Download Very High Sensitivity Somatic Mutation Detection

Very High Sensitivity Somatic Mutation Detection using ICE COLD-PCR
and BLOCker Sequencing
G. Wu, B. Legendre, R. Lin, Y. Shi, K. Echtenkamp, S. Jansen, S. Peterson, E. Comtois, J. Sommer, P. Eastlake and K. Richardson
Transgenomic, Inc., 12325 Emmet Street, Omaha, NE.
ICHG2011
Poster # 930F
®
the power of discovery
ABSTRACT
RESULTS
INTRODUCTION
ICE COLD-PCR
Limit of detection of KRAS exon 2 ICE COLD-PCR: Dilution
series using KRAS wild-type and mutant plasmids
1/20
1/100
1/200
After regular PCR
1/10,000
49:G12A
24:G12V
24:G12V
G12D
35:G12V
15:G12C
15:G12C
23:G12C
23:G12C
29:G12C
EGFR exon 19 deletion detected by ICE COLD-PCR: Mock
CTCs (PC9Cells) were spiked into blood and isolated by the
ScreenCell filtration device
Control: E746_A750:c.22 35-2249del15 (1 mutant in 1000 wild-type DNAs)
ScreenCell
Filtration Device
ICE COLD-PCR with RS-oligo
Mock CTC prep (10 PC9 cells): E746_A750 c.2235-2249del15
ICE COLD-PCR with RS-oligo
BLOCker Sequencing
RD10-498 (Prostate): G12D
50:50 mixture of WT: G12R (c.G>C) mutant
Amt
BLOCker
Oligo
Forward WT Blocking Oligo
Reverse WT Blocking Oligo
0 nM @ Tc
50 nM @ Tc
Matched
Tumor
+ BLOCker
75 nM @ Tc
Matched
Tumor
- BLOCker
ICE COLD-PCR
Extraction
T
5’
5’
T
A
5’
C
G
5’
C
5’
STEP 1:
Denature
5’
T
ss
A
5’ ss
C
ss
G
5’ ss
5’
C
5’
ss
C
G
C
5’
5’
ds
PO4
A
5’
3. ICE COLD-PCR Provides a cost-effective, sensitive approach to mutational
analysis where the data generated is a Sanger sequencing electropherogram.
5’
ds
C
C
5’
PO4
ss
PO4
PO4
PO4
Denature
STEP 3: Differentially
Denature at Tc
Final
Extension
C
5’
Sequencing
5’
G
C
5’
A
C
5’
C
5’
PO4
T
5’
5’
PO4
WT
5’
C
5’
Exp. Amp
C
5’
G
C
4. BLOCker sequencing is a modification of standard Sanger sequencing which
can increase the sensitivity of mutation detection following standard PCR by
at least 10 fold (1% as opposed to 10%)
5’
ds
PO4
5’ ss
C
5. BLOCker sequencing can enhance the sensitivity of mutation
analysis following ICE COLD-PCR amplification by at least 2 fold
PO4
5’
Linear Amp
5’
ss
G
C
PO4
A
C
G
5’
MUT
ss
A
STEP 5:
Extension
5’
C
5’
5’
5’
T
A
T
5’
PO4
Amp Primer 1
Amp Primer 2
5’
T
5’
5’
60 cell lines panel.
2. ICE COLD-PCR methodologies are suited for detecting somatic mutations in
patient FFPE tissues, serum (or plasma) and CTCs using standard molecular
biology laboratory equipment
STEP 2:
Hybridize at 70°C
Preamplification PCR
5’
CONCLUSIONS
Figure 2: Two types of Heteroplasmy detected in NCI1. ICE COLD-PCR is a highly sensitive methodology for detection of very low
level mutations (0.1%-0.05%)
METHODS
5’
Plasma
+ BLOCker
Plasma
- BLOCker
100 nM @ Tc
BLOCker Sequencing is an approach which selectively blocks the sequencing of wildtype DNA and allows samples with lower concentrations of mutant DNA to be
detected using standard Sanger Sequencing methods. This methodology is similar in
concept to ICE COLD-PCR in that there is a BLOCking oligo added to the cycle
sequencing reaction which is complementary to the wild-type sequence. Low levels of
somatic mutations can be directly sequenced following standard PCR amplification.
After ICE COLD-PCR
49:G12A
29:G12C
ICE COLD-PCR without RS-oligo
COamplification at Lower Denaturation temperatures PCR (COLD-PCR) technology
is a significant new discovery from Dr. Mike Makrigiorgos’ lab at the Dana Farber
Cancer Institute for highly sensitive detection of mutations. One iteration of COLDPCR, ICE COLD-PCR (Improved and Complete Enrichment), results in the highest
mutation enrichment rates while capturing all mutations (Tm lowering, Tm neutral, Tm
raising, insertions, deletions, indels, etc). ICE COLD-PCR uses a reference sequence
oligonucleotide (RS-oligo) which is complimentary to one strand of the wild-type
sequence containing some modified nucleosides. Incorporation of these modified
bases results in more rapid and stable hybridization to the complimentary wild-type
sequence, and results in higher Tm’s for wild-type:RS-oligo versus mutant:RS-oligo
duplexes. This allows sufficient Tm variation so that amplification of the mutant
sequence will be enriched as compared to the wild-type sequence. There is sufficient
enrichment of mutant sequences to allow the detection of the mutation using standard
Sanger sequencing protocols.
1/2000
35:G12V
ICE COLD-PCR without RS-oligo
High sensitivity detection of cancer biomarkers facilitates early diagnosis and
provides insight for intervention including stratification for drug treatment and
monitoring changes in tumor mutation profiles by analyzing DNA isolated from
serum/plasma or circulating tumor cells.
1/1000
G12R
WT
KRAS exon 2 mutation detected in
DNA isolated plasma of NSCLC patients
G13D
Transgenomic has developed very high sensitivity methods for detecting somatic
mutations notably in the discipline of cancer therapy where key genetic changes are
associated with EGFR antagonists ’ effectiveness, e.g. in the genes EGFR, KRAS,
PIK3CA, BRAF, p53 and NRAS. The novel techniques of ICE COLD-PCR and
BLOCker Sequencing preferentially enrich mutant alleles compared to wild-type
alleles. These are not allele-specific techniques; therefore all mutations, both known
and unknown, are enriched and identified in a single reaction. Together these
methods allow mutations at concentrations as low as 0.01-0.05% in background of
wild-type to be confirmed by sequencing. Additionally ICE COLD-PCR and BLOCker
Sequencing require no special equipment using only standard DNA thermal cyclers
and Sanger sequencing equipment. Data will be presented showing how these
methods allow DNA sequencing confirmation of somatic mutations in samples such as
low tumor-load biopsies, formalin-fixed paraffin-embedded slides, fine-needle
aspirates, circulating tumor cells and circulating free tumor DNA in plasma and serum.
These methods can also be used to confirm low signal Pyrosequencing and nextgeneration deep-sequencing results. Using these techniques routine, rapid, simple
and inexpensive detection of somatic mutations in (1) cancer patients ’ plasma or
serum or; (2) samples where very limited tumor tissue is available, offers a noninvasive option for cancer biomarker detection as well as monitoring remission,
relapse and emergence of resistance mutations post-treatment. Finally ICE COLDPCR and BLOCker Sequencing can be applied to analysis of mixed viral infections
and mitochondrial heteroplasmies.
Mutant strands
available
for amplification
STEP 4:
Anneal Amplification
Primers
6. BLOCker sequencing can be used as a stand alone or in conjunction with
other techniques to provide a cost-effective, tiered, sensitive approach to
mutational analysis where the data generated is a Sanger sequencing
electropherogram.
PO4
RS-oligo
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