Developing a multiplex next generation sequencing assay to ...€¦ · 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%

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


Oncomine cfDNA Breast Assay


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


Oncomine Lung cell free Total Nucleic

Acid Assay

11 genes, hotspots, indels, CNVs, Fusions


Oncomine cfTNA PanCaner Assay

(In Development)


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)


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


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


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




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

=~


Testing Limits of Detection with Higher amount of input cfDNA

1 ng cfDNA – 0.6% 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


• 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


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


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


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


Oncomine Knowledgebase Reporter

Quickly create custom, lab-generated reportsOncomine Knowledgebase

Reporter


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:


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.

Documents

Developing a multiplex next generation sequencing assay to ...€¦ · 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%