1 For Research Use Only. Not for use in diagnostic procedures. The world leader in serving scienceFor Research Use Only. Not for use in diagnostic procedures.
Dumitru Brinza, Ph.D
Clinical Next-Generation Sequencing Division
Developing a multiplex next generation sequencing assay to study highly clonal tumor samples
2 For Research Use Only. Not for use in diagnostic procedures.
1 – 25 Genes > 100 Genes26 – 100 Genes
Oncomine Comprehensive Assay
143 genes, hotspots, indels, CNVs,
Fusions
Ion AmpliSeq Comprehensive
Cancer Panel
400 genes
Oncomine Focus Assay
50 DNA genes + 23 RNA genes;
hotspots, indels, CNVs, Fusions
Ion AmpliSeq Cancer
Hotspot Panel v2
50 genes; hotspots, indels
Oncomine cfDNA Lung Assay
11 genes, hotspot driver mutations
Oncomine BRCA Research Assay
Somatic mutations + germline
Ion AmpliSeq AML Research Panel
19 genes
Oncomine Myeloid Research Panel
coming in September 2017
Oncomine cfDNA Colon Assay
14 genes, hotspot driver mutations
Oncomine cfDNA Breast Assay
10 genes, hotspot driver mutations
Oncomine Immune Response
Research Assay
395 genes, expression assay
Ion AmpliSeq Custom Panels
Oncomine Childhood Cancer
Research Panel
(In Development)
Thermo Fisher Scientific’s NGS Oncology Portfolio
Category
Solid tumor profiling
Heme-Onc profiling
Liquid biopsy
cfDNA solutions
Immuno-Oncology
(IO) solutions
Custom Oncology
solutions
Oncomine Tumor Mutation Burden
Research Assay
(In Development)
Oncomine Immune Repertoire Research
Assay
Enhanced Oncomine cfDNA Breast Assay
10 genes, hotspots, indels, novel TP53, CNVs
coming in September 2017
Oncomine Lung cell free Total Nucleic
Acid Assay
11 genes, hotspots, indels, CNVs, Fusions
coming in September 2017
Oncomine cfTNA PanCaner Assay
(In Development)
3 For Research Use Only. Not for use in diagnostic procedures.
Lab-created
Report
Oncomine™
Knowledgebase
Reporter
Oncomine cfTNA/cfDNA Assays – 2x8hr Clinical Research Workflow
Template PrepcfTNA/cfDNA
IsolationSequencing
MagMAX™ cfTNA
Isolation Kit
Ion S5™ and Ion
S5™ XL SystemsAlso enabled on Ion
PGM™ and Ion Proton™
Systems
Analysis
Variant caller in
Torrent Suite™ and
Ion Reporter™
Software
Library Prep
Oncomine™ cfDNA
or cfTNA Assays
Ion Chef™ System
Single blood tube Refined variant data
cfTNA and cfDNA Assays include library prep and analysis
• Compatible with cfTNA, cfDNA and FFPE input amounts down to 1ng
• Validated limit of detection (with 20ng input) down to:
• Validated using a single tube of blood
• Targeted, tumor type-specific panels allow maximum multiplexing, driving down sequencing costs
• In development, PanCancer Assay allows to maximize content
• Integrated Analysis and Reporting Software
Hotspot (SNV,Indel) TP53 whole target (SNV,Indel) RNA Fusions CNV
0.1% 0.5% 1% 2%(CN >10) or 5%(CN >4)
4 For Research Use Only. Not for use in diagnostic procedures.
Core Technology and Variant Analysis Method
cfDNA molecule with tumor variant Wild type cfDNA molecule
Variant
Gene Specific Primer
Molecular tagsAssay
Analysis
Amplification, tagging
and sequencing
Variant calling
5 For Research Use Only. Not for use in diagnostic procedures.
NEW Oncomine™ Lung & Breast cfTNA/cfDNA – September 2017
The Oncomine cfTNA and cfDNA Assays are designed to detect primary tumor drivers and resistance mutations identified
by the Oncomine™ Knowledgebase, a cancer genomics data resource, and reviewed by our trained professionals.
ENHANCED CONTENT
• Amplicons: 58
• Key hotspot mutations in 11 genes
• Increase in hotspot SNVs & indels
• Fusions (49) – ALK, RET, ROS1
• CNV – MET
• MET exon 14 skipping (3)
• Single library (DNA & RNA)
SNV
• LOD down to 0.1% with 20 ng input
• Same sensitivity & specificity
• 6-plex on a Ion S5 530™ Chip
• 24-plex on a Ion S5 540™ Chip
Oncomine™ Lung cell free
Total Nucleic Acid Assay
ALK
BRAF
EGFR
ERBB2
KRAS
MAP2K1
MET
NRAS
PIK3CA
ROS1
TP53
Enhanced Oncomine™ Breast v2
cfDNA Assay
AKT1
EGFR
ERBB2
ERBB3
ESR1
FBXW7
KRAS
PIK3CA
SF3B1
TP53
ENHANCED CONTENT
• Amplicons: 76
• Key hotspot mutations in 10 genes
• Increase in hotspot SNVs & indels
• CNVs – CCND1, ERBB2, FGFR1
• More complete coverage of TP53
• Single library to detect SNVs and
CNVs
SNV
• LOD down to 0.1% with 20 ng input
• Same sensitivity & specificity
• 5-plex on a Ion S5 530™ Chip
• 20-plex on a Ion S5 540™ Chip
6 For Research Use Only. Not for use in diagnostic procedures.
Biological Limitations of cfDNA Analyses
Limit of Detection 1% 0.1% 0.05%
cfDNA (ng) 1 20 30
Mutant Molecules 3 6 4
“Usable” Mutant Molecules 1-2 3 2*
Biological limit of detection with cfDNA template
*Below 0.05%, there may not be any molecules present for analysis even with large input amounts
Oncomine cfDNA Assays enable detection of somatic mutations
down to biological limitation of the sample
=~
10 mL blood 20 ng cfDNA 6 mutant molecules
present at 0.1%
Specificity >99% at 0.1% LOD
=~
7 For Research Use Only. Not for use in diagnostic procedures.
Testing Limits of Detection with Higher amount of input cfDNA
1 ng cfDNA – 0.6% LOD
5 ng cfDNA – 0.25% LOD
10 ng cfDNA – 0.15% LOD
20 ng cfDNA – 0.1% LOD
40 ng cfDNA – 0.05% LOD
100ng cfDNA – 0.01% LOD
0,60%0,40%0,25%
0,15%
0,11%
0,09%
0,05%
0,01%
0
10
20
30
40
50
60
70
80
90
100
0
20.000
40.000
60.000
80.000
100.000
120.000
0,00% 0,20% 0,40% 0,60%
Limit of Detection
PUSH THE LIMIT
ng o
f in
pu
t cfD
NA
Min
imu
m r
ea
d c
ove
rage
*Data based on fragmented Acrometrix Oncology Hotspot Control diluted into fragmented gDNA from
the background GM24385 to create mixtures with variants at 0.01% to 1% frequency
Oncomine cfDNA Assays maintain sensitive performance
with larger amount of input cfDNA
Specificity >99% at 0.1% and 0.01% LOD
8 For Research Use Only. Not for use in diagnostic procedures.
• Acute myeloid leukemia (AML) – clones at frequency <0.01% commonly
represent a significant, if not the main, clone at the time of relapse.
• 7 Million White and 5 Billion Red Blood Cells per 1mL of blood – input DNA is
not a limiting factor
Acute myeloid leukemia (AML)
Ion S5 sequencer Oncomine Lung cfDNA Panel 540 Chip
(~80 million reads)
300,000 copies (1000ng)
gDNA or cfDNA control
Detected >250,000 ssDNA molecules
Experimental setup:
Demonstrated feasibility for 0.001% LOD
9 For Research Use Only. Not for use in diagnostic procedures.
Sample EGFRE746_A750d
elELREA
EGFRL858R
EGFRT790M
EGFRV769_D770i
nsASV
KRASG12D
NRASA59T
NRASQ61K
PIK3CAE545K
0.1% HDX 0.06 0.17 0.06 0.10 0.22 0.17 0.15 0.10
1% HDX 0.72 1.07 0.75 0.74 1.14 1.15 1.15 2.29
5% HDX 4.52 4.86 6.32 3.97 6.34 6.11 6.94 5.29
100% WT 0 0 0 0 0 0 0 0
Variants Called from Horizon cfDNA Multiplex Reference Set
Expected
allelic
frequency
Detected allelic frequency (%)
by Oncomine Lung cfDNA Assay Workflow
Limit of Detection 0.1%
Average Sensitivity 94.8%
Average Specificity 99.8%
Onconetwork Consortia Report
10 For Research Use Only. Not for use in diagnostic procedures.
Retrospective Research study with Oncomine Lung cfDNA Assay
Presented at IAP / ESP Congress, September 29th 2016, by Jose Luis Costa
0.08%
0.15%
2.55%
0.07%
0.65%
0
0,5
1
1,5
2
2,5
3
Dx 1 2 3 4 5 6 7 8 9 10 11 12 13
AF
(%
)
Month
cfDNA alterations
EGFR del15
KRAS
EGFR T790M
0
11 For Research Use Only. Not for use in diagnostic procedures.
Oncology Informatics – Providing Sample-to-Answer Workflow
Prioritize variants, find the relevant few
Streamlines sequencing
runs and creates BAM files
Start your targeted
sequencing analysis
Sequencing reads to BAM file
Link variants to relevant,
published evidence
Quickly create custom,
lab-generated reports
VCF file to PDF report
Calls variants,
identifies and annotates
cancer drivers
Identify likely driver variants
BAM file to VCF file
Torrent Suite™ Software Oncomine Knowledgebase
ReporterIon Reporter Software
with Oncomine™ plug-in
What does
it do?
What is it?
Benefit
Data
journey
Create custom reportsSequencing analysis Annotate and filter
For Research Use only. Not for use in diagnostic procedures.
Oncomine Knowledgebase
Reporter
12 For Research Use Only. Not for use in diagnostic procedures.
Oncomine Knowledgebase Reporter
Quickly create custom, lab-generated reportsOncomine Knowledgebase
Reporter
13 For Research Use Only. Not for use in diagnostic procedures.
In Development Pan-Cancer v4 Gene List 08-02-2017
Hotspot Genes
Tumor
Suppressor
Genes
Copy
Number
Genes
Gene
Fusions
AKT1
ALK
ARAF
BRAF
CHEK2
CTNNB1
DDR2
EGFR
ERBB2
ERBB3
ESR1
FGFR1
FGFR2
FGFR3
FGFR4
FLT3
GNA11
GNAQ
GNAS
HRAS
IDH1
IDH2
KIT
KRAS
MAP2K1
MAP2K2
MET
MTOR
NRAS
NTRK1
NTRK3
PDGFRA
PIK3CA
RAF1
ROS1
SF3B1
SMAD4
SMO
APC
ATM
FBXW7
PIK3R1
PTCH1
PTEN
TP53
TSC1
TSC2
CCND1
CCND2
CCND3
CDK4
CDK6
EGFR
ERBB2
FGFR1
FGFR2
FGFR3
MET
MYC
ALK
BRAF
ERG
ETV1
FGFR2
FGFR3
MET
NTRK1
NTRK3
RET
ROS1
Assay Configuration Unique Genes DNA RNA
Pan-Cancer v4 TNA (DNA + RNA) 56 53 11Covering:
• Key hotspot mutations
• Fusions
• CNVs
• MET exon 14 skipping
Single Pool design (DNA & RNA)
SNV
• LOD down to 0.1% with 20 ng
input
• Same Sensitivity & Specificity
Whole target SNV/Indel calling at
0.5%
CNV fold change 1.2-1.4X
Expect 4 libraries on a 540 chip:
14 For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures. © 2016
Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the
property of Thermo Fisher Scientific and its subsidiaries.