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 15
Digital Counting: How it Works (1 of 2) Target-specific Capture Probe Target-specific Reporter Probe Half Site 50 bases Half Site 50 bases Barcode Biotin
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 Bind to Surface Immobilize and Align Image Surface Count Codes Target nucleic acid Capture and Reporter Probes ( CodeSet )
7 ncounter Assay Hybridize CodeSet to RNA Remove Excess Bind to Surface Immobilize and Align Image Surface Count Codes Hybridized mrna Excess Probes Removed
8 ncounter Assay Hybridize CodeSet to RNA Remove Excess Bind to Surface Immobilize and Align Image Surface Count Codes Hybridized Probes Bind to Cartridge Bottom of cartridge is coated with streptavidin
9 ncounter Assay Hybridize CodeSet to RNA Remove Excess Bind to Surface Immobilize and Align Image Surface Count Codes Immobilize and Align for Image Collecting and Barcode Counting +
10 ncounter Assay Hybridize CodeSet to RNA Remove Excess Bind to Surface Immobilize and Align Image Surface Count Codes One Coded Reporter = One RNA Molecule
11 ncounter Assay Hybridize CodeSet to RNA Remove Excess Bind to Surface Immobilize and Align Image Surface Count Codes Codes are Counted and Tabulated One Count = One Gene
Digital and Automated Workflow 5 min HANDS-ON Step 1 OVERNIGHT 5 min HANDS-ON Step 2 2.5 3.0 HOURS, AUTOMATED 5 min HANDS-ON Step 3 3 4.5 HOURS, AUTOMATED ncounter Prep Station ncounter Digital Analyzer 1 Hybridize 2 Purify 3 Count Flexible sample input requirements Only 4 pipetting steps/sample No amplification (except for single-cell) 800 hybridizations in a single tube Sensitive Precise Quantitative Simple
ncounter: Versatile and Validated Platform Gene Expression Copy Number Variation Single Cell Expression Multiple Applications mirna Expression Gene Fusions Proteins (In Early Access) Over 850 Peer-reviewed Publications 254 186 105 44 8 17 2009 2010 2011 2012 2013 2014 13 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. NanoString Confidential.
ncounter Product Portfolio: ncounter Reagents Advanced Disease Research Enable Clinical Testing Global Diagnostic Kits Advance Disease Research Enable Clinical Testing Globalize Diagnostic Kits Since 2009 Commercial Release: February 11, 2014 FDA-cleared: September 9, 2013 CodeSets Custom-built assays Standardized panels PanCancer pathways PanCancer immune profiling ncounter Elements Components to Develop Assays Registered with FDA Flexible Format 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 Researchers publish ncounter-based subtyping assay for Diffuse Large B Cell Lymphoma (DLBCL) May 2014 NanoString licenses DLBCL IP June 2014 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 16 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 17
Targeted drugs in ABC type DLBCL 18 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 19 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) Untreated DLBCL Central pathology confirmation ABC Select by GEP in real time GCB, unclassified R Lenalidomide 15 mg x 14 days + R-CHOP21 6 cycles* n=280 Placebo x 14 days + R-CHOP21 6 cycles* n=280 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 23
Development of CDx: An Involved, Cross-Functional Effort Development Manufacturing Clinical Regulatory G&A Reagent Development Manufacturing Transfer Protocol Development 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 = j a j X 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) Testing Feasibility testing Development & Verification testing Reagent Development Accuracy of Algorithm > 500 assays (multiple lots of critical reagents) LLMPP Lymph 2Cx > 1000 assays run (multiple lots of critical reagents) Develop: 51 samples Verify: 68 samples Reproducibility 528 assays across 44 tissue samples 160 assays across 20 tissue samples Precision RNA input 176 assays (44 RNA samples x 3 kit lots) 15 RNA samples Input range of 62.5 1500 ng 120 assays (10 RNA samples x 3 kit lots x 2 users) 20 RNA samples Input range of 62.5 1000 ng 26
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 28
Project ChinOOK and the NanoString team