Download DLBCL PAC Pitch - World CDx Boston 2016

Developing an Accurate and Precise Companion
Diagnostic Assay for Targeted Therapies in DLBCL
James Storhoff, Ph.D.
Senior Manager, Diagnostic Test Development
World Cdx, Boston, Sep. 10th
Molecules That Count®
© 2012 NanoString® Technologies, Inc. All rights reserved.
Presentation Overview
Introduction to nCounter platform
Discovery of a gene signature in DLBCL
Lymphoma Subtyping Test: A Cdx IUO assay developed at
NanoString and implemented as part of a global phase III trial in
collaboration with Celgene
nCounter: The only direct, digital, nucleic-acid
counting technology
Molecular Barcoding
• Novel chemistry invented in Leroy Hood’s lab at Institute of Systems Biology
• Probes up to 800 genes simultaneously
• Digital gene expression applied to biological pathways
Single molecule fluorescent barcodes,
each attached to an individual nucleic acid
molecule
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Digital Counting: How it Works (1 of 2)
Target-specific Capture Probe
Half Site
50 bases
Biotin
Target-specific Reporter Probe
Half Site
50 bases
Barcode
Digital Counting: How it Works (2 of 2)
Half Site
50 bases
Half Site
50 bases
Barcode
Biotin
Measured Nucleic Acid
6
nCounter® Assay
Hybridize
CodeSet
to RNA
Remove
Excess
Reporters
Target nucleic acid
Bind
Reporters
to Surface
Immobilize
and Align
Reporters
Image
Surface
Count
Codes
Capture and Reporter Probes
(“CodeSet”)
7
nCounter® Assay
Hybridize
Hybridize
CodeSet
CodeSet
to RNA
RNA
to
Remove
Excess
Reporters
Bind
Reporters
to Surface
Hybridized mRNA
Immobilize
and Align
Reporters
Image
Surface
Excess Probes Removed
Count
Codes
8
nCounter® Assay
Hybridize
Hybridize
CodeSet
CodeSet
to RNA
RNA
to
Remove
Excess
Reporters
Bind
Reporters
to Surface
Immobilize
and Align
Reporters
Image
Surface
Count
Codes
Hybridized Probes Bind to Cartridge
Bottom of cartridge is coated with
streptavidin
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nCounter® Assay
Hybridize
Hybridize
CodeSet
CodeSet
to RNA
RNA
to
Remove
Excess
Reporters
Bind
Reporters
to Surface
Immobilize
and Align
Reporters
Image
Surface
Count
Codes
Immobilize and Align Reporters for Image Collecting and Barcode Counting
−
+
10
nCounter® Assay
Hybridize
Hybridize
CodeSet
CodeSet
to RNA
RNA
to
Remove
Excess
Reporters
Bind
Reporters
to Surface
Immobilize
and Align
Reporters
Image
Surface
One Coded Reporter = One RNA Molecule
Count
Codes
11
nCounter® Assay
Hybridize
CodeSet
to RNA
Remove
Excess
Reporters
Bind
Reporters
to Surface
Immobilize
and Align
Reporters
Image
Surface
Codes are Counted and Tabulated
One Count = One Gene
Count
Codes
Digital and Automated Workflow
5 min
HANDS-ON
Step
1
OVERNIGHT
5 min
HANDS-ON
Step
2
2.5 – 3.0 HOURS, AUTOMATED
nCounter® Prep Station
1
Hybridize
Flexible sample input requirements
Only 4 pipetting steps/sample
No amplification (except for single-cell)
800 hybridizations in a single tube
2
Purify
5 min
HANDS-ON
Step
3
3 – 4.5 HOURS, AUTOMATED
nCounter® Digital Analyzer
3
Count
Sensitive
Precise
Quantitative
Simple
nCounter: Versatile and Validated Platform
Multiple
Applications
Multiple
Applications
Gene Expression
miRNA Expression
Copy Number
Variation
Gene Fusions
Single Cell
Expression
Proteins
(In Early Access)
Over 850 Peer-reviewed Publications
254
186
105
8
2009
17
2010
44
2011
The nCounter Dx Analysis System is FDA 510(k) cleared for use with the Prosigna Breast Cancer Prognostic Gene Signature Assay.
To date, it has not been cleared by the FDA for other indications or for use with other assays.
13
NanoString Confidential.
2012
2013
2014
nCounter Product Portfolio: nCounter Reagents
Advanced Disease Research
Advance Disease Research
Since 2009
CodeSets
Custom-built assays
Standardized panels
PanCancer pathways
PanCancer immune
profiling
Enable Clinical Testing
Enable Clinical Testing
Commercial Release:
February 11, 2014
nCounter Elements™
Components to Develop Assays
Registered with FDA
Flexible Format
Global Diagnostic Kits
Globalize Diagnostic Kits
FDA-cleared:
September 9, 2013
Prosigna™
Prosigna Breast Cancer Assay
IVD Test Kit
CE Marked & launched in EU,
Australia, Canada, Hong Kong,
New Zealand, Israel, Turkey
Discovery to Companion Dx: NanoString LST
January 2014
May 2014
June 2014
Researchers publish nCounter-based
subtyping assay for
Diffuse Large B Cell Lymphoma
(DLBCL)
NanoString licenses
DLBCL IP
Collaboration with Celgene to support
development of REVLIMID as treatment
for patients with DLBCL announced
21
DLBCL molecular subtypes first identified in 2000
Observed two dominant gene expression
patterns in a set of DLBCL tumors
First pattern clustered with gene expression from
Germinal Center B-Cells
Second pattern clustered gene expression from
Activated Blood B-Cells
Subtypes reflected the “Cell of Origin”
“Activated B-Cell-Like” or ABC
“Germinal Center B-Cell-Like” or GCB
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Alizadeh; Nature 2000; 403,503-511
The cell-of-origin is prognostic in R-CHOP treated
patients
Data show that ABC have worse outcome relative to GCB-type tumors
when treated with R-CHOP
Lenz; NEJM 2008; 359, 2313-2323
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Targeted drugs in ABC type DLBCL
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Roschewski; Nature Reviews in Clinical Oncology 2014; 11, 12-23
Cell-of-Origin subtypes have the potential to be
predictive for benefit of Lenalidomide
 ABC/non-GCB subtype exhibits largest response to Lenalidomide
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Nowakowski et al; JCO; 2014; 33(3):251-7
Implementation of Cell-of-Origin in the clinic was
limited by gene expression profiling platforms
DNA microarrays require fresh frozen tissue
Clinical samples are FFPE
DNA microarrays often exhibit site-to-site reproducibility
Bias from enzymatic reactions
Bias from operators
In an effort to develop assays that could be translated into the
clinic, researchers evaluated FFPE-based nCounter and IHC
assays
Lymph2Cx Overview
20-Gene Signature for COO Classification
FFPE-Compatible COO Classifiers
IHC-based COO Assay
Misclassification Rate:
6 – 17%*
*Depends on interpretation method
Established Prognostic Utility
nCounter-based COO Assay
nCounter-based
Lymph2Cx Assay
Misclassification Rate:
2%
Lymph2Cx – Concordance Between Independent
Laboratories
98% for biopsies with “definitive COO”
95% for all biopsies
Scott et al; Blood; 2014
Clinical Study Design DLC-002 (ROBUST)
Lenalidomide 15 mg x 14 days + R-CHOP21
6 cycles*
n=280
ABC
Untreated
DLBCL
Central pathology
confirmation
R
Placebo x 14 days + R-CHOP21
6 cycles*
n=280
Select by GEP in
real time
GCB,
unclassified
Ineligible
Global Phase III study (CTG: NCT02285062)
Select ABC subtype of DLBCL using nCounter gene expression profiling assay
Lock down assay and algorithm prior to trial
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Development of CDx: An Involved, Cross-Functional Effort
Development
Manufacturing
Clinical
Reagent Development Manufacturing Transfer Protocol Development
Regulatory
G&A
Pre-IDE
Alliance Management
Assay Development
Reagent Production
Assay SOP transfer
IDE submission
Quality Control
Process Validation
Site qualification
PMA submission
Manufacturing Transfer
System installation
ExUS for global study
Algorithm Training
User training
Software Development
Physician training
Site monitoring
Lymphoma Subtyping Test Overview for DLC-002 (ROBUST)
Extract RNA from
FFPE slide mounted
tissue sections
Process Patient Tumor RNA & RNA
Reference Sample on nCounter Dx
Analysis System
Capture patient gene
expression profile
Apply Software Algorithm (Embedded)
Data QC
𝑳𝑷𝑺 =
𝒂𝒋 𝑿𝒋
𝒋
Assign
Subtype
LPS: Linear Predictor Score; aj = weight for gene j, Xj = normalized gene expression value for gene j
NanoString Confidential.
Feasibility, Dev. & Verification Testing Overview
(Wallden et. al, JCO, 33, 2015; abst 8536)
Feasibility testing
Development &
Verification testing
Reagent
Development
> 500 assays
(multiple lots of critical reagents)
> 1000 assays run
(multiple lots of critical reagents)
Accuracy of
Algorithm
LLMPP Lymph 2Cx
Develop: 51 samples
Verify: 68 samples
528 assays across 44 tissue samples
160 assays across 20 tissue samples
Precision
176 assays
(44 RNA samples x 3 kit lots)
120 assays
(10 RNA samples x 3 kit lots x 2 users)
RNA input
15 RNA samples
Input range of 62.5 – 1500 ng
20 RNA samples
Input range of 62.5 – 1000 ng
Testing
Reproducibility
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Conclusions – LST CDx on nCounter platform
The NanoString nCounter platform is well-suited to multiplexed genomic CDx
The NanoString LST is being deployed in a pivotal phase III clinical trial
From Publication to Clinical Study Initiation in ~250 Business Days
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Project ChinOOK and the NanoString team