NGS solutions for research and clinical applications in ... · PDF fileNGS solutions for research and clinical applications in oncology. 2 ... by Oncomine Focus sample tissue hotspots

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The world leader in serving science

NGS solutions for research and

clinical applications in oncology

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Routine Biomarker Analysis

• Limited sample volumes

• Multiple biomarkers for one disease indication

• Reflex testing is too slow

• Growing list of biomarkers changing rapidly

• Fixed assay needed for approval processes

Current methods to test samples against various

biomarkers are slow and often require more tumour

sample than available

Today’s Challenges

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Ion Torrent™ Next-Generation Sequencing

• Detection of multiple biomarkers in one test from one sample, in

one streamlined workflow¹

• Accurate and reproducible detection of variants

• Hotspots, SNPs, indels, CNVs and gene fusions in a single

workflow for DNA & RNA

• Fast turn-around time

• From 10 ng of DNA or RNA from FFPE tissue

• Already implemented in clinical research and early development of

future targeted therapies2

• First CE IVD solution for routine diagnostics available

• Oncomine Solid Tumour DNA kit*

• Oncomine Solid Tumour Fusion Transcript kit*

*CE marked and registered in accordance with in vitro Diagnostic Medical Devices Directive (98/79/EC).

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Ion Torrent NGS Solutions

for Cancer Research

From Transcriptome

to Oncomine Assays

For Research Use Only. Not for use in diagnostic procedures.

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Ion Torrent NGS panels for cancer research

• Ion AmpliSeq™ BRCA1 and BRCA2 Panel

• Ion AmpliSeq™ Colon and Lung Cancer Research

Panel v2

• Ion AmpliSeq™ RNA Fusion Lung Cancer Research

Panel

• Ion AmpliSeq™ Comprehensive Cancer Panel

• Ion AmpliSeq™ Cancer Hotspot Panel v2

• Ion AmpliSeq™ Transcriptome

• Oncomine™ Focus Assay

• Oncomine™ Comprehensive Assay

For research use only. Not for use for diagnostic procedures

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Human Gene Expression with Ion AmpliSeq Transcriptome and Ion 540 Chip

For Research Use only. Not for use in diagnostic procedures.

Samples • Four resected invasive ductal breast carcinoma research samples

• Starting input 10ng of FFPE RNA

Optimum panel design & performance • >20,800 genes on Ion 540 Chip (8 barcoded samples per chip)

• 13,740 mean genes per sample detected

• >0.99 pairwise replicate correlation

• 0.984 gene count replicate correlation

High quality results • Top 50 most variable genes with differential expression show

clear clustering by sample type as well as by sub-ype

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THE ONCOMINE™

KNOWLEDGEBASE

Pre-NGS sample prep:

• Custom / pre-designed

panels

• Hundreds of gene variants

per sample in single run

• Low input amounts

Ion AmpliSeq™

Technology*

One of the world’s largest curated cancer genomic database:

• Public sources

• Peer reviewed literature

• Published clinical research

trials

Compendia

Bioinformatics

Our Ion Torrent NGS Cancer Research Tools

*For Research Use Only. Not for use in diagnostic procedures.

Ion Torrent™ NGS platform:

• Minimal sample input

• Fast turnaround times

• Enables reproducible results

• Enables accurate performance

• All in a single run

Ion Torrent™

Systems*

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Hotspot Mutations 8,000 exomes + COSMIC database

Hotspot enrichment analysis

Deleterious Mutations 8,000 exomes, COSMIC database

Indel + nonsense enrichment analysis

High-Level CNVs 30,000 array-based genomes

Minimal common region analysis

Gene Fusions 6,000 transcriptomes + COSMIC

Proprietary fusion analysis

One of he world’s largest curated cancer genomic database, gathered from

public sources, peer reviewed literature, and published clinical research trials

The Oncomine Knowledgebase

The Oncomine™

Knowledgebase


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Bladder

Breast

Colorectal

Endometrial

Esophageal

Gastric

GIST

Glioblastoma

Head and Neck

Kidney

Liver

Melanoma

Mesothelioma

NSCLC

Osteosarcoma

Ovarian

Pancreas

Prostate

Basal Cell

SCLC

Sarcoma

Testicular

Thyroid

Solid Tumor Driver Variant Map

Copy gain Copy loss Hotspot Deleterious Gene fusion Legend: 0.01 >0.30

ALK EGFR

KRAS

TP53 PIK3CA

ERBB2

NTRK1, RET, ROS1

BRAF

NRAS

KIT

CTNNB1

CCND1

MYC

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Oncomine Assays: Solid Tumor Cancer Research NGS Assays Designed for Translational and Clinical Research

Benefits:

• Enables detection of relevant hotspots, SNVs, indels, CNVs and gene fusions in a

single workflow

• Intelligent NGS content design-based on the Oncomine Knowledgebase

• Minimal sample input required per run—making it well suited for analysis of

precious tumor samples

• Easy-to-perform workflow with fast turnaround time—get your NGS data

easily and quickly

• Service offerings to meet your clinical research needs

Detects variants in 52 solid tumor genes

that are associated with current

oncology drugs and backed by

published evidence

Detects variants in 143 unique genes

relevant to published trials

Oncomine™ Focus Assay: Oncomine™ Comprehensive Assay:


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Oncomine™ Focus Assay: Content Summary

Categorized by somatic alteration type

Hotspot genes

Focal CNV gains

Fusion drivers

Intelligent design includes hotspot SNV and indels, CNVs and gene fusions

52 genes

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19

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Categorized by potential relevance

Labels

Guidelines

Drug Targets

Clinical Trials

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9

43

>400(US)

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Run Sequence

Prepare Library

Sample Input

Analyze Data

Prepare Template

Oncomine Focus™ Assay Workflow

Oncomine

Focus™ Assay

FFPE material

including fine

needle aspirates,

needle biopsy

Oncomine™

Knowledgebase

Reporter

Ion Select™ 318

Chip

Ion OneTouch™

Select Template

200 Kit

Six samples, one run, to access hotspots, SNVs, indels,

CNVs and gene fusions for 52 solid tumor genes


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Cohort analysis

• 127 FFPE tumor samples analyzed across 4 different

laboratories

• Demographics:

• Median age: 58 (range 18-86)

• Male 52%, Female 48%

• Ethnicity: 69% Asian, 29% Caucasian, 2% Unknown

• Sample characteristics:

• Tissue of origin: Lung 19%, Ovary 12%, Stomach 12%, Brain 11%,

Kidney 8%, Cervix 8%, Skin 8%, Esophagus 5%, Pancreas 4%, Uterus

4%, Colon 3%, Prostate 3%, Liver 3%

• Tumor grade:

• G1 3%, G2 30%, G3 15%, G4 52%

• Well-Differentiated 6%, Well to Moderate 9%, Moderately Differentiated 50%,

Moderate to Poor 20%, Poorly Differentiated 15%

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Examples of Recurrent Somatic Mutations detected by Oncomine Focus

sample tissue hotspots CNVs fusions 1193124B Brain MET p.M1268T, IDH1 p.R132H CDK6, PDGFRA, KIT PTPRZ1-MET.P1M2

1199440B Brain EGFR p.A289T EGFR EGFR-EGFR.E1E8

1188945B Brain IDH1 p.R132H CDK4

1199191B Brain GOPC-ROS1.G8R35.COSF1139

1181647B Brain EGFR-EGFR.E1E8

1181219B Brain BRAF p.V600E

1181943B Brain IDH1 p.R132H

313774A1 Breast ERBB2 p.L755S, ERBB2 p.G776C, PIK3CA p.M1043V

308452A4 Breast PIK3CA p.E545K

1182647B Colon KRAS p.G12D, PIK3CA p.E545K

1185114B Colon KRAS p.G12D, PIK3CA p.E545K

1186528B Colon BRAF p.V600E

1187394B Lung MET p.T1010I, KRAS p.G12C

1200313B Lung CTNNB1 p.D32N, KRAS p.G12D

1194253B Lung PIK3CA p.E545K PIK3CA

1195523B Lung SDC4-ROS1.S2R32.COSF1265

1196001B Lung MET-MET.M13M15

1196650B Lung NRAS p.Q61L

1199311B Lung EGFR p.E746_A750del

1199087B Lung CCND1

1180121B Lung MYC

1196647B Lung PIK3CA

365 Skin NRAS p.Q61K, CTNNB1 p.G34R CCND1

367 Skin CDK4 p.R24C, BRAF p.V600E

370 Skin NRAS p.Q61K CCND1

364 Skin BRAF p.V600E

369 Skin BRAF p.V600E

366 Skin FGFR4


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Oncomine™ Focus Assay: Reproducible Hotspot detection

Cancer type, sample, gene, variant


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Cancer type, gene, variant

Oncomine™ Focus Assay: Reproducibility and consistency relative to Sanger


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CNV detection by Oncomine™ Focus Assay*: Comparison to FISH

• 20 FFPE tumor samples

representing 8 different cancer

types

• Tumor fraction range 40-100%

• Validated FISH assays for 10

different gene targets

• Samples run at 4 different labs

from different sections of the

same block

• 97% sensitivity (67/69) to detect

CN ≥ 8; 94% (81/86) sensitivity to

detect CN ≥ 6 using the 5% CI ≥ 4

Good concordance between CN estimates produced by FISH or NGS


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Gene Fusion detection by Oncomine™ Focus Assay*

• Targeted fusion breakpoint

assays

• Alignment of reads to

reference sequences

Sensitive and specific detection of fusion breakpoints *For Research Use Only. Not for use in diagnostic procedures.

EML4-ALK.

E6aA20.

AB374361

CCDC6-RET.

C1R12.

COSF1271

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Oncomine™ Focus Assay: Gene Fusions

Driver Partner Gene Variants

(N)

ALK A2M, ACTG2, ATIC, C2orf44, CARS, CLIP4, CLTC, DCTN1, EML4, GTF2IRD1, HIP1, KIF5B, KLC1, MEMO1, NCOA1, PRKAR1A, PTPN3, RANBP2, SEC31A, SMEK2, STRN, TFG, TPM1, TPM3, TPM4, TPR, TRAF1, VCL

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RET ACBD5, AFAP1, AKAP13, CCDC6, CUX1, ERC1, FKBP15, GOLGA5, HOOK3, KIAA1468, KIF5B, KTN1, NCOA4, PCM1, PRKAR1A, RUFY2, SPECC1L, TBL1XR1, TRIM24, TRIM27, TRIM33

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ROS1 CCDC6, CD74, CEP85L, CLIP1, CLTC, ERC1, EZR, GOPC, HLA-A, KDELR2, KIAA1598, LRIG3, MSN, MYO5A, PPFIBP1, PWWP2A, SDC4, SLC34A2, TFG, TPM3, ZCCHC8

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NTRK1 BCAN, CD74, CEL, DYNC2H1, IRF2BP2, LMNA, MPRIP, NFASC, RNF213, SQSTM1, SSBP2, TFG, TPM3, TPR 19

NTRK2 AFAP1, AGBL4, NACC2, QKI, SQSTM1, TRIM24, VCL 7

NTRK3 BTBD1, COX5A, ETV6 6

FGFR1 BAG4, ERLIN2, TACC1 4

FGFR2 AFF3, BICC1, CASP7, CCAR2, CIT, MGEA5, OFD1, SLC45A3, TACC3 10

FGFR3 AES, BAIAP2L1, ELAVL3, TACC3 26

BRAF AGTRAP, AKAP9, CDC27, FAM131B, FCHSD1, KIAA1549, PAPSS1, SLC45A3, SND1, TAX1BP1, TRIM24 15

MET BAIAP2L1, C8orf34, CAPZA2, OXR1, PTPRZ1, TFG, TPR, MET exon 14 deletion 10

other drivers: EGFR, ERG, ETV1, ETV4, ETV5, FGFR1, RAF1, PPARG, ABL1, AKT3, AXL, ERBB2, PDGFRA 36

total targeted assays 271

expression imbalance assays for ALK, RET, ROS1, NTRK1 8

expression controls 5


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A targeted, multi-biomarker assay that enables detection of

variants in 52 genes relevant to solid tumors

Oncomine™ Focus Assay*

Intelligent NGS content design:

• Designed for translational & clinical research, includes 52

key cancer genes associated with current oncology drugs

and published evidence

• Includes relevant hotspots, SNVs, indels, CNVs and gene

fusions in a single workflow for DNA and RNA

• Content driven by the Oncomine™ Knowledgebase;

curated cancer genomics database

Sensitive tumor analysis:

• Enables detection using low input amounts including FFPE

fine needle aspirates and core needle biopsies

• Deliver results on more of your FFPE samples


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Proposed Plans for IVD Product Development

Oncomine™ Focus

Assay* RUO

Early Development

Oncomine™ Universal

Dx Test IUO

Registration Trials

Oncomine™ Universal

Dx Test CE IVD

Companion Diagnostics


*The content provided herein may relate to products that have not been officially released and is subject to change without notice.

In cooperation with pharma Industry –

Validating the assay on clinical trial samples for

therapy selection (on PGMDx, as a full workflow )

2015 2016

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Oncomine Comprehensive Assay* Content Summary


Categorized by somatic alteration type

Hotspot genes

Focal CNV gains

Full CDS for DEL

mutations & CNV loss

Fusion drivers

143 genes: Several used in multiple applications

(hotspot, CNV, driver fusion)

143 genes

73

49

26

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Categorized by potential relevance

12

9

72

>450(US)

Labels

Guidelines

Drug Targets

Clinical Trials

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NCI Match Trial

1Mickey Williams, PhD, Frederick National Laboratory for Cancer Research

Overview

Goals

Goals Approach

• Nationwide study open to

~2,400 NCTN sites

• 3-years, 3,000 samples

• Multiple clinical laboratories

sequencing tumor samples

• Multiple basket study design

• Overcome drug

development challenges

with clinical trial

enrollment & patient

matching

• Avoid undue costs & time–

to– enrollment process

• A clinical research study

using Ion Torrent™

instrumentation and

Oncomine® reagents

• NCI validation performed

in alignment with FDA

requirements for

investigational use1

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Publication: Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors.


• Performance characterized with

molecular standards and > 300 FFPE

samples

• NGS results matched those of

validated single gene tests

• Additional relevant variants detected

• Results validate the multiplexed

PCR-based RNA-seq approach for

detecting targeted gene fusions

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Oncomine™ Comprehensive

Assay*

Relative Performance

Gene Test Molecular Pathology Results Sensitivity Specificity

BRAF

V600 BRAF: 63 samples

• 29 positive (26 V600E, 2 T599dup, 1 V600K)

• 34 negative

• Melanoma, Colorectal adenocarcinoma, Lung

adenocarcinoma, Endometrial adenocarcinoma

100% (29/29)

100% (34/34)

EGFR

L858R &

Exon 19 del

EGFR: 22 samples • 3 positive (2 Exon 19 del, 1 L858R)

• 19 negative

• Lung adenocarcinoma, Sarcomatoid carcinoma

100% (3/3)

100% (19/19)

KRAS

G12, G13,

Q61

KRAS: 21 samples • 11 positive (1 G12A, 2 G12 D, 5 G12V, 2 G13D, 1 Q61K)

• 10 negative

• Colorectal adenocarcinoma, Small bowel adenocarcinoma

100% (11/11)

100% (10/10)

n=103


Comparison to Verified Single Gene Test Results

Hovelson et al., 2015. Development and validation of a scalable next-generation sequencing

system for assessing relevant somatic variants in solid tumors. Neoplasia 17:385-399.

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Oncomine™

Knowledgebase

Reporter

Ion Reporter™

Workflow*

Lab Generated

Report*

Research

Laboratory

Oncomine Knowledgebase Reporter


Genetic variants

Associated published evidence

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Relevant variants detected by the Oncomine™ Comprehensive Panel*

Tumor Sample Types Analyzed Using the Oncomine™ Comprehensive Panel*

*For Research Use Only. Not for use in diagnostic procedures. Hovelson et al., 2015. Development and validation of a scalable next-generation sequencing

system for assessing relevant somatic variants in solid tumors. Neoplasia 17:385-399.

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Demonstration of the Oncomine Comprehensive Assay analyzed on the Ion S5 System

Samples used: Sample 1: 80 / 20 mix, AcroMetrix™ Oncology Hotspot Control (DNA) / Mixture of three

lung cancer cell lines (RNA)

Sample 2: 80 / 20 mix, CRL-1619 cell line (DNA) / Mixture of 3 lung tumor cell lines (RNA)

Sample 3: 80 / 20 mix, HCC1143D cell line (DNA) / normal colon cell line (RNA)

Reagents used: • Oncomine Comprehensive Assay Primers and Library Kit

• Ion S5 Series Template and Sequencing Reagents

Panel metrics • 143 gene targets (2,738 amplicons, 3 pools) to DNA & RNA (fusion transcripts)

• Run on an Ion 530 Chip (3 samples per chip)

Mutation Detection Results • Highly concordant results across Ion Torrent platforms

• Gene Fusions: 100% of fusions were detected

• SNVs: 99.6% of SNVs were detected

• No false positive SNV calls were generated

Note: Demonstration for feasibility and baseline performance only. Not a study for clinical validation.

For Research Use only. Not for use in diagnostic procedures.

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Summary: Oncomine NGS Assays for Research

Utilize low sample input—enabling you

to access results for more of your samples

Intelligent NGS content design backed

by the Oncomine™ Knowledgebase and

confirmed with pharma partners

Single workflow to analyze 52 or 143

solid tumor genes for biomarker analysis

Designed to meet your needs for biomarker analysis

3-4 FFPE slides, compatible with fine

needle aspirates and core biopsies

For research use only. Not for use for diagnostic procedures

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Oncomine Solid Tumor

Kits* for Clinical Diagnostics

Oncomine™ Solid Tumour DNA Kit*

Oncomine™ Solid Tumour Fusion*

Transcript Kit


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Oncomine Solid Tumour Kits Offering

• RNA/DNA analysis in one workflow from sample to result typically

in one day

• Requires as little as 10 ng of DNA or RNA, which can be obtained

from as few as 1–2 FFPE slides

• Targeted approach allows accurate detection of relevant somatic

alterations in solid tumors with evidence linking alterations to targeted

therapies

• Designed in cooperation with group of leading cancer clinicians:

OncoNetwork

• CE-IVD* ready-to-use, flexible solution

• Manufactured under GMP and ISO 13485


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Goal: Develop a complete solution for colon and lung cancer biomarker DNA and RNA

analysis that is efficient, not subjective, and works with limited FFPE samples

OncoNetwork Consortium Designed with input from a global team of leading cancer clinicians

Radboud University

Medical Center

Trinity College

ARUP Institute

Kinki

University

Viollier Université Paris

Descartes

Warwick

Medical

School

IPATIMUP

Portugal

Queen’s University

ARC-NET

University of

Verona

Centro Ricerche

Oncologiche

Customer Design Requirements

DNA Analysis RNA Analysis

Low input DNA requirement Reduced cost/complexity and good concordance with FISH

FFPE sample compatibility Quantitative, reliable results using low input of FFPE RNA

Selective gene content View multiple fusions simultaneously

Easy to implement Easy to interpret results without the need for a bioinformatician


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96 test kit

Consists of 6 packs, 16 tests each

and it is possible to run 1–16

samples at once

Contains primers, library

reagents and equalizer

Manufactured according to GMP and ISO 13485 standards

High level of Quality Control helps provide confidence in

reproducibility, accuracy, and consistent quality of results

CE-IVD* Ready-to-Use Solution


In compliance with current

European In Vitro Diagnostic

Directive (IVDD)

Helps simplify in-house

validation for accreditation

purposes

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Customer evaluation of the

Oncomine CE IVD Solid Tumour kits*


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Multi-Centric Evaluation of the CE IVD kits in Spain

Aim of this study was to demonstrate the applicability of the Oncomine™ CE-IVD kits in

the clinical diagnostic routine of the pathology labs with the objective to reduce the time

for patient’s diagnosis, the hands-on work and increasing the results sensitivity

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Study Design

Results

• 46 samples (lung and colon CA and melanoma)

• Provided by 8 laboratories running routine solid tumour biomarker testing

• 6 previously characterized (by FISH or PCR), anonymized FFPE samples from

each laboratory

• 2 for fusion transcript and 4 for gene mutation analysis

• 3 section of each samples were prepared and always one anlysed in 3 laboratories

• Hospital Ramon y Cajal – Madrid (not experienced in NGS)

• Hospital 12 de Octubre – Madrid (not experienced in NGS)

• Laboratorio de Dianas Terapeuticas – Madrid (experienced in NGS)

• Very strong inter-laboratory concordance of results obtained, with the reproducibility

between 96% and 100%

• All the expected variants (32) were identified and 23 potentially pathogenic extra

variants were found. With the DNA kit

• All fusion transcripts were detected with a very high sensitivity when sequencing

results reach the defined threshold of quality to be analysed. Just 1 false negative was

found.

• Using the kits in diagnostic routine increases laboratory productivity while achieving

quicker time to results and higher sensitivity

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Inter-Laboratory Concordance

DNA mutation analysis RNA fusion transcript analysis


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Accreditation of of NGS workflow for routine diagnosis Cochin Hospital, Paris, France

Aim of the study was evaluation of the performance of the Oncomine Solid

Tumour DNA kit* in order to prepare aaccreditation of NGS workflow for

routine diagnosis in melanoma according to ISO15189.

Study Design

• 163 analysis in 10 runs (8 or 16 samples on 316 or 318 chip)

• 2 runs with the same libraries (stored 4°C for 2 months or -20°C for one month)

• Materials:

• 15 IQC : commercial DNAs with known BRAF and NRAS genotypes (Horizon

Diagnostics (AmpliTech): FFPE DNAs

• 20 DNAs from inter-laboratory comparison testing (Dr. Hélène Blons, HEGP

Hospital, Paris)

• 1 negative extraction control (blank paraffin control block)

• 20 EEQ from external quality assessment (EQA)

• Parameters Evaluated:

• Repeatability and Reproducibility

• Inter-operator variability

• Analytical sensitivity and specificity


• Limit of detection

• Contamination

• Robustness

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Accreditation of of NGS workflow for routine diagnosis Cochin Hospital, Paris, France

• The expected

performances of the Oncomine™ Solid Tumour DNA Kit have been experimentally confirmed in a routine diagnostic laboratory for hotspot mutation detection in melanoma.

• The same experiments will be used to extend the validation to hotspot mutation detection in lung and colon cancers.

Parameters

Repeatability √

Reproducibility √

Inter-operator variability √

Analytical sensitivity and specificity √

Limit of detection √

Contamination √

Robustness √

Conclusion


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Summary

Actionable, content-driven targeted NGS will transform

the future of cancer care

Thermo Fisher Scientific leads the way with the

market-leading targeted NGS solution for research and clinical

applications based on the Ion Torrent™ platforms

Ion S5™ System*

Ion Proton ™ System*

Ion PGM™ Dx System** with

combined function software Ion PGM™ System*

*

*For research use only. Not for use for diagnostic procedures

**CE marked and registered in accordance with in vitro Diagnostic Medical Devices Directive (98/79/EC).

NGS solutions for research and clinical applications in ... · PDF fileNGS solutions for research and clinical applications in oncology. 2 ... by Oncomine Focus sample tissue hotspots

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