Oncomine Immune Response Research Assay

An assay to profile the tumor microenvironment

Janice Khor
Product Manager
Life Sciences Solutions, Southeast Asia

For Research Use Only. Not for use in diagnostic procedures. The world leader in serving science
Improved survival over existing therapies drive increased interest in immuno-oncology research

Checkpoint therapy market: revenue

forecast scenario analysis, global,
20152020
30
25
25
21

Revenue (USD billions)

20
20 139%
CAGR
15 14

10
10
7
6
5 3
2

0
2015 2018 2020
Pessimistic Frost & Sullivan Optimistic
Frost & Sullivan, July 2016
Sharma P, Allison JP. Cell, 161: 205214, 2015

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

Todays challenges in immuno-oncology research
Predictive biomarkers
Stratification of clinical trial samples

Potential to respond

IHC

What makes stratification a challenge?

Current PD-1, PD-L1 IHC assays are

insufficient

Current knowledge of MOA is incomplete

IHC assays (PD-L1) have been shown to

lack reproducibility and are not scalable
beyond 12 assays per tumor tissue
sample Lower potential to
respond

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

Consensus from immuno-oncology community on the multi-marker approach

Potential to respond

Oncomine Immune Response

Research Assay

Lower potential to
respond

Tumor microenvironment
Adapted from Upreti et al., Transl Canc Res 2:4 (2013)

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

Gene expression assay based on Ion AmpliSeq technology

Assay includes library prep and analysis

RNA Extraction Ion AmpliSeq Templating Sequencing Analysis

from FFPE Library Creation

RecoverAll Oncomine
Total Nucleic Acid Immune Ion Chef Ion Torrent
Ion S5 System
Isolation Kit for Response System Suite Plug-in
FFPE Research Assy

Two-day workflow*

High sensitivity to low expressing genes, broad dynamic range, and with
excellent reproducibility Multiplexing
per chip

Includes panel and reagents for library construction in manual or automated

mode, and a corresponding analysis tool
530: 8 samples
Enabled on the Ion Chef System with Ion S5 System Sequencing, and the Ion
OneTouch 2 System with Ion PGM System sequencing
318: 4 samples

* Two-day workflow requires overnight templating run

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

Panel content is carefully selected and curated to meet customer needs

Current research National Cancer Pharma Oncomine

publications Center Japan customers and Knowledgebase
collaborations
Annotation Database DAVID Oncomine KOLs
clinicaltrials.gov

Immune checkpoint pathways and targets

Interferon signaling genes Tumor markers, tumor antigens

Cytokine signaling genes Proliferation markers

T cell & B cell signaling genes

Markers of lymphocyte subsets
Housekeeping genes

Activation )
proliferatio
n
cytotoxicity
CD ) )
8+ T
cell

MOA study Biomarker Identification Drug combinations

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

Oncomine Immune Response Research Assay panel content

Function # Genes Function # Genes

395 genes Antigen presentation 3 B cell marker 11
Antigen processing 19 Dendritic cell 7
394 primer sets Innate immune response 11 Dendritic cell, macrophage 6
36 functional annotation Leukocyte inhibition 2 Helper T cells 8
Leukocyte migration 5 Macrophage 5
groups Lymphocyte activation 2 Myeloid marker 7
Lymphocyte regulation Lymphocyte development 3 Neutrophil 5
Cytokine signaling Lymphocyte infiltrate 46 NK activation 8
Lymphocyte markers B cell receptor signaling 3 NK cell marker 4
Checkpoint pathway T cell receptor signaling 6 T cell differentiation 2
Tumor characterization T cell regulation 9
Housekeeping TCR coexpression 19 Checkpoint pathway 30
PD-1 signaling 9
Chemokine signaling 10 Drug target 21
Cytokine signaling 15
Interferon signaling 8 Adhesion, migration 14
Type I interferon signaling 8 Apoptosis 4
Type II interferon signaling 23 Proliferation 10
Tumor antigen 17
Housekeeping 11 Tumor marker 27

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

Targeted panel offers significantly higher sensitivity over transcriptome
Oncomine Immune Response Research
Range of expression across 8 NSCLC tumor samples Assay
Ion AmpliSeq Transcriptome Human
Gene Expression Kit

More reads
across low
expressors

Subset of key genes

associated with immune
response research

Genes associated with checkpoint pathways Genes associated TIL* Genes associated with
with drug targets nterferon signaling

* Tumor-infiltrating lymphcytes. Genes associated with T cell activation

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

 CRC samples: 10% of genes detected only with targeted panel
Oncomine assay

Units on both axes are log2(RPM). Each plot shows the expression of the same gene
either on Immune Response or the transcriptome panel
Data show that Ion AmpliSeq Transcriptome scales linearly between two panel sizes but
the Oncomine Immune Response Research Assay offers more robust estimate for
Transcriptome expression

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

 Oncomine assay covers key pathways more deeply than competitive assays

141 Unique genes on Oncomine Immune

Response Panel cover key pathways
Oncomine Immune
Response Research Type II interferon signaling Macrophage
Assay Type I interferon signaling Lymphocyte infiltrate
Tumor marker, stemness Lymphocyte development
Tumor marker Leukocyte migration
Tumor antigen Leukocyte inhibition
TCR coexpression Interferon signaling
511 254 141 T cell regulation, trafficking Innate immune response
T cell regulation Housekeeping
T cell receptor signaling Drug target
T cell differentiation Dendridic cell
Proliferation Cytokine signaling
Nanostring PD-1 signaling, tumor marker Chemokine signaling
PD-1 signaling Checkpoint pathway
nCounter PanCancer Immune
NK cell marker B cell marker
Profiling assay NK activation Apoptosis
Neutrophil Antigen processing
Myeloid marker, stem cell Adhesion, migration
Myeloid marker, MDSC
Myeloid marker

Strong emphasis on lymphocyte infiltrate markers

TCR co-expressing genes (derived from Oncomine Knowledge
Base) facilitates discovery research
Higher focus on genes characterizing tumor and tumor antigen

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

Comparison study with a similar assay on the market

A head-to-head study was RNA

Quality
conducted on 10 samples Sample Tissue RNA Method Score
Breast CR-4-1493-1 Breast Qiagen RNEasy 5.016
Head and neck (H&N)
CR-4-1296-7 Breast Qiagen RNEasy 3.635
Melanoma
NSCLC CR-4-1258-2 Breast Qiagen RNEasy 1.247
RCC NP-13634-3 H&N Qiagen RNEasy 7.418
nCounter Pancancer panel runs CR-9-1251-6 H&N Qiagen RNEasy 4.076
CR-9-1270-3 H&N Qiagen RNEasy 4.573
performed by a third-party CRO
NP-16332-5 Melanoma Recover All 3.57
Oncomine Immune Response 131503T2(6) Melanoma Qiagen RNEasy 8.111
Research Assay runs performed CR-19-1452-8 NSCLC Recover All 0.374
NP-16585-4 RCC Recover All 4.225
by Thermo Fisher Scientific
A subset of samples were run in
replicates to assess repeatability
RNA quality
Good
Average
Below average

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

20% higher dynamic range for Oncomine Immune Response Research Assay
Distribution of gene expression

nCounter
Nanostring
Oncomine
OIRRA
0.15
Immune
Response
Research
Assay
Fraction of genes

0.10
0.05
0.00

-5 0 5 10 15 20
Gene expression in log2
Gene expression in log2
Each line represents the distribution of genes in 1 run
Distribution of gene expression shows the expression all of genes in each sample
Dynamic range is estimated by the highest and lowest genes detected in each sample

Clear signal-to-noise ratio (bimodal distribution)

Helps researchers make more confident calls

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

2040% increase in overall sensitivity
Sensitivity of gene panels Oncomine Immune
100 Response Research Assay
nCounter

80
% genes detected

60

40

20

0
CR.4.1258.2

CR.4.1258.2.1

CR.4.1296.7

CR.9.1251.6

CR.9.1251.6.1

CR.9.1251.6.2

CR.9.1270.3

CR.9.1270.3.1

NP.13634.3.A.1

NP.16332.5

NP.16332.5.1

X131503T26.A.1

NP.16585.4.A.1
CR.4.1493.1.A

NP.13634.3.A

NP.13634.3.B

X131503T26.A

X131503T26.B

CR.19.1452.8.A

NP.16585.4.A

NP.16585.4.B
Sample

NSCLC
Breast Head & Neck Melanoma RCC

With sequencing information of individual reads, the Oncomine Immune Response

Research Assay offers high specificity, which translates to high sensitivity of gene
detection

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

 Higher sensitivity to genes of interest
Oncomine Immune Response Reearch Assay
Range of expression across 10 pancancer research samples nCounter

Subset of key genes

associated with
immune response
research

Genes associated with T cell Genes associated with checkpoint pathways

receptor signaling

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

Preliminary indicator that may help improve stratification of samples
Oncomine Immune Response Research Assay nCounter PanCancer Panel

1-correlation
Correlation Correlation
0.00 0.10 0.20 0.30 0.00 0.10 0.20

100
Melanoma_X131503T26_R4 H&N_NP.13634.3.A
Melanoma

95
Melanoma_X131503T26.rep_R1 H&N_NP.13634.3.B

98
Melanoma_X131503T26_R1 NSCLC_CR.19.1452.8.A

100
Melanoma_NP.16332.5_R1 NSCLC_CR.19.1452.8.B
Melanoma_NP.16332.5_R4 RCC_NP.16585.4.A
NSCLC NSCLC_CR.19.1452.8_R4 RCC_NP.16585.4.B

69

au
RCC_NP.16585.4_R2 100 64 NSCLC_CR.19.1100.2.A
C
RC

Nanostring
RCC_NP.16585.4_R1 NSCLC_CR.19.1100.2.B
RCC_NP.16585.4_R4 H&N_CR.9.1280.6

OIRRA
H&N_CR.9.1251.6_R2 H&N_CR.9.1251.6
97

64
70

H&N_CR.9.1251.6_R1 H&N_CR.9.1270.3
H&N_CR.9.1251.6_R4 71 Melanoma_X131503T26.A
100
H&N

H&N_NP.13634.3_R4 Melanoma_X131503T26.B
94

H&N_NP.13634.3_R1 Melanoma_NP.16332.5
60

H&N_NP.13634.3.rep_R1 Breast_CR.4.1493.1.A
75
100

H&N_CR.9.1270.3_R1 Breast_CR.4.1493.1.B
H&N_CR.9.1270.3_R4 Breast_CR.4.1296.7
Breast_CR.4.1296.7_R4 Breast_CR.4.1292.3
83
Breast

Breast_CR.4.1493.1_R4
87

Breast_CR.4.1258.2
84

Breast_CR.4.1258.2_R1 Breast_CR.4.1484.1
Breast_CR.4.1258.2_R4

Oncomine Immune Response Research Assay:

Offers clearer distinction between cancer types when clustered by all genes on each panel
Content offers better tumor characterization

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

Begin with a highly sensitive biomarker profiling assay to identify signatures of interest

CTA and/CDx test generation may follow after markers of response

Intended application are identified using the Oncomine Immune Response Research Assay

Biomarker profiling Clinical trial assay CDx

RUO RUO IVD

PROFILING SELECTION TESTING
~400 genes ~ >40 genes ~ 2040 genes

Immune response multimarker assay helps

accelerate development of companion diagnostic tests

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

Oncomine Assays to help advance precision oncology

Oncology Research
Specimen

Tumor focused + Immune focused

Oncomine Tumor Oncomine Immune

Profiling Assays Oncology Assays
Multi-gene assays for Multi-gene assays for
solid tumor molecular immune response
characterization characterization

Oncomine Comprehensive Assay Oncomine Immune Response

Oncomine Focus Assay Research Assay
Oncomine Colon and Lung Assay
Oncomine cfDNA Assays
Driving important
decisions in clinical
cancer research

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

Summary

Highly curated content to assess tumor microenvironment

and identify predictive markers for drug response
An assay optimized for FFPE sample with input
requirement of as little as 10 ng total RNA
High sensitivity to low expressing genes, and broad
dynamic range with excellent reproducibility
Automated NGS workflow with a fast turn-around time

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

A Mission We are Proud of

We enable our customers to make the world

healthier, cleaner and safer.

19 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 unless otherwise specified. RNeasy is a trademark of Qiagen Group. nCounter is a trademark of NanoString
Technologies, Inc.

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

Three verified workflows

Input Library Generation Templating Sequencing Analysis

Oncomine
OneTouch 2 Ion 318 Chip
Immune
Hi-Q View 1-4 libraries
Response
Templating Kit
Research Assay
Kit (32 Libraries)

Analysis
solution with
10 ng Torrent
total 3 SuiteTM
RNA Software

Oncomine Immune
Ion Chef System Ion 520 Chip 1-4 libraries
Response Research
520/530 Templating Ion 530 Chip 2-8 libraries
Assay Kit Chef Ready
Kit
(32 Libraries)

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

Gene function: Oncomine Immune Response Research Assay and nCounter
Pancancer panel

Transporter Functions, TNF Superfamily

Transporter Functions The functional categories of these genes
TNF Superfamily
TLR are well represented in current
T-Cell Functions, Regulation
T-Cell Functions Oncomine Immune Response panel
Senescence, T-Cell Functions
Senescence
Regulation, TNF Superfamily Regulation, Cell function, Transporter
Regulation, T-Cell Functions
Regulation, Pathogen Defense functions are deemed too general or not
Regulation
Pathogen Defense
Too general sufficient relevant
NK Cell Functions, Regulation
NK Cell Functions
Microglial Functions, TLR
Macrophage Functions
Leukocyte Functions
Interleukins
Cytokines, TNF Superfamily
Cytokines, T-Cell Functions
Cytokines, Regulation, Pathogen Defense
Cytokines, Regulation
Cytokines, Macrophage Functions, Regulation
Cytokines, Interleukins, Regulation, T-Cell Functions
Cytokines, Interleukins
Cytokines
Complement
Chemokines, TNF Superfamily
Chemokines, T-Cell Functions
Chemokines, Regulation
Chemokines, Interleukins
Chemokines, Complement
Chemokines
Cell Functions, T-Cell Functions
Included
Cell Functions, Cytotoxicity
Cell Functions
Cell Cycle, Regulation, Senescence
Too general
Cell Cycle, Regulation
Cell Cycle
B-Cell Functions, T-Cell Functions
B-Cell Functions, Cytokines, Interleukins
B-Cell Functions
B-cell Functions
Antigen Processing, Cell Cycle, Regulation Most are annotated with innate
Antigen Processing
Adhesion, Regulation
Adhesion, Cell Functions
223 or adoptive response
Adhesion
No further annotation for
functional category

Number of genes
0

10

20

30

40

50

60

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

Linearity and LOD determined with limiting dilution
Objective:
Determine linearity of fold-change estimation
Samples and experimental design:
Mix HL-60 and Normal lung research samples at 4 concentrations

Sample Ratio (20ng input)

Leukemic cell line
1 4 9 19
(HL-60)
Normal Lung
1 1 1 1
(AM7972)

2 runs total consisting of 3 replicates of each mixing proportion on both runs

4 mixing proportions * 3 replicates * 2 runs = 24 samples total
Compare expected fold change (dilution*fold change in pure samples) and
Observed assay readouts to determine sensitivity and specificity of differential
expression.

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

Linearity in fold change estimates

Linearity of absolute fold change estimates Linearity of relative fold change estimates
Observed

Observed f1<f2<f3..<f395

Expected Expected

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

LOD of 2 fold and 4 fold differential expression

False False
Observed Expected True neg positive negative True pos TP/(TP+FN) TN/(TN+FP)
titration
abs fc absolute both titration sig not.sig
q_value titration fc pure not sig pure not.sig pure sig both sig Sensitivity Specificity
0.05 0 0 14 12 6 270 97.8% 53.8%
0.001 0 0 29 34 7 232 97.1% 46.0%
0.05 0 2 20 73 0 209 100.0% 21.5%
0.001 0 2 35 67 1 199 99.5% 34.3%
0.05 2 2 93 0 51 158 75.6% 100.0%
0.001 2 2 95 7 49 151 75.5% 93.1%
0.05 0 4 20 136 0 146 100.0% 12.8%
0.001 0 4 36 123 0 143 100.0% 22.6%
0.05 2 4 131 25 13 133 91.1% 84.0%
0.001 2 4 131 28 13 130 90.9% 82.4%
0.05 4 4 155 1 13 133 91.1% 99.4%
0.001 4 4 155 4 13 130 90.9% 97.5%

2 fold differential expression difference can be detected with >75% sensitivity and >99% specificity
4 fold differential expression difference can be detected with >90% sensitivity and >99% specificity

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

qPCR concordance: 8 NSCLC FFPE + 2 controls
log2(RPM)

Ct

HL-60 Normal
lung

Gene tested: MLANA, TFRC,

CD52, CXCL9, ID2, DDX58, TBP,
CD2, GZMA, IL7R, IFNG,
CDKN3, CD28, TNF, CTLA4, IL6,
TNFRSF14, GUSB, PMEL, LCN2,
KLRG1, FOXP3

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