`
`Comparison of the Short-Term Biological Effects of 7a-[9-(4,4,5,5,5-
`
`pentafluoropentylsulfinyl)-nonyl] estra-1,3,5, (10)—triene-3,17B-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
`Departn‘ent of Surgery, Nottingham City Hospital, Nottingham, United Kingdom [J. F. R.]; Tenovus Centre for Cancer Research, Welsh School of Pharnacy, Cardiff, Wales
`CF10 3XF [R.|.N., J.M.WG.]; Department of Surgery, South Mancheéer University Hospital, Manchester M20 8LR, United Kingdom [N. J. 8.]; Clinical Research
`Departrrent, Christie Hospital National Health Service Trust, Manchester M20 4BX, United Kingdom [E.A.]; Department of Biochem'stry, Royal Marsden Hospital, London
`3N3 6JJ, United Kingdom [M. Do.]; Bristol Royal Infirrrary, Bristol D52 8HW, United Kingdom [Z. R.]; Castle Hill Hospital Cottingham, East Yorkshire HU16 5JQ, United
`Kingdom [J. N. F.]; ASraZeneca, Maoclesfield SK10 4TF, United Kingdom [A.W, A. E.W, C. M.]; and Departn‘ent of Surgery, Western General Hospital, Edinburgh EH4 2XU,
`Scotland [M. Di.]
`
`ABSTRACT
`
`INTRODUCTION
`
`7o;-[9—(4,4,5,5,S—Pentafluoropentylsulf'myl)—nonyl]estra—1,3,5, (10)—triene—
`3,17B—diol (ICI 182,780; Faslodex) is a novel steroidal antiestrogen. This
`partially blind, randomized, nrulticenter study compared the effects of
`single doses of long—acting ICI 182,780 with tamoxifen or placebo on
`estrogen receptor (ERa) and progesterone receptor (PgR) content, Ki67
`proliferation—associated antigen labeling index (Ki67LI), and the apo—
`ptotic index in the primary breast tumors of postmenopausal women.
`Previously untreated patients (stages Tl—T3; ER—positive or —unknown)
`were randomized and received a single i.m. dose of ICI 182,780 50 mg
`(n = 39), ICI 182,780 125 mg (n = 38), or ICI 182,780 250 mg (n = 44) or
`oral tamoxifen 20 mg daily (n = 36) or matching tamoxifen placebo
`(n = 43) for 14—21 days before tumor resection surgery with curative
`intent. The ER and PgR H—scores, together with the Ki67LI were deter—
`mined immunohistochemically in the matched pretreatment biopsy and
`the posttreatment surgical specimens. The apoptotic index was deter—
`mined by terminal de0xynucleotidyltransferase—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—deperrdent 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 = 0.026; 125 mg, P = 0.006; 250 mg, P = 0.0001), and for ICI 182,780
`250 mg compared with tamoxifen (P = 0.024). For PgR H—score, there
`were statistically significant reductions after treatment with ICI 182,780
`125 mg (P = 0.003) and 250 mg (P = 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 tamoxifen—
`treated group. All doses of ICI 182,780 significantly reduced Ki67LI
`values compared with placebo (ICI 182,780 50 mg, P = 0.046; 125 mg,
`P = 0.001; 250 mg, P = 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 ERa in breast tumor cells
`in a dose—dependent marmer by down—regulating ER protein concentra—
`tion. 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.
`
`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).
`2To whom requests for reprints should be addressed. at Department of Surgery,
`Nottingham City Hospital, Hucknall Road, Nottingham, NG5 lPB, United Kingdom.
`
`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 honnone—sensitive
`breast cancer. In premenopausal women these include reducing cir-
`culating 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 antiestro-
`gen 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 irr-
`creased 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 (1 1).
`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, cross-
`resistance 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. l). 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 ER-
`agonist activity.
`ICI 182,780 has a binding affinity for the ER that is ~100 times
`
`3 The abbreviations used are: ER, estrogen receptor(s)', SERNI, selective estrogen
`receptor modulator; ICI 182,780, 7a-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)-nonyl]estra-
`l,3,5. (lO)-triene-3,l7B-diol; PgR. progesterone receptor(s)', Ki67LL Ki67 proliferation-
`associated antigen labeling index; AI, apoptotic index; DAB, diaminobenzidine tetrahy-
`drochloride', ANCOVA, analysis of covariance; ICA. irnmunocytochemical assay; N'RS,
`normal rabbit serum.
`6739
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`
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`
`
`SHORT-TERM EFFECTS OF 1C1 182,780 IN PRIMARY BREAST CANCER
`
`Tamoxifen
`NMEz
`
`ICI 182,780
`
`0H
`
`~(‘Ci—iflasmcHammers
`
`H0
`
`- 7
`
`Fig. 1. Chemical structures of the nonsteroidal SERM, tamoxifen, and of the novel
`nonagonist (pure) antiestrogen, 1C1 182,780.
`
`greater than that of tamoxifen (l7), and in animal models, 1C1 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). 1C1 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, 1C1 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 estrogerr—regulated gene (34), so drugs with
`estrogerric activity will increase its expression. Accordingly, tamox-
`ifen has been shown to increase PgR levels (35), whereas initial work
`on primary breast tumors found that a short-acting formulation of 1C1
`182,780 reduced PgR levels (30), suggesting that it
`is devoid of
`estrogerr-agonist activity and may have a different mechanism of
`action to that of tamoxifen. Additional evidence that 1C1 182,780 and
`tamoxifen have different underlying modes of action comes from
`studies showing that tamoxifen-resistant tumors remain sensitive to
`1C1 182,780 treatment in vitro (18, 19), in vivo (36, 37), and in the
`clinic (38—40).
`1C1 182,780 has antiproliferative effects, as assessed by immuno-
`histochemical detection of the Ki67 proliferation-associated antigen
`(30—32). Previous small clinical studies have suggested that both
`tamoxifen and 1C1 182,780 increase apoptosis in primary human
`breast cancer (33).
`The study reported here represents the first direct randomized
`comparison of the short-temr biological effects of 1C1 182,780 (50
`mg, 125 mg, or 250 mg as a single im. 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 dose-
`response effect of ICT 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 ERa (referred to as ER for the
`remainder of this paper) and PgR H—scores, Ki67L1, and the A1.
`
`itant therapy, demography, current medical conditions, hematology, and bio-
`chemistry 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 X log/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 14721 days (n = 37); or tamoxifen
`placebo, once daily p.o. for 14721 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 prerandornization 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 parafiin wax for sectioning and subsequent
`analysis of biological markers.
`
`Drug Administration
`
`Long acting ICI 182,780 (AstraZeneca, Macclesfield, United Kingdom) was
`administered by i.r’n. injection into the gluteus maxirnus muscle. Patients were
`randomized to receive 50 mg of 1C1 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 adminis-
`tered 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
`
`PATIENTS AND METHODS
`
`ER. ERa expression was assessed at the Tenovus Centre for Cancer
`Research, Cardiff, Wales, on sections cut from the formalin-fixed, paraffin-
`ernbedded tissue specimens described above. All mounted sections were dried
`overnight at 60°C before being dewaxed and relrydrated to PBS (pH 7277.4).
`Two hundred and one women with primary breast cancer participated in a
`Endogenous peroxidase activity was quenched by incubation in hydrogen
`multicenter, randomized, partially blinded study. The administration of tamox-
`peroxide (0.5% in methanol) for 10 min and then rinsing in running tap water
`ifen and tamoxifen placebo was double blind, and the administration of ICI
`for 5 min and in PBS for 5 min. Then sections were enzyme-digested in a bath
`182,780 (at one of three doses) was open. Postmenopausal women with
`of 0.02% Pronase E (Sigma Chemical Co., Poole, United Kingdom) in PBS at
`histologically proven primary breast cancer awaiting tumor resection were
`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.,
`Glostrup, Denmark) in PBS was applied to the sections and then “tapped off”
`randomization. Prestudy assessments included past medical history, concom-
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`
`
`SHORT-TERM EFFECTS OF 1C1 182,780 IN PRIMARY BREAST CANCER
`
`Table 1 ER and PgR satus of tunnrs—per-protoool patients
`1C1 182,780
`1C1182,780
`1C1 182,780
`
`Characteristic
`Placebo
`50 mg
`125 mg
`250 mg
`Tamoxifen
`ER status
`29 (69.0)
`33 (86.8)
`34 (89.5)
`32 (74.4)
`27 (81.8)
`Positive
`n (%)
`8 (19.0)
`4 (10.5)
`1 (2.6)
`6 (14.0)
`4 (12.1)
`Negative
`5 (11.9)
`1 (2.6)
`3 (7.9)
`5 (11.6)
`2 (6.1)
`Unknown
`28 (66.7)
`29 (76.3)
`29 (76.3)
`29 (67.4)
`21 (63.6)
`Positive
`PgR status
`10 (23.8)
`Negative
`n (%)
`7 (18.4)
`5 (13.2)
`9 (20.9)
`9 (27.3)
`
`Unknown
`4 (9.5)
`2 (5.3)
`4 (10.5)
`5 (11.6)
`3 (9.1)
`
`before incubation overnight at room temperature with the primary antibody
`(diluted 1:2), which was the rat antihuman ERa antibody (Clone P1222)
`supplied in the ER-ICA kit by Abbott Laboratories (North Chicago, IL).
`Sections were washed in PBS (5 X 4 min) and then a secondary biotinylated
`sheep arrtirat
`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 X 4 min) before the avidin-biotin-
`horseradish peroxidase complex (Dako Ltd.) diluted 1:120 in PBS was added
`for 60 min with additional washing afterward in PBS (5 X 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 X 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 X 3 min).
`The sections were counterstained with 0.5% methyl green for 5 min, washed
`in distilled water (2 X 3 min), dehydrated, cleared, and mounted for exami-
`nation by light microscopy.
`ERa 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 oftwo people (J. M. W. G. and R. I. N.)
`using the dual viewing attachinth of a light microscope. Overall staining was
`assessed at X 10, and a representative area was viewed at X40 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 X %
`i/
`)
`i
`(1 X% i) i (2X% i
`i) 1 (3X% 1
`i
`i)].Avalueof>0implies
`an ER-positive state with a range of 07300.
`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.
`
`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 X 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 X 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 X 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 counter-
`stained with 20% hernatoxylin for 375 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).
`Al. The Al was measured using the terminal deoxynucleotidyltransferase-
`mediated 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
`ul/slide of a reaction mixture containing 0.75 121 of terminal deoxynucleoti-
`dyltransferase, 0.50 [21 of biotinylated 16dUTP, 10 11.1 of 50 mM cobalt
`chloride, and 20 121 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 horse-
`radish peroxidase-conjugated streptavidin (Dako Ltd.) diluted 1:4000 in
`PBS + 1% BSA + 0.5% Tween 20. Another three washes in PBS/Tween 20
`
`Kifi7 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-l anti-Ki67
`antibody supplied by Coulter Electronics (Luton, United Kingdom). Briefly,
`slides were dewaxed and rehydrated to PBS (pH 7.6). Endogenous peroxidase
`
`preceded development with 0.05% DAB and 0.07% imidazole for 30 s and
`then 10 min of incubation with 100 [11 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,
`
`Clinical disease staging
`n (%)
`
`Table 2 Dermgraphic characterisics of ER—ptive per-protocol patients
`1C1 182,780
`1C1 182,780
`1C1 182,780
`
`Characteristic
`Placebo
`50 mg
`125 mg
`250 mg
`Tamoxifen
`Age (yr)
`29
`33
`34
`32
`27
`n
`65.9
`69.2
`68.7
`66.1
`68.7
`lVlean
`9.2
`8.4
`7.3
`8.3
`8.4
`SD
`5 (17.2)
`2 (6.1)
`5 (14.7)
`2 (6.3)
`6 (22.2)
`T1
`10 (34.5)
`11 (33.3)
`10 (29.4)
`12 (37.5)
`9 (33.3)
`T2
`1 (3.4)
`3 (9.1)
`1 (2.9)
`0 (0.0)
`1 (3.7)
`T3
`13 (44.8)
`17 (51.5)
`18 (52.9)
`18 (56.3)
`11 (40.7)
`Not T4a
`6 (20.7)
`6 (18.2)
`8 (23.5)
`9 (28.1)
`6 (22.2)
`G1
`Tumor grade at surgeryb
`14(483)
`16 (48.5)
`15 (44.1)
`16 (50.0)
`13 (48.1)
`G2
`n (%)
`7 (24.1)
`G3
`9 (27.3)
`9 (26.5)
`6 (18.8)
`7 (25.9)
`Gx 1(3.7) 2(69) 2 (6.1) 2 (5.9) 1 (3.1)
`
`
`
`
`
`5Unable to categorize. but definitely not T4.
`b G1, well-differentiated; G2, moderately differentiated; G3, poorly differentiated; GX, unassessable.
`6741
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`SHORT—TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
`
`Pretreatment
`
`Post-treatment
`
`
`
`aE
`
`. iE
`
`"
`“E
`3:H
`
`MU_
`
`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 1C1
`182,780 (250, mg; Ail, tamoxifen (Bi, and tamoxifen
`placebo (03. ER immunopositivity appears as a
`brown nuclear signal against a background of the
`green nuclear counterstain. Photographs supplied
`by R. I. N.
`
`=
`.2
`"51'
`a
`a
`E"
`
`o
`.9
`3
`.5fl-
`
`counterstained with hematorIylin in bluedtap 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
`
`addition, any patients in the per—protocol population with a
`value for a tumor tissue marker were also excluded from the analysis
`for that particular marker. The ANCOVA allowed an overall assess—
`ment of differences between each dose of ICI 182,780 and tamoxifen
`and each do so of ICI 182,780 and placebo. A test for overall treatment
`
`Ovarall treatment afloat P = 0.0003
`F a 0.049
`
`r
`P=0.0001
`E = dome
`1%
`P=o.nzs
`
`I
`
`NS
`
`12“
`
`
`
`i
`‘
`NS[—1
`I
`¥
`-_
`
`E
`
`E
`
`5 80
`1-
`
`100
`+1
`60
`oE 40
`
`This trial was an exploratory trial, so the minimum power required
`for statistical testing
`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 toler—
`ability and pharmacokinetic data (pharmacokinetic data are not pre—
`sented in this paper). This “per protocol” analysis included only tho sc
`patients who received the full course of treatment, completed the end
`of treatment assessment for the primary end point, and had no signif—
`icant 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—sccre, 0.4 for PgR H-score; 4.5 for Ki67; and 0.2 for apoptosis. To
`allow for ER—ngR—negative tumors, a total of 201 patients were
`recruited and ~40 were randomized to each treatment group.
`The
`end point data were assessed statistically using
`GOVA 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, K167, and AI, and patients who
`Fig. 3. Posttreah’nent mean ER H—scores after a single im. injection of 50 mg, 125 mg,
`
`or 250 mg of 1C1 182,780 or Tamoxifen 20 mg once daily 13.0. or tamomfen placebo.
`were PgR—negative were excluded from the analysis of PgR. In
`6742
`
`20
`
`mm
`
`:o
`
`o
`
`
`Placebo
`50 mg
`125 mg
`250 mg
`Tamoxifen
`([1 =29)
`ICI 132730 ICI 182,780
`101 182,780
`(n = 25)
`(n: 31)
`(n = 32)
`(n: 32)
`
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`AstraZeneca Exhibit 2030 p. 4
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`
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`SHORT-TERM EFFECTS OF 1C1 182,780 IN PRIMARY BREAST CANCER
`
`Table 3 Ser‘ary of results for ER H-soore
`1C1 182,780
`1c1 182,780
`1C1 182,780
`
`Placebo
`50 mg
`125 mg
`250 mg
`Tamoxifen
`n
`29
`31
`32
`32
`25
`Pretreatment mean H-score
`125
`136
`124
`1 13
`123
`Percentage change (posttreatment)
`713
`739
`750
`759
`736
`Overall treatment effect
`P : 0.0003
`Treatment difference vs placebo (95% CI)
`NA5
`
`729 (757, 70.2)
`760 (786, 734)
`747 (774. 721)
`730 (757, *4)
`P : 0.0485
`P : 0.0001
`P : 0.0006
`P : 0.0255
`Treatment difference VS tamoxifen (95% C1)
`29 (0.2, 57)
`*2 (729, 26)
`719 (746, 9)
`732 (759, *4)
`NA
`
` P : 0.0485 P : 0.8955 P : 0.1833 P : 0.0239
`
`
`
`
`ENA, not applicable.
`
`Overall treatment effect P = 0,0001
`
`RESULTS
`
`80
`
`P = "-0002
`
`
`
`Mean:1.-
`
`1SEM
`
`Placebo
`(n = 28)
`
`Patient Characteristics. A total of 201 postmenopausal women
`P =°-°°9°
`100 A (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
`l‘““"""—"|'
`treatment, and 10 patients withdrew from the trial. The withdrawal
`rates were similar for the 1C1 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 per-
`protocol population, 155 were ER-positive. Groups were well bal-
`anced 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 1C1
`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. 1C1 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 ofICI 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 1C1 182,780 and tamoxifen
`did reach significance (P = 0.0239).
`PgR Expression. Analysis of the PgR H—scores showed a signif-
`icant 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
`1C1 182,780, with the 125 mg and 250 mg doses of 1C1 182,780
`producing significantly greater reductions in PgR H—score than pla-
`cebo. Tamoxifen caused a significant increase in PgR H—score com-
`pared 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 Ki67L1 showed a significant overall treat-
`ment effect (P = 0.0029). The posttreatment mean Ki67LIs are shown
`in Fig. 5 and the summary ofresults are shown in Table 5. 1C1 182,780
`
`250 mg
`125 mg
`50 mg
`lCl 182,780
`lCl 182,780
`lCl 182.780
`(n = 29)
`(n = 29)
`(n = 29)
`Fig. 4. Posttreatment mean PgR H-scores after a single i.m. injection of 50 mg, 125 mg,
`or 250 mg of 1C1 182,780 or tamoxifen 20 mg once daily p.o. or tamoxifen placebo.
`
`Tamoxifen
`(n = 21)
`
`effect was undertaken. If this was significant at the 5% level, then the
`following pairwise comparisons were made: 1C1 182,780 50 mg
`versus placebo; 1C1 182,780 125 mg versus placebo; 1C1 182,780 250
`mg versus placebo and 1C1 182,780 50 mg versus tamoxifen; 1C1
`182,780 125 mg versus tamoxifen; and 1C1 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 Al, 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 i 1 SE by treatment
`group for each end point are also presented.
`
`Table 4 Sammy of results for PgR H-soore
`1C1 182,780
`1C1 182,780
`1C1 182,780
`
`Placebo
`50 mg
`125 mg
`250 mg
`Tamoxifen
`n
`28
`29
`29
`29
`21
`Pretreatment mean H-score
`30
`47
`28
`33
`49
`Percentage change (posttreatment)
`+43
`712
`752
`767
`+63
`Overall treatment effect
`P : 0.0001
`Treatment difference vs. placebo (95% C1)
`NAa
`
`27 (7, 47)
`735 (753, 717)
`728 (746, 710)
`714 (732, 5)
`P : 0.0090
`P : 0.0002
`P : 0.0030
`P : 0.1455
`Treatment difference vs. tamoxifen (95% C1)
`727 (747, *7)
`740 (760, 721)
`755 (775, 734)
`762 (782, 742)
`NA
`
`
` P : 0.0090 P : 0.0001 P : 0.0001 P : 0.0001
`
`
`
`7 NA, not applicable.
`
`6743
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`
`AstraZeneca Exhibit 2030 p. 5
`
`
`
`SHORT-TERM EFFECTS OF 1C1 182,780 IN PRIMARY BREAST CANCER
`
`Overall treatment effect P = 0.2382
`
`produced dose-dependent reductions in the Ki67L1 such that the
`Ki67LI at each dose of 1C1 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 1C1 182,780.
`AI. There was no statistically significant overall treatment effect
`on the A1 (P = 0.2382). There was no difference in A1 between any
`dose of 1C1 182,780 and tamoxifen compared with control. Posttreat—
`ment mean values for apoptosis are shown in Fig. 6, and the summary
`of results are shown in Table 6.
`
`Drug Tolerabflity. In general, all of the drugs were well tolerated.
`The most commonly reported adverse event were those relating to
`breast surgery.
`
`Mean11SEM
`
`DISCUSSION
`
`This study provides the first direct comparison of 1C1 182,780 with
`tamoxifen and placebo 011 the biological end points of breast tumor
`ERa and PgR content, Ki67 labeling, and A15 in p