Estrogen Receptor (ER ) Level but Not Its ER Cx Variant Helps to Predict Tamoxifen Resistance in Breast Cancer

Vol. 10, 5769–5776, September 1, 2004 Clinical Cancer Research 5769

Estrogen Receptor ␤ (ER␤) Level but Not Its ER␤cx Variant Helps to Predict Tamoxifen Resistance in Breast Cancer

؍ Majida Esslimani-Sahla,1,2 total population was positively correlated with ER␤cx (r Joelle Simony-Lafontaine,2 Andrew Kramar,3 0.63, P < 0.001), and was independent of the other param- 2 3 eters. In a multivariate analysis, ER␤ expression was the ؍ ,Roselyne Lavaill, Caroline Mollevi 4 4 most important variable (P 0.001), followed by SBR grade .(0.016 ؍ and MIB-1 (P ,(0.008 ؍ Margaret Warner, Jan-Åke Gustafsson, and (I؉II versus III; P 1 Henri Rochefort To conclude, tamoxifen resistance is associated with 1Endocrinologie mole´culaire et cellulaire des cancers (U 540), Institut classical variables of aggressive tumors (high SBR grade, National de la Sante´et de la Recherche Me´dicale (INSERM), 2 3 proliferation index, and tumor size) but not with node inva- Montpellier, France; Departments of Pathology and Biostatistics, ␤ Cancer Center Val d’Aurelle, Montpellier, France; and 4Department siveness. Low ER level is an additional independent of Medical Nutrition and Biosciences, Karolinska Institute, Novum, marker, better than ER␣ level, to predict tamoxifen resist- Huddinge, Sweden ance.

ABSTRACT INTRODUCTION The antiestrogen tamoxifen, a major endocrine therapy Tamoxifen is one of the first-line adjuvant therapy options of estrogen receptor (ER)-positive breast cancer, is never- in women with ER-positive breast cancer. However, in 30 to theless inefficient in 30 to 40% of cases for unknown rea- 40% of cases, these tumors relapse within 5 years of tamoxifen sons. We retrospectively studied 50 ER-positive primary treatment, which requires the cessation of the regimen and the breast carcinomas. All of the patients had received tamox- initiation of a second-line therapy. The mechanism of tamoxifen ifen as the only adjuvant therapy. They were divided into resistance in ER-positive breast cancer is unknown despite two groups depending on whether they relapsed within 5 extensive studies (1–3). Tamoxifen either is inactive and unable years (16 tamoxifen-resistant cases) or did not relapse within to block the mitogenic effect of estrogen and growth factors or 5 years (34 tamoxifen-sensitive cases). The expression of behaves as an agonist that stimulates the growth of cancer cells total ER␤ protein, and of ER␤cx protein, was estimated and induces growth-associated genes, as shown in different cell anonymously in formalin-fixed, paraffin-embedded tumor lines selected for their ability to grow with this antiestrogen (4, sections, by using specific antibodies and quantifiying nu- 5). This estrogenic effect of tamoxifen can be blocked by pure clear immunostaining with a computer image analyzer. All antiestrogens (6). of the tumors were found to be HER-2/neu-negative by It has been established, however, both in cell lines (7, 8) immunohistochemistry. and in patients (9), that tamoxifen is mostly active in ER- Univariate analysis showed that Scarff-Bloom-Rich- positive breast cancer, and that the assay of ER in cytosol or in ardsson grade modified by Elston (SBR grade; P < 0.001), tumor section is the first predictive marker used in practice to .(guide the clinicians in defining systemic therapy (10 ؍ and MIB-1 proliferation index (P ,(0.042 ؍ tumor size (P 0.02) were significantly higher in tamoxifen-resistant tu- The recent discovery of a second ER, named ER␤ (11), and mors. A low level of total ER␤, whether in percentage of of several of its variants, raised the question of the relative value positive cells or in quantitative immunocytochemical (QIC) of ER␣ and ER␤ in predicting tamoxifen resistance or sensitivity in breast cancer patients. ER␤ binds antiestrogens and their ؍ score, was also associated with tamoxifen resistance (P 0.004). ER␤cx expression and lymph node status were sim- hydroxylated metabolites (12) with a higher affinity than does ilar between the two groups. The expression of ER␤ in the ER␣ (13). Both the full-length ER␤ (ER␤1) and its COOH- terminally truncated splice variant (ER␤cx or ER␤2), which is unable to bind tamoxifen, have been found in breast cancer (14, 15). They are able to act as dominant negative of ER␣ after Received 2/27/04; revised 4/29/04; accepted 5/12/04. heterodimerization (16), but their significance in antiestrogen Grant support: Supported by INSERM, the Ligue Nationale Contre le resistance is controversial. It has been proposed that the action cancer, Comite´De´partemental de l’Herault (to M. Esslimani-Sahla) and of tamoxifen on ER␤ stimulates tumor growth via AP-1 inter- by grants from The Swedish Cancer Society and KaroBio AB (to J-A. actions (17). Conversely, ER␤ could inhibit the agonist activity Gustafsson). of tamoxifen for instance on AF-1, the activating domain of The costs of publication of this article were defrayed in part by the ␣ ␤ payment of page charges. This article must therefore be hereby marked transcription of ER (18, 19). Finally, ER might have no value advertisement in accordance with 18 U.S.C. Section 1734 solely to in predicting tamoxifen efficacy or resistance. indicate this fact. To discriminate among these possibilities, we have quan- Requests for reprints: Henri Rochefort, Endocrinologie mole´culaire et tified anonymously by immunohistochemistry the expression of cellulaire des cancers (U540) INSERM, 60 rue de Navacelles, 34090 ␤ ␤ Montpellier, France. Phone: 33-467043760; Fax: 33-467540598; E- total ER protein and its variant ER cx in 50 archival ER- mail: [email protected]. positive breast carcinomas, which had been treated by tamoxifen ©2004 American Association for Cancer Research. as the only adjuvant therapy, and we have compared their value

in tamoxifen-resistant and tamoxifen-sensitive tumors, defined Table 1 Comparison of clinical and histopathologic characteristics according to the presence or absence of relapse within 5 years of between tamoxifen-resistant and tamoxifen-sensitive patients standard tamoxifen therapy (20). Resistant cases Sensitive cases P value Patients Number 16 34 MATERIALS AND METHODS Age, median (range), y 68 (43–88) 63 (36–80) Ͼ0.05 Patient Selection. The files of 850 patients with primary Menopausal status Ͼ breast carcinomas treated during 1992, at the Val d’Aurelle Pre 3 (18.8%) 4 (11.8%) 0.05 Post 13 (81.3%) 30 (88.2%) Cancer Center in Montpellier, France, were considered for this Therapy study. Patients were selected according to the following criteria: Surgery (a) absence of neoadjuvant therapy; (b) tumor diameter greater Radical 12 (75%) 17 (50%) Ͼ0.05 than 1 cm allowing biochemical assay; (c) ER-positive tumor Conservative 4 (25%) 17 (50%) Ն Radiotherapy 9 (56.3%) 28 (82.2%) 0.082 according to cytosolic radioligand assay ( 10 fmol/mg protein); Tumor (d) adjuvant therapy exclusively by tamoxifen for 5 years (20 Histologic type mg/d); (e) availability of paraffin blocks for analysis; and (f) IDC 14 (87.5%) 31 (93.9%) Ͼ0.05 complete clinical data and sufficient follow-up. The tamoxifen- ILC 2 (12.5%) 2 (6.1%) resistant patients were defined as those patients who recurred SBR grading* 0.001 I 0 (0%) 11 (32.4%) while on adjuvant tamoxifen therapy (up to 5 years). The II 3 (18.8%) 16 (47.1%) tamoxifen-sensitive patients were defined as those patients who III 13 (81.3%) 7 (20.6%) had not recurred while on tamoxifen therapy during 5 years. Tumor size 0.042 T 8 (50%) 17 (50%) Only 50 cases of 850 could be included in this study with 16 1 T 5 (31.3%) 17 (50%) tamoxifen-resistant cases and 34 tamoxifen-sensitive cases. His- 2 T3–T4 3 (18.8%) 0 (%) topathologic grading of tumor was obtained according to Scarff- Cytosolic receptor levels, Bloom-Richardsson (SBR) modified by Elston (21, 22). Nodal fmol/mg status was obtained by histologic analysis of at least eight ER, mean 116.5 122.1 Ͼ0.05 Ͼ axillary nodes. Menopausal status was determined by clinical PgR, mean 107.5 149.5 0.05 Nodal status† and hormonal analysis. Ͼ pN0 7 (43.8%) 21 (61.8%) 0.05 Immunohistochemical Assay. All of the tumor sam- pN1 9 (56.3%) 13 (38.2%) ples were fixed in formalin-alcohol solution and embedded in NOTE. Patient and tumor characteristics are presented as percent- paraffin. The archived breast cancer specimens were studied ages for categorical variables and as means and medians (range) for by immunohistochemistry. The pathologist (ME-S) was continuous variables. P value was obtained by Fisher’s exact test for categorical variables and by two-sample Wilcoxon test for all continu- blinded to the patient characteristics. Immunostaining was Ͻ ␤ ous variables; P 0.05 was considered statistically significant (bold performed with ER antibodies obtained in Dr. J-Å. Gustafs- type). son’s laboratory (Department of Medical Nutrition and Bio- Abbreviations: IDC, invasive ductal carcinoma; ILC, invasive lob- sciences, Karolinska Institute, Novum, Huddinge, Sweden). ular carcinoma. The chicken polyclonal ER␤ 503 IgY antibodies recognize * Histopathologic grading of Scarff-Bloom-Richardsson (SBR) total ER␤ proteins (both full-length ER␤ and its splice vari- modified by Elston. † Histopathologic nodal status: pN , absence of nodal metastasis, ants) and have been previously validated for immunohisto- 0 pN1, metastasis of one or more nodes. chemistry (23, 24), including validation by protein extinction with authentic ER␤ protein (23). The ER␤cx polyclonal antibodies were raised in sheep against the 14-amino-acid peptides of the COOH-terminal region: MKMETLLPEAT- with the automated Dako Autostainer (code no. K0675); and MEQ. Analysis was also performed by ER␣ (clone 6F11, 3Ј,3Ј-diaminobenzidine tetrahydrochloride (DAB) was used Novocastra, United Kingdom), progesterone receptor [PgR as a chromogen. The immunohistochemical procedure for (clone PgR 636, Dako)], Ki67 (clone MIB-1, Dako), and two total ER␤ marker was described previously (23). A similar HER2/neu (c-ErbB2) markers [polyclonal A0485 (Dako, protocol was performed for polyclonal sheep ER␤cx anti- Denmark) and monoclonal CB11 (Novocastra)]. Adjacent body (1:300 dilution), except for the use of an appropriate sections of 5 ␮m each were deparaffinized in xylene and secondary biotinylated antisheep antibody (Santa Cruz Bio- rehydrated with graded EtOH concentrations. Before stain- technology, Santa Cruz, CA). Negative controls were per- ing, a heat epitope retrieval procedure was performed. Sec- formed by the replacement of primary antibody by IgY tions were pretreated by pressure cooking for 15 minutes in nonspecific serum (Nordic, Netherlands) for ER␤, mouse EDTA buffer (pH 7) for ER␤ and ER␤cx, and by waterbath IgG1 nonspecific serum (X0931, Dako) for ER␣, PgR, and for 40 minutes at 95° for the other markers, with citrate MIB-1 markers, with similar protein concentrations. Positive buffer (pH 6) for ER␣, PgR, and HER2/neu, and Tris-EDTA external controls were used in each experiment, sections of buffer (pH 8) for MIB-1. For ER␣ (1:50 dilution), PgR OVCAR cells, pellet-embedded in paraffin, were used for (1:100 dilution), MIB-1 (1:100 dilution), and c-ErbB2 (1:500 ER␤, and a positive breast cancer sample was used for each dilution for polyclonal antibody and 1:800 dilution for CB11 other marker. Adjacent normal breast tissue was also used as antibody), immunohistochemical labeling with the “Dako an internal control for ER␣, PgR, MIB-1, and ER␤.ER␤cx LSABR 2 System-HRP” was performed at room temperature specificity of immunostaining was established by preincubat-

Fig. 1 A, immunohistochemistry in adjacent serial sections of two breast invasive ductal carcinomas: the first, a to c, tamoxifen-sensitive case. A-a, a strong nuclear expression of ER␤ total protein; A-b, nonspecific IgY serum (Nordic); A-c, a low proliferation index (Ki-67, clone MIB-1, Dako); the second, d to e, tamoxifen-resistant case, relapsing after 28 months. A-d, low nuclear ER␤ expression; A-e, high expression of ER␣ (clone 6F11, Novocastra); A-f, high MIB-1 proliferation index. B,ER␤cx-staining specificity in adjacent serial sections of two invasive breast carcinomas, different nuclear ER␤cx immunostaining in invasive cancer cells; B-c, DCIS, nuclear ER␤cx immunostaining in adjacent ductal ,ء ,from those of A. B-a and B-c carcinoma in situ. B-a, blue arrow, nuclear ER␤cx immunostaining in stromal cells; red arrows, nuclear ER␤cx immunostaining in inflammatory cells. B-b and d, staining specificity is evidenced by extinction experiment after adding a 10-fold excess of ER␤cx protein (b) and by using preimmune serum (d). ing the sheep polyclonal ER␤cx antibody with a 10-fold Quantitative Method. Quantification was performed excess of ER␤cx peptide. It was also shown with pre-ad- with a computerized image analyzer (Samba 2005 TITN, Alca- sorbed ER␤cx antiserum (1:2100 dilution), and with preim- tel, Grenoble, France) as described previously (23). Ten to mune sheep serum for ER␤cx (1:5000 dilution). In each twelve microscopic fields (G200) of invasive tumor, represent- ER␤cx experiment, pre-immune sheep serum was used, in ative of all surface cut, were analyzed for ER␣,-␤, and -␤cx and addition to a positive external control (breast cancer tissue for PgR. The highly stained fields were chosen for MIB-1 overexpressing ER␤cx). Archival material of mammary tu- proliferation marker assessment. Results were expressed as the mor recurrence and/or metastasis was obtained for six resist- percentage of nuclear-stained epithelial cells, or as a quantitative ant patients and was analyzed with the same markers. immunocytochemical (QIC) score [(percentage of surface

Table 2 Comparison of immunohistochemical variables in invasive tumors between tamoxifen-resistant and -sensitive patients Resistant cases Sensitive cases P value ER␤ % stained nuclei median (range)* 41.8 (0–86.8) 76.4 (7.5–98.6) 0.004 Ն70% expression 4 (25%) 21 (62%) 0.015 QIC score,† median (range) 12.5 (0–39) 29.5 (1.8–93) 0.006 ER␤cx ‡ % stained nuclei median (range)* 30.4 (1.8–58.2) 32.7 (6.7–67.7) Ͼ0.05 Ͼ30% expression 7 (54%) 17 (52%) Ͼ0.05 QIC score,† median (range) 2.9 (0.2–32) 4.3 (0.9–16) Ͼ0.05 ER␤ϪER␤cx‡ (% stained nuclei), median (range)* 12.4 (3.3–47.3) 33.7 (0.8–75.3) 0.015 ER␣ (% stained nuclei) Median (range)* 31.2 (0.4–87.2) 54.5 (0–91.8) Ͼ0.05 Ͼ50% expression 6 (38%) 18 (53%) Ͼ0.05 ER␣/ER␤ ratio, % stained nuclei, median (range)* 1.01 (0–4.5) 0.63 (0–12.2) Ͼ0.05 PgR, % stained nuclei Median (range)* 13 (0–86.1) 24.5 (0–79.1) Ͼ0.05 Ͼ20% expression 7 (44%) 19 (56%) Ͼ0.05 MIB-1, % stained nuclei Median (range)* 21.9 (0.3–45.7) 7 (0–31.2) 0.020 Proliferation index Ͻ10% (low) 6 (37.5%) 23 (67.6%) 0.01 10–19% (moderate) 1 (6.3%) 6 (17.6%) Ն20% (high) 9 (56.3%) 5 (14.7%) NOTE. P value was obtained by Fisher’s exact test for categorical variables and two-sample Wilcoxon test for all continuous variables; P Ͻ 0.05 was considered statistically significant (bold type). All cases were ERϩ by radioligand and HER2neuϪ * Percentage stained nuclei quantified by computer image analyzer. † QIC score obtained by computer image analyzer by assessment of the percentage of positive nuclei and intensity of staining. ‡ In three resistant cases and one sensitive case, tumor materials were exhausted and ER␤cx analysis was not realized.

stained in epithelial cells) ϫ (mean staining intensity) ϫ 10] RESULTS expressed in arbitrary units (AU). The percentage of nuclear Clinical and Histopathologic Characteristics of Tamox- staining of negative control was usually nil and, when weak, ifen-resistant and Tamoxifen-sensitive Patients. Two groups was subtracted. A semiquantitative method was performed for of patients were compared according to the occurrence of re- c-erbB-2 membrane staining, according to the Dako Hercept lapse within 5 years of tamoxifen therapy. The 16 tamoxifen Test scoring. resistant cases relapsed within a median of 3 years from surgery Statistical Methods. All of the parameters were ana- (range 14–56 months). Among the 34 tamoxifen sensitive cases, lyzed by continuous values and by expression status. Receptor 4 patients relapsed after 80 months, and the other 30 patients status were defined taking as cutoff points the median values were alive and disease free at a median follow up of 9.4 years observed in the 50 ER-positive cases (70% for ER␤, 30% for (range 60–128 months). Most clinical and pathologic character- ER␤cx, 50% for ER␣, and 20% for PgR). Because the sensi- istics were not different in the resistant and sensitive groups tivity of the assay may vary according to the receptors, these (Table 1). The only differences were SBR grading and tumor values may not indicate their relative level in the tumor. Uni- size, which were more elevated in resistant cases. variate analysis comparing resistant and sensitive cases were Immunohistochemical Staining of ER␤ and ER␤cx in performed by Fisher’s exact test for categorical variables and by Resistant and Sensitive Tumors. As shown in Fig. 1A-a, the two-sample Wilcoxon test for all continuous parameters. ER␤ immunoreactivity was detected in the nuclei of invasive The Wilcoxon test and the Spearman correlation coefficient breast cancer cells, where brown staining was totally abolished were used to evaluate the relationship between ER␤ and ER␤cx with an excess of antigen (23). Since the cytoplasmic staining expression with the other parameters. P values Ͻ 0.05 were was not fully abolished, only the nuclear staining was quanti- considered statistically significant. The multivariate analysis fied. The absence of cross-reactivity between ER␤ and ER␣ was carried out in two steps by first introducing all of the antibodies was also confirmed as shown in Fig. 1A-d and -e. immunohistochemical variables in a stepwise backward logistic Nuclear intensity and staining distribution of invasive tumors regression model (25) Significant clinical variables were then were variable according to the patient. Staining distribution was introduced to investigate the relationships with the immunohis- either diffuse in all of the tumor, or was focal and generally tochemical variables. Statistical significance was measured by localized in tumoral islets at the periphery of the tumor. Fig. 1A the likelihood ratio test. Odds ratios were used to summarize the shows a typical example of a tamoxifen-sensitive (Fig. 1A- a, -b, effects. Statistical analyses were performed with Stata software and -c) and tamoxifen-resistant (Fig. 1A-d, -e, and -f) invasive (StataCorp, College Station, TX; ref. 26). ductal breast carcinoma, with similar SBR grade (II) and size

(pT2) and without nodal invasiveness. In the tamoxifen-sensitive case, there was a strong expression of ER␤ (Fig. 1A-a), with a low MIB-1 proliferation rate (Fig. 1A-c). The tamoxifen- resistant case showed a low ER␤ expression (Fig. 1A-d) con- trasting with a high ER␣ level (Fig. 1A-e), and high MIB-1 staining (Fig. 1A-f). ER␤ nuclear staining was also detected in epithelial and myoepithelial cells of normal mammary glands, in stromal and inflammatory cells. ER␤cx immunostaining is shown in Fig. 1B. The nuclear staining mostly observed in cancer cells (Fig. 1B-a and -c) contrasted with a weak cytoplasmic staining. Nuclear staining was also observed in some stromal endothelial cells and inflammatory cells such as lymphocytes and macrophages (Fig. 1B-a). The specificity of the ER␤cx immunostaining was evidenced by three criteria: (a) nuclear signal was removed by adding a 10-fold excess of the antigen (Fig. 1B-b), (b) nuclear signal was removed by using an ER␤cx pre-adsorbed antiserum (not shown), and (c) nuclear signal was removed by using the preimmune serum (Fig. 1B-d). ER␤cx reactivity varied according to patients (Table 2 and Fig. 3). As shown in Table 2, total ER␤ level was significantly higher in sensitive tumors than in resistant cases, when comparing the percentage of stained nuclei or QIC score. The same ␤ ␤ difference was found with continuous values (Fig. 2A) or status Fig. 3 Total ER protein and ER cx protein levels were positively correlated (Spearman correlation, r, ϭ 0.63; P Ͻ 0.001) in adjacent expression taking the median as a cutoff level of 70% of stained sections of the same breast cancer. The percentage of stained nuclei for nuclei. Unlike total ER␤,ER␤cx level (either with percentage or ER␤cx was always inferior to that of total ER␤ protein quantified in adjacent sections of the same tumors.

with QIC score) did not differ between resistant and sensitive tumors. The difference between total ER␤ and ER␤cx was significantly higher in sensitive tumors. The difference in ER␣ levels between the two groups was not significant (Fig. 2). However the ER␣-positive tumors (Ն50% of stained nuclei) were mostly seen in tamoxifen-sensitive patients. The ER␣/ER␤ ratio, estimated in adjacent sections of each tumor, and PgR expression were not different between the two groups. The proliferation rate assessed by MIB-1 was greater in the resistant group (P ϭ 0.01), with 63% of resistant cases expressing more than 10% of stained nuclei as compared with 32% of sensitive cases. We found no HER2/neu (c-erbB2) overexpression in any of the 50 tumors, which is consistent for ER-positive tumors. We found no significant variation in ER␤,ER␣, PgR, and MIB-1 levels between the primary tumor and recurrence or metastasis for the same patient, but the number (six cases) was too small to reach a conclusion. ER␤ Correlations With the Other Variables. ER␤ expression was independent of all parameters, including PgR (Table 3). It was correlated only with ER␤cx expression (Spear- man correlation coefficient, r, ϭ 0.63, P Ͻ 0.001). The percent- ␤ ␤ ␣ age of ER -positive cells was always superior to the percentage Fig. 2 ER ,ER protein levels, and MIB-1 proliferation index were ␤ analyzed by immunohistochemical staining and were quantified by of ER cx-positive cells (Fig. 3). Interestingly, MIB-1 was in- computer image analyzer in percentage of positive cells. Significant versely correlated with ER␣ level (P ϭ 0.003) but not with ER␤ differences between tamoxifen-resistant (R) and tamoxifen-sensitive (S) levels. A positive correlation was observed, however, between tumors were evaluated with the two-sample Wilcoxon test. Total ER␤ ER␤ expression and MIB-1 proliferation index in the tamox- protein values were significantly higher in sensitive cases. The differ- ifen-resistant tumors (r ϭ 0.51, P ϭ 0.04), but no relationship ence in ER␣ values between the two groups was not significant. MIB-1 was significantly higher in tamoxifen-resistant tumors. Bars, median was found in the tamoxifen-sensitive group. All 14 patients with values. a low MIB-1 proliferation index (Ͻ10%) and a high ER␤ status

(Ն70%) were tamoxifen sensitive (Fig. 4). ER␤cx expression was associated with total ER␤ expression but was independent of all other variables. Multivariate Analysis and Predictive Variables of Re- sistance to Tamoxifen. In the univariate analysis (Tables 1 and 2), SBR grade was found to be the most discriminant variable between the two groups of resistant and sensitive cases (P ϭ 0.001), followed by ER␤ expression (P ϭ 0.004), MIB-1 proliferation index (P ϭ 0.02), and tumor size (P ϭ 0.042). ER␣ expression and nodal status were not significant. In the multivariate analysis, SBR grade (IϩII versus III), MIB-1 proliferation index, and ER␣ and ER␤ expression were introduced in a multivariate logistic regression model (25) on a continuous scale (Table 4). Tumor size had no predictive value and was not

Table 3 Distribution of ER␤ status as a function of clinicopathologic and immunohistochemical variables Negative ER␤ Positive ER␤ expression expression Variables (Ͻ70%) (Ն70%) P value No. of patients 25 25 Age, median (range), y 64 (43–79) 63 (36–88) Ͼ0.05* Menopausal status Ͼ Pre 4 3 0.05† Fig. 4 A slight positive correlation between total ER␤ protein and Post 21 22 MIB-1 proliferation index in adjacent sections of the same tumors was Therapy found in resistant (R) tumors (r ϭ 0.51; P ϭ 0.04), but not in the Surgery Ͼ sensitive (S) group nor in the overall population. The group with high Radical 13 16 0.05† ER␤ protein levels (Ն70% of stained nuclei) and low MIB-1 prolifer- Conservative 12 9 Ͻ Ͼ ation rate ( 10%) contains almost exclusively tamoxifen-sensitive tu- Radiotherapy 18 19 0.05† mors; P ϭ 0.001, according to Fisher’s exact test. Histologic type IDC 22 23 Ͼ0.05† ILC 3 1 SBR grading ␤ I47Ͼ0.05† included in the model. ER expression was the most important II 10 9 independent variable (P ϭ 0.001), followed by SBR grade (P ϭ III 11 9 0.008) and MIB-1 proliferation index (P ϭ 0.016), whereas Tumor size ER␣ expression was at the limit of statistical significance (P ϭ T 14 11 Ͼ0.05† 1 0.060). According to this model, 43 (86%) of the 50 patients T 913 2 were correctly classified. The sensitivity and specificity were 81 T3–T4 21 Nodal status and 88%, respectively. The positive and negative predictive Ͼ pN0 15 13 0.05† values were 76 and 91%, respectively, assuming a prevalence pN1 10 12 rate of resistance equal to 32% (16 of 50). On the basis of ER␤cx, % Median (range) 17.2 (1.8–58.2) 38.0 (10.9–67.7) 0.002* expression status, the logistic regression model identified SBR Ͼ30% expression 8 (35%) 16 (70%) 0.038† grade (P ϭ 0.003), followed by ER␤ (P ϭ 0.013) and MIB-1 ER␣,% (P ϭ 0.032). ER␣ level was not significant. In a regrouping of Median (range) 44.3 (0.4–91.8) 53.0 (0–84.7) Ͼ0.05* the four variables, grade III tumors with elevated MIB-1 pro- Ͼ Ͼ 50% expression 11 (44%) 13 (52%) 0.05† liferation index and low ER␤ level were at a greater risk for PgR, % Median (range) 14.3 (0–86) 25.6 (0–72) Ͼ0.05* tamoxifen-resistance. Ͼ20% expression 10 (40%) 16 (64%) Ͼ0.05† ER, fmol/mg DISCUSSION Median (range) 79 (28–441) 111 (37–375) Ͼ0.05* Ͼ100 10 (43%) 12 (52%) Ͼ0.05† In addition to classical prognostic variables associated with PgR, fmol/mg aggressive tumors, such as histologic SBR grade and tumor size, Median (range) 52 (0–442) 83 (0–576) Ͼ0.05* the level of ER␤ determined by immunohistochemistry in a Ͼ Ͼ 20 10 (40%) 16 (64%) 0.05† population of ER-positive tumors treated by tamoxifen was MIB-1, % Median (range) 6.2 (0–35.9) 9.5 (0–45.7) Ͼ0.05 found to be the major variable in predicting tamoxifen sensitiv- Ն10% 10 (40%) 11 (44%) Ͼ0.05† ity. ER␣ had a lower value, and ER␤cx had no value. This Note. P Ͻ 0.05 was considered statistically significant (bold type). should clarify the significance of the cytosolic radioligand assay * Wilcoxon test of ER (10) on which most of the clinical studies allowing † Fisher’s exact test. introduction of this marker to predict breast cancer response to

Table 4 Multivariate analysis of predictive factors of tamoxifen was supported by a recent study on 242 breast cancers (33). The resistance reasons for these discrepancies is unknown and could be due to 95% confidence different methods used for quantification and/or different sets of Variables Odds ratio interval P value patients. ER␤ expression* 0.949 0.92–0.98 0.001 Our results do not exclude the involvement of other entities SBR grade (IϩII versus III) 11.881 1.57–89.70 0.008 able to induce tamoxifen resistance, such as an increased ex- MIB-1 proliferation index* 1.108 1.00–1.22 0.016 pression of HER-2/neu (34) and an altered expression of coac- ␣ ER expression* 0.968 0.93–1.00 0.060 tivator (35) or corepressor (36). However they strongly suggest NOTE. The analysis was as described in Materials and Methods that the level of ER␤ in breast epithelial cancer cells contributes Ͻ according to the logistic regression model (25). P 0.05 was consid- better than the level of ER␣ in predicting tamoxifen-sensitivity ered statistically significant (bold type). * Coded as a continuous variable. of breast cancer patients. This should stimulate both large-scale clinical studies before entering ER␤ assay into clinical practice and basic studies to define the biological significance of the association between ER␤ level and tamoxifen responsiveness of antiestrogen therapy were based (9, 10). According to this pilot breast cancer. study, which should be confirmed prospectively on a larger ␤ scale, the assay of ER by immunohistochemistry is better than ACKNOWLEDGMENTS that of ER␣ in guiding the clinician, at least in HER2/neu- We thank Drs. Philippe Rouanet, Bernard Saint-Aubert, Jean Gre- negative tumors. The few studies on the clinical value of ER␤ in nier, Franc¸ois Quenet, and G. Romieu (from the CRLC Val d’Aurelle, terms of prediction of response to tamoxifen have been contro- Montpellier) for supplying clinical data, and Jean-Yves Cance for pre- versial. Our results agree with others reporting an association paring the figures. between ER␤ and response to tamoxifen treatment (27, 28). They disagree. however. with the proposal that ER␤ overexpression is associated with tamoxifen resistance (29), and that REFERENCES the tamoxifen/ER␤ complex increases expression of AP-1- 1. Katzenellenbogen BS. Antiestrogen resistance: mechanisms by which cancer cells undermine the effectiveness of endocrine therapy. controlled genes involved in cell proliferation (17). Whether J Natl Cancer Inst (Bethesda) 1991;83:1433–5. ␤ ER actively protects breast cancer cells against tamoxifen- 2. Osborne C. Tamoxifen in the treatment of breast cancer. N Engl resistance is unknown. One possible mechanism, however, J Med 1998;339:1609–18. could be a dominant-negative effect of ER␤ after heterodimer- 3. Ali S, Coombs CR. Endocrine-responsive breast cancer and strate- ization (16) inhibiting the tamoxifen agonist activity of ER␣ via gies for combatting resistance. Nat Rev Cancer 2002;2:101–12. the AF-1 domain (18, 19). 4. Westley B, May FEB, Brown AMC, et al. Effects of antiestrogens on We have not discriminated between initial and acquired the estrogen-regulated pS2 RNA, and the 52- and 160-kilodalton pro- tamoxifen resistance, the median time for relapse being 3 years; teins in MCF7 cells and two tamoxifen resistant sublines. J Biol Chem 1984;259:10030–5. some resistant cases could be secondary to the selection of 5. Nawata H, Bronzert D, Lippman ME. Isolation and characterization cancer cells stimulated for growth by tamoxifen acting as an of tamoxifen-resistant cell line derived from MCF-7 human breast agonist via ER␣. The four patients who recurred more than 1 cancer cells. J Biol Chem 1981;256:5016–21. year after the 5 years’ therapy were included in the tamoxifen- 6. Howell A, DeFriend DJ, Robertson JF, et al. Pharmacokinetics, sensitive group, with the assumption that these breast cancers pharmacological and anti-tumour effects of the specific anti-estrogen were initially responsive to tamoxifen. When considering these ICI 182780 in women with advanced breast cancer. Br J Cancer 1996; 74:300–8. four patients as tamoxifen resistant, the multivariate analysis ␤ ϭ 7. Coezy E, Borgna JL, Rochefort H. Tamoxifen and metabolites in gave a similar significance for ER expression (P 0.012). MCF7 cells: correlation between binding to estrogen receptor and inhi- Among the classical markers of aggressiveness (SBR bition of cell growth. Cancer Res 1982;42:317–23. grade, tumor size, MIB-1 proliferation), only lymph node inva- 8. Bardon S, Vignon F, Derocq D, Rochefort H. The antiproliferative siveness was not associated with tamoxifen resistance. This is in effect of tamoxifen in breast cancer cells: mediation by the estrogen agreement with studies showing that node-positive tumors re- receptor. Mol Cell Endocrinol 1984;35:89–96. spond as well as node-negative tumors to tamoxifen therapy 9. Systemic treatment of early breast cancer by hormonal, cytotoxic, or (30) and that cancer cells, having migrated to lymph nodes, immune therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75000 women. Early Breast Cancer Trialists’ retain the same antiestrogen responsiveness as the primary Collaborative Group. Lancet 1992;339:1–15, 71–85. tumor. 10. Mc Guire WL. Current status of estrogen receptors in human breast The fact that ER␤cx expression in breast cancer is not cancer. Cancer (Phila) 1975;36:638–44. predictive of tamoxifen resistance in our study, suggests that the 11. Gustafsson JA. Estrogen receptor ␤-a new dimension in estrogen full-length ER␤-1, or another ER␤ variant, may be involved in mechanism of action. J Endocrinol 1999;163:379–83. tamoxifen sensitivity. It is not excluded, however, that ER␤cx 12. Borgna JL, Rochefort H. Hydroxylated metabolites of tamoxifen plays a role in the initial tamoxifen resistance as suggested by are formed in vivo and bound to estrogen receptor in target tissues. J Biol Chem 1981;256:859–68. studies in which tamoxifen responsiveness was evaluated after 3 13. Kuiper GGJM, Carlsson B, Grandien K, et al. Comparison of the months of neo-adjuvant therapy (31). The absence of correlation ligand binding specificity and transcript tissue distribution of estrogen of ER␤ with other classic prognostic parameters further sup- receptor ␣ and ␤. Endocrinology 1997;138:863–70. ports its interest for breast cancer monitoring. The absence of 14. Saunders PTK, Millar MR, Williams K, Macpherson S, Bayne C, correlation with PgR disagrees with other studies (29, 32) but O’Sullivan C, Anderson TJ, Groome NP, Miller WR. Expression of

oestrogen receptor beta (ER␤1) protein in human breast cancer biopsies. 26. StataCorp. Stata Statistical Software: release 7.0. College Station, Br J Cancer 2002;86:250–256. TX: Stat Corporation; 2001. 15. Palmieri C, Cheng GJ, Saji S, et al. Estrogen receptor beta in breast 27. Mann S, Laucirica R, Carlson N, et al. Estrogen receptor ␤ expres- cancer. Endocr-Rel Cancer 2002;9:1–13. sion in invasive breast cancer. Hum Pathol 2001;32:113–8. 16. Pettersson K, Delaunay F, Gustafsson JA. Estrogen receptor ␤ acts 28. Murphy LC, Leygue E, Niu Y, Snell L, Ho SM, Watson PH. as a dominant regulator of estrogen signaling. Oncogene 2000;19: Relationship of coregulator and estrogen receptor isoform expression to 4970–8. de novo tamoxifen resistance in human breast cancer. Br J cancer 17. Paech K, Webb P, Kuiper GGJM, et al. Differential ligand activa- 2002;87:1411–6. tion of estrogen receptors ER␣ and ER␤ at AP-1 sites. Science (Wash 29. Speirs V, Malone C, Walton DS, Kerin MJ, Atkin SL. Increased DC) 1997;277:1508–10. expression of receptor ␤ mRNA in tamoxifen-resistant breast cancer 18. Berry M, Metzger D, Chambon P. Role of the two activating patients. Cancer Res 1999;59:5421–4. domains of the estrogen receptor in the cell-type and promoter-context- 30. Pritchard K. Effects on breast cancer: clinical aspects. In: Lindsay dependent agonistic activity of the antiestrogen 4-hydroxytamoxifen. R, Dempster DW, Jordan VC, editors. Estrogens and antiestrogens. EMBO. J 1990;9:2811–8. Philadelphia: Lippincott-Raven Publishers; 1997. p. 175–210. 19. Delaunay F, Pettersson K, Tujague M, Gustafsson JA. Functional 31. Saji S, Omoto Y, Shimizu C, et al. Expression of estrogen receptor differences between the amino-terminal domains of estrogen receptors ␣ (ER) ␤cx protein in ER␣ positive breast cancer. Specific correlation and ␤. Mol Pharmacol 2000;58:584–90. with progesterone receptor. Cancer Res 2002;62:4849–53. 20. Davidson NE, Levine M. Breast cancer consensus meetings: vive la 32. Skrilis GP, Munot K, Bell SM, et al. Reduced expression of estro- difference. J Clin Oncol 2002;20:1719–20. gen receptor ␤ in invasive breast cancer and its re-expression using 21. Bloom HJG, Richardson WW. Histological grading and prognosis DNA methyltransferase inhibitors in a cell line model. J Pathol 2003; in breast cancer. Br J Cancer 1957;11:359–77. 201:213–20. 22. Elston, C. Grading of invasive carcinoma of the breast. In: Page D, 33. Fuqua SAW, Schiff R, Parra I, et al. Estrogen receptor ␤ protein in Andreson T, editors. Diagnostic histopathology of the breast. Edin- human breast cancer: correlation with clinical tumor parameters. Cancer burgh: Churchill Livingstone; 1987. p. 300–11. Res 2003;63:2434–9. 23. Roger P, Esslimani Sahla M, Makela S, Gustafsson JA, Baldet P, 34. Osborne CK, Bardou V, Hopp TA, et al. Role of the estrogen Rochefort H. Decreased expression of estrogen receptor ␤ protein in pro- receptor coactivator AIB-1 (SRC-3) and HER-2/neu in tamoxifen re- lliferative preinvasive mammary tumors. Cancer Res 2001;61:2537–41. sistance in breast cancer. J Natl Cancer Inst (Bethesda) 2003;95:353–61. 24. Saji S, Jensen EV, Nilsson S, Rylander T, Warner M, Gustafsson 35. Shang Y, Brown M. Molecular determinants for the tissue speci- JA. Estrogen receptors ␣ et ␤ in the rodent mammary gland. Proc Natl ficity of SERMs. Science (Wash DC) 2002;295:2465–8. Acad Sci USA 2000;97:337–42. 36. Jepsen K, Hermanson O, Onami TM, et al. Combinatorial roles of 25. Hosmer DW and Lemeshow SL. Applied logistic regression. In: the nuclear receptor corepressor in transcription and development. Cell John Wiley & Sons, editors. Wiley Interscience. New York; 1989. 2000;102:753–63.

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Majida Esslimani-Sahla, Joelle Simony-Lafontaine, Andrew Kramar, et al.

Clin Cancer Res 2004;10:5769-5776.

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