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
Endocrinologie moléculaire et cellulaire des cancers (U 540), Institut
National de la Santé et de la Recherche Médicale (INSERM),
Montpellier, France; Departments of 2Pathology and 3Biostatistics,
Cancer Center Val d’Aurelle, Montpellier, France; and 4Department
of Medical Nutrition and Biosciences, Karolinska Institute, Novum,
Huddinge, Sweden
total population was positively correlated with ER␤cx (r ⴝ
0.63, P < 0.001), and was independent of the other parameters. In a multivariate analysis, ER␤ expression was the
most important variable (P ⴝ 0.001), followed by SBR grade
(IⴙII versus III; P ⴝ 0.008), and MIB-1 (P ⴝ 0.016).
To conclude, tamoxifen resistance is associated with
classical variables of aggressive tumors (high SBR grade,
proliferation index, and tumor size) but not with node invasiveness. Low ER␤ level is an additional independent
marker, better than ER␣ level, to predict tamoxifen resistance.
ABSTRACT
INTRODUCTION
The antiestrogen tamoxifen, a major endocrine therapy
of estrogen receptor (ER)-positive breast cancer, is nevertheless inefficient in 30 to 40% of cases for unknown reasons. We retrospectively studied 50 ER-positive primary
breast carcinomas. All of the patients had received tamoxifen as the only adjuvant therapy. They were divided into
two groups depending on whether they relapsed within 5
years (16 tamoxifen-resistant cases) or did not relapse within
5 years (34 tamoxifen-sensitive cases). The expression of
total ER␤ protein, and of ER␤cx protein, was estimated
anonymously in formalin-fixed, paraffin-embedded tumor
sections, by using specific antibodies and quantifiying nuclear immunostaining with a computer image analyzer. All
of the tumors were found to be HER-2/neu-negative by
immunohistochemistry.
Univariate analysis showed that Scarff-Bloom-Richardsson grade modified by Elston (SBR grade; P < 0.001),
tumor size (P ⴝ 0.042), and MIB-1 proliferation index (P ⴝ
0.02) were significantly higher in tamoxifen-resistant tumors. A low level of total ER␤, whether in percentage of
positive cells or in quantitative immunocytochemical (QIC)
score, was also associated with tamoxifen resistance (P ⴝ
0.004). ER␤cx expression and lymph node status were similar between the two groups. The expression of ER␤ in the
Tamoxifen is one of the first-line adjuvant therapy options
in women with ER-positive breast cancer. However, in 30 to
40% of cases, these tumors relapse within 5 years of tamoxifen
treatment, which requires the cessation of the regimen and the
initiation of a second-line therapy. The mechanism of tamoxifen
resistance in ER-positive breast cancer is unknown despite
extensive studies (1–3). Tamoxifen either is inactive and unable
to block the mitogenic effect of estrogen and growth factors or
behaves as an agonist that stimulates the growth of cancer cells
and induces growth-associated genes, as shown in different cell
lines selected for their ability to grow with this antiestrogen (4,
5). This estrogenic effect of tamoxifen can be blocked by pure
antiestrogens (6).
It has been established, however, both in cell lines (7, 8)
and in patients (9), that tamoxifen is mostly active in ERpositive breast cancer, and that the assay of ER in cytosol or in
tumor section is the first predictive marker used in practice to
guide the clinicians in defining systemic therapy (10).
The recent discovery of a second ER, named ER␤ (11), and
of several of its variants, raised the question of the relative value
of ER␣ and ER␤ in predicting tamoxifen resistance or sensitivity in breast cancer patients. ER␤ binds antiestrogens and their
hydroxylated metabolites (12) with a higher affinity than does
ER␣ (13). Both the full-length ER␤ (ER␤1) and its COOHterminally 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
heterodimerization (16), but their significance in antiestrogen
resistance is controversial. It has been proposed that the action
of tamoxifen on ER␤ stimulates tumor growth via AP-1 interactions (17). Conversely, ER␤ could inhibit the agonist activity
of tamoxifen for instance on AF-1, the activating domain of
transcription of ER␣ (18, 19). Finally, ER␤ might have no value
in predicting tamoxifen efficacy or resistance.
To discriminate among these possibilities, we have quantified anonymously by immunohistochemistry the expression of
total ER␤ protein and its variant ER␤cx in 50 archival ERpositive breast carcinomas, which had been treated by tamoxifen
as the only adjuvant therapy, and we have compared their value
Majida Esslimani-Sahla,1,2
Joelle Simony-Lafontaine,2 Andrew Kramar,3
Roselyne Lavaill,2 Caroline Mollevi,3
Margaret Warner,4 Jan-Åke Gustafsson,4 and
Henri Rochefort1
1
Received 2/27/04; revised 4/29/04; accepted 5/12/04.
Grant support: Supported by INSERM, the Ligue Nationale Contre le
cancer, Comité Départemental de l’Herault (to M. Esslimani-Sahla) and
by grants from The Swedish Cancer Society and KaroBio AB (to J-A.
Gustafsson).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Henri Rochefort, Endocrinologie moléculaire et
cellulaire des cancers (U540) INSERM, 60 rue de Navacelles, 34090
Montpellier, France. Phone: 33-467043760; Fax: 33-467540598; Email: henri.rochefort@montp.inserm.fr.
©2004 American Association for Cancer Research.
5770 ER␤ in Breast Cancer and Tamoxifen Resistance
in tamoxifen-resistant and tamoxifen-sensitive tumors, defined
according to the presence or absence of relapse within 5 years of
standard tamoxifen therapy (20).
MATERIALS AND METHODS
Patient Selection. The files of 850 patients with primary
breast carcinomas treated during 1992, at the Val d’Aurelle
Cancer Center in Montpellier, France, were considered for this
study. Patients were selected according to the following criteria:
(a) absence of neoadjuvant therapy; (b) tumor diameter greater
than 1 cm allowing biochemical assay; (c) ER-positive tumor
according to cytosolic radioligand assay (ⱖ10 fmol/mg protein);
(d) adjuvant therapy exclusively by tamoxifen for 5 years (20
mg/d); (e) availability of paraffin blocks for analysis; and (f)
complete clinical data and sufficient follow-up. The tamoxifenresistant patients were defined as those patients who recurred
while on adjuvant tamoxifen therapy (up to 5 years). The
tamoxifen-sensitive patients were defined as those patients who
had not recurred while on tamoxifen therapy during 5 years.
Only 50 cases of 850 could be included in this study with 16
tamoxifen-resistant cases and 34 tamoxifen-sensitive cases. Histopathologic grading of tumor was obtained according to ScarffBloom-Richardsson (SBR) modified by Elston (21, 22). Nodal
status was obtained by histologic analysis of at least eight
axillary nodes. Menopausal status was determined by clinical
and hormonal analysis.
Immunohistochemical Assay. All of the tumor samples were fixed in formalin-alcohol solution and embedded in
paraffin. The archived breast cancer specimens were studied
by immunohistochemistry. The pathologist (ME-S) was
blinded to the patient characteristics. Immunostaining was
performed with ER␤ antibodies obtained in Dr. J-Å. Gustafsson’s laboratory (Department of Medical Nutrition and Biosciences, Karolinska Institute, Novum, Huddinge, Sweden).
The chicken polyclonal ER␤ 503 IgY antibodies recognize
total ER␤ proteins (both full-length ER␤ and its splice variants) and have been previously validated for immunohistochemistry (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: MKMETLLPEATMEQ. Analysis was also performed by ER␣ (clone 6F11,
Novocastra, United Kingdom), progesterone receptor [PgR
(clone PgR 636, Dako)], Ki67 (clone MIB-1, Dako), and two
HER2/neu (c-ErbB2) markers [polyclonal A0485 (Dako,
Denmark) and monoclonal CB11 (Novocastra)]. Adjacent
sections of 5 ␮m each were deparaffinized in xylene and
rehydrated with graded EtOH concentrations. Before staining, a heat epitope retrieval procedure was performed. Sections were pretreated by pressure cooking for 15 minutes in
EDTA buffer (pH 7) for ER␤ and ER␤cx, and by waterbath
for 40 minutes at 95° for the other markers, with citrate
buffer (pH 6) for ER␣, PgR, and HER2/neu, and Tris-EDTA
buffer (pH 8) for MIB-1. For ER␣ (1:50 dilution), PgR
(1:100 dilution), MIB-1 (1:100 dilution), and c-ErbB2 (1:500
dilution for polyclonal antibody and 1:800 dilution for CB11
antibody), immunohistochemical labeling with the “Dako
LSABR 2 System-HRP” was performed at room temperature
Table 1 Comparison of clinical and histopathologic characteristics
between tamoxifen-resistant and tamoxifen-sensitive patients
Resistant cases Sensitive cases P value
Patients
Number
16
Age, median (range), y
68 (43–88)
Menopausal status
Pre
3 (18.8%)
Post
13 (81.3%)
Therapy
Surgery
Radical
12 (75%)
Conservative
4 (25%)
Radiotherapy
9 (56.3%)
Tumor
Histologic type
IDC
14 (87.5%)
ILC
2 (12.5%)
SBR grading*
I
0 (0%)
II
3 (18.8%)
III
13 (81.3%)
Tumor size
T1
8 (50%)
T2
5 (31.3%)
T3–T4
3 (18.8%)
Cytosolic receptor levels,
fmol/mg
ER, mean
116.5
PgR, mean
107.5
Nodal status†
pN0
7 (43.8%)
pN1
9 (56.3%)
34
63 (36–80)
⬎0.05
4 (11.8%)
30 (88.2%)
⬎0.05
17 (50%)
17 (50%)
28 (82.2%)
⬎0.05
31 (93.9%)
2 (6.1%)
⬎0.05
0.082
0.001
11 (32.4%)
16 (47.1%)
7 (20.6%)
0.042
17 (50%)
17 (50%)
0 (%)
122.1
149.5
21 (61.8%)
13 (38.2%)
⬎0.05
⬎0.05
⬎0.05
NOTE. Patient and tumor characteristics are presented as percentages for categorical variables and as means and medians (range) for
continuous variables. P value was obtained by Fisher’s exact test for
categorical variables and by two-sample Wilcoxon test for all continuous variables; P ⬍ 0.05 was considered statistically significant (bold
type).
Abbreviations: IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma.
* Histopathologic grading of Scarff-Bloom-Richardsson (SBR)
modified by Elston.
† Histopathologic nodal status: pN0, absence of nodal metastasis,
pN1, metastasis of one or more nodes.
with the automated Dako Autostainer (code no. K0675); and
3⬘,3⬘-diaminobenzidine tetrahydrochloride (DAB) was used
as a chromogen. The immunohistochemical procedure for
total ER␤ marker was described previously (23). A similar
protocol was performed for polyclonal sheep ER␤cx antibody (1:300 dilution), except for the use of an appropriate
secondary biotinylated antisheep antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Negative controls were performed by the replacement of primary antibody by IgY
nonspecific serum (Nordic, Netherlands) for ER␤, mouse
IgG1 nonspecific serum (X0931, Dako) for ER␣, PgR, and
MIB-1 markers, with similar protein concentrations. Positive
external controls were used in each experiment, sections of
OVCAR cells, pellet-embedded in paraffin, were used for
ER␤, and a positive breast cancer sample was used for each
other marker. Adjacent normal breast tissue was also used as
an internal control for ER␣, PgR, MIB-1, and ER␤. ER␤cx
specificity of immunostaining was established by preincubat-
Clinical Cancer Research 5771
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
from those of A. B-a and B-c, ⴱ, nuclear ER␤cx immunostaining in invasive cancer cells; B-c, DCIS, nuclear ER␤cx immunostaining in adjacent ductal
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
excess of ER␤cx peptide. It was also shown with pre-adsorbed ER␤cx antiserum (1:2100 dilution), and with preimmune sheep serum for ER␤cx (1:5000 dilution). In each
ER␤cx experiment, pre-immune sheep serum was used, in
addition to a positive external control (breast cancer tissue
overexpressing ER␤cx). Archival material of mammary tumor recurrence and/or metastasis was obtained for six resistant patients and was analyzed with the same markers.
Quantitative Method. Quantification was performed
with a computerized image analyzer (Samba 2005 TITN, Alcatel, Grenoble, France) as described previously (23). Ten to
twelve microscopic fields (G200) of invasive tumor, representative of all surface cut, were analyzed for ER␣, -␤, and -␤cx and
for PgR. The highly stained fields were chosen for MIB-1
proliferation marker assessment. Results were expressed as the
percentage of nuclear-stained epithelial cells, or as a quantitative
immunocytochemical (QIC) score [(percentage of surface
5772 ER␤ in Breast Cancer and Tamoxifen Resistance
Table 2
Comparison of immunohistochemical variables in invasive tumors between tamoxifen-resistant and -sensitive patients
ER␤
% stained nuclei
median (range)*
ⱖ70% expression
QIC score,† median (range)
ER␤cx ‡
% stained nuclei
median (range)*
⬎30% expression
QIC score,† median (range)
ER␤ ⫺ ER␤cx‡ (% stained nuclei), median (range)*
ER␣ (% stained nuclei)
Median (range)*
⬎50% expression
ER␣/ER␤ ratio, % stained nuclei, median (range)*
PgR, % stained nuclei
Median (range)*
⬎20% expression
MIB-1, % stained nuclei
Median (range)*
Proliferation index
⬍10% (low)
10–19% (moderate)
ⱖ20% (high)
Resistant cases
Sensitive cases
P value
41.8 (0–86.8)
4 (25%)
12.5 (0–39)
76.4 (7.5–98.6)
21 (62%)
29.5 (1.8–93)
0.004
0.015
0.006
30.4 (1.8–58.2)
7 (54%)
2.9 (0.2–32)
12.4 (3.3–47.3)
32.7 (6.7–67.7)
17 (52%)
4.3 (0.9–16)
33.7 (0.8–75.3)
⬎0.05
⬎0.05
⬎0.05
0.015
31.2 (0.4–87.2)
6 (38%)
1.01 (0–4.5)
54.5 (0–91.8)
18 (53%)
0.63 (0–12.2)
⬎0.05
⬎0.05
⬎0.05
13 (0–86.1)
7 (44%)
24.5 (0–79.1)
19 (56%)
⬎0.05
⬎0.05
21.9 (0.3–45.7)
6 (37.5%)
1 (6.3%)
9 (56.3%)
7 (0–31.2)
23 (67.6%)
6 (17.6%)
5 (14.7%)
0.020
0.01
NOTE. P value was obtained by Fisher’s exact test for categorical variables and two-sample Wilcoxon test for all continuous variables; P ⬍ 0.05
was considered statistically significant (bold type). All cases were ER⫹ by radioligand and HER2neu⫺
* 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) ⫻ (mean staining intensity) ⫻ 10]
expressed in arbitrary units (AU). The percentage of nuclear
staining of negative control was usually nil and, when weak,
was subtracted. A semiquantitative method was performed for
c-erbB-2 membrane staining, according to the Dako Hercept
Test scoring.
Statistical Methods. All of the parameters were analyzed by continuous values and by expression status. Receptor
status were defined taking as cutoff points the median values
observed in the 50 ER-positive cases (70% for ER␤, 30% for
ER␤cx, 50% for ER␣, and 20% for PgR). Because the sensitivity of the assay may vary according to the receptors, these
values may not indicate their relative level in the tumor. Univariate analysis comparing resistant and sensitive cases were
performed by Fisher’s exact test for categorical variables and by
the two-sample Wilcoxon test for all continuous parameters.
The Wilcoxon test and the Spearman correlation coefficient
were used to evaluate the relationship between ER␤ and ER␤cx
expression with the other parameters. P values ⬍ 0.05 were
considered statistically significant. The multivariate analysis
was carried out in two steps by first introducing all of the
immunohistochemical variables in a stepwise backward logistic
regression model (25) Significant clinical variables were then
introduced to investigate the relationships with the immunohistochemical variables. Statistical significance was measured by
the likelihood ratio test. Odds ratios were used to summarize the
effects. Statistical analyses were performed with Stata software
(StataCorp, College Station, TX; ref. 26).
RESULTS
Clinical and Histopathologic Characteristics of Tamoxifen-resistant and Tamoxifen-sensitive Patients. Two groups
of patients were compared according to the occurrence of relapse within 5 years of tamoxifen therapy. The 16 tamoxifen
resistant cases relapsed within a median of 3 years from surgery
(range 14 –56 months). Among the 34 tamoxifen sensitive cases,
4 patients relapsed after 80 months, and the other 30 patients
were alive and disease free at a median follow up of 9.4 years
(range 60 –128 months). Most clinical and pathologic characteristics were not different in the resistant and sensitive groups
(Table 1). The only differences were SBR grading and tumor
size, which were more elevated in resistant cases.
Immunohistochemical Staining of ER␤ and ER␤cx in
Resistant and Sensitive Tumors. As shown in Fig. 1A-a,
ER␤ immunoreactivity was detected in the nuclei of invasive
breast cancer cells, where brown staining was totally abolished
with an excess of antigen (23). Since the cytoplasmic staining
was not fully abolished, only the nuclear staining was quantified. The absence of cross-reactivity between ER␤ and ER␣
antibodies was also confirmed as shown in Fig. 1A-d and -e.
Nuclear intensity and staining distribution of invasive tumors
were variable according to the patient. Staining distribution was
either diffuse in all of the tumor, or was focal and generally
localized in tumoral islets at the periphery of the tumor. Fig. 1A
shows a typical example of a tamoxifen-sensitive (Fig. 1A- a, -b,
and -c) and tamoxifen-resistant (Fig. 1A-d, -e, and -f) invasive
ductal breast carcinoma, with similar SBR grade (II) and size
Clinical Cancer Research 5773
(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 tamoxifenresistant case showed a low ER␤ expression (Fig. 1A-d) contrasting 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
expression taking the median as a cutoff level of 70% of stained
nuclei. Unlike total ER␤, ER␤cx level (either with percentage or
Fig. 2 ER␤, ER␣ protein levels, and MIB-1 proliferation index were
analyzed by immunohistochemical staining and were quantified by
computer image analyzer in percentage of positive cells. Significant
differences between tamoxifen-resistant (R) and tamoxifen-sensitive (S)
tumors were evaluated with the two-sample Wilcoxon test. Total ER␤
protein values were significantly higher in sensitive cases. The difference in ER␣ values between the two groups was not significant. MIB-1
was significantly higher in tamoxifen-resistant tumors. Bars, median
values.
Fig. 3 Total ER␤ protein and ER␤cx protein levels were positively
correlated (Spearman correlation, r, ⫽ 0.63; P ⬍ 0.001) in adjacent
sections of the same breast cancer. The percentage of stained nuclei for
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 (ⱖ50% 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 ⫽ 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 (Spearman correlation coefficient, r, ⫽ 0.63, P ⬍ 0.001). The percentage of ER␤-positive cells was always superior to the percentage
of ER␤cx-positive cells (Fig. 3). Interestingly, MIB-1 was inversely correlated with ER␣ level (P ⫽ 0.003) but not with ER␤
levels. A positive correlation was observed, however, between
ER␤ expression and MIB-1 proliferation index in the tamoxifen-resistant tumors (r ⫽ 0.51, P ⫽ 0.04), but no relationship
was found in the tamoxifen-sensitive group. All 14 patients with
a low MIB-1 proliferation index (⬍10%) and a high ER␤ status
5774 ER␤ in Breast Cancer and Tamoxifen Resistance
(ⱖ70%) 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 Resistance 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 ⫽ 0.001), followed by ER␤ expression (P ⫽ 0.004), MIB-1
proliferation index (P ⫽ 0.02), and tumor size (P ⫽ 0.042). ER␣
expression and nodal status were not significant. In the multivariate analysis, SBR grade (I⫹II 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
Variables
No. of patients
Age, median (range), y
Menopausal status
Pre
Post
Therapy
Surgery
Radical
Conservative
Radiotherapy
Histologic type
IDC
ILC
SBR grading
I
II
III
Tumor size
T1
T2
T3–T4
Nodal status
pN0
pN1
ER␤cx, %
Median (range)
⬎30% expression
ER␣, %
Median (range)
⬎50% expression
PgR, %
Median (range)
⬎20% expression
ER, fmol/mg
Median (range)
⬎100
PgR, fmol/mg
Median (range)
⬎20
MIB-1, %
Median (range)
ⱖ10%
Negative ER␤
expression
(⬍70%)
Positive ER␤
expression
(ⱖ70%)
P value
25
64 (43–79)
25
63 (36–88)
4
21
3
22
⬎0.05†
13
12
18
16
9
19
⬎0.05†
22
3
23
1
⬎0.05†
4
10
11
7
9
9
⬎0.05†
14
9
2
11
13
1
⬎0.05†
15
10
13
12
⬎0.05†
17.2 (1.8–58.2)
8 (35%)
38.0 (10.9–67.7)
16 (70%)
44.3 (0.4–91.8)
11 (44%)
53.0 (0–84.7)
13 (52%)
⬎0.05*
⬎0.05†
14.3 (0–86)
10 (40%)
25.6 (0–72)
16 (64%)
⬎0.05*
⬎0.05†
79 (28–441)
10 (43%)
111 (37–375)
12 (52%)
⬎0.05*
⬎0.05†
52 (0–442)
10 (40%)
83 (0–576)
16 (64%)
⬎0.05*
⬎0.05†
6.2 (0–35.9)
10 (40%)
9.5 (0–45.7)
11 (44%)
⬎0.05
⬎0.05†
⬎0.05*
⬎0.05†
0.002*
0.038†
Note. P ⬍ 0.05 was considered statistically significant (bold type).
* Wilcoxon test
† Fisher’s exact test.
Fig. 4 A slight positive correlation between total ER␤ protein and
MIB-1 proliferation index in adjacent sections of the same tumors was
found in resistant (R) tumors (r ⫽ 0.51; P ⫽ 0.04), but not in the
sensitive (S) group nor in the overall population. The group with high
ER␤ protein levels (ⱖ70% of stained nuclei) and low MIB-1 proliferation rate (⬍10%) contains almost exclusively tamoxifen-sensitive tumors; P ⫽ 0.001, according to Fisher’s exact test.
included in the model. ER␤ expression was the most important
independent variable (P ⫽ 0.001), followed by SBR grade (P ⫽
0.008) and MIB-1 proliferation index (P ⫽ 0.016), whereas
ER␣ expression was at the limit of statistical significance (P ⫽
0.060). According to this model, 43 (86%) of the 50 patients
were correctly classified. The sensitivity and specificity were 81
and 88%, respectively. The positive and negative predictive
values were 76 and 91%, respectively, assuming a prevalence
rate of resistance equal to 32% (16 of 50). On the basis of
expression status, the logistic regression model identified SBR
grade (P ⫽ 0.003), followed by ER␤ (P ⫽ 0.013) and MIB-1
(P ⫽ 0.032). ER␣ level was not significant. In a regrouping of
the four variables, grade III tumors with elevated MIB-1 proliferation index and low ER␤ level were at a greater risk for
tamoxifen-resistance.
DISCUSSION
In addition to classical prognostic variables associated with
aggressive tumors, such as histologic SBR grade and tumor size,
the level of ER␤ determined by immunohistochemistry in a
population of ER-positive tumors treated by tamoxifen was
found to be the major variable in predicting tamoxifen sensitivity. ER␣ had a lower value, and ER␤cx had no value. This
should clarify the significance of the cytosolic radioligand assay
of ER (10) on which most of the clinical studies allowing
introduction of this marker to predict breast cancer response to
Clinical Cancer Research 5775
Table 4 Multivariate analysis of predictive factors of tamoxifen
resistance
Variables
Odds ratio
ER␤ expression*
SBR grade (I⫹II versus III)
MIB-1 proliferation index*
ER␣ expression*
0.949
11.881
1.108
0.968
95% confidence
interval
P value
0.92–0.98
1.57–89.70
1.00–1.22
0.93–1.00
0.001
0.008
0.016
0.060
NOTE. The analysis was as described in Materials and Methods
according to the logistic regression model (25). P ⬍ 0.05 was considered statistically significant (bold type).
* Coded as a continuous variable.
antiestrogen therapy were based (9, 10). According to this pilot
study, which should be confirmed prospectively on a larger
scale, the assay of ER␤ by immunohistochemistry is better than
that of ER␣ in guiding the clinician, at least in HER2/neunegative tumors. The few studies on the clinical value of ER␤ in
terms of prediction of response to tamoxifen have been controversial. Our results agree with others reporting an association
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
the tamoxifen/ER␤ complex increases expression of AP-1controlled genes involved in cell proliferation (17). Whether
ER␤ actively protects breast cancer cells against tamoxifenresistance is unknown. One possible mechanism, however,
could be a dominant-negative effect of ER␤ after heterodimerization (16) inhibiting the tamoxifen agonist activity of ER␣ via
the AF-1 domain (18, 19).
We have not discriminated between initial and acquired
tamoxifen resistance, the median time for relapse being 3 years;
some resistant cases could be secondary to the selection of
cancer cells stimulated for growth by tamoxifen acting as an
agonist via ER␣. The four patients who recurred more than 1
year after the 5 years’ therapy were included in the tamoxifensensitive group, with the assumption that these breast cancers
were initially responsive to tamoxifen. When considering these
four patients as tamoxifen resistant, the multivariate analysis
gave a similar significance for ER␤ expression (P ⫽ 0.012).
Among the classical markers of aggressiveness (SBR
grade, tumor size, MIB-1 proliferation), only lymph node invasiveness was not associated with tamoxifen resistance. This is in
agreement with studies showing that node-positive tumors respond as well as node-negative tumors to tamoxifen therapy
(30) and that cancer cells, having migrated to lymph nodes,
retain the same antiestrogen responsiveness as the primary
tumor.
The fact that ER␤cx expression in breast cancer is not
predictive of tamoxifen resistance in our study, suggests that the
full-length ER␤-1, or another ER␤ variant, may be involved in
tamoxifen sensitivity. It is not excluded, however, that ER␤cx
plays a role in the initial tamoxifen resistance as suggested by
studies in which tamoxifen responsiveness was evaluated after 3
months of neo-adjuvant therapy (31). The absence of correlation
of ER␤ with other classic prognostic parameters further supports its interest for breast cancer monitoring. The absence of
correlation with PgR disagrees with other studies (29, 32) but
was supported by a recent study on 242 breast cancers (33). The
reasons for these discrepancies is unknown and could be due to
different methods used for quantification and/or different sets of
patients.
Our results do not exclude the involvement of other entities
able to induce tamoxifen resistance, such as an increased expression of HER-2/neu (34) and an altered expression of coactivator (35) or corepressor (36). However they strongly suggest
that the level of ER␤ in breast epithelial cancer cells contributes
better than the level of ER␣ in predicting tamoxifen-sensitivity
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
breast cancer.
ACKNOWLEDGMENTS
We thank Drs. Philippe Rouanet, Bernard Saint-Aubert, Jean Grenier, François Quenet, and G. Romieu (from the CRLC Val d’Aurelle,
Montpellier) for supplying clinical data, and Jean-Yves Cance for preparing the figures.
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