Download 2422/9 Simultaneous detection of activating somatic DNA mutations

Simultaneous detection of activating somatic DNA mutations and expressed fusion transcripts from lung tumor FFPE samples
# 2422/9
A. McGarry Houghton1, Gavin Meredith2, Julia Kargl1,3, Jill McKay-Fleisch2, P. Martin Ross2, Anisha Kharkia2, Afshin Mashadi-Hossein2, Dae Kim2, Joseph Beechem2.
1Fred
Hutchinson Cancer Research Center, 1100 Fairview Ave. N. Seattle, WA 98109; 2NanoString Technologies, Inc. 530 Fairview Ave. N. Seattle, WA 98109
Abstract
SNV Panel multiplex detection of 5% alleles from FFPE
Worldwide, lung cancer is the most commonly diagnosed form of cancer with a survival rate among the lowest.
Combined, somatic mutations (in the form of SNVs and InDels) and gene fusions, account for the majority of
interpretable and actionable genomic alterations. Importantly, this typically requires the analysis of DNA and
RNA from limited amounts of FFPE-preserved specimens. Currently, these analyses typically require complex
sample pre-processing for assay on separate platforms or separate complex library preparation methods for
assessment by high throughput sequencing. To provide a unified and simpler alternative, NanoString’s
molecular barcoding technology has been modularized to permit simultaneous digital measurement of cancerrelevant targets that span these two analyte classes. Novel ‘SNV’ probes enable sensitive and specific
identification of DNA mutant allele sequences down to a level of detection of 5% from 5 ng of FFPE-extracted
genomic DNA. Fusion transcripts are detected with 5’/3’ imbalance probes and toehold-mediated junction
probes. This dual analyte workflow requires just a single 5-10 micron section of FFPE tissue and provides
sample-to-answer results with approximately 5 minutes of hands-on time per sample after nucleic acid
extraction.
To demonstrate utility, 45 lung cancer samples were assayed simultaneously with an SNV panel that targets >100
solid tumor somatic mutations and a lung cancer fusion gene panel that provides general evidence of ALK, RET,
and ROS1 gene fusion events along with specific detection of 35 unique fusion transcripts that correspond to
known break-points. In this particular cohort, 19 samples were positive for activating KRAS SNVs (one of which
was also positive for an activating STK11 variant), 3 were positive for activating EGFR mutations including two
SNVs and an 18-base InDel, one was positive for an activating NRAS mutation and one was positive for an
activating KIF5B16:RET12 fusion transcript. Positive mutation calls obtained with the SNV panel could only be
confirmed by whole-exome sequencing (average depth of 100X) for 19 of 25 variants detected; however, ultradeep (average depth of 4400X) targeted sequencing revealed that the 5 additional panel-detected mutations
were, in fact, present along with one apparent false positive call. With DNA from fresh frozen tissue and
measured against the sequencing datasets, the SNV panel provided 100% sensitivity, specificity, accuracy and
precision for all variants present at 5% or greater allele frequency. SNV detection calls were 95.7% concordant
between tumor-matched FFPE and fresh frozen samples. The KIF5B_15:RET_12 fusion event was also confirmed
by genomic DNA sequencing. Combined, these results show that these two important classes of activating
mutations can be readily and efficiently assayed together on a NanoString nCounter® system (for research use
only).
FFPE gDNA: EGFR
Reference Standard
FFPE gDNA: KRAS
Reference Standard
5%
Sample ID
5%
5%
Reporter tag
Probe T
Capture probe
•
DNA target fragment of interest
•
•
•
•
Source
Solid Tumor
Fusion by Lung
SNVs
Gene Fusion Panel Diagnosis
Detected
assay
NGT-ALK-006 PrecisionMed
none
NGT-ALK-008 PrecisionMed
none
B140001262 NWBioTrust
none
No specific ALK fusion
transcript detected
Interphase FISH
Comments
NSCLC, adeno ALK fusion positive
POSITIVE for a missing signal of 5' ALK
Positive by 3 methods
ALK 3'/5' imbalance NSCLC, adeno ALK fusion positive
POSITIVE for rearrangement of ALK at
2p23
Positive by 3 methods
EML4_13:ALK_20
KIF5B_15:RET_12
NSCLC, adeno
IHC results
KIF5B_15:RET_12
transcript detected
none
none
Sanger Seq. confirmed
(see below)
The Vantage 3D™ Lung Fusion Panel includes 63 probes: 24 for positional gene expression imbalance detection of ALK, RET, and ROS1 derived
transcripts, 35 for specific fusion detection that also covers 2 NTRK1 variants. Fusion probe counts for two IHC- and FISH-verified ALK fusion
positive FFPE samples obtained from PrecisionMed (Solana Beach, CA) and one unverified sample from NWBioTrust (Seattle, WA) are shown.
The KIF5B_15:RET_12 fusion event was subsequently verified by Sanger sequencing of PCR-amplified genomic DNA (below).
KIF5B_15:RET_12 fusion verified
by Sanger sequencing
Each SNV Probe is an oligonucleotidebased construct with:
Probe S
In order to estimate mutational
burden, whole exome
sequencing (WES, 111X
average depth) was used to
survey the mutations present in
a cohort of lung cancer cases.
Both fresh-frozen and FFPEpreserved tissue samples were
available. Genomic DNA from
fresh-frozen samples was used
for WES library preparation
and it was also used in the
SNV panel assay for 42 of 45
tumor samples.
EML4_13:ALK_20
transcript detected
nCounter® Vantage 3D™ Probe Design for Specific SNV Detection
•
Imbalance
probes
detect
presence
of a fusion
Specific
fusion
transcript
detection
67%
5%
Simultaneous, Sensitive Detection of SNVs & Fusion Transcripts via Vantage 3D™
Assays Verifies and Augments Sequencing Data
Lung Cancer Gene Fusions detected from FFPE samples
5%
Probe S with two ‘short’ arms  basepairs contiguously with the DNA target
strand
Reporter tag  uses existing NanoString
optical fluorescent barcodes
A single mismatch on one arm inhibits
hybridization of SNV probe S
Detection of an SNV results from counting
the SNV assigned barcode
A simple hybridization protocol precedes
nCounter quantification
Each assay use probes for the reference
and the variant allele
Simultaneous SNV and Fusion Detection on a Positive Control
5%
SNV detection results
Separately, 2 FFPE sections
from each sample was used to
isolate DNA and RNA using a
Qiagen AllPrep DNA/RNA
FFPE Kit. For these samples
yields, determined by Qubit
assay, ranged from 680-5900
ng (avg: 2300 ng) for gDNA
and from 2.1-10.5 µg (avg: 5.3
µg) for total RNA per section.
The combined 3D Biology™
SNV + Fusion detection assay
workflow was used for all 45
FFPE tumors.
Lung Tumor
Gene
B140001390
B140001383
B150001509
B140001157
B140000283
B150000039
B140000090
B140000885
B150001490
B140001139
B140000270
B150001359
B140000892
B140002445
KRAS
KRAS
EGFR
KRAS
KRAS
EGFR
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
B150001491
COSMIC ID of
Base
variant
change(s)
COSM516
COSM522
COSM6224
COSM522
COSM520
COSM6224
COSM521
COSM521
COSM520
COSM520
COSM554
COSM520
COSM521
COSM516
FreshFrozen
Tumor SNV
panel call
Variant freq: Variant freq:
FFPE Tumor
Fusion
Targeted NGS WES (111X
SNV panel call
Detected
(4720X avg)
avg)
34G>T
35G>C
2573T>G
35G>C
35G>T
2573T>G
35G>A
35G>A
35G>T
35G>T
183A>C
35G>T
35G>A
34G>T
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
51.2%
48.9%
48.7%
44.4%
43.1%
39.3%
38.3%
33.4%
32.8%
32.5%
31.1%
23.3%
20.8%
20.6%
53.2%
51.5%
47.9%
45.8%
38.7%
37.6%
36.0%
30.0%
21.4%
35.1%
34.9%
15.9%
19.5%
18.2%
none
none
none
none
none
none
none
none
none
none
none
none
none
none
STK11 COSM1523962 465-2A>T
Detected TP
Detected TP
17.4%
9.8%
none
Detected TP
Detected FP
9.7%
Undetected
Undetected
Undetected
none
none
Detected TP
15.7%
19.0%
none
KRAS
CTNNB1
B140002436
EGFR
B140001273
B140001176
B140001275
B140001269
B150001547
B140001377
B140001231
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
NRAS
COSM555
COSM5662
183A>T
Detected TP
110C>T Undetected TN
2240_2257
COSM12370
Detected TP
del18
COSM520
35G>T
Detected TP
COSM521
35G>A
Detected TP
COSM516
34G>T
Detected TP
COSM527
37G>T
Detected TP
COSM516
34G>T
Detected TP
COSM516
34G>T
Not Testable
COSM573
38G>A
Not Testable
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
Detected TP
None
Not Testable
Detected
None
None Detected
Detected
11.2%
10.3%
9.9%
7.9%
5.8%
19.3%
not tested
Undetected
none
5.3%
none
11.5%
none
Undetected
none
Undetected
none
Undetected
none
2.40%
none
None
KIF5B:RET
Detected
None
none
Detected
Samples that yielded positive
SNV or fusion transcript
not tested
B140001262 none
none
NA
detection calls with the
nCounter assays are shown at
21 more
not tested
none
none
NA
left. A subset of samples that
tumors
produced discordant mutational
profiling data between the WES and nCounter® assays were used to prepare amplicon libraries for “Deep” targeted NGS sequencing. In all cases
tested, the variants detected by the SNV assay from fresh/frozen tumors were verified to be ‘True Positives’ and all variants detected by WES
were confirmed by the nCounter® Vantage 3D™ DNA SNV Solid Tumor Panel assay. Five of the 24 (~20%) verified mutations detected with the
SNV panel failed to be detected by WES. SNV detection was >95% concordant between matched fresh/frozen and FFPE samples.
One sample, B140001262, was verified to harbor a detected KIF5B:RET fusion (see above and sequencing trace at left).
RNA and Protein Expression Profiling with Vantage 3D™ Assays
5%
PanCancer Pathways RNA
MUTEGFR
MUTKRAS
Vantage 3D™ Protein Solid Tumor
PanCancer Immune RNA
MUTEGFR
MUTKRAS
MUTEGFR
MUTKRAS
p=0.01
5%
p=0.01
p=0.01
p=0.05
p=0.05
p=0.05
3D Biology™ Workflow: Simultaneous DNA SNV & Fusion Detection
Extract and
quantify
nucleic acids
Hybridize in
parallel overnight
(16 hr)
Pool
hybridizations &
load nCounter
cartridge
Count on
nCounter
system
Obtain
detection
calls
Imbalance probes detect presence of multiple fusions
50%
In addition to SNV and Fusion profiling with Vantage 3D™ Assays, the gene expression profiles of the 45 lung samples were determined
using 770 RNA in the PanCancer Pathways Panel and 770 RNA in the PanCancer Immune Profiling Panel, plus 28 total and phosphoproteins in the Vantage 3D™ Protein Solid Tumor panel. This analysis is ongoing and initial results suggest that MUTEGFR tumors show a
more clustered and distinct phenotype than MUTKRAS samples when compared to all other samples. Combined with the SNV and Fusion
profiling, this 3D Biology™ approach allows for more comprehensive molecular profiling of these precious samples.
SNV probe counts
Input mass
Panel
5 ng*
DNA
Fresh/
Frozen
or
nCounter
DNA
SNV
Solid
Tumor
Panel
Fusion probe counts
RNA
FFPE
nCounter
Lung
Fusion
50-300 ng Panel
Conclusions
Specific fusion transcript detection
12 samples
per cartridge
* The SNV detection assay requires 18-20 cycles of pre-amplification by PCR.
The Vantage 3D™ DNA SNV Solid Tumor Panel permits detection of multiple DNA mutations
down to 5% minor allele frequency from 5ng of FFPE-extracted gDNA. These detection plots
show the 104 variants assayed. Detection calls (colored boxes and text) are based on three
criteria: mean log2(fold-change), above reference control samples ≥ 2 (red-dashed line),
estimated p-value ≤ 0.01 (confidence interval = box width), and variant probes counts ≥ 200.
For gDNA extracted from commercial FFPE control samples (HD300 and HD301) from
Horizon Discovery, plc, every expected variant is detected. Allele frequencies are indicated
as percentages. Results were generated with an nSolver™ Analysis Software 4.0 Advanced
Analysis module (alpha version).
•
•
•
•
•
•
As proof-of-principal for the 3D Biology™ workflow to simultaneously detect SNVs and fusion transcripts that are associated with lung cancer,
DNA and RNA were extracted from a single 10 micron section of a commercially obtained SNV & Fusion positive FFPE-preserved sample
(HD-784; Horizon Discovery). DNA and RNA were processed per the workflow at left on an nCounter® MAX system. Analysis of the
nCounter digital count data indicates the presence of KRAS 38G>A and PIK3CA 3140A>G SNVs and ALK, RET, and ROS1 fusion transcripts.
NanoString’s new SNV probe technology enables sensitive and specific detection of somatic (5% allele frequency) SNVs, MNVs, and InDels.
The Vantage 3D™ DNA SNV Solid Tumor Panel assay is optimized for very small amounts (5 ng) of FFPE gDNA.
The Vantage 3D™ SNV assay workflow is simple and requires minimal hands-on time.
3D Biology™ Enabled: Simultaneous detection of DNA and RNA variants and of RNA and protein expression profiling can be performed on
nCounter® systems.
The 3D Biology™ workflow is especially useful for augmenting and verifying somatic cancer-driver mutations profiled by discovery datasets.
For more information, please visit 3d.nanostring.com
Acknowledgements
Data and results cited in this study were funded, in part, as follows: Fred Hutchinson Cancer Research Center, STTR Oncoscape Award, NIH/NCI. JK received support through
grant EU-FP7-PEOPLE-2012-IOF 331255
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
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March 2017