frederique penault llorca : prosigna : un test décentralisé apporte t il une valeur...
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Centre de Lutte contre le Cancer d'Auvergne Clermont-Ferrand - France -
Centre Jean Perrin
Un test dcentralis apporte t il une valeur ajoute aux quipes doncologie mdicale dans les cancers du sein en
situa8on adjuvante ? Reproduc8bilit et abilit des rsultats
Frdrique Penault-Llorca, MD, PhD
CENTRALIZED APPROACH
The Oncotype DX Assay
Genomic Health, Inc.
OncotypeDX (Genomic Health, USA)
HR+ / HER2- , T1-3, N-/N+ FFPE specimens
qRT-PCR 21 GENES
PROLIFERATION, OESTROGENE, HER2, INVASION (16 GENES) + REFS (5 GENES)
CENTRALIZED TEST
(recurrence score) RS Late recurrence (10 years)
Benefit from adjuvant TT PROGNOSTIC AND PREDICTIVE
LOW RISK :
+ HORMONOTHERAPY / - CHEMOTHERAPY
INTERMEDIATE RISK : DISCUSSION
HIGH RISK : + HORMONOTHERAPY / + CHEMOTHERAPY
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The Oncotype DX assay is analy3cally validated
Elements of analy8c valida8on Analy3cal sensi3vity (limits of detec3on and quan3ta3on)
Assay precision and linear dynamic range Analy3cal reproducibility PCR amplica3on eciency Sample and reagent stability Reagent calibra3on Instrument valida3on and calibra3on
Chau CH, et al. Clin Cancer Res. 2008;14(19):5967-5976.
Analytical validation is the assessment of assay performance characteristics and the optimal conditions to
generate accuracy, precision and reproducibility
MammaPrint
Agendia, Inc.
MammaPrint (Agendia, NL)
HR+ ET HR - / HER2- , T < 5cm, N 3
Fresh frozen=> FFPE DNA array
70 GENES CELL CYCLE/ PROLIFERATION
SIGNAL TRANSDUCTION INVASION, METASTASIS, ANGIOGENESIS
CENTRALIZED TEST
RECENTLY ADAPTATED TO FFPE
Group of genes ( signatures )
EARLY RECURRENCE (Dg < 5 ans) PROGNOSTIC
GOOD SIGNATURE : LOW RISK
POOR SIGNATURE : HIGH RISK
HR+& HR-
70 Gene Assay FFPE Uncertainty FFPE tumor 3ssue xa3on
causes RNA to degrade, the accuracy of microarray tes3ng depends on keeping the tumor RNA intact
The 70 gene assay analy3cal validity tests were performed on fresh frozen 3ssue (current method in the FDA label)
The 70 gene assay is now available in paran as part of the SYMPHONY tests; however, adequate valida3on of this method is not documented in the public literature
h\p://www.agendia.com Scicchitano MS, et al. J. Histochem. Cytochem. 2006; 54 (11): 12291237. 8
Procedure
FFPE Fill the form Send block, slides or tumor
sample to the central lab with a dedicated box
Results within 8 days via e-mail
DECENTRAL GENE EXPRESSION ANALYSIS
Prosigna PAM50 ROR NanoString nCounter
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Development of Prosigna is Based on PAM50 Gene Signature
2000 Researchers first describe
breast cancer intrinsic subtypes based on microarray
experiments
2009 Researchers first describe PAM50 gene expression
signature
2010 NanoString exclusively licenses
PAM50 gene expression signature
2012/13 Prosigna launches after receiving CE Mark for Europe & Israel; FDA 510k clearance in US
PAM50 developed by a consor3um of four academic breast cancer experts Charles Perou, PhD, University of North Carolina Dr. Ma\ Ellis, Washington University School of Medicine Torsten Nielsen, MD, PhD, Pathologist, BC Cancer Agency Philip Bernard, MD, University of Utah / Huntsman Cancer Ins3tute
Source: Molecular portraits of breast cancer. Nature. 2000 May 25;. Source: Supervised Risk Predictor of Breast Cancer Based on Intrinsic Subtypes, JCO.2009
Overview of Principles: Design Advantages of nCOUNTER Direct detec8on (no amplica3on of target)
Designed for short sequences ~100 bp
Digital coun8ng results in excellent analy8cal performance
Highly sensi3ve and precise Wide dynamic range (5 logs)
Automated processing Internal controls Mul8plexed
Capture Probe
Reporter Probe
Target
Target-Probe Complex
SOLUTION HYBRIDIZATION
REMOVE EXCESS PROBE IMMOBILIZE/ALIGN
DIGITAL COUNT
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Intrinsic Subtype: Organizing Framework for Breast Cancer
Supported by The Cancer Genome Atlas Study1
Endocrine therapy alone Luminal A
1. Comprehensive molecular portraits of breast cancer. Nature. 2012 Oct 4;490(7418):61-70. 2. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen InternaQonal Expert Consensus on the Primary
Therapy of Early Breast Cancer 2013 Annals of Oncology Advance Access published August 4, 2013
Diverse genetic and epigenetic alterations converge phenotypically into the four main breast
cancer subtypes defined by PAM50
Endorsed in 2013 St. Gallen Guidelines2
If HER2, endocrine +/- cytotoxic therapy If HER2+, cytotoxics + an3-HER2 +
endocrine Could include anthracyclines and taxanes
Luminal B
Cytotoxics + an3-HER2 Could include anthracyclines and taxanes
HER2 enriched
Cytotoxics therapy alone, poten3ally including anthracyclines, taxanes and analkyla3ng agent
Do not rou3nely use cispla3n or carbopla3n Basal-like
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Three Elements of Prosigna Breast Cancer Assay
Hardware: nCounter Analysis System
Consumable: Prosigna Kits
SoYware: Prosigna Report
Prep Sta8on
Digital Analyzer
Includes: 50 gene-based CodeSet with 8 controls Other consumables required for assay CE Marked Roche RNA isola3on kit sold separately
nCounter Analysis System and Prosigna Breast Cancer Assay Kit received FDA 510K clearance in 2013 and CE Marked in 2012
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Prosigna Tests Formalin-Fixed Paraffin-Embedded Samples
H&E stain to iden3fy tumor area and
cellularity
Tumor area transposed to unstained slides and macrodissected
RNA extracted Block selected
Specimen Attribute Requirement Tissue input Viable invasive breast carcinoma (ductal, lobular, mixed, or NOS/NST)
Tissue input format Macrodissected 10-micron-thick slide-mounted tissue sections
Minimum tumor size 4 mm2 tumor area
Minimum tumor cellularity 10% within tumor area
Minimum RNA amount 125 ng (12.5 ng/l)
Tissue area 100mm2 1 slides required
20 99mm2 3 slides required
4 19mm2 6 slides required
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Simple and fast workflow is well suited for qualified clinical laboratories
Simple Prosigna Workow Enables Decentralized Tes3ng Model
1
nCounter Prep Station nCounter Digital Analyzer
Hybridize 2 Purify 3 Count
Step 3 3 4.5 HOURS, AUTOMATED
5 min
HANDS-ON Step 2 2.5 3.0 HOURS, AUTOMATED
5 min
HANDS-ON Step 1 12 HOURS OR OVERNIGHT
5 min
HANDS-ON
PAM50 ROR by NanoString nCounter
Extract RNA from FFPE
tumor sample
Run RNA & PAM50 CodeSet on nCounter Analysis System
Capture paQent expression prole
Calculate Risk of Recurrence (ROR) Score
Determine Intrinsic Subtype through Pearsons CorrelaQon to Centroids
4 or 10 samples Overnight incubation
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PAM50 Algorithm Generates a Prosigna Score for Each Patient
Gene expression data are weighted with clinical variables to determine an integer score from 0 through 100 (ROR/Prosigna Score) indica3ve of the probability of distant recurrence
ROR is based on the similarity of the gene expression prole to intrinsic subtypes, prolifera3on score, and tumor size
Assay requires input of gross tumor size and nodal status Determine intrinsic subtype through Pearsons correla8on to centroids
ROR = aRLumA+ bRLumB+
cRHer2e+
dRBasal+
eP+
fT
Pearsons correla3on to centroids
Calcula8ng ROR (Prosigna Score)
Pa8ent expression prole
Prosigna centroids
Prolifera3on score Gross tumor size >2cm
Gnant M, et al. SABCS 2012; poster P2-10-02.
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Patient Report Output: Page 1
Patient report: Identifying information
Assay description: Describes components of the Prosigna assay
Risk of Recurrence: Patient specific ROR is reports based on Prosigna algorithm. The ROR ranges from 0 to 100
Probability of distant recurrence: This section provides the correlation of the ROR with a specific likelihood of distant recurrence at 10 years, based on the average 10-year distant recurrence rate for that ROR in the clinical trial population. The probability of distant recurrence at 10 years increases continuously with an increase in ROR
Designed as a tool for pa8ent/oncologist communica8on
CE-IVD-marked
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Patient Report Output: Page 2
Description of validation studies
Distant recurrence by subtype
Risk curves by study
Provided as detailed background on valida8on studies for oncologist
CE-IVD-marked
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Highlights of Prosigna Report
Prosigna Output: Risk Interpreta3on and Categoriza3on by Nodal Status
Risk classica3on guidelines are provided based on cutos related to clinical outcome in the tested pa3ent popula3ons:
10-year probability of distant recurrence of < 10% is considere