[CANCER RESEARCH 61, 673976746. September 15, 2001]
`
`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 Rwarch, Welsh School of Pharn‘aw, Cardiff, Wales
`CF10 3XF [R.|.N., J.M.WG.]; Department of Surgery, South Mancheser University Hospital, Mancheder M20 8LR, United Kingdom [N. J. 8.]; Clinical Research
`Departn‘ent, Christie Hospital National Health Service Trus, Manchester M20 4BX, United Kingdom [E.A.]; Departn‘ent of Biochem'stry, Royal Marsden Hospital, London
`SAB 6JJ, United Kingdom [M. Do.]; Bristol Royal Infirnary, Bristol DSZ 8HW, United Kingdom [Z. R.]; Casle Hill Hospital Cottingham, East Yorkshire HU16 5JQ, United
`Kingdom [J. N. F.]; AdraZeneca, Maoclesfield 3(10 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
`
`7n;-[9—(4,4,5,5,5—Pentafluoropentylsulf'myl)—n0nyl]estra—1,3,5, (10)—triene—
`3,17B—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 (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 iminunohistochemically in the matched pretreatment biopsy and
`the posttreatment surgical specimens. The apoptotic index was deter—
`mined by terminal de0xynucleotidyltransferase—mediated dUTP—hiotin
`nick end labeling on the same samples. The effects of treatment 011 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 = 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 manner 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 1PB, 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 hormone-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 arornatase 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 in-
`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), fomrerly 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); SERM, selective estrogen
`receptor modulator; ICI 182,780, 7oz—[9-(4,4,5,5,5-pentafiuoropentylsulfmy1)-nony1]estra-
`1,3,5. (10)-triene-3,l7B-diol; PgR. progesterone receptor(s); Ki67LL Ki67 proliferation-
`associated antigen labeling index; AI, apoptotic index; DAB, diaminobenzidine tetrahy-
`drochioride; ANCOVA, analysis of covariance; ICA. immunocytochemical assay; N'RS,
`normal rabbit serum.
`6739
`
`Downloaded from cancerres.aam’journalscrg on July 29, 2014. © 2001 American Association for Cancer Research.
`
`AstraZeneca Exhibit 2030 p. 1
`InnoPharma Licensing LLC v. AstraZeneca AB IPR2017-00904
`Fresenius-Kabi USA LLC v. AstraZeneca AB IPR2017-01910
`
`

`

`SHORT-TERM EFFECTS OF 1C1 182,780 IN PRIMARY BREAST CANCER
`
`Tamoxifen
`”Mag
`
`ICI 182,780
`
`0H
`
`~(‘Ci—izigsmcHammers
`
`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 dirnerization, 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 estrogen-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
`estrogen-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-term biological effects of 1C1 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 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 replacenrent therapy
`within 4 weeks of starting the trial; (b) baseline hematology or clinical
`chemistry outside the normal range; (0) 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 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 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.m. 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 tanroxiferr 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 Terrovus 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 7.2774).
`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 nrin 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
`staining, a blocking reagent, comprising 20% normal swine serum (Dako Ltd. ,_
`informed consent and underwent an initial eligibility screen in the week before
`randomization. Prestudy assessments included past medical history, concom-
`Glostrup, Denmark) in PBS was applied to the sections and then “tapped off”
`6740
`
`Downloaded from cancerres.aacrj‘ournalscrg on July 29, 2014. © 2001 American Association for Cancer Research.
`
`AstraZeneca Exhibit 2030 p. 2
`
`

`

`SHORT-TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
`
`Table 1 ER and PgR satus of tunnrs—per-protocol patients
`ICI 182,780
`ICI 182,780
`ICI 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 ovemight 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 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 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 attachment of a light microscope. Overall staining was
`assessed at X10, 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 (1X% i) i (2X% i
`i) i (3X% i
`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.
`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
`
`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% hematoxylin 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 AI 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 12.1 of 50 mM cobalt
`chloride, and 20 1.21 of reaction buffer (1 M sodium cacodylale + 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
`preceded development with 0.05% DAB and 0.07% imidazole for 30 s and
`then 10 min of incubation with 100 pd 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—poutive per-protocol patients
`ICI 182,780
`ICI 182,780
`ICI 182,780
` Characteristic Placebo 50 mg 125 mg 250 mg Tamoxifen
`
`
`
`
`
`Age (yr)
`n
`29
`33
`34
`32
`27
`Mean
`65.9
`69.2
`68.7
`66.1
`68.7
`SD
`9.2
`8.4
`7.3
`8.3
`8.4
`T1
`5 (17.2)
`2 (6.1)
`5 (14.7)
`2 (6.3)
`6 (22.2)
`T2
`10 (34.5)
`11 (33.3)
`10 (29.4)
`12 (37.5)
`9 (33.3)
`T3
`1(34)
`3 (9.1)
`1 (2.9)
`0 (0.0)
`1 (3.7)
`Not T4a
`13 (44.8)
`17 (51.5)
`18 (52.9)
`18 (56.3)
`11 (40.7)
`G1
`6 (20.7)
`6 (18.2)
`8 (23.5)
`9 (28.1)
`6 (22.2)
`Tumor grade at surgeryb
`G2
`14(483)
`16 (48.5)
`15 (44.1)
`16 (50.0)
`13 (48.1)
`n (%)
`G3
`7 (24.1)
`9 (27.3)
`9 (26.5)
`6 (18.8)
`7 (25.9)
`Gx 1(37) 2(69) 2 (6.1) 2 (5.9) 1 (3.1)
`
`
`
`
`
`5 Unable to categorize but definitely not T4.
`b G1, well-differentiated; G2, moderately differentiated; G3, poorly differentiated; GX, unassessable.
`6741
`
`Downloaded from cancerres.aacrjournaiscrg on July 29, 2014. © 2001 American Association for Cancer Research.
`
`AstraZeneca Exhibit 2030 p. 3
`
`

`

`SHORT-TERM EFFECTS OF ICI WEI/$0 IN PRIMARY BREAST CANCER
`
`Pretreatment
`
`Post-treatment
`
`
`
`
`
`[CI182,780(250mg)
`
`Fig. 2. Comparison of ER expresgion in a biopsy
`sample taken pretreatment with that from a sample
`taken from the same turner after treatment with 101
`182,783 (250 mg; Ail, tamosnfen (B), and tamoxifen
`placebo (Gj. ER iimnunopositivity appears as a
`erWn nuclear signal against a background of the
`green nuclear counterstam. Photographs supplied
`by R. L N.
`
`=
`.2.
`fi
`a
`a
`E"
`
`.8
`3
`.5fl-
`
`
`
`counterstained with hematoxylin in blood 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
`
`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 ANCQVA allowed an oVerall assess—
`ment 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.
`
`12°
`
`NS
`
`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 toler—
`ability and pharmacokinetie data (pharmacokinetie data are not pre—
`sented in this paper). This “per protocol” analysis included only those
`patients who received the full come 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, AstraZencoa.
`It was calculated that ~30 patients/group were needed to detect the
`following differences betvveen 161 182,780 and the comparator with
`80% power using a him-sided significance level of 5%: 10.3. for ER
`H—score; 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 primary end point data were nosessed statistically using
`ANGOVA according to treatment received with terms included in the
`model for treannent, center, and the baneline tumor marker value.
`Patients in the per-protocol population. who were FIR—negative were
`excluded from the analysis of ER, K167, and AI, and patients who
`Fig. 3. Postncatrnent mean ER H—s’coros after a single im. injection of 50 mg, 125 mg,
`or 250 mg of 1C1 182,780 or Tamoxifen 20 mg once daily po. or tamoxifen placebo.
`were PgR—ncgative were excluded from the analysis of PgR. In
`6742
`
`Ovemll treatment em P = 0.0003
`new
`
`9:0.0001
`s
`
`‘
`
`= .
`F
`J—l
`P=o.nzs
`
`
`
`
`r
`[N8l—l
`I
`¥
`,.
`
`100 w%
`60
`
`5 80
`Ill
`V-
`m
`+1
`2m
`
`0E
`
`a
`
`Placebo '
`(n = 29)
`
`125 mg
`50' mg
`ICI 132130 ac: 132,780
`(n= 31)
`(n = 32)
`
`250 7mg
`10: 182,780
`(n = 32)
`
`Tamoxifen
`(n = ’25}
`
`Downloaded from on ceneemetfleumefleo-r‘g; on July 29, .2014. © 2001 American Association for Cancer Research.
`
`AstraZeneca Exhibit 2030 p. 4
`
`

`

`SHORT-TERM EFFECTS OF 1C1 182,780 IN PRIMARY BREAST CANCER
`
`Table 3 Ser’ary of results for ER H-wore
`ICI 182,780
`101 182,780
`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
`r—flgflomi
`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 of 1C1 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 1C1 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 Ki67L1s are shown
`in Fig. 5 and the summary of results 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
`Ki67L1 and Al, the data showed evidence of nonnor‘mality, 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 Sinnery of results for PgR H-wore
`ICI 182780
`1C1 182,780
`ICI 182.780
`
`Placebo
`50 mg
`125 mg
`250 mg
`Tamoxifen
`n
`28
`29
`29
`29
`21
`Pretreatment mean H-score
`3’0
`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 (*46, 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
`
`Downloaded from cancerres.aacrj‘ournalsorg on July 29, 2014. © 2001 American Association for Cancer Research.
`
`AstraZeneca Exhibit 2030 p. 5
`
`

`

`SHORT-TERM EFFECTS OF ICI 182,780 IN PRIMARY BREAST CANCER
`
`Overall treatment effect P = 0.2382
`
`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. 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.
`
`Mean1’1SEM
`
`DISCUSSION
`
`This study provides the first direct comparison of ICI 182,780 with
`tamoxifen and placebo on the biological end points of breast tumor
`ERoz and PgR content, Ki67 labeli