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
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
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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
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
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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
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
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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
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
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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
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
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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
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
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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
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
Count codes
Hybridized mRNA Excess Reporters
nCounter Assay
HybridizeReporter to RNA
Automated Process
nCounter Prep Station
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
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
HybridizeReporter to RNA
Automated Process
nCounter Prep Station
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
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
HybridizeReporter to RNA
Automated Process
nCounter Digital Analyzer
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
Count codes
nCounter Assay
Code Gene Count
ESR1 3
ERBB2 1
MKI67 2
Codes are counted and tabulated
HybridizeReporter to RNA
Automated Process
nCounter Digital Analyzer
Removeexcessreporters
Bindreporterto surface
Immobilize and align reporter
Image surface
Count codes
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
8
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
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
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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)
PAM50 identifies a very low risk group: even when there is nodal involvement!(Gnant M et al. platform presentation ASCO June 2, 2013)
(21% of patients)
PAM50 identifies a very low risk group: even when there is nodal involvement!(Gnant M et al. platform presentation ASCO June 2, 2013)
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
10
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
T Nielsen: Breast cancer subtyping (Nanostring)
June 6, 2013
11
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