1
Oncotype DX® breast cancer assay:
quantitative Single-Gene Testing
Quantitative hormone-receptor analysis
2
What role does Single-Gene Testing
in the Oncotype DX® assay play
in adjuvant therapy decision-making?
3
Single-Gene Testing and
the Oncotype DX® Assay
The Predictive Utility of ER and HER2 Status
ER Testing Methodology: IHC
HER2 Testing Methodologies: IHC and FISH
Single-Gene Testing in the Oncotype DX Assay
Degree of Concordance in ER Status
Between IHC and the Oncotype DX Assay
Degree of Concordance in HER2 Status
Among IHC, FISH, and the Oncotype DX Assay
4
Clinicopathological factors correlated with
prognosis and/or response to therapy
Factor1,2
Information provided1,2
Patient age
Associated with aggressiveness of cancer
Tumor size
Directly correlates with survival
Tumor grade
Predicts relapse and prognosis
Axillary node status
Number and extent of nodal involvement strongly predict
survival
ER/PR status
Correlates with survival; predicts response to hormonal
therapy
HER2 status
Correlates with relapse and survival; predicts response to
chemotherapy and anti-HER2–targeted therapy
ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2
ER/PR and HER2 status can predict response to therapy
1. Bundred J. Cancer Treat Rev. 2001;27:137.
2. Kapoor A, et al. Expert Rev Anticancer Ther. 2005;5:269.
5
Milestones in the development of ER
and HER2 as predictive biomarkers
ER
Ovary
removal
linked to
improved
prognosis
1890
Estrogen
receptor
(ER)
discovered
1960
Tamoxifen
approved by
FDA for
metastatic
breast
cancer
1970
Monoclonal
antibody
technique
developed
to detect ER
1980
HER2
HER2
gene
cloned
ER gene
Cloned
1990
HER2 overexpression
linked to
breast
cancer
progression
Jordan VC. Cancer Res. 2009;69:1243.
Breast Cancer Campaign. Available at: www.breastcancercampaign.org. Accessed June 23, 2009.
NSABP B-14:
tamoxifen
confers
improved
outcomes on
ER+ patients
2000
Trastuzumab approved by
FDA for HER2+ advanced
disease; IHC and FISH
assays approved to test for
HER2 status from patient
samples
6
ER status is predictive of response
to tamoxifen therapy
Risk of breast cancer death
Risk of recurrence
HR
HR
ER-poor
1.04
1.04
ER-positive
0.59
0.66
ER-unknown
0.69
0.80
0
0.5
Tamoxifen better
1.0
1.5
2.0
Tamoxifen worse
Early Breast Cancer Trialists’ Collaborative Group. Lancet. 2005;365:1687-1717.
0
0.5
Tamoxifen better
1.0
1.5
2.0
Tamoxifen worse
7
Anti-HER2 therapy provides
significant benefit in HER2+ patients
100
Chemotherapy + trastuzumab (n = 235)
Chemotherapy alone (n = 234)
Survival (%)
80
60
40
Median 25.1 months
Median 20.3 months
20
0
P = 0.046
5
15
25
35
Months after enrollment
Slamon DJ, et al. N Engl J Med. 2001;344:783.
45
8
Single-Gene Testing and
the Oncotype DX® Assay
The Predictive Utility of ER and HER2 Status
ER Testing Methodology: IHC
HER2 Testing Methodologies: IHC and FISH
Single-Gene Testing in the Oncotype DX Assay
Degree of Concordance in ER Status
Between IHC and the Oncotype DX Assay
Degree of Concordance in HER2 Status
Among IHC, FISH, and the Oncotype DX Assay
9
10
Controversies in IHC testing
of ER in breast cancer
Parameter
Controversy
Tissue
sample1
•Surgeons’ choice of tissue sample (eg, biopsy or resection) may vary by
time to optimal fixation
Sample
processing1-4
•The preanalytical process, including time to fixation, duration in fixative,
and choice of fixative is not standardized, which may lead to falsenegative IHC results
Staining
protocol1
•More than 3 primary antibodies are in use by labs for tumor IHC testing
•Choice of primary and secondary antibodies, antigen retrieval and
detection systems vary by laboratory
Threshold
for positivity1
•Cutoffs for determining positivity are not standardized and vary by
laboratory or by pathologist
•Cutoff used should be clinically validated for the primary antibody used
Quantification
of ER expression1,2
•Studies have shown that ER status by IHC is bimodal, either negative or
positive, not semiquantitative
•Other evidence supports ER status as a continuous variable
1. Gown AM. Mod Pathol. 2008;21(suppl 2):S8-S15; 2. Nadji M, et al. Am J Clin Pathol. 2005;123:21-27;
3. Goldstein NS, et al. Am J Clin Pathol. 2003;120:86-92; 4. Khoury T, et al. Mod Pathol. 2009;22(11):1457-1467.
11
Single-Gene Testing and
the Oncotype DX® Assay
The Predictive Utility of ER and HER2 Status
ER Testing Methodology: IHC
HER2 Testing Methodologies: IHC and FISH
Single-Gene Testing in the Oncotype DX Assay
Degree of Concordance in ER Status
Between IHC and the Oncotype DX Assay
Degree of Concordance in HER2 Status
Among IHC, FISH, and the Oncotype DX Assay
12
Immunohistochemistry semiquantitates
HER2 protein expression
IHC 0
IHC 1+
IHC 2+
IHC 3+
• IHC is scored on a 0-3+ scale, based on staining intensity and completeness of membrane staining
• ASCO/CAP HER2+: 3+ staining in > 30% of cells
Wolff AC, et al. J Clin Oncol. 2007;25:118-145.
HercepTest [package instructions]. Available at: www.dakousa.com.
Herceptin® [website]. Available at: http://www.herceptin.com/hcp/HER2-testing/interpreted-results.jsp.
13
Sources of HER2 IHC testing
variability affect results
Parameter
Source of variability
Choice of IHC test1
•No single standardized IHC method used
Sample
processing1-3
•The preanalytical process, including time to fixation, duration in fixative,
and choice of fixative is not standardized, which may lead to falsenegative IHC results
Staining
protocol1
•Numerous commercial antibodies are available that recognize various
HER2 epitopes
•Differences in staining characteristics of these many antibodies have been
documented
•Methods for antigen retrieval may vary by laboratory
Control
samples1
•Standardized negative and positive control samples for IHC have only
recently become available and are not in wide use
Laboratory
proficiency1
•Only 30% of laboratories participate in external proficiency testing
•Few laboratories validate a test procedure before offering the test
•Less than half of laboratories that use FDA-approved tests actually follow
the approved instructions
1. Wolff AC, et al. J Clin Oncol. 2007;25:118-145.
2. Goldstein NS, et al. Am J Clin Pathol. 2003;120:86-92.
3. Khoury T, et al. Mod Pathol. 2009;22(11):1457-1467.
14
HER2 Gene
HER2 m
FISH test measures HER2
gene amplification
Normal
copy #
HER2 Gene
Normal
amount
HER2 mRNA
Normal
copy #
Normal
amount
Amplified
copy #
Increased
amount
Amplified
copy #
HER2 Prote
Increased
Normal
amount
amount
Overexpression
• HER2 is detected with a red probe. A green probe specific to the centromeric region of
chromosome 17 (CEP17) serves as an internal control.
• ASCO/CAP guidelines: HER2 amplification defined as a mean ratio of ≥ 2.2 HER2 gene copies (red)
to CEP17 copies (green)
Wolff AC, et al. J Clin Oncol. 2007;25:118-145.
Herceptin® [website]. Available at: http://www.herceptin.com/hcp/HER2-testing/interpreted-results.jsp.
15
Sources of HER2 FISH testing
variability affect results
Parameter
Source of variability
Sample fixation1-3
•Length of fixation and type of fixative are not standardized
Centromeric
control1,2
•Use of CEP17 centromeric control is not uniform, with some assays lacking
this internal control
•In 2-9% of breast cancers, the centromeric region of chromosome 17 is
amplified
Equivocal
results1,2
•Test results that show equivocal amplification of HER2 must be subject to
increased or repeated counting, or confirmation by a second reader, which
may introduce inter-reader variability
Technical
feasibility1,2
•Proficiency in performing the assay and interpreting the results is variable
among pathologists
1. Wolff AC, et al. J Clin Oncol. 2007;25:118-145.
2. Sauter G, et al. J Clin Oncol. 2009;27:1323-1333.
3. Khoury T, et al. Mod Pathol. 2009;22(11):1457-1467.
16
Single-Gene Testing and
the Oncotype DX® Assay
The Predictive Utility of ER and HER2 Status
ER Testing Methodology: IHC
HER2 Testing Methodologies: IHC and FISH
Single-Gene Testing in the Oncotype DX Assay
Degree of Concordance in ER Status
Between IHC and the Oncotype DX Assay
Degree of Concordance in HER2 Status
Among IHC, FISH, and the Oncotype DX Assay
17
Oncotype DX® testing provides quantitative
RT-PCR measurement of ER, PR, HER2
Provides additional quantitative
insight into the biology of
individual tumors
ER score
PR score
HER2 score
18
The Oncotype DX® assay uses
RT-PCR technology
Polymerization
Forward
Primer
Reporter
R
Probe
Quencher
Q
Reverse
Primer
• RT-PCR provides
> 65,000-fold range
of measurement
– Maximizes ability to
discriminate the full range of
gene expression differences
among individual samples
R
Q
Strand displacement
and cleavage of probe
• RT-PCR reactions
can be repeated with high
quantitative precision
– Provides required reliability for
individualized reporting
Q
R
Polymerization
completion
and signal detection
Cronin M, et al. Am J Pathol. 2004;164:35-42.
• RT-PCR works well with
RNA from formalin-fixed
paraffin-embedded tissue
19
PR expression by RT-PCR
(relative to reference genes; log 2)
Overall
PR Range
1000-fold
Continuous measurement of ER/PR is
reflective of tumor biology
12
NSABP B-14 (N = 645)
ER+ PR+
ER- PR+
11
10
9
8
7
6
ER+ Range
200-fold
5
4
Overall
ER Range
3000-fold
3
2 ER- PR2
3
4
ER+ PR5
6
7
8
9
10
11
12
13
14
ER expression by RT-PCR (relative to reference genes; log 2)
Reproducibility of the assay has a standard deviation of less than 0.4 units
Data on file compiled from Paik S, et al. N Engl J Med. 2004;351:2817-2826.
20
Oncotype DX® clinical validation:
NSABP B-14
• Objective: Prospectively validate Recurrence Score® value
as predictor of distant recurrence in N–, ER+ patients
Placebo—not eligible
Randomized
Registered
Tamoxifen—eligible
Tamoxifen—eligible
• Multicenter study with prespecified 21-gene assay,
algorithm, endpoints, analysis plan
Paik et al. N Engl J Med. 2004;351:2817-2826.
Quantitative ER expression is not strongly prognostic
but is predictive of tamoxifen benefit in ER+ patients
Proportion without distant recurrence
1.0
0.9
Large tamoxifen benefit
in highest / mid-ER tertiles
0.8
Little tamoxifen benefit
in lowest ER tertile
0.7
0.6
Quantitative ER expression in ER+, placebo-treated
patients is not strongly prognostic (P = 0.54)
0.5
0.4
Placebo – highest ER tertile
Placebo – mid ER tertile
Placebo – lowest ER tertile
Tamoxifen – highest ER tertile
Tamoxifen – mid ER tertile
Tamoxifen – lowest ER tertile
0.3
0.2
0.1
0.0
0
2
4
6
8
Years
Baehner FL, et al. SABCS 2006. Abstract #510.
10
N
105
117
113
99
88
94
12
14
16
21
22
Single-Gene Testing and
the Oncotype DX® Assay
The Predictive Utility of ER and HER2 Status
ER Testing Methodology: IHC
HER2 Testing Methodologies: IHC and FISH
Single-Gene Testing in the Oncotype DX Assay
Degree of Concordance in ER Status
Between IHC and the Oncotype DX Assay
Degree of Concordance in HER2 Status
Among IHC, FISH, and the Oncotype DX Assay
23
E2197: study design overview
ECOG 2197
Patients with
0-3 positive
nodes
(N = 2872)
SUB ANALYSIS
4 × 3-week cycles
Doxorubicin 60 mg/m2
Cyclophosphamide 600 mg/m2
(Hormonal therapy if HR+)
Case-cohort sample (N = 776)
• Patients with tissue samples and
RT-PCR obtained
• Had recurrence (n = 179)
• Had no recurrence (n = 597)
4 × 3-week cycles
Doxorubicin 60 mg/m2
Docetaxel 60 mg/m2
(Hormonal therapy if HR+)
Primary analysis
• Determine whether the Recurrence
Score® value can be used to predict
chemotherapy benefit for a particular
group
Goldstein LJ, et al. J Clin Oncol. 2008;26(25):4063-4071.
24
E2197: quantitative ER
by Oncotype DX® and central IHC
8
Overall
concordance
between IHC and
RT-PCR was 93%
ER+
6
5
4
3
2
ER–
ER status by IHC
7
14% of ER– cases
by IHC are ER+ by
RT-PCR
1
0
2
3
7 8 9 10 11 12 13
4 5 6
ER–
ER+
ER status by Oncotype DX assay
Badve SS, et al. J Clin Oncol. 2008;26:2473-2481.
Corr. = 0.85
P < 0.0001
25
The Kaiser Permanente study:
study design overview
Study design
Matched case-control
Study
population
(N = 4964)
Kaiser Permanente patients < 75 years old in 14
Northern California hospitals diagnosed with nodenegative breast cancer 1985-1994, no adjuvant
chemotherapy
Cases: Deaths from breast cancer (n = 220)
Controls: Randomly selected, matched on age, race,
diagnosis year, KP facility, tamoxifen (n = 570)
Data sources
Cancer registry, medical records, archived diagnostic
slides, and tumor blocks
Habel LA, et al. Breast Cancer Res. 2006;8(3):R25.
26
Overall
concordance
between IHC and
RT-PCR was 96%
ER+
3+
2+
1+
20% of ER– cases
by IHC are ER+ by
RT-PCR
ER–
ER status by IHC
Kaiser: quantitative ER by
Oncotype DX® and central IHC
0
2
3
4
5
ER–
6
7
8
9
10
11
12
13
ER+
ER status by Oncotype DX assay
Baehner FL, et al. ASCO Breast Cancer Symposium. 2007; Abstract 88.
14
15
Corr. = 0.85
P < 0.0001
27
Genomic Health now accepts all appropriate*
early-stage breast cancer samples
Analysis by IHC
Quantitative
analysis
by RT-PCR
Report
generation
ER–†
ER+
(including ER uncertain)
ER+
by RT-PCR
ER–
by RT-PCR
ER+
by RT-PCR
ER–
by RT-PCR
YES
(Recurrence
Score®)
YES
(Recurrence
Score)
YES
(Recurrence
Score)
NO
FAILED
SPECIMEN
*Decision to submit samples for testing based on medical necessity as determined by the referring physician.
†Excludes TAILORx and selected investigator-sponsored trials, which would be treated like ER+ by IHC.
28
Single-Gene Testing and
the Oncotype DX® assay
The Predictive Utility of ER and HER2 Status
ER Testing Methodology: IHC
HER2 Testing Methodologies: IHC and FISH
Single-Gene Testing in the Oncotype DX Assay
Degree of Concordance in ER Status
Between IHC and the Oncotype DX Assay
Degree of Concordance in HER2 Status
Among IHC, FISH, and the Oncotype DX Assay
29
NSABP B-31/NCCTG 9831: significant rates of
discordance between local and central testing
Study
NSABP B-31
NCCTG 9831
No. of cases
retested
104
119
Paik S, et al. J Natl Cancer Inst. 2002;94:852-854.
Roche PC, et al. J Natl Cancer Inst. 2002;94:855-857.
Method of
retesting
Concordant
cases, n (%)
Discordant
cases, n (%)
IHC
84 (81)
20 (19)
FISH
82 (79)
22 (21)
IHC
88 (74)
31(26)
Approximately 20%
of HER2 testing
may be inaccurate
30
High degree of concordance
between RT-PCR and FISH for HER2
HER2 concordance 2 × 2
Equivocal cases excluded by both assays (according to ASCO/CAP guidelines)
Central IHC+
Central IHC–
Total
Oncotype® DX+
94 (78%)
4 (1%)
98
Oncotype DX–
27 (22%)
439 (99%)
466
121
443
564
ECOG 2197*
Total
CONCORDANCE 95%* [95% CI (92%, 96%) Kappa 83%, 95% CI (77%, 88%)]
Concordance calculated as (94 + 439)/564
Kaiser Study*
Central FISH+
Central FISH–
Total
Oncotype DX+
55 (98%)
11 (3%)
66
Oncotype DX–
1 (2%)
408 (97%)
409
56
419
475
Total
CONCORDANCE 97%* [95% CI (96%, 99%), Kappa 89%, 95% CI (82%, 95%)]
Concordance calculated as (55 + 408)/475
Sparano JL, et al. ASCO Breast Cancer Symposium. 2008; Abstract 13.
Baehner FL, et al. ASCO Breast Cancer Symposium. 2008; Abstract 41.
Wolff AC, et al. J Clin Oncol. 2007;25:118-45.
*See Appendix (slides 34-39) for in-depth information on these studies
DX®
Single-Gene Testing in the Oncotype
assay addresses limitations with current
methodologies
• Both IHC and FISH are associated with variability that
can affect the accuracy of test results.
• The impact of variability can be minimized by
“normalization” strategies used in quantitative gene
expression assessment as performed by quantitative
RT-PCR in the Oncotype DX assay.
• By minimizing variability, hormone-receptor status can
be more accurately reported, and treatment decisions
that depend on ER or HER2 status can be made with
greater confidence.
31
DX®
Single-Gene Testing in the Oncotype
assay addresses limitations with current
methodologies
• There is a high degree of overall concordance between
local and central IHC and central RT-PCR for ER, PR,
and hormone-receptor status.
• The relatively high incidence of IHC-negative hormonereceptor status positive by RT-PCR is notable and
deserves further study.
• Quantitative RT-PCR using the Oncotype DX assay is an
alternative method for determining hormone-receptor
status.
32
DX®
Single-Gene Testing in the Oncotype
assay addresses limitations with current
methodologies
• For HER2 status, there is a high degree of overall
concordance between central FISH for gene amplification and
central RT-PCR for quantitative gene expression.
• For HER2 status, there is a high degree of overall
concordance between central IHC for protein expression and
central RT-PCR for quantitative gene expression.
• Quantitative RT-PCR by the Oncotype DX assay for HER2
status is an alternative to FISH.
• Quantitative Single-Gene Testing results may be used by
local pathology laboratories as an external concordance
standard for ER, PR, and HER2.
33
34
Appendix
Concordance between IHC, FISH, and
RT-PCR for HER2 in the E2197 and
Kaiser Permanente studies
35
E2197: moderate degree of concordance
between IHC and RT-PCR for HER2
ECOG E2197
Central
IHC+
Central
IHC Equiv.
Central
IHC–
Total
Oncotype DX®+
94 (70%)
0
4 (< 1%)
98
Oncotype DX Equiv.
13 (10%)
10 (6%)
3 (< 1%)
26
Oncotype DX–
27 (20%)
165 (94%)
439 (99%)
631
Total
134
175
446
755
CONCORDANCE 72%*
Sparano JL, et al. ASCO Breast Cancer Symposium. 2008; Abstract 13.
Baehner FL, et al. ASCO Breast Cancer Symposium. 2008; Abstract 41.
*Concordance calculated as (94 + 10 + 439)/755
36
E2197: moderate degree of concordance
between IHC and RT-PCR for HER2 (N = 755)
27 (20%) IHC 3+
cases negative by
RT-PCR
Pos.
HER2
Central IHC
27
13
Equiv.
175
Neg.
corr. = 0.62*
P < 0.001
6
7
8
9
11
12
HER2
RT-PCR reference-normalized CT
10
Sparano JL, et al. ASCO Breast Cancer Symposium. 2008; Abstract 13.
Baehner FL, et al. ASCO Breast Cancer Symposium. 2008; Abstract 41.
13
14
4 (< 1%) IHC
negative cases
positive by RT-PCR
15
*Spearman rank correlation
37
E2197: majority of HER2 equivocal cases
by IHC are HER– by RT-PCR and FISH
Pos
HER2
central IHC
3
Color coded by FISH status
Red = FISH positive
Green = FISH equivocal
Blue = FISH negative
Grey = FISH unknown
Eq
13
Neg
6
7
8
Baehner, FL. USCAP 2009; abstract 115.
9
10
11
12
13
HER2 (normalized expression units)
14
15
RT-PCR and FISH
results for IHC
equivocal cases
indicate that the
majority of the
cases are HER2–
(79% by both FISH
& RT-PCR)
38
Kaiser: moderate degree of concordance
between FISH and RT-PCR for HER2
Includes positive, equivocal & negative cases in analysis
Central
FISH+
Central
FISH Equiv.
Central
FISH–
Total
Oncotype DX®+
55 (92%)
1 (10%)
11 (2%)
67
Oncotype DX Equiv.
4(7%)
5 (50%)
79 (16%)
88
Oncotype DX–
1 (2%)
4 (40%)
408 (82%)
413
Total
60
10
498
568
Concordance 82%* [95% CI (79%, 85%)]
Weighted Kappa 63% [95% CI (55%, 70%)]
Baehner FL, et al. ASCO Breast Cancer Symposium. 2008; Abstract 41.
Wolff AC, et al. J Clin Oncol. 2007;25:118-45.
*Concordance calculated as (55 + 5 + 408)/568
39
Kaiser: moderate degree of concordance
between FISH and RT-PCR for HER2
HER2_3 RT-PCR (CT) by FISH
12
11
One case (2%) was HER2
gene amplified by FISH but
negative by RT-PCR
10
9
FISH
8
7
6
5
4
3
2
1
0
7
8
8
10
11
12
13
14
15
16
11 cases (3%) were HER2+
by RT-PCR but negative for
HER2 gene amplification
by FISH
RT-PCR reference-normalized CT
Corr. = 0.4542
P < .0001
Baehner FL, et al. ASCO Breast Cancer Symposium. 2008; Abstract 41.
Wolff AC, et al. J Clin Oncol. 2007;25:118-45.