Download Accurate Quantification of HER2 Gene Amplification Using

AACR 2014
Poster #1521
Accurate Quantification of HER2 Gene Amplification
Using QuantStudioTM 3D Digital PCR System
Kelly Li, Devin Do, Patricia Hegerich, Nivedita Majumdar, David Keys, Stephen Jackson, Junko Stevens and Caifu Chen
Life Science Solutions Division, Life Technologies, A part of Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080
RESULTS
Human epidermal growth factor receptor 2 (HER2) gene amplification (increasing
copy number), not only is a strong indicator for tumor progression/poor prognosis
but also presents as an effective drug target. Accurate measurement of HER2
copy number (CN) is so critical that it determines patient qualification for HER2targeted therapy with Trastuzumab (Herceptin) for early or advanced breast
cancer. Currently, in situ hybridization-based and immunohistochemical analysis
are commonly used, but they can be subjective, lengthy, labor intensive and less
quantitative, especially for samples whose HER2/CEP17 ratio fall into the
equivocal range (1.8-2.2). Life Technologies offers TaqMan® Copy Number
Assays for copy number analysis on real-time qPCR platform, but qPCR also
becomes less quantitative for CN calls reach higher than 4, due to its relative
quantitation (RQ) methodology. Recently launched QuantStudioTM 3D (QS3D)
Digital PCR System provides a new platform with absolute quantification capability
to address these challenges. We have developed a copy number application on
the dPCR system and we can calculate copy number based on absolute
quantitation exported from QS3D Analysis Suite software. We demonstrate that
the system not only can quantify gene copy numbers from zero to eight with high
accuracy and precision but also is able to differentially detect heterogeneous
samples with 5% resolution. We also present our technical assessment for
quantifying HER2 copy number using breast cancer FFPE samples and our data
show high concordance with results from SISH method. As a robust platform, the
dPCR system not only leverages our existing TaqMan® CNV assays but also
enables customers to measure higher CN with absolute accuracy, simple workflow
and fast turnaround time.
The workflow for QuantStudio™ 3D Digital PCR System and its 20K chip is shown
in Figure 1. For copy number analysis, 14.5 ul volume of dPCR reaction mix,
including QS3D dPCR master mix, a FAM-labeled TaqMan® CNV assay for target
of interest and VIC-labeled RNaseP reference assay (P/N 4400294) and gDNA, is
loaded onto a chip. The amount of gDNA used usually targets range of 2002,000 copies/ul on the chip and adjustment for input DNA may be required
depending gene copy number to be detected. PCR thermalcycling condition and
other details can be found in QS3D User Guide. After PCR, chips are read on
QS3D dPCR Instrument and based on absolute quantitation exported from QS3D
Analysis Suite software, copy number is calculated by 2 times the ratio of FAMlabeled target and VIC-labeled RNaseP reference.
Regular gDNA samples are purchased from Coriell Repository. TaqMan® CNV
assays for CCL3L1 (Hs03198166_cn) and for HER2 (Hs00817646_cn) are from
Life Technologies. For qPCR, FAM-labeled TaqMan CNV assay for target of
interest was duplexed with VIC-labeled RNaseP CN reference assay, and copy
number is determined by a relative quantitation (RQ) based-∆∆Ct method, as
described in TaqMan® CNV assay protocol.
1. Mix
2. Load
3. Amplify
4. Read
• Sealed consumables
• Limited hands-on
• Minimal sample loss
B. QuantStudio™ 3D Digital PCR 20K Chip
• Interrogated volume similar to
real-time PCR
• Hexagonal packing enables
20,000 wells / 10×10 mm2 chip
• Each reaction well is isolated
from its neighbors
60 µ m
Figure 4. Testing TaqMan Copy Number (CNV) Assay for HER2 Gene by qPCR
A
9
Fig.2A The limitation of qPCR for CNV
detection is that it measurement lose
accuracy when copy number is above 4
due to its relative quantitation method.
We demonstrate here that the dPCR
can accurately measure copy number
from 0 to 8 for CCL3L1 gene using nine
gDNA samples with known copy
number for the target. For each
sample, 6-8 replicates were run and
their standard deviation is shown in the
error bars. The CV for each sample is
within the range of 0.1-0.25%.
8
Copy Number
7
6
5
4
3
2
1
Samples
8
4
2
1
0.5
CN by dPCR 0.8 1.1 1.1 1.4 1.4 1.5 1.6 1.6 1.7 1.7 1.8 1.8 1.9 2.0 2.0 2.1 2.1 2.2 2.2 2.3 2.4 2.5 2.7 2.8 3.0 3.2 3.5 4.7 5.0 5.2 5.6 6.8 6.9 7.3 7.4 8.2 9.7 9.8 10.521.022.424.631.1
SISH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
HER2 gene amplification was analyzed for 43 breast cancer FFPE samples using QuantStudioTM 3D dPCR
system. HER2 copy number was determined by ratio of absolute quantitation of FAM-labeled HER2 target and
VIC-labeled RNaseP reference exported from QS3D Analysis Suite software. Each sample was also analyzed
by SISH. For SISH, HER2 gene amplification was defined as positive(P) or negative (N) according to
ASCO/CAP 2007 guideline (HER2/C17 ratio >2.2 as positive or <1.8 as negative, in between as equivocal).
The high concordance between the two completely different technologies (dPCR and SISH) is shown. The red
line is drawn roughly CN=4.4 corresponding to the cutoff of HER2/C17 ratio (2.2) for calling positive in SISH.
CONCLUSIONS
• The QuantStudio™ 3D Digital PCR System provides a sensitive, accurate, robust
technology with a simple workflow for copy number analysis. Copy number can be
calculated based the absolute quantitation exported from QS3D Analysis Suite software. It
is a reliable platform for measuring gene amplification and copy number variation (CNV).
2
• We can accurately measure copy number from 0 to 8 with high precision (with CV of
1.0%-2.5%) using CCL3L1 as a model gene and the samples with known copy number for
the gene.
1
0
gDNA
Figure 5 HER2 Copy Number Determination Using FFPE Samples and Two Distinct
Copy Number Reference Assays with dPCR
B
6
Fig.2B shows high precision of copy
number (CN) measurement from individual
replicate chips. Measured CN is plotted
against expected CN for each of a given
sample (shown in Fig.2A). The blue dots
are CN from individual chips and red
cross is mean of all the measurement for
the given sample. The grey shaded
rectangular bars are their standard
deviations, and the dotted line are 99%
prediction interval. Copy number is
calculated from absolute quantitation
reported by QS3D Analysis Suite .
16
3
Copy Number
Figure 2. QuantStudioTM 3D Digital PCR Can Accurately Quantify Higher Copy Number
32
4
To ensure high performance of
TaqMan CNV assay for HER2,
we first tested it with qPCR
using a panel of 94 gDNAs from
Coriell. Four replicates were
run for each sample. HER2
copy number was determined
by a relative quantitation (RQ)
based-∆∆Ct method. All the
samples are expected to have 2
copies of the gene and the
HER2 CNV assay shows the
robust performance cross the
gDNA panel.
NA10859_1
NA10859_2
NA10859_3
NA10859_4
NA14474
NA14476
NA17102
NA17103
NA17104
NA17105
NA17106
NA17107
NA17108
NA17109
NA17110
NA17111
NA17112
NA17113
NA17114
NA17115
NA17116
NA17117
NA17118
NA17119
NA17120
NA17121
NA17122
NA17123
NA17124
NA17125
NA17126
NA17127
NA17128
NA17129
NA17130
NA17131
NA17132
NA17134
NA17136
NA17137
NA17139
NA17140
NA17144
NA17147
NA17148
NA17149
NA17155
NA17188
NA17194
NA17201
NA17202
NA17203
NA17204
NA17205
NA17206
NA17207
NA17208
NA17209
NA17210
NA17211
NA17212
NA17213
NA17214
NA17215
NA17216
NA17217
NA17220
NA17221
NA17223
NA17225
NA17226
NA17227
NA17228
NA17230
NA17231
NA17232
NA17235
NA17237
NA17239
NA17240
NA17241
NA17242
NA17245
NA17247
NA17251
NA17253
NA17254
NA17255
NA17258
NA17259
NA17260
NA17261
NA17262
NA17263
Fig. 1A illustrates the workflow of QuantStudioTM 3D (QS3D) dPCR system. The steps are simple and
straightforward with minimal hands-on requirement. The system consists of an autoloader, a modified
thermocycler 9700 and a reader (QS3D dPCR Instrument). Copy number is calculated from absolute
quantitation reported by the cloud-based QS3D Analysis Suite software .
Fig. 1B shows a 20K chip used in the dPCR system. PCR reaction mix (volume of 14.5 ul) is loaded onto a
chip (roughly 0.8nl/well).
Copy Number by dPCR (log2 ) **
Fig. 3 demonstrates high sensitivity
of 5% or even lower of the dPCR
system in differentially detecting CN
in heterogeneous samples. Two
samples from Coriell (NA 17258 and
NA 17132) have known 2 or 3 copy
number for CCL3L1 gene. The 2
samples with CN of 2 and 3 are
mixed at different proportion to
generate the predicted CN at 0.1
incremental changes between CN 2
and 3 ( like 2.0. 2.1, 2.2…3.0). CN
for the mixed samples is calculated
from absolute quantitation reported
by the QS3D Analysis Suite.
Measured CN is plotted against
predicted CN and the legends are the
same as Fig. 2.
Sealed Consumables
0
MATERIALS AND METHODS
Figure 6. HER2 Copy Number Analysis of Breast Cancer FFPE Samples
by dPCR and SISH
A. Workflow
INTRODUCTION
Gene amplification is a common genetic abnormality in many types of cancers and
has been implicated in playing important roles in cancer development. HER2
gene amplification occurring in about 10-30% of breast cancer cases, is strongly
associated with aggressive tumor progression and poor prognosis, and also an
effective therapeutic target. Breast cancer patients with HER2+ are eligible for
Herceptin treatment. Therefore, accurately determining HER2 gene amplification
is essential yet challenging for cancer researchers and clinicians. Currently, IHC
(immunohistochemical analysis), FISH (fluorescence in situ hybridization) or SISH
(silver in situ hybridization) are routinely used. However, the challenge is that
these methods can be laborious and less quantitative, especially when CN falls
into equivocal range. In the study, we aim to 1). demonstrate the utility of
QuantStudio™ 3D Digital PCR for quantifying DNA copy number with high
precision, accuracy, resolution, 2). show its application in measuring HER2 gene
amplification in breast cancer FFPE samples, and its high concordance with
current method (SISH ).
Figure 3. High Sensitivity of dPCR in Quantifying Copy Number in Heterogeneous Samples
Figure 1. QuantStudioTM 3D Digital PCR System
CN_using RNaseP Ref
5
CN_using TERT Ref
Copy Number**
ABSTRACT
4
3
2
1
0
30
11
6
14
10
7
Samples
3-2
3-1
19-1
19-2
It is a concern of using a
single copy number
reference assay with
cancer samples due to
cancer genome instability.
We tested HER2 copy
number by dPCR with
randomly selected 8
breast cancer FFPE
samples using both
RNaseP and TERT CN
reference assays. Two of
the samples (#3 and #19)
were run in duplicate
chips. **CN is calculated
based on absolute
quantitation reported by
QS3D Analysis Suite.
• We also demonstrate the ability of QS3D to differentially detect copy number differences
in heterogeneous samples with high sensitivity of 5% or even lower.
• We have assessed HER2 gene amplification in 43 breast cancer FFPE samples and high
concordance is achieved compared to SISH results from the same set of samples.
ACKNOWLEDGEMENTS
We thank Bruno Ping, Molecular Diagnostics Department at Royal Surrey County Hospital,
Guildford, United Kingdom for sharing the breast cancer FFPE samples and their SISH
data.
TRADEMARKS/LICESING
© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of
Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a
registered trademark of Roche Molecular Systems, Inc., used under permission and
license.
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
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