1. qBiomarker™ Somatic Mutation PCR Arrays: A Novel Tool
for Pathway-Focused Cancer Mutation Profiling
Samuel Long, Min You, Liang Wang, Xiaofang Jiang and Yexun Wang
QIAGEN, 6951 Executive Way, Frederick, MD 21703
Assay Differentiation Window (ADW), Assay Efficiency
and Assay Detection Limit for 4 Pathway Arrays
Recent advances in sequencing technology have led to a significant
increase in the number of known cancer somatic mutations. Translating
such information into therapeutic benefits requires the molecular
characterization of all major human tumor types and the advent of
research tools that enable interrogation of multiple mutations in multiple
cancer genes simultaneously, with high accuracy and scalability.
A new system of real-time PCR based somatic mutation arrays has
been developed as a pathway-focused cancer mutation profiling tool.
By combining allele specific amplification (ARMS®) and 5’ hydrolysis
probe detection, the PCR assays on these arrays are designed to
detect as little as 1% somatic mutation in a background of wild type
genomic DNA. Each array contains assays for detecting the most
frequent and functionally verified mutations for multiple genes within a
specific pathway that has been implicated in a variety of cancers. For
example, the PCR panels cover pathways such as the EGFR, PDGFR
and c-Met pathways, each on separate, ready-for-sample PCR plates.
Each targeted mutation on the array is carefully chosen from curated,
comprehensive somatic mutation databases (including the COSMIC
database) and peer-reviewed scientific literature.
The qBiomarker Somatic Mutation PCR Arrays, with their
comprehensive content coverage, are an ideal tool for studying
mutations in the context of whole pathway and have the potential for
discovering and verifying biomarkers for a variety of human cancers.
KRAS
c.34G>T
p.G12C
BRAF
c.1391G>T
p.G464V
EGFR
c.2573T>G
p.L858R
KRAS
c.38G>A
p.G13D
Signal generated on
mutant template
Signal generated on
WT gDNA template
All assays on the arrays have close to 100% PCR efficiency. The following is a summary
of average ADW and assay efficiencies on the 4 qBiomarker™ Somatic Mutation PCR
arrays.
Figure #1
EGFR pathway
In this study, data is presented to demonstrate the consistency and
reliability of array performance using a variety of sample types (fresh,
frozen or FFPE samples from cell lines and tissues) and under a large
range of sample quality conditions (such as FFPE tumor samples from
different sources). In addition, some of the results were further verified
by DNA sequencing using Pyrosequencing ®.
Somatic Mutation Array Performance in FFPE Samples
The assay differentiation window was defined as the Ct difference between the signals
generated on wildtype genomic DNA background and on 100% mutant template by
a mutation assay. Assays were verified to have a minimum differentiation window
of 8 but are usually higher. The BRAF V600E assay ADW is shown as follows:
Fluorescence (dR)
Abstract
ERBB2 pathway
PDGFR pathway
Average ADW
11.15
12.33
11.41
FLT3 pathway
11.73
Average efficiency
104.7%
107.1%
102.3%
In an off-site beta-testing project, the human EGFR pathway somatic mutation array was
used to profile somatic mutations in 4 FFPE cell line samples, a wildtype FFPE placenta
sample (control), and 9 lung adenocarcinoma FFPE tissue samples (Asterand) of mainly
(8/9) Caucasian origin with mixed gender and smoking status. The array detected BRAF
and KRAS mutations in the FFPE MDA-MB-231 cell line, which were independently
confirmed by DxS KRAS mutation kit and Pyrosequencing. The array also detected one
KRAS somatic mutation and one EGFR somatic mutation in two FFPE lung
adenocarcinoma samples. The EGFR mutation rate is in agreement with expected
EGFR mutation rate (~10%) in lung cancer adenocarcinoma samples of such origin.
102.5%
The majority of assays have mutation detection limit of 1% or lower. Shown below
is the detection limit performance data for the BRAF V600E mutation assay. A series
of 10 ng genomic DNA samples, which contain genomic DNA from A375 cell line
(containing BRAF V600E mutation) mixed with WT genomic DNA at different ratios, were
tested on Somatic Mutation Assay for BRAF V600E with or without whole genome
amplification (with QIAGEN Repli-g UltraFast kit). Mutation detection limit for this assay
is determined to be ~2%.
Somatic Mutation PCR Array Data Confirmed by
Pyrosequencing
EGFR
BRAF
CHART or PICTURE
39
No WGA
WGA
KRAS
37
35
Ct
qBiomarker Mutation PCR Array Technology Principles
• Allele-specific PCR amplification with ARMS (amplification refractory mutation system)
primers
33
31
29
• Mutant amplicon detection by hydrolysis probe
27
Additional
mismatch
Mutation of
interest
25
1
2
4
8
10
20
50
Human EGFR pathway somatic mutation PCR array detected BRAF, EGFR and KRAS
mutations in FFPE lung adenocarcinoma samples (Cybrdi) of Asian, female, non-smoker
origin. EGFR somatic mutations occurred in 30% of the samples, as expected. Sample
quality varies by 8 Ct as measured by gene copy assays on the array.
100
% mutant DNA
Mutant template
296-1 KRAS c.35G>T 25%
Somatic Mutation Assay Cross-reactivity Performance
Cross-reactivity tests were performed for all assay clusters containing assays that
potentially recognize another assay’s template due to imperfect base-pairing
and read-through. Shown below are the cross-reactivity test results (represented by
Ct between the Ct obtained for a mutation assay with a template and the Ct obtained
for the mutation assay with its own template) for the KRAS Codon 12 and 13 cluster
mutation assays. A Ct=0 (cells highlighted in yellow) is set for a mutation assay with its
own template. Cells containing Ct<6 values are highlighted in blue. The results indicate
that the G12V assay shows significant cross-reactivity with the G12R mutant template,
and the G13V assay shows moderate cross-reactivity with the G13A mutant template.
Wildtype template
Sample requirement
• 5-10 ng genomic DNA isolated from fresh tissues/cells for each array OR
• 200-500 ng DNA from FFPE sections for each array
Template => p.G12S p.G12R p.G12C p.G12D p.G12A p.G12V p.G13S p.G13R p.G13C p.G13D p.G13A p.G13V
Extraction
(opt.)Amplification
DNA mini or
FFPE kit
Detection
30 min
Analysis
qPCR Array
and Assay
REPLI-g for
Fresh sample
1.5hr
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
Template
2hr
10-20 min
=>
Assay
Somatic Mutation PCR Array Workflow
p.G12S
p.G12R
p.G12C
p.G12D
p.G12A
p.G12V
p.G13S
p.G13R
p.G13C
p.G13D
p.G13A
p.G13V
0.00
7.20
9.06
7.60
10.10
7.19
10.55
10.88
8.54
7.72
9.04
7.96
7.42
0.00
6.46
7.60
10.10
3.87
10.00
9.81
7.97
7.47
8.82
7.90
7.42
6.48
0.00
7.60
9.48
7.17
10.55
10.80
8.56
7.63
8.80
7.96
7.42
8.31
8.26
0.00
10.10
7.01
9.05
10.21
7.87
7.62
8.35
7.96
7.42
9.18
7.49
7.60
0.00
6.99
10.11
10.93
8.14
7.59
8.21
7.96
7.42
9.56
7.88
7.60
9.31
0.00
7.54
10.77
9.16
7.58
8.99
7.96
7.42
9.82
9.20
7.60
10.10
7.19
0.00
8.62
7.50
8.02
9.04
7.96
7.42
9.26
9.20
7.59
9.02
7.19
9.46
0.00
6.99
7.78
9.04
7.96
7.42
9.51
9.20
7.60
10.10
6.98
10.13
9.59
0.00
7.68
7.43
7.96
7.42
9.67
9.20
7.46
10.10
7.19
9.80
10.31
8.06
0.00
6.88
7.96
7.42
9.40
9.20
7.60
10.10
7.05
10.55
9.99
8.97
7.21
0.00
5.24
7.42
9.43
9.20
7.60
10.10
7.19
9.96
10.94
8.83
7.97
8.46
0.00
Human EGFR Pathway Somatic Mutation PCR Array Layout
Methods
1
2
3
4
5
6
7
8
9
10
11
12
AKT1
B
BRAF
BRAF
BRAF
BRAF
BRAF
BRAF
BRAF
BRAF
EGFR
EGFR
EGFR
p.E17K
p.G464V
p.G466V
p.G469A
p.L597V
p.V600M
p.V600E
p.V600A
p.V600G
p.G719S
p.G719C
p.G719A
EGFR
A
EGFR
EGFR
EGFR
EGFR
EGFR
EGFR
EGFR
EGFR
EGFR
EGFR
EGFR
p.E746_A750
del
p.E746_A750
del
p.E746_T751
del
p.E746_T751
>A
p.L747_A750
>P
p.E746_S752
>D
p.L747_E749
del
p.L747_S752d
el
p.L747_T751
del
p.S768I
p.V769_D770
insASV
p.D770_N771
insG
EGFR
E
EGFR
EGFR
EGFR
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
p.T790M
p.L858M
p.L858R
p.L861Q
p.Q61R
p.Q61L
p.Q61H
p.G12S
p.G12R
p.G12C
p.G12D
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
HRAS
HRAS
p.G12A
p.G12V
p.G13S
p.G13R
p.G13C
p.G13D
p.G13A
p.G13V
p.Q22K
p.Q61K
p.Q61R
HRAS
HRAS
HRAS
HRAS
HRAS
HRAS
NRAS
NRAS
NRAS
p.G13R
p.G13C
p.Q61K
p.Q61P
p.Q61R
NRAS
NRAS
NRAS
NRAS
NRAS
NRAS
MEK1
MEK1
MEK1
p.G12A
p.G13R
p.G13D
p.G13A
p.G13V
p.A18T
Q56P
K57N
D67N
PIK3CA
PIK3CA
PIK3CA
PIK3CA
PIK3CA
PTEN
PTEN
PTEN
PTEN
NRAS
c.182A>T
p.Q61L
PIK3CA
c.3140A>G
p.H1047R
p.P539R
p.E542K
p.E545K
p.E545G
p.E545D
p.H1047R
p.H1047L
p.R130*
276-2 KRAS c.35G>T 35%
3237-1 EGFR c.2236_2250del15 20%
266-1 EGFR c.2236_2250 deletion 12%
317-2 KRAS c.35G>C 10%
KRAS G12 somatic mutations and EGFR deletion mutations in these samples were
confirmed by Pyrosequencing. Representative pyrograms are shown.
Conclusions
• Utilizing allele-specific PCR amplification with ARMS primers
combined with probe detection, assays on qBiomarker™ Somatic
Mutation PCR arrays achieve sufficient specificity and sensitivity
for detecting as low as 1% mutant alleles in wild type genomic DNA
background.
• Simple workflow and sample requirement allows fresh, frozen or
FFPE samples from a variety of sources and with a wide range of
quality to be routinely profiled in any laboratory with access to
real-time PCR instruments; sample input requirement is small (as
little as 200 ng per sample/array).
• Comprehensive content coverage on each array allows for
studying mutations in the context of a pathway and enables
discovering and verifying clinical biomarkers for a variety of
human cancers involving the selected signaling pathway.
Bibliography
PIK3CA
c.1633G>A
p.E545K
• Mutation profiling results obtained on qBiomarker™ Somatic
Mutation PCR Array have been confirmed by Pyrosequencing.
PTEN
p.Q61H
p.G12S
p.G12R
p.G12C
p.G12D
p.G12V
p.R233*
p.R173C
p.R173H
p.R130Q
p.R130G
AKT1
PTEN
H
KRAS
c.38G>A
p.G13D
P124L
PIK3CA
KRAS
c.35G>T
p.G12V
MEK1
p.G12D
BRaf
c.1799T>A
p.V600E
p.Q61L
NRAS
p.G12S
PIK3CA
G
KRAS
c.38G>A
p.G13D
NRAS
NRAS
F
KRAS
c.34G>A
p.G12S
p.Q61L
HRAS
BRAF
c.1391G>T
p.G464V
HRAS
HRAS
D
EGFR
p.H773_V774
insH
KRAS
C
Somatic Mutation Array Performance in Cancer Cell Lines
256-1 KRAS c.35G>A 17%
BRAF
EGFR
KRAS
HRAS
NRAS
MEK1
PIK3CA
PTEN
SMPC
SMPC
Copy
Number
Assay
Copy
Number
Assay
Copy
Number
Assay
Copy
Number
Assay
Copy
Number
Assay
Copy
Number
Assay
Copy
Number
Assay
Copy
Number
Assay
Copy
Number
Assay
Positive PCR
Control
Positive PCR
Control
Mutations were chosen from curated, comprehensive somatic mutation databases and
peer-reviewed scientific literature. Assays were designed to detect the most frequent,
functionally verified, and biologically significant mutations in the particular pathway. The
content of the human EGFR pathway array is presented above.
References
• Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Newton CR,
Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Nucleic Acids Res.
1989 Apr 11;17(7):2503-16
200 ng of gDNA from WT or well-characterized cancer cell lines was profiled on the
human EGFR somatic mutation PCR array. All previously identified mutations in the EGFR
pathway in these cell lines were called correctly using the Ct method.
A01-H01: mutation assays; H02-H10: gene copy assays; SMPC: PCR control assay
• Targeting the EGFR and the PKB pathway in cancer. Klein S, Levitzki A. Curr Opin Cell Biol. 2009 Apr;
21(2):185-93
• Update on HER-2 as a target for cancer therapy: intracellular signaling pathways of ERBB2/HER-2 and family
members. Olayioye MA. Breast Cancer Res. 2001; 3(6):385-9.
• Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations in acute
promyelocytic leukemia: a systematic review. Beitinjaneh A, Jang S, Roukoz H, Majhail NS. Leuk Res. 2010
Jul; 34(7):831-6.
• Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumours. Lasota J,
Miettinen M. Histopathology. 2008 Sep; 53(3):245-66.
AACR Abstract # 1157
The qBiomarker Somatic Mutation PCR Arrays are intended for molecular biology applications. They are neither intended for the
diagnosis, prevention, or treatment of a disease, nor have been verified for such use either alone or in combination with other products.
Corresponding author email: Samuel.Long@qiagen.com
Sample & Assay Technologies