Original Article

Recommendations for Validating Estrogen and

Progesterone Receptor Immunohistochemistry Assays
Patrick L. Fitzgibbons, MD; Douglas A. Murphy, MT; M. Elizabeth H. Hammond, MD; D. Craig Allred, MD; Paul N. Valenstein, MD

Context.Estrogen receptor and progesterone receptor

Nstatus
is assessed on all newly diagnosed, invasive breast
carcinomas and in recurrences to determine patient
% to 20%
% of
eligibility for hormonal therapy, but 10%
estrogen receptor and progesterone receptor test results
are discordant when tested in multiple laboratories.
Objective.To define the analytic (technical) validation
requirements for estrogen receptor and progesterone
receptor immunohistochemistry assays used to select
patients for hormonal therapy.
alidation of a clinical laboratory test means confirmaV
tion, through a defined process, that the test performs
as intended or claimed. Proper validation provides
reasonable, but not absolute, assurance that a test is
performing as anticipated. There is no single, universally
acceptable procedure for validating clinical laboratory
tests. The design of a validation protocol requires
professional judgment, and validation schemes must take
into account the tests intended use, other claims made
about the test, and risks that may prevent the test from
meeting performance claims.
This article provides guidance on analytic (technical)
validation procedures that we believe should be used by
laboratories offering estrogen receptor (ER) and progesterone receptor (PgR) assays by immunohistochemical
(IHC) methods. We describe minimal procedures for
initially validating the tests before they are placed in
clinical service. We also discuss labeling requirements
(language) applicable to reporting and to claims a
laboratory may choose to make about its assays. A
separate guideline1 describes required elements of an
ongoing quality management program for hormonereceptor testing by IHC methods, including daily quality
control testing, external proficiency testing, and general
Accepted for publication February 2, 2010.
From the Department of Pathology, St Jude Medical Center, Fullerton,
California (Dr Fitzgibbons); the Surveys Department, College of
American Pathologists, Northfield, Illinois (Mr Murphy); the Department
of Pathology, Intermountain Healthcare, University of Utah School of
Medicine, Salt Lake City (Dr Hammond); the Department of Pathology
and Immunology, Washington University School of Medicine, St Louis,
Missouri (Dr Allred); and the Department of Pathology, St Joseph Mercy
Hospital, Ann Arbor, Michigan (Dr Valenstein).
The authors have no relevant financial interest in the products or
companies described in this article.
Reprints: Patrick L. Fitzgibbons, MD, Department of Pathology, St
Jude Medical Center, 101 E Valencia Mesa Dr, Fullerton, CA 92835
(e-mail: pfitz@stjoe.org).
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Arch Pathol Lab MedVol 134, June 2010

Data Sources.Literature review and expert consensus.

Conclusions.A standardized process for initial test
validation is described. We believe adoption of this process
will improve the accuracy of hormone-receptor testing,
reduce interlaboratory variation, and minimize falsepositive and false-negative results. Required ongoing assay
assessment procedures are also described.
(Arch Pathol Lab Med. 2010;134:930935)

controls applied to laboratory personnel, equipment,

reagents, and other aspects of laboratory service.
USE OF IHC HORMONE-RECEPTOR TESTING
Estrogen receptor and PgR status is assessed in all
newly diagnosed, invasive breast carcinomas and in
recurrences to determine patient eligibility for adjuvant
hormonal therapy.2 There is a substantial survival benefit
from tamoxifen and aromatase inhibitors, but only among
patients with ER-positive tumors.35 Accurate classification of hormone-receptor status is, therefore, critical to
ensure patients receive appropriate therapy.
Immunohistochemistry is currently the most commonly
used method for determining ER and PgR status because
of its relatively low cost, its applicability to routinely
processed and archival tissue samples, and importantly,
its use in evaluating small cancers to ensure that only
invasive tumor cells are assessed. This guideline describes
validation procedures for ER and PgR IHC assays that are
used to predict response to tamoxifen and aromatase
inhibitors (predictive markers). Validation procedures are
designed to reasonably confirm that a new test performs
this task as well as existing validated assays. The
procedures described in this guideline are not adequate
to demonstrate that a new assay is superior to existing
assays; such claims require additional validation procedures.
Risks of IHC Hormone-Receptor Testing
Patients with breast cancer who are misclassified as
having ER-negative tumors are denied the potential
benefit of hormonal treatment, whereas those who are
misclassified as having ER-positive tumors will be
exposed unnecessarily to the risks and costs of ineffectual
treatment and potentially being denied the benefit of other
treatments. Other risks from hormonal treatment include
a decrease in bone density with an increased fracture risk,
Validating ER and PgR Immunohistochemistry AssaysFitzgibbons et al

Table 1.

Acceptable Validation of Hormone-Receptor Assaysa

ER and PgR IHC assays not subjected to direct clinical validation may be validated by showing 90% agreement for positive results and 95%
agreement for negative results with any of the following:
1. Testing performed on the same blocks in another laboratory that has directly validated its assay against clinical outcome
2. Testing performed on the same blocks using a previously validated ligand binding assay
3. Testing performed on the same blocks in another laboratory that provides written attestation that it is in conformance with ASCO/CAP
testing requirements and is using one of the following:
a. An FDA-approved assay that has been fully validated using an 80-specimen challenge set as described in Table 3, or
b. An LDT or LMT that has been validated according to all other requirements set forth in this document
4. Testing performed on the same blocks in another laboratory that uses an alternative, clinically validated method for measuring hormonereceptor expression (eg, a gene-expression assay)17
5. Testing performed on one of the following:
a. Tissue challenges used in a formal PT program, provided that each case used in the validation study was graded by the PT vendor
and $50 laboratories are included in the participants peer group, or
b. Validation tissues provided by an organization such as the CAP or the NIST, with established ER and PgR status determined through
IHC testing using a technically validated assay24
Abbreviations: ASCO, American Society of Clinical Oncology; CAP, College of American Pathologists; ER, estrogen receptor; FDA, US Food and
Drug Administration; IHC, immunohistochemical; LDT, laboratory-developed test; LMT, laboratory-modified test; NIST, National Institute of
Standards and Technology; PgR, progesterone receptor; PT, proficiency testing.
a

In the case of unmodified, FDA-cleared or FDA-approved ER and PgR IHC assays, an alternative verification procedure may be specified in the
FDA-approved or FDA-cleared package insert.

an increase in the risk of thromboembolic events, and an

increase in the risk of uterine cancer.6
Although IHC test methods have improved with the
widespread adoption of automated staining platforms, the
development of more sensitive antibody clones and
detection systems, and the use of US Food and Drug
Administration (FDA)approved test kits,7 results are still
affected by factors such as delayed or inadequate fixation,
nonoptimized antigen retrieval, and nonstandardized
interpretation of findings or reporting of results.811 Initial
validation and periodic or ongoing reassessment of IHC
ER assays provide reasonable assurance that manageable
factors that affect test accuracy and clinical usefulness are
properly controlled.
Special Considerations in IHC Hormone-Receptor Testing
The test-validation procedures described in this guideline are designed to provide reasonable assurance that
false-positive and false-negative test results are minimized. Because patients with even low levels of hormonereceptor expression (ie, 1%) may respond to hormonal
therapy,1220 these procedures are intended to ensure that
ER assays can accurately identify tumors with weak ER
expression.
The validation of any clinical assay requires that results
be compared with a standard. Some clinical assays may be
validated with certified reference material with known
reactivity that is traceable to an authoritative standard, but
such materials are not currently available for ER IHC. For
ER and PgR assays, which are primarily used as predictive
markers, the ideal standard is a clinical demonstration that
the test accurately identifies patients who will benefit from
hormonal therapy; however, few laboratories have the
resources available to validate their assay with reference
to clinical outcome. This validation protocol was designed
for assays that are used to guide therapeutic decision
making but are not being evaluated directly against a
clinical standard. Some of the procedures described in this
protocol may be unnecessary if direct clinical validation is
done, but additional requirements for clinical validation
may be applicable.
The approach to validation used in this guideline relies
on comparing the assays results with results obtained by
another laboratory using a testing method that has been
Arch Pathol Lab MedVol 134, June 2010

validated against clinical outcome or against proficiencytesting material that has been validated by showing
consensus results among multiple laboratories in a peer
group (which must include laboratories with validated
assays). Acceptable approaches are listed in Table 1.
Assay validation that relies on comparison with another
unvalidated assay is not sufficient.21,22
The level of agreement required for validating a new
assay (90% agreement for positive specimens and 95%
agreement for negative specimens) has been achieved in
several studies that compared local laboratory and central
laboratory ER results.16,17 If the positive samples in the
validation set are enriched with weakly positive specimens,
as we propose, we believe that laboratories with at least 90%
positive agreement at the time of assay validation under
these conditions are likely to detect considerably more than
90% of ER-positive specimens in clinical practice.
The laboratory director is responsible for ensuring that
all validation steps have been performed according to
these guidelines.
VALIDATION REQUIREMENTS
Initial Validation
Initial validation or verification of ER and PgR IHC
assays must be successfully completed before the tests can
be placed into clinical service.
An IHC predictive-marker assay includes a defined set
of test conditions (reagents, equipment, specimen types,
and standard operating procedures) and an ongoing
quality management regimen that includes, as applicable,
routine quality control, periodic assay recalibration,
employee-competency testing, external proficiency testing, and laboratory inspection. During the interval when a
test is being validated, the test conditions and ongoing
quality-management regimen should be the same as the
conditions and quality-management regimen that will be
used once the assay is placed in clinical service.
Laboratories that intend to use specialized techniques
for scoring, such as image analysis, must use those same
techniques in the validation study.
To ensure that the validation study assesses betweenrun variation, the laboratory validating its assay should
not test all specimens in the validation set on the same

Validating ER and PgR Immunohistochemistry AssaysFitzgibbons et al

931

Table 2. Recommendations for Initial Test

Verification of US Food and Drug Administration
(FDA)Cleared Assaysa
Procedure

Acceptable Outcome

The laboratory should

Agreement must be $90% for
compare the results from
positive results and $95% for
testing $20 positive and
negative results; positive
$20 negative specimens
results are defined as $1%
using one of the methods
immunoreactive cells
described in Table 1b; $5
of the positive specimens
should be weakly positive
(1%10%), and #10
specimens should be tested
in any one run
or
or
Any verification procedure
Acceptable outcome described
described in an FDAin an FDA-cleared or FDAcleared or FDA-approved
approved package insert for
package insert for the test
the test system
system
Any pathologist who
Each pathologist must
interprets ER and PgR IHC
demonstrate #2 incorrect
results but did not
assessments in the 40-slide
participate in the validation
challenge set; an incorrect
procedure described above
assessment is any specimen
must have his or her skill
with $1% immunoreactive
validated by examining
cells reported as negative, or
$20 positive and $20
any specimen with ,1%
negative specimens; at least
immunoreactive cells reported
some of the positive
as positive
specimens must be weakly
positive; slides from the
initial assay validation may
be used for this skillvalidating procedure.
Abbreviations: ER, estrogen receptor; IHC, immunohistochemical; PgR,
progesterone receptor; PT, proficiency testing.
a
Applies to unmodified, FDA-cleared or FDA-approved, ER and PgR IHC
assays. Validation specimens for FDA-cleared or FDA-approved assays
may be obtained from the assay manufacturer if the assays FDA-approved
package insert indicates that manufacturer-supplied specimens may be
relied on to verify performance of the assay. A verification study does not
need to be repeated if a previous study conforming to the manufacturers
FDA-cleared recommendations or to this guideline has been completed
and the records are available for review by external inspectors.
b
Laboratories with assays already in clinical service (and used on
$200 specimens) may use the results of previously analyzed cases in
their validation study, provided that the previously analyzed cases
have been tested using one of the methods listed in Table 1. Those
using historic results of PT challenges should include the same
number of cases as specified above. If the PT program has, for
example, 20 annual challenges, results for $2-years continuous
participation (40 challenges) would be required.

day. These specimens should be run in batches on

multiple days, with multiple testing personnel, when
possible.
FDA-Cleared Assays.Laboratories with assays cleared
or approved by the FDA must verify the performance
specifications stated by the manufacturer. Manufacturers
of FDA-approved or cleared test kits may provide the user
with FDA-approved or cleared recommendations and
directions for verifying that the kit is performing according to the manufacturers specification. Usually this is
performed by testing known positive and negative
samples that either are supplied by the manufacturer or
have been tested by a validated reference-laboratory
method.22 Recommended verification procedures for these
assays are described in Table 2.
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Arch Pathol Lab MedVol 134, June 2010

For laboratories with unmodified FDA-cleared or FDAapproved ER or PgR assays that were in clinical service
before the publication of the American Society of Clinical
Oncology/College of American Pathologists Guideline Recommendations for Immunohistochemical Estrogen/Progesterone
Receptor Testing in Breast Cancer1 and that have been used
on at least 200 clinical specimens, the verification
procedure may include the results of previously analyzed
cases; however, laboratories introducing an assay after
publication of the guidelines must complete the verification study before the test is placed in service.
A verification study does not need to be repeated if a
previous study conforming to the manufacturers FDAcleared recommendations or with this guideline has been
completed and the records are available for review by
external inspectors. Laboratories that cannot document
initial test verification of an FDA-cleared or FDAapproved assay must complete a new verification study
to show that the test performs as intended.
Laboratory-Developed and Laboratory-Modified Assays.
Laboratories must validate laboratory-developed tests
(LDTs) and any FDA-cleared or FDA-approved laboratory-modified tests (LMTs). Recommended validation
procedures for ER or PgR LDTs or LMTs are described in
Table 3. For laboratories with assays that were in clinical
service before the publication of the American Society of
Clinical Oncology/College of American Pathologists testing
guidelines1 and that have been applied to at least 200 clinical
specimens, the validation set may include the results of
previously analyzed cases provided that the previously
analyzed cases have been tested using one of the methods
listed in Table 1; however, laboratories introducing an LDT
or LMT after publication of the guidelines must complete
the validation study before the test is placed in service.
A validation study does not need to be repeated if a
previous validation study conforming to this guideline
has been completed and the records are available for
review by external inspectors. Laboratories that cannot
document initial validation of an LDT or LMT must
complete a new validation study to show that the test
performs as intended.
Changes in Test Methods
All assays must be revalidated whenever there is a
significant change to the test system, such as a change in
the primary antibody clone, introduction of new antigenretrieval or immunohistochemistry detection systems, or a
significant relaxation of ongoing quality-management
procedures. Assay revalidation after significant changes
should meet the requirements specified in Table 3.
Ongoing Assessment
Regardless of the methodology used, all laboratories
with validated ER and PgR IHC assays must periodically
reassess the assays to ensure that their analytic sensitivity
has not drifted. Required ongoing assay assessment
procedures are described in Table 4. Ongoing assay
reassessment does not require repeating the same procedures used for initial test validation.
LABELING AND REPORTING
Laboratories using LDT and LMT assays must append a
statement to each IHC result indicating that the assay was
developed and its performance characteristics determined
by [name of laboratory]. Laboratories should not make
Validating ER and PgR Immunohistochemistry AssaysFitzgibbons et al

Table 3. Recommendations for Initial Test Validation

of all Laboratory-Developed and LaboratoryModified Assaysa
Procedure

Table 4.

Procedure

Acceptable Outcome

Monitor overall positive and

negative ER rates (trend
analysis), calculated at
least twice annually

The laboratory should

Agreement must be $90% for
compare the results of
positive results and $95% for
testing $40 positive and
negative results; positive
$40 negative specimens
results are defined as $1%
using one of the methods
immunoreactive cells
described in Table 1b; $10 of
the positive cases should be
weakly positive (1%10%),
and #20 specimens should
be tested in any one run
Any pathologist who interprets Each pathologist must
ER and PgR IHC results but
demonstrate #2 incorrect
did not participate in the
assessments in the 40-slide
validation procedure
challenge set; an incorrect
described above must have
assessment is any specimen
his or her skill validated by
with $1% immunoreactive
examining $20 positive and
cells reported as negative, or
$20 negative specimens; at
any specimen with ,1%
least some of the positive
immunoreactive cells
specimens must be weakly
reported as positive
positive; slides from the
initial assay validation may
be used for this procedure
Abbreviations: ER, estrogen receptor; IHC, immunohistochemical; PgR,
progesterone receptor; PT, proficiency testing.
a

Applies to all laboratory-developed or laboratory-modified assays and to

any assay in which there is a major change to the test system, such as a
change in the primary antibody, in the antigen-retrieval system, or in the
antigen-detection system (but does not apply to new reagent lots or other
minor modifications). A validation study does not need to be repeated if a
previous validation study conforming to this guideline has been
completed and the records are available for review by external inspectors.
b
Laboratories with assays already in clinical service (and used on $200
specimens) may use the results of previously analyzed cases in their
validation study, provided that the previously analyzed cases have been
tested using one of the methods listed in Table 1. Those using historic
results of PT challenges should include the same number of cases as
specified above. If the PT program has, for example, 20 annual
challenges, results for $4-years continuous participation (80 challenges)
would be required.

claims that their ER or PgR assays are superior to other

assays, unless such claims have been specifically validated
by comparison with clinical outcome.
DOCUMENTATION
Records of method validation must be maintained while
the test is in service and for at least 2 years after the method
is no longer used for clinical testing. Records of microscopist
validation must be maintained for the same time period.
DEFINITIONS
. Analyte-Specific Reagent.Antibodies, both polyclonal and monoclonal, specific-receptor proteins, ligands,
nucleic acid sequences, and similar reagents that,
through specific binding or chemical reaction with
substances in a specimen, are intended for use in a
diagnostic application for identification and quantification of an individual chemical substance or ligand in
biological specimens [21CFR864.4020(a)].
. FDA-Cleared Test.A test that has been cleared by the
FDA after analysis of data showing substantial performance equivalence to other tests being marketed for the
same purpose. Such tests typically follow the 510(k)
approval route [21CFR807].
Arch Pathol Lab MedVol 134, June 2010

Recommendations for Ongoing (Periodic)

Reassessment for All Assays
Acceptable Outcome

Overall ER2 rate should be

,30%. If $30%, correlate ER
results with age and histologic
parameters (ie, grade and
histologic type)
N About 80% of invasive
carcinomas in women older
than 65 y should be ER+12; if
ER2 rate among patients older
than 65 y is .20%, repeat the
validation procedure
described in Table 3
N Nearly all low-grade breast
carcinomas should be ER+25;
if ER2 rate among low-grade
carcinomas is $5%, repeat
the validation procedure
described in Table 3
Concordance should be $95%
for both ER and PgR results

Monitor concordance
between ER and PgR
results and those of gene
expression analyses (if
gene expression analysis
performed)
Demonstrate successful
Laboratories must achieve 90%
performance in an external
correct responses on graded
PT program for each
PT challenges
marker
Monitor ER and PgR results Acceptable variation among
by pathologist (calculated
pathologists should be
at least semiannually)
established by the laboratory
director

Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; PT,

proficiency testing.

. FDA-Approved Test.A test that is classified as a class

III medical device and that has been approved by the
FDA through the premarket approval process
[21CFR814.3].
. Laboratory-Modified Test.An FDA-cleared or FDAapproved test that is modified by a clinical laboratory
but not to a degree that changes the stated purpose of
the test, the approved test population, the specimen
type, the specimen handling, or claims related to the
interpretation of results.
. Laboratory-Developed Test.A test developed within
a clinical laboratory that is performed by the laboratory
in which the test was developed and is neither FDAcleared nor FDA-approved.
# Note.All LMTs are, by definition, LDTs. An LDT
may or may not employ analyte-specific reagents,
research-use only reagents, or investigationaluse only reagents; the types of reagents and devices
employed does not affect whether a test is classified as an LDT. A laboratory is considered to have
developed a test if the test procedure or implementation of the test was created by the laboratory performing the testing, irrespective of whether
fundamental research underlying the test was
developed elsewhere or whether reagents, equipment, or technology integral to the test was
purchased, adopted, or licensed from another entity.
. Test Validation.Confirmation through a defined
process that a test performs as intended or claimed.

Validating ER and PgR Immunohistochemistry AssaysFitzgibbons et al

933

Note.There is no single universally accepted

procedure for validating tests. The process for
validating tests must take into account the purpose
for which a test is intended, claims made about the
test, and the risks that may prevent the test from
serving its intended purpose or meeting performance claims. Even FDA-approved and FDAcleared tests require limited revalidation in clinical
laboratories (a process often referred to as verification) to establish that local implementation of the test
can reproduce a manufacturers validated claims.
Tests that use reagents or equipment that have not
been validated typically pose increased risks that
require more extensive validation, as do tests used
in more loosely controlled settings. The determination of whether a test has been adequately validated
requires professional judgment.
. Test Verification.An abbreviated process through
which a clinical laboratory establishes that its implementation of an FDA-approved and FDA-cleared test
performs in substantial conformance to a manufacturers stated claims.
. Analytic Validity.A tests ability to accurately and
reliably measure the analyte (the measurand) of interest.
The elements of analytic validity include the following,
as applicable:
# Accuracy.The closeness of agreement between the
average value obtained from a large series of
measurements and the true value of the analyte.
# Precision.The closeness of agreement between
independent results of measurements obtained
under stipulated conditions.
# Analytic Sensitivity.For quantitative and semiquantitative tests, analytic sensitivity is the lowest
amount of an analyte in a sample that can be
detected with (stated) probability. With respect to
hormone-receptor testing, analytic sensitivity refers
to the lowest amount of the receptor protein that can
be detected by the assay.
# Analytic Specificity.The ability of a test to measure
solely the analyte.
# Note.Analytic validity is expressed in the context
of a defined set of test conditions (including
standard operating procedures and permissible
specimen types) and an ongoing quality-management regimen (including, as applicable, routine
quality control, periodic assay recalibration, and
external proficiency testing or alternative external
testing). If the test conditions or quality-management regimen changes, the analytic validity of a test
may change.
. Clinical Validity.A tests ability to detect or predict a
disorder, a prognostic risk, or another condition or to
assist in the management of patients. The elements of
clinical validity include the following, as applicable:
# Clinical Sensitivity (Clinical Detection Rate).The
proportion of individuals with a disorder, prognostic risk, or condition that is detected by the test. For
hormone-receptor testing, clinical sensitivity refers
to the ability of the assay to correctly identify
patients who are eligible for hormonal therapy.
# Clinical Specificity.The proportion of individuals
without a disorder, prognostic risk, or condition that
is excluded by the test. For ER and PgR testing,
#

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Arch Pathol Lab MedVol 134, June 2010

Table 5.

Example of a Concordance Study

Reference Assay, No.

Test

Result

Positivea

Negativeb

New assay

Positive
Negative

38
2

1
39

Total

40

40

Positive percentage of agreement (new assay/validated assay) 5 38/

(38 + 2) 5 38/40 3 100 5 95.0%.
b
Negative percentage of agreement (new assay/validated assay) 5 39/
(39 + 1) 5 39/40 3 100 5 97.5%.

clinical specificity refers to the ability of the assay to

correctly identify patients who are not likely to
benefit from hormonal therapy.
# Reference Limits.A value or range of values for an
analyte that assists in clinical decision making.
Reference values are generally of 2 types: reference
intervals and clinical decision limits. A reference
interval (or reference range) is the range of test values
expected for a designated population of individuals.
This may be the central 95% interval of the distribution of values from individuals who are presumed to
be healthy (or have normal results). For some analytes
that reflect high-prevalence conditions (such as
cholesterol), significantly fewer than 95% of the
population may be healthy. In such a case, the
reference interval may be something other than the
central 95% of values. A clinical decision limit represents the lower or upper limit of a test value at which a
specific clinical diagnosis is indicated or a specified
course of action is recommended.
. Clinical Utility.The clinical usefulness of the test.
The clinical utility is the net balance of risks and
benefits associated with using a test in a specific clinical
setting. Clinical utility does not take into consideration
the economic cost or economic benefit of testing and is
to be distinguished from cost-benefit and cost-effectiveness analysis. Clinical utility focuses entirely on the
probabilities and on the magnitude of the clinical
benefit and clinical harm that result from using a test
in a particular clinical context.
# Note 1.The qualities listed above represent the
primary performance measurements that are used to
describe the clinical capabilities of a test. Other
measures of clinical validity may be applicable in
particular circumstances.
# Note 2.Clinical validity is expressed in the context
of a defined test population and a defined testing
procedure. If the test population changes (eg, a
change in the prevalence of disease) or the testing
procedure changes, the clinical validity of a test may
change.
. Percent Agreement.The proportion of specimens that
produce the same result when tested twice (eg, results
that are either both positive or both negative). The term
is generally used when the reference method is
acknowledged to be imperfect and the true result is
not known with high confidence. In this situation the
terms sensitivity and specificity are not appropriate to
describe the comparative results.
# Positive Percent Agreement.Refers to the percentage
of agreement among specimens that test positive
with the reference assay.
Validating ER and PgR Immunohistochemistry AssaysFitzgibbons et al

Negative Percent Agreement.Refers to percentage of

agreement among specimens that test negative with
the reference assay.23

An example of a concordance study illustrating calculations with positive and negative percentages of agreement
is provided in Table 5.
References
1. Hammond MEH, Hayes D, Dowsett M, et al. American Society of Clinical
Oncology/College of American Pathologists guideline recommendations for
immunohistochemical testing of estrogen/progesterone receptor testing in breast
cancer. Arch Pathol Lab Med. 2010;134(6):907922.
2. Harris L, Fritsche H, Mennel R, et al. American Society of Clinical Oncology
2007 update of recommendations for the use of tumor markers in breast cancer. J
Clin Oncol. 2007;25(33):52875312.
3. Early Breast Cancer Trialists Collaborative Group (EBCTCG). Effects of
chemotherapy and hormonal therapy for early breast cancer on recurrence and
15-year survival: An overview of the randomised trials. Lancet. 2005;365(9472):
16871717.
4. Clark GM, McGuire WL, Hubay CA, Pearson OH, Carter AC: The
importance of estrogen and progesterone receptor in primary breast cancer.
Prog Clin Biol Res. 1983;132E:183190.
5. Ravdin PM, Green S, Dorr TM, et al: Prognostic significance of progesterone
receptor levels in estrogen receptor-positive patients with metastatic breast
cancer treated with tamoxifen: results of a prospective Southwest Oncology
Group study. J Clin Oncol. 1992;10(8):12841291.
6. Visvanathan K, Chlebowski RT, Hurley P, et al. American Society of Clinical
Oncology clinical practice guideline update on the use of pharmacologic
interventions including tamoxifen, raloxifene, and aromatase inhibition for breast
cancer risk reduction. J Clin Oncol. 2009;27(19):32353258.
7. Gown AM. Current issues in ER and HER2 testing by IHC in breast cancer.
Mod Pathol. 2008;21(suppl 2):S8S15.
8. Rhodes A, Jasani B, Balaton AJ, Miller KD. Immunohistochemical demonstration of oestrogen and progesterone receptors: correlation of standards achieved
on in house tumours with that achieved on external quality assessment material in
over 150 laboratories from 26 countries. J Clin Pathol. 2000;53(4):292301.
9. Rhodes A, Jasani B, Barnes DM, Bobrow LG, Miller KD. Reliability of
immunohistochemical demonstration of oestrogen receptors in routine practice:
interlaboratory variance in the sensitivity of detection and evaluation of scoring
systems. J Clin Pathol. 2000;53(2):125130.
10. Rhodes A, Jasani B, Balaton AJ, et al. Study of interlaboratory reliability
and reproducibility of estrogen and progesterone receptor assays in Europe:
documentation of poor reliability and identification of insufficient microwave
antigen retrieval time as a major contributory element of unreliable assays. Am J
Clin Pathol. 2001;115(1): 4458.
11. Leake R, Barnes D, Pinder S, et al. Immunohistochemical detection of
steroid receptors in breast cancer: a working protocol. J Clin Pathol. 2000;53(8):
634635.
12. Harvey JM, Clark GM, Osborne CK, et al. Estrogen receptor status by
immunohistochemistry is superior to the ligand binding assay for predicting
response to adjuvant endocrine therapy in breast cancer. J Clin Oncol. 1999;
17(5):14741481.

Arch Pathol Lab MedVol 134, June 2010

13. Cheang MC, Treaba DO, Speers CH, et al. Immunohistochemical

detection using the new rabbit monoclonal antibody SP1 of estrogen receptor
in breast cancer is superior to mouse monoclonal antibody 1D5 in predicting
survival. J Clin Oncol. 2006;24(36):56375644.
14. Dowsett M, Allred C, Knox J, et al. Relationship between quantitative
estrogen and progesterone receptor expression and human epidermal growth
factor receptor 2 (HER-2) status with recurrence in the Arimidex, Tamoxifen,
Alone or in Combination trial. J Clin Oncol. 2008;26(7):10591065.
15. Mohsin SK, Weiss H, Havighurst T, et al. Progesterone receptor by
immunohistochemistry and clinical outcome in breast cancer: a validation study.
Mod Pathol. 2004;17(12):15451554.
16. Phillips T, Murray G, Wakamiya K, et al. Development of standard
estrogen and progesterone receptor immunohistochemical assays for selection of
patients for antihormonal therapy. Appl Immunohistochem Mol Morphol. 2007;
15(3):325331.
17. Badve SS, Baehner FL, Gray RP, et al. Estrogen- and progesterone-receptor
status in ECOG 2197: comparison of immunohistochemistry by local and central
laboratories and quantitative reverse transcription polymerase chain reaction by
central laboratory. J Clin Oncol. 2008;26(15):24732481.
18. Regan MM, Viale G, Mastropasqua MG, et al. Re-evaluating adjuvant
breast cancer trials: assessing hormone receptor status by immunohistochemical
versus extraction assays. J Natl Cancer Inst. 2006;98(21):15711581.
19. Viale G, Regan MM, Maiorano E, et al. Prognostic and predictive value of
centrally reviewed expression of estrogen and progesterone receptors in a
randomized trial comparing letrozole and tamoxifen adjuvant therapy for
postmenopausal early breast cancer: BIG 1-98. J Clin Oncol. 2007;25(25):
38463852.
20. Viale G, Regan MM, Maiorano E, et al. Chemoendocrine compared with
endocrine adjuvant therapies for node-negative breast cancer: predictive value of
centrally reviewed expression of estrogen and progesterone receptors. J Clin
Oncol. 2008;26(9):14041410.
21. Goldstein NS, Hewitt SM, Taylor CR, et al. Recommendations for
improved standardization of immunohistochemistry. Appl Immunohistochem
Mol Morphol. 2007;15(2):124133.
22. Immunology Branch, Division of Clinical Laboratory Devices, Office of
Device Evaluation. 3.9: manufacturers recommendations for verification of IHC
performance by the user. In: Guidance for Submission of Immunohistochemistry
Applications to the FDA. Center for Devices and Radiological Health, US
Food and Drug Administration; 1998. http://www.fda.gov/MedicalDevices/
DeviceRegulationandGuidance/GuidanceDocuments/ucm094002.htm. Accessed
February 1, 2010.
23. Diagnostic Devices Branch, Division of Biostatistics, Office of Surveillance and Biometrics 4.1: benchmark and study population recommendations.
In: Statistical Guidance on Reporting Results From Studies Evaluating Diagnostic
Tests. Center for Devices and Radiological Health, US Food and Drug Administration; 2007. http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/
GuidanceDocuments/ucm071148.htm. Accessed February 1, 2010.
24. Allred DC, Carlson RW, Berry DA, et al. NCCN Task Force report:
estrogen receptor and progesterone receptor testing in breast cancer by
immunohistochemistry. J Natl Compr Canc Netw. 2009;7(suppl 6):S1S21.
25. Dunnwald LK, Rossing MA, Li CI. Hormone receptor status, tumor
characteristics, and prognosis: a prospective cohort of breast cancer patients.
Breast Cancer Res 2007;9(1):R6.

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