Download Comparison of the Short-Term Biological Effects

[CANCER RESEARCH 61, 6739 – 6746, September 15, 2001]
Comparison of the Short-Term Biological Effects of 7␣-[9-(4,4,5,5,5pentafluoropentylsulfinyl)-nonyl]estra-1,3,5, (10)-triene-3,17␤-diol
(Faslodex) versus Tamoxifen in Postmenopausal Women with
Primary Breast Cancer1
John F. Robertson,2 Robert I. Nicholson, Nigel J. Bundred, Elizabeth Anderson, Zenon Rayter, Mitchell Dowsett,
John N. Fox, Julia M. W. Gee, Alan Webster, Alan E. Wakeling, Charles Morris, and Michael Dixon
Department of Surgery, Nottingham City Hospital, Nottingham, United Kingdom [J. F. R.]; Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff, Wales
CF10 3XF [R. I. N., J. M. W. G.]; Department of Surgery, South Manchester University Hospital, Manchester M20 8LR, United Kingdom [N. J. B.]; Clinical Research
Department, Christie Hospital National Health Service Trust, Manchester M20 4BX, United Kingdom [E. A.]; Department of Biochemistry, Royal Marsden Hospital, London
SW3 6JJ, United Kingdom [M. Do.]; Bristol Royal Infirmary, Bristol DS2 8HW, United Kingdom [Z. R.]; Castle Hill Hospital Cottingham, East Yorkshire HU16 5JQ, United
Kingdom [J. N. F.]; AstraZeneca, Macclesfield SK10 4TF, United Kingdom [A. W., A. E. W., C. M.]; and Department of Surgery, Western General Hospital, Edinburgh EH4 2XU,
Scotland [M. Di.]
ABSTRACT
INTRODUCTION
7␣-[9-(4,4,5,5,5-Pentafluoropentylsulfinyl)-nonyl]estra-1,3,5, (10)-triene3,17␤-diol (ICI 182,780; Faslodex) is a novel steroidal antiestrogen. This
partially blind, randomized, multicenter study compared the effects of
single doses of long-acting ICI 182,780 with tamoxifen or placebo on
estrogen receptor (ER␣) and progesterone receptor (PgR) content, Ki67
proliferation-associated antigen labeling index (Ki67LI), and the apoptotic index in the primary breast tumors of postmenopausal women.
Previously untreated patients (stages T1–T3; ER-positive or -unknown)
were randomized and received a single i.m. dose of ICI 182,780 50 mg
(n ⴝ 39), ICI 182,780 125 mg (n ⴝ 38), or ICI 182,780 250 mg (n ⴝ 44) or
oral tamoxifen 20 mg daily (n ⴝ 36) or matching tamoxifen placebo
(n ⴝ 43) for 14 –21 days before tumor resection surgery with curative
intent. The ER and PgR H-scores, together with the Ki67LI were determined immunohistochemically in the matched pretreatment biopsy and
the posttreatment surgical specimens. The apoptotic index was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin
nick end labeling on the same samples. The effects of treatment on each of
these parameters were compared using analysis of covariance. ICI 182,780
produced dose-dependent reductions in ER and PgR H-scores and in the
Ki67LI. The reductions in ER expression were statistically significant at
all doses of ICI 182,780 compared with placebo (ICI 182,780 50 mg,
P ⴝ 0.026; 125 mg, P ⴝ 0.006; 250 mg, P ⴝ 0.0001), and for ICI 182,780
250 mg compared with tamoxifen (P ⴝ 0.024). For PgR H-score, there
were statistically significant reductions after treatment with ICI 182,780
125 mg (P ⴝ 0.003) and 250 mg (P ⴝ 0.0002) compared with placebo. In
contrast, tamoxifen produced a significant increase in the PgR H-score
relative to placebo, and consequently, all doses of ICI 182,780 produced
PgR values that were significantly lower than those in the tamoxifentreated group. All doses of ICI 182,780 significantly reduced Ki67LI
values compared with placebo (ICI 182,780 50 mg, P ⴝ 0.046; 125 mg,
P ⴝ 0.001; 250 mg, P ⴝ 0.0002), but there were no significant differences
between any doses of ICI 182,780 and tamoxifen. ICI 182,780 did not alter
the apoptotic index when compared with either placebo or tamoxifen.
Short-term exposure to ICI 182,780 reduces the ER␣ in breast tumor cells
in a dose-dependent manner by down-regulating ER protein concentration. The reductions in tumor PgR content by ICI 182,780 demonstrate
that ICI 182,780, unlike tamoxifen, is devoid of estrogen-agonist activity.
Reductions in tumor cell proliferative activity (as indicated by Ki67LI)
show that ICI 182,780 is likely to have antitumor activity in the clinical
setting.
Estrogens act as endocrine growth factors for at least one-third of
breast cancers (1), and their effects are mediated via the ER3 pathway.
Several approaches have been adopted to treat hormone-sensitive
breast cancer. In premenopausal women these include reducing circulating estrogen by ovarian ablation or by inhibiting ovarian estrogen
production. In postmenopausal women, the mainstays of therapy are
the prevention of estrogen binding to its receptor using an antiestrogen
or lowering estrogen levels with aromatase inhibitors. The antiestrogen tamoxifen is the most widely used hormonal treatment for all
stages of breast cancer (2). However, tamoxifen possesses partial
agonist activity which has positive effects on bone (3, 4) and blood
lipids (5), but which also has unwanted side effects, including increased endometrial proliferation (6), a small increase in the risk of
endometrial cancer (7–9), tumor flare at the start of treatment (10),
and tamoxifen-mediated tumor stimulation upon progression (11).
Currently, there are two other clinically available nonsteroidal,
mixed agonist/antagonist antiestrogens, toremifene, which is used in
the treatment of breast cancer (12), and raloxifene, which is being
used in the management of osteoporosis (13). These two agents,
together with tamoxifen, comprise a group of compounds that are
described as SERMs (14). No new SERM has yet provided significant
advantages over tamoxifen in the treatment of breast cancer in terms
of either efficacy or tolerability, and all SERMs discovered to date
show some degree of partial agonist activity. Furthermore, crossresistance between the new SERMs and tamoxifen may limit their
application in advanced disease after adjuvant tamoxifen treatment
(15). Despite the potential advantages of the partial agonist properties
of the SERMs, a drug that acts as a nonagonist (pure) antiestrogen
may be an important step toward improving breast cancer treatment
(16).
Fulvestrant (Faslodex), formerly known as ICI 182,780, is a novel
estrogen antagonist that, unlike tamoxifen, has no estrogen-agonist
activity (Fig. 1). Preclinical and early clinical studies (17– 40) suggest
that ICI 182,780 has biological effects indicative of improved clinical
efficacy in the treatment of breast cancer. The main features are ER
down-regulation, antiproliferative activity, induction of apoptosis,
lack of cross-resistance with tamoxifen, and the absence of ERagonist activity.
ICI 182,780 has a binding affinity for the ER that is ⬃100 times
Received 11/9/00; accepted 7/25/01.
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.
1
This trial was sponsored and funded by AstraZeneca Pharmaceuticals (Macclesfield,
United Kingdom).
2
To whom requests for reprints should be addressed, at Department of Surgery,
Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, United Kingdom.
3
The abbreviations used are: ER, estrogen receptor(s); SERM, selective estrogen
receptor modulator; ICI 182,780, 7␣-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)-nonyl]estra1,3,5, (10)-triene-3,17␤-diol; PgR, progesterone receptor(s); Ki67LI, Ki67 proliferationassociated antigen labeling index; AI, apoptotic index; DAB, diaminobenzidine tetrahydrochloride; ANCOVA, analysis of covariance; ICA, immunocytochemical assay; NRS,
normal rabbit serum.
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SHORT-TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
Fig. 1. Chemical structures of the nonsteroidal SERM, tamoxifen, and of the novel
nonagonist (pure) antiestrogen, ICI 182,780.
greater than that of tamoxifen (17), and in animal models, ICI 182,780
markedly attenuates the ability of the ER to activate or inhibit gene
transcription (20 –22). Several different mechanisms may underlie this
effect, including impaired dimerization, increased ER turnover, and
disrupted nuclear localization (23–25). ICI 182,780 treatment blocks
the uterotrophic effects of ER agonists (estrogens) and of partial
agonists such as tamoxifen (26 –28) and raloxifene (29) and reduces
ER levels in the tumors of women with primary breast cancer (30).
Therefore, ICI 182,780 seems to act as an ER down-regulator, because
it functionally blocks the ER and reduces cellular ER levels such that
the receptor is rendered unavailable or unresponsive to estrogen or
estrogen agonists.
The PgR gene is an estrogen-regulated gene (34), so drugs with
estrogenic activity will increase its expression. Accordingly, tamoxifen has been shown to increase PgR levels (35), whereas initial work
on primary breast tumors found that a short-acting formulation of ICI
182,780 reduced PgR levels (30), suggesting that it is devoid of
estrogen-agonist activity and may have a different mechanism of
action to that of tamoxifen. Additional evidence that ICI 182,780 and
tamoxifen have different underlying modes of action comes from
studies showing that tamoxifen-resistant tumors remain sensitive to
ICI 182,780 treatment in vitro (18, 19), in vivo (36, 37), and in the
clinic (38 – 40).
ICI 182,780 has antiproliferative effects, as assessed by immunohistochemical detection of the Ki67 proliferation-associated antigen
(30 –32). Previous small clinical studies have suggested that both
tamoxifen and ICI 182,780 increase apoptosis in primary human
breast cancer (33).
The study reported here represents the first direct randomized
comparison of the short-term biological effects of ICI 182,780 (50
mg, 125 mg, or 250 mg as a single i.m. injection) with tamoxifen (20
mg/day p.o. for 14 –22 days) and tamoxifen placebo in women with
primary breast cancer. It is also the first investigation of any doseresponse effect of ICI 182,780 and the first time that the biological
effects of the clinical trials formulation (250 mg) have been assessed.
The end points of the trial were ER␣ (referred to as ER for the
remainder of this paper) and PgR H-scores, Ki67LI, and the AI.
itant therapy, demography, current medical conditions, hematology, and biochemistry screening.
Patients were included if they were postmenopausal (⬎12 months since the
last menstrual period and/or had castrate levels of follicle-stimulating hormone
⬎40 IU/liter) and had a clinically staged, histologically confirmed T1, T2 or T3
primary breast cancer. They had to be fit for surgery within 1 month and have
a tumor large enough to provide sufficient biopsy samples. Patients were
ER-positive or -unknown at entry to the trial. The study was approved by the
Ethics Committees of all centers.
Patients were not eligible for the study if they had evidence of metastatic
disease or had received any prior treatment for their primary tumor. Other
exclusion criteria were: (a) treatment with hormone replacement therapy
within 4 weeks of starting the trial; (b) baseline hematology or clinical
chemistry outside the normal range; (c) risk of human immunodeficiency virus,
hepatitis B, or hepatitis C transmission; (d) history of disease affecting steroid
metabolism; (e) bleeding diathesis or thrombocytopenia (platelets
⬍100 ⫻ 109/liter); or any other reason that could jeopardize the protocol.
Treatment with drugs known to affect sex hormone status could not be
commenced during the trial (e.g., ketoconazole or prednisolone), although the
patient could continue to receive such drugs if they were being taken before the
study and the patient’s hormone status was stable.
Patients were randomized to one of the following treatments: single i.m.
dose of ICI 182,780 50 mg (n ⫽ 40), 125 mg (n ⫽ 40), and 250 mg (n ⫽ 41);
tamoxifen, 20 mg, once daily p.o. for 14 –21 days (n ⫽ 37); or tamoxifen
placebo, once daily p.o. for 14 –21 days (n ⫽ 43). Patients were scheduled for
tumor resection surgery with curative intent between day 15 and day 22 after
the start of treatment. On the day of surgery, patients were reassessed for
concomitant therapy, concomitant conditions, hematology, and biochemistry.
All patients returned for postsurgical assessment on day 57.
Tumor Sampling
The Tru-cut/core biopsy taken at the first clinic attendance for diagnostic
purposes was used as the prerandomization tumor sample. Where possible, a
minimum of three cores was taken, sufficient to provide material for the three
laboratories. The posttreatment specimen was obtained at definitive surgical
resection. All of the tissue samples were fixed in 3.7% formalin immediately
after removal, then embedded in paraffin wax for sectioning and subsequent
analysis of biological markers.
Drug Administration
Long acting ICI 182,780 (AstraZeneca, Macclesfield, United Kingdom) was
administered by i.m. injection into the gluteus maximus muscle. Patients were
randomized to receive 50 mg of ICI 182,780 (1 ml), 125 mg of ICI 182,780
(2.5 ml), or 250 mg of ICI 182,780 (5 ml). Tamoxifen was supplied as
Nolvadex tablets containing 20 mg of tamoxifen (AstraZeneca) and administered at a dose of 20 mg/day. The tamoxifen placebo tablet (AstraZeneca)
matched the 20 mg tamoxifen tablet. Both tamoxifen and tamoxifen placebo
were administered p.o.
Adverse Events Monitoring
Adverse events (defined as the development of a new medical condition or
the deterioration of a preexisting medical condition subsequent to or during
exposure to the trial medications) were monitored throughout the study.
Patients were followed up for adverse events for 57 days postdosing.
Analysis of Tumor Marker Expression
ER. ER␣ expression was assessed at the Tenovus Centre for Cancer
Research, Cardiff, Wales, on sections cut from the formalin-fixed, paraffinembedded tissue specimens described above. All mounted sections were dried
Two hundred and one women with primary breast cancer participated in a overnight at 60°C before being dewaxed and rehydrated to PBS (pH 7.2–7.4).
multicenter, randomized, partially blinded study. The administration of tamox- Endogenous peroxidase activity was quenched by incubation in hydrogen
ifen and tamoxifen placebo was double blind, and the administration of ICI peroxide (0.5% in methanol) for 10 min and then rinsing in running tap water
182,780 (at one of three doses) was open. Postmenopausal women with for 5 min and in PBS for 5 min. Then sections were enzyme-digested in a bath
histologically proven primary breast cancer awaiting tumor resection were of 0.02% Pronase E (Sigma Chemical Co., Poole, United Kingdom) in PBS at
recruited to the study from June 1997 to May 1999. Each woman gave written
37°C before being rinsed as described previously. To block the nonspecific
informed consent and underwent an initial eligibility screen in the week before staining, a blocking reagent, comprising 20% normal swine serum (Dako Ltd.,
randomization. Prestudy assessments included past medical history, concom- Glostrup, Denmark) in PBS was applied to the sections and then “tapped off”
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PATIENTS AND METHODS
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SHORT-TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
Table 1 ER and PgR status of tumors—per-protocol patients
Characteristic
ER status
n (%)
PgR status
n (%)
Positive
Negative
Unknown
Positive
Negative
Unknown
Placebo
ICI 182,780
50 mg
ICI 182,780
125 mg
ICI 182,780
250 mg
Tamoxifen
29 (69.0)
8 (19.0)
5 (11.9)
28 (66.7)
10 (23.8)
4 (9.5)
33 (86.8)
4 (10.5)
1 (2.6)
29 (76.3)
7 (18.4)
2 (5.3)
34 (89.5)
1 (2.6)
3 (7.9)
29 (76.3)
5 (13.2)
4 (10.5)
32 (74.4)
6 (14.0)
5 (11.6)
29 (67.4)
9 (20.9)
5 (11.6)
27 (81.8)
4 (12.1)
2 (6.1)
21 (63.6)
9 (27.3)
3 (9.1)
before incubation overnight at room temperature with the primary antibody
(diluted 1:2), which was the rat antihuman ER␣ antibody (Clone H222)
supplied in the ER-ICA kit by Abbott Laboratories (North Chicago, IL).
Sections were washed in PBS (5 ⫻ 4 min) and then a secondary biotinylated
sheep antirat immunoglobulin (Amersham Life Science Ltd., Amersham,
United Kingdom) diluted 1:500 in 20% normal swine serum was applied for 60
min. Sections were washed again in PBS (5 ⫻ 4 min) before the avidin-biotinhorseradish peroxidase complex (Dako Ltd.) diluted 1:120 in PBS was added
for 60 min with additional washing afterward in PBS (5 ⫻ 4 min). Then the
DAB chromogen was applied (as supplied in the Abbott ER-ICA kit) to the
sections and left for 10 min before rinsing in distilled water (2 ⫻ 3 min).
Staining was enhanced by treating the sections with 0.5% copper sulfate in
0.85% sodium chloride for 8 min and rinsing in distilled water (2 ⫻ 3 min).
The sections were counterstained with 0.5% methyl green for 5 min, washed
in distilled water (2 ⫻ 3 min), dehydrated, cleared, and mounted for examination by light microscopy.
ER␣ immunopositivity appeared clearly as a brown nuclear signal in tumor
epithelial cells against a background of green nuclear counterstain. Tumor
epithelial cell ER content in the pre- and posttreatment specimens for each
patient was assessed by the consensus of two people (J. M. W. G. and R. I. N.)
using the dual viewing attachment of a light microscope. Overall staining was
assessed at ⫻10, and a representative area was viewed at ⫻40 to assess the
number of positive tumor cell nuclei and staining intensity. The percentages of
positive tumor epithelial cells in each staining intensity category (i.e., negative
⫺/⫺; very weak ⫹/⫺; weak ⫹; moderate ⫹⫹; and strong ⫹⫹⫹) were
recorded for each sample, and positive-control breast cancer samples of known
ER positivity were included in every assay to monitor assay performance.
Results were expressed as the ER H-score where: H-score ⫽ [(0.5 ⫻ %
⫹/⫺) ⫹ (1 ⫻ % ⫹) ⫹ (2 ⫻ % ⫹⫹) ⫹ (3 ⫻ % ⫹⫹⫹)]. A value of ⬎0 implies
an ER-positive state with a range of 0 –300.
PgR Expression. Levels of PgR in sections from the same samples were
also assessed by the Tenovus Centre for Cancer Research, Cardiff, Wales. The
assay procedure was similar to that used to detect ER, except that the primary
anti-PgR antibody (Clone KD68) was that supplied by Abbott Laboratories in
the PgR-ICA kit, as was the DAB chromogen. In this assay the primary
antibody was diluted 1:4, and no enzyme retrieval was used. Results were
expressed as the PgR H-score, using the same equation as that used to calculate
the ER H-score.
Ki67 Proliferation-associated Antigen Expression. Ki67 antigen was
assessed on sections of the pre- and posttreatment tissue specimens at the
Christie Hospital, Manchester, United Kingdom, using the MIB-1 anti-Ki67
antibody supplied by Coulter Electronics (Luton, United Kingdom). Briefly,
slides were dewaxed and rehydrated to PBS (pH 7.6). Endogenous peroxidase
was quenched using hydrogen peroxide (0.2%) in methanol for 10 min. The
sections were then rinsed in water and PBS and microwaved (800 W) in 10 mM
citrate buffer (pH 6.0) at power 7 for 15 min after boiling point was reached.
After cooling for 20 min, sections were washed in PBS and nonspecific
binding was blocked with 10% NRS in 0.5% casein/PBS containing 4 drops/ml
of the avidin block supplied by Vector Laboratories (Peterborough, United
Kingdom) for 15 min. The primary antibody was then applied at a dilution of
1:50 in 10% NRS/0.5% casein/PBS containing 4 drops/ml of biotin block
(Vector Laboratories), and the sections were incubated for 80 min at room
temperature. After washing in PBS (2 ⫻ 5 min), the secondary biotinylated
rabbit antimouse antibody (DAKO E413; Dako Ltd., Ely, United Kingdom)
was applied at a dilution of 1:300 in 10% NRS/0.5% casein/PBS for 40 min,
and after washing in PBS (2 ⫻ 5 min), the avidin biotinylated enzyme complex
reagent (Vectastain ABC Elite kit; Vector Laboratories) was applied for 40
min. After the final PBS wash (2 ⫻ 5 min), incubation with the DAB
chromogen (“SigmaFast” 3,3-diaminobenzidine tablet set; Sigma Chemical
Co.-Aldrich Company, Poole, United Kingdom) was performed for 8 min at
room temperature before a wash in distilled water. Samples were counterstained with 20% hematoxylin for 3–5 min, dehydrated, cleared, and mounted
for examination by light microscopy. Results were expressed as the Ki67LI
(the percentage of positively stained nuclei calculated after counting at least
1000 tumor cells).
AI. The AI was measured using the terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling assay at the Royal Marsden Hospital,
London, United Kingdom. After dewaxing and rehydration to deionized water,
endogenous peroxidases were quenched with hydrogen peroxide (1%) in PBS
for 15 min and washing three times in deionized water. Then sections were
digested in 0.5% pepsin (pH 2) for 30 min at 37°C in a humidified chamber.
Digestion was terminated, and sections were rinsed for 1 min and washed five
times for 5 min each in deionized water. Then sections were washed twice in
Tris-buffered saline (pH 7.6) for 5 min and incubated for 1 h at 37°C in 100
␮l/slide of a reaction mixture containing 0.75 ␮l of terminal deoxynucleotidyltransferase, 0.50 ␮l of biotinylated 16dUTP, 10 ␮l of 50 mM cobalt
chloride, and 20 ␮l of reaction buffer (1 M sodium cacodylate ⫹ 125 mM
Tris-HCl ⫹ 1.25 mg/ml BSA in deionized water). After washing twice in
deionized water and three times in PBS, sections were incubated with horseradish peroxidase-conjugated streptavidin (Dako Ltd.) diluted 1:4000 in
PBS ⫹ 1% BSA ⫹ 0.5% Tween 20. Another three washes in PBS/Tween 20
preceded development with 0.05% DAB and 0.07% imidazole for 30 s and
then 10 min of incubation with 100 ␮l of 1% hydrogen peroxide. Sections were
washed in running tap water for 5 min and then immersed in 0.5% copper
sulfate plus 0.9% sodium chloride in deionized water for 1 min. DAB was then
inactivated with chloros and the sections were washed in running tap water,
Table 2 Demographic characteristics of ER-positive per-protocol patients
Characteristic
Age (yr)
Clinical disease staging
n (%)
Tumor grade at surgeryb
n (%)
a
b
n
Mean
SD
T1
T2
T3
Not T4a
G1
G2
G3
GX
Placebo
ICI 182,780
50 mg
ICI 182,780
125 mg
ICI 182,780
250 mg
Tamoxifen
29
65.9
9.2
5 (17.2)
10 (34.5)
1 (3.4)
13 (44.8)
6 (20.7)
14 (48.3)
7 (24.1)
2 (6.9)
33
69.2
8.4
2 (6.1)
11 (33.3)
3 (9.1)
17 (51.5)
6 (18.2)
16 (48.5)
9 (27.3)
2 (6.1)
34
68.7
7.3
5 (14.7)
10 (29.4)
1 (2.9)
18 (52.9)
8 (23.5)
15 (44.1)
9 (26.5)
2 (5.9)
32
66.1
8.3
2 (6.3)
12 (37.5)
0 (0.0)
18 (56.3)
9 (28.1)
16 (50.0)
6 (18.8)
1 (3.1)
27
68.7
8.4
6 (22.2)
9 (33.3)
1 (3.7)
11 (40.7)
6 (22.2)
13 (48.1)
7 (25.9)
1 (3.7)
Unable to categorize, but definitely not T4.
G1, well-differentiated; G2, moderately differentiated; G3, poorly differentiated; GX, unassessable.
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SHORT-TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
Fig. 2. Comparison of ER expression in a biopsy
sample taken pretreatment with that from a sample
taken from the same tumor after treatment with ICI
182,780 (250 mg; A), tamoxifen (B), and tamoxifen
placebo (C). ER immunopositivity appears as a
brown nuclear signal against a background of the
green nuclear counterstain. Photographs supplied
by R. I. N.
counterstained with hematoxylin in blued tap water (30 s), dehydrated, cleared,
and mounted for examination by light microscopy. Results were expressed as
the percentage of apoptotic cells in 3000 tumor cells.
Statistical Analysis
This trial was an exploratory trial, so the minimum power required
for statistical testing was set at 80%. The four end points (surrogate
tumor tissue markers) were considered equally important, so all were
classed as primary end points. The secondary end points were tolerability and pharmacokinetic data (pharmacokinetic data are not presented in this paper). This “per protocol” analysis included only those
patients who received the full course of treatment, completed the end
of treatment assessment for the primary end point, and had no significant protocol deviations or violations. All analyses were carried out
by the Biometrics Group, AstraZeneca.
It was calculated that ⬃30 patients/group were needed to detect the
following differences between ICI 182,780 and the comparator with
80% power using a two-sided significance level of 5%: 0.3 for ER
H-score; 0.4 for PgR H-score; 4.5 for Ki67; and 0.2 for apoptosis. To
allow for ER-/PgR-negative tumors, a total of 201 patients were
recruited and ⬃40 were randomized to each treatment group.
The primary end point data were assessed statistically using
ANCOVA according to treatment received with terms included in the
model for treatment, center, and the baseline tumor marker value.
Patients in the per-protocol population who were ER-negative were
excluded from the analysis of ER, Ki67, and AI, and patients who
were PgR-negative were excluded from the analysis of PgR. In
addition, any patients in the per-protocol population with a missing
value for a tumor tissue marker were also excluded from the analysis
for that particular marker. The ANCOVA allowed an overall assessment of differences between each dose of ICI 182,780 and tamoxifen
and each dose of ICI 182,780 and placebo. A test for overall treatment
Fig. 3. Posttreatment mean ER H-scores after a single i.m. injection of 50 mg, 125 mg,
or 250 mg of ICI 182,780 or Tamoxifen 20 mg once daily p.o. or tamoxifen placebo.
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SHORT-TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
Table 3 Summary of results for ER H-score
Placebo
a
n
Pretreatment mean H-score
Percentage change (posttreatment)
Overall treatment effect
Treatment difference vs. placebo (95% CI)
29
125
⫺13
P ⫽ 0.0003
NAa
Treatment difference vs. tamoxifen (95% CI)
29 (0.2, 57)
P ⫽ 0.0485
ICI 182,780
50 mg
ICI 182,780
125 mg
ICI 182,780
250 mg
Tamoxifen
31
136
⫺39
32
124
⫺50
32
113
⫺59
25
123
⫺36
⫺30 (⫺57, ⫺4)
P ⫽ 0.0255
⫺2 (⫺29, 26)
P ⫽ 0.8955
⫺47 (⫺74, ⫺21)
P ⫽ 0.0006
⫺19 (⫺46, 9)
P ⫽ 0.1833
⫺60 (⫺86, ⫺34)
P ⫽ 0.0001
⫺32 (⫺59, ⫺4)
P ⫽ 0.0239
⫺29 (⫺57, ⫺0.2)
P ⫽ 0.0485
NA
NA, not applicable.
RESULTS
Fig. 4. Posttreatment mean PgR H-scores after a single i.m. injection of 50 mg, 125 mg,
or 250 mg of ICI 182,780 or tamoxifen 20 mg once daily p.o. or tamoxifen placebo.
effect was undertaken. If this was significant at the 5% level, then the
following pairwise comparisons were made: ICI 182,780 50 mg
versus placebo; ICI 182,780 125 mg versus placebo; ICI 182,780 250
mg versus placebo and ICI 182,780 50 mg versus tamoxifen; ICI
182,780 125 mg versus tamoxifen; and ICI 182,780 250 mg versus
tamoxifen. A supplementary comparison of tamoxifen versus placebo
was also undertaken. For ER and PgR, the comparisons are presented
as treatment differences with 95% confidence intervals. The mean
change from baseline was also calculated for each treatment group
and expressed as a percentage of the baseline mean value. For both
Ki67LI and AI, the data showed evidence of nonnormality, so all
values were log- (base e) transformed for the ANCOVA analysis, and
the comparisons are, therefore, presented as treatment ratios with 95%
confidence intervals. In addition, the median change from baseline
was calculated for each treatment group and expressed as a percentage
of the baseline median value. Plots of means ⫾ 1 SE by treatment
group for each end point are also presented.
Patient Characteristics. A total of 201 postmenopausal women
(mean age, 67.6 years; range: 48 – 86 years) were randomized into the
trial, and 190 completed the trial. One patient did not take any trial
treatment, and 10 patients withdrew from the trial. The withdrawal
rates were similar for the ICI 182,780 groups (1/treatment group) but
four patients withdrew from the tamoxifen treatment group and three
from the tamoxifen placebo group. Of those patients in the perprotocol population, 155 were ER-positive. Groups were well balanced with respect to age, disease stage, and tumor grade at surgery.
The ER and PgR status of the tumors at study entry are given in Table
1. The demographic characteristics of the ER-positive per-protocol
patients in the five treatment groups are summarized in Table 2.
ER Expression. Treatment of ER-positive tumors with ICI
182,780 resulted in a marked reduction of nuclear ER content that
could easily be seen under the light microscope (Fig. 2). This was
confirmed by statistical analysis of the ER H-score, which showed a
significant overall treatment effect (P ⫽ 0.0003). The posttreatment
mean ER H-scores are shown in Fig. 3, and the summary of results are
shown in Table 3. ICI 182,780 produced a dose-dependent reduction
in the ER H-scores, and all doses significantly reduced the ER H-score
compared with placebo. The reduction in ER H-scores seen at the
lower doses of ICI 182,780 (50 mg and 125 mg) were not statistically
significantly different from those caused by tamoxifen, although the
comparison between the 250-mg dose of ICI 182,780 and tamoxifen
did reach significance (P ⫽ 0.0239).
PgR Expression. Analysis of the PgR H-scores showed a significant overall treatment effect (P ⫽ 0.0001). Posttreatment mean PgR
H-scores are shown in Fig. 4, and the summary of results is shown in
Table 4. There was a dose-dependent reduction in PgR H-score with
ICI 182,780, with the 125 mg and 250 mg doses of ICI 182,780
producing significantly greater reductions in PgR H-score than placebo. Tamoxifen caused a significant increase in PgR H-score compared with placebo; consequently, each dose of ICI 182,780 resulted
in a PgR H-score that was significantly lower than that of tamoxifen.
Ki67LI. Analysis of the Ki67LI showed a significant overall treatment effect (P ⫽ 0.0029). The posttreatment mean Ki67LIs are shown
in Fig. 5 and the summary of results are shown in Table 5. ICI 182,780
Table 4 Summary of results for PgR H-score
Placebo
n
Pretreatment mean H-score
Percentage change (posttreatment)
Overall treatment effect
Treatment difference vs. placebo (95% CI)
Treatment difference vs. tamoxifen (95% CI)
a
28
30
⫹43
P ⫽ 0.0001
NAa
⫺27 (⫺47, ⫺7)
P ⫽ 0.0090
ICI 182,780
50 mg
ICI 182,780
125 mg
ICI 182,780
250 mg
Tamoxifen
29
47
⫺12
29
28
⫺52
29
33
⫺67
21
49
⫹63
⫺14 (⫺32, 5)
P ⫽ 0.1455
⫺40 (⫺60, ⫺21)
P ⫽ 0.0001
⫺28 (⫺46, ⫺10)
P ⫽ 0.0030
⫺55 (⫺75, ⫺34)
P ⫽ 0.0001
⫺35 (⫺53, ⫺17)
P ⫽ 0.0002
⫺62 (⫺82, ⫺42)
P ⫽ 0.0001
27 (7, 47)
P ⫽ 0.0090
NA
NA, not applicable.
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SHORT-TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
produced dose-dependent reductions in the Ki67LI such that the
Ki67LI at each dose of ICI 182,780 was significantly lower than that
in the placebo group. There were no significant differences in Ki67
labeling between tamoxifen and any dose of ICI 182,780.
AI. There was no statistically significant overall treatment effect
on the AI (P ⫽ 0.2382). There was no difference in AI between any
dose of ICI 182,780 and tamoxifen compared with control. Posttreatment mean values for apoptosis are shown in Fig. 6, and the summary
of results are shown in Table 6.
Drug Tolerability. In general, all of the drugs were well tolerated.
The most commonly reported adverse event were those relating to
breast surgery.
DISCUSSION
This study provides the first direct comparison of ICI 182,780 with
tamoxifen and placebo on the biological end points of breast tumor
ER␣ and PgR content, Ki67 labeling, and AIs in primary breast
cancer.
Previous preclinical and early clinical studies have indicated that
ICI 182,780 is an estrogen antagonist that is devoid of estrogenagonist activity and which has a mode of action that is different from
tamoxifen—ICI 182,780 down-regulates the ER, rendering it unavailable or unresponsive to estrogen or estrogen agonists (30). Accordingly, the data reported here show that ICI 182,780 produced a
marked dose-dependent reduction in ER expression that was significantly different from placebo at all doses. Although the standard dose
of tamoxifen (20 mg/day for 14 –21 days p.o.) significantly reduced
ER expression compared with placebo, the ER suppression caused by
the 250 mg dose of ICI 182,780 was significantly greater than that
Fig. 5. Ki67. Posttreatment mean values after a single i.m. injection of 50 mg, 125 mg,
or 250 mg of ICI 182,780 or tamoxifen 20 mg once daily p.o. or tamoxifen placebo.
Fig. 6. Apoptosis. Posttreatment mean values after a single i.m. injection of 50 mg, 125
mg, or 250 mg of ICI 182,780 or tamoxifen 20 mg once daily p.o. or tamoxifen placebo.
caused by tamoxifen. The present study does not provide information
on the biological processes responsible for the reductions in ER
content with ICI 182,780. However, data from preclinical and early
clinical studies using a short-acting formulation of ICI 182,780 indicate that it reduces ER content more rapidly than does tamoxifen
(through reduced half-life) and causes a greater turnover of the ER
receptor (25, 32) but not of ER mRNA (41).
This study also measured tumor PgR content. PgR is an estrogenregulated gene (34), so a decrease in ER content and activity would be
expected to decrease PgR expression. A previous study with a shortacting formulation of ICI 182,780 in primary breast cancer found that
it did, indeed, reduce PgR levels (30). The present study confirmed
that the long-acting form of ICI 182,780 also causes a dose-dependent
reduction in PgR expression that is significantly different from placebo for the 125 mg and 250 mg doses. As anticipated, tamoxifen
increased PgR expression, a finding that can be attributed to its partial
agonist effects (3, 6 –11, 18, 19, 36, 37), and all doses of ICI 182,780
produced significant reductions in PgR expression compared with
tamoxifen. These data confirm that the mechanism of ICI 182,780
action is different from that of tamoxifen, and also that it lacks
estrogen agonist activity. Whether or not these differences in mechanism of action translate to clinically beneficial outcomes will be
confirmed in the ongoing clinical program comparing ICI 182,780
with tamoxifen in postmenopausal women with advanced breast
cancer.
Given the differences in biological findings with respect to ER and
PgR, it was important to consider whether the observed results may
have been influenced by either tamoxifen or ICI 182,780 being at
less-than-optimal exposure levels during the time course of the study.
Although no definitive data are available on this, nor was pharmacokinetic data collected in this study, it is known that the time to steady
state for tamoxifen is ⬃4 weeks (42) and that for ICI 182,780 is 3– 6
Table 5 Summary of results for Ki67LI
Placebo
n
Pretreatment median value
Percentage change (posttreatment)
Overall treatment effect
Treatment ratio vs. placebo (95% CI)
Treatment ratio vs. tamoxifen (95% CI)
a
27
14.5
⫹3
P ⫽ 0.0029
NAa
1.557 (1.084, 2.234)
P ⫽ 0.0169
ICI 182,780
50 mg
ICI 182,780
125 mg
ICI 182,780
250 mg
Tamoxifen
28
13.1
⫺25
28
12.5
⫺28
29
10.4
⫺49
22
14.1
⫺22
0.710 (0.507, 0.994)
P ⫽ 0.0460
1.106 (0.778, 1.572)
P ⫽ 0.5725
0.571 (0.407, 0.801)
P ⫽ 0.0014
0.889 (0.623, 1.269)
P ⫽ 0.5139
0.531 (0.382, 0.740)
P ⫽ 0.0002
0.828 (0.582, 1.178)
P ⫽ 0.2900
0.642 (0.447, 0.922)
P ⫽ 0.0169
NA
NA, not applicable.
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SHORT-TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
trials will determine whether the unique mode of action of ICI
182,780 translates into clinical benefits for patients with breast cancer.
Table 6 Summary of results for apoptotic index
Placebo
n
28
Pretreatment median
0.59
value
Percentage change
⫹15
(posttreatment)
Overall treatment
P ⫽ 0.2382
effect
ICI 182,780 ICI 182,780 ICI 182,780
50 mg
125 mg
250 mg
Tamoxifen
28
0.55
⫺4
30
0.62
⫹5
31
0.56
⫹16
26
0.64
ACKNOWLEDGMENTS
⫺5
months (AstraZeneca; data on file). During the time course of this
study, both tamoxifen and ICI 182,780 would have been at submaximal exposure levels. On the basis of the PgR data alone, it is clear that
at their clinically used dose, both ICI 182,780 at 250 mg and tamoxifen at 20 mg, are present at sufficiently high exposure levels to elicit
their marked, but different, biological effects.
In estrogen-dependent tumors, tamoxifen has been shown to cause
tumor shrinkage and produce objective responses (43). Therefore, at
the cellular level, antiestrogens might be expected to reduce proliferation and increase cell death (apoptosis). In support of this, other
studies have shown antiproliferative effects in primary breast cancer
with both tamoxifen (31) and the short acting formulation of ICI
182,780 (30, 32), and that both tamoxifen and ICI 182,780 may
induce apoptosis (33, 44).
As expected, in the present study, both tamoxifen and ICI 182,780
decreased the Ki67LI. ICI 182,780 produced a dose-dependent reduction in Ki67 labeling, with all three doses being significantly different
from placebo. There were no significant differences in tumor Ki67LI
between any dose of ICI 182,780 and tamoxifen. The dose-dependent
reduction in Ki67 labeling indicates that ICI 182,780 produces antiproliferative effects even after the short-term exposure used in this
study. ICI 182,780 might have been expected to produce a more
marked antiproliferative effect than tamoxifen in the light of data
demonstrating different modes of action (18, 19, 36, 37) and data
suggestive of good response rates and prolonged duration of action in
the early Phase II study (38 – 40).
The AI is a measure of the number of tumor cells induced to
undergo programmed cell death. In the present study, neither ICI
182,780 nor tamoxifen increased the AI when compared with placebo.
This lack of effect of either agent on apoptosis is surprising in view of
the earlier work showing that treatment with the short-acting formulation of ICI 182,780 significantly increased the AI in primary breast
tumors (33).
The observed lack of a difference between the effects of ICI
182,780 and tamoxifen on Ki67LI, and of either compound on AI,
may be a reflection of the study design. The (single) sampling time
may not have coincided with maximal antiproliferative or apoptotic
activity, or the treatment duration was insufficient for an effect on
apoptosis to become apparent. The precise relationship between dose
exposure and the pharmacodynamic effects remains to be determined,
but the current study shows that, even with a 50-mg dose, sufficient
ICI 182,780 was reaching the tumor to lead to marked biological
effects upon ER and PgR.
Overall, the data on ER and PgR, together with findings from
earlier studies, demonstrate that the mode of action and the biological
effects of ICI 182,780 in breast cancer are distinct from those of the
prototype SERM tamoxifen, and they support the concept that ICI
182,780 represents a novel class of antiestrogen—an ER down-regulator—that has estrogen-antagonist effects upon human breast tumor
cells in vivo. The clinical efficacy of ICI 182,780 is being evaluated
in Phase III clinical trials versus anastrozole (Arimidex) and tamoxifen in postmenopausal women with advanced breast cancer. These
The United Kingdom centers and principal investigators at those centers are as
follows: Nigel Bundred, South Manchester University Hospital, Manchester,
United Kingdom; Martin Cooper, Royal Devon and Exeter Hospital, Exeter,
United Kingdom; Michael Dixon, Edinburgh Breast Unit, Western General
Hospital, Edinburgh, Scotland; Prof. Oleg Eremin, Aberdeen Royal Infirmary,
Aberdeen, United Kingdom; John N. Fox, Castle Hill Hospital, Cottingham, East
Yorkshire, United Kingdom; Clive Griffiths, Royal Victoria Infirmary, NewcastleUpon-Tyne, United Kingdom; Chris Holcombe, Royal Liverpool University Hospital, Liverpool, United Kingdom; Stanley Kohlhardt, Royal Hallamshire Hospital,
Sheffield, United Kingdom; Mark Lansdown, St. James’ University Hospital,
Leeds, United Kingdom; Professor Robert E. Mansel, University of Wales College
of Medicine, Cardiff, Wales; William Odling-Smee, Belfast City Hospital, Belfast,
Northern Ireland; Arnie Purushotham, Western Infirmary, Glasgow, Scotland;
Zenon Rayter, Bristol Royal Infirmary, Bristol, United Kingdom; Prof. John F. R.
Robertson, Nottingham City Hospital, Nottingham, United Kingdom; John Sainsbury, Huddersfield Royal Infirmary, Huddersfield, United Kingdom; Alistair M.
Thompson, Ninewells, Hospital and Medical School, Dundee, Scotland.
We acknowledge the clinical and technical assistance of S. Kyme and P.
Finlay, Tenovus Centre for Cancer Research, Cardiff, Wales; T. McCready,
Castle Hill Hospital, Cottingham, East Yorkshire, United Kingdom; J. Salter
and M. Hills, Royal Marsden Hospital, London, United Kingdom; L. Barr, A.
Baildam, and M. McDonnell, South Manchester University Hospital,
Manchester, United Kingdom; and A. Cramer, Christie Hospital National
Health Service Trust, Manchester, United Kingdom.
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Comparison of the Short-Term Biological Effects of 7α
-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)-nonyl]estra-1,3,5,
(10)-triene-3,17 β-diol (Faslodex) versus Tamoxifen in
Postmenopausal Women with Primary Breast Cancer
John F. Robertson, Robert I. Nicholson, Nigel J. Bundred, et al.
Cancer Res 2001;61:6739-6746.
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