Interview
Immunohistochemistry for Oestrogen
and Progesterone Receptors
Connection: Can oestrogen receptor sta-
tus of invasive carcinoma of the breast be
reliably assessed on core needle biopsy
(CNB)?
In a recent audit, we found agreement be-
tween the oestrogen receptor status as-
sessed on core biopsy and surgical specimen
in 99% of tumours (1). Such a high level of
agreement is possible because of the bimodal
distribution of oestrogen receptor expression
in breast cancers. Most mammary carcino-
mas are either completely negative or show
convincing expression (2,3). We found only
4% of tumours with an H score between 1
and 49. Standardisation of fixation, a sensi-
tive immunohistochemistry method with good
choice of primary antibody, antigen retrieval,
and visualisation methods and controls are vi-
tal to achieving reliable assessment of oestro-
gen receptor status. A weakly positive exter-
nal control is particularly important as the most
frequent problem is failure to detect weakly
positive tumours. Advice about methods that
others have found successful are available
from external quality assurance schemes
such as UK NEQAS (4) and NordiQC (5).
Connection: Can you use CNB to dictate
the patients response to anti-hormonal
therapy?
The major factor predictive of response of an
invasive carcinoma of the breast to hormonal
31 | Connection 2008
Dr. Andrew Lee
Consultant Histopathologist,
Nottingham City Hospital, UK
therapy, such as tamoxifen, is the oestrogen
receptor status. As discussed above, this can
be reliably assessed on core biopsy.
Connection: How about progesterone re-
ceptor status? Is it true that the results are
less reliable for progesterone receptor as-
sessment? Why?
The level of agreement between progesterone
receptor assessment of invasive mammary
carcinomas on core biopsy and surgical speci-
men is consistently less than for oestrogen re-
ceptor. This is true even in centres with excel-
lent agreement for oestrogen receptor status.
A likely explanation is that, although the dis-
tribution of progesterone receptor expression
in breast cancers is bimodal, there is a higher
proportion of weakly positive carcinomas with
heterogeneous staining (3). In another recent
audit, we found 14% of invasive carcinomas
had an H score for progesterone receptor
between 1 and 49 (compared with 4% for
oestrogen receptor).
Connection: Did you evaluate human epi-
dermal growth factor receptor-2 (HER2)
status?
We have not compared HER2 status on core
biopsy and surgical specimens. The literature
is less extensive than for oestrogen recep-
tor, but suggests that reliable assessment
of HER2 can be made on core biopsy. The
level of agreement between core biopsy and
surgical specimens is less than for oestrogen
receptor. This is not surprising as there is
not such a clear cutoff between positive and
negative compared with oestrogen receptor.
Connection: After CNB, how is the tissue
fixed? Is it fixed in 10% neutral-pH, phos-
phate buffered formalin? How long is the
tissue fixed? Are there variations between
laboratorie?
The core biopsies in our hospital are fixed in
4% phosphate-buffered formaldehyde (10%
formalin) for a minimum of eight hours and
processed overnight. There is evidence that
a minimum of six to eight hours fixation is
needed for consistent immunohistochemical
staining for oestrogen receptor (6). There is
variation in the choice of fixative and duration
of fixation between laboratories.
Connection: Is antigen retrieval by micro-
wave the standard method for CNB tis-
sues? Some pathologists use the pressure
cooker method. Why?
The choice of pre-treatment is determined by
the antibody that is being used, so the same
pre-treatment is used for core biopsies and ex-
cision specimens. kn;Heat-mediated antigen
retrieval, either microwave or pressure cooker,
works well for both the oestrogen and proges-
terone receptor antibodies that we use. There
is some evidence that pressure cooking pro-
duces slightly stronger results.
Connection: What is the difference between
the H score and the Allred score? Which is
better? What do you prefer? Why are you
not using the Allred scoring system?
Both methods combine assessment of per-
centage of tumour cell nuclei staining and the
intensity of staining. The H score is the sum
of the percentage of weakly stained cells, the
percentage of moderately stained cells multi-
plied by two, and the percentage of strongly
staining cells multiplied by three. This gives
a range of scores from 0 to 300. The Allred
score is the sum of a score for percentage of
cells staining (no staining = 0, staining in <1%
of cells = 1, 1 to 10% = 2, 10 to 33% = 3, 33
to 67% = 4 and 67% to 100% = 5) and a score
for intensity (absent = 0, weak = 1, moderate
= 2, strong = 3). The possible scores are 0
and 2 to 8.
I prefer the H score as it gives a more detailed
assessment. The Allred score gives too much
emphasis to the weakly positive group: Most
tumours score 0, 7 or 8 (ref 2).
There are other scoring methods. The Quick
score is the sum of a score for percentage of
cells staining (no staining = 0, staining in 1 to
25% of cells = 1, 26 to 50% = 2, 51 to 75% = 3
and 76% to 100% = 4) and a score for intensi-
ty (absent = 0, weak = 1, moderate = 2, strong
= 3). Other alternatives are to use a cutoff of
1% or 10% staining of any intensity.
Connection: Is the H score, the universal
system for the assessment of the intensity
and distribution of positivity on sections
stained for oestrogen receptor (ER)?
There is no universal system for scoring.
Connection: What cutoff should be used
for scoring oestrogen receptors?
There is no consensus on what cutoffs should
be used for scoring hormone receptors.
The chance of response to endocrine therapy
has been studied in patients with metastatic
breast cancer. Tumours with no expression
of oestrogen receptor rarely respond to endo-
crine treatment, and tumours with convincing
expression of oestrogen receptor have a good
chance of responding to such treatment. It is
possible to divide tumours into negative and
positive with significantly different chances of
response as long as the cutoff is somewhere
between the group with no staining and the
bulk of tumours with a convincingly positive
staining. Barnes et al (7) assessed eight dif-
ferent methods of categorising tumours with
between 31 and 81% of tumours classified
as positive; all showed a significant relation-
ship with response to endocrine treatment.
This negative/positive categorisation is an
oversimplification. There is evidence that the
level of expression relates to the chance of
response (7, 8).
There are far fewer data on the choice of cut-
off in the adjuvant setting. Sadly, a large study
by Harvey et al (9) reused frozen samples re-
ferred from other centres that were pulverised
and later fixed with less control over specimen
quality. The high proportion of weakly posi-
tive tumours probably reflect the sub-optimal
methods used
Tumours that are positive for both oestrogen
and progesterone receptor are more likely to
respond to endocrine therapy than tumours
that are oestrogen receptor positive and pro-
gesterone receptor negative. However, the
levels of oestrogen receptor tend to be higher
in the double positive group.
In conclusion, one cannot be dogmatic about
exactly where the cutoff or cutoffs should be.
Large studies in both adjuvant and metastatic
settings are needed. With large studies it may
be possible to give reliable estimates of the
chance of response according to different de-
grees of hormone receptor expression.
Connection: How do laboratories report
their results? Do they just say positive or
negative or report on the actual score (and
the cutoff used)?
With no consensus on the cutoffs that should
be used it is scarcely surprising that there is
wide variation in the way that hormone recep-
tors are reported.
We report the H score and percentage of posi-
tive nuclei and categorise tumours as nega-
tive (H score 0 to 9), weakly positive (H score
10 to 49) and positive (H score 50 to 300). It is
useful to report the score as different cutoffs
are useful in different clinical circumstances.
We tend to use the lower cutoff for decisions
about adjuvant endocrine treatment and the
higher cutoff when considering primary endo-
crine treatment without surgery.
Connection: Is it true that three to six sepa-
rate core needle insertions are needed to
obtain a sufficient sample of breast tissue?
Of these, how many cores do laboratorie
stain for ER/PR? How do they report on the
results, if more than one core is stained?
Do they report each core separately or
just report a positive result, if one of those
cores are stained?
The number of samples varies between cen-
tres. Our routine practice for mass lesions
visible on ultrasound lesions (including most
invasive carcinomas) is to perform one or two
ultrasound-guided core biopsies. These are
usually embedded in one block.
Connection: Which is better: Core or exci-
sion biopsy? Is there a trend towards great-
er accuracy with larger gauge samples in
the CNB procedure?
Core biopsies have more reliable fixation. The
main reason that we assess hormone recep-
tor status on core biopsy is that the result is
available earlier. This is particularly useful,
if primary hormonal therapy is being con-
sidered. Good results can also be achieved
in excision specimens, if attention is paid to
good fixation - we routinely receive all speci-
mens fresh and incise the tumour. One ad-
vantage of surgical specimens is that there
is almost always an internal control present.
Another advantage is that very occasionally a
tumour shows marked variation in expression
of hormone receptors in different areas. Such
heterogeneity of expression may not be ap-
parent in the core biopsy (1).
It is possible to achieve excellent results with
standard 14 gauge needle core biopsy. I am
not aware of any data on larger gauge core
biopsies, but it is unlikely to offer a significant
advantage over standard gauge core biopsy.
Connection: When laboratories get a false-
positive or false-negative result, the impact
for the patient is enormous. What is the rea-
son for the slow progress towards ER/PR
standardisation?
The methods used in many laboratories have
arisen in an ad hoc way. A possible reason for
lack of standardisation is the work required.
Connection 2008 | 32
Connection: Can you comment on the bur-
den in the laboratory, if one changes from
a current ER/PR assay to a standardised
ER/PR assay?
Important decisions in standardising these
assays are the choice of fixation, processing,
pre-treatment, primary antibody and visuali-
sation method. These choices can be helped
by consulting external quality assurance
schemes (4,5), which provide details of which
methods have worked well. As mentioned ear-
lier to the answer to the first question, a weak
positive external control is important. It can
take a while to find a suitable control as only
a few percent of tumours are weakly positive.
There is obviously work involved in ensuring
that a new method works satisfactorily, but
being a member of an external quality assur-
ance scheme can provide helpful support in
both establishing and maintaining a high qual-
ity service. I am fortunate to work in a depart-
ment with an excellent immunohistochemistry
laboratory that is able to do this work. An alter-
native is to use a commercially available stan-
dardised kit. Such kits mean that one does not
have to make the choices mentioned above,
but one must follow the instructions carefully,
and one still has to ensure that the method is
working well.
Connection: What is your opinion regard-
ing the use of ER/PR pharmDxTM Kit as an
aid in the prognosis and management of
breast cancer? How do you compare this
kit to your own method both in relation to
clinical value and ease-of-use? Can kits
standardise ER/PR - since there are so
many methods available?
We compared the staining for oestrogen and
progesterone receptor using the pharmDxTM
Kits and our standard methods, which have
been developed over a number of years. The
results were very similar, but the staining was
slightly stronger with the pharmDxTM method
for oestrogen receptor, which led to a slightly
higher positive rate (1% increase). Both meth-
ods produce satisfactory results for clinical
use. The immunohistochemical methods are
33 | Connection 2008
both straightforward to use. The pharmDxTM
method has fewer steps. However, it does
require pre-treatment with a specific Pascal
pressure cooker, which can take a limited
number of slides per batch and is slower to
use than microwave pre-treatment. Develop-
ing in-house methods requires more work to
validate and to ensure standardisation, but is
cheaper. One suggestion that we would have
to improve the pharmDxTM Kits would be the
addition of a weakly positive control to the
negative and strongly positive ones.
Tests that have evolved over time, such as
immunohistochemistry for hormone receptors,
tend to have a variety of methodologies and
as a consequence are more difficult to stan-
dardise. Nevertheless, the key principles of
validation and standardisation of therapeutic
assays remain paramount. Laboratories with
limited expertise in assay development should
consider the use of standardised kits. Such
kits can improve the immunohistochemical
part of the assay, but the questions of scoring
discussed above still remain.
Acknowledgements: I would like to acknowl-
edge John Ronan who runs our excellent
immunohistochemistry laboratory and other
colleagues in the department who have also
contributed to the audits mentioned above,
including Claire Paish, Shaffiq Gill, Zsolt Hodi
and Ian Ellis.
References
1. Hodi Z, Chakrabarti J, Lee AHS, et al. The reli
ability of assessment of oestrogen receptor
expression on needle core biopsy specimens
of invasive carcinomas of the breast. J Clin Pathol
2007;60:299-302.
2. Collins LC, Botero ML, Schnitt SJ. Bimod-
al frequency distribution of estrogen receptor
immunohistochemical staining results in breast
cancer: an analysis of 825 cases. Am J Clin-
Pathol 2005;123:16-20.
3. Nadji M, Gomez-Fernandez C, Ganjei-Azar P, et
al. Immunohistochemistry of estrogen and pro-
gesterone receptors reconsidered: experi-
ence with 5,993 breast cancers. Am J Clin Pathol
2005;123:21-27.
4. http://www.uknequasicc.ucl.ac.uk
5. http://www.nordiqc.org
6. Goldstein NS, Ferkowicz M, Odish E, et al. Mini-
mum formalin fixation time for consistent estrogen
receptor immunohistochemical staining of
invasive breast carcinoma. Am J Clin Pathol
2003;120:86-92.
7. Barnes DM, Harris WH, Smith P, Millis RR, Rubens
RD. Immunohistochemical determination of
oestrogen receptor: comparison of different
methods of assessment of staining and correla-
tion with clinical outcome of breast cancer
patients. Br J Cancer 1996; 74: 1445-1451
8. Elledge RM, Green S, Pugh R, et al. Estrogen
receptor (ER) and progesterone receptor (PgR),
by ligand-binding assay compared with ER, PgR
and pS2, by immuno-histochemistry in predicting
response to tamoxifen in metastatic breast
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Cancer 2000; 89: 111-117
9. Harvey JM, Clark GM, Osborne CK, et al. Estro-
gen receptor status by immunohistochemistry is
superior to the ligand-binding assay for predict-
ing response to endocrine therapy in breast
cancer. J Clin Oncol 1999:17;1474-81