HER2 Testing and Current Knowledge on the Biology of HER2-Positive Tumors

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HER2-Positive Breast Cancer: Applying the Latest Developments to Clinical Practice

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Faculty
Michael F. Press, MD, PhD
Harold E. Lee Chair in Cancer Research Professor of Pathology Department of Pathology University of Southern California, Norris Comprehensive Cancer Center Los Angeles, California

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Disclosure
Michael F. Press, MD, PhD, has disclosed that he has received consulting fees from GlaxoSmithKline, Halozyme, and Roche and funds for contracted research from Genentech and Ventana Medical Systems.

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HER2 Testing and Current Knowledge on the Biology of HER2-Positive Tumors

Background: human epidermal growth factor receptor, type 2 (HER2) gene amplification is directly correlated with overexpression in frozen samples
HER2 testing: evaluation of IHC and FISH in molecularly characterized samples Concordance of IHC and FISH IHC: false-negatives and false-positives

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HER2 Biology: Localization of HER2 Gene on Chromosome 17

Normal interphase nucleus and metaphase spread

HER2/neu/c-erbB-2 Chromosome 17 centromere

17

17

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HER2 Biology: HER2 Gene Is Amplified in 25% of Breast Cancers

Slamon DJ, et al. Science. 1987;235:177-182.

HER2/neu/c-erbB-2 Chromosome 17 centromere

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HER2 Biology: Correlation of HER2 Gene Amplification With Overexpression

12.5 kb -

Southern
(DNA)
4.4 kb p185 -

Northern
(mRNA)

Western
(protein)

Frozen IHC
27%
Slamon D, et al. Science. 1989;244:707-712.

63%

% Women

10%

Amplification Level :

> 10 5 - 10 2-5 1

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HER2 Biology: Single Copy Overexpression

IHC

H&E

Southern blot

FISH

Slamon D, et al. Science. 1989;244:707-712. Pauletti G, et al. Oncogene. 1996;13:63-72.

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HER2 Biology: HER2 Gene Amplification Is Responsible for Overexpression

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Fluorescence in situ Hybridization

Key Features: Probes Direct labeled HER2 sequence

Chromosome 17 centromere
Interpretation Signal enumeration Ratio of HER2:Chr 17 signals

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HER-2 Gene Assessment by FISH

< 2.0 Not Amplified (FISH-)

2.0 Amplified (FISH+)

FISH ratio = HER2 gene copies / chromosome 17 centromere copies

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IHC: HER2 Overexpression Detection by Immunohistochemistry

0 1+

2+

3+

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Evaluation of HER2 Testing in Paraffin-Embedded Tissues Correlation of HER2/neu Gene Amplification With Overexpression in Frozen and FFPE Tissues
Amplification Level : 12.5 kb -

> 10 5 - 10 2-5 1

Southern
(DNA) 4.4 kb p185 -

Northern
(mRNA)

Western
(protein)

Multitumor Paraffin-Embedded Tissue Block

Frozen Paraffin-Embedded IHC Tissue (protein)

% Women

Frozen Tissue
Slamon D, et al. Science. 1989;244: 707-712.

27%

63%

Press MF, et al. Cancer Res. 1993;53:4960-4970. Press MF, et al. Cancer Res. 1994;54:2771-2777 Press MF, et al. J Clin Oncol. 2002;20:3095-3105.

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Comparison of 6 Different HER2 Assays in HER2 Molecularly Characterized Breast Cancers

FISH Ventana IHC Ventana FISH Vysis IHC DAKO IHC R60 IHC 10H8

Press MF, et al. J Clin Oncol. 2002;20:3095-3105.

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Selection of Assay Method for Clinical Trials Samples Comparison of FISH and IHC Assay Results in Cancers With Known HER2/neu Gene Status
Assay
FISH (Vysis) FISH (Ventana) IHC-R60 IHC-10H8

#Amplified or #Over/#Low
41/76 44/73 39/78 38/79

Sensitivity
95% 95% 91% 88%

Specificity
99% 96% 100% 100%

Accuracy
97% 96% 97% 96%

IHC-DAKO IHC-Ventana

30/87 31/86

70% 72%

100% 100%

89% 90%

43 amplified breast cancers and 74 nonamplified breast cancers.

Press MF, et al. J Clin Oncol. 2002;20:3095-3105.

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ASCO-CAP Guideline Testing Algorithm for HER2 Testing by IHC

3+ Breast cancer specimen (invasive component)
HER2 testing by validated IHC assay for HER2 protein expression

1+

Positive for HER2 protein expression IHC 3+ (defined as uniform intense membrane staining of > 30% of invasive tumor cells)

Equivocal for HER2 protein expression IHC 2+

Negative for HER2 protein expression IHC 0 or 1+

Test with validated assay for HER2 gene amplification

Positive for HER2 gene amplification Treatment with trastuzumab or lapatinib

Equivocal HER2 gene amplification (patients with HER2/CEP17 ratio 2.0 were eligible for the adjuvant trastuzumab trails)

Negative for HER2 gene amplification No targeted therapy

Wolff AC, et al. J Clin Oncol. 2007;25:118-145.

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Concordance Study Results: GNE Trials 1:1 Population 0

FISH + 207

CTA-IHC 1+ 2+ 28 67

3+
21

7
3%

2
7%

21
24%

176
89%

Amplification rate

3.7%
Overall concordance between FISH and IHC results was 82% (95% CI: 78%-85%; P < .0004)
Dybdal N, et al. Breast Cancer Res Treat. 2005;93:3-11.

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Local IHC Compared to Central FISH (79% Concordance) Central Screening of HER2 Status for Entry to Adjuvant Trastuzumab Trial (BCIRG006) Local IHC 0 1+ 2+ 3+ 538 20 4% 230 15 6% 4.3%
Press MF, et al. Clin Cancer Res. 2005;11:6598-6607.

165 33 17%

90 316 78%

73%

Central FISH
35/384 = 9.1% +

23%

Amplification rate

N = 1407

Overall concordance between FISH and IHC was 79% (95% CI: 77%-81%)

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Prevalence of HER2 Gene Amplification (%) in Each IHC Immunostaining Category (0, 1+, 2+ and 3+) by Study*
HER2 Gene Amplification Rate according to IHC Score 0 1+ 2+ 3+ Number IHC Method Reference

0%
1.8% 3.5% 0% 0% 3.8% 0% 0.7% 4.2% 1.1% 0%

0%

17%
35.9%

89%
100% 99% 91.4% 100% 100% 89.8% 94.1% 49% 89.7% 90%

100
750 2857 189 170 198 119 426 102 2279 360

DAKO HercepTest
DAKO Ab, Unspecified DAKO HercepTest DAKO A0485 Ab Homebrew Ab DAKO HercepTest DAKO HercepTest DAKO HercepTest DAKO HercepTest DAKO HercepTest DAKO HercepTest

Hoang et al, Am J Clin Pathol, 2000

Ridolfi et al, Mod Pathol, 2000 Simon et al, JNCI, 2001 Wang et al, Am J Clin Pathol, 2001 Kobayashi et al, Hum Pathol, 2002 McCormick et al, Am J Clin Pathol, 2002 Roche et al, JNCI, 2002 Dowsett et al, J Pathol, 2003 Hammock et al, Hum Pathol, 2003 Lal et al, Am J Clin Pathol, 2004 Mrozkowiak et al, Pol J Pathol, 2004

66.2% 2.2% 5.7% 8.5% 0%

97.1% 38.2% 18.2% 42.2% 0% 48.1% 6.1%

3.1%

26.5% 20%

0%
2.8% 3% 6.9% 2.4% 3.6% 12.5% 0% 2.7% 8.7% 6.1% 6.7% 0% 21.4% 7%

15%
17% 24% 31.8% 72% 16.7% 7% 12.2% 36.1% 36.1%

79%
91.6% 89% 90% 100% 78.1% 52.4% 91.6% 90.1% 90.1%

600
2913 529 114 215 2249 108 289

DAKO HercepTest
DAKO A0485 Ab Clinical Trials Assay DAKO HercepTest DAKO HercepTest DAKO HercepTest DAKO HercepTest DAKO HercepTest

Varshney et al, Am J Clin Pathol, 2004

Yaziji et al, JAMA, 2004 Dybdal et al, Br Ca Res Treatment, 2005 Ellis et al, J Clin Pathol, 2005 Lottner et al, J Pathol, 2005 Press et al, Clin Cancer Res, 2005 Ciampa et al, Appl Im Mol Morphol, 2006 Hofmann et al, J Clin Pathol, 2008

Weighted Average Percentages 16,142 Weighted Average Percentages

*IHC and FISH in at least 100 cases.

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Prevalence of HER2 Gene Amplification (%) in Each IHC Immunostaining Category (0, 1+, 2+ and 3+) by Study (2)*
HER2 Gene Amplification Rate according to IHC Score 0 1+ 2+ 3+ Number IHC Method Reference

0%
1.60% 12.5% 3.3% 0% 2.6%

8.30%

23%

56.3%

661

DAKO HercepTest

Rasmussen et al Acta Oncol., 2008

34.29%
68.6%
57.9% 3.30% 4.80% 15.20% 28.10%

86.00%
96.3%
95.2% 84.1% 93.8%

697
171
100 200 950

A0485 antibody (Dako) Grimm et al, AJCP, 2010

3B5 antibody
DAKO HercepTest 4B5 antibody

Panjwani et al, Indian J Med Res., 2010

Tsuda et al, BMC Cancer, 2010 Lambein et al, J Clin Pathol., 2011

A0485 antibody (Dako) Park et al, Cancer, 2011

0%
12.8%

3.17%

21.51%
43.8%

90.98%
97.8% 100.0% 88.4% 97.3%
89.65%

681
291 216 543 125
4635

Dako Herceptest

Jorgenson et al., AJCP, 2011

Bernasconi et al, BrCa ResTreat., DAKO antibody, A0485 2012

CB11 antibody CB11 antibody DAKO HercepTest
Average Percentages Average Percentages

0% 3.4% 0%

10.0% 7.1% 12.5%

7.02% 7.55%

25.0% 49.2% 76.5%

40.28%

Martin et al, Patholog Res Int., 2012 Lee et al, Arch Med Res., 2012 Kiyose et al, Pathol Int., 2012

0.86%

*IHC and FISH in at least 100 cases.

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ASCO-CAP Guidelines Algorithm for HER2 Testing by FISH

Breast cancer specimen (invasive component)
HER2 testing by validated FISH assay for HER2 gene amplification

FDA approved a HER2-toCEP17 ratio 2.0 (or an average HER2 copy number of > 4.0) as HER2 amplified
Negative for HER2 gene amplification (FISH ratio > 1.8 or HER2 gene copy < 4.0)

Positive for HER2 gene amplification (FISH ratio > 2.2 or HER2 gene copy > 6.0)

Equivocal for HER2 gene amplification (FISH ratio 1.8-2.2 or HER2 gene copy 4.0-6.0*)

Count additional cells for FISH or retest, or test with HER2 IHC

Treatment with trastuzumab or lapatinib

Equivocal HER2 gene amplification result (patients with HER2/ CEP17 ratio 2.0were eligible for the adjuvant trastuzumab trails

No targeted therapy

ASCO-CAP guidelines create an equivocal zone (1.802.20) without providing any supportive data Considerable data and the FDA consider this zone to be the area around the cut-off of amplification where additional cells should be scored

Wolff AC, et al. J Clin Oncol. 2007;25:118-145.

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HER2 FISH Ratios in 2502 Consecutive Cases Screened for Entry to Clinical Trials: Equivocal Cases Only a Minute Assessment Problem
10.00 9.00 8.00 HER2 FISH Ratios 7.00 6.00 5.00 4.00 3.00
2.20

Plot of FISH Ratios

2.00 1.00 0.00

1.80

Sauter G, et al. J Clin Oncol. 2009;27:1323-33.

Cases

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Correlation of PFS With Response to Lapatinib Based on HER2 FISH Status in HVLab vs Academic Lab
Blinded FISH analysis by large, commercial, high-volume laboratory

HER2 Status Positive Negative

Number 255 86

HR 0.47 0.54

95% CI 0.32-0.67 0.30-0.99

P Value < .001 .046

Medical technician assessment of FISH status

Blinded FISH analysis by small, academic laboratory

Positive Negative

271 47

0.46 0.94

0.33-0.65 0.39-2.28

< .001 .888

Board-certified pathologist assessment of FISH status

Press MF, et al. Clin Cancer Res. 2008;14:7861-7870.

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Frequency and Distribution of HER2 FISH Ratios Between 2 Central Laboratories

HER2 ratio = 4.0 (n = 160)

High Volume Lab: Med Tech Assessment

Academic Lab: Pathologist Assessment

Press MF, et al. Clin Cancer Res. 2008;14:7861-7870.

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Inclusion of Chromosome 17 Centromere Alpha Satellite DNA in the HER2 Amplicon

Troxell ML, et al. Am J Clin Pathol. 2006;126:709-716.

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Use of Alternative Control Genes to Confirm HER2 Gene Amplification

SMS CEP17HER2
RARA

Troxell ML, et al. Am J Clin Pathol. 2006;126:709-716.

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HER2 FISH Ratios in 2502 Consecutive Cases Screened for Entry to Clinical Trials
Plot of FISH Ratios 10.00 9.00 8.00

HER2 FISH Ratios

7.00 6.00 5.00 4.00 3.00

2.20

2.00 1.00 0.00

1.80

23.1 = 2.0 11.5

Cases

Sauter G, et al. J Clin Oncol. 2009;27:1323-33.

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HER2 Gene Amplification by FISH Compared With HER2 Protein by IHC IHC-HercepTest
0 1+ 2+ 3+

FISH 34/331 (10%) +

237 13 5%

99 21 21 18% 9%

36 39 52%

8 258 97%

53%
47%

Amplification rate

N = 711

Overall concordance between FISH and IHC results was 89% (95% CI: 87%-91%).

P < .0001
Press MF, et al. Clin Cancer Res. 2008;14:7861-7870.

HER Tyrosine Kinase Inhibitors: Lapatinib

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PFS for Women in EGF30001 and EGF100151 Trials

HER2 FISH Positive

HER2 IHC 3+

HER2 FISH Negative and IHC 0

L+C or L+P C or P alone

Lapatinib with chemotherapy is active in patients with HER2 FISH+ or IHC3+

Press MF, et al. Clin Cancer Res. 2008;14:7861-7870.

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PFS for Women in EGF30001 and EGF100151 Trials

HER2 amplification was a determinant of lapatinib efficacy

FISH Positive and IHC 2+

Similar results for FISH-positive, IHC 0 or 1+ breast cancers (HR, 0.34; P = 0.033).
Press MF, et al. Clin Cancer Res. 2008;14:7861-7870.

FISH Negative and IHC 1+, 2+ or 3+

L+C or L+P C or P alone

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Improved Outcome Is Associated With Entire Spectrum of HER2 Amplification Ratios (FISH)
FISH Ratio < 2.0 2.00-4.99 5.00-6.99 7.0-10.0 10.0 Number 390 59 89 106 92 HR 1.09 0.44 0.45 0.58 0.39 95% CI 0.86-1.37 0.24-0.83 0.25-0.81 0.33-1.02 0.22-0.68 P Value .486 .010 .007 .059 .001

Analysis of all cases in the ALab for patients in either clinical trial.

Press MF, et al. Clin Cancer Res. 2008;14:7861-7870

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Low Level HER2 Gene Amplication Appears to Respond to Trastuzumab Treatment: FISH Ratio 2.0-2.2
100 90 80 DFS (%) 70 60 50 40 0 1 2 3 4 5 Time From Registration (Yrs) 6 FDA only ACT Ref ACT+H HR: 0.60; 95% CI: 0.12-3.13; P = .55 ASCO/CAP ACT Ref ACT+H HR: 0.59; 95% CI: 0.48-0.73; P < .001

Perez EA, et al. J Natl Cancer Inst. 2012;104:159-162.

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