Download 3D Biology Technology on the nCounter Platform SNV Performance

Oncology Biomarker Development: Simultaneous Digital Counting
of Nucleic Acids and Proteins at 800-plex
NanoString Technologies 530 Fairview Avenue North, Seattle, WA 98109
3D Biology™ Case Study: Drug Response by BRAF Genotype
Abstract
With the growth of biomarker discovery, identification of novel cancer signatures has revealed the limitations of relying on a single analyte class to characterize tumors or predict
their responses to therapies. NanoString® has developed its Vantage 3D™ product line to meet the need for simultaneous analysis of DNA, RNA, and proteins. Based on singlemolecule optical barcoding technology, NanoString’s products are designed to consume as little as 15,000-25,000 cells while utilizing the experimental power of 800-plex, crossanalyte quantification. We present two case studies of 3D Biology in action. First, we demonstrate that our RNA and protein detection chemistries work in concert with our novel
SNV-specific probes for in-depth characterization of genotype-specific expression changes in a cell culture model of BRAF inhibition. Second, we show that the 30-plex
nCounter® Vantage 3D™ Protein Solid Tumor Signaling Pathways Panel can be used in conjunction with the 770-plex PanCancer Pathways Panel to monitor expression
regulation and protein phosphorylation within clinically relevant samples, including FFPE tissues. Together, these examples highlight the rich data sets and robust discovery
potential provided by a 3D Biology approach to cancer biomarker research.
Raf/MEK/ERK and PI3K/AKT/mTOR signaling pathways
BRAF genotype-specific gene signatures of drug response
1 6 G E NE E X P RE S S I O N S I G NATURE *
EGF
RESPONSE SCORE
50
3D Biology Technology on the nCounter Platform
3D Biology Workflow
WT: SKMEL28 cells
75
SNV Probe Design
25
Heterozygous mutant: RPMI-7951 cells
0
-25
Homozygous mutant: SW48 cells
-50
cell
membrane
Perfect match
-75
0
5
10
15
HOURS OF TREATMENT WITH VEMURAFENIB
20
WT/V600E
V600E/V600E
WT/WT
Single mis-match
•
•
Protein Probe Design
*Gene Signature: CCND1, DUSP4, DUSP6, ETV1, FOS, FOSL1, HES1, HMGA2, HPGD, ID2, MAP3K, MYC,
NR4A3, PPARGC1A, SPRY1, SPRY2
See also Joseph et al. PNAS 2010 107: 14903-908
~50% cutaneous melanomas harbor BRAF mutations
BRAF V600E comprises ~90% of BRAF mutations
3D Biology of drug response with simultaneous DNA/RNA/protein profiling
RNA or DNA Detection
DNA SNV
Genotype
25
RNA
Expression
3D Biology of drug response with simultaneous DNA/RNA/protein profiling
Drug
Treatment
Protein
Expression
WT BRAF Genotype
V600E/V600E Genotype
Endogenous Nucleic
Acid
The nCounter platform can be used to digitally count DNA, RNA, and proteins from a single sample
by using fluorescent optical barcodes that attach to a probe, which then binds directly to the
analyte of interest. Our probes have been adapted to allow single base pair resolution and protein
detection.
+ vem
Photocleavable Linker
+ vem + tram
SNV Performance
Confidence
Primary
Antibody
Confidence
Unique
ssDNA
Tag
RNA Expression
Protein Detection
50%
99%
+ vem
Variants present at 1-10% levels are detected from 5 ng of FFPE DNA
-8 -4 -2 0 2 4 8 -8 -4 -2 0 2 4 8
fold change with drug
fold change with drug
+ vem + tram
+ vem
Here the sample was 5 ng of
genomic DNA extracted from
FFPE sections purchased from
Horizon Discovery containing
10 different variants ranging in
frequency from 1% to 10%. All
assayed variants are detected
with > 95% confidence (p <
0.05 confidence threshold
indicated by the dashed pink
line).
Vemurafenib shows remarkably few off-target gene expression
effects in cells with WT BRAF V600 alleles
+ vem + tram
In a single nCounter lane, 6 SNVs, 180 transcripts, and 16 protein species are quantified simultaneously.
The heatmaps represent average levels from biological triplicate samples after normalization to controls.
Single and combination drug treatment was for 8 hours measured against treatment with vehicle (DMSO)
alone.
HLA-DRA
drug +
Phosphorylation changes with combination drug treatment
0
-1
-2
-3
+ vem
+ combo
+ vem
WT/WT
+ combo
+ vem
V600E/WT
p-AKT/AKT
p-MEK/MEK
+ combo
V600E/V600E
p-ERK/ERK
HLA-DRA
drug +
CD73
+
2
1
-1
-2
-3
p-EGFR/EGFR
p-ERK/ERK
p-S6/S6
In WT cells, the degree of phosphorylation of S6 and Erk, a direct substrate of Mek, is reduced by exposure
to the Mek inhibitor trametinib in addition to the B-Raf V600E inhibitor vemurafenib. In cells carrying the
V600E mutation, S6 phosphorylation appears to be reduced in an allele-dose dependent manner, specifically
in response to vemurafenib,
p-AKT/AKT
p-S6/S6
p-MEK/MEK
After 8 hours, inhibition of the Ras/Raf/Mek/Erk pathway by
vemurafenib reduces the degree of downstream substrate S6
phosphorylation in BRAF V600E mutant cells 4.8-fold. In
contrast, the relative phosphorylation of the upstream receptor
EGFR increases in cells with WT BRAF V600 alleles
3D Biology Case Study: Profiling Melanoma FFPE Samples
11 reference alleles
all detected in sample
26 COSMIC variant alleles assayed.
16/16 detected in sample.
Somatic variant frequency (%) listed
log2 fold-change with drug
Using commercially available human
genomic DNA (Horizon Discovery plc)
of 16 solid-tumor associated SNVs at
somatic frequencies (2.4% and 4.8%
in blue). Upon assay with an ‘alpha’
version of the nCounter SNV panel,
all 16 variants are detectable with
counts levels that are 3-fold greater
than counts obtained from the
negative control reference sample
and with an estimated confidence of >
99%. Count-level fold-change over
reference (in log2 units) is indicated
by the number in each red box. The
estimated count variance in each
assay is reflected by the size of the
boxes. The 11 reference alleles’
probe counts are grouped at right.
CD73
+
CD73, a cell-surface enzyme that is immune suppressive, is
upregulated in cells with mutant BRAF V600E alleles
Protein Phosphorylation
log2 fold-change in ratio
1
High confidence mutation detection in low frequency samples
Digital Counts
Down-regulated
8000
Digital Counts
Up-regulated
Protein Expression
EGFR
p-EGFR
Syk
p-Syk
Akt
p-Akt
pPras40
p-cRaf
Mek
p-Mek
Erk
p-Erk
S6
p-S6
Her2
Gsk-3b
8000
3D Biology on FFPE with simultaneous DNA/RNA/protein profiling
BRAF genotype-specific gene signatures at baseline
Protein Performance
Equivalence between single-plex and multiplex assay format
3D protein signal on FFPE
BRAF status: V600E
Other SNV:
APC R876
V600E
wt
wt
wt
wt
wt
wt
wt
NRASQ61K
CCND1
18
DUSP4
16
DUSP6
R² = 0.9825
14
ETV1
12
FOS
10
8
6
6
8
10
12
14
Single Plex Assay (log2 Signal)
16
FOSL1
RNA:Protein Solid Tumor
Signaling Pathways panel for FFPE
(770 RNA, 30 Proteins)
Multiplexed Assay (log2 Signal)
20
V600E
18
Protein detection shows high correlation (R2>0.98) when comparing single plex versus multiplex analysis. Additionally,
protein signal is identical when run as a stand alone assay or in combination with nucleic acid analysis
Benchmarking protein results shows high concordance
HES1
HMGA2
HPGD
ID2
MAPK3
MYC
NR4A3
Lysate
PPARGC1A
6
R² = 0.9713
5
AKT
p-AKT
S6
SPRY1
p-S6
SPRY2
4
3
0
-6
-4
-2
-1
0
2
4
Counts
Counts
1
150000
100000
5000
50000
0
6
0
p-AKT
A431 + SS
-2
SS + CA
P-S6
SS + EGF
A431 + SS
5000
Counts
-3
-4
-5
ELISA
(log2 fold change)
Counts
nCounter
(log2 fold change)
10000
2
4500
4000
A431 + SS
SS + CA
SS + EGF
2600
2500
2400
2300
2200
AKT
Protein detection is highly correlated with gold standard techniques for protein
analysis from lysates. Antibody performance was compared using ELISA versus
NanoString in addition to western analysis versus NanoString.
SS + CA
26 genes were profiled for SNV status followed by expression profiling of 770 RNA and 30
proteins on six melanoma FFPE samples. Expression data clusters by BRAF V600E
mutational status and NRAS and APC mutational status, two additional SNVs detected in
samples 3 and 4.
Gene Signature from above at baseline shows heterogeneity among samples. NRAS mutant shows large
expression changes (decrease) compared to WT. Additionally, MAPK3 shows little change in expression
among samples despite changes in phosphorylation seen below.
See also Joseph et al. PNAS 2010 107: 14903-908
Phopspho-protein profiling at baseline
Conclusions
S6
SS + EGF
A431 + SS
SS + CA
SS + EGF
0.5
FFPE
S/N (Error Bars = 2 SD)
10.0
R² = 0.9472
9.5
9.0
8.5
80
60
Phosho/total protein
(Counts)
100
10.5
IHC
(Arb. Units Log10)
The nCounter® Vantage 3D™ portfolio powered by 3D Biology technology allows for simultaneous
profiling of DNA, RNA, and protein
•
SNV probes also permit detection of somatic variants with 5% allele frequency from as little as 5 ng fresh
or FFPE gDNA.
•
NanoString’s Protein Solid Tumor assays for lysate and FFPE show high correlation to gold standard
techniques, specifically western, ELISA, and IHC, respectively.
•
NanoString’s multi-plex Protein Solid Tumor assays show high correlation to detection in single-plex
assays and when run alone versus in combination with RNA and RNA + DNA for a full 3D experiment.
•
Complex biology can be profiled simultaneously using Vantage 3D assays and the case studies presented
demonstrate this capability both on cell lysates and FFPE samples allowing you to see more biology with
less sample.
•
For more information, visit booth 512 and 3D.nanostring.com
0.4
Phospho-Targets Across Treatments
P-AKT P31
P-c-Raf P09
P-PDK1 P25
P-AmpK P18
40
HELA +PI -λPhosph
HELA -PI +λPhosph
HELA -PI +λPhosph
7.5
2.5
3.0
3.5
4.0
4.5
20
S/N (Error Bars = 2 SD)
P-GSK P21
Protein detection is highly correlated with gold standard
techniques for IHC analysis from FFPE. Specifically,
upregulation of phospho-targets is seen after stimulation
and can be blocked by treatment with lambda phosphatase.
15
wt
APC R876
NRASQ61K
wt
wt
V600E
V600E
V600E
wt
wt
wt
Genotype
P-ERK P14
10
wt
P-Mek P26
pS6/S6
pERK/ERK
pAKT/AKT
P-pras P13
5
0
HELA -PI -λPhosph
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
www.nanostring.com | [email protected] |
0
Phospho-Targets Across Treatments
5.0
nCounter
(Counts Log10)
0.2
0.1
HELA -PI -λPhosph
8.0
2.0
0.3
20
0
1.5
•
@nanostringtech
© 2016 NanoString Technologies, Inc. All rights reserved. Patents pending.
NanoString, NanoString Technologies, the NanoString logo, nCounter, 3D Biology, and Vantage 3D are registered
trademarks or trademarks of NanoString Technologies, Inc., in the United States and/or other countries.
HELA +PI -λPhosph
HELA -PI +λPhosph
HELA -PI +λPhosph
Protein targets drastically changed by treatment above show consistent profiles across
genotype at baseline in FFPE samples. Interestingly, the sample with an activating NRAS
mutation shows increased ERK phosphorylation despite not change ERK RNA highlighting
the importance of integrated DNA, RNA, protein and phospho-protein profiling.
AUTHORS: Elizabeth Manrao, Dae Kim, Gavin Meredith, P. Martin Ross, Jill McKay-Fleisch, Erin Piazza, Celine Ngouenet,
Mingdong Liu, Patrick Danaher, Gokhan Demirkan, Brian Filanoski, Brian Birditt, Gary Geiss and Joseph Beechem
Superscripts
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