Special Article
Recommendations for Human Epidermal Growth Factor
Receptor 2 Testing in Breast Cancer
American Society of Clinical Oncology/College of American Pathologists
Clinical Practice Guideline Update
Antonio C. Wolff*, M. Elizabeth H. Hammond*, David G. Hicks*, Mitch Dowsett*, Lisa M. McShane*, Kimberly H. Allison,
Donald C. Allred, John M.S. Bartlett, Michael Bilous, Patrick Fitzgibbons, Wedad Hanna, Robert B. Jenkins, Pamela B. Mangu,
Soonmyung Paik, Edith A. Perez, Michael F. Press, Patricia A. Spears, Gail H. Vance, Giuseppe Viale, and Daniel F. Hayes*
Purpose. To update the American Society of Clinical
Oncology (ASCO)/College of American Pathologists (CAP)
guideline recommendations for human epidermal growth
factor receptor 2 (HER2) testing in breast cancer to
improve the accuracy of HER2 testing and its utility as a
predictive marker in invasive breast cancer.
Methods. ASCO/CAP convened an Update Committee
that included coauthors of the 2007 guideline to conduct a
systematic literature review and update recommendations
for optimal HER2 testing.
Results. The Update Committee identified criteria and
areas requiring clarification to improve the accuracy of
HER2 testing by immunohistochemistry (IHC) or in situ
hybridization (ISH). The guideline was reviewed and
approved by both organizations.
Recommendations. The Update Committee recommends
that HER2 status (HER2 negative or positive) be determined
in all patients with invasive (early stage or recurrence) breast
cancer on the basis of one or more HER2 test results
(negative, equivocal, or positive). Testing criteria define
HER2-positive status when (on observing within an area of
tumor that amounts to >10% of contiguous and homoge-
neous tumor cells) there is evidence of protein overexpres-
sion (IHC) or gene amplification (HER2 copy number or
HER2/CEP17 ratio by ISH based on counting at least 20 cells
within the area). If results are equivocal (revised criteria),
reflex testing should be performed using an alternative assay
(IHC or ISH). Repeat testing should be considered if results
seem discordant with other histopathologic findings. Labo-
ratories should demonstrate high concordance with a
validated HER2 test on a sufficiently large and representative
set of specimens. Testing must be performed in a laboratory
accredited by CAP or another accrediting entity. The Update
Committee urges providers and health systems to cooperate
to ensure the highest quality testing.
(Arch Pathol Lab Med. 2014;138:241–256; doi: 10.5858/
arpa.2013-0953-SA)
I
n 2007, a joint Expert Panel convened by the American
Society of Clinical Oncology (ASCO) and the College of
American Pathologists (CAP) met to develop guidelines for
when and how to test for the human epidermal growth
factor receptor 2 (HER2) gene (also referred to as ERBB2),
1,2
which is amplified and/or overexpressed in approximately
15% to 20% of primary breast cancers. Since then, minor
clarifications and updates to the ASCO/CAP HER2 testing
guideline have been issued.
3–5
A detailed rationale for this
full 2013 update, as well as additional background infor-
mation, is available in Data Supplement 1.
In 2012, ASCO and CAP convened an Update Committee
to conduct a formal and comprehensive review of the peer-
reviewed literature published since 2006 and to revise the
guideline recommendations as appropriate. Since publica-
tion of the 2007 guideline, new diagnostic strategies, like
measures of HER2 amplification by bright-field in situ
hybridization, DNA expression by microarray, or mRNA
American Society of Clinical Oncology Clinical Practice Guide-
line Committee approval: April 26, 2013; College of American
Pathologists approval: June 21, 2013.
Published as an Early Online Release October 7, 2013.
*Steering Committee member.
This guideline was developed through a collaboration between the
American Society of Clinical Oncology and the College of American
Pathologists and has been published jointly by invitation and consent
in both the Journal of Clinical Oncology and the Archives of
Pathology & Laboratory Medicine. It has been edited in accordance
with the standards established at the Journal of Clinical Oncology.
Copyright Ó 2013 American Society of Clinical Oncology and
College of American Pathologists. All rights reserved. No part of this
document may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopy, recording, or
any information storage and retrieval system, without written
permission by the American Society of Clinical Oncology or College
of American Pathologists.
Editor’s Note: This article summarizes the Recommendations for
Human Epidermal Growth Factor Receptor 2 Testing in Breast
Cancer: American Society of Clinical Oncology/College of American
Pathologists Clinical Practice Guideline Update and provides the
updated recommendations with brief discussions of the relevant
literature for each. Additional information, including extensive Data
Supplements, used by the Update Committee to formulate these
recommendations is available at www.asco.org/guidelines/her2.
Author affiliations appear at the end of this article.
Authors’ disclosures of potential conflicts of interest and author
contributions are found at the end of this article.
Corresponding author: American Society of Clinical Oncology,
2318 Mill Rd, Suite 800, Alexandria, VA 22314; e-mail: guidelines@
asco.org.
Arch Pathol Lab Med—Vol 138, February 2014 ASCO/CAP HER2 Testing Guideline Update—Wolff et al 241
The Bottom Line
ASCO Guideline Update
Recommendations for HER2 Testing in Breast Cancer: ASCO/CAP Guideline Update
Intervention
Recommendations for HER2 testing in breast cancer
Target Audience
Medical oncologists, pathologists, and surgeons
Key Recommendations for Oncologists
Must request HER2 testing on every primary invasive breast cancer (and on metastatic site, if stage IV and if specimen available)
from a patient with breast cancer to guide decision to pursue HER2-targeted therapy. This should be especially considered for a
patient who previously tested HER2 negative in a primary tumor and presents with disease recurrence with clinical behavior
suggestive of HER2-positive or triple-negative disease.
Should recommend HER2-targeted therapy if HER2 test result is positive, if there is no apparent histopathologic discordance with
HER2 testing (Tables 1 and 2), and if clinically appropriate. If the pathologist or oncologist observes an apparent histopathologic
discordance after HER2 testing, the need for additional HER2 testing should be discussed.
Must delay decision to recommend HER2-targeted therapy if initial HER2 test result is equivocal. Reflex testing should be performed
on the same specimen using the alternative test if initial HER2 test result is equivocal or on an alternative specimen (Tables 1 and 2).
Must not recommend HER2-targeted therapy if HER2 test result is negative and if there is no apparent histopathologic discordance
with HER2 testing (Tables 1 and 2). If the pathologist or oncologist observes an apparent histopathologic discordance after HER2
testing, the need for additional HER2 testing should be discussed.
Should delay decision to recommend HER2-targeted therapy if HER2 status cannot be confirmed as positive or negative after
separate HER2 tests (HER2 test result or results equivocal). The oncologist should confer with the pathologist regarding the need for
additional HER2 testing on the same or another tumor specimen.
If the HER2 test result is ultimately deemed to be equivocal, even after reflex testing with an alternative assay (ie, if neither test is
unequivocally positive), the oncologist may consider HER2-targeted therapy. The oncologist should also consider the feasibility of
testing another tumor specimen to attempt to definitely establish the tumor HER2 status and guide therapeutic decisions. A clinical
decision to ultimately consider HER2-targeted therapy in such cases should be individualized on the basis of patient status
(comorbidities, prognosis, and so on) and patient preferences after discussing available clinical evidence.
Key Recommendations for Pathologists
Must ensure that at least one tumor sample from all patients with breast cancer (early-stage or metastatic disease) is tested for either
HER2 protein expression (IHC assay) or HER2 gene expression (ISH assay) using a validated HER2 test.
In the United States, the ASCO/CAP Guideline Update Committee preferentially recommends the use of an assay that has received FDA
approval, although a CLIA-certified laboratory may choose instead to use a laboratory-developed test (LDT). In this case, the analytic
performance of the LDT must be prospectively validated in the same clinical laboratory that will perform it, and the test must have
documented analytic validity (CAP guidance document). Bright-field ISH assays must be initially validated by comparing them with an
FDA-approved FISH assay.
Must report a HER2 test result as positive if: (a) IHC 3þ positive or (b) ISH positive using either a single-probe ISH or dual-probe
ISH (Table 1; Figs 1 to 3). This assumes that there is no apparent histopathologic discordance observed by the pathologist (Table 2).
Must report a HER2 test result as equivocal and order reflex test on the same specimen (unless the pathologist has concerns about
the specimen) using the alternative test if: (a) IHC 2þ equivocal or (b) ISH equivocal using single-probe ISH or dual-probe ISH
(Table 1; Figs 1 to 3). This assumes that there is no apparent histopathologic discordance observed by the pathologist (Table 2). Note
that there are some rare breast cancers (eg, gland-forming tumors, micropapillary carcinomas) that show IHC 1þ staining that is
intense but incomplete (basolateral or U shaped) and that are found to be HER2 amplified. The pathologist should consider also
reporting these specimens equivocal and request reflex testing using the alternative test.
Must report a HER2 test result as negative if a single test (or all tests) performed on a tumor specimen show: (a) IHC 1þ negative or
IHC 0 negative or (b) ISH negative using single-probe ISH or dual-probe ISH (Table 1; Figs 1 to 3). This assumes that there is no
apparent histopathologic discordance observed by the pathologist (Table 2).
Must report a HER2 test result as indeterminate if technical issues prevent one or both tests (IHC and ISH) performed on a tumor
specimen from being reported as positive, negative, or equivocal. This may occur if specimen handling was inadequate, if artifacts
(crush or edge artifacts) make interpretation difficult, or if the analytic testing failed. Another specimen should be requested for
testing, if possible, and a comment should be included in the pathology report documenting intended action.
Must ensure that interpretation and reporting guidelines for HER2 testing are followed (Table 1; Data Supplements 7, 8, 9, and 10).
Should interpret bright-field ISH on the basis of a comparison between patterns in normal breast and tumor cells, because artifactual
patterns may be seen that are difficult to interpret. If tumor cell pattern is neither normal nor clearly amplified, test should be
submitted for expert opinion.
Should ensure that any specimen used for HER2 testing (cytologic specimens, needle biopsies, or resection specimens) begins the
fixation process quickly (time to fixative within 1 hour) and is fixed in 10% neutral buffered formalin for 6 to 72 hours and that
routine processing, as well as staining or probing, is performed according to standardized analytically validated protocols.
Should ensure that the laboratory conforms to standards set for CAP accreditation or an equivalent accreditation authority, including
initial test validation, ongoing internal quality assurance, ongoing external proficiency testing, and routine periodic performance
monitoring.
If an apparent histopathologic discordance is observed in any HER2 testing situation (Table 2), the pathologist should consider
ordering additional HER2 testing and conferring with the oncologist, and should document the decision-making process and results
in the pathology report. As part of the HER2 testing process, the pathologist may pursue additional HER2 testing without conferring
with the oncologist.
Although categories of HER2 status by IHC or ISH can be created that are not covered by these definitions, in practice they are
uncommon and if encountered should be considered IHC equivocal or ISH equivocal.
242 Arch Pathol Lab Med—Vol 138, February 2014 ASCO/CAP HER2 Testing Guideline Update—Wolff et al
expression reverse-transcriptase polymerase chain reaction,
have been introduced into practice, and the Update
Committee felt these required evidence-based review. The
Update Committee wishes to re-emphasize that it is
important that any new test methodology, for the same
clinical use, be compared with a reference test that assays for
the same analyte and for which there are high levels of
evidence that use of the test leads to clinical benefit for the
patient (ie, clinical utility). It is the opinion of the Update
Committee that there is insufficient evidence to support use
of mRNA or DNA microarray assays to determine HER2
status in unselected patients (Data Supplement 2A).
Further experience with established HER2 assays also led
to the identification of unusual HER2 genotypic abnormal-
ities, like aneusomy of chromosome 17 (polysomy and
monosomy), colocalization of HER2 and CEP17 signals that
affect HER2/CEP17 ratio in dual-signal in situ hybridization
(ISH) assays, and genomic heterogeneity. Limited retro-
spective data on the clinical significance of these abnormal-
ities in completed prospective trials also guided the
discussions that were part of this guideline update.
6–22
Some of these issues are discussed in Data Supplements
2B and 2C and in a separate review article by Hanna et al.
23
During the deliberations, the Update Committee was
concerned about false-negative and false-positive HER2
assessments. For example, a false-negative test result could
lead to denial of trastuzumab treatment for a patient who
could benefit from it. False-positive results could lead to the
administration of potentially toxic, costly, and ineffective
adjuvant HER2-targeted therapy for 1 year.
24–27
The Update
Committee considered mandatory testing of all HER2-
negative tests (Data Supplement 2D) and addressed also a
narrower set of scenarios that may on occasion be observed
with dual-signal ISH assays (Data Supplement 2E; Inter-
pretation Criteria If Using a Dual-Signal HER2 Assay and
Average HER2 Copy Number ,6 Signals Per Cell).
Trastuzumab had previously been shown to improve
progression-free survival and overall survival when com-
bined with chemotherapy in the metastatic setting.
28
Since
2005, several of the first-generation adjuvant trials have
been updated and have confirmed the disease-free and
overall survival benefit offered by 1 year of trastuzumab
administered with or after adjuvant chemotherapy.
29–31
Prospective randomized trials, first reported in abstract
form in late 2012, seem to suggest that 12 months is the
optimal duration of adjuvant trastuzumab therapy.
Other HER2-targeted drugs (eg, the kinase inhibitor
lapatinib,
32
the antibody pertuzumab,
33
and the antibody-
drug conjugate ado-trastuzumab emtansine [T-DM1]
34
)
have been approved for the treatment of HER2-positive
metastatic breast cancer. At the same time, data show that
lapatinib (when added to paclitaxel)
35
and pertuzumab (as a
single agent)
36
offer no clinical benefit in patients with
HER2-negative metastatic disease. These new HER2-tar-
geted drugs are now being tested in the adjuvant setting,
including in studies evaluating their adjuvant role alone or
in dual-antibody regimens without concomitant or sequen-
tial chemotherapy. Compared with regimens already in use,
the newer agents are as or more expensive, and they may be
associated with other dose-limiting toxicities, such as skin
and GI tract toxicities with lapatinib and liver toxicities with
ado-trastuzumab emtansine.
37
Therefore, the need for an updated ASCO/CAP guideline
on accurate HER2 testing to ensure that the right patient
receives the right treatment is now more critical than
ever.
22,24–27,38
Since the publication of the 2007 HER2 testing
guideline, CAP has observed a remarkable uptake of
proficiency testing (Fig 4),
5
with nearly 1,500 laboratories
currently participating. CAP has also observed fewer
laboratories experiencing deficiencies on laboratory inspec-
tion. Indirect evidence suggests that the performance of
laboratories that conduct HER2 testing in the United States
and elsewhere is improving.
39–42
Available evidence and
experience since 2007 reinforce the importance of robust
validation of new assays by laboratories before clinical
implementation, as well as their ongoing monitoring, and
the value of various external quality assurance schemes
adopted in many countries.
METHODS
The HER2 testing Update Committee (Appendix Table A1,
online only at www.asco.org/guidelines/her2) met 3 times via
Webinars coordinated by its Steering Committee to review the data
published from January 2006 to January 2013 and to revise the
recommendations. Additional data were gathered from in-press
publications and personal correspondence with researchers to
address the issue of mandatory testing if a test result is 0 or 1þ.
Draft manuscripts were circulated by e-mail, and the Update
Committee approved the final manuscript. This guideline was
reviewed by external reviewers and approved by the ASCO Clinical
Practice Guideline Committee and relevant CAP entities.
Literature Search Strategy
The MEDLINE and the Cochrane Collaboration Library elec-
tronic databases were searched with the date parameters of January
2006 through January 2013 for articles in English. The MEDLINE
search terms are included in Data Supplement 3, and a summary of
the literature search results is provided in Data Supplement 4.
Inclusion and Exclusion Criteria
Articles were selected for inclusion in the systematic review of
the evidence if they met the following criteria: (1) the study
compared, prospectively or retrospectively, fluorescent ISH (FISH)
and immunohistochemistry (IHC) results or other tests; described
technical comparisons across various assay platforms; examined
potential testing algorithms for HER2 testing; or examined the
The Bottom Line (Continued)
Methods
Systematic review and analysis of the medical literature were conducted by the 2013 Update Committee.
Additional Information
The revised recommendations and a brief summary of the literature and analysis are provided in this article. Data Supplements
including clinical tools and resources can be found at http://www.asco.org/guidelines/her2 and at http://www.cap.org. Patient
information is available at http://www.cancer.net. ASCO and CAP believe that cancer clinical trials are vital to inform medical
decisions and improve cancer care, and that all patients should have the opportunity to participate.
Arch Pathol Lab Med—Vol 138, February 2014 ASCO/CAP HER2 Testing Guideline Update—Wolff et al 243
correlation of HER2 status in primary versus metastatic tumors
from the same patients; (2) the study population consisted of
patients with a diagnosis of invasive breast cancer; or (3) the
primary outcomes included the negative predictive value (NPV) or
positive predictive value (PPV) of ISH and IHC assays used to
determine HER2 status, alone and in combination; negative and
positive concordance across platforms; and accuracy in determining
HER2 status and benefit from anti-HER2 therapy and in
determining sensitivity and specificity of individual tests. Consid-
eration was given to studies that directly compared results across
assay platforms.
Studies were not limited to randomized controlled trials but also
included other study types, including cohort designs, case series,
evaluation studies, and comparative studies. The Update Committee
also reviewed other testing guidelines and proficiency strategies of
various US and international organizations, including unpublished
data. Letters, commentaries, and editorials were reviewed for any
new information. Case reports were excluded. The clinical questions
addressed in the update are available in Data Supplement 5.
This information was used to help the Update Committee
develop new algorithms (for pathologists and oncologists) for
testing, specify testing requirements and exclusions, and facilitate
the necessary quality assurance monitoring that will make HER2
testing less variable and ensure more analytic consistency between
laboratories. The term ratio, as used in the guideline recommen-
dations and algorithms, always applies to the HER2/CEP17 ratio,
which means the ratio of HER2 signals per cell (numerator) over
CEP17 signals per cell (denominator).
ASCO Guideline Disclaimer
The clinical practice guideline and other guidance published
herein are provided by ASCO to assist practitioners in clinical
decision making. The information herein should not be relied on as
being complete or accurate, nor should it be considered as inclusive
of all proper treatments or methods of care or as a statement of the
standard of care. With the rapid development of scientific
knowledge, new evidence may emerge between the time informa-
tion is developed and when it is published or read. The information
is not continually updated and may not reflect the most recent
evidence. The information addresses only the topics specifically
identified herein and is not applicable to other interventions,
diseases, or stages of diseases. This information does not mandate
any particular course of medical care. Furthermore, the information
is not intended to substitute for the independent professional
judgment of the treating physician, because the information does
not account for individual variation among patients. Recommen-
dations reflect high, moderate, or low confidence that the
recommendation reflects the net effect of a given course of action.
The use of terms like must, must not, should, and should not
indicate that a course of action is recommended or not
recommended for either most or many patients, but there is
latitude for the treating physician to select other courses of action in
individual cases. In all cases, the selected course of action should be
considered by the treating physician in the context of treating the
individual patient. Use of the information is voluntary. ASCO
provides this information on an as-is basis and makes no warranty,
express or implied, regarding the information. ASCO specifically
disclaims any warranties of merchantability or fitness for a
particular use or purpose. ASCO assumes no responsibility for
any injury or damage to persons or property arising out of or
related to any use of this information or for any errors or omissions.
CAP Guideline Disclaimer
Clinical practice guidelines reflect the best available evidence and
expert consensus supported in practice. They are intended to assist
physicians and patients in clinical decision making and to identify
questions and settings for further research. With the rapid flow of
scientific information, new evidence may emerge between the time
a practice guideline or consensus statement is developed and when
it is published or read. Guidelines and statements are not
continually updated and may not reflect the most recent evidence.
Guidelines and statements address only the topics specifically
identified therein and are not applicable to other interventions,
diseases, or stages of diseases. Furthermore, guidelines and
statements cannot account for individual variation among patients
and cannot be considered inclusive of all proper methods of care or
exclusive of other treatments. It is the responsibility of the treating
physician, relying on independent experience and knowledge, to
determine the best course of treatment for the patient. Accordingly,
adherence to any practice guideline or consensus statement is
voluntary, with the ultimate determination regarding its application
to be made by the physician in light of each patient’s individual
circumstances and preferences. CAP makes no warranty, express or
implied, regarding guidelines and statements and specifically
excludes any warranties of merchantability and fitness for a
particular use or purpose. CAP assumes no responsibility for any
injury or damage to persons or property arising out of or related to
any use of this statement or for any errors or omissions.
Guideline and Conflicts of Interest
The Update Committee was assembled in accordance with CAP
and ASCO Conflicts of Interest Management Procedures for
Clinical Practice Guidelines (ASCO procedures are summarized
at http://www.asco.org/guidelinescoi). Members of the Update
Committee completed the ASCO disclosure form, which requires
disclosure of financial and other interests that are relevant to the
subject matter of the guideline, including relationships with
commercial entities that are reasonably likely to experience direct
regulatory or commercial impact as the result of promulgation of
the guideline. Categories for disclosure include employment
relationships, consulting arrangements, stock ownership, honorar-
ia, research funding, and expert testimony. In accordance with the
procedures, the majority of the members of the Update Committee
did not disclose any such relationships.
RECOMMENDATIONS
CLINICAL QUESTION 1
What is the optimal testing algorithm for the assessment
of HER2 status?
Literature Update and Discussion
The Update Committee found more than 70 new
publications that informed a revision of the testing algorithms
contained in the original 2007 guideline. At the time of the
original guideline, significant concern existed about false-
positive HER2 test results. Guideline recommendations
emphasized those changes that would mitigate false posi-
tives, particularly relating to issues of specimen fixation and
pathologist interpretation.
39,43–47
Preliminary data from an
ongoing prospective study seem to suggest that the frequency
of false-positive test results may have diminished, in that the
concordance between local testing in laboratories throughout
the United States and confirmatory central HER2 testing at
the Mayo Clinic (Rochester, MN) for the ALTTO (Adjuvant
Lapatinib and/or Trastuzumab Treatment Optimization
HER2 Adjuvant Trial) trial showed that less than 6% of
patients initially considered eligible were not subsequently
centrally confirmed as being HER2 positive.
48
On the other end of the spectrum, clinical experience and
recent literature have indicated that false-negative HER2 test
results must also be considered. The Update Committee was
sensitive to the concerns that surfaced after the publication of
the 2007 guideline about the very small number of patients
potentially affected by the recommendation to consider as
HER2 positive only those tumors with more than 30% of
cells (or .10% to 30% if HER2 amplified by FISH) with
diffuse and intense circumferential staining.
49
Therefore, the
244 Arch Pathol Lab Med—Vol 138, February 2014 ASCO/CAP HER2 Testing Guideline Update—Wolff et al
Update Committee decided to revert to the previously used
IHC criterion of more than 10% cells staining for HER2,
which had been used as an entry criterion for eligibility for
the first generation of prospective randomized trials of
adjuvant trastuzumab.
18,22,49–53
The rationale for this recom-
mendation by the Update Committee is detailed in Data
Supplement 1. Aside from the very small number of patients
affected (as few as 0.15% of all newly diagnosed patients, as
previously discussed),
5
the Update Committee was also of
the opinion that improvements in analytic performance of
HER2 testing in clinical practice since 2007 have further
reduced the already small number of patients potentially at
risk of receiving a false-negative test result.
Testing is now recommended for primary, recurrent, and
metastatic tumors.
19,35,45,54–63,64
Tissue from the primary
tumor can be obtained through a core needle biopsy, as
well as from an incisional and excisional surgical proce-
dure.
65
Metastases can be biopsied from chest wall, regional
lymph nodes, or distant organs.
66–74
It is essential to ensure
that time to fixation (cold ischemic time) and time in fixative
(which has increased from 6 to 48 hours to 6 to 72 hours in
this update on the basis of available data and to conform
with the ASCO/CAP estrogen receptor [ER]/progesterone
receptor [PgR] testing guideline
75,76
) are recorded and
considered in defining the test result. More detail about
preanalytic issues is available in Data Supplement 6.
In summary, if available, perform the first test in the core
biopsy specimen in a patient with newly diagnosed breast
cancer. If the test result is clearly positive or clearly negative
as defined in Table 1, no retesting is needed. If the test is
negative and there is apparent histopathologic discordance
(Table 2), or if specimen handling has not been in
accordance with guideline recommendations, a section of
the tumor from the excisional specimen should be tested. If
this result is positive, no further testing is needed. However,
if the test is negative and there remains significant clinical
concern about the result after consultation between the
pathologist and the medical oncologist, it may be appro-
priate to repeat the test in a different block from the
patient’s tumor. If all three tests are negative, no additional
testing is recommended.
Data Supplement 7 is a table of IHC Interpretation
Criteria, and Data Supplement 8 provides ISH Interpretation
Criteria. Both of these Data Supplements expand on details
provided in Table 1.
The Update Committee clarified several issues in the
update on the basis of recently published literature. The
recommendations in Table 1 reflect the Update Commit-
tee’s interpretation of the new data on polysomy,
heterogeneity in ISH, types of assays, and methods of
analysis
10–14,19–21,45,67,69,79–135
for inclusion in this update.
See Data Supplement 2 for an extensive discussion of these
issues.
A list of US Food and Drug Administration (FDA) approved
assays is available at http://www.accessdata.fda.gov/scripts/
cdrh/devicesatfda/index.cfm?start_search¼1&search_term¼
HER2&approval_date_from¼&approval_date_to¼07/14/
2013&sort¼approvaldatedesc&pagenum¼10 (last checked
July 14, 2013). The product package inserts for trastuzumab
and pertuzumab prepared by the FDA indicate that ‘‘HER2
testing should be performed using US Food and Drug
Administration-approved tests by laboratories with demon-
strated proficiency.’’
77,78
HER2 Assay Exclusions
Each assay type has diagnostic pitfalls to be avoided. The
Update Committee agreed that there were situations in
which one assay type was preferred because of assay or
sample considerations. Exclusion criteria to perform or
interpret an IHC or any ISH assay for HER2 are unchanged
but can be viewed in the original guideline.
1,2
The
pathologist who reviews the histologic findings should
determine the optimal assay (IHC or ISH) for determination
of HER2 status.
Algorithms for HER2 Testing by IHC and ISH
Algorithms for evaluation of HER2 protein expression by
IHC and HER2 amplification by single-probe or dual-probe
ISH are presented in Figures 1, 2, and 3.
CLINICAL QUESTION 2
What strategies can help ensure optimal performance,
interpretation, and reporting of established assays?
Literature Update and Discussion
Testing analytic validation requirements.—The Update
Committee reviewed new papers and reports on strategies
to ensure optimal performance, interpretation, and report-
ing of assays.
16,22,100,136,137
Most new HER2 assays have been
submitted to the FDA for premarket approval review as class
III devices in view of their use for therapy selection.
Although a new HER2 assay ideally should have its clinical
utility validated using specimens from prospective thera-
peutic trials that tested the effects of anti-HER2 therapy, the
Update Committee recognizes that the rarity of these
valuable specimens requires that new HER2 assays be
approved on the basis of concordance studies comparing
them with other established HER2 tests. Consequently, it is
important that tissues selected for such concordance studies
come from datasets that include a broad representation of
patients with breast cancer in whom HER2-positive status
will be observed in approximately 15% to 20%.
Ongoing competency assessment.—The Update Com-
mittee urges ongoing competency assessment as a part of
every laboratory’s internal quality assessment program. The
competency of the laboratory professionals and pathologists
interpreting assays must be continuously addressed as
required under the Clinical Laboratory Improvements
Amendments (CLIA 88). The acceptable performance
standard for such competency tests remains the same as
in the original guideline.
Reporting requirements.—Data Supplements 9 and 10
are tables of reporting elements for IHC and reporting
elements for ISH, respectively. Some changes have been
made to the reporting elements for IHC and ISH to ensure
that they are in accordance with the revised recommenda-
tions. In addition, a disclaimer statement is required if the
specimen handling requirements are not met.
New interpretation requirements relate to the definition of
tumor samples with genomic heterogeneity as well as the
examination of specimens and interpretation of results in
these samples. No specific requirements were added for
designation of polysomy by ISH. Laboratories should
maintain documentation of their quality assurance practices
and ensure that such documentation is available for
inspection.
Regulatory framework.—The regulatory framework
remains the same as discussed in the original guideline.
Arch Pathol Lab Med—Vol 138, February 2014 ASCO/CAP HER2 Testing Guideline Update—Wolff et al 245
![Figure 2. Algorithm for evaluation of human epidermal growth factor receptor 2 (HER2) gene amplification by in situ hybridization (ISH) assay of the invasive component of a breast cancer specimen using a single-signal (HER2 gene) assay (single-probe ISH). Amplification in a single-probe ISH assay is defined by examining the average HER2 copy number. If there is a second contiguous population of cells with increased HER2 signals per cell, and this cell population consists of more than 10% of tumor cells on the slide (defined by image analysis or visual estimation of the ISH or immunohistochemistry [IHC] slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this cell population and also reported. Although categories of HER2 status by ISH can be created that are not covered by these definitions, in practice they are rare and if encountered should be considered ISH equivocal (see Data Supplement 2E). NOTE: the final reported results assume that there is no apparent histopathologic discordance observed by the pathologist. (*) Observed in a homogeneous and contiguous population.](/figures/figure-2-algorithm-for-evaluation-of-human-epidermal-growth-as7s4xoj.webp)