Analytically robust expression Outline of profiles from ... · cancer 2) Subtyping by immunohistochemistry 3) Subtyping by qRT‐PCR 4) Nanostring techology 5) Development for clinical

T Nielsen: Breast cancer subtyping (Nanostring)

June 6, 2013

1

Analytically robust expression profiles from FFPE sections using Nanostring technology

Torsten O. Nielsen, MD/PhD, FRCPC Professor of Pathology and Laboratory Medicine

University of British Columbia, Vancouver, Canada

1COI statement: Dr. Nielsen is a coinventor of the PAM50 test, which has been licensed to Nanostring

Outline of Presentation

1) Intrinsic molecular subtypes of breast cancer

2) Subtyping by immunohistochemistry

3) Subtyping by qRT‐PCR

4) Nanostring techology

5) Development for clinical use

classification of breast cancerusing gene expression profile microarrays

Modified from Perou, 2010, Cold Spring Harb Perspect Biol. DOI: 10.1101/cshperspect.a003293

Key points about expression profile intrinsic subtypes

• Unsupervised biological signature: genes selected because they discriminate intrinsic patterns, not clinical outcome categories

• Breast cancers more clearly segregate this way than other common carcinomas

• Patterns hold up on multiple platforms and independent investigators

Outcome-based Classifiers: • Oncotype Recurrence Score• Mammaprint• Endopredict• Breast Cancer Index

Biology-based Classifers:Microarray-based• Intrinsic subtyping• Stromal signatures• Genomic Grade Index

NextGen Sequencing Based• Aparicio integrated groups


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The Major Intrinsic Biological Subtypes

Lum A Lum B HER2E Basal

+ + ‐ ‐

‐ + + +

‐ +/‐ + ‐

‐ ‐ ‐ +

Estrogen Response genes:ESR1, PGR, GATA3, FOXA1

Proliferation genes:MKI67, CCNB1, CENPF, FOXA1, MYBL2, ORC6L

HER2‐associated:ERBB2, GRB7

Basal‐like markers: KRT5, KRT17, ERBB1,

TRIM29, CRYAB

HER2E Breast Cancer

• 10-15% of cases are HER2E

• well-studied; identifiable by FISH and IHC (as well as PAM50)

• very aggressive natural history

• responds to anthracyclines, taxanes, trastuzumab & new anti-HER2 agents

ER HER2PR CK5/6EGFR Ki67

Luminal A Breast Cancer

• most common type: ~40-50%. Older pts.

• express ER-associated proteins, and respond to endocrine therapy

• antiapoptotic phenotype: BCL2, slow growing, eventually metastasizes (to nodes and bone), good 5- and 10-year outcomes

• do NOT express proliferation or HER2-associated genes, chemotx may not benefit

Luminal B Breast Cancer• ~25-35% of cases (depends on study popn)

• includes cases which co-express hormone receptors and HER2, and ER positive cases with high proliferation

• Disease specific survival hazard ratio ~ 2.0 vs Luminal A

• may benefit from both hormonal and chemotherapy

ER HER2PR CK5/6EGFR Ki67

Basal Like Breast Cancer hallmarks

• do NOT express estrogen receptor nor ER associated genes (includingPR, GATA3, BCL2, FOXA1)

• do NOT express HER2 nor associated genes (e.g. GRB7)

ER HER2PR CK5/6

Genome-wide Profiling:Problems & Limitations

• Costs high for equipment

• Data processing & analysis is complex

• Needs special tissue handling

• Does not work well on FFPE


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• format for ready application integration into hospital lab testing• main format of archival tissue linked to existing outcome data

• Immediate clinical applicability

• cf. to diagnostic gold standard

• Morphology confirms tumor & pattern

• tissue from standard handling

immunohistochemistry

Subtyping in a clinical setting

+

+

+‐ ‐ ‐

‐ ‐

+ +/‐

≤13%

>13%

‐>20%

+/‐

breast cancer prognosis (DSS) by subtypeBCCA 4000 case series, diagnosed 1987‐1992

intrinsic subtype is also prognostic for local and regional relapses, post lumpectomy and post mastectomy…

LumA

LumB

basal

TNP non-basal

HER2Lum/HER2

subtypemedian

survival w/ mets (yr)

most common site

of met

other sites

@↑risk

Lum A 2.2 bone

Lum B 1.6 bone

HER2E 0.7 bonebrain, liver,

lung

basal 0.5 lung brain


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“Triple Negative” Breast Cancer:a heterogenous entity

PRESENTED BY: Maggie Cheang, Aleix Prat, and Chuck Perou

• clinical assays of convenience (ER/PR/HER2) are not great at predicting molecular subtype– TNBC as defined at local hospitals

– compared to expression profile

Survey of Basal Biomarkers• 72 biomarkers were drawn from (1) existing markers, (2) review

of published literature, and (3) gene expression profile data.

INPP4B neg

(1) existing markerse.g.ER negPR negHER2 negCK5/6EGFR

(2)

(3)

Won JR et al., Modern Pathol. 2013

Biomarker Sensitivity Specificity Odds Ratio

Neg INPP4B 61.1 98.6 108.4

Nestin 54.1 95.8 26.7

Neg ER 92.1 67.6 24.3

CK5 70.6 90.3 22.4

c‐Kit 42.4 96.8 22.1

p16 78.8 83.9 19.3

Fascin 57.9 92.9 17.9

…etc.

Biomarker Sensitivity Specificity Odds Ratio

Neg INPP4B 61.1 98.6 108.4

Nestin 54.1 95.8 26.7

Neg ER 92.1 67.6 24.3

CK5 70.6 90.3 22.4

c‐Kit 42.4 96.8 22.1

p16 78.8 83.9 19.3

Fascin 57.9 92.9 17.9

…etc.

LIMITATIONS OF IMMUNOHISTOCHEMISTRY

• Semiquantitative at best

• Limited capacity to subtype accurately (70-80%)

• Quality control is difficult


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Expression profiling of

FFPE cancer

specimens at the RNA

level 16 genes (+ 5 controls), derived from published expression profiles – fitted for prognosis in ER(+), node (-)

Tissue sent to central lab (California) as not FDA approved

$4000 / test (but cost effective)

PAM50 qPCR panel

basal

proliferation

estrogen

HER2

ES

R1

ERBB2

“luminal”

“HER2E”“Basal”

Cohort: ER+, tamoxifen-treated breast cancers from BC: outcomes by PAM50-assigned intrinsic subtype and Risk Of Relapse

Relapse Free Survival


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PAM50• Assigns subtype on paraffin blocks

• Assigns a Risk Of Relapse (ROR) –similar to recurrence score

• Identifies very low risk women: <10% relapse even without chemotherapy

• Works better than standard clinical grading and immunohistochemistry

predicting tamoxifen benefit in MA.12Chia SK, Nielsen TO & colleagues Clinical Cancer Research 2012

N = 398

predicting anthracycline benefit in MA.5Cheang MC, Nielsen TO & colleagues Clinical Cancer Research 2012

node (+) women, randomized to adjuvant CMF vs. CEF (anthracycline substitution)

HE

R2E

basa

l

relapse-free survival overall survival

HER2E subtype: CEF is superior

Luminal and basal: no significant differences

Basal: trend to CMF actually being better

N = 476

CMF

CEF

predicting benefit of dose-dense chemotxCALGB 9741: N = 1321 node (+); AC/T q2wk vs q3wk

intrinsic subtype by PAM50 remains prognostichowever

interaction test by treatment arm is negative: subtype does not predict value of q2wk (dose-dense) over q3wk (standard cycles) chemotx

Data generation by Nielsen, Perou, Ellis. Data handling by M Liu, W Barry, B Pitcher (Alliance central office)

No cDNA synthesis, cloning, or amplification, no enzymesDigital readout: 1 count = 1 mRNA; detection limit ~2000 molecules; range 106

100 ng FFPE total RNA can measure > 800 genesCompared to PCR: lower effort, little increased cost with increased numbers of genes

Nanostring nCounter Analysis System

Geiss G, Nature Biotechnology2008; 26:317-25

An easy and reliable way to measure RNA on standard pathology (FFPE) tissues:

nCounter Probe Sets

• Gene specific oligos are 35-50 bases long: “capture” has biotin tag, “reporter” has gene-specific fluorscent bar code• One mRNA template in specimen = one pairing of capture probe to reporter = one fluorescent bar code signal to count


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nCounter Assay

mRNA Capture & Reporter Probes

HybridizeCodeSet to RNA

Automated Process

Removeexcessreporters

Bindreporterto surface

Immobilize and align reporter

Image surface

Count codes

1. Isolate RNA from FFPE section, and mix with code set

nCounter Assay

HybridizeReporter to RNA

Automated Process

nCounter Prep Station




Image surface

Count codes

Hybridized mRNA Excess Reporters

nCounter Assay


Automated Process





Image surface

Count codes

Hybridized Probes Bind to Cartridge

Surface of cartridge is coatedwith streptavidin

3. Biotin on capture probe binds avidin on slide

nCounter Assay


Automated Process





Image surface

Count codes

Immobilize and align reporter for image collecting and barcode counting

4. Electric current lays down probe-RNAs flat in straight lines

nCounter Assay

Image Surface

One coded reporter = 1 mRNA


Automated Process

nCounter Digital Analyzer




Image surface

Count codes

nCounter Assay

Code Gene Count

ESR1 3

ERBB2 1

MKI67 2

Codes are counted and tabulated


Automated Process

nCounter Digital Analyzer




Image surface

Count codes


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Procedure of Nanostring RNA assay

HE slides reviewed by a pathologist

Unstained slides macrodissected to remove non-tumor tissue,

and total RNA extracted

Hybridization of RNA and codeset

Nanostring assay conducted on 250ng extracted RNA using the NanoString nCounter Analysis system

Prep station: 2.5 hour Digital analyzer: 4-6 hourDay 1: macrodissection Day 2: RNA isolation Hybridization: overnight

Analytical Validity: measurement precision, across observers and sites (clinical labs)

Clinical Validity: demonstration of independent value for prognosis or prediction on large clinical specimen series

EGAPP criteriaEvaluation of Genomic Applications in

Practice and Prevention

44

Clinical Utility = Analytical Validity + Clinical Validity

43 breast cancer specimens

3 sites / 3 operators / 3 machines

97-100% technical success rate

97% subtype concordance

Standard deviation of ROR < 2.9

Presented March 2013 at USCAP meeting in Baltimore…

Nanostring PAM50 assay: analytical reproducibility

1. Prognosis in ER positive breast cancer – including for late recurrence events, and node (+) cases

2. Predicting value of specific chemotherapy regimens

Nanostring PAM50 assay: clinical validity

Nanostring PAM50 test, applied to the ABCSG8 breast cancer clinical trial

presented by Gnant & Nielsen at SABCS December 2012

1671 patients re-consented for long term follow-up (or deceased)Median follow up 11 years1620 blocks collected:

25 (1.5%) insufficient cancer in block on path review73 (4.5%) insufficient RNA isolated44 (2.7%) failed QC specs for Nanostring device (i.e. control genes)

Overall, 1478 patients (91.2%) were informative for PAM50

ABCSG8 (N= 3714)

Postmenopausal ER+ women, grade 1 or 2 breast cancer

No chemotherapy(Dubsky PC et al., JCO 2012)

all patients receive 5 yrs hormone tx

RORRisk Group

Estimated % with no recurrence at 10 yearsPatients (%)

Events by 10 years

498 (34%) 15 96.7% (94.6-98.0)

478 (32%) 35 91.3% (88.1-93.8)

502 (34%) 87 79.9% (75.7-83.4)

Total 1,478 (100%) 137Dis

tant

re

laps

e-fr

ee s

urvi

val

yr yr

Nanostring PAM50 on ABCSG8: results

PAM50 adds prognostic information beyond Clinical Treatment Score in all patients: Likelihood ratio test LR 2 = 54, P <0.0001

multi-analytical gene-expression test performed on-site in a hospital lab


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Nanostring PAM50 test applied to the ATAC breast cancer clinical trial

1007 patients, adjuvant tamoxifen or anastrazole: Dowsett et al., in press

PAM50 has much more clinical predictive value than OncotypeDX for likelihood of distant recurrencein ER+, node (-)

Also: fewer cases classified as “intermediate risk” with PAM50 (22%, vs 27% by RS)

Long term Aromatase inhibitors

• Trials including all ER(+) women support long term (>10 yr) use of aromatase inhibitors ... due to risk of late relapses

• Problems: noncompliance, cost, risk of osteoporosis

• ATAC trial: not prognostic for late relapse = IHC4 test, Oncotype Recurrence Score

• PAM50 identifies a group of 37% of women who do not need long term AI

PAM50 identifies a very low risk group: even when there is nodal involvement!(Gnant M et al. platform presentation ASCO June 2, 2013)

(40% of patients)


(21% of patients)



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Different Assays Track the Same Biology

IHCER+ PR+ HER2-

Ki67 lowother ER+

ER-HER2+

Triple neg. or

basal

Mamma-Print (array)

good prog

poor prognosis

Oncotype (qRT-PCR)

low RS high Recurrence Score

Intrinsic / PAM50

Lum A Lum B HER2 basal← (ROR high) →(ROR low)

good prognosis group: may not need chemotherapypoor prognosis groups: what about prediction? … which

chemotherapy regimen is best, for which patients?

Gemcitabine in metastatic breast cancer

• Scandinavian Breast Group 0102: patients with advanced, metastatic breast cancer randomized to standard docetaxel, vs. gemcitabine + (lower dose) docetaxel

• Nielsen D et al JCO 2011: no clinically meaningful benefit from gemcitabine

• BUT … not analyzed by subtype … in vitro data suggests basals are most susceptible to gemcitabine …

Charlotte L. T. Jørgensen, Torsten O. Nielsen, Karsten D. Bjerre, Shuzhen Liu, Brett Wallden, Eva Balslev, Dorte L. Nielsen and Bent Ejlertsen

DBCG translationalstudy population

(n = 273)

DBCG / PAM50 translational

study population(n = 270)

FFPE samples not available / unsuitable for PAM50

(n = 64)

RNA extraction unsuccessful(n = 3)

PAM50 successful(n = 270)

DBCG clinical trial population

(N = 337)Assays run in Vancouver; all statistical analyses prespecified and performed independently at DBCG

Primary hypothesis: basal breast cancers will have a better outcome in the gemcitabine arm

17%

36%31%

16%

Nanostring PAM50 profiling of SBG0102

%

predicting gemcitabine benefit in SBG0102Tykjaer-Jorgensen C et al. SABCS 2013

metastatic breast cancer, randomized to docetaxel vs. gemcitabine + docetaxel

N = 270, PAM50 by Nanostring …

trial overall = no significant differences

but… by subtype… basals do muchbetter on gemcitabine

effect masked by opposite finding in HER2

still need to validate in a second similar trial, to reach level 1 evidence…

Active development projects

Prediction for anthracycline benefit

New breast cancer signatures: predicting responsiveness to radiation therapy

Signatures in other cancers: hepatocellular carcinoma (186 genes), medulloblastoma (22 genes) …

Other Nanostring uses: quantifying miRNA, DNA copy number, fusion genes, ChIP assays. Does not detect point mutations.

Clinical Meaning Of Intrinsic Subtypes

Lum A Lum B HER2 basal

hormonal tx + + - -trastuzumab - - + -prognosis + - - -

cyclophosphamide, taxanes - + + +anthracyclines - + + -

gemcitabine - - - +light blue = shown by IHC, microarray and/or PCRdark blue = confirmed by Nanostring PAM50


June 6, 2013

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1. Validated test with completely pre-defined classifier

2. Analysis plan prespecified in writing

3. Results of analysis still need to be validated on a second, similar clinical trial dataset

to obtain Level 1 Evidence from archived tissue from completed

Phase III trials

JNCI 101:1446-52 | November 4, 2009

Level 1 evidence

Lum A Lum B HER2 basal

hormonal tx + (short term)

+ (long term)

- -

trastuzumab - - + -prognosis + - - -

cyclophosphamide, taxanes - + + +anthracyclines - + + -

gemcitabine - - - +

issue completed underway

antiestrogen MA.12, ATAC, ABCSG8

anthracyclines MA5 DBCG 89D

taxanes GEICAM 9906 CALGB 9344

gemcitabine SBG0102 ?

dose dense chemo CALGB 9741 MA21

need for XRT OCOG/BC DBCG 82C

Plans: generate Level 1 evidence for the PAM50 assay’s Clinical Utility

collaborations involve Canadian, US, Danish, Spanish and Austrian clinical trial groups (so far…)

PAM50 Nanostring intrisic subtype test: current status

• Received CE mark for use in Europe

• Registry studies for FDA approval complete & submitted (decision pending)

• marketed as Prosigna test

Conclusions

1) Molecular intrinsic subtypes have become a fundamental concept in understanding breast cancer biology, diagnosis and treatment

2) Translating from discovery on microarrays into available, valid clinical tests takes >10 years

3) Nanostring‐based version of test is analytically valid. Tells us who needs therapy (level 1 evidence), & may predict which therapy is best (confirmatory studies on clinical trials pending)

acknowledgements

UBC & BCCA

• Blake Gilks

• David Huntsman

• Stephen Chia

• Karen Gelmon

• Sam Leung

• Doris Gao

• Jennifer Won

• David Voduc

• Christine Chow

• Charlotte Tykjaer‐

Jorgensen (DBCG)

• Tinne Laurberg

(Aarhus)

COLLABORATING NETWORK

• Chuck Perou (UNC‐Chapel Hill)

• Matthew Ellis (Washington U)

• Philip Bernard (University of Utah)

• S. Ferree, J. Storhoff (Nanostring)

• Lois Shepherd (NCICCTG)

• Mitch Dowsett (UK), M Gnant (Austria)

• Bent Ejlertsen (Denmark)

FUNDING

• US Nat’l Institute of Health

• CBCF (BC/Yukon); BCRF

• Michael Smith Foundation for Health Research

• Nanostring technologies

Documents

Analytically robust expression Outline of profiles from ... · cancer 2) Subtyping by immunohistochemistry 3) Subtyping by qRT‐PCR 4) Nanostring techology 5) Development for clinical