284 Am J Clin Pathol 2010;134:284-292
284 DOI: 10.1309/AJCPUQB18XZOHHBJ
© American Society for Clinical Pathology
Anatomic Pathology / Concordance in HER2 Testing
Achieving 95% Cross-Methodological Concordance
in HER2 Testing
Causes and Implications of Discordant Cases
Erin E. Grimm, MD, Rodney A. Schmidt, MD, PhD, Paul E. Swanson, MD,
Suzanne M. Dintzis, MD, PhD, and Kimberly H. Allison, MD
Key Words: ASCO/CAP guidelines; HER2 testing; Breast cancer; HER2 discordance; HER2 validation; Fluorescence in situ
A b s t r a c t
We were interested in determining our concordance
between fluorescence in situ hybridization (FISH) and
a previously validated immunohistochemical HER2
assay to identify possible reasons for discordance
and to determine if all reasons for discordance
were addressed by the American Society of Clinical
Oncology/College of American Pathologists guidelines.
We reviewed 697 cases (2004-2007) in which
HER2 immunohistochemical and FISH testing were
concurrently done. Overall concordance between
nonequivocal immunohistochemical and FISH results
was 96%. Of the 19 discordant cases, 13 (68%)
were interpreted as positive immunohistochemically
but negative by FISH. The primary reason for this
discordance was immunohistochemical interpretation.
Weak stain intensity, granular staining, and
interpretation in areas of crush artifact were identified
as the most common issues. Of the 6 cases interpreted
as immunohistochemically negative and FISH-
positive, 2 were from patients known to be receiving
trastuzumab at the time of biopsy, 1 was very close to
the FISH equivocal category, and 4 cases had fewer
than 1.5 CEP17 signals per cell (1 patient in this group
was also receiving trastuzumab). Focusing on issues
with HER2 immunohistochemical interpretation can
improve concordance rates for immunohistochemically
positive cases, but biologic reasons may explain some
discordant immunohistochemically negative cases.
The human epidermal growth factor receptor 2 protein
(ERBB2, CERB-B2, or HER2) is overexpressed in 10% to
25% of all breast carcinomas.1-4 The HER2 protein, a trans-
membrane tyrosine kinase receptor, is part of a complex path-
way of signal transduction that affects cell proliferation, sur-
vival, motility, and adhesion.5 Breast carcinomas with HER2
overexpression have increased proliferation, altered motility
and invasive potential, regional and distant metastases, accel-
erated angiogenesis, and reduced apoptosis.5 Amplification
of the ERBB2 (HER2) gene is the primary mechanism of
HER2 protein overexpression in most cases.6-8 Clinically,
HER2 overexpression is associated with a more aggressive
clinical course and increased resistance to nonanthracycline-,
nontaxane-containing chemotherapy regimens.9 However,
overall and recurrence-free survival improve with regimens
that include anthracycline-based chemotherapy and HER2-
targeted antibody therapies like trastuzumab and lapatinib.
Correct assessment of HER2 status in breast carcinoma is
therefore essential in guiding therapy-related decisions.
Three central methods to evaluate HER2 status are
currently in clinical use: immunohistochemical analysis,
fluorescence in situ hybridization (FISH), and chromogenic
in situ hybridization (CISH). Immunohistochemical analysis
detects the amount of HER2 protein at the cell membrane in
a semiquantitative manner: incomplete or weak labeling of
cell membranes typically equates with fewer than 185,000
receptors at the cell surface and no detectable amplification
of the HER2 gene; strong, circumferential membrane stain-
ing correlates with more than 500,000 receptors at the cell
surface, a level of expression almost always associated with
Upon completion of this activity you will be able to:
• list key American Society of Clinical Oncology/College of American
Pathologists recommendations for HER2 test performance,
interpretation, and validation.
• calculate assay concordance and how to perform validation of an
HER2 testing methodology.
• list the primary causes of HER2 immunohistochemical (IHC) and
fluorescence in situ hybridization discordance.
• identify the main reasons for interpreter error in HER2 IHC testing and
apply this knowledge to improve HER2 IHC stain interpretation.
The ASCP is accredited by the Accreditation Council for Continuing
Medical Education to provide continuing medical education for physicians.
The ASCP designates this educational activity for a maximum of 1 AMA PRA
Category 1 Credit ™ per article. This activity qualifies as an American Board
of Pathology Maintenance of Certification Part II Self-Assessment Module.
The authors of this article and the planning committee members and staff
have no relevant financial relationships with commercial interests to disclose.
Questions appear on p 341. Exam is located at www.ascp.org/ajcpcme.
Am J Clin Pathol 2010;134:284-292 285
285 DOI: 10.1309/AJCPUQB18XZOHHBJ 285
© American Society for Clinical Pathology
Anatomic Pathology / Original Article
HER2 amplification.10 FISH uses a fluorescent-labeled DNA
probe to detect the relative copy numbers of the HER2 gene
per cell. While less sensitive to technical factors that influ-
ence the reliability of immunohistochemical analysis (such
as tissue fixation), FISH is more time- and labor-intensive
and requires the use of a fluorescence microscope. CISH,
like FISH, detects the HER2 gene copy number but uses a
chromogenic detection method scored using standard bright-
field microscopy. In current practice, immunohistochemical
analysis is commonly used as the initial test for HER2 status,
with FISH and CISH reserved for assessment of equivocal
(2+) immunohistochemical results. However, studies indicate
that as many as 20% of immunohistochemically positive (3+)
breast carcinomas do not exhibit HER2 gene amplification in
FISH assays performed at a centralized testing facility.11,12
In an attempt to further standardize HER2 testing and
reduce discordance between laboratories and testing methods,
the American Society of Clinical Oncology (ASCO) and the
College of American Pathologists (CAP) published guideline
recommendations for HER2 testing in 2007.9 These guide-
lines established standard testing and interpretive criteria
for immunohistochemical analysis and FISH. Through these
guidelines and enforcement of new inspection checklist items
for laboratory practice, the CAP has effectively required all
CAP-accredited laboratories performing HER2 testing to doc-
ument 95% overall concordance with an external or internal
validated method (the former typically the same basic testing
platform, such as immunohistochemical analysis vs immuno-
histochemical analysis; the latter more often cross-platform,
such as immunohistochemical analysis vs FISH) for non-
equivocal results. So that we might gain a better understand-
ing of how laboratories may improve concordance between
in-house immunohistochemical and FISH methods for HER2
status, we investigated the causes of immunohistochemical-
FISH discordance in our own practice.
Materials and Methods
After University of Washington Medical Center (UWMC;
Seattle) Human Studies approval, we reviewed the HER2
status of 697 cases of breast carcinoma at UWMC on which
immunohistochemical and FISH testing were performed
between September 2004 and December 2007 (inclusive).
Overall concordance rates between immunohistochemical and
FISH results were determined, and all discordant cases were
reviewed. Reasons for discordance were identified with an
eye to improving performance of immunohistochemical and
FISH methods and overall concordance between them.
The study population included cases referred directly
for FISH testing (without prior HER2 testing by immunohis-
tochemical analysis) as well as “in-house” cases at UWMC in
which HER2 immunohistochemical analysis was performed
and results were reported as equivocal. Our internal qual-
ity assurance protocol requires that all referral HER2 FISH
cases also have immunohistochemical analysis for HER2
performed concurrently. The results of the latter immunohis-
tochemical tests are recorded in our pathology database but
are not reported. Cases without immunohistochemical and
FISH results were excluded from further analysis.
Immunohistochemical analysis was performed using the
avidin-biotin-peroxidase method (VectaStain Elite, Vector
Labs, Burlingame, CA). Three 4-μm tissue sections cut from
the formalin-fixed, paraffin-embedded tissue block were
used. All slides were baked for 20 minutes at 60°C, dewaxed
in xylene, and rehydrated through graded alcohols to buffer
before immersion in a solution of 3% hydrogen peroxide for
10 minutes to block endogenous peroxidase.
The first slide was run without heat-induced epitope
retrieval (HIER). This “non-HIER” slide was blocked in nor-
mal goat serum (dilution 1:100; catalog No. S1000, Vector
Labs) for 10 minutes and then incubated with the primary
anti-HER2 antibody (polyclonal antibody, dilution 1:200;
catalog No. A0485, DAKO, Carpinteria, CA) for 40 minutes
at room temperature in a moist chamber. The second slide
was run with HIER, 0.05 mol/L citrate buffer (pH 6.0), for 15
minutes at 98°C in a commercial microwave (model H2800,
Energy Beam Sciences, East Granby, CT), cooled to room
temperature (for 20 minutes), and then incubated with the
primary antibody (diluted 1:400) in a manner otherwise iden-
tical to the non-HIER slide. The third slide, a methodological
(negative) control, was handled the same as the HIER slide,
except that the primary antibody was replaced by normal rab-
bit serum (dilution 1:8,000; MP BioMedicals, Solon, OH).
All slides were then subjected to sequential 30-minute
immersion in biotinylated horse antirabbit antibody (1:500
dilution; Vector Labs) and avidin-biotin-peroxidase (1:100
dilution; catalog No. PK-6100, Vector Labs), followed by
chromogenic development, using 3,3ʹ-diaminobenzidine tet-
rahydrochloride hydrate (catalog No. D5637-10G, Sigma,
St Louis, MO) as the chromogen and hydrogen peroxide
(3%; catalog No. 3306HP0316, MediChoice, Mechanicsville,
VA) as the substrate. After hematoxylin counterstaining,
slides were rinsed and dehydrated through graded alcohols
to xylene and coverslipped using an automated coverslipper.
Appropriate external positive control tests were performed
concurrently with all HIER and non-HIER test slides.
Final HER2 immunohistochemical interpretation in cases
requested for immunohistochemical testing was performed
by members of a dedicated breast pathology service or by
the immunohistochemical laboratory director. The HER2
immunohistochemical stains performed for quality assurance
purposes in cases sent for FISH only were interpreted by the
pathologist signing out the FISH results. HercepTest (DAKO)
292 Am J Clin Pathol 2010;134:284-292
292 DOI: 10.1309/AJCPUQB18XZOHHBJ
© American Society for Clinical Pathology
Grimm et al / Concordance in HER2 Testing
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