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1592 Arch Pathol Lab MedVol 127, December 2003 ER/PR Immunohistochemistry in Metastatic Breast CancerNash et al

Table 1. Estrogen Receptor (ER) and ProgesteroneReceptor (PR) Immunoreactivity in Tumors in the

Liver

Tumor Type ER, No. (%) PR, No. (%)

Breast (n 17) 6 (35) 5 (29)Hepatocellular carcinoma

(n 14) 0 1 (7)Biliary (n 16) 0 5 (31)Esophageal/gastric (n16) 0 2 (13)Colorectal (n 14) 0 0

Pancreatic (n 15) 0 1 (6)

graded ethanol solutions to water. All slides were quenched for5 minutes in a 3% hydrogen peroxide solution in methanol to

block for endogenous peroxidase. Antigen retrieval was per-formed by a heat method in which the specimens were placed ina citric acid solution (Target Retrieval Solution, pH 6.1; Dako Cy-tomation, Carpinteria, Calif), for 30 minutes at 94C using a veg-etable steamer. Slides were then placed on a Dako Autostainerfor use with immunohistochemistry and stained with the ER(clone 1D5, 1:50, Dako Cytomation A/S, Glostrup, Denmark)and PR (clone 1A6:1:10, Dako Cytomation, Carpinteria, Calif) an-tibodies. The detection system used was a labeled streptavidin-

biotin complex. This method is based on the consecutive appli-cation of (1) a primary antibody against the antigen to be local-

ized, (2) a biotinylated linking antibody, (3) enzyme-conjugatedstreptavidin, and (4) substrate chromogen (diaminobenzidine).Tissues were avidin and biotin blocked prior to the applicationof the biotinylated secondary reagent. Slides were then counter-stained in Richard Allen hematoxylin, dehydrated through grad-ed ethanol solutions, and cover-slipped. The positive control wasa known ER- and PR-positive infiltrating ductal breast adenocar-cinoma in the liver. For the negative control, nonimmune mouseimmunoglobulin 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 aspositive or negative. Greater than 1% nuclear staining was con-sidered positive. Additionally, the tumors were evaluated on he-matoxylin-eosinstained sections, and each was graded as well,moderately, or poorly differentiated. All slides were reviewed by2 pathologists.

RESULTS

Table 1 shows ER and PR immunoreactivity data for allthe tumors studied. Estrogen receptor immunoreactivitywas seen exclusively in MBA, but only 6 (35%) of 17 casesof MBA demonstrated immunoreactivity with ER. Figure1 shows an MBA that was positive for ER and negativefor PR. Metastatic breast adenocarcinoma, as well as CC,HCC, and metastatic adenocarcinoma from esophagus,stomach, and pancreas, showed immunoreactivity withPR in some cases. Figure 2 shows a poorly differentiatedgastric adenocarcinoma that was positive with PR andnegative with ER. When subdivided according to degreeof differentiation, positive staining with PR was nearly as

frequent in poorly differentiated metastatic adenocarci-nomas (3/16, 19%) as in poorly differentiated MBAs (2/8, 25%; Table 2).

COMMENT

In most cases, the site of origin for tumors in the livercan be determined by clinical history and radiographicfindings. In some instances, the pathologist may be calledon to help determine the primary site for metastatic tu-mors in the liver. The determination of site of origin basedon morphologic findings can be difficult in some cases,particularly with small biopsies. Metastatic breast adeno-

carcinoma commonly involves the liver, and the histologicdistinction of breast adenocarcinoma from other metastat-ic tumors in the liver can be challenging when the patienthas no known history of breast adenocarcinoma, or whena previously diagnosed breast cancer is not available forcomparison. Estrogen receptor and PR immunohistochem-istry has been used by some in clinical practice to establishthe diagnosis of MBA to the liver. Positive staining witheither or both steroid receptor markers may be interpretedby some as consistent with breast origin.

Estrogen and progesterone receptors have been docu-mented in numerous locations throughout the body, in-cluding breast, ovary, uterus, colon, esophagus, stomach,liver, pancreas, skin, pituitary, and bone.2,3,69,13,15 Receptorsin these sites serve a variety of physiologic functions. Es-trogen receptors and PRs have also been documented inmany neoplasms from various locations, including breast,liver, pancreas, colon, ovary, skin adnexa, and uterus.514,16

Recently ERs have been subdivided into and sub-types.2,17 The function of the subtype is unclear, withmost of the physiologic effects of estrogen being attributedto its activity at the receptor. Currently, the immunohis-tochemical stains used are specific for the subtype. Thepresence of physiologic hormone receptors in multiple an-

atomic sites, as well as their expression in different neo-plasms, compounds the difficulty of relying on their pres-ence or absence (as determined by immunohistochemicalmethods) in metastatic lesions to delineate tumor site oforigin.

In breast cancer, the ER/PR status of the tumor is usefulfor both prognosis and therapy, with more chemothera-peutic options available to patients with hormone recep-torpositive tumors.18,19 Breast adenocarcinoma has beenshown to be positive for ER in 24% to 63% of cases andpositive for PR in 9% to 37% of cases. 14,20,21 Breast adeno-carcinoma may demonstrate immunophenotypic variabil-ity in its expression of ER and PR, with differences de-pendent on histologic grade, histologic subtype, antibodyclone applied, and immunohistochemical techniques used.

These factors limit the sensitivity of these markers for ex-cluding MBA in cases of unknown primary site.

We investigated the utility of ER and PR immunohis-tochemical staining in tumors in the liver, including HCC,CC, and metastatic adenocarcinoma from several sites,such as breast, esophagus/stomach, colorectum, and pan-creas. Our series of cases showed excellent specificity ofER immunostaining for MBA when compared to extra-mammary metastatic carcinomas in the liver and HCC re-gardless of the degree of differentiation. However, ER ex-hibited poor sensitivity for MBA, staining only 35% ofthese cases. Progesterone receptor staining was neithersensitive nor specific, and decorated metastatic lesions ofboth breast and nonbreast origin.

The determination of tumor site of origin is the mostchallenging in poorly differentiated tumors; therefore, im-munohistochemistry tends to be used most often in thesecases. In our series of cases, PR staining was seen in poor-ly differentiated tumors of breast, colorectal, esophageal/gastric, and hepatocellular origin. In the HCC cases, noneof the well-differentiated to moderately differentiated tu-mors showed PR staining, while 25% of the poorly differ-entiated cases showed PR positivity. Nearly 50% of theMBAs we examined were poorly differentiated, and ofthese, only 25% showed PR immunoreactivity. Progester-one receptor staining was nearly as frequent in poorly dif-

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Arch Pathol Lab MedVol 127, December 2003 ER/PR Immunohistochemistry in Metastatic Breast CancerNash et al 1593

Figure 1. A, Moderately differentiated breast adenocarcinoma metastatic to the liver (hematoxylin-eosin, original magnification100). B, Nuclearimmunopositivity for estrogen receptor in metastatic breast adenocarcinoma (original magnification 200). C, Metastatic breast adenocarcinomashowing negativity for progesterone receptor (original magnification 200).

Figure 2. A, Poorly differentiated gastric adenocarcinoma metastatic to the liver (hematoxylin-eosin, original magnification 100). B, Poorlydifferentiated gastric adenocarcinoma showing nuclear positivity for progesterone receptor in the majority of cells (original magnification 200).C, Poorly differentiated gastric adenocarcinoma negative for estrogen receptor (original magnification 200).

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1594 Arch Pathol Lab MedVol 127, December 2003 ER/PR Immunohistochemistry in Metastatic Breast CancerNash et al

Table 2. Progesterone Receptor (PR) Immunostainingin Poorly Differentiated Tumors in the Liver

Tumor Type

PoorlyDifferentiated

Tumors,No. (%)

PR Stainingin Poorly

DifferentiatedTumors,No. (%)

Breast (n 17) 8 (47) 2 (25)Hepatocellular carcino-

ma (n 14) 4 (29) 1 (25)

Biliary (n 16) 2 (13) 1 (50)Esophageal/gastric

(n 16) 4 (25) 1 (25)Colorectal (n 14) 1 (7) 0Pancreatic (n 15) 5 (33) 0

ferentiated nonbreast carcinomas (3/16, 19%) as it was inpoorly differentiated MBAs (2/8, 25%). The lack of spec-ificity with PR in poorly differentiated tumors severelylimits the utility of PR as a marker of MBA, particularlyfor those poorly differentiated cases in which this distinc-tion is most important.

Some investigators have concluded that ER and PR stain-

ing in MBA is fairly specific, yet lacks sensitivity.14,2023

However, steroid receptor positivity has been documentedin a number of other nonmammary tumors. As many as28% (30/108) of gastric carcinomas in one study were im-munoreactive with ER.12 In the same study, it was notedthat the predominant grade of gastric carcinoma stainingfor ER was poorly differentiated.12 Colorectal adenocarci-noma has also been shown to mark with ER in up to 46%of cases.10,15 Estrogen receptor and PR immunopositivityin colorectal adenocarcinomas has been shown to posi-tively correlate with higher stage disease.15

In our study, we did not demonstrate ER positivity inany of the 16 esophageal/gastric or 14 colorectal carci-nomas. This is in contrast to the findings of some earlierstudies, which showed 28% and up to 46% ER staining in

gastric and colorectal carcinomas, respectively.10,12,15 Dif-ferences in methodology likely explain some of the ap-parent discrepancies in staining results. Discrepanciesmay be encountered with the use of ER and PR immu-nohistochemistry owing to differences in tissue process-ing/preparation, antigen retrieval techniques, and the an-tibody clone applied. Some variability may be encoun-tered with different histologic grades of tumors. One re-port used radiolabeled receptor-ligand assays to quantifyER cytosolic and/or nuclear positivity,10 while anotherused a different anti-ER antibody clone (H222) than wasused in our study.12 In a study of the expression of ERimmunopositivity of primary lung adenocarcinomas, ERwas 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 anti-body clones that were apparently similar to ours notedsome ER positivity in colorectal carcinomas, whereas wedid not.15 This discrepancy could be due to differences intumor grade. The variability in the findings with differentantibody clones and tumor grades highlights the potentialhazards in the reliance on a single antibody such as ERto be highly specific for MBA.

Immunohistochemistry for ER and PR has been dem-onstrated to be helpful in distinguishing the site of tumor

origin when used together with other markers. Estrogenand progesterone receptors were shown to be helpfulwhen added to a panel of immunomarkers, including thy-roid transcription factor-1 for the distinction of breast ver-sus lung adenocarcinoma in pleural- or pericardial-de-rived effusion specimens.25 The utility of ER and PR hasbeen documented when used in a larger panel of immu-nomarkers, including gross cystic disease fluid protein-15(GCDFP-15), S100 protein, carcinoembryonic antigen, pla-cental alkaline phosphatase, CD15, epithelial membrane

antigen, and cytokeratins (AE1/AE3, MAK-6, CAM 5.2)for distinguishing MBA from primary pulmonary neo-plasms in a study of 30 cases of primary and metastaticadenocarcinoma in the lung.26 For the differentiation ofmetastatic adenocarcinoma to the brain, ER and GCDFP-15 have been shown to be specific, but not sensitive forbreast carcinoma, while PR demonstrated good sensitivityand lacked specificity.23 In a study comparing MBA andother metastatic adenocarcinomas of unknown primarysites, immunostaining for GCDFP-15 in conjunction withER and/or PR positivity allowed greater sensitivity (83%)and specificity (93%) for adenocarcinomas of the breastversus bronchogenic, pancreatic, colonic, gastric, renal,and ovarian adenocarcinomas than ER and/or PR alone.20

Immunohistochemistry can be very useful to help de-termine the site of origin in some liver tumors when pan-els of immunomarkers are used. However, results shouldbe interpreted cautiously and in conjunction with clinicaland radiographic data. Not all MBAs in the liver displayimmunoreactivity with ER and/or PR, and adenocarci-nomas of extramammary origin can show ER and PR pos-itivity. The distinction of poorly differentiated tumors inthe liver creates the greatest diagnostic challenge, and im-munoreactivity with ER and/or PR should not be reliedon in these cases to distinguish site of origin for metastatictumors to the liver.

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