The Utility of Estrogen Receptor and Progesterone

Receptor Immunohistochemistry in the Distinction of

Metastatic Breast Carcinoma From Other
Tumors in the Liver
Jason W. Nash, DO; Carl Morrison, MD; Wendy L. Frankel, MD

Context.The distinction of metastatic breast adenocarcinoma (MBA) to the liver from hepatocellular carcinoma
(HCC), cholangiocarcinoma (CC), and metastatic adenocarcinoma from other sites may require the use of immunohistochemistry. The use of antibodies directed against estrogen receptor (ER) and progesterone receptor (PR) has
been suggested to help make this distinction.
Objective.To examine the utility of ER and PR immunohistochemistry in the distinction of MBA from HCC, CC,
and other metastatic adenocarcinomas in the liver.
Methods.Ninety-two previously characterized hepatic
neoplasms were identified, including HCC (n 5 14), CC (n
5 16), and metastatic tumors from breast (n 5 17), colorectal (n 5 14), pancreatic (n 5 15), and esophageal/gastric (n 5 16) origins. For all cases of metastatic tumor, the
primary tumor was reviewed to verify the diagnosis. All
tumors were graded as well, moderately, or poorly differentiated. Estrogen receptor and PR immunohistochemical
staining was performed on all cases and evaluated by 2
pathologists.

Results.Immunoreactivity for ER was identified only in

MBA, with 6 (35%) of 17 cases positive. Positive immunoreactivity for PR was not restricted to MBA, but was seen
in HCC, CC, esophageal/gastric, and pancreatic metastases. Positive immunostaining with PR was nearly as frequent in poorly differentiated carcinomas of nonbreast origin (3/16 cases, 19%) as in poorly differentiated breast
carcinomas (2/8 cases, 25%).
Conclusion.Progesterone receptor exhibited poor
specificity and sensitivity for the distinction of MBA from
HCC, CC, and other metastatic adenocarcinomas. Estrogen
receptor exhibited poor sensitivity for MBA, although the
specificity was good. The finding that PR positivity was
present with a similar frequency in poorly differentiated
tumors of breast and nonbreast origin limits the usefulness
of this marker. Therefore, ER and PR staining have limited
utility in the distinction of MBA from HCC, CC, and other
metastatic adenocarcinomas in the liver.
(Arch Pathol Lab Med. 2003;127:15911595)

Given the wide distribution of ER and PR in a variety

of neoplastic and nonneoplastic tissues, the utility of these
markers may be limited in distinguishing metastatic
breast adenocarcinoma (MBA) from tumors of nonbreast
origin in the liver. We examined the immunoreactivity
with ER and PR of HCC and metastatic adenocarcinomas
to the liver from various sites, including breast, to evaluate
the usefulness of these stains in determining the site of
origin for tumors in the liver.

he determination of estrogen (ER) and progesterone receptor (PR) status has become part of the standard
workup for breast carcinoma. Information regarding steroid receptor status is used therapeutically and prognostically. Some pathologists in clinical practice have attempted to use immunohistochemistry for ER and PR to determine whether breast is the site of origin for metastatic
adenocarcinoma to the liver.
Estrogen receptors have been documented in nonneoplastic tissues other than breast, including liver, pancreas,
ovary, stomach, skin, and colon.14 Many neoplasms have
been shown to express ERs, including breast carcinoma;
hepatocellular carcinoma (HCC); cerebral meningioma;
nonsmall cell lung carcinoma; papillary thyroid carcinoma; pancreatic, colonic, and gastric adenocarcinoma;
and skin adnexal tumors.414 Progesterone receptors have
also been reported in tissues other than breast.69,13,15
Accepted for publication July 24, 2003.
From the Department of Pathology, The Ohio State University, Columbus.
Reprints: Wendy L. Frankel, MD, Department of Pathology, E-401
Doan Hall, 410 W Tenth Ave, Columbus, OH 43210-1228 (e-mail:
frankel-1@medctr.osu.edu).
Arch Pathol Lab MedVol 127, December 2003

MATERIALS AND METHODS

The archival files of the Department of Pathology, The Ohio
State University, Columbus, were searched, and 92 previously
characterized hepatic neoplasms were identified, including HCC
(n 5 14), cholangiocarcinoma (CC) (n 5 16), and metastatic adenocarcinomas from breast (n 5 17), colorectal (n 5 14), pancreatic (n 5 15), and esophageal/gastric (n 5 16) origins. All breast
adenocarcinomas were of infiltrating ductal type. Consecutive
cases were selected in a retrospective fashion from 1987 to 2000.
For all cases of metastatic tumor, the primary tumor was reviewed to verify the original diagnosis.
Immunoperoxidase staining was performed on formalin-fixed,
paraffin-embedded tissue cut at 4 mm and placed on positively
charged slides. Slides were then placed in a 608C oven for 1 hour,
cooled, and deparaffinized and rehydrated through xylenes and

ER/PR Immunohistochemistry in Metastatic Breast CancerNash et al 1591

COMMENT
In most cases, the site of origin for tumors in the liver
can be determined by clinical history and radiographic
findings. In some instances, the pathologist may be called
on to help determine the primary site for metastatic tumors in the liver. The determination of site of origin based
on morphologic findings can be difficult in some cases,
particularly with small biopsies. Metastatic breast adeno-

carcinoma commonly involves the liver, and the histologic

distinction of breast adenocarcinoma from other metastatic tumors in the liver can be challenging when the patient
has no known history of breast adenocarcinoma, or when
a previously diagnosed breast cancer is not available for
comparison. Estrogen receptor and PR immunohistochemistry has been used by some in clinical practice to establish
the diagnosis of MBA to the liver. Positive staining with
either or both steroid receptor markers may be interpreted
by some as consistent with breast origin.
Estrogen and progesterone receptors have been documented in numerous locations throughout the body, including breast, ovary, uterus, colon, esophagus, stomach,
liver, pancreas, skin, pituitary, and bone.2,3,69,13,15 Receptors
in these sites serve a variety of physiologic functions. Estrogen receptors and PRs have also been documented in
many neoplasms from various locations, including breast,
liver, pancreas, colon, ovary, skin adnexa, and uterus.514,16
Recently ERs have been subdivided into a and b subtypes.2,17 The function of the b subtype is unclear, with
most of the physiologic effects of estrogen being attributed
to its activity at the a receptor. Currently, the immunohistochemical stains used are specific for the a subtype. The
presence of physiologic hormone receptors in multiple anatomic sites, as well as their expression in different neoplasms, compounds the difficulty of relying on their presence or absence (as determined by immunohistochemical
methods) in metastatic lesions to delineate tumor site of
origin.
In breast cancer, the ER/PR status of the tumor is useful
for both prognosis and therapy, with more chemotherapeutic options available to patients with hormone receptorpositive tumors.18,19 Breast adenocarcinoma has been
shown to be positive for ER in 24% to 63% of cases and
positive for PR in 9% to 37% of cases.14,20,21 Breast adenocarcinoma may demonstrate immunophenotypic variability in its expression of ER and PR, with differences dependent on histologic grade, histologic subtype, antibody
clone applied, and immunohistochemical techniques used.
These factors limit the sensitivity of these markers for excluding MBA in cases of unknown primary site.
We investigated the utility of ER and PR immunohistochemical staining in tumors in the liver, including HCC,
CC, and metastatic adenocarcinoma from several sites,
such as breast, esophagus/stomach, colorectum, and pancreas. Our series of cases showed excellent specificity of
ER immunostaining for MBA when compared to extramammary metastatic carcinomas in the liver and HCC regardless of the degree of differentiation. However, ER exhibited poor sensitivity for MBA, staining only 35% of
these cases. Progesterone receptor staining was neither
sensitive nor specific, and decorated metastatic lesions of
both breast and nonbreast origin.
The determination of tumor site of origin is the most
challenging in poorly differentiated tumors; therefore, immunohistochemistry tends to be used most often in these
cases. In our series of cases, PR staining was seen in poorly differentiated tumors of breast, colorectal, esophageal/
gastric, and hepatocellular origin. In the HCC cases, none
of the well-differentiated to moderately differentiated tumors showed PR staining, while 25% of the poorly differentiated cases showed PR positivity. Nearly 50% of the
MBAs we examined were poorly differentiated, and of
these, only 25% showed PR immunoreactivity. Progesterone receptor staining was nearly as frequent in poorly dif-

1592 Arch Pathol Lab MedVol 127, December 2003

ER/PR Immunohistochemistry in Metastatic Breast CancerNash et al

Table 1. Estrogen Receptor (ER) and Progesterone

Receptor (PR) Immunoreactivity in Tumors in the
Liver
Tumor Type

ER, No. (%)

PR, No. (%)

Breast (n 5 17)
Hepatocellular carcinoma
(n 5 14)
Biliary (n 5 16)
Esophageal/gastric (n 5 16)
Colorectal (n 5 14)
Pancreatic (n 5 15)

6 (35)

5 (29)

0
0
0
0
0

1
5
2
0
1

(7)
(31)
(13)
(6)

graded ethanol solutions to water. All slides were quenched for

5 minutes in a 3% hydrogen peroxide solution in methanol to
block for endogenous peroxidase. Antigen retrieval was performed by a heat method in which the specimens were placed in
a citric acid solution (Target Retrieval Solution, pH 6.1; Dako Cytomation, Carpinteria, Calif), for 30 minutes at 948C using a vegetable steamer. Slides were then placed on a Dako Autostainer
for use with immunohistochemistry and stained with the ER
(clone a1D5, 1:50, Dako Cytomation A/S, Glostrup, Denmark)
and PR (clone 1A6:1:10, Dako Cytomation, Carpinteria, Calif) antibodies. The detection system used was a labeled streptavidinbiotin complex. This method is based on the consecutive application of (1) a primary antibody against the antigen to be localized, (2) a biotinylated linking antibody, (3) enzyme-conjugated
streptavidin, and (4) substrate chromogen (diaminobenzidine).
Tissues were avidin and biotin blocked prior to the application
of the biotinylated secondary reagent. Slides were then counterstained in Richard Allen hematoxylin, dehydrated through graded ethanol solutions, and cover-slipped. The positive control was
a known ER- and PR-positive infiltrating ductal breast adenocarcinoma in the liver. For the negative control, nonimmune mouse
immunoglobulin G1 was used in place of the primary antibody.
The positive and negative controls stained appropriately.
Nuclear staining for ER and PR was qualitatively recorded as
positive or negative. Greater than 1% nuclear staining was considered positive. Additionally, the tumors were evaluated on hematoxylin-eosinstained sections, and each was graded as well,
moderately, or poorly differentiated. All slides were reviewed by
2 pathologists.

RESULTS
Table 1 shows ER and PR immunoreactivity data for all
the tumors studied. Estrogen receptor immunoreactivity
was seen exclusively in MBA, but only 6 (35%) of 17 cases
of MBA demonstrated immunoreactivity with ER. Figure
1 shows an MBA that was positive for ER and negative
for PR. Metastatic breast adenocarcinoma, as well as CC,
HCC, and metastatic adenocarcinoma from esophagus,
stomach, and pancreas, showed immunoreactivity with
PR in some cases. Figure 2 shows a poorly differentiated
gastric adenocarcinoma that was positive with PR and
negative with ER. When subdivided according to degree
of differentiation, positive staining with PR was nearly as
frequent in poorly differentiated metastatic adenocarcinomas (3/16, 19%) as in poorly differentiated MBAs (2/
8, 25%; Table 2).

Figure 1. A, Moderately differentiated breast adenocarcinoma metastatic to the liver (hematoxylin-eosin, original magnification 3100). B, Nuclear
immunopositivity for estrogen receptor in metastatic breast adenocarcinoma (original magnification 3200). C, Metastatic breast adenocarcinoma
showing negativity for progesterone receptor (original magnification 3200).
Figure 2. A, Poorly differentiated gastric adenocarcinoma metastatic to the liver (hematoxylin-eosin, original magnification 3100). B, Poorly
differentiated gastric adenocarcinoma showing nuclear positivity for progesterone receptor in the majority of cells (original magnification 3200).
C, Poorly differentiated gastric adenocarcinoma negative for estrogen receptor (original magnification 3200).

Arch Pathol Lab MedVol 127, December 2003

ER/PR Immunohistochemistry in Metastatic Breast CancerNash et al 1593

Table 2. Progesterone Receptor (PR) Immunostaining

in Poorly Differentiated Tumors in the Liver

Tumor Type

Breast (n 5 17)
Hepatocellular carcinoma (n 5 14)
Biliary (n 5 16)
Esophageal/gastric
(n 5 16)
Colorectal (n 5 14)
Pancreatic (n 5 15)

Poorly
Differentiated
Tumors,
No. (%)

PR Staining
in Poorly
Differentiated
Tumors,
No. (%)

8 (47)

2 (25)

4 (29)
2 (13)

1 (25)
1 (50)

4 (25)
1 (7)
5 (33)

1 (25)
0
0

ferentiated nonbreast carcinomas (3/16, 19%) as it was in

poorly differentiated MBAs (2/8, 25%). The lack of specificity with PR in poorly differentiated tumors severely
limits the utility of PR as a marker of MBA, particularly
for those poorly differentiated cases in which this distinction is most important.
Some investigators have concluded that ER and PR staining in MBA is fairly specific, yet lacks sensitivity.14,2023
However, steroid receptor positivity has been documented
in a number of other nonmammary tumors. As many as
28% (30/108) of gastric carcinomas in one study were immunoreactive with ER.12 In the same study, it was noted
that the predominant grade of gastric carcinoma staining
for ER was poorly differentiated.12 Colorectal adenocarcinoma has also been shown to mark with ER in up to 46%
of cases.10,15 Estrogen receptor and PR immunopositivity
in colorectal adenocarcinomas has been shown to positively correlate with higher stage disease.15
In our study, we did not demonstrate ER positivity in
any of the 16 esophageal/gastric or 14 colorectal carcinomas. This is in contrast to the findings of some earlier
studies, which showed 28% and up to 46% ER staining in
gastric and colorectal carcinomas, respectively.10,12,15 Differences in methodology likely explain some of the apparent discrepancies in staining results. Discrepancies
may be encountered with the use of ER and PR immunohistochemistry owing to differences in tissue processing/preparation, antigen retrieval techniques, and the antibody clone applied. Some variability may be encountered with different histologic grades of tumors. One report used radiolabeled receptor-ligand assays to quantify
ER cytosolic and/or nuclear positivity,10 while another
used a different anti-ER antibody clone (H222) than was
used in our study.12 In a study of the expression of ER
immunopositivity of primary lung adenocarcinomas, ER
was noted to be present in many lung adenocarcinomas
when using the 6F11 (Ventana Medical Systems, Tucson,
Ariz) clone, while no ER staining was noted using the 1D5
(Dako) clone.24 One study that used methods and antibody clones that were apparently similar to ours noted
some ER positivity in colorectal carcinomas, whereas we
did not.15 This discrepancy could be due to differences in
tumor grade. The variability in the findings with different
antibody clones and tumor grades highlights the potential
hazards in the reliance on a single antibody such as ER
to be highly specific for MBA.
Immunohistochemistry for ER and PR has been demonstrated to be helpful in distinguishing the site of tumor
1594 Arch Pathol Lab MedVol 127, December 2003

origin when used together with other markers. Estrogen

and progesterone receptors were shown to be helpful
when added to a panel of immunomarkers, including thyroid transcription factor-1 for the distinction of breast versus lung adenocarcinoma in pleural- or pericardial-derived effusion specimens.25 The utility of ER and PR has
been documented when used in a larger panel of immunomarkers, including gross cystic disease fluid protein-15
(GCDFP-15), S100 protein, carcinoembryonic antigen, placental alkaline phosphatase, CD15, epithelial membrane
antigen, and cytokeratins (AE1/AE3, MAK-6, CAM 5.2)
for distinguishing MBA from primary pulmonary neoplasms in a study of 30 cases of primary and metastatic
adenocarcinoma in the lung.26 For the differentiation of
metastatic adenocarcinoma to the brain, ER and GCDFP15 have been shown to be specific, but not sensitive for
breast carcinoma, while PR demonstrated good sensitivity
and lacked specificity.23 In a study comparing MBA and
other metastatic adenocarcinomas of unknown primary
sites, immunostaining for GCDFP-15 in conjunction with
ER and/or PR positivity allowed greater sensitivity (83%)
and specificity (93%) for adenocarcinomas of the breast
versus bronchogenic, pancreatic, colonic, gastric, renal,
and ovarian adenocarcinomas than ER and/or PR alone.20
Immunohistochemistry can be very useful to help determine the site of origin in some liver tumors when panels of immunomarkers are used. However, results should
be interpreted cautiously and in conjunction with clinical
and radiographic data. Not all MBAs in the liver display
immunoreactivity with ER and/or PR, and adenocarcinomas of extramammary origin can show ER and PR positivity. The distinction of poorly differentiated tumors in
the liver creates the greatest diagnostic challenge, and immunoreactivity with ER and/or PR should not be relied
on in these cases to distinguish site of origin for metastatic
tumors to the liver.
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