Accurate detection of low-frequency somatic mutations as well as low level structural variants such as copy number variation (CNV) in circulating cell-free DNA (cfDNA) using blood samples from subjects previously diagnosed with cancer provides a potential non-invasive approach to monitor cancer status and evaluate cancer evolution in the future. We have previously reported the Oncomine™ Breast cfDNA Assay enables detection of somatic mutations in plasma down to a level of 0.1% variant allelic frequency in breast cancer relevant genes. Here we extend this technology to simultaneously detect single nucleotide variants (SNVs) as well as copy number variation (CNV) from a single cfDNA sample.

1. Thermo Fisher Scientific • Street Address • City, ST ZIP Code • thermofisher.com
Yanchun Li, Jian Gu, Jeff Schageman, Dima Brinza, Kris Lea, Madhu Jasti, Priyanka K. Kshatriya, Varun Bagai, Kelli S. Bramlett.
Thermo Fisher Scientific, 2130 Woodward St, Austin, Texas, USA, 78744
Ion Torrent™ Next Generation Sequencing – Detect 0.1% Low
Frequency Somatic Variants and Copy Number Variations
simultaneously in Cell-Free DNA
RESULTS
ABSTRACT
Genetic variation information from cell-free DNA (cfDNA) will
provide valuable opportunities in cancer research and impact
future oncology treatment and diagnosis. We have previously
reported the Oncomine™ Breast research cfDNA Assay
enables detection of somatic mutations in plasma down to a
level of 0.1% variant allelic frequency in breast cancer relevant
genes. Here we extend this technology to simultaneously
detect single nucleotide variants (SNVs) as well as structural
variants such as copy number variation (CNV) from a single
cfDNA sample. cfDNA was extracted from purchased normal
donor blood plasma using MagMAX™ Cell-Free DNA Isolation
Kit.
To evaluate sensitivity and specificity of SNV detection, we
used reference DNA containing 0.1% or 0.5% variant allelic
frequency based on fragmented AcroMetrixTM Oncology
Hotspot Control material that mimicked the size of cfDNA (
MM). The variant frequencies of the reference DNA were
verified with dPCR. For CNV sensitivity and specificity
evaluation, cfDNA from CNV positive cell lines were isolated
and titrated into normal donor plasma cfDNA background and
copy number status was verified with dPCR. Libraries were
prepared manually, templated with Ion Chef automated
templating instrument (Thermo Fisher Scientific) and
sequenced with Ion S5 Sequencing instrument (Thermo Fisher
Scientific). Data analysis was performed using the
variantCaller plugin in Torrent Suite™ Software for SNV target
and Ion Reporter for CNV analysis.
The update Oncomine™ Breast cfDNA Assay v2 including
CNV analysis extend our current Oncomine™ Breast cfDNA
Assay from low frequency SNV detection to both SNV and
CNV detection. The new panel covers 152 hotspots in breast
cancer related genes (AKT1, EGFR, ERBB2, ERBB3, ESR1,
FBXW7, KRAS, PIK3CA, SF3B1, TP53) plus 3 CNV regions
(ERBB2, FGFR1 and CCND1) commonly related to breast
cancers. Additionally it extended the coverage of tumor
suppressor gene TP53 to more than 80% coding region. This
new breast panel is optimized for low allelic frequency
detection at 0.1% in hotspot region and CNV detection as low
as 1.2 fold amplification (equivalent to 10% tumor frequency at
3 fold) from cfDNA.
The Oncomine™ Breast cfDNA Assay v2 provides an easy,
quick and reliable solution for researchers in detecting both low
frequencies SNV and CNV simultaneously in cfDNA.
INTRODUCTION
Accurate detection of low-frequency somatic mutations as well
as low level structural variants such as copy number variation
(CNV) in circulating cell-free DNA (cfDNA) using blood
samples from subjects previously diagnosed with cancer
provides a potential non-invasive approach to monitor cancer
status and evaluate cancer evolution in the future. We have
previously reported the Oncomine™ Breast cfDNA Assay
enables detection of somatic mutations in plasma down to a
level of 0.1% variant allelic frequency in breast cancer relevant
genes. Here we extend this technology to simultaneously
detect single nucleotide variants (SNVs) as well as copy
number variation (CNV) from a single cfDNA sample.
MATERIALS AND METHODS
Plasma preparation: Blood samples were collected into BD K2
EDTA tubes and Streck Cell-Free DNA BCT tubes following
manufacturer’s instructions by a third-party vendor. Blood
samples were shipped overnight on cold gel packs to the lab.
Immediately after receiving the samples, plasma from blood
sample was obtained by centrifugation at 1600 x g for 10 min
at 4C, followed by another spin at 6,000 x g for 30 min at 4 C
to remove any residual blood cells.
cfDNA isolation: cfDNA was isolated from ~4-mL of plasma
using MagMAX Cell-Free DNA Isolation Kit following
alternative protocol in User Guide.
CNV reference control :
cfDNA from CNV positive cell lines were isolated and titrated
into normal donor plasma cfDNA background and copy number
status was verified with dPCR.
Library preparation: OncomineTM Breast cfDNA Assay v2 was
used to prepare libraries from cfDNA following standard user
guide.
Templating and Sequencing: Ion ChefTM System and Ion 530TM
Kit-Chef were used for template preparation, followed by
sequencing on Ion S5 system using Ion 530 chips. Generally,
5-plex library pool ran on one Ion 530 chip.
Data analysis: Data analysis was performed in Torrent SuiteTM
5.4 and Ion Reporter 5.6.
Table 1. Comparison of Oncomine™ Breast cfDNA
Assay v1 and v2
Library preparation
with OncomineTM
cfDNA Breast Assay
MagMax
cell free DNA
extraction
single blood
tube
Sequencing of
sample on Ion S5
Sequencer
Analyze data in Ion
TSS/IR software to
generate test report
Figure 1. Workflow for cfDNA analysis using
Oncomine Breast cfDNA Assay
A31183: Oncomine Breast cfDNA
Assay ( 2016)
,
A35865: Oncomine Breast cfDNA
Assay v2 ( 2017)
12 Genes
10 Genes with key hotspot mutations
76 Amplicons
>152 Hotspot SNVs and Indels
CNVs CCND1, ERBB2, FGFR1
detect fold change 1.2-1.4x
TP53 More complete coverage
0.5% LOD on de novo sequencing
Single library (SNVs and CNVs)
SNV LOD down to 0.1% with 20 ng input
Same sensitivity & specificity
5-plex on Ion 530™ Chip
20-plex on Ion 540™ Chip
AKT1
CCND1
EGFR
ERBB2
ERBB3
ESR1
FBXW7
FGFR1
KRAS
PIK3CA
SF3B1
TP53
AKT1
EGFR
ERBB2
ERBB3
ESR1
FBXW7
KRAS
PIK3CA
SF3B1
TP53
cfDNA
10 Genes
10 Genes with key hotspot mutations
26 Amplicons
152 Hotspot SNVs and Indels
Critical hotspots include:
• AKT1: E17K
• ERBB2: mutations associated with
sensitivity to anti-ERBB2 therapies
• ESR1: mutations associated with
anti-estrogen resistance
• PIK3CA: E545K, H1047R
• TP53: mutations associated with
loss of function
12-plex on Ion 530™ Chip
48-plex on Ion 540™ Chip
user
Median
Read
Cov
Median
Mol
Cov
LOD % TP FP
sensitivity specificity
User 1 32940 5427
0.0276 -
0.0332
17 0 100.0% 100.0%
User 2 36469 4781
0.0314 -
0.0367
17 0 100.0% 100.0%
User 3 22582 3920
0.0383 -
0.0424
17 0 100.0% 100.0%
User 4 21609 3831
0.0392 -
0.044
17 0 100.0% 100.0%
average 100.0% 100.0%
Sample
Median
Read
Cov.
Median
Mol.
Cov.
LOD% TP FP Sensitivity Specificity
User 1
rep 1
45,034 5,417
0.0277 -
0.0331
22 0 88.0% 100.0%
User 1
rep 2
44,462 5,408
0.0277 -
0.0328
24 0 96.0% 100.0%
User 1
rep 3
45,893 5,427
0.0276 -
0.0337
21 0 84.0% 100.0%
User 1
rep 4
48,288 5,292
0.0283 -
0.0333
22 1 88.0% 99.2%
User 2
rep 1
49,191 5,561
0.027 -
0.0327
25 0 100.0% 100.0%
User 2
rep 2
46,807 5,436
0.0276 -
0.0324
24 0 96.0% 100.0%
User 2
rep 3
50,753 5,578
0.0269 -
0.0325
22 0 88.0% 100.0%
User 2
rep 4
47,493 5,531
0.0271 -
0.0325
25 0 100.0% 100.0%
User 3
rep 1
45,252 5,718
0.0262 -
0.0314
23 0 92.0% 100.0%
User 3
rep 2
41,377 5,466
0.0274 -
0.0315
22 0 88.0% 100.0%
User 3
rep 3
46,337 5,644
0.0266 -
0.0309
25 0 100.0% 100.0%
User 3
rep 4
50,863 6,002
0.025 -
0.0307
25 1 100.0% 99.2%
User 4
rep 1
47,939 5,772
0.026 -
0.0303
25 0 100.0% 100.0%
User 4
rep 2
48,282 5,542
0.0271 -
0.032
24 0 96.0% 100.0%
User 4
rep 3
39,658 5,185
0.0289 -
0.0328
22 0 88.0% 100.0%
User 4
rep 4
48,464 5,720
0.0262 -
0.0306
21 0 84.0% 100.0%
Average 93.0% 99.9%
Sample ID MAPD (QC)
ERBB2
1.3X
FGFR1
1.3X
CCND1
1.4X
user1-rep1 0.081 1.31 1.35 1.46
user1-rep2 0.071 1.29 1.26 1.41
user1-rep3 0.079 1.29 1.26 1.45
user1-rep4 0.08 1.31 1.32 1.5
user1-rep5 0.124 1.35 1.31 1.41
user2-rep1 0.1 1.31 1.26 1.4
user2-rep2 0.146 1.26 1.3 1.54
user2-rep3 0.079 1.33 1.3 1.39
user2-rep4 0.075 1.23 1.31 1.46
user2-rep5 0.098 1.3 1.29 1.42
user3-rep1 0.106 1.31 1.35 1.46
user3-rep2 0.078 1.29 1.26 1.41
user3-rep3 0.07 1.29 1.26 1.45
user3-rep4 0.113 1.31 1.32 1.5
user3-rep5 0.1 1.35 1.31 1.41
user4- rep1 0.126 1.33 1.27 1.5
user4- rep2 0.119 1.34 1.24 1.47
user4- rep3 0.124 1.31 1.27 1.45
user4- rep4 0.095 1.32 1.26 1.35
user4- rep5 0.216 1.3 ND ND
Average Sensitivity 100% 96% 96%
Sample ID MAPD (QC) CNV Call
EDTA-cfDNA-S1 0.112 ND
EDTA-cfDNA-S2 0.103 ND
EDTA-cfDNA-S3 0.116 ND
EDTA-cfDNA-S4 0.105 ND
EDTA-cfDNA-S5 0.145 ND
Streck-cfDNA-S1 0.09 ND
Streck-cfDNA-S2 0.103 ND
Streck-cfDNA-S3 0.094 ND
Streck-cfDNA-S4 0.122 ND
Streck-cfDNA-S5 0.11 ND
Average Specificity 100%
Input
Median
Read
Cov
Median
Mol
Cov
ERBB2
1.2x
Median
Read
Cov
Median
Mol
Cov
CCND1
1.4x
20ng 56046 3565 1.19 40033 2938 1.52
10ng 48250 1830 1.22 35028 1497 1.52
5ng 32952 874 1.17 29069 764 1.43
2ng 17760 361 1.19 19450 340 1.49
1ng 13551 290 ND 11720 220 1.58
CN target
Taqman
Assay
dPCR Average STD
Oncomine
Breast
cfDNA
assay
CCND1
C1 7.92
7.4 1.19
6.54
C2 5.95
C3 8.72
6.17
C4 7.02
CCND1
titration
C1 1.83
1.79 0.26
1.89
C2 1.43
C3 2.04
2
C4 1.85
FGFR1
F1 4.82
5.54 0.98
5.53
F2 4.67
F3 6.76
5.13
F4 5.91
FGFR1
titration
F1 1.37
1.59 0.16
1.48
F2 1.64
F3 1.76
1.49
F4 1.59
CONCLUSIONS
The Oncomine™ Breast cfDNA Assay v2 provides an easy,
quick and reliable solution for researchers in detecting both
low frequencies SNV and CNV simultaneously in cfDNA.
 SNVs/short indels, an LOD of 0.1% can be achieved with
sensitivity of >90% and specificity of >99%
 For TP53 mutations(~80% coverage of full length gene), an
LOD of 0.5% can be achieved
 For ERBB2 and FGFR1 CNV targets, detection as low as
1.2-fold amplification can be achieved and for CCND1 CNV
target, detection as low as 1.4-fold amplification can be
achieved with sensitivity of >90% and specificity of >99%
TRADEMARKS/LICENSING
For Research Use Only. Not for use in diagnostic
procedures.
© 2017 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.
We utilized 0.1% fragmented AcroMetrix Oncology Hotspot control
material (MM) to determine SNV sensitivity and specificity. The allelic
frequency of 0.1% MM was verified with dPCR assay. Sensitivity and
specificity measurements were carried out at two independent test sites
with multiple independent instruments (five S5 sequencing systems and
five Ion Chef templating instruments) and four individuals users. Following
equations were used to calculate sensitivity and specificity:.
SENSITIVITY = TP/(TP+FN)%
SPECIFICITY = TN/(TN+FP)%
The Oncomine™ Breast cfDNA
Assay v2 is a content-optimized
solution for NGS cfDNA liquid
biopsy research. It includes both
library prep and analysis solutions
that enable a blood sample to
report workflow completed in 2
days.
The Oncomine™ Breast cfDNA Assay v2 enables the analysis of:
previous v1 content with new addition of Copy Number genes
CCND1, ERBB2, FGFR1 and a extended coverage in tumor
suppressor gene TP53 (~80% full length coding region )
Table 2 Detection Sensitivity and Specificity for SNV using
0.1% MM
Table 4 Detection Sensitivity for ERBB2, FGFR1 and CCND1
copy number using CNV positive cfDNA control
Table 6 cfDNA input s study for ERBB2 and CCND1 CN
detection
Sensitivity and specificity evaluation for extended TP53 coverage
utilized 0.5% fragmented AcroMetrix Oncology Hotspot control material
(allelic frequencies verified with dPCR assay) following previously
described multi- sites/equipments/users tests.
To evaluate ERBB2, FGFR1 and CCND1 CN status, we titrated cfDNA
isolated from CN positive cell lines harbored either ERBB2 or
FGFR1/CCND1 into normal donor plasma cfDNA background down to
1.3~1.4 fold with dPCR verified CN status . Sensitivity evaluation
followed previously described multi-sites/equipments/users tests. ND:
not detected.
Table 5 Detection Specificity for ERBB2, FGFR1 and CCND1
copy number using cfDNA from normal donor plasma
Specificity of CN detection used normal donor cfDNA to minimize
cell line cfDNA background noise . ND: not detected.
Table 3 Detection Sensitivity and Specificity for extended
TP53 SNV coverage using 0.5% MM
CNV positive control cfDNA (ERBB2 1.2X fold and CCND1 1.4X fold) were
used to test input range from 1ng-20ng. The assay could detect ERBB2
1.2X as low as 2ng cfDNA and CCND1 1.4X at 1ng cfDNA
Table 7 CNV positive cfDNA control verified with
orthogonal dPCR assay
dPCR assays were used to quantify FGFR1 and CCND1 CN status
using multiple TaqMan® probes targeting to the specific genes. dPCR
assay showed consistent CN calling results as Oncomine™ Breast
cfDNA Assay v2. ND: not detected.