What Prosigna and other (molecular …...Assay Chemistry Overview Advantages: Direct detection No bias due to amplification of target Molecular counting No bias due to fluorescence

What Prosigna and other (molecular

multiparameter) tests tell us about

breast cancer.

John Bartlett, PhD, FRCPath

ONTARIO INSTITUTE FOR CANCER RESEARCH

What is Prosigna?

What is a multiparameter test?

What does Prosigna tell us about breast cancer?

What do other MMTs tell us about breast cancer?

Aren’t they all the same?

Can we predict treatment response?

Can we do better?

What is PROSIGNA?

3


What is Prosigna?

The Prosigna Breast Cancer Prognostic Gene Signature Assay is a

qualitative in vitro diagnostic tool that utilizes multiparameter

gene expression data weighted together with clinical variables to

generate a risk category and numerical score to assess a

patient’s risk of distant recurrence of disease at 10 years in

postmenopausal women with node-negative (Stage I or II) or

node-positive (Stage II), hormone receptor-positive (HR+) breast

cancer.


Multiparameter Tests – made simple?

• Multiparameter tests

– Measure multiple

factors which impact

outcome.

• Mathematical

modelling

• Prediction of

outcome

∑ (n-k) (n-k-1)

(n-k-2) … (n-k-

r+2) x k x (1)

n (n-1) (n-2) (n-

r+2) (n-r+1) (r-1)


Nottingham prognostic index

A mathematically derived multi-parameter prognostic test.

9 prognostic markers included in a single test

Age, Menopausal status, size (cm), lymph node stage,

tumour grade, “cell reaction”, sinus histiocytosis, ER,

adjvuant therapy

3 were independently prognostic in multivariate analysis

Size, grade, stage

Index is weighted – size is relatively less impactful than

grade/nodal status:

0.2xsize (cm) + grade(1-3)+ nodal status (0, 1-3, 4+ nodes).sinus histiocytosis =lymph nodes that contain a prominent population of histiocytes


Nottingham Prognostic Index (NPI)

Blamey et al. Eur J Cancer. 2007;43:1548


Simple really….

Multiparameter tests assess

different aspects of

clinical/pathological features of

cancer and using

mathematically derived

formulae produce a compound

assessment of risk/benefit to

guide treatment decisions.

What about PROSIGNA?

9


PROSIGNA: A 50 gene signature to sub-type and assess residual

relapse risk in breast cancer

2000Researchers first

describe breast cancer intrinsic subtypes based

on microarray experiments

2009Researchers first describe “PAM50” gene expression

signature

2010NanoString exclusively licenses PAM50 gene expression signature

2013NanoString began

marketing Prosigna in Europe, Israel and US

after receipt of CE Mark and 510(k)

clearance


PROSIGNA: How does it actually work?

Assay Chemistry Overview

Advantages:

Direct detectionNo bias due to amplification of target

Molecular countingNo bias due to fluorescence intensity

Wide dynamic range (5 logs)Accurate measurement of gene expression

Internal assay controlsEnsure data quality for every test result

Automated processingImproves reproducibility between

laboratories

11

Capture Probe

Reporter Probe

mRNA Target

Target-Probe Complex

Solution Hybridization

Prep Station:Remove Excess ProbeImmobilize/Align

DigitalAnalyzer: Counting mRNA


Count

Simple Workflow Enables Decentralized Testing Model

Simple and fast workflow is well suited for qualified clinical laboratories

1

nCounter® Prep Station nCounter® Digital Analyzer

Hybridize 2 Purify

Step 33 – 4.5 HOURS, AUTOMATED

Minimal

HANDS-ONStep 2

2.5 – 3.0 HOURS, AUTOMATED

Minimal

HANDS-ONStep 1

OVERNIGHT

Minimal

HANDS-ON

3


And a Risk Estimate for Each Patient

Estimate 10-Year Risk of Distant Recurrence With Endocrine Therapy Alone

= aRLumA +

bRLumB +

cRHer2e +

dRBasal +

eP +

fT

Pearson’s correlation to centroids

Calculate Prosigna Score

Patient expression

profile

Prosigna centroids

Proliferation scoreTumor size

Prosigna Score

1 Prosigna Score is also referred to as the ROR score in the literature

Gene expression data are weighted with clinical variables to determine the

Prosigna score1, an integer score from 0 through 100 indicative of the probability of

distant recurrence

Prosigna Score is based on the similarity of the gene expression profile to intrinsic

subtypes, proliferation score, and tumor size

What does Prosigna tell us

about breast cancer?

14


ATAC: 10-year predicted risk of DR using ROR Score

Predicted 10-year

risk of distant

recurrence (%)

03

6

% of cases

ROR Score

0 20 40 60 80 100

02

04

06

08

01

00

Node Negative1-3 Positive Nodes4+ Positive Nodes95% CI


DBCG Comprehensive Cohort Study:

10-year Distant Recurrence Analysis

Cumulative Incidence by Risk Group Cumulative Incidence for 1 Positive Node

Ejlertsen B et al. J Clin Oncol 33, 2015 (suppl; abstr 513) 2015.; Lænkholm A et al. J Clin Oncol 33, 2015 (suppl; abstr 546) 2015.

Nodal Status Risk Category10-Year DR [95%

CI]

P values

Any Diff.Diff. from

Int

All Patients

High 20.8 [18.3-23.4]

<0.0001

<0.0001

Intermediate 9.6 [7.4-12.2]

Low 4.3 [2.9-6.2] 0.0005

Node-Negative

High 18.5 [14.9-22.4]

<0.0001

<0.0001

Intermediate 7.3 [4.8-10.5]

Low 4.9 [2.8-7.8] 0.1543

1-Positive Node

High 21.0 [15.9-26.6]

<0.0001

0.0202

Intermediate 14.9 [9.9-20.9] -

Low 3.6 [1.7-6.5] 0.0001


Prosigna Utility Summary

Selecting Patients for Informing Chemotherapy Decisions

Node Negative Patients: Prosigna can identify a low risk group consisting of

50% of the patient population that have a probability of 10-year distant

recurrence < 4%

Node Positive Patients: Prosigna can identify patients with limited nodal

involvement who have a very low absolute risk of distant recurrence (< 5%)

Neoadjuvant Chemotherapy Response: The Prosigna score is significantly

associated with response to neoadjuvant chemotherapy with anthracyclines and

taxanes. Low risk patients have a very low response rate

Selecting Patients for Informing Extended Endocrine Therapy Decisions

Late Recurrence: Prosigna has been validated to predict recurrence in

years 5-10 (late distant recurrence) in > 2,000 patients

Prosigna is not cleared for use to select patients for therapy

Parker et al. JCO 2009; 27(8): 1160-1168; Prat et al. Clin Cancer Res 2016 Feb 1;22(3):560-6.; Fyles A, et al. ASCO-BC 2013 oral presentationEjlertsen B et al. J Clin Oncol 33, 2015 (suppl; abstr 513) 2015; Lænkholm A et al. J Clin Oncol 33, 2015 (suppl; abstr 544) 2015;Lænkholm A et al. J Clin Oncol 33, 2015 (suppl; abstr 546) 2015.


Simple really….

Prosigna provides an estimate of risk of recurrence (ROR) following optimal treatment (endocrine therapy)

“Residual risk” vs “Prognosis”

Risk estimates include risk of “late recurrence”

Node-ve and limited node +ve patients

Potential to predict chemotherapy response?

NB: PROSIGNA is a validated

PROGNOSTIC signature it is not

currently validated as a predictive

signature in any setting.

18

What do other multiparameter molecular

tests tell us about breast cancer?

19


MMTs – multiple variations on a theme…

20

Test Technology Parameters OutputValidation population

Oncotype DX RT-PCR 21 genes /RNA risk score /catCT benefit

ER+ (N0)

MammaPrint array 70 genes /RNA risk cat ER+/- (N0/N1)

BluePrint array 80 genes /RNA Subtype unrestricted

Prosigna(PAM50)

nCounter 50 genes /RNA sub-type/ risk score

ER+ (N0/N1-2) post menopause

BCI RT-PCR 11 genes /RNA risk score /catET benefit

ER+ (N0)

EndoPredict RT-PCR 11 genes/ RNA risk score ER+ (N0/N1)

Mammostrat IHC 5 proteins risk score ER+ (N0)

IHC4 IHC 4 proteins risk score ER+, post men.


TransATAC Study Comparing Signatures for Breast Cancer Recurrence

DR free (%) in years 0-10 node-negative*

Dis

tan

t re

cu

rre

nc

e f

ree

(%

)

0 2 4 6 8 10Follow-up time [years]

10

08

06

0

BCI

EPclin

10

08

06

0

3.9%

19.3%

27.3%

6.6%

22.1%

61.8% patients

72.6% patients

27.4% patients

24.2% patients

14.0% patients

10

08

06

0

ROR3.0%

14.1%

33.4%

53.8% patients

30.1% patients

16.1% patients

100

80

60

RS

16.7%27.2%

5.9% 63.3% patients

26.4% patients10.3% patients

DR risk % Patients % in risk groups

*TransATAC data is not part of the US-cleared Prosigna Intended Use

*Comprehensive Comparison of Prognostic Signatures for Breast Cancer Recurrence in TransATAC presentation; SABCS Dec, 2016



DR free (%) in years 0-10 node-positive*

Dis

tan

t re

cu

rre

nc

e f

ree

(%

)

23.8%

33.1%

50.6%

5.6%

37.2%

49.3% patients

18.9% patients

81.1% patients

32.6% patients

18.1% patients

0 2 4 6 8 10Follow-up time [years]

10

08

06

0

BCI

0.0%

20.7%

39.1%

6.6% patients

25.6% patients

67.8% patients

10

08

06

0

ROR

34.7%

48.8%

26.2% 57.7% patients

31.7% patients

10.6% patients

10

08

06

0

RS

10

08

06

0

EPclin






DR free (%) in years 5-10 node-negative*



Dis

tan

t re

cu

rre

nc

e f

ree

(%

)

Follow-up time [years]

5 6 7 8 9 10

80

90

100 EPclin

80

90

100 BCI

2.5%

14.4%15.9%

4.3%

14.6%

63.6% patients

73.5% patients

26.5% patients

23.6% patients12.9% patients

80

90

100 ROR

1.4%

10.0%

23.2%

54.6% patients

30.8% patients

14.6% patients

80

90

100 RS

9.6%

16.1%

4.8% 65.6% patients

25.1% patients

9.4% patients



MULTI-parameter assays evaluated in OPTIMA prelim

n=302

Equal allocation to

each arm

All patients were tested

with Oncotype DX

Additional tests

OICR

IHC4-DAB

Prosigna

Agendia

MammaPrint

BluePrint

Genoptix

IHC4-Aqua

Stratifyer

MammaTyper

24

Risk score lowintermediatehigh

Risk of Recurrence Score

Subtyping

Risk score lowhigh

Subtyping Luminal A/ BHer2 Enriched

Basal

Subtyping Luminal ALuminal B (int/hi)Her2 EnrichedBasal

Risk score lowintermediatehigh

lowintermediatehigh

Luminal A/BHer2 EnrichedBasal

PAM50


Marked differences between existing commercial diagnostic

risk tests;

Similar performance in the quantity of risk information provided;

Which test is most effective is unknown;

The large number of discrepant cases indicates that there are

opportunities for significant improvement in the development

of residual risk tests.

Summary of pathology results

26


What about prediction:

Chemotherapy

Oncotype Dx?

Prosigna?

Extended Endocrine therapy

Breast Cancer Index

Targeted therapies

95 Gene signature

27


Prat et al. suggests Prosigna may predict response to neo-

adjuvant Chemotherapy

Prosigna assay tissue input

optimized for core-needle

biopsies

Prediction of neo-adjuvant

chemotherapy response

Consistent with other

outcomes

(e.g. Oncotype Dx)

Wide confidence intervals.

Prat et al. Clin Cancer Res. 2015 Jul 7. pii:

clincanres.0630.2015.


Breast Cancer Index (BCI) Clinical Assay combines

Two Biomarkers

29

Breast Cancer Index

BCI PredictiveIndividualized Prediction of Likelihood of

Benefit from Extended Endocrine Therapy

BCI PrognosticIndividualized Risk of Cumulative

Overall (0-10 yr) and Late Recurrence (5-10 yrs)

Numerical result on a continuous curve

Algorithmic combination of MGI (Proliferation

Pathway) and H/I (Estrogen Signaling Pathway)

Genes: BUB1B, CENPA, NEK2, RACGAP1, RRM2,

HoxB13/IL17BR

Binary result: High/Low

Quantitative molecular assessment of estrogen

signaling pathways

Genes: HoxB13/IL17BR (H/I ratio)


Summary: Predictive

H/I shown to be a significant predictor of endocrine benefit in 3

randomized trial cohorts with significant interaction P-value

between BCI and treatment

30

Study Cohort

Treatment Predictive analysisInteraction P value

Stockholm

(n=600)1

Adjuvant tamoxifen

vs untreated

H/I High HR: 0.35 (0.19-0.65); p=0.0005

H/I Low HR: 0.67 (0.36-1.24), p=0.20.003

TransATAC(n=665)2

Adjuvant anastrozole vs tamoxifen

H/I High HR: 0.51 (0.27-0.97); p=0.04H/I Low HR: 1.33 (0.65-2.71), p=0.4

0.004

MA.17(n=249)3

Extended letrozole vs placebo

H/I High OR: 0.33 (0.15-0.73); p=0.006H/I Low OR: 0.58 (0.25-1.36), p=0.21

0.03

Results suggest generalizability as an endocrine response biomarker

1. Zhang Y, et al. Clin Cancer Res. 2013;19(15):4196-205. 2. Sgroi D, et al. Lancet Oncol. 2013 Oct;14(11):1067-76. 3. Sgroi et al, J Natl Cancer Inst. 2013;105:1036-1042


TEAM Trial Design

This presentation is the intellectual property

of author/presenter. Contact at

[email protected] for permission to

reprint and/or distribute.

4500 samples banked in a central research biorepository.


Recapitulation of Multiparametric Test Clinical Utility in TEAM

32

OncotypeDx-25-like

MammaPrint-like Genomic Grade Index-like

Prosigna-like

mRNA-IHC4

Intrinsic Subtyping

Bayani and Yao et al.,(2017) npj Breast Cancer


Identification of a 95-gene signature of residual risk

following endocrine treatment

33

10-Year Risk Score

TRA

ININ

G

VA

LID

ATI

ON

Genomic Grade Index-like (AUC=0.67)

IHC4-Protein (AUC=0.68)

Prosigna-like (AUC=0.70)

OncotypeDx-like (AUC=0.71)

IHC4-mRNA (AUC=0.72)

MammaPrint-like (AUC=0.72)

95-Gene Risk Signature (AUC=0.76)

Bayani and Yao et al.,(2017) npj Breast Cancer


Functional gene interaction analyses

34Bayani and Yao et al.,(2017) npj Breast Cancer


Overall Summary:

Multiparameter gene expression profiling provides robust,

reproducible information on residual risk post endocrine

therapy.

Clinical parameters still add value!

Several tests are validated for residual risk assessment, all

have similar performance.

There is considerable variation in test performance at the

individual patient level.

Few tests have data on prediction – none are yet fully

validated for prediction.

There remains room for improvement!

35


Acknowledgements

36

OICR

Transformative Pathology

John Bartlett

Jane Sun

Melanie Spears

Mary Anne Quintayo

Fu Yan

Cheryl Crozier

Taryne Chong

Nicola Little

Linda Liao

Ilinca Lungu

Dan Dion

Minalini Lakshman

Informatics and BioComputing

Paul Boutros

Cindy Yao

Syed Haider

Drug Discovery

Michael Prakesch

FACIT

TEAM Trial Group

Cassandra L. Brookes (UK)

Cornelis J.H. van de Velde (Netherlands)

Annette Hasenburg (Germany)

Dirk G. Kieback (Germay)

Christos Markopoulos (Greece)

Luc Dirix (Belgium)

Caroline Seynaeve (Netherlands)

Daniel Rea (UK)

OPTIMA Trial Group

Andrea Marshall

Janet A. Dunn

Amy Campbell

Peter S. Hall

Christopher J. Poole

David A. Cameron

Helena M. Earl

Daniel W. Rea

Iain R. Macpherson

Peter Canney

Adele Francis

Christopher McCabe

Sarah E. Pinder

Luke Hughes-Davies

Andreas Makris

Robert C. Stein

University of Edinburgh

Carrie Cunningham

Monika S. Sobol

Tammy Piper

Documents

What Prosigna and other (molecular …...Assay Chemistry Overview Advantages: Direct detection No bias due to amplification of target Molecular counting No bias due to fluorescence