Somatic variant detection in oncology research may provide insight into drug response and helps identify new therapeutic targets in the future. A key limitation to this process is the availability of tumor tissue that adequately represents the current disease status. Development of a robust method for detecting mutational status from a blood sample would enable analysis of tumor changes over time, which is impractical using tissue samples. A workflow is presented for analyzing somatic variants in circulating tumor cell (CTC) samples using next-generation sequencing (NGS).

1. Gavin Meredith, Christopher Davies, Yongming Sun, Kristina Giorda, Chaitali Parikh, and Warren Tom (Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 94080 USA)
Cristian Ionescu-Zanetti, Michael Schwartz, Andrea Fan, Christine Fu, and Tony Tran (Fluxion Biosciences, 385 Oyster Point Blvd., Suite #3, South San Francisco, CA 94080 USA)
FIGURE 2. FLUXION IsoFlux™ TUMOR CELL ENRICHMENT
FROM BLOOD
ABSTRACT
Introduction:
Somatic variant detection in oncology research may provide insight into drug
response and helps identify new therapeutic targets in the future. A key limitation to
this process is the availability of tumor tissue that adequately represents the current
disease status. Development of a robust method for detecting mutational status
from a blood sample would enable analysis of tumor changes over time, which is
impractical using tissue samples. A workflow is presented for analyzing somatic
variants in circulating tumor cell (CTC) samples using next-generation sequencing
(NGS).
Methods:
CTC enrichment - CTCs were enriched from whole blood using an
immunomagnetic microfluidic capture device (IsoFlux System) and a combination of
EpCAM and EGFR capture antibodies. Recovered cells were purity-enhanced,
lysed, and DNA was amplified.
NGS – Libraries were produced with the Cancer Hotspot Panel v2 (Thermo Fisher
Scientific) that includes coverage of 50 tumor-associated genes. The Ion PGMTM
utilizes semiconductor next-generation sequencing to rapidly sequence and detect
somatic variants.
Variant analysis - A custom data analysis workflow is described, including sequence
alignment, variant filtering, annotation, and report generation.
Results and Discussion:
The somatic variant detection workflow demonstrated analytical sensitivity down to
3 cells / mLblood. In spike-in experiments, a titrated range of tumor cell line (MDA-
MB-231) was added to peripheral whole blood (0-180 cells spiked per 8mL tube).
Average cell recovery for this low-EpCAM expressing, mesenchymal-like cell line
was 66% (range: 54-73%). Average tumor cell purity was 12% (range: 5-22%).
Analysis of mutations in the target cell-line demonstrated detection of KRAS (8/8
samples), TP53 (7/8 samples), BRAF (8/8 samples) and PDGFRA (8/8) at 97%
sensitivity and 97% PPV. Results were confirmed with a sensitive, allele-specific
qPCR assay. Analysis of a cohort of 10 clinical research samples demonstrated
somatic variant detection in a majority of the samples with no false positives coming
from 5 normal controls.
CONCLUSIONS
• The combination of Fluxion IsoFLuxTM, Ion AmpliSeqTM and the Ion PGMTM
System is a complete solution for translational oncology research studies.
• Fluxion IsoFluxTM System resulted in tumor cell purities of 5-22% when starting
with 2-12 breast tumor cells per ml of blood. CTC recovery rates were 53-72%
using the IsoFluxTM System.
• The detection of four know variants across a range of cell concentrations
demonstrated 97% sensitivity and 97% positive prediction value (PPV).
• Analysis using Ion ReporterTM v4.4 software with enhanced sensitivity
parameters enable variant calls down to a 1.0% allele ratio compared to
standard settings.
• For more information regarding this presentation, please contact Rob Bennett
(rob.bennett@thermofisher.com).
ACKNOWLEDGEMENTS
We would like to thank Dr. Ajjai Alva (University of Michigan), and Drs. Terence
Friedlander and Pamela Paris (UCSF) for providing samples for this study.
REFERENCES
1. For more information please visit www.ampliseq.com
2. For more information on Ion Torrent™ Systems, please visit
www.iontorrent.com
For Research Use Only. Not for use in diagnostic procedures.
Somatic variant detection from circulating tumor cells using targeted
next-generation sequencing
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.comFor Research Use Only. Not for use in diagnostic procedures.
FIGURE 7. CTC CAPTURE AND SEQUENCING RESULTS USING
BLADDER CANCER RESEARCH SAMPLES
FIGURE 7. CTCs from neoadjuvant (before cystectomy) and metastatic bladder
cancer research samples were enriched using the IsoFluxTM system with EpCAM
antibodies. Variants were detected using Ion ReporterTM v4.4 software with
enhanced variant calling parameters. All 3 expected variants in the 2 positive
controls were detected, with no false positives were detected in the negative
controls. For these clinical research samples, 4/8 (50%) had at least one somatic
variant detected.
LIBRARY PREPARATION AND SEQUENCING METHODS
Genomic DNA was amplified using whole genome amplification (Repli-g™, Qiagen).
Ion AmpliSeq™ libraries (Figure 1) were prepared using 10 ng DNA input with the Ion
AmpliSeq™ Cancer Hotspot Panel v2 (PN 4475346) and the Ion AmpliSeq™ Library
Kit 2.0 (PN 4475345) according to the product manual. The library was purified with
AMPure™ XT beads and quantitated by qPCR. A dilution of the library was then used
to template Ion Sphere™ Particles (ISPs) using the Ion OneTouch™ 2 instrument (PN
4474779). Enriched ISPs were loaded onto a 318™ Chip v2 and sequenced for 500
flows using the Ion PGM™ Sequencing 200 Kit v2 (PN 4482006). Analysis was
performed with the Ion Torrent Suite™ Software v.4.2 and Ion Reporter™ v4.4.
SAMPLE
IDENTIFIER
CTC COUNT CTC PURITY
SOMATIC VARIANTS DETECTED
(allelic frequency)
Healthy #1 0 0% None
Healthy #2 0 0% None
Healthy #3 0 0% None
Healthy #4 0 0% None
Neoadjuvant #1 40 10% FGFR2 (1%)
Neoadjuvant #2 100 19% PDGFRA (1%)
Neoadjuvant #3 20 12% None
Neoadjuvant #4 21 8% EGFR (6%)
Metastatic #1 154 25% JAK2 (12%)
Metastatic #2 34 4% None
Metastatic #3 11 15% None
Metastatic #4 8 N/D None
MDA-MB-231
Spike In (Pos
Control) #1 N/D N/D KRAS (6%), BRAF (7%), TP53 (12%)
MDA-MB-231
Spike In (Pos
Control) #2 60 8% KRAS (4%), BRAF (6%), TP53 (11%)
Proof of Principle Experimental Design (Spike-In Study)
Mixture study of MDA-
MB-231 cells into
healthy donor whole
blood
triplicate IsoFlux™
capture
0 - 314 CTCs in 14 mL
whole blood
Enumerate
spiked cells vs.
total nuclei
CTC Recovery Rates:
53-72% (32-169 CTCs)
CTC Purity: 5-22%
single
WGA with
REPLI-g™
duplicate
Cast-PCR™
KRAS G13D
Ion
AmpliSeq™
CHPv2
Sample Processing
Cell Enrichment
Cell Retrieval
IsoFlux™ Isolation
Magnetic bead based positive selection
Current Applications
• Enumeration
• Immunofluorescence
• In situ hybridization
• Mutation detection
• Gene expression
• Next Gen sequencing
FIGURE 1. ION AMPLISEQ™ CANCER HOTSPOT PANEL V2
FIGURE 1. The Ion AmpliSeqTM technology utilizes a technology to rapidly
enrich hundreds to thousands of mutations at low allele frequencies with very
low sample input requirements (10ng or less) and minlmal hands on time. The
Ion AmpliSeqTM technology can support multiplex amplification reactions from
12-24,000 amplicons in a single reaction. The Ion AmpliSeqTM Cancer Hotspot
Panel v2 (CHPv2) uses a single-tube assay to detect 739 mutations in 190
amplicons covering relevant regions of key tumor genes.
FIGURE 2. The IsoFluxTM System utilizes magnetic beads targeted towards
antigens expressed on the cell surface. Following removal of the red blood
cells by Ficoll, the remaining monocyte fraction is incubated with IsoFluxTM
magnetic beads functionalized with one or more types of antibodies (e.g.
EpCAM, EGFR, etc.). Cells with magnetic beads are then isolated by a
magnet using the IsoFluxTM cartridge.
FIGURE 3. MDA-MB-231 PROOF-OF-PRINCIPLE STUDY
FIGURE 4. MDA-MB-231 AND PBMC GENOTYPES
FIGURE 4. The breast tumor cell line MDA-MB-321 and peripheral blood
mononuclear cells (PBMCs) have different variants at the above positions,
which allows the prediction of allelic frequencies in the spike-in experiments.
The allelic positions shown for PDGFRA, BRAF, KRAS, and TP53 are
detectable by the Ion AmpliSeqTM Cancer Hotspot Panel.
FIGURE 5. Allele ratios (%) for all detected polymorphisms are shown for
each of the estimated CTC purities. Low frequency variants were detected
using Ion ReporterTM v4.4 software with enhanced variant calling
parameters. Results from two independent sites are shown.
FIGURE 5. RESULTS SUMMARY OF SPIKE-IN EXPERIMENTS
FIGURE 6. CONCORDANCE OF KNOWN VARIANTS IN MDA-MB-231
Site 1 Variant Frequency
Site2VariantFrequency
FIGURE 6. The same analytical samples were sequenced at two facilities,
Thermo Fisher Scientific and the UCSF Genome Center Core. Concordance of
the variant calling frequencies from both sites was very high ( R2=0.94).
MDA-MB231 Spike-In PBMA
GENE CHR POS REF ALT REF COUNT ALT COUNT REF ALT
PDGFRA 4 55152040 C T 2391 1331 C -
BRAF 7 140481417 C A 1161 1755 C -
KRAS 12 25398281 C T 986 1924 C -
TP53 17 7577099 C T 14 2462 C -