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Anatomic Pathology / ER Phenotype Is Stable in Breast Cancer

Immunohistochemically Determined Estrogen Receptor Phenotype Remains Stable in Recurrent and Metastatic Breast Cancer Carmen Gomez-Fernandez, MD,1 Yahya Daneshbod, MD,2 Mehdi Nassiri, MD,1 Clara Milikowski, MD,1 Consuelo Alvarez, MD,1 and Mehrdad Nadji, MD1 Key Words: Estrogen receptor; Immunohistochemistry; Breast cancer; Metastatic breast carcinoma DOI: 10.1309/AJCPD1AO3YSYQYNW

Abstract We evaluated the estrogen receptor (ER) phenotype of recurrent and/or metastatic breast cancers and compared it with the ER status of the primary tumor in 278 cases. All patients had undergone surgical excision of the primary tumor, followed by observation only or treatment modalities that included radiation and/or chemotherapy or hormonal therapy. The immunohistochemical expression of ER was evaluated by using monoclonal antibody ER-1D5 and heatinduced antigen retrieval. At diagnosis, 165 patients had locoregional disease and 7 had distant metastases. Local recurrences and/or distant metastases occurred from 2 months to 21 years after the diagnosis of the primary tumor. Overall, 159 primary tumors (57.2%) were positive for ER. In 269 cases (96.8%), the ER status of the primary and metastatic tumors was the same. In 9 patients with ER+ primary tumors, the metastases were ER–. There were no ER– primary tumors with ER+ metastases. The time to distant metastasis was significantly longer for ER+ tumors. The immunohistochemically determined ER phenotype of primary breast cancers remains stable in most recurrent and metastatic disease.

© American Society for Clinical Pathology

The question of whether locoregional recurrences or distant metastases of breast cancer represent selective clones of tumor cells with phenotypes that render growth advantage has not been adequately answered. The estrogen receptor (ER) is expressed by approximately two thirds of newly diagnosed breast cancers. It remains unclear whether the ER phenotype of primary breast tumors undergoes a natural biologic drift or is altered as a direct consequence of therapy in recurrent and metastatic disease. Discordant results for ER have been described between primary and metastatic breast cancers.1-4 Acquired resistance to endocrine therapy has been variably attributed to loss of the ER.4 However, the lack of a uniform, standardized method for the assessment of ER may have contributed to the discrepancies observed between the ER content of primary and metastatic breast cancers. In this study, we retrospectively reviewed the expression of ER in primary breast carcinomas, axillary lymph nodes, locoregional recurrences, and distant metastases from 278 patients, using the monoclonal antibody ER-1D5 and formalin-fixed, paraffin-embedded tissue samples.

Materials and Methods We retrieved 278 primary infiltrating mammary carcinomas with their respective axillary lymph node dissection, locoregional recurrence, and distant metastases specimens from the surgical pathology files of the University of Miami, Jackson Memorial Hospital, Miami, FL. Cases were selected for the presence of local recurrence and/or distant metastases. All patients had undergone surgical excision of the primary breast carcinomas with axillary node dissections. Follow-up

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Am J Clin Pathol 2008;130:879-882 DOI: 10.1309/AJCPD1AO3YSYQYNW

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included observation only or various combinations of hormonal therapy, radiation therapy, and chemotherapy. All specimens were fixed in 10% neutral buffered formalin for 12 to 48 hours and processed by conventional overnight processing. Slides were reviewed, and the presence of invasive carcinoma was confirmed in all cases. For primary breast carcinomas, blocks of tumor that contained normal or nonneoplastic mammary epithelium were preferentially selected. Immunohistochemical analysis for ER was performed on 3- to 4-µm paraffin sections using the monoclonal antibody ER-1D5, heat-induced antigen retrieval, and the L-SAB detection system (DAKO, Carpinteria, CA). The stepwise procedure is summarized in zTable 1z. Immunohistochemically stained slides were evaluated for the presence of a positive nuclear reaction. Because a positive reaction for ER-1D5 is typically homogeneous and diffuse, scoring for the percentage of positive cells was not necessary. The intensity of positive nuclear reactions was evaluated against the reaction in respective internal control samples whenever available or the external positive control sample. Negative reactions for ER were validated against the positive reaction of the internal control sample in mammary epithelium whenever possible.

Results All 278 patients had primary breast carcinomas with local recurrence and/or distant metastatic disease. At diagnosis, 165 patients had positive axillary lymph nodes and 7 had distant metastases. Locoregional recurrence in the ipsilateral breast, chest wall, and skin occurred from 2 months to 7 years later. Distant metastases in bone, brain, female genital tract, gastrointestinal tract, kidney, liver, lung, gallbladder, and serosal surfaces occurred up to 21 years after the primary diagnosis. In 269 patients (96.8%), the ER status of the primary and metastatic tumors remained the same, ie, diffusely positive

or completely negative. Of the primary breast tumors, 159 (57.2%) were positive for ER. The immunohistochemical reaction for ER was generally diffuse and uniform. Positive intranuclear staining was observed in more than 90% of the tumor cells. In primary breast carcinomas, the intensity of the positive reaction was equal to or greater than that of the adjacent nonneoplastic mammary epithelial cells. Variation in the intensity of the reaction among tumor cells was minimal. In sections of tumor that had been inadequately fixed, a gradation of staining intensity was observed, with the periphery of the tissue section demonstrating a stronger reaction for ER, relative to the center of the section. For 150 ER+ primary tumors, there was complete concordance with the expression of ER in the lymph nodes and recurrent and/or metastatic disease. In the remaining 9 ER+ primary tumors, the metastases (8 in axillary lymph nodes and 1 in bone) were ER–. However, owing to the lack of internal control at these sites, we were unable to determine if the metastases were truly ER– or represented false-negative results owing to inadequate fixation. All 119 ER– breast cancers had concordant results in nodal, recurrent, and/or metastatic disease samples. There were no ER– primary tumors with ER+ metastases zTable 2z. The mean time to distant metastases was significantly longer for ER+ than for ER– tumors (7.2 vs 2.6 years, respectively; P < .01; 2-tailed t test) zTable 3z.

Discussion The expression of ER is routinely assayed in all newly diagnosed breast cancers. The presence of ER predicts an increased likelihood of initial response to endocrine therapy. Acquired resistance to endocrine therapy, in particular to tamoxifen, has been attributed by some to a drift in the ER phenotype as the tumor progresses. Reports of loss of ER expression in recurrent and metastatic breast cancer

zTable 1z Stepwise Procedure for Estrogen Receptor Staining in This Study* 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. *

Cut paraffin sections at 3.0-4.0 µm. Melt paraffin by placing slides in a 58°C oven for 30 minutes or a 37°C oven overnight; dewax in xylene. Rehydrate slides in decreasing ethanol grades. Block endogenous peroxidase by a 6% solution of hydrogen peroxide in water (3.0 min, room temperature). Place slides in target retrieval solution (catalog No. S1699, DAKO, Carpinteria, CA) and heat at 90°C in a vegetable steamer for 10 min. Block endogenous biotin by the biotin-blocking reagent (catalog No. X0590, DAKO). Incubate with primary antibodies, ER-1D5 (dilution 1:25), 22 minutes at room temperature (DAKO). Add the linking solution, biotinylated antimouse antibody, incubate for 22 minutes (catalog No. K0690, DAKO). Add streptavidin-peroxidase conjugate and incubate for 22 min (catalog No. K0690, DAKO). Place slides in diaminobenzidine solution for 10 min (catalog No. K3468, DAKO). Apply 1% cupric sulfate (1.0 min, room temperature) to intensify nuclear signal; counterstain with 0.2% fast green (2.0 s). Dehydrate in increasing grades of ethanol, clear in xylene, and mount.

All washes and dilutions are made with tris(hydroxymethyl)aminomethane-buffered saline (S1968, DAKO). Steps 6 through 10 are carried out in an automated instrument (Autostainer Plus, DAKO).

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have lent support to the concept that acquired resistance to tamoxifen may be secondary to the selection of ER– clones in disease progression.1,3,4 Before ascribing reported changes in ER status to inherent biologic changes in breast carcinomas, the potential variables that may influence the measurement of ER should be considered. Initial reports of phenotypic drift of the ER were based on dextran charcoal assays of fresh tumor tissue that required unoccupied ligand binding domains of the ER protein for accurate measurement.1-3 ER+ breast carcinomas were reported to convert to ER– phenotypes following tamoxifen therapy, whereas progesterone receptor (PR) expression remained the same.5 It soon became evident, however, that an ER– result on the recurrent or metastatic ER+ tumor represented a false-negative result owing to occupancy of the ligand-binding domain of the ER by tamoxifen.5-7 Even with the clarification of the confounding effects of tamoxifen therapy, the concept of an ER+ tumor converting to a more aggressive ER– phenotype still exists. The current standard of care includes the use of immunohistochemical analysis in routinely fixed paraffin-embedded tissue samples for the detection of ER in breast cancer. Lack of standardization of the technique exists in part because of various preanalytic factors, including type and duration of fixation and the use of a number of different antibodies for the detection of the receptor protein. Much is known today about the structure and function of the ER protein. Different antibodies react against different portions or epitopes of the ER protein, and, hence, familiarity with the antibodies used for the immunohistochemical assessment of ER is essential for understanding their impact on test results. ER proteins exist as 2 isoforms, α and β, with relatively similar structures but different functions. Although both are expressed in normal mammary tissue, it is the ER-α isoform that has the critical role in malignant transformation and progression. Human ER-β is somewhat homologous to ER-α in the ligand-binding domain. However, the N-terminal regions that contain the ligand-independent functions of these 2 proteins are quite distinct.8,9 As a consequence, some antibodies against the ER may recognize ER-α but not ER-β, and others may recognize both. For example, the ER antibody 6F11 reacts against ER-α and ER-β and, thus, detects ER in lung cancers that may express high levels of ER-β.10 The monoclonal antibody ER-1D5, on the other hand, is a mouse monoclonal antibody that reacts with the A/B region of the N-terminal domain specific to ER-α. Lack of reactivity of this antibody with ER-β makes it ideal for the distinction of metastatic ER+ breast carcinoma in lung from a primary lung cancer. The existence of splice variants of the ER-α protein is well documented.8,11,12 The most common form of the truncated ER protein lacks the ligand-binding domain but maintains the well-characterized, constitutively active, ligand-independent © American Society for Clinical Pathology

zTable 2z Correlation Between ER Status of Primary Breast Cancer and Locoregional Recurrences and Distant Metastases

Recurrence and Metastasis

Primary Breast Carcinoma

ER+ (n = 159) ER– (n = 119)

ER+ 150 0

ER– 9 119

ER, estrogen receptor.

zTable 3z Relationship Between Primary ER Status and Time to Metastasis ER Status

Time to Distant Metastasis (mean) (y)

ER+ ER–

1-21 (7.2) 2-4 (2.6)

ER, estrogen receptor.

transcriptional domain located in the N terminus. This variant protein is detectable with ER-1D5, whereas it may be missed with assays that require an intact ligand-binding domain. It is possible that continued use of tamoxifen and/or biologic drift selects for tumor cells with these variant ER proteins that are present but undetectable with antibodies that require an intact tertiary structure. Immunohistochemical analysis detects only the presence of the ER protein and not its function. A truncated ER protein may still be biologically present and even functional but immunohistochemically undetectable, depending on the antibody used. This phenomenon may, in part, explain the negative ER results observed with some ER antibodies in tumors that express PR. With ER-1D5, we have yet to encounter an ER–, PR+ breast cancer in well-fixed tissue.13 The epitope identified by ER-1D5 is relatively sensitive to fixation, and, hence, false-negative results owing to inadequate fixation and/or decalcification may occur.13 This may be difficult to assess in metastatic foci because there are no internal positive control cells to be evaluated. With the antibodies that recognize epitopes that are less fixation-sensitive, this may not be a major issue. For example, the rabbit monoclonal anti-ER antibody SP1 is reported to have an affinity for the ER protein that is 8 times higher than that of ER-1D5 and may, therefore, be more sensitive, particularly in cases in which epitope fixation is compromised.14 As a measure of the presence or absence of the ER protein, the use of ER-1D5 is of great value in the detection of recurrent and/or metastatic breast cancer. The immunophenotypic concordance for the expression of ER, regardless of therapy, can be quite helpful, in particular for cases in which the morphologic changes following therapy

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make the comparison between the primary tumor and the metastasis more challenging. The phenotypic expression of ER as a stable marker for breast cancer can also be used as a diagnostic tool of great value for patients with metastatic disease from unknown primary tumors. Although the presence of ER predicts an increased likelihood of response to hormonal therapy, its immunohistochemical expression merely reflects the presence and not the function of the receptor protein or the likelihood of response to therapy. Until a more biologically meaningful predictor of response to endocrine therapy for breast cancer is found, immunohistochemical analysis for ER remains the “best available” test. Finally, the time to metastasis observed for patients with ER+ tumors was greater than that for patients with ER– tumors. The ER+ phenotype remained stable for patients who had metastasis up to 21 years after the diagnosis of the primary tumor. Patients who experienced late recurrence were more likely to have tumors initially phenotypically positive for ER. Thus, an ER– tumor in a patient with a remote history of ER+ breast cancer should suggest a second primary event. From the 1Department of Pathology, University of Miami Miller School of Medicine, Miami, FL; and 2Daneshbod Pathology Laboratories, Shiraz, Iran. Address reprint requests to Dr Nadji: Dept of Pathology, University of Miami Miller School of Medicine, Miami, FL 33136; [email protected].

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3. Hull DF, Clark GM, Osborne CK, et al. Multiple estrogen receptor assays in human breast cancer. Cancer Res. 1982;43:413-416. 4. Kuukasjarvi T, Kononen J, Helin H, et al. Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy. J Clin Oncol. 1996;14:2584-2589. 5. Johnston SRD, Saccani-Jotti G, Smith E, et al. Changes in estrogen receptor, progesterone receptor, and pS2 expression in tamoxifen-resistant human breast cancer. Cancer Res. 1995;55:3331-3338. 6. Encarnacion CA, Ciocca DR, McGuire WL, et al. Measurement of steroid hormone receptors in breast cancer patients on tamoxifen. Breast Cancer Res Treat. 1993;26:237-246. 7. Robertson JFR. Oestrogen receptor: a stable phenotype in breast cancer. Br J Cancer. 1996;73:5-12. 8. Osborne CK. Steroid receptors in breast cancer management. Breast Cancer Res Treat. 1998;51:227-238. 9. Sommer S, Fuqua SAW. Estrogen receptor and breast cancer. Semin Cancer Biol. 2001;11:339-352. 10. Dabbs DJ, Landreneau RJ, Liu Y, et al. Detection of estrogen receptor by immunohistochemistry in pulmonary adenocarcinoma. Ann Thorac Surg. 2002;73:403-406. 11. McGuire WL, Chamness GC, Fuqua SAW. Estrogen receptor variants in clinical breast cancer. Mol Endocrinol. 1991;5:1571-1577. 12. Fuqua SAW, Wolf DM. Molecular aspects of estrogen receptor variants in breast cancer. Breast Cancer Res Treat. 1995;35:233-241. 13. Nadji M, Gomez-Fernandez C, Ganjei-Azar P, et al. Immunohistochemistry of estrogen and progesterone receptors reconsidered: experience with 5,993 breast cancers. Am J Clin Pathol. 2005;123:21-27. 14. Huang Z, Zhu W, Szekeres G, et al. Development of new rabbit monoclonal antibody to estrogen receptor. Appl Immunohistochem Mol Morphol. 2005;13:91-95.
