DNA
Selectively Denature
Mismatched Sequences at
Th Critical
The
C iti l Temperature
T
t
(Tc)
(T )
MT
95oC
MT
WT
WT
95oC
RS
WT
WT
MT
MT
Reduce Temperature:
Cross-Hybridize
Tc
RS
MT
RS
RS
MT
Reduce Temperature for
Primer Annealing
g to Both
Strands of the
Mutant Sequences
WT
WT
RS
WT
WT
MT
RS
Reference Sequence Oligonucleotide f
li
l id
Enables Improved Hybridization Kinetics
©Transgenomic‐2013
Exclusive license from Dr. Mike Makrigiorgos, DFCI
MT
ICE COLD-PCR Selectively Amplifies
Mutant DNA “PCR
PCR on Steroids”
Steroids
Standard PCR
ICE COLD‐PCR
# PCR Prroducts
Wild‐type
Mutants
Wild‐type
Mutants
Number of PCR Cycles
Number of PCR Cycles Enriching the Mutant Population = Increasing the Sensitivity
-- For Use in ANY Downstream Analysis -©Transgenomic‐2013
Exclusive license from Mike Makrigiorgos, DFCI
ICE COLD-PCR Selectively Amplifies
Mutant DNA “PCR
PCR on Steroids”
Steroids
Standard PCR
ICE COLD‐PCR
# PCR Prroducts
Mutants
Wild‐type
Number of PCR Cycles
Number of PCR Cycles Enriching the Mutant Population = Increasing the Sensitivity
-- For Use in ANY Downstream Analysis -©Transgenomic‐2013
Exclusive license from Mike Makrigiorgos, DFCI
ICE COLD-PCR: Enhanced Mutation Detection:
Multiple Sample Types – Multiple Platforms
ICE COLD-PCR
works with DNA from
any sample type:








Tissue (Biopsy)
FFPE
Blood
cfDNA
fDNA
CTCs
FNAs
Urine
Sputum
DHPLC
HRM
COBAS
ICE
COLDPCR
Pyro-
Sanger
BEAMing
NGS
©Transgenomic‐2013
Digital
PCR
ICE COLD-PCR: Enhanced Mutation Detection:
Multiple Sample Types – Multiple Platforms
ICE COLD-PCR
works with DNA from
any sample type:








Tissue (Biopsy)
FFPE
Blood
cfDNA
fDNA
CTCs
FNAs
Urine
Sputum
DHPLC
HRM
COBAS
ICE
COLDPCR
Pyro-
Sanger
BEAMing
NGS
©Transgenomic‐2013
Digital
PCR
Enhanced Mutation Detection Assays for DNA
from FFPE
FFPE, cfDNA or Other Body Fluids
Increasing level of Mutation Detection
Sanger
BLOCker + Sanger
ICE COLD-PCR + Sanger
NGS
ICE COLD-PCR + NGS
0
Limit of Detection (Percent)
 Sanger: 15% to 0.1%: ~ 150-fold enrichment in LOD
 NGS: 2.5% to 0.01%: ~ 250- fold enrichment in LOD
©Transgenomic‐2013
ICE COLD-PCR: One Assay – Multiple Mutations
Sanger Sequence Detection of
Any Mutation in the Sample
WT
WT
0.5% G12C
0.5% G13C
0.5% G12R
0.5% G13S
0.5% G12S
0.5% G13D
0.5% G12V
100
90
80
70
60
50
40
30
20
10
0
5%
G12A

0.5% G12A
G12C
42 Allele-Specific Probes Required
OR
 1 ICE COLD-PCR Assay
©Transgenomic‐2013
1%
0.5%
0.1%
0.05%
Percent Mutant in Starting DNA
G12D
G12R
42 Point mutations in KRAS codons 12 & 13
THEREFORE:

0.5% G12D
Limit of Sanger Sequencing Detection of
KRAS Mutations Following ICE COLD-PCR
Codon Codon
12
13
(GGT) (GGC)
Percent Mutan
nt Peak
Codon Codon
12
13
(GGT) (GGC)
G12S
G12V
G13C
0.01%
G13D
G13S
ICE COLD-PCR: One Assay – Multiple Mutations
50
EGFR Deletions
47
45
Varriant Frequenc
cy
40
35
ICE COLD-PCR + NGS:
1% Deletion Sample
30
ICE COLD-PCR + NGS:
5% Deletion Sample
27
25
29
24
20
20
15
14
10
5
41
NGS Only:
1% and 5% Deletion Sample
15
13
10
9
6
4
7
6
8
7
4
4
0
 Deletions are difficult to detect on NGS platforms
 Over 60 Relevant EGFR Exon 19 Deletions in NSCLC Patient Tumors
THEREFORE:
 60 Allele-specific probes required
©Transgenomic‐2013
OR
 1 ICE COLD-PCR Assay for of all Exon 19 Deletions
11
13
6
ICE COLD-PCR Detects Somatic Mutations in
cfDNA from Plasma (Merck: 53 NSCLC Patients)
Patient A
Codon
12
Patient B
Codon
13
Standard
PCR
Codon
12
Codon
13
Standard
PCR
ICECOLD
ICECOLD
~10%
~95%
G12C (GGT>TGT)
Historical Published Results:
KRAS mutation data for DNA from
FFPE sections
• Standard PCR:~25-30% mutant
0%
~85%
G12C (GGT>GAT)
Study Results 53 NSCLC Patients:
KRAS mutation data from plasma cfDNA
• Standard PCR: 11% (6/53)
• ICE COLD-PCR: 25% (13/53)
ICE COLD-PCR detects low level mutations in cfDNA (“Liquid Biopsy”)
©Transgenomic‐2013
ICE COLD-PCR Detection of Mutations in cfDNA:
Impact on Cancer Management
Tumorr Growth
Progressive Disease
No
Monitoring
No Monitoring
ICE COLD‐PCR Analysis Tumor
D t t d
Detected
Stable Disease
Monitoring
Surveillance
Time
Treatment Started
©Transgenomic‐2013
Overall Project Goals
 Prove ICE COLD-PCR as a relevant tool to assist in
improving patient outcome
 Concordance studies between mutations detected in
t
tumor
and
d iin cfDNA
fDNA
 Partnering with world-class oncology organizations for
clinical expertise
 All “Li
“Liquid
id Bi
Biopsies”
i ”
 First target cfDNA from plasma
©Transgenomic‐2013
ICE COLD-PCR Research Collaborations
RESEARCH COLLABORATIONS ARE ONGOING IN VARIOUS TYPES OF CANCER
AT THE MOST PRESTIGIOUS INSTITUTIONS WORLDWIDE
PROVIDE BLOOD/PLASMA SAMPLES FOR PROOF THAT ICE COLD-PCR
CAN BE A RELEVANT TOOL TO ASSIST IN IMPROVING PATIENT OUTCOME
THIS VALIDATION PROJECT PROVIDES THE DATA TO SHOW THE CLINICAL IMPORTANCE AND
CORRELATION FOR BLOOD SAMPLES COMPARED TO TISSUE SAMPLES AS A PROOF OF CONCEPT FOR THE
MUTATION DETECTION IN “LIQUID BIOPSIES” ©Transgenomic‐2013
Project 1: Application of ICE-COLD-PCR to the
Detection of Rare NRAS Mutations in
Myelodisplastic Syndrome (MDS) (Mike Makrigiorgos, DFCI)
International Prognostic Scoring System (IPSS): basis for treatment guidelines
Patients in same IPSS risk group have varied clinical outcomes
 Additional p
prognostic
g
indicators needed
 Previous studies suggested NRAS mutations could be such an indicator
Current Study:
295 Patients in the low/intermediate IPSS risk group
Sanger Sequence analysis of
Conventional Methods (LOD >5%)
Fast-COLD-PCR (LOD 2-5% or 0
5-2%)
0.5-2%)
ICE COLD-PCR (LOD 0.1-0.5%)
What level of mutations is clinically significant?
Murphy et.al. 2013. Leukemia 27:2067‐2111
DANA‐FARBER/BRIGHAM AND WOMEN’S
©Transgenomic‐2013
C A N C E R C E N T E R
ICE COLD-PCR Analysis of NRAS Mutations:
Impact on Overall Survival Lower IPSS Risk
MDS(Mike Makrigiorgos, DFCI)
Overall Survival in Lower IPSS Risk MDS
Overall Surviival (%)
O
NRAS Mutant >5% MAF (n=8) –----conventio
Conventional Methods
fast COLD
----fast-COLD
NRAS Mutant 2-5% MAF (n=3) – fast COLD-PCR
----fast-COL
NRAS Mutant 0.5-2% MAF (n=7) – fast COLD-PCR
NRAS Mutant 0.1-0.5% MAF (n=6) – ---ICE-CO
ICE COLD-PCR
NRAS Wild-Type (n=271)
0.1‐0.5%
00.1
1-00.5%
5%
> 2%
>2%
0.5-2%
0.5‐2%
Years
NO MUTAT
No Mutation
“………Multivariable analysis that
considered IPSS risk group, sex, age
and the mutation status of 16 other
recurrently mutated genes
demonstrates that the prognostic
significance
s
g ca ce o
of NRAS
S mutations
utat o s
detected by fast-COLD-PCR is
independent of other known risk
factors ……… As the technical limit
of sensitivity should preferably be
lower than the clinically relevant
threshold detection of NRAS with
threshold,
ICE COLD-PCR followed by
determination of whether the
mutation is clinically relevant (higher
or lower than 0.5%) would be
appropriate.”
Murphy et.al. 2013. Leukemia
27:2067-2111
Ability to differentiate mutation load enables patient treatment decisions
which in turn means better patient outcome.
DANA‐FARBER/BRIGHAM AND WOMEN’S
©Transgenomic‐2013
C A N C E R C E N T E R
Project 1 Conclusions
ICE COLD-PCR Analysis:
 Helps determine subgroups of MDS patients with differing
responses.
 Enables patient stratification for treatment options.
 Provides critical information from tissues of MDS patients
for clinical treatment plans
plans.
DANA‐FARBER/BRIGHAM AND WOMEN’S
©Transgenomic‐2013
C A N C E R C E N T E R
MD ANDERSON: FAST FACTS
FY 2014
Total patients served (2012)
~115,000
New patients served (2012)
~38 000
~38,000
Admissions
Total visits
27,905
1,338,706
Department of Investigational Cancer Therapeutics: Mission
Therapeutics: Mission
To translate laboratory discoveries and clinical
observations into hypothesis-driven clinical
trials leading to targeted, tailored and
personalized cancer treatments
Liquid Biopsies
 Cell‐free (cf) DNA  Plasma
 Serum
 Other liquid material
 Circulating tumor cells (CTC)
 Exosomes
Liquid Biopsies: Sensitivity Requirements
 Human cell contains 6pg of DNA
 “Average” advanced cancer has about 15‐20ng of DNA per ml of plasma
 “Average” advanced cancer has less than 10 CTCs per 7.5 ml of blood
 Plasma has less DNA than serum; however, a lot of DNA in serum samples is from leukocytes  Because only proportion of cfDNA originates from the tumor sensitivity requirements are from 1 mutant in allele in 1,000 to 10,000 of wild‐type
MD Anderson and Transgenomic Project:
ICE COLD‐PCR and plasma cfDNA testing for mutations ICE COLD‐PCR and plasma cfDNA
testing for mutations
in KRAS and BRAF •
Objectives (75 patients with advanced cancers and known tumor tissue BRAF
Objectives
(75 patients with advanced cancers and known tumor tissue BRAF or or
KRAS mutation status)
– To measure the degree of concordance between results of ICE COLD‐PCR mutation analysis of BRAF and KRAS mutations from cfDNA and tumor tissue (obtained and tested as standard of care) in patients with advanced cancers.
– To assess BRAF and KRAS mutations in cfDNA at multiple time‐points, at baseline before treatment, then up to monthly, and at the time of disease progression in patients with advanced cancers treated with anticancer therapy.
d
d
t t d ith ti
th
– To assess treatment outcomes in analyzed patients with advanced cancers
– Assay Protocol:
•
•
•
•
Receipt of 3 – 4 mL frozen plasma
Isolation of cfDNA
Perform ICE COLD‐PCR Assays for KRAS and BRAF
Sanger Sequence Analysis
Concordance Analysis: cfDNA
y
vs. tissue
BRAF
KRAS
AACR‐EORTC‐NCI 2013
Longitudinal Assessment of cfDNA
g
BRAF Mutations AACR‐EORTC‐NCI 2013
Longitudinal Assessment of cfDNA
g
KRAS Mutations AACR‐EORTC‐NCI 2013
Q
Quantification of Mutation in cfDNA
74 year old female with
appendiceal
di
l carcinoma
i
and BRAF V600E mutation
0 20
0.20
0.15
0.10
11/1/2…
10/1/2…
9/1/2013
8/1/2013
7/1/2013
6/1/2013
5/1/2013
4/1/2013
3/1/2013
2/1/2013
1/1/2013
0.05
Amount Mutant in cfDNA (%)
Amount Mutant in cfDNA (%)
ICE COLD-PCR Mutation Frequency (%)
ICE COLD-PCR Mutation Frequency (%)
0.00
Relative Amountt Mutant
R
in cfDNA (%
%)
0.25
12/1/2…
9/5/2013
8/5/2013
7/5/2013
6/5/2013
5/5/2013
4/5/2013
3/5/2013
2/5/2013
0
1/5/2013
12/5/20…
11/5/20…
10/5/20…
1
Progressive
Disease
0.30
11/1/2…
2
BRAFi
based
therapy
0.35
10/1/2…
3
90
80
70
60
50
40
30
20
10
0
9/1/2012
4
IC
CE COLD-PCR Mutation
Frequency (%
%)
5
Relative A
Amount
Mutant in cffDNA (%)
6
9/5/2012
ICE COLD-PCR Mutation
Frequency
y (%)
BRAFi
Progressive
based
Disease
therapy
BRAFi
based
therapy
Progressive
Disease
90
80
70
60
50
40
30
20
10
0
46 year old female with
melanoma
l
and
d
BRAF V600E mutation
Project 2 CONCLUSIONS
 Liquid biopsies have a potential to further advance personalized medicine by offering a source of easily obtainable material for mutation analysis
 Liquid biopsies can help to determine prognosis, detect early recurrence and disease progression  Liquid biopsies can be used for monitoring of molecular profile in patients undergoing cancer therapy to detect specific mechanisms of resistance
 N
Novel multiplex technologies capable of testing for multiple mutations need to be l
lti l t h l i
bl f t ti f
lti l
t ti
dt b
developed
 Barriers to implementation of next‐generation sequencing need to be overcome Barriers to implementation of next‐generation sequencing need to be overcome
 Novel sources of DNA (urine, exosomes, CTCs) need to be investigated
Overall Survival (%)
O
Overall Survival in Lower IPSS Risk MDS
----conventio
NRAS Mutant >5% MAF
(n=8) – Conventional Methods
----fast-COLD
NRAS Mutant 2-5% MAF
(n=3) – Fast COLD-PCR
----fast-COL
NRAS Mutant 0.5-2% MAF---ICE-CO
(n=7) – Fast COLD-PCR
NRAS Mutant
M t t 0.1-0.5%
0 1 0 5% MAF ((n=6)
6) – ICE COLD-PCR
COLD PCR
NRAS Wild-Type (n=271)
01 1‐0
0.1
5%
0.1-0.5%
0
0 0.5%
5%
> 2%
>2%
NO MUTAT
No Mutation
0.5-2%
0.5‐2%
Y
Years
Patient
#
Tissue
Surrogate
Response
Monitoring
Monitoring
&
Surveillance
5
6
2
3
4
1
7
8
©Transgenomic‐2013
Disease
Erdheim‐Chester
Papillary Thyroid Cancer
Melanoma
Melanoma
Melanoma
Melanoma
Appendiceal
Carcinoma
Colorectal Carcinoma
Baseline cfDNA
Mutations
Follow up cfDNA
Mutation
Treatment Response
BRAF V600E
Treatment prior to baseline cfDNA
No
BRAF V600E
Wild‐Type
SD
BRAF V600E
No
BRAF V600E
Wild‐Type
SD
BRAF V600E
BRAF V600E
BRAF V600E
BRAF V600E
No
No
No
Yes
BRAF V600E Wild‐Type
BRAF V600E Wild‐Type
BRAF V600E Wild‐Type
Wild‐Type BRAF V600E
KRAS G13D
Yes
Wild‐Type
KRAS G13D
PD
KRAS G12D
Y
Yes
Wild T
Wild‐Type
KRAS G12D
PD
Tissue Mutation
DANA‐FARBER/BRIGHAM AND WOMEN’S
C A N C E R C E N T E R
SD
SD
SD
PD
Overall Survival (%)
O
Overall Survival in Lower IPSS Risk MDS
----conventio
NRAS Mutant >5% MAF
(n=8) – Conventional Methods
----fast-COLD
NRAS Mutant 2-5% MAF
(n=3) – Fast COLD-PCR
----fast-COL
NRAS Mutant 0.5-2% MAF---ICE-CO
(n=7) – Fast COLD-PCR
NRAS Mutant
M t t 0.1-0.5%
0 1 0 5% MAF ((n=6)
6) – ICE COLD-PCR
COLD PCR
NRAS Wild-Type (n=271)
Projects 1 and 2 Summary

ICE COLD-PCR can be used for the analysis of mutations in any tissue.

ICE COLD-PCR is especially
p
y suited0.1-0.5%
analysis
y
of mutations in any
y
01for
1‐0
5%
00.1
0 0.5%
5%
“Liquid Biopsy” sample:

MUTAT
No Mutation
> 2% by
As a Tissue “Surrogate”
offering aNO
source
of easily obtainable material
0.5-2%
0.5‐2%
>2%
for mutation analysis and treatment determination.

As a Surveillance Method by helping
to determine prognosis,
Treatment Baseline Follow up prior to Patient recurrence andTissue Treatment detect early
disease
progression.
cfDNA
cfDNA
Disease

Y
Years
#
Mutation
Response
baseline Mutations
Mutation
cfDNA the molecular profile of mutations
As a Monitoring Method for assessing
5
Erdheim‐Chester
BRAF V600E
BRAF V600E Wild‐Type
SD
in patients
undergoing
cancer
therapyNoto detect:
Tissue
Papillary Thyroid Surrogate
6
BRAF V600E
No
BRAF V600E Wild‐Type
SD

Specific Cancer
mechanisms of resistance
2
Melanoma
BRAF V600E
No
BRAF V600E Wild‐Type
SD

Occurrences
of
new
mutations
Response
3
Melanoma
BRAF V600E
No
BRAF V600E Wild‐Type
SD
Monitoring
4
Melanoma
BRAF V600E
No
BRAF V600E Wild‐Type
SD
1
Melanoma
BRAF V600E
Yes
Wild‐Type BRAF V600E
PD
Appendiceal
Monitoring
7
KRAS G13D
Yes
Wild‐Type
KRAS G13D
PD
Carcinoma
&
Surveillance
Colorectal 8
KRAS G12D
Y
Yes
Wild T
Wild‐Type
KRAS G12D
PD
Carcinoma
©Transgenomic‐2013
DANA‐FARBER/BRIGHAM AND WOMEN’S
C A N C E R C E N T E R
ICE COLD-PCR
COLD PCR Paired with NGS:
Future Directions
©Transgenomic‐2013
ICE COLD-PCR: Impact on Cancer Management
Tumorr Growth
Progressive Disease
No
Monitoring
No Monitoring
ICE COLD‐PCR Analysis Tumor
D t t d
Detected
Stable Disease
Monitoring
Surveillance
Time
Treatment Started
©Transgenomic‐2013
Phase
II Goal
ICE COLD-PCR:
Impact on Cancer Management
Tumorr Growth
A novel multiplex technology capable
Progressive No
Monitoringdetecting
No Monitoring
of the simultaneously
Disease
ICE COLD‐PCR Multiple Mutations in
Analysis Multiple Genes for
Multiple Samples
Tumor
Stable D t t d
Detected
Disease
att th
the level
l
l off sensitivity
iti it
Monitoring
needed for use in a
Surveillance
“Liquid
Time Biopsy”
Treatment Started
©Transgenomic‐2013
Rapid & Focused Development Plan:
Multiplexed ICE COLD-PCR
COLD PCR Paired with NGS
Development  Multiplex ICE COLD-PCR
assays for lung
cancer, CRC, melanoma,
etc.
 Analyze using Ion Torrent
or MiSeq (FDA approved)
 Partnering with MD
Anderson and others for
clinical validation studies
 Partnering with Pharma for
clinical trials to companion
diagnostics
Performance Target
 Focus on cfDNA from
lung cancer, CRC and
melanoma
 Increased sensitivity over
current NGS detection
 Compatible with any
current NGS instrument
Benefits
 Better diagnosis &
personalized drug therapy
 Faster diagnosis
turnaround
 Achieve optimal therapeutic
benefits
 No need for tissue biopsies
 C
Correlation
l ti with
ith patient
ti t
treatment decisions
 Continual patient
monitoring
 Correlate with patient
outcome (biopsies)
 Personalized Cancer
Management
g
“LIQUID BIOPSY” Non‐invasive, Rapid and Easy Detection of Low Level Mutations ©Transgenomic‐2013
Technology Comparison Summary
Mutant Mutants known Detected
Mutants Mutants
# Assays for Detected Detected in 20 Detecting 20 Assay/Probe Exons
Mutations
# Probes to Combination detect 20 with Mutations
Sequencing Average LOD of Assay Sanger
No ALL Unlimited N/A
1
0
N/A
15%
NGS
No ALL ++++
++++
1
0
N/A
4‐5%
Surveyor
No ALL Unlimited N/A
1
0
Yes 2%
ICP (NGS) (
) No ALL +++ ++++
1
0
Yes 0.01%
ALL +++ +++++ 1
0
Yes 0.01%
Probe Specific
1
20
20
20
No
0.01%
Probe Specific
1
20
20
20
No
0.01%
MX ICP No (NGS) Emulsion
Yes
PCR
C + Flow Cytometry
Digital PCR
PCR Yes
 Multiplexed ICE COLD-PCR:
 One Assay; All Mutations with LODs ~0.01%
 Supercharges Any NGS or Other Downstream Analysis
©Transgenomic‐2013
Multiple Samples, Multiple Amplicons – 1 NGS Analysis for
Standard PCR or ICE COLD-PCR of cfDNA from Plasma
90
83
ICP + NGS
77
70
60
cfDNA Sample Number;
Gene Region; Mutation
56
50
48
40
35
30
35
26
12
12
12
7
10
Sample 1
Sample 9
Sample 8
Sample 7
Sample 7
Sample 6
Sample 5
Sample 5
Sample 4
Sample 3
Sample 2
Sample 1
8
0
6
6
12
Sample 1
10
11
Sample 1
20
Sample 1
LOD (Va
ariant Frequ
uency %)
80
NGS ONLY
87
 Analysis of 3 Genes/5 Exons/12 Plasma Samples
 NGS: only 3 Mutations detected in 2/12 Samples
 ICE COLD-PCR paired with NGS: 15 Mutations Detected in 12 Samples
©Transgenomic‐2013
MX ICE COLD-PCR + NGS: Ideally Suited for Mutation
Detection in both Oncogenes and Tumor Suppressor Genes
(COSMIC Database)
(COSMIC Database)
Oncogenes:
Few Mutations
U
Usually
ll L
Localized
li d Sit
Sites
©Transgenomic‐2013
Ins/Del Com
mplex
Amino
Acid
Ins/Del Com
mplex
Amino
Acid
Su
ubstitutions
TP53
Su
ubstitutions
KRAS
Tumor Suppressor Genes:
Large Numbers of Mutations
Diffuse sites
MX ICE COLD-PCR + NGS: Ideally for Mutation
Detection in Oncogenes
g
and Tumor Suppressor
pp
Genes
60
Oncogenes (eg. KRAS)
 Few Mutations
 Usually Localized Sites
 High Percentage of Mutations
in Limited Set of Cancers
Percent Mutants
50
40
30
Tumor Suppressor
T
S
Genes
G
(eg.
(
TP53)
 Large Numbers of Mutations
 Diffuse Sites
 High Percentage of Mutations
in Many
y Cancers
20
10
0
Tumor Tissue
 ICE COLD-PCR Assays require no further development as new relevant
mutations are validated
 All mutations detected, broad applicability for oncogenes and tumor
suppressor genes
©Transgenomic‐2013
Custom TP53 NGS Assay: Complete Exon Coverage
139
Exons 2 3
50
4
100
150
5
200
6
250
7
8
300
9
350
10
0
Commercial
NGS Panel
Transgenomic
Custom TP53
NGS Panel
Point
Mutations
Amino Acids
11
50
100
150
200
250
300
350
Complex
Mutations
2
Insertions/
Deletions
Amino Acids
Confirmed Somatic Mutations of the TP53 gene (COSMIC
( gene Database)
Confirmed
Somatic Mutations of the TP53
(COSMIC database)
Confirmed Somatic Mutations of the TP53 gene (COSMIC database)
50
100
150
200
250
300
350
 One NGS Assay : All TP53 Mutations; LOD 3% (currently)
 With Addition of ICE COLD-PCR; Projected LOD 0.01%
©Transgenomic‐2013
ICE COLD-PCR Assay for TP53 Exon 5
ICE COLD-PCR paired with Sanger
Cell Line K562
WT for TP53 at R175
Cell Line K562
+ 1% R175H
Wild-Type
c. CGC>CAC; p.R175H
ICE COLD-PCR Paired with NGS:
Increased Sensitivity for
Detection of Low Level
Mutations in TP53
ICE COLD-PCR
COLD PCR paired
i d with
ith NGS
Chrom
chr17
h
Gene Target ID
Sym
CHP2_TP53
CHP2 TP53
7578406 TP53
_4
Position
©Transgenomic‐2013
Type Zyg
Ref
Var
SNP
C
T
Het
Var
Freq
8.72
P‐value
1.00E
1.00E‐
10
Coverage Ref Cov
34708
Var
Cov
31537 3026
Utility of MX ICE COLD-PCR Paired with NGS:

There are more than 15 tumor types that have druggable mutation
targets amenable to MX ICE COLD-PCR.
COLD PCR.

There are more than 250 early stage clinical trials examining
druggable mutation targets amenable to MX ICE COLD-PCR.

Collection of sera/plasma is becoming commonplace in clinical trial
protocols. These “Liquid Biopsy” samples will be used for:


Mutation characterization of clinically actionable and resistance mutations

Identification of mutations that may become important as future biomarkers

Longitudinal assessment of treatment regimes

Surveillance of disease progressions
p g
Clinical results would validate the utility of MX ICE COLD-PCR in
Cancer Management for patients.
©Transgenomic‐2013
MX ICE COLD-PCR: Clinical Product Concepts
p
Product Type
Liquid Biopsy Test **
Screening Test
Targeted Drug Test
Monitoring Test
Surveillance Test
Description
Targeted to cancers and patients where tissue samples are
unavailable or can’t be taken easily or efficiently
Multiple amplicons from different genes and different cancer indications
Multiple amplicons from different genes and targeted towards specific
cancer drug
Replica of above tests but looking at monitoring treatment and or
progression
Replica of above tests but looking at on-going level of mutations over long
periods at even lower levels
** First target test, which will drive adoption and will be accepted rapidly by Oncologists/Pathologist ©Transgenomic‐2013
Clinical Applications
 Detection of oncogenic mutations and molecular targets in plasma cfDNA when tissue is in short supply (approximately 10‐20% of patients with metastatic cancer)
 Monitoring of pertinent oncogene mutant allele fraction in serial Monitoring of pertinent oncogene mutant allele fraction in serial
plasma cfDNA samples as a surrogate marker for response to anticancer therapy
 Detection of emerging mutations in serial plasma cfDNA samples leading to secondary resistance to anticancer therapy  Detection of low frequency mutations in the tumor tissue to determine possible mechanisms of future resistance
determine possible mechanisms of future resistance
ICE COLD-PCR Conclusions
Compelling
C
lli d
data
t
 cfDNA as a sample source for ICE COLD-PCR
 Platform independent
 Mutation independent
 LOD of ICE COLD-PCR paired with NGS ~0.01%
Development of MX ICE COLD-PCR
COLD PCR allows sensitive mutation analysis for :
 Multiple Mutations
 Multiple Genes
 Multiple
p Samples
p
Ongoing clinical validations
 EGFR, KRAS, BRAF, NRAS, TP53…
The 'Perfect Pairing’: ICE COLD-PCR with cfDNA
for Highly Sensitive Mutation Detection
©Transgenomic‐2013
Thank You
ICE COLD‐PCR: A Valid Assay for Patient Monitoring and Surveillance
Phase I: Utility as a tumor tissue surrogate 1 “Liquid Biopsy” Surrogate for Cancers without Available Tissue 2 “Monitoring” for Treatment Response
o to g o eat e t espo se
3
“Monitoring” & “Surveillance” to assist in patient treatment plan
©Transgenomic‐2013
ICE COLD-PCR: One Assay – Multiple Mutations
50
EGFR Deletions
47
45
Varriant Frequenc
cy
40
35
ICE COLD-PCR + NGS:
1% Deletion Sample
30
ICE COLD-PCR + NGS:
5% Deletion Sample
27
25
29
24
20
20
15
14
10
5
41
NGS Only:
1% and 5% Deletion Sample
15
13
10
9
6
4
0
©Transgenomic‐2013
7
6
8
7
4
4
11
13
6
Multiple Samples, Multiple Amplicons – 1 NGS Analysis for
Standard PCR or ICE COLD-PCR of cfDNA from Plasma
90
83
ICP + NGS
77
70
60
cfDNA Sample Number;
Gene Region; Mutation
56
50
48
40
35
30
35
26
12
12
12
7
10
Sample 1
Sample 9
Sample 8
Sample 7
Sample 7
Sample 6
Sample 5
Sample 5
Sample 4
Sample 3
Sample 2
Sample 1
8
0
6
6
12
Sample 1
10
11
Sample 1
20
Sample 1
LOD (Va
ariant Frequ
uency %)
80
NGS ONLY
87
 Analysis of 3 Genes/5 Exons/12 Plasma Samples
 NGS: only 3 Mutations detected in 2/12 Samples
 ICE COLD-PCR paired with NGS: 15 Mutations Detected in 12 Samples
©Transgenomic‐2013
ICE COLD-PCR
T
A
5’
5’
C
G
5’
STEP 1:
Denature
5’
C
5’
STEP 2 :
Hybridize
PO4
5’
T
ss
5’ ss
5’
A
C
G
5’ ss
5’
T
ss
5’
C
G
C
A
C
ss
5’
ss
5’
C
5’
C
5’
XXXXXX
G
C
5’
5’
5’
PO4
A
C
5’
5’
PO4
5’
Extension
5’
T
A
5’
Amp
p Primer 2
C
PO4
RS- oligo
©Transgenomic‐2013
5’
5’
5’
5’
PO4
MUT
5’
C
5’
Linearr Amp Exp. Amp
C
G
XXXXXX
C
STEP 5 :
T
A
C
G
ss
ss
G
C
5’
PO4
A
T
5’
5’
T
C
5’
C
5’
Amp Primer 1
ds
PO4
5’
5’
KEY
5’
PO4
STEP 3: Differentially
Denature at Tc
Final
Extension
5’
ds
PO4
Denature
Mutation Confirmation
 Sanger
 NGS
 Any other platform
5’
PO4
ds
5’ ss
5
C
PO4
Mutant strands
available
for amplification
STEP 4 :
Anneal
Amplification Primers
PO4
WT
Exclusive license from Dr. Mike Makrigiorgos, DFCI
Longitudinal Assessment of cfDNA
g
BRAF Mutations AACR‐EORTC‐NCI 2013
Longitudinal Assessment of cfDNA
g
KRAS Mutations AACR‐EORTC‐NCI 2013
Q
Quantification of Mutation in cfDNA
74 year old female with
appendiceal
di
l carcinoma
i
and BRAF V600E mutation
0 20
0.20
0.15
0.10
11/1/2…
10/1/2…
9/1/2013
8/1/2013
7/1/2013
6/1/2013
5/1/2013
4/1/2013
3/1/2013
2/1/2013
1/1/2013
0.05
Amount Mutant in cfDNA (%)
Amount Mutant in cfDNA (%)
ICE COLD-PCR Mutation Frequency (%)
ICE COLD-PCR Mutation Frequency (%)
0.00
Relative Amountt Mutant
R
in cfDNA (%
%)
0.25
12/1/2…
9/5/2013
8/5/2013
7/5/2013
6/5/2013
5/5/2013
4/5/2013
3/5/2013
2/5/2013
0
1/5/2013
12/5/20…
11/5/20…
10/5/20…
1
Progressive
Disease
0.30
11/1/2…
2
BRAFi
based
therapy
0.35
10/1/2…
3
90
80
70
60
50
40
30
20
10
0
9/1/2012
4
IC
CE COLD-PCR Mutation
Frequency (%
%)
5
Relative A
Amount
Mutant in cffDNA (%)
6
9/5/2012
ICE COLD-PCR Mutation
Frequency
y (%)
BRAFi
Progressive
based
Disease
therapy
BRAFi
based
therapy
Progressive
Disease
90
80
70
60
50
40
30
20
10
0
46 year old female with
melanoma
l
and
d
BRAF V600E mutation