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© 2019 College of American Pathologists

 Review Article
Breast Cancer Biomarkers
Challenges in Routine Estrogen Receptor, Progesterone Receptor, and HER2/neu
Evaluation
Julie M. Jorns, MD
 Context.—Evaluation of estrogen receptor (ER), proges-
terone receptor (PR), and HER2/neu (HER2) biomarkers is
standard of care for all cases of newly diagnosed invasive,
recurrent, and metastatic breast cancer. Repeat analysis is
also performed in select cases per College of American
Pathologists/American Society of Clinical Oncology guide-
lines and other clinical indications. However, in specific
scenarios, preanalytic and analytic variables may pose
distinct challenges to testing.
Objective.—To provide a review of select challenges in
the testing of commonly performed breast cancer bio-
E strogen receptor (ER), progesterone receptor (PR) and
HER2/neu (HER2) status have proven prognostic and
therapeutic significance in patients with breast cancer.1 In
general, good-prognosis, well-differentiated invasive carci-
nomas of luminal A type have the highest levels of ER/PR
expression, often with strong, diffuse tumor cell positivity.
Conversely, HER2-positive tumors are more aggressive and
more frequently higher grade with high proliferative rate.
However, the introduction of HER2-targeted therapy has
resulted in improved outcomes, with 22% to 63% of patients
achieving pathologic complete response after neoadjuvant
chemotherapy (NAC) with HER2-directed therapies.2–8
Additionally, ER/PR and HER2 biomarkers may change
with disease progression and in response to therapy.9–11
Thus, College of American Pathologists (CAP)/American
Society of Clinical Oncology (ASCO) guidelines recommend
ER, PR, and HER2 analysis for all new diagnoses of primary,
recurrent, and metastatic breast cancer.12,13
In current practice, ER and PR are typically assessed by
immunohistochemical (IHC) staining, evaluating protein
expression. Just 1% tumor staining is often deemed positive
because early studies such as that by Harvey et al14 showed
Accepted for publication May 7, 2019.
From the Department of Pathology, Medical College of Wisconsin,
Milwaukee.
The author has no relevant financial interest in the products or
companies described in this article.
Presented in part at the 11th Annual Midwestern Conference:
Update Course in Surgical Pathology; September 14–16, 2018;
Milwaukee, Wisconsin.
Corresponding author: Julie M. Jorns, MD, Department of
Pathology, Medical College of Wisconsin, 9200 W Wisconsin Ave,
Milwaukee, WI 53226 (email: jjorns@mcw.edu).
Arch Pathol Lab Med
markers ER, PR, and HER2 and outline best practices for
overcoming these challenges.
Data Sources.—Review of College of American Pathol-
ogists/American Society of Clinical Oncology recommen-
dations, current literature and personal experience of the
author.
Conclusions.—Attention must be given to specimen
handling to ensure accurate ER, PR, and HER2 biomarker
assessment and appropriate management of breast cancer
patients.
(Arch Pathol Lab Med. doi: 10.5858/arpa.2019-0205-RA)
improved disease-free survival, even at low levels, but with
better disease-free survival correlating with higher expres-
sion. Of note, this study14 focused on patients who received
adjuvant endocrine therapy alone. The 1% threshold for
positivity has been challenged, though, as in the study by
Fujii et al,15 which found that patients with ER/PR low-
positive (1%–9%) (and HER2-negative) tumors who under-
went NAC derived little benefit from adjuvant endocrine
therapy, behaving more like those with triple (ER, PR, and
HER2)–negative tumors. Example cases with variable ER
IHC staining are shown in Figure 1, A through D.
HER2 is assessed by IHC to evaluate protein expression
and/or by in situ hybridization (ISH) methods that evaluate
gene amplification. Current CAP/ASCO guidelines do not
recommend which test to run primarily but recommend
reflexing to the other method when the first provides an
equivocal result.13 However, because of faster turnaround
time and lower cost, most laboratories start with IHC and
reflex to ISH. HER2 reporting has changed in different
iterations of CAP/ASCO guidelines. Immunohistochemical
staining is graded in a semiquantitative manner, with no (0)
to incomplete, faint (1þ) membrane staining being negative;
incomplete and/or weak/moderate (2þ) membrane staining
being equivocal; and complete, intense (3þ) membrane
staining being positive. Current thresholds have established
that more than 10% of tumor cells must have staining for
interpretation in a category (eg, just 5% of tumor cells with
complete, intense membrane staining would constitute a 2þ
equivocal and not a positive result).13 In previous CAP/
ASCO guidelines this threshold was 30% of tumor cells.16
Example cases with variable HER2 IHC staining are shown
in Figure 2, A through D.
Although HER2 ISH can be done by single-probe assay,
currently most laboratories use a dual-probe method,
Challenges in Routine ER, PR, and HER2—Jorns 1
Figure 1. Variation in estrogen receptor staining in invasive breast cancers, ranging from negative (0%) (A) to focal, low positive (1% to 9%) (B),
patchy positive (10%) (C), and diffuse positive (100%) (D) (immunohistochemistry, original magnification 3200).
allowing quantification of HER2 and control (typically
CEP17) signals, with HER2:CEP17 ratio of 2 or greater and
4 or more HER2 copies/cell (group 1) constituting an
amplified or positive result and HER2:CEP17 ratio less than
2 and average HER2 signals/cell less than 4 (group 5)
constituting a nonamplified or negative result. Other HER2
ISH groups 2 (HER2:CEP17 ratio 2 and average HER2
signals/cell ,4), 3 (HER2:CEP17 ratio ,2 and average HER2
signals/cell 6) and 4 (HER2:CEP17 ratio ,2 and average
HER2 signals/cell .4 but ,6) are now interpreted in
combination with IHC to give a definitive positive or
negative classification. Of note, in the typical algorithm of
reflexing only IHC (2þ)–equivocal cases, this would result in
group 3 having positive and group 2 and 4 having negative
overall HER2 status.17 Example amplified/positive (group 1)
and nonamplified/negative (group 5) cases of HER2
fluorescence ISH are shown in Figure 3, A and B.
Current ER, PR, and HER2 CAP/ASCO guidelines have
well-outlined, comprehensive standards for evaluation that
allow laboratories to develop standard protocols that
frequently result in timely, accurate reporting of results.
However, there are relatively common scenarios in which
preanalytic and analytic variables pose challenges to
accurate evaluation.
2 Arch Pathol Lab Med
PREANALYTIC VARIABLES
Warm Ischemia Time
Warm ischemia is the state in which a tumor has
experienced loss of blood supply but remains in the warm
environment of the body and undergoes enzymatic degra-
dation. Prolonged warm ischemia time is more often seen
with difficult, protracted surgical procedures or can be seen
in portions of tumors that undergo rapid enlargement,
where blood supply has been outgrown. Because warm
ischemia is difficult to control or measure, effects of tumor
prolonged warm ischemia time are less understood.
Cold Ischemia Time
Cold ischemia time (CIT) is the time from tumor removal
from the patient until exposure to formalin fixation. Delay to
formalin fixation, or prolonged CIT, has shown to cause
decline in ER, PR, and HER2 in numerous studies, with
increasing possibility for status change, or false-negative
results, with increasing delay to formalin fixation.18–22 For
ER/PR, false negatives secondary to prolonged CIT appear
most likely to occur in tumors with low levels of expression
(Figure 4, A and B).23
The CAP/ASCO optimal handling requirements state that
CIT should be recorded and recommend CIT of 1 hour or
less.12 However, some authors have suggested that CIT of
Challenges in Routine ER, PR, and HER2—Jorns
Figure 2. Variation in HER2/neu immunohistochemical staining in invasive breast cancers, ranging from 0 (A) to 1þ (B) (negative) to 2þ
(equivocal) (C) and 3þ (positive) (D) for overexpression (immunohistochemistry, original magnification 3200).
up to 4 hours may be acceptable, especially if the specimen
is chilled, as biomarker changes are minimal if CIT is within
this timeframe.21,22,24,25
For core biopsy specimens, CIT is usually within minutes,
but optimally should be recorded on the specimen
requisition for quality assurance. For resection specimens,
recording of accurate CIT requires knowledge of 2 specific
time points: excision time, or time of removal from the
patient, and time in formalin, or time that the tumor is
exposed to formalin. Of note, time in formalin is not
accurate when a large specimen is simply placed in a
container of formalin, as tumor within resection specimens
is typically surrounded by a layer of normal breast tissue that
is only very slowly penetrated by formalin. Protocols for
rapid specimen delivery, special triaging or grossing, and/or
injection of the tumor with formalin can minimize CIT.26–28
Despite these protocols, optimal CIT is often more difficult
to achieve for resection specimens because of delays in
specimen delivery from the operating room and handling in
the laboratory.
Fixatives
ER, PR, and HER2 studies are generally validated for use
only with formalin-fixed, paraffin-embedded tissue that has
been fixed in 10% neutral buffered formalin between 6 and
72 hours; when there is any deviation from this, it must at
least be documented.29 Prolonged fixation has been shown
to cause loss of biomarker expression, with the potential for
Arch Pathol Lab Med
false-negative results. Inconsistencies in ER, PR, and HER2
expression have also been seen with formalin fixation of less
than 6 hours.30
In recent years there has also been significant work in
validating and optimizing methods that allow for accurate
evaluation of breast biomarkers using alcohol-based and
dried smears used in cytology.31–33 Additionally, decalcifica-
tion agents, which are notable for their deleterious effects on
fluorescence ISH studies, fortunately only appear to have
modest negative effects on ER, PR, and HER2 IHC.34
However, when a patient has a history of breast cancer
and/or breast cancer metastasis is high on the differential
diagnosis, cytology material can be preferentially reserved
for cell block preparation. Likewise, if a bone metastasis is
favored, there is often tissue that, because of replacement by
carcinoma, can and should be preferentially processed
without decalcification to avoid potential false-negative or
indeterminate (failed test) results.
ANALYTIC VARIABLES
General
Laboratories performing ER, PR, and HER2 studies should
meet accreditation guidelines. Proper optimization of
antibodies and validation should be done prior to perform-
ing patient testing. Quality should be assessed via review of
internal and external controls. External proficiency testing
should also be performed to ensure accurate testing and
Challenges in Routine ER, PR, and HER2—Jorns 3
Figure 3. Example cases of HER2/neu fluorescence in situ hybridization with nonamplified (group 5) (A) and amplified (group 1) (B) results
(original magnification 3600).

interpretation. Additionally, CAP proficiency standards
outline recording of the percentage of ER- and HER2-
positive results, as a comparison with published bench-
marks (eg, having no more than 30% of tumors being ER
negative). Notably, if the laboratory falls outside of
established benchmarks, further investigation may be done
to assess if the deviation is appropriate or inappropriate. For
example, a greater frequency of high-grade, ER-negative
cancers may be appropriate at an institution with a younger,
minority-enriched population.
Controls
When evaluating ER/PR IHC, internal control elements
are often present in primary breast specimens. Internal as
well as external controls should be evaluated alongside all
patient tumors to help ensure appropriate results, especially
when negative. If there is control failure, the assay should be
repeated. In patients with specific well-differentiated inva-
sive carcinomas (Figure 5, A through D), the negative ER/PR
result (or the diagnosis/grade) should always be questioned
and repeat ER/PR and/or additional studies are warranted.
For HER2 studies, there is often no internal positive
control, and external controls are more relied upon. Of note,
high-grade ductal carcinoma in situ is frequently HER2
positive and thus may serve as a positive control. However,
care must be taken to evaluate HER2 in invasive carcinoma,
not ductal carcinoma in situ, which it is frequently
intermixed with, as HER2 status has prognostic and
therapeutic significance only in the setting of invasive
carcinoma.

Figure 4. Example case with conversion of estrogen receptor staining from low positive (A) on biopsy to negative (B) on resection with cold
ischemia time of more than 8 hours (immunohistochemistry, original magnification 3200).

4 Arch Pathol Lab Med Challenges in Routine ER, PR, and HER2—Jorns
Figure 5. Examples of well-differentiated invasive tubular (A), ductal (B), mucinous (C), and lobular (D) breast cancer subtypes, which are nearly
always estrogen receptor positive (hematoxylin and eosin, original magnification 3200).

SPECIFIC SCENARIOS should be consideration of features and primary tumor

Testing Multiple Tumors
In the most recent (2010) CAP/ASCO ER/PR guidelines,
recommendations for patients with multiple synchronous
tumors state that ‘‘testing should be performed on at least
one of the tumors, preferably the largest.’’12 However,
testing is warranted on smaller tumors with different
histology and/or grade, especially if it is likely to result in
a meaningfully different biomarker profile (for which there
Arch Pathol Lab Med
results).35
Retesting Resection Specimens
ER, PR, and HER2 testing can usually be done reliably on
biopsy material, providing valuable information to guide
clinical therapy, especially in the decision to treat with NAC
or to proceed to surgery first. However, retesting of tumor in
resection material is relatively frequently performed. Specific
Challenges in Routine ER, PR, and HER2—Jorns 5
CAP/ASCO indications for repeat testing include biomark-
er/histology discordance, small focus, indeterminate or
equivocal result, and lack of internal control (ER/PR) on
biopsy, among others.12,13
As noted previously, retesting of resection tumors can be
problematic, as false negatives can occur because of
prolonged CIT, especially at low/borderline biomarker
levels, when specimen delivery and triage protocols are
not in place.
Biomarker conversion of ER/PR and/or HER2 after NAC
in patients without pathologic complete response has been
shown to have prognostic significance, with worse overall
survival for patients converting to triple-negative status.36
However, decision to retest in the setting of residual tumor
after NAC should be based on tumor characteristics (pre-
NAC and post-NAC) and multidisciplinary discussion.
DISCUSSION
Accurate ER, PR, and HER2 evaluation is imperative for
appropriate patient diagnosis and management. Detailed
requirements and guidelines for testing help ensure accurate
evaluation. However, certain variables, such as alternate
fixatives/processing techniques, prolonged CIT, and short-
ened or prolonged formalin fixation can result in false-
negative or indeterminate results. Fortunately, understand-
ing of these variables and the scenarios in which they are
likely to occur, as well as coordination between clinical and
laboratory providers, can help circumvent biomarker mis-
classification and ensure accurate patient results.
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