Breast Cancer Res Treat (2012) 133:237-246
`DOI 10.1007/s10549-011-1947-7
`
`CLINICAL TRIAL
`
`Dose-dependent change in biomarkers during neoadjuvant
`endocrine therapy with fulvestrant: results from NEWEST,
`a randomized PhaseII study
`
`Irene Kuter - Julia M. W. Gee - Roberto Hegg « Christian F. Singer - Rajendra A. Badwe-
`Elizabeth S. Lowe » Ugochi A. Emeribe - Elizabeth Anderson « Francisco Sapunar °
`Pauline Finlay + Robert I. Nicholson « José Bines - Nadia Harbeck
`
`Received: 23 December 2011/ Accepted: 26 December 2011/Published online: 28 January 2012
`© Springer Science+Business Media, LLC. 2012
`
`Abstract NEWEST (Neoadjuvant Endocrine Therapy for
`Womenwith Estrogen-Sensitive Tumors)is the first study to
`compare biological and clinical activity of fulvestrant 500
`versus 250 mg in the neoadjuvant breast cancer setting. We
`hypothesized that fulvestrant 500 mg may be superior to
`250 mg in blocking estrogen receptor (ER) signaling and
`growth. A multicenter, randomized, open-label, Phase II
`study was performed to compare fulvestrant 500 mg
`(500 mg/month plus 500 mg on day 14 of month 1) versus
`fulvestrant 250 mg/month for 16 weeks prior to surgery in
`postmenopausal women with ER+ locally advanced breast
`cancer. Core biopsies at baseline, week 4, and surgery were
`
`
`On behalf of the NEWEST Investigators.
`The details of the investigators participating in the study are given in
`Appendix.
`
`Electronic supplementary material The online version of this
`article (doi: 10.1007/s10549-011-1947-7) contains supplementary
`material, which is available to authorized users.
`
`
`I. Kuter (4)
`Massachusetts General Hospital, Professional Office Building
`228, 55 Fruit Street, Boston, MA 02114, USA
`e-mail: ikuter@partners.org
`
`J. M. W. Gee - P. Finlay - R. I. Nicholson
`Tenovus Centre for Cancer Research, Welsh School of
`Pharmacy, Cardiff University, Cardiff, Wales, UK
`
`assessed for biomarker changes. Primary endpoint: change
`in Ki67 labeling index (LI) from baseline to week 4 deter-
`mined by automated computer imaging system (ACIS).
`Secondary endpoints: ER protein expression and function;
`progesterone receptor (PgR) expression; tumor response;
`tolerability. ER and PgR were examined retrospectively
`using the H score method. A total of 211 patients were
`randomized
`(fulvestrant 500mg: a= 109;
`250 mg:
`n = 102). At week 4, fulvestrant 500 mg resulted in greater
`reduction of Ki67 LI and ER expression versus 250 mg
`(—78.8 vs. —474% |p < 0.0001] and —25.0 vs. —13.5%
`[p = 0.0002], respectively [ACIS]); PgR suppression was
`not significantly different (—22.7 vs. —17.6; p = 0.5677).
`However, H score detected even greater suppression of ER
`(—50.3 vs. —13.7%; p < 0.0001) and greater PgR suppres-
`sion (—80.5 vs. —46.3%; p = 0.0018) for fulvestrant 500
`versus 250 mg. At week 16, tumorresponse rates were 22.9
`and 20.6% for fulvestrant 500 and 250 mg, respectively,
`with considerable decline in all markers by both ACIS and
`
`E. S. Lowe - U. A. Emeribe
`
`AstraZeneca, Wilmington, DE, USA
`
`E. Anderson - F. Sapunar
`Formerly AstraZeneca Pharmaceuticals, Macclesfield, UK
`
`J. Bines
`Instituto de Cancer, Rio de Janeiro, Brazil
`
`R. Hegg
`School of Medicine, University of Sao Paulo and Hospital Pérola
`Byington, Sao Paulo, Brazil
`
`N. Harbeck
`Frauenklinik der Technischen Universitat Miinchen, Munich,
`Germany
`
`C. F. Singer
`Division of Special Gynaecology, Medical University of Vienna,
`Vienna, Austria
`
`R. A. Badwe
`
`Tata Memorial Hospital, Mumbai, India
`
`Present Address:
`N. Harbeck
`
`Breast Centre, Department of Obstetrics and Gynaecology,
`University of Cologne, Cologne, Germany
`
`g) Springer
`
`AstraZeneca Exhibit 2077 p. 1
`InnoPharma Licensing LLC v. AstraZeneca AB IPR2017-00904
`Fresenius-Kabi USA LLCv. AstraZeneca AB IPR2017-01910
`
`

`

`238
`
`Breast Cancer Res Treat (2012) 133:237-246
`
`H score. No detrimental effects on endometrial thickness or
`
`bone markers and no new safety concerns were identified.
`This providesthe first evidence of greater biological activity
`for fulvestrant 500 versus 250 mg in depleting ER expres-
`sion, function, and growth.
`
`Keywords Estrogen receptor-positive breast cancer -
`Fulvestrant 500 mg - Faslodex® - Neoadjuvant-
`Biomarkers
`
`Introduction
`
`Endocrine therapy is commonly used in the neoadjuvant
`setting to attempt to downstage large primary tumors and
`permit breast conserving surgery [2]. This setting also
`enables assessment of tumor responsein situ and allows for
`further tailoring of subsequent adjuvant therapy based on
`the biological characteristics of the individual tumor.
`Fulvestrant is an estrogen receptor (ER) antagonist with
`no known agonist effects. Data from the recent COmparisoN
`of Fulvestrant In Recurrent or Metastatic breast cancer
`
`with fulvestrant 500 mg and fulvestrant 250 mg in terms of
`biological activity (Ki67 LI, ER,
`and PgR),
`tumor
`response, and tolerability in postmenopausal women with
`locally advanced breast cancer.
`Imaging
`The ChromaVision™ Automated Cellular
`System (ACIS) used in this study is an image analysis
`system that can detect and count individual pixels of two
`chromogen colors used to stain histological sections. The
`use of automated image analysis systems has become more
`frequent over recent years, although it has never previously
`been used for biomarker measurementin a fulvestrant trial
`
`setting. Therefore, we also used an established manual
`scoring method (7 score). Since the H score method has
`been used effectively in previous fulvestrant studies and
`those of other endocrine agents [12], its use in the present
`study enabled subsequent cross-study comparisons to be
`made whenconsidering the effects of fulvestrant observed
`here in the neoadjuvant setting.
`
`Patients and methods
`
`(CONFIRM) study showed that a high-dose regimen of
`Study design and patients
`fulvestrant 500 mg was associated withasignificantly longer
`progression-free survival than the 250 mg regimen (hazard
`NEWEST (Neoadjuvant Endocrine Therapy for Women
`ratio [HR] = 0.80, 95% confidence interval [CI] 0.68-0.94;
`with Estrogen-Sensitive Tumors; 9238IL/0065) was a
`p = 0.006), corresponding to a 20% reduction in the risk of
`randomized, open-label, multicenter, Phase
`JI
`study
`progression [5]. These data led to the approval of fulvestrant
`involving postmenopausal women with newly diagnosed,
`500 mg (500 mg on day 0, 14, 28, and every 28 days
`ER-positive,
`locally advanced breast cancer who had
`thereafter) for the treatment of postmenopausal womenwith
`received no prior breast cancer treatment (NCT0093002).
`locally advanced or metastatic breast cancer who have pro-
`Eligible patients (intent-to-treat
`[ITT] population) were
`gressed or recurred after prior anti-estrogen therapy. In the
`randomly assigned to receive neoadjuvant treatment with
`first-line setting, the randomized Phase II Fulvestrant fIRst-
`either fulvestrant 500 mg/month (plus 500 mg on day14 of
`month 1) or
`fulvestrant 250 mg/month for 16 weeks
`line Study comparing endocrine Treatments (FIRST) study
`demonstrated that fulvestrant 500 mg isat least as effective
`immediately before surgery.
`Womenhad to be postmenopausal (>60 years old, or age
`as anastrozole in terms of clinical benefit
`(odds ratio
`[OR] = 1.30, 95% CI 0.72-2.38; p = 0.386), and has a
`>45 years with amenorrhea for >12 months, or follicle-
`similar objective responserate (36.0 vs. 35.5%, respectively)
`stimulating hormone and estradiol levels within postmeno-
`[11]. In a preplanned follow-up analysis reporting mature
`pausal range, or prior bilateral oophorectomy). Other key
`data, time to progression was 23.4 months for fulvestrant
`inclusion criteria were: histologically or cytologically con-
`500 mg compared with
`13.1 months
`for
`anastrozole
`firmed invasive breast cancer; ER-positive disease as
`(HR = 0.66, 95% CI 0.47-0.92; p = 0.01) [13].
`determined locally; operable or potentially operable locally
`Two presurgical studies have previously shown that
`advanced tumor (T», 3, 4p, No-3, Mo); tumor size >2 cm;
`treatment with fulvestrant
`leads
`to a dose-dependent
`willingness to undergo biopsy procedures and surgery; and
`downregulation of ER, depletion of the ER-regulated pro-
`World Health Organization performance status 0-2. Key
`tein progesterone receptor (PgR), and reduction in prolif-
`exclusion criteria were: any previous treatment for breast
`erative activity as indicated by the Ki67 labeling index (LD
`cancer; inoperability; multifocal disease (>2 major tumor
`with doses up to 250 mg [4, 12]. It was expected, therefore,
`nodules); presence of metastatic disease; other current
`malignancyor prior malignancy within the previous 3 years;
`that neoadjuvant therapy with a high-dose regimen of ful-
`vestrant 500 mg would further increase biological activity
`abnormal laboratory values; any severe concurrent condi-
`on ER expression, function, and growth.
`tion; history of bleeding diathesis or need for long-term anti-
`Against this background, the current study was designed
`coagulant therapy; or treatment with a non-approved or
`to evaluate the effects of neoadjuvant endocrine therapy
`experimental drug within 4 weeks of randomization.
`
`g) Springer
`
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`
`

`

`Breast Cancer Res Treat (2012) 133:237-246
`
`239
`
`All patients provided written informed consent prior to
`registration. The study was conducted in accordance with
`the Declaration of Helsinki and with local ethics committee
`
`approval at each participating center (36 centers in Austria,
`Brazil, Germany, India,
`the United Kingdom, and the
`United States).
`
`Treatment
`
`Eligible patients were randomized 1:1 to receive either
`fulvestrant 500 mg or fulvestrant 250 mg for 16 weeks
`preceding the surgery. Fulvestrant 500 mg was given as
`two 5-mL intramuscular (IM) injections, one in each but-
`tock, on days 0, 14, 28, and every 28 days thereafter for
`16 weeks. Fulvestrant 250 mg was given as one 5-mL IM
`injection, in the buttock, on days 0, 28, and every 28 days
`thereafter for 16 weeks. Patients in the fulvestrant 250 mg
`atm did not receive additional (fulvestrant placebo) injec-
`tions. At the completion of 16 weeks of treatment, patients
`underwentdefinitive surgery (lumpectomy or mastectomy).
`
`Study objectives
`
`The primary objective of the study was to compare the
`effects of fulvestrant 500 and 250 mg on expression of the
`proliferation marker Ki67 after 4 weeks of treatment.
`Secondary objectives included: effects on ER and PgR
`expression,
`tumor response, and tolerability; effects on
`endometrial thickness and uterine dimensions; effects on
`serum markers of bone turnover (bone-specific alkaline
`phosphatase [ALP], C-terminal
`telopeptides of type-1
`collagen [CTX-1], and procollagen type 1 N propeptide
`[PINP]); and downstaging assessed by a comparison of the
`actual surgery performed at 16 weeks with the likely sur-
`gery predicted at study entry. Ki67 index and ER and PgR
`expression were also assessed at 16 weeks to monitor for
`sustained fulvestrant activity.
`
`MIB-1 anti-Ki67 antibody, the 1D5 anti-ER antibody or the
`PgR 636 anti-PgR antibody (all supplied by Dako, Ely, UK).
`Binding of the primary antibodies was visualized using an
`avidin-biotin complex and the chromogen 3,3’-diamino-
`benzide. The sections were lightly counterstained with
`hematoxylin before being dehydrated and mounted. Quality
`control slides were included in all assays to ensure consis-
`tency.
`In the first
`instance,
`the immunohistochemical
`staining of the tissues was assessed using the ChromaVi-
`sion’ ACIS. This system detects and determines the
`intensity and counts individual pixels of the two chromogen
`colors used in the immunohistochemical procedures (in this
`case, brown = positive; blue = negative). Wherever pos-
`sible, ten representative fields across each tumor specimen
`were scored; in cases whereten fields could not be obtained,
`every available tumor cell was includedin the analysis. The
`Ki67 LI was defined as the percentage of tumorcell nuclei
`positively stained with intensity above a predetermined
`threshold. In the case of ER and PgR, the meanintensity as
`well as the percentage of positively stained nuclei was
`calculated and combined to produce a proprietary histo-
`score. ER and PgR expression were also assessed retro-
`spectively on the same stained tissue samples using the
`Hscore method whichis derived by microscopic assessment
`of the percentage of tumor cells in each of five staining
`categories
`(negative, very weak, weak, moderate and
`strong) to give an H score ranging from 0 to 300 [8, 12]. This
`assessment was performedat the Tenovus Centre for Cancer
`Research by two experienced observers (JMWG and PP)
`who were blinded to the ACIS and clinical outcome data
`
`and reached a consensus for each slide. Sequential samples
`from each patient were evaluated at the same time to ensure
`comparative assessment of tumorhistology wherever pos-
`sible. To ensure the analysis was robust, only paired sam-
`ples for both the ACIS and H score methods were included.
`Any samples with non-specific staining or unacceptably low
`cellularity were eliminated from analysis.
`
`Study assessments
`
`Assessment of clinical response
`
`Assessment of biomarkers
`
`During the 16-week treatment phase, patients underwent
`clinical breast examination every 4 weeks. Tumor volume
`was measured by 3D ultrasound at baseline, week 4, and
`Core biopsies, using an 11- to 14-gauge needle, were taken
`after 16 weeks of treatment before definitive surgery.
`at baseline, at week 4, and at surgery (week 16). These
`Optional
`tumor measurements by magnetic resonance
`tumor cores were routinely formalin-fixed and paraffin-
`imaging (MRI) were obtained at baseline and 16 weeks.
`embedded locally, with central
`immunohistochemical
`assessment of changes in Ki67, ER, and PgR expression at
`Tumor response was defined as complete response (dis-
`appearance ofall lesions), partial response (>65% reduc-
`each time point using well-established methods[7]. Briefly,
`5-~um sections of pre- and post-treatment tissue samples
`
`tion in tumor volume by 3D_ultrasound), disease
`were dewaxed in xylene and rehydrated through graded
`progression (>73% increase in tumor volume), or stable
`alcohols after which endogenous peroxidase was blocked.
`disease (neither partial response nor disease progression)
`Following heat-mediated antigen retrieval and blocking of
`[17]. Objective responders were those patients with a
`non-specific binding, the sections were incubated with the
`complete response or partial response.
`
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`
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`
`

`

`240
`
`Statistical analysis
`
`Sample size calculation was based on the primary endpoint.
`
`Based on a 5.36% (+0.616) reduction in Ki67 values fol-
`lowing treatment with fulvestrant 250 mg in Study 018
`(which compared the short-term biological effects of ful-
`vestrant vs. tamoxifen) [12], a sample size of 80 patients
`per group would provide 80% powerto detect a difference
`of 0.274 in log-transformed Ki67 values at 4 weeks for
`fulvestrant 500 mg relative to 250 mg at the two-sided,
`p = 0.05 significance level. Data for the efficacy endpoints
`were analyzed and summarized on an ITT basis. Treatment
`differences in Ki67 LI between fulvestrant 500 and 250 mg
`were assessed using analysis of variance (ANOVA),
`modeling natural log-transformed changes from baseline
`Ki67 LI to Ki67 LI at week 4. Also, a post hoc ANOVA
`was used to assess the effects of fulvestrant 500 mg and
`fulvestrant 250 mg on ER and PgR expression derived by
`the ACIS method. For easier interpretation of the data,
`treatment effects (least squares mean and CIs) were back-
`transformed and expressed as percentages. Mean percent-
`age changes in H scores were calculated from baseline to
`weeks 4 and 16 using the manually derived score data.
`Differences in tumor response were analyzed using logistic
`regression. The safety population consisted of all patients
`whoreceived at least one dose of study drug. Only patients
`with a baseline endometrial thickness <5 mm were inclu-
`ded in the statistical analysis of this safety endpoint.
`
`Tolerability
`
`The frequency and severity of adverse events (AEs) were
`recorded throughout the study and up to 8 weeks after the
`last
`injection. Changes
`from baseline in endometrial
`thickness
`and uterine dimensions were
`assessed at
`
`16 weeks using transvaginal ultrasound(in all patients with
`an intact uterus). Patients with apparent thickening of the
`endometrium (>5 mm) or with suspicious ovarian findings
`were referred to a gynecologist
`for advice, but were
`allowed to continue the study unless the investigator
`decided otherwise. Serum was collected for analysis of
`bone CTX-1 (a marker of bone resorption) and of both
`ALP and PINP (markers of bone formation), which were
`assessed twice at baseline (before randomized treatment),
`then every 4 weeks until surgery.
`
`Results
`
`Patients
`
`Breast Cancer Res Treat (2012) 133:237-246
`
`receive fulvestrant 250 mg. The first subject was enrolled
`on 7 February 2005 and the last subject completed the
`study on 9 July 2007. Patient disposition throughout the
`study is shown in Supplemental Fig. 1. Overall, 99.0% of
`patients had ER-positive disease and only one patient in
`each group had unknown ERstatus. Patient demographics
`and characteristics
`at baseline were similar between
`
`groups, as outlined in Table 1. The mean age of patients
`enrolled was 67 years and 85.3% were Caucasian.
`
`Biological activity
`
`Fulvestrant 500 mg reduced mean Ki67 LI to a signifi-
`cantly greater extent
`than fulvestrant 250 mg (mean
`
`Table 1 Patient demographics and characteristics at baseline
`
`Mean age, years (range)
`Age category, n (%)
`<65 years
`>65years
`Race (%)
`Caucasian
`Black
`Oriental
`Other
`
`WHOperformance status (%)
`Unknown
`0
`1 or 2
`
`
`
`ER/PgRstatus (%)
`ER+/PgR+
`ER+/PgR—
`ER or PgR unknown
`Primary tumor stage (%)
`T2
`T3/T4b
`Unknown
`
`Tumorgrade (%)
`1
`2
`3
`
`Fulvestrant,
`500 mg
`(a = 109)
`
`Fulvestrant,
`250 mg
`(n = 102)
`
`66.9 (47-94)
`
`66.8 (47-87)
`
`46 (42.2
`63 (57.8)
`
`92 (84.4)
`5 (4.6)
`1 (0.9)
`11 (0.1)
`
`2 (1.9)
`19 (17.4)
`88 (80.7)
`
`76 (69.7)
`23 (21.1)
`10 (9.2)
`
`53 (48.6)
`55 (50.5)
`1 (0.9)
`
`12 (11.0)
`56 (51.4)
`18 (16.5)
`
`44 (43.1)
`58 (56.9)
`
`88 (86.3)
`3 (2.9)
`3 (2.9)
`8 (7.8)
`
`2 (2.0)
`16 (15.7)
`84 (82.4)
`
`72 (70.6)
`20 (19.6)
`10 (9.8)
`
`51 (0.0)
`50 (49.0)
`1 (1.0)
`
`9 (8.8)
`52 (51.0)
`21 (20.6)
`
`23 (21.1)
`
`Unassessable, missing or not done
`Intact uterus, (%)
`82 (80.4)
`87 (79.8)
`Yes
`14 (13.7)
`16 (14.7)
`No
`
`Unknown 6 (5.9) 6 (5.5)
`
`
`20 (19.6)
`
`A total of 211 women were included in the study; 109 were
`randomized to receive fulvestrant 500 mg and 102 to
`
`ER estrogen receptor, PgR progesterone receptor, WHO World Health
`Organization
`
`g) Springer
`
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`
`

`

`Breast Cancer Res Treat (2012) 133:237-246
`
`241
`
`
`Week 4
`Week 16
`
`=
`
`
`
`
`
`
`
`
`n=63
`
`n=60
`p< 0.0001
`
`n= $i
`
`n= 26
`p=0.1269
`
`10-4
`-20 4
`
`83
`
`oS
`Ze
`oo 2-
`DQ
`G2 40-5
`S6QO
`3 gt 507
`= =
`-60 4
`oO
`Oe
`=
`-70 4
`= 3
`g
`
`B04]
`“90 —
`
`
`
`
`Fulvestrant 500 mg
`
`
`
` Fulvestrant 250 mg
`
`
`
`Fig. 1 Effects of fulvestrant 500 mg and fulvestrant 250 mg on Ki67
`labeling index after 4 and 16 weeks of treatment (intent-to-treat).
`ACIS Automated Cellular Imaging System
`
`Table 2 Effects of fulvestrant 500 mg and fulvestrant 250 mg on
`Ki67 labeling index after 4 weeks of treatment (ITT)
`Fulvestrant
`Fulvestrant
`
`500 mg
`(a = 109)
`
`250 mg
`(n = 102)
`
`Evaluable patients*,
`Meanpercent reduction from
`baseline
`
`60
`—78.8
`
`63
`ATA
`
`95% CI
`Absolute reduction from
`baseline
`95% CI
`—15.7 to -18.8
` —6.3 to —13.6
`p value” <0.0001
`
`
`
`—70.8 to —84.6 —28.6 to —61.3
`-17.5
`—10.5
`
`Ki67 labeling index was determined by ChromaVision™ Automated
`Cellular Imaging System (ACIS)
`CI confidence interval, 777 intent-to-treat
`* Patients for whom data were available at both baseline and 4-week
`time. points
`> From ANOVA, modeled on the natural log-transformed change
`from baseline with treatment as a model term
`
`vs. —47.4%,
`from baseline: —78.8
`change
`percent
`p <0.0001) after 4 weeks of treatment (Fig. 1; Table 2).
`This corresponded with a significantly greater reduction in
`mean ER expression at week 4 for fulvestrant 500 mg
`compared with fulvestrant 250 mg using both ACIS and
`H scoring methods (Fig. 2a, b; Table 3). However,
`the
`magnitude of reduction caused by fulvestrant 500 mg
`detected by H score (—50.3%) was greater
`than that
`detected by ACIS (—25.0%). At week 16, reductions in
`mean Ki67 LI (—77.4 vs. —62.8%; Fig. 1) as well as mean
`ER expression by ACIS (—36.5 vs. —31.3%; Fig. 2a) and
`H score (—45.2. vs. —56.1%; Fig. 2b) were observed for
`both fulvestrant 500 mg and fulvestrant 250 mg, but the
`differences between the doses were not significant at this
`
`longer treatment time point. Fulvestrant 500 mg reduced
`mean PgR expression to a greater extent than fulvestrant
`250 mg at week 4 (Fig. 2c, d; Table 3). These differences
`reached statistical significance using the H score method
`(—80.5 vs. —46.3%; p = 0.0018; Table 3; Fig. 2d) but
`were not statistically significant according to ACIS data
`(—22.7 vs. —17.6%; Table 3; Fig. 2c), At week 16,
`decreases in PgR were observed relative to baseline, but
`there was no significant difference in PgR expression for
`fulvestrant 500 mg compared with fulvestrant 250 mg
`using either ACIS (—29.2 vs. —30.5%; Fig. 2c) or H score
`methods (—88.0 vs. —84.5%; p = 0.6445; Fig. 2d).
`
`Clinical activity
`
`At weeks 4 and 16, tumor response rates in the ITT popu-
`lation were numerically higher with fulvestrant 500 mg than
`with fulvestrant 250 mg (17.4 vs. 11.8% at week 4 and 22.9
`vs. 20.6% at week 16, respectively) (Table 4). In a post hoc
`analysis of evaluable patients with a baseline and a 16-week
`assessment (n = 69 in both arms), tumor response rates were
`36.2 and 30.4% for fulvestrant 500 mg and fulvestrant 250
`mg, respectively (Table 4). Overall, only 13% of evaluable
`patients progressed during the 16 weeks of therapy (fulve-
`strant 500 mg m = 8; fulvestrant 250 mg 2 = 10).
`
`Tolerability
`
`In total, 208 patients were eligible for assessment of tol-
`erability. Both treatments were well
`tolerated over the
`16-week treatment period. Treatment-related AEs were
`experienced by 37.4 and 30.7% of patients and treatment-
`related serious AEs by 0.9 and 3.0% of patients in the
`fulvestrant 500 mg
`and
`fulvestrant 250 mg groups,
`respectively. Only two AEs (one per group) led to with-
`drawal; neither was thought to be treatment-related (one
`transient ischemic attack; one pulmonary embolism). One
`patient randomized to fulvestrant 250 mg experienced an
`AE leading to death during the posttreatment follow-up
`period that was also not considered to be treatment-related
`(cause of death unknown, possibly cardiac-related). The
`most common AEsare described in Table 5.
`
`Both doses of fulvestrant reduced endometrial thickness,
`with changes after 16 weeks of treatment similar between
`fulvestrant 500 mg and fulvestrant 250 mg groups (Sup-
`plemental Table 1). Serum bone marker levels were similar
`within and between the two groups throughout the study,
`with neither dose producing substantial changesin anyof the
`three bone markers assessed (ALP, CTX, and PINP) (Sup-
`plemental Fig. 2). Few patients reported receiving prior
`medications
`(bisphosphonates, corticosteroids, hormone
`replacement therapy) that might confound interpretation of
`bone or endometrial data (fulvestrant 500 mg: 8 patients;
`
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`
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`
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`

`242
`
`Breast Cancer Res Treat (2012) 133:237-246
`
`_~»=
`
`300
`250
`
`100
`50
`
`200
`
`
`
`
`==
`150
`z
`=
`
`
`
`
`
`
`
`
`
`
`ms
`
`
`+
`
`
`
`
`
`ee
`
`Fulvestrant 500 mg
`Fulvestrant 250 mg
`
`
`
`
`
`
`
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`n=108 n=101 n=60 n=63 n=26 n=31
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`Time
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`Fulvestrant 250 mg
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`n=108 nm=101 n=59
`n=63 m=23 n=25
`0 weeks
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`Time
`
`***19 < 0.001 (fulvestrant 500 mg vs 250 mg)
`
`***9 < 0.001 (fulvestrant 500 mg vs 250 mg)
`
`fulvestrant500mgand250mg(ACIS)
`fulvestrant500mgand250mg(ACIS) E
`
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` Fulvestrant 250 mg
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`n=76 n=72 n=43 n=45 n=19 n=21
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`Fulvestrant 250 mg
`
`(Hscore)
`
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` fulvestrant500mgand250mg
`
`
`
`
`Time
`
`Time
`
`**9 < 0.01 (fulvestrant 500 mg vs 250 mg)
`
`Fig. 2 Effects of fulvestrant 500 mg and fulvestrant 250 mg after 4
`and 16 weeks of treatment
`(intent-to-treat) om: estrogen-receptor
`expression by a ACIS and b #H score, and progesterone receptor
`
`expression by ¢ ACIS and d H score. ACIS Automated Cellular
`Imaging System; ER estrogen receptor; PgR progesterone receptor.
`Horizontal line in box plots represents the median value
`
`fulvestrant 250 mg: 13 patients). Overall, AEs were con-
`sistent with the knowntoxicity profile of fulvestrant and no
`new safety concerns were identified.
`
`Discussion
`
`NEWEST isthe first study to compare both the biological
`and clinical activity of fulvestrant 500 and 250 mg regi-
`mens in the neoadjuvant breast cancer setting. Fulvestrant
`500 mg reduced mean Ki67 LI to a significantly greater
`extent than fulvestrant 250 mg at week 4 (p < 0.0001)—the
`primary endpoint. There was also a significantly greater
`reduction in ER (p <0.0001) and PgR (p = 0.0018)
`expression at week 4 with fulvestrant 500 mg using the
`H score method. These data provide the first indication that
`fulvestrant 500 mg has
`significantly greater biological
`activity on proliferative activity and ER and PgR expression
`than fulvestrant 250 mg in this setting. The results are
`consistent with the dose-dependent clinical effect
`for
`
`fulvestrant, with increased clinical efficacy recently repor-
`ted for the higher fulvestrant 500 mg dose regimen[5, 14].
`At week 16, similar degrees of reduction in Ki67 LI, ER,
`and PgR expression were observed with both doses. How-
`ever, the lower numbers of paired samples available at this
`time point (16-31; Fig. 2) and the resulting loss of statis-
`tical power to detect a treatment difference may be con-
`founding the current analysis. This study required adequate
`tumor biopsies to be taken at the specified time points
`(baseline, week 4, and at surgery), and only paired samples
`were analyzed. Since many centers were reluctant to permit
`taking more than two or three cores at each time point, and
`some cores contained limited tumor material, biomarkers
`could unfortunately not be measured on every patient. at
`every time point. In addition, 18 patients in the fulvestrant
`500 mg group and 23 in the fulvestrant 250 mg group did
`not undergo surgery at week 16 as per protocol, which also
`contributed to the low numberof paired samples available.
`The greater effect of fulvestrant 500 mg on Ki67, ER,
`and PgR at 4 weeks may beat least partly attributable to
`
`g) Springer
`
`AstraZeneca Exhibit 2077 p. 6
`
`

`

`Breast Cancer Res Treat (2012) 133:237-246
`
`243
`
`
`Table 3 Effects of fulvestrant 500 mg and fulvestrant 250 mg on ER and PgR expression after 4 weeks of treatment (ITT)
`ACIS
`HT score
`
`
`ER
`
`Evaluable patients*, n
`Mean percent reduction from baseline
`95% CI
`
`p value?
`PgR
`Evaluable. patients", 2
`Meanpercent reduction from baseline
`95% CI
`
`Fulvestrant
`
`500 mg
`(n = 109)
`
`Fulvestrant
`
`250 mg
`(n = 102)
`
`Fulvestrant
`
`500 mg
`(n = 109)
`
`60
`—25.0
`—21.0 to —28.8
`
`0.0002
`
`43
`—22.,7
`—9.5 to —33.9
`
`63
`—13.5
`—9.0 to —17.8
`
`45
`—17.6
`—4.0 to —29.3
`
`58
`—50.3
`—39.9 to —58.9
`
`<0.0001
`
`31
`—80.5
`—69.5 to —87.6
`
`Fulvestrant
`
`250 mg
`(n = 102)
`
`60
`—13.7
`—4.0 to —28.4
`
`34
`—46.3
`—17.5 to —65.0
`
`
`
`0.5677p value” 0.0018
`
`
`
`ACIS Automated Cellular Imaging System, C/ confidence interval, ER estrogen receptor, /7TT intent-to-treat, PgR progesterone receptor
`* Patients for whom data were available at both baseline and 4-week time points
`» Prom ANOVA, modeled on the natural log-transformed. change from baseline with treatment as a model term
`
`Table 4 Tumor response by 3D ultrasound (ITT)
`
`Fulvestrant 250 mg Odds ratio (CI)
`
`Fulvestrant 500 mg
`3
`and p value
`ITT population
`(n = 102)
`
`ITT population
`(n = 109)
`
`Evaluable for
`response
`
`Evaluable for
`response
`
`Time ofassessment
`
`Response n (%)
`
`Week 4
`
`Not determined*
`
`Objective response”
`Stable disease
`
`Disease progression
`Not evaluable
`
`12 (11.0)
`
`19 (17.4)
`65 (59.6)
`
`7 (6.4)
`6 (5.5)
`
`97 (100)
`(19.6)
`
`(67.0)
`(7.2)
`(6.2)
`
`69 (100)
`(36.2)
`
`7 (6.9)
`
`12 (11.8)
`
`77 (75.5)
`6 (5.9)
`0 (0.0)
`
`33 (32.4)
`
`21 (20.6)
`
`95 (100)
`(12.6)
`
`(81.1)
`(6.3)
`(0.0)
`
`69 (100)
`(30.4)
`
`1.68 (0.77-3.70)
`
`p = 0.1933°
`
`1.30 (0.64-2.64)
`
`p = 0.4705°
`
`Week 16
`
`Not determined*
`
`40 (36.7)
`
`Objective response”
`Stable disease
`
`25 (22.9)
`32 (29.4)
`
`(55.1)
`38 (37.3)
`(46.4)
`(14.5)
`10 (9.8)
`(11.6)
`8 (7.3)
`Disease progression
`
`
`0 (0.0)(5.8) (0.0)
`4 (3.7)
`Not evaluable
`
`CT confidence interval, /TT intent-to-treat
`* For patients who did not have data at baseline or time of assessment; "patients with complete or partial responses(all but one patient (250 mg
`group, week 16) had partial responses); “from logistic regression, modeled on objective response rate, with treatment as a model term
`
`the loading element of the fulvestrant 500 mg regimen,
`since steady-state plasma levels should have been reached
`at 4 weeks, whereas steady-state levels would not have
`been reached with fulvestrant 250 mg at this time [10].
`Here, we used two methods (ACIS and 7 score) of
`assessing ER and PgR expression. While interpretation of
`H score results is dependent.on the experience andability of
`the assigned experts, automated systems such as ACIS have
`been developed with a view to reducing intra- and inter-
`observer variability. However,
`the use of the H score
`
`technique does enable comparison with previous fulvestrant
`studies that have also used this methodology successfully.
`Interestingly, our analyses showed significant differences
`in sensitivity between ACIS and 7 score. Automated scoring
`by ACIS reduced the spread of individual measurements,
`making it difficult
`to detect a significant difference in
`treatment effect or to discriminate between fulvestrant
`
`doses, particularly for PgR. In contrast, H score data had a
`wider spread measured on a continuous scale encompassing
`both percentage positive and intensity (without athreshold
`
`g) Springer
`
`AstraZeneca Exhibit 2077 p. 7
`
`

`

`244
`
`Breast Cancer Res Treat (2012) 133:237-246
`
`(occurring in >5% of
`Table 5 Most common adverse events
`patients)
`
`Fulvestrant 500 mg
`(x = 107) %
`
`Fulvestrant 250 mg
`(a = 101) %
`
`Hotflash
`
`Injection-site pain
`Fatigue
`Nausea.
`Headache
`
`Hypertension
`Procedural pain
`Pain in extremity
`Cough
`Diarrhea
`
`Vomiting
`Peripheral edema
`Postoperative wound
`infection
`
`15 (14.0)
`
`16 (15.0)
`15 (14.0)
`11 (10.3)
`10 (9.3)
`
`T (6.5)
`8 (7.5)
`5 (4.7)
`8 (7.5)
`6 (5.6)
`
`3 (2.8)
`6 (5.6)
`5 (4.7)
`
`10 (9.9)
`
`4 (4.0)
`5 (5.0)
`T (6.9)
`8 (7.9)
`
`8 (7.9)
`5 (5.0)
`7 (6.9)
`4 (4.0)
`6 (5.9)
`
`8 (7.9)
`5 (5.0)
`6 (5.9)
`
`6 (5.9)
`4 (3.7)
`Back pain
`
`Pyrexia 2 (2.0) 7 (6.5)
`
`
`for positivity), and so differences at each time point could
`be more easily detected, with increased discrimination
`between doses. This is consistent with findings reported
`from a previous study (018) involving fulvestrant and using
`H score, where dose-dependent reductions in ER (—39, —
`50, and —59%) and PgR (—12, —52, and —67%) expression
`were reported 2—3 weeks after single doses of fulvestrant
`(50, 125, and 250 mg, respectively) [12]. However,
`the
`magnitude of reduction in ER (—59%) and PgR (—67%)
`expression induced by fulvestrant 250 mg in Study 018 was
`greater than that reported with fulvestrant 250 mg at week 4
`in this study (—13.5 and —46.3% for ER and PgR,respec-
`tively). This is undoubtedly due to differences in baseline
`H score values, which were much higher in NEWEST
`compared with Study 018, and which can beattributed to
`the increased sensitivity of the more recent immunohisto-
`chemical methods for determining ER and PgR status.
`These differences have impact on the magnitude of change
`in expression levels detected and therefore on any sub-
`sequent cross-trial comparisons made. Nonetheless, the data
`reported here support the hypothesis that higher fulvestrant
`doses lead to greater ER downregulation and subsequently
`decreased ER function in postmenopausal women with
`hormone-receptor positive breast cancer. Despite the high
`sensitivity and reproducibility of results reported previously
`with ACIS in both breast [1, 18] and colorectal tissue [9],
`our results suggest that H scoring may be a more suitable
`approach for examining changes in ER and PgR expression
`associated with neoadjuvant endocrine treatment.
`
`g) Springer
`
`In ter