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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

5

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

12

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

16

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

17

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

19

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.

20

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

22

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