WO2008009927A1 - Use of fulvestrant and an aromatase inhibitor for treating breast cancer - Google Patents

Abstract

The invention relates to a method of treatment of breast cancer which comprises administration of fulvestrant and simultaneous, separate or sequential treatment with an aromatase inhibitor, for example anastrozole, to a patient in need thereof wherein the patient has hormone receptor positive early breast cancer.

Description

USE OF FULVESTRANT AND AN AROMATASE INHIBITOR FOR TREATING BREAST CANCER

The invention relates to a method of treatment of breast cancer which comprises administration of fulvestrant and simultaneous, separate or sequential treatment with an aromatase inhibitor to a patient in need thereof wherein the patient has hormone receptor positive early breast cancer.

Clinical science is seeking to find the optimum drug regimen for breast cancer therapy. Fulvestrant (FASLODEX ™) is presently approved for the treatment of hormone receptor positive advanced breast cancer with disease progression following antioestrogen therapy. The approved dose is 250mg per month administered by intramuscular injection. Fulvestrant is not approved for treatment of early breast cancer.

Results of the ATAC (Arimidex™, Tamoxifen, Alone or in Combination) trial tested an aromatase inhibitor (Arimidex™, non-proprietary name = anastrozole) and an antioestrogen (tamoxifen) as combined or separate agents in over 9000 postmenopausal women with localised hormone receptor positive breast cancer. The combined agents showed low efficacy and anastrozole alone proved to be the optimum therapy (Lancet 2005, vol 365, 60-62).

Therefore there is a need to find an improved combination therapy for early breast cancer.

According to one aspect the present invention provides a method of treatment of breast cancer which comprises administration of fulvestrant and simultaneous, separate or sequential treatment with an aromatase inhibitor to a patient in need thereof wherein the patient has hormone receptor positive early breast cancer.

Preferably fulvestrant is administered as a 450-550 mg intramuscular injection monthly. Preferably one additional 450-550 mg dose is administered at day 14 of the first month. Preferably the dose of fulvestrant is about 500mg. Preferably the patient has hormone receptor positive early breast cancer and a biomarker indicative of a poor prognosis. Preferably the biomarker indicative of a poor prognosis is the presence of disseminated tumour cells in the bone marrow. Preferably the aromatase inhibitor is anastrozole, exemestane or letrozole. Preferably the aromatase inhibitor is anastrozole. Preferably the patient is a postmenopausal female. Another aspect of the present invention provides fulvestrant for use in treatment of hormone receptor positive early breast cancer in a patient wherein the patient is receiving simultaneous, sequential or separate treatment with an aromatase inhibitor.

Another aspect of the present invention provides fulvestrant in a package together with instructions for its use for treatment of hormone receptor positive early breast cancer with simultaneous, sequential or separate treatment with an aromatase inhibitor.

Another aspect of the present invention provides the use of fulvestrant for manufacture of a medicament for treatment of hormone receptor positive early breast cancer in a patient wherein the patient is receiving simultaneous, sequential or separate treatment with an aromatase inhibitor.

Another aspect of the present invention provides the use of fulvestrant for manufacture of a medicament in a package together with instructions for its use for treatment of hormone receptor positive early breast cancer with simultaneous, sequential or separate treatment with an aromatase inhibitor.

"Early breast cancer" is defined as cancer confined to the breast and/or local lymph nodes without inflammatory or metastatic breast cancer patients who have had their primary treatment with surgery, radiotherapy and/or chemotherapy and also those who are suitable for hormonal treatment before this happens (also called "neoadjuvant") or as a sole form of treatment. In another embodiment "early breast cancer" is defined as cancer confined to the breast and/or local lymph nodes without inflammatory or metastatic breast cancer patients who have had their primary treatment with surgery, radiotherapy and/or chemotherapy.

The invention is exemplified by the following non-limiting Example.

Example 1

A randomised Phase II Study comparing anastrozole and fulvestrant to anastrozole for adjuvant treatment of postmenopausal patients with early breast cancer

Objectives

• To compare the frequency of events (presence of DTC, clinical recurrence and / or death) after 12 months of randomised treatment (primary)

• To assess the safety of the anastrozole-fulvestrant combination (secondary) • To assess the frequency of events (presence of DTC, clinical recurrence and / or death) after 24 months of randomised treatment (secondary)

• To explore the clinical utility of comparing the numbers of DTC in patients before and after 12 and 24 months of randomised treatment by evaluating the degree of any DTC reduction in relation to clinical recurrence and / or death (exploratory)

• To explore the clinical utility (occurrence versus absence of clinical event) of a molecular approach for the assessment of disseminated ribonucleotides in BM as compared to immunocytochemistry in BM before and after 12 and 24 months of randomised treatment (exploratory)

• To explore the clinical utility (occurrence versus absence of clinical event) of a molecular approach for the assessment of circulating ribonucleotides in blood as compared to immunocytochemistry in BM before and after 12 and 24 months of randomised treatment (exploratory)

• To explore the clinical utility (occurrence versus absence of clinical event) of a molecular approach for the assessment of circulating methylated DNA in blood and BM as compared to immunocytochemistry in BM before and after 12 and 24 months of randomised treatment (exploratory)

• To explore the expression of hormone receptors, HER2 oncoprotein and broad stem cell characteristics of DTC before and after 12 and 24 months of randomised treatment (exploratory)

Study design

Multi-centre, open-label, randomised, controlled phase II proof of concept clinical trial. Target patient population

Adult postmenopausal (i.e., female) patients (n=176) with hormone-receptor positive non- metatstatic primary breast cancer and presence of DTC in BM at the time point of decision to administer adjuvant endocrine therapy. Investigational product, dosage and mode of administration

Combination of anastrozole 1 mg daily, orally, and fulvestrant 500 mg intramuscular injection monthly with one additional 500 mg dose at day 14 of the first month.

Comparator, dosage and mode of administration

Anastrozole 1 mg daily, orally Duration of treatment

• Duration of randomised treatment for this trial is 24 months

• Duration of SAE / AE reporting is 27 months and is concluded at the End-of-Study (EOS) visit (3 months ±4 weeks after the end of randomised treatment).

• Follow-up treatment is at the discretion of the treating physician. Continuation of anastrozole is recommended for a total of 5 years for patients in both treatment groups. Continuation of anastrozole-fulvestrant combination is not permitted and neither drug will be provided as study medication after the 2 year study period

Outcome variables 1. Efficacy

Primary outcome variable:

• Frequency of events (DTC-positive BM specimens, clinical recurrence, death) after 12 months of randomised treatment

Secondary outcome variable:

• Frequency of events (DTC-positive BM specimens, clinical recurrence, death) after 24 months of randomised treatment

Exploratory outcome variables:

• Number of DTC in BM and frequency of clinical recurrence / death after 12 and 24 months of randomised treatment • Frequency of molecular DTC markers (ribonucleotides) in BM and circulating tumour cell (CTC) markers (ribonucleotides, methylated DNA) in peripheral blood before and after 12 and 24 months of randomised treatment

• Frequency of expression of hormone receptors, HER2 oncoprotein and broad stem cell characteristics of DTC before and after 12 and 24 months of randomised treatment

2. Safety

Secondary outcome variable:

Frequency of National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE Version 3) grade 3 or 4 Adverse Events (AE), and Serious Adverse Events (SAE) after 12 and 24 months of randomised treatment. All adverse events will be recorded.

Statistical methods

Randomisation after stratification for country, hormone-receptor expression, any preceding chemotherapy; standard summary statistics of outcome variables; intention-to-treat analysis of efficacy of randomised treatment; summary of safety data of all patients who received at least one dose of trial therapy; odds ratios, associated confidence intervals and 2-sided p-values to compare "success" of randomised treatment; Kaplan-Meier survival estimates (log-rank test); multiple Cox proportional hazard regression model; linear regression, kappa coefficient.

LIST OF ABBREVIATIONS AND DEFINITION OF TERMS

Abbreviation or Explanation special term

ABCSG Austrian Breast and Colorectal Cancer Study Group

AE Adverse event (see definition in Section 4.7.1.1)

AP Alkaline phosphatase

ALT Alanine aminotransferase

AI Aromatase inhibitor

ASA Acetylsalicylic Acid (aspirin)

ASCO American Society of Clinical Oncology

AST Aspartate aminotransferase

Assessment An observation made on a variable involving a subjective judgment

AUC Area under the plasma concentration time curve from zero to infinity

BM Bone marrow

BMA Bone marrow aspiration

BP Blood pressure

CI Confidence interval

^trough Trough concentration ^max Maximum concentration

Clinical recurrence Occurrence of locally, regionally recurrent breast cancer and / or distant metastases after primary surgical, systemic and / or radiologic treatment

CR Complete response

CRA Clinical Research Associate

CRF Case Report Form

CRO Contract Research Organisation

CT Computerised tomography Abbreviation or Explanation special term

CTC Circulating tumour cells d day

DCIS Ductal carcinoma in situ

DHEA Dihydroepiandrosterone

DIC Disseminated intravascular coagulation

DTC Acronym for presence of immunocy tochemically positive cells in bone marrow aspirate after exclusion of immunostained cells with clear haematopoietic morphology

ECG Electrocardiogram Endpoint A status of the patient that constitutes the 'endpoint' of a patient's participation in a clinical study and that is used as the final outcome.

EOS End-of-Study-Treatment ER Oestrogen receptor

Event Presence of DTC after 12 months AND/OR a clinical event, defined as any clinical recurrence and/or death from any cause within 12 months (see definition in Section 4.6)

FDA Food and Drug Administration

FFPE Formalin-fixed, paraffin-embedded

FSH Follicle-stimulating hormone

Fulvestrant 500 mg Fulvestrant 500 mg im on days 0, 14 (±3 days), 28 (±3 days) and every 28 regime (±3 days)

GCP Good Clinical Practice

HDPE High density polyethylene

HER-2 Human epithelial growth factor receptor 2

HR Hormone receptor

HRT Hormone replacement therapy

ICH International Conference on Harmonisation Abbreviation or Explanation special term

IEC Independent Ethics Committee im Intramuscular

INR International normalized ratio

IRB Institutional Review Board

L Litre

LA Long-acting

LH Luteinising hormone

LH-RH Luteinising hormone-releasing hormone

LMWH Low Molecular Weight Heparin

Measurement An observation made on a variable using a measurement device.

MedDRA Medical dictionary for Regulatory Activities mg Milligram mL Millilitre monthly 28 ±3 days

MRI Magnetic resonance imaging

NCI CTCAE National Cancer Institute Common Terminology Criteria for Adverse

Events

NCR No carbon required

NE Not Evaluable

OAE Other significant Adverse Event (i.e., an adverse event of special interest in this clinical development; see definition in Section 4.7.1.1.). AstraZeneca drug safety physicians will perform the classification of OAEs after the study is complete.

OR Objective Response PD Progressive disease PFS Pre-filled syringes Abbreviation or Explanation special term

PgR Progesterone receptor po By mouth

PK Pharmacokinetics

PP Per protocol

PR Partial response

Principal Investigator A person responsible for the conduct of a clinical study at an investigational study centre. Every investigational study centre has a principal investigator.

RT-PCR Reverse-transcriptase polymerase chain reaction

SAE Serious adverse event (see definition in Section 4.7.1.1).

SAP Statistical analysis plan sd Standard deviation

SD Stable disease

SERM Selective Oestrogen Receptor Modulator

SGOT Serum glutamate oxalacetate transaminase

SGPT Serum glutamate pyruvate transaminase

SOP Standard operational procedure

TTP Time to progression

ULRR Upper limit of reference range

WHO World Health Organisation

1 BACKGROUND

Breast cancer and hormones

Breast cancer is one of the most common female cancers and the most common cause of cancer deaths in women. It comprises 18% of all female cancers worldwide (McPherson et al, 2000). The incidence varies among populations with about half of all cases occurring in North America and Western Europe. Oestrogen acts as an endocrine growth factor for at least one third of breast cancers. It has long been acknowledged that many cancers are hormone dependent and that hormonal manipulation can affect the progress of the disease. The effects of hormonal manipulation on metastatic breast cancer were observed as early as 1896 following removal of the oestrogenic stimulus by bilateral oophorectomy (Beatson, 1896). The most important factor determining response to hormonal manipulation is the presence of the oestrogen receptor in the target tissue (Fisher et al, 2001).

The anti-oestrogen tamoxifen (AstraZeneca, Nolvadex™) is the most widely used hormonal treatment for breast cancer in both pre- and postmenopausal women. This drug has been used to treat patients with breast cancer in advanced disease, as an adjuvant therapy after surgery, and for the treatment of ductal carcinoma in situ (DCIS), and also to reduce the risk of breast cancer development in women at high risk (Fisher et al, 1998). Despite its demonstrated efficacy in these patient populations, de novo resistance or acquired resistance may occur after prolonged treatment limiting the effectiveness of tamoxifen in many patients. In some patients the disease progresses during therapy because tumour growth may also be stimulated by tamoxifen (Weibe et al, 1993).

Because the major source of oestrogen in postmenopausal women is aromatase-mediated conversion of circulating androstenedione to estrone in peripheral tissues, an alternative approach to management of breast cancer has been the use of aromatase inhibitors (AI). The development of oral, selective, non-steroidal AI led to the introduction of well-tolerated agents with clear evidence of clinical benefit. The third generation oral AIs, i.e., anastrozole (Arimidex™, AstraZeneca), letrozole (Femara™, Novartis, USA), and the steroidal, type I inhibitor, exemestane (Aromasin™, Pfizer, USA), have now been tested in phase III trials where each has been shown to be more effective than the progestin megestrol acetate (Megace™, Bristol-Myers Squibb, USA) (Buzdar et al, 1998, Buzdar et al, 2001, Dombernowsky et al, 1998, Kaufman et al, 2000) and two have been shown to be more effective than tamoxifen (Bonneterre et al, 2001, Mouridsen et al, 2001). Anastrozole greatly reduces estradiol levels in both serum and tumours in postmenopausal women leading to clinical benefits in women with hormone-responsive breast cancer (Geisler et al, 1996, 2001). Since its introduction, anastrozole has displaced older therapies (i.e., progestins, androgens) and become widely used in patients whose disease has progressed on anti-oestrogen therapy (Buzdar et al, 1998). Studies showing a survival advantage for patients treated with anastrozole over the progestin megestrol acetate have made selective nonsteroidal AIs the treatment of choice. At 31 months follow-up, anastrozole (1 mg) demonstrated a significant survival advantage over megestrol acetate (Buzdar et al, 1998, Messori et al, 2000). AIs are now considered established first- and second-line hormonal agents.

For the adjuvant treatment of early stage breast cancer, data from four published (Jakesz et al, 2005; Coombes et al, 2004; Goss et al, 2003; Baum et al, 2002) and one orally reported trial (IBCSG 18-98/BIG 1-98 trial by Thurlimann et al, 2005) are currently available. Irrespective of the potential differences between the drugs used in these trials, a common result is the improved therapeutic index of aromatase-inhibitors over that of tamoxifen. Although to date no survival benefit has been demonstrated, the strong evidence for improved disease-free and recurrence-free survival data prompted the Intenational St. Gallen Consensus Panel to propose the use of aromatase-mhibitors in the clinical situations tested in the respective trials. Breast cancer and occult haematogenous tumour cell spread

Overall and for several subgroups, the adverse prognostic influence of the presence of DTC on clinical patient outcome indices, including overall, disease-free and distant metastasis-free survival, has been conclusively shown in a pooled analysis of individual patient data (Braun et al, 2005). In this study, data from the nine studies involving 4703 breast cancer patients with stage I-III disease (Pierga et al, 2004; Wiedswang et al, 2003; Gebauer et al, 2001; Gerber et al, 2001; Braun et al, 2000a; Mansi et al, 1999; Diel et al, 1996) had been included and data on long-term outcome had been made available. Conclusive data of level-I evidence show that the presence of DTC in the BM at the time of diagnosis of breast cancer is associated with a poor prognosis.

The actual presence of tumour cells outside the primary tumour and in organs relevant for subsequent metastasis formation, such as bone and BM, would serve three purposes that could be clinically useful: i) as unambiguous evidence for an early occult spread of tumour cells (Braun et al, 2005; Pierga et al, 2004; Wiedswang et al, 2003; Gebauer et al, 2001; Gerber et al, 2001; Braun et al, 2000a; Mansi et al, 1999; Diel et al, 1996); ii) as a relevant risk factor for subsequent metastasis and, thus, a poor prognosis (Braun et al, 2005; Pierga et al, 2004; Wiedswang et al, 2003; Gebauer et al, 2001; Gerber et al, 2001; Braun et al, 2000a; Mansi et al, 1999; Diel et al, 1996); and iii) as a marker for monitoring treatment susceptibility (Braun & Naume, 2005; Janni et al, 2005; Wiedswang et al, 2004). Finally, genotyping and phenotyping of CTC and DTC should provide detailed insight into the metastatic process and permit direct exploration of targeted treatment strategies (Pantel & Brakenhoff, 2004). In the light of the overall performance of current standard adjuvant treatment regimens, the need for better predictive markers is frequently addressed. Such markers are needed to improve selection of both low-risk patients with no need to up-scale systemic adjuvant treatment and high-risk patients with a clear need for improved control of microscopic residual disease, since they frequently recur despite the administration of recommended standard therapy. The pooled analysis conclusively demonstrated that the presence of DTC predicts poor outcome of patients treated with (inadequate?) adjuvant chemotherapy or endocrine therapy (Braun et al, 2005). Therefore, we clearly need studies that will permit to the use of individualized treatment based on the individual risk of harbouring DTC at relevant metastasis sites. In the long run, only such studies will enable us to alter consensus treatment recommendations toward individualized therapy. The currently available data strongly support the view that DTCs and CTCs are candidate surrogate markers suitable for various clinical settings and that these options in principle should be tested in proof-of-principle clinical trials, as presented here.

Recent studies on hormone therapy of patients with a history of receptor-positive breast cancer found that aromatase inhibitor treatment, therapy prolongation beyond 5 years and alteration after 2-3 years are beneficial strategies, which reduce the overall rate of recurrences (Jakesz et al, 2005; Coombes et al, 2004; Goss et al, 2003; Baum et al, 2002). A diagnostic test specifically designed for the selection of patients, who could benefit from either prolonged or altered hormonal treatment would be most helpful. Again, presence of CTC or DTC, or both, might be an indicator for both patients who have not responded to the previous treatment and are likely to recur in the near future. Rationale for this study

The search for an anti-oestrogen which is devoid of the agonist activity of tamoxifen and which can effectively block oestrogen receptor (ER) activity resulted in the discovery and clinical development of fulvestrant (Faslodex™). Fulvestrant is a new ER antagonist, without known agonistic properties, that downregulates cellular levels of the ER in a dose-dependent manner (Howell et al, 2000, Robertson et al, 2001, Wakeling et al, 1991). Fulvestrant is well- tolerated and has demonstrated efficacy in women whose breast cancer has progressed following tamoxifen therapy (Howell et al, 2002. Osborne et al, 2002). The results from two phase III trials showed that fulvestrant, given at a dose of 250 mg every 28 days, was at least as effective for both Time To Progression (TTP) and Objective Response (OR) as anastrozole (1 mg/day).

The 250 mg dose and the 28 days schedule first received regulatory approval in the USA, and has since been approved in 35 markets worldwide, including Canada and the European Union. However, evidence from a number of studies suggests that higher doses of fulvestrant may enhance efficacy further (see Section 3.2).

Patients with DTC represent a subgroup of patients in high-risk for relapse, which justifies that they should be suitable for initial testing of fulvestrant in the adjuvant setting. Furthermore, as both the detection of DTC during and persistence of DTC after therapy identifies patients with poor prognosis, DTC can be tested as a surrogate marker for subsequent clinical relapses and consequently for evaluation of the efficacy of the anastrozole-fulvestrant combination.

In contrast to anti-tumour strategies involving chemotherapy regimens, which have been reported to be rather ineffective in DTC elimination in very high-risk patients (Braun et al, 2000b), targeted and more tumour-cell specific approaches, including antibodies (Braun et al, 1999) and bisphosphonates (Rack et al. ASCO 2004), have shown more promising results in eliminating DTC. Completely antagonising the oestrogen receptor represents an improved targeted and, hence, specific approach to eliminate DTC as compared to the sole targeting the peripheral aromatase to prevent aromatase-mediated production of estrone.

2. STUDY OBJECTIVES

2.1 Primary objective

2.1.1 Efficacy (presence / absence of DTC after 12 months and outcome)

The primary objective of this study is to compare the frequency of events (i.e., presence of DTC in BM, clinical recurrence and/or death) in patients treated with anastrozole-fulvestrant combination as compared to that in patients treated with anastrozole alone by assessment of the BM status (i.e., the presence or absence of DTC) and the occurrence of clinical recurrences and/or deaths after 12 months (with a diagnostic window of ±3 weeks) of randomised treatment.

2.2 Secondary objectives 2.2.1 Safety

A secondary objective of this study is to assess the safety of the anastrozole-fulvestrant combination by evaluation of the rate of NCI CTCAE grade 3 and 4 adverse events (AE) and serious AE (SAE) after 12 and 24 months (with a diagnostic window of ±3 weeks) of randomised treatment. All adverse events will be recorded. 2.2.2 Efficacy (presence / absence of DTC after 24 months and outcome)

A secondary objective of the study is to compare the frequency of events (i.e., presence of DTC in BM, clinical recurrence and/or death) in patients treated with anastrozole-fulvestrant combination as compared to that in patients treated with anastrozole alone by assessment of the BM status (i.e., the presence or absence of DTC) and the occurrence of clinical recurrences and/or deaths after 24 months (with a diagnostic window of ±3 weeks) of randomised treatment.

2.3 Exploratory objectives

2.3.1 Efficacy (Outcome / survival and number of DTC after 12 and 24 months)

An exploratory objective of the study is to compare the numbers of DTC in patients before and after 12 months ±3 weeks and 24 months ±3 weeks of treatment with anastrozole- fulvestrant combination to that in patients before and after 12 months ±3 weeks and 24 months ±3 weeks of treatment with anastrozole by evaluating the degree of any DTC reduction in relation to clinical recurrences and/or deaths.

2.3.2 Molecular DTC assessment

An exploratory objective of the study is to investigate the clinical utility (occurrence versus absence of clinical event) of a molecular approach for the assessment of disseminated ribonucleotides by utilizing a multi-marker RT-PCR and its concordance with the standard immunocytocheniical assay for the detection of DTC before and after 12 and 24 months of randomised treatment.

2.3.3 Molecular CTC assessment

An exploratory objective of the study is to investigate the clinical utility (occurrence versus absence of clinical event) of a molecular approach for the assessment of circulating ribonucleotides by utilizing a multi-marker RT-PCR and its concordance with the standard immunocytochemical assay for the detection of DTC before and after 12 and 24 months of randomised treatment.

2.3.4 Molecular assessment of circulating methylated DNA

An exploratory objective of the study is to explore the clinical utility (occurrence versus absence of clinical event) of a molecular approach for the assessment of circulating tumour- cell derived methylated nucleotides (presence versus absence of ribonucleotides; e.g., RASSFl) and its concordance with the standard immunocytochemical assay for the detection of DTC before and after 12 and 24 months of randomised treatment. 2.3.5 Phenotyping and genotyping of DTC

An exploratory objective of the study is to investigate the expression of hormone receptors, HER2 oncoprotein and broad stem cell characteristics (e.g., CD44, CD24) of DTC. In selected cases, broad amplification of DTC genome is attempted to assess genetic changes, including loss of heterozygosity. Other translational research projects may be initiated.

3. STUDY PLAN AND PROCEDURES 3.1 Overall study design and flow chart

Figure 1 displays a brief overview of the study design.

This is a multi centre, open label, randomised phase II trial on postmenopausal patients with hormone-receptor positive, non-metastatic primary breast cancer with DTC in BM to compare the efficacy of anastrozole-fulvestrant combination in eliminating DTC to that of anastrozole alone. Between 600 to 1,000 patients (50 to 300 in each country) will be screened for the presence of DTC, and of those with DTC, approximately 176 will be subsequently

randomised (allowing for 26% dropouts) to detect, with a 2-sided alpha = 0.05, a 75% reduction in DTC-positive cases with / without clinical evidence of any disease recurrence or death during 12 months of anastrozole-fulvestrant treatment and a 50% reduction DTC- positive cases with / without clinical evidence of any disease recurrence or death during 12 months of anastrozole treatment. DTC-negative patients will excluded from the trials and will immediately leave the trial after screening and assessment of a DTC-negative BM result. Treatment period for the comparison of anastrozole-fulvestrant combination and anastrozole alone is 24 months. For this period, SAE / AE reporting, close clinical documentation every 3 months and efficacy assessment by biomarker testing (bone marrow aspiration, peripheral blood draws) after 12 and 24 months is performed. An End-of-Study-Treatment visit is scheduled at 3 months (±4 weeks) after the last fulvestrant injection, which is 27 months (±4 weeks) after the start of randomised treatment. Table 1 — Study plan

a Cycle refers to fulvestrant injection q28±3 days, except first cycle with an additional application on day 14 and/or dispensing of anastrozole tablets; check, evaluation of safety and efficacy; EOS, End-of-Study visit (also if a patient is withdrawn from / discontinues study except for clinical events). b Chemistry (total bilirubin, AST, ALT, AP, creatinine), Haematology (neutrophils, platelets), Clinical Visits with assessment of disease status, vital signs (blood pressure, heart rate, temperature), weight, height, WHO performance status and AEs are scheduled every 3 months; additional blood test to be done as clinically indicated c May be replaced by chest and abdomen CT scan; additional radiologic imaging to be done as clinically indicated; radiologic imaging for absence of distant metastases may be performed after receipt of the DTC-positive bone marrow result that qualifies for study enrolment and randomisation; after randomisation patients must be treated within 7 days. d Bone scan only when clinical symptoms are present. e If patient is DTC-positive from the screening sample but requires further surgery, the screening period or the interval between randomisation and starting treatment can be increased by 2 weeks.

3.2 Rationale

3.2.1 Rationale for study design, doses and control groups

3.2.1.1 Rationale for the dose of fulvestrant

Despite advances in the treatment of postmenopausal women with hormone receptor positive breast cancer (Jakesz et al, 2005; Coombes et al, 2004; Goss et al, 2003; Baum et al, 2002), a relatively similar (though increasingly smaller) fraction of patients will consistently experience disease recurrence over time and progress from this relapse to often fatal distant metastasis. The development of more effective hormonal strategies is therefore still needed.

• Fulvestrant at a dose of 250 mg every 28 days is the first oestrogen receptor antagonist shown to be at least as effective as a third-generation aromatase inhibitor in the second- line treatment of advanced breast cancer (Howell et al, 2002, Osborne et al, 2002). In these studies, overall survival was also similar between the fulvestrant and anastrozole treatment arms (Pippen et al, 2003). By using the fulvestrant 500 mg regimen, both earlier achievement and higher levels of steady-state plasma concentrations may contribute to lower treatment failures, especially if given in addition to peripheral aromatase-inhibition.

• The pharmacokinetic (PK) modelling (see Figure 2) providing evidence that an increase in fulvestrant dose and dosing schedule will result in a:

• decreased time to steady state plasma levels • higher AUC and C_max

• higher trough level and therefore maintenance of a higher exposure throughout the dosing interval

At present, no dose-ranging trials to assess efficacy beyond the 250 mg every 28 days dosing schedule have been completed. The delivery of a higher dose of fulvestrant in a 5 mL volume is currently constrained by the limits of the formulation, and what is perceived as being a clinically acceptable schedule of administration. However, because of the favourable safety profile of fulvestrant, administration of a higher dose (volume) may be acceptable if it is paired with greater efficacy. The delivery of a higher dose, as predicted by pharmacologic modelling, may lead to the rapid achievement of steady state (and maintenance of C_trOugh)- In this study, fulvestrant will be administered at a dose of 500 mg every 28 days plus an additional 500 mg dose on day 14 of the first month only in an attempt to decrease the time to achieve steady-state levels and increase efficacy. Phase III pharmacokinetic (PK) sampling data has demonstrated that a 250 mg dose administered every 28 days requires 3 to 6 months (90 to 180 days) before approaching steady-state drug concentrations. Earlier achievement of steady-state plasma concentrations may be important to avoid earlier "progression events". The expected mean peak plasma concentration (C_max) is approximately 27 ng/niL, around day 32. Over the next 10 months, C_max will be expected in the range of 26 — 28 ng/mL. The trough plasma concentrations at steady state are expected to be approximately 15 ng/mL compared to approximately 7 ng/mL for the 250 mg every 28 days injection.

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Figure 2 represents population-predicted profiles of fulvestrant 250 mg treatment and the planned 500 mg treatment regimen

3.2.1.2 Rationale for the choice of comparator As mentioned in Section- 1.1.1, anastrozole at a dose of 1 mg/day is an established treatment for postmenopausal patients with hormone-receptor expressing primary breast cancer.

3.2.1.3 Rationale for the choice of the surrogate marker DTC

As mentioned in Section 1.1.1, anastrozole at a dose of 1 mg/day as compared to tamoxifen at a dose of 20 mg/day reduces but does not abolish breast cancer recurrence. Therefore, a subgroup among anastrozole-treated patients may be considered at (high-)risk of breast cancer recurrence despite an effective treatment. This interpretation leads to the question if addition of an oestrogen antagonist, such as fulvestrant (as originally intended by the combination of anastrozole and tamoxifen in the ATAC trial), will be able to modify such a high-risk status. A large pooled analysis of 4703 patients with breast cancer has conclusively shown that presence of DTC is associated with an adverse prognostic influence on patient outcome overall and in the patient subgroup that received endocrine treatment alone (Braun et al, 2005b); several smaller trials have demonstrated that persistence of DTC after (neo-)adjuvant therapy is associated with an increased risk of tumour relapse and reduced survival (Braun et al, 2000b; Wiedswang et al, 2004; Janni et al, 2005). We hypothesise therefore that presence of DTC may be used i) as a selection criterion of high-risk patients at the time of decision about adjuvant treatment, and ii) as a surrogate marker of therapeutic efficacy of the applied treatment in patients with no clinically detectable evidence of disease.

3.3 Selection of study population

3.3.1 Study selection record

Before entering the study, patients will be assessed to ensure that they meet the eligibility criteria. Patients not meeting these criteria should not be entered into the study. The investigator must keep a record of patients who were considered for enrolment but were never enrolled, e.g., patient screening log indicating the reason why they were not enrolled. This information is necessary to establish that the patient population was selected without bias. The patient-screening log should be filed in the Investigator Study File at each centre. Note: The patient's signed and dated informed written consent must be obtained before conducting any procedure specifically for the study.

3.3.2 Inclusion criteria

For inclusion in the study, patients must fulfil all of the following criteria:

1. Provision of written informed consent: signed and dated Informed Consent Form prior to commencement of any specific protocol procedures

2. Histological confirmation (core needle biopsy or tumour excision) of breast cancer staged (c/p)Tl-4a-c N0-3 Gl-3 MO

3. No signs of distant metastasis in

• Chest X-ray and liver ultrasound (OR chest and abdomen CT scan)

• Whole body bone scan

• Medical history and physical examination

• Routine laboratory evaluation as outlined in section 4.7.2

4. Documented positive hormone receptor status (ER +ve and/or PgR +ve) of paraffin- embedded primary tumour tissue (core needle biopsy or tumour excision), according to the local laboratory parameters. A positive hormone receptor status in fine needle aspiration may be used for pre-selection of patients for intraoperative Screening BM aspiration; a positive hormone receptor status of the tumour excision is required for inclusion in the study. 5. Postmenopausal woman, defined as a woman fulfilling any one of the following criteria:

• Age >60 years;

• Amenorrhoea >12 months with an intact uterus and > 1 intact ovaries;

• Having undergone a bilateral oophorectomy;

• For patients with at least one ovary but without a uterus, FSH and estradiol in the postmenopausal range;

• For patients previously treated with an LHRH antagonist, the last dose must have been 4 months prior to randomisation, menses must not have restarted and FSH and estradiol must be in the postmenopausal range;

• Patients who have received adjuvant or neoadjuvant chemotherapy must have met one of the above criteria for postmenopausal status prior to that chemotherapy (subgroup for which pregnancy testing might be considered).

6. WHO performance status 0, 1 or 2.

7. At Screening Phase (within 35 days prior to randomisation) ^l

• Physical examination without clinically significant abnormalities

• Bone marrow aspiration with "DTC-positive" immunocytochemical result

• Chest X-ray, liver ultrasound, (or CT chest and abdomen) and bone scan without signs of metastatic breast cancer

• Laboratory requirements (at a clinically relevant time point prior to specific protocol procedures; i.e. for example 3-4 weeks after any preceding anti- tumour treatment, including chemotherapy, neoadjuvant Herceptin therapy):

• Neutrophils > 1.5 x 109/1, platelets > 100 x 109/1

• Total bilirubin < 1 x ULN (except patients with confirmed Gilbert's syndrome ² who may be included in the study) • AST (SGOT) and ALT (SGPT) < 1.5x ULN

• Alkaline phosphatases < 1 x ULN.

In case of abnormal values, the liver function tests have to be repeated and meet above criteria within 3 days before study treatment.

• Creatinine < 1.1 x ULN

8. At randomisation (endocrine treatment should start within the following 7 days) ³

• Final disease stage of completely resected pTl-4a-c NO-3 Gl-3 MO breast cancer

• Positive (ER +ve and/or PgR +ve) hormone receptor status of the formalin- fixed, paraffin-embedded tumour excision / mastectomy.

¹ Patients who fulfill inclusion criteria listed under point 1-7 are eligible for Screening Phase BM aspiration (with the exception that radiologic imaging for absence of distant metastases may be performed after receipt of the DTC-positive bone marrow result that qualifies for study enrolment and randomisation).

² Chronic intermittent mild predominately unconjugated hyperbilirubinemia in the absence of evidence of hemolysis or other underlying liver pathology

FFoorr rraannddoommiissaattiioonn,, ffiinnaall ddiisseeaassee ssttaaggee,, rraaddiologic imaging for absence of distant metastases and hormone receptor status are required in addition to points 1-7.

3.3.3 Exclusion criteria

Any of the following is regarded as a criterion for exclusion from the study:

1. Inflammatory (pT4d) and / or metastatic breast cancer (Ml)

2. Current or prior malignancy within previous 5 years (other than breast cancer or adequately treated non melanoma skin cancer or in-situ carcinoma of the cervix)

3. Premenopausal women

4. Planned adjuvant trastuzumab treatment, and preceding or concurrent systemic antitumour therapy; EXCEPT: • Patient had received adjuvant chemotherapy and has a DTC-positive Screening Phase BM aspiration in local anaesthesia up to 8 weeks after but not less than 2 weeks of completion of chemotherapy;

• Patient had received prior neoadjuvant chemotherapy with or without trastuzumab with an incomplete response (e.g.PR, SD, PD) for assessment of hormone-receptor expression of the residual tumour and has a DTC-positive Screening Phase BM aspiration at or after definitive primary surgery up to 8 weeks after but not less than 2 weeks of completion of chemotherapy, and no continuation of trastuzumab treatment after surgery is planned;

5. Immunocytochemical BM screening: "DTC-negative"

Note: patient is no longer subject of this trial and may enter other clinical trial

6. Treatment with a non-approved, investigational or experimental drug within 28 days before randomisation

7. History of bleeding diathesis (i.e., vW syndrome, disseminated intravascular coagulation [DIC], clotting factor deficiency) or long-term anticoagulant therapy (other than antiplatelet therapy and low dose warfarin with INR < 2 — see Section 3.7)

8. History of hypersensitivity to active or inactive excipients of fulvestrant, aromatase inhibitors or castor oil

9. Any severe concomitant condition which makes it undesirable for the patient to participate in the trial or which would jeopardize compliance with the trial protocol, e.g., uncontrolled cardiac disease or uncontrolled diabetes mellitus

10. Involvement in the planning and conduct of the study (applies to ABCSG staff, AstraZeneca staff or staff at the study site)

11.Previous enrolment or randomisation of treatment in the present study

3.3.4 Restrictions

1. Patients who are blood donors should not donate blood during the study and for 12 weeks following their last dose of study treatment 2. Patients who have confirmed disease recurrence must be discontinued from their randomised treatment

3. Concomitant treatments listed in Section 3.7.

3.3.5 Discontinuation of patients from treatment or assessment

3.3.5.1 Criteria for discontinuation

Patients may be discontinued from study treatment and assessments at any time. Specific reasons for discontinuing a patient from this study are:

• Any disease recurrence at any time during the 24 months of study treatment

• Voluntary discontinuation by the patient who is at any time free to discontinue her participation in the study, without prejudice to further treatment

• Safety reasons as judged by the investigator, ABCSG and/or AstraZeneca

• Severe non-compliance to protocol as judged by the investigator, ABCSG and/or AstraZeneca

• Incorrect enrolment of the patient (i.e., the patient does not meet the required inclusion/exclusion criteria)

• Patient lost to follow-up

• Any other reasons not listed above as per investigator discretion. The reason must be adequately documented.

3.3.5.2 Procedures for discontinuation

Patients who discontinue should always be asked about the reason(s) for their discontinuation and about the presence of any adverse events. If possible, they should be seen and assessed by an investigator. The Case Report Form (CRF) form for Study Discontinuation needs to be filled in within 3 months after discontinuation, which includes assessment of vital signs, weight, adverse events, reason(s) for the discontinuation. If a patient discontinues randomised treatment prior to efficacy evaluation, and does not withdraw consent, she should continue to be followed up for clinical recurrence. AEs should be reported for up to 3 months after the last injection of fulvestrant or the last tablet of anastrozole. The patient should return any investigational products.

3.4 Treatments

3.4.1 Identity of investigational product and comparators

Fulvestrant (Faslodex™) as a 5% w/v solution in clear neutral glass pre-filled syringes (PFS). Each syringe will contain 250 mg of fulvestrant in 5 niL (Formulation Number F6521). The constituents of the solution are as follows: fulvestrant, ethanol 96%, benzyl alcohol, benzyl benzoate and castor oil.

Anastrozole (Arimidex™) 1 mg white film coated tablets (Formulation number Fl 1292). The constituents of each tablet are as follows: anastrozole, lactose monohydrate, macrogol, magnesium stearate, hypromellose, povidone, sodium starch glycollate and titanium dioxide. Each fulvestrant pre-filled syringe will be packed into a light excluding carton. Anastrozole 1 mg tablets will be packed into tamper evident, high density polyethylene (HDPE) bottles with an induction seal child resistant cap. Each bottle will contain 35 tablets (sufficient for 28 days plus 7 days overage).

3.4.2 Doses and treatment regimens

Patients will be randomised to receive either fulvestrant 500 mg regime [fulvestrant 500 mg im on days 0, 14 (±3), 28 (±3) and every 28 (±3) days thereafter] plus anastrozole 1 mg po once daily or, anastrozole 1 mg po once daily. Study treatment is given for 24 months. In calculating days of treatment administration, the first day of treatment is considered as day 0. Therefore, if injections are given on a Monday, the Monday four weeks later will be considered day 28; a window of ±3 days is acceptable.

Fulvestrant will be provided as 250 mg in 5 mL as a pre-filled syringe. Each dose of fulvestrant will be administered as 500 mg, that is, two 5 mL intramuscular injections, one in each buttock. Injections will be given as 500 mg on days 0, 14 (±3 days), 28 (±3 days) and every 28 (±3 days) thereafter.

Anastrozole 1 mg will be administered as one tablet daily. Each bottle will contain 35 tablets, allowing for 28 days treatment, plus an additional 7 days if a patients' visit is delayed. Study treatments must be administered within 7 days of randomisation and will continue until disease progression or until considered by the investigator not in the best interest of the patient, (whichever occurs first). Reasons for treatment discontinuation will be collected. Otherwise study treatment will cease after 2 years of treatment. 3.4.3 Labelling

Labelling of the investigational product will be performed in accordance with GMP, Good Manufacturing Practice.

3.4.4 Storage

AU investigational products must be kept in a secure place under appropriate storage conditions. The storage conditions for the drugs are shown on the Clinical Trial Label affixed to each level of packaging.

3.7 Pre-study, concomitant and post-study treatment(s)

All prior treatments for cancer and all drugs given to, or taken by, the patient at entry and during the study must be clearly documented on the appropriate CRF page. The following restrictions apply:

1. Concomitant anticancer treatments, (including concurrent trastuzumab treatment), except for postoperative radiotherapy of breast/chest wall with or without regional lymph nodes, are not permitted during the study. Such treatments are prohibited even if they were given for another indication (e.g., megestrol acetate for appetite stimulation, methotrexate for rheumatological disorders). Preceding adjuvant / neoadjuvant chemotherapy but not endocrine therapy are permitted (see Sections 3.3.2 and 3.3.3). Neoadjuvant trastuzumab treatment is allowed, but concurrent adjuvant trastuzumab treatment is prohibited.

2. Bisphosphonate therapy, either before or after randomisation, for the treatment of osteoporosis is permitted during the study.

3. Sex hormone containing drugs such as hormone-replacement therapy (HRT), progestational agents (megestrol acetate), DHEA, other androgens (e.g., oxandrolone) and SERMs (eg raloxifene (Evista)) are not permitted during the study. In cases where patients suffer severe menopausal symptoms, management with non-hormonal agents, eg, clonidine or venlafaxine, is recommended. In cases of atrophic vaginitis the use of non-hormonal vaginal moisturising or lubricating gels or creams is recommended. Use of oestrogen- containing vaginal creams or other topical preparations is not allowed on the study, but use of controlled-release vaginal rings (e.g. Estring(R)) may be considered by and at the investigator's discretion in severe cases or where all other treatment possibilities have been exhausted and bearing in mind current advice on their use in combination with AIs. 4. In addition, other drugs than those mentioned above which may affect sex hormone status or disease response, such as systemic ketoconazole, systemic corticosteroids and adrenocortical suppressants are not allowed to begin after randomisation in to the study. However, the patient can continue to receive such drugs if they were taken before randomisation and the investigator is satisfied that the patient's hormonal status is stable. Hormone antagonists and related agents (eg. soy isoflavones) are not allowed.

Topical applications, inhaled sprays, eye drops, local injections and mouth- washes (if not swallowed) containing corticosteroids or ketoconazole are permitted during the study.

5. Patients receiving long-term anti-coagulant therapy with warfarin are ineligible for the study unless they are receiving low dose warfarin and have an INR < 2. The INR should be checked to ensure that it is < 2 prior to each injection. If the INR is >2, the injections may be withheld until the INR has returned to < 2. It is advised to apply direct pressure to the injection site in these patients.

6. Patients who need to begin anti-coagulant therapy while receiving study treatment may be treated, at the discretion of the investigator, with low molecular weight heparin (LMWH). The LMWH should be temporarily discontinued 12-24 hours prior to each injection and then resumed 12-24 hours later (depending on the particular LMWH used). There is an increased risk of haemorrhage in these patients and the investigator should decide whether that risk is outweighed by the possible benefits of continued treatment. It is advised to apply direct pressure to the injection site in these patients.

Anticoagulant therapy should be driven by the indication for which it is being administered.

7. Patients receiving antiplatelet therapy (ASA, ticlopidine, clopidogrel etc.) may be at increased risk of bleeding from intramuscular injection. The investigator should decide whether that risk is outweighed by the possible benefits of continued treatment. It is advised to apply direct pressure to the injection site in these patients. Therapeutic warfarin with its target INR > 2 is not allowed; subcutaneous application of fractionated heparins may be considered an alternative treatment (if appropriate for the underlying disease) to facilitate study enrolment. Subsequent treatment for breast cancer will be at the discretion on the investigator. Continuation of anastrozole is recommended for a total of 5 years for patients in both treatment groups. Continuation of anastrozole-fulvestrant combination is not permitted and neither drug will be provided as study medication after the 2 year study period. It is planned to clinically follow patients until the data cut-off date of 60 months after the last patient has been recruited and the date for this is estimated to be approximately the end of 2011. This procedure is not part of this trial and will be executed according to a separate study document, for which patients are eligible who have completed 24 months of randomised treatment as defined in this study document.

With the use of the surrogate marker DTC in BM, the fast assessment of superior efficacy may induce subsequent design of a phase III trial to examine the improved clinical outcome of patients using anastrozole-fulvestrant combination.

4. MEASUREMENTS OF STUDY VARIABLES AND DEFINITIONS OF OUTCOME VARIABLES

4.1 Primary and secondary variables Primary outcome variables for efficacy are:

• DTC in BM aspirates obtained prior to randomisation and after 12 months (±3 week diagnostic window)

• Any recurrence of disease, including ipsilateral intramammary (=local) relapse (Note: secondary contralateral breast cancer is not considered as recurrence), ipsilateral regional lymph node metastasis (including supraclavicular metastasis), any osseus and visceral distant metastasis at 12 months (±3 week diagnostic window)

• Death of any cause 12 months (±3 week diagnostic window)

Variables for safety are:

• Every NCI CTCAE grade 3 or 4 AE (e.g. including clinical fractures) observed after randomisation and during the treatment with anastrozole and fulvestrant as compared to anastrozole alone • Every SAE observed after randomisation and during the treatment with anastrozole and fulvestrant as compared to anastrozole alone

Secondary and explorative outcome variables for efficacy are:

• DTC in BM aspirates obtained prior to randomisation and after 24 months (±3 week diagnostic window)

• Any recurrence of disease (as stated above) or death at 24 months (±3 week diagnostic window)

• Number of DTC in BM aspirates obtained prior to randomisation and after 12 and 24 months (each ±3 week diagnostic window)

• Molecular assessment of DTC (i.e., disseminated ribonucleotides) in BM aspirates obtained prior to randomisation, after 12 and 24 months (each ±a 3 week diagnostic window)

• Molecular assessment of CTC (i.e., circulating ribonucleotides) in peripheral blood draws obtained prior to randomisation, after 12 and 24 months (each ±3 week diagnostic window)

• Molecular assessment of mDNA (e.g., methylated RASSF-I) in BM aspirates and peripheral blood obtained prior to randomisation, after 12 and 24 months (each ±a 3 week diagnostic window)

• Assessment of the expression of hormone receptors, HER2 oncoprotein and broad stem cell characteristics of DTC before and after 12 and 24 months of randomised treatment

4.2 Screening and demographic measurements

Please refer to the Study Plan (Table 1) for the list of procedures and assessments to be performed at screening and their relative timings prior to randomisation. Before entering the study, patients will be assessed to ensure they meet the eligibility criteria (see Section 3.3.2 and Section 3.3.3). Patients not meeting these criteria should not be entered into the study. Written informed consent must be obtained prior to any study specific assessments. Each patient will undergo screening procedures within 5 weeks prior to randomisation. The eligibility data (for details, see Section 3.3.2 and Section 3.3.3) listed below will be collected on the relevant CRF. The patient should be treated within 7 days following randomisation if all criteria listed below are fulfilled. If a patient is DTC-positive from the screening sample but requires further surgery, the screening period or the interval between randomisation and starting treatment can be increased by 2 weeks.

• Oestrogen and progesterone receptor status in FFPE tissue, obtained:

• At diagnosis by core needle-biopsy prior to definitive surgery as first therapeutic activity

• After neoadjuvant therapy at definitive surgery

• HER-2 status (where available)

• Metastasis screening

• chest X-ray, liver ultrasound (or chest and abdomen CT scan), and

• bone scan

• each after neoadjuvant chemotherapy or

• each prior to definitive surgery as first therapeutic action or

• each after adjuvant chemotherapy

• Bone marrow status with the number of detected DTC, obtained:

• At or within 4 weeks after definitive surgery

• At definitive surgery within 2-8 weeks after preceding neoadjuvant chemotherapy

• Within 2-8 weeks after adjuvant chemotherapy

• Previous breast cancer therapy

• Type, dosing, number of cycles of neoadjuvant chemotherapy ±trastuzumab

• Type, dosing, number of cycles of adjuvant chemotherapy • WHO performance status

• Laboratory Haematology and Biochemistry

The demographic and medical history data listed below will be collected on the relevant CRF:

• Date of birth and race

• Significant medical and surgical history

• Concurrent medical conditions

• Smoking history (Current, Previous or Never)

• ECG

• Blood pressure, heart rate, weight and height

• Concurrent medications (see Section 3.7).

• All concomitant medication/treatment will be recorded at entry. All changes will be recorded until the patient stops randomised study treatment.

• Subsequent visits (but not those visits for fulvestrant injections) should occur within ±3 weeks of the protocolled visit times. Also, assessment of efficacy with metastasis screening and BM aspiration after the first year of treatment will be done at the fourth visit after 12 months of treatment with a diagnostic window of ±3 weeks. .

4.6 Efficacy measurement and variables

DTC

In comparison to time of diagnosis, presence / number of DTC in BM (also see Table 4) is determined

• After 12 months of randomised treatment OR at the time of any disease recurrence during randomised treatment

• After 24 months of randomised treatment OR at the time of any disease recurrence during randomised treatment (if recurrence-free after 12 months) CTC

In comparison to time of diagnosis, presence / number of CTC (i.e., circulating ribonucleotides) in peripheral blood (also see Table 4) is determined after 12 and 24 months mDNA

In comparison to time of diagnosis, presence of mDNA in BM and peripheral blood (also see Table 4) is determined after 12 and 24 months

Event

An event for the primary and secondary efficacy endpoint is defined as:

• Presence of DTC after 12 and 24 months

• Clinical event: any disease recurrence, death from any cause

Table 1 — Scale and range of efficacy outcome biomarker variables

Variable Target Scale Range Endpoint

Disseminated Cytokeratin, Pos / neg l-∞ / 0 primary / tumor cells (DTC) EpCAM secondary

Disseminated Multiple markers Pos / neg >cut-off/ < cut-off exploratory ribonucleotides

Circulating Multiple markers Pos / neg >cut-off/ < cut-off exploratory ribonucleotides

Methylated DNA RASSF-IA Pos / neg Present / absent exploratory

4.6.1 Events

The primary efficacy variable is an "event" as defined in Section 4.6. It constitutes of DTC measurement after 12 months of randomised treatment (as described in Section 4.6.2) AND / OR occurrence of a clinical event (as described in Section 4.6.3); either one alone is considered an event, despite that BM aspiration and peripheral blood draw are to be performed in the case of a clinical event (i.e., any disease recurrence of death). Events are measured as described in Sections 4.6.2 and 4.6.3. 4.6.2 Disseminated tumour cells in bone marrow

4.6.2.1 Methods of assessment

Bone marrow aspirates (preferably 5 mL up to a maximum of 10 mL from each iliac crest; also see Table 5) are obtained and shipped to the Innsbruck laboratory in charge of the central workup and analysis of the samples according to the standard operational procedure (SOP) attached in a Supplement to this protocol. Bone marrow aspirations are performed within 4 weeks prior to randomisation (as specified in Section 4.2) for screening and after 12 and 24 months of randomised treatment OR at the time of a clinical event. Of note, recurrent and recurrence-free patients will have three BM aspirates per protocol. Of further note, each BM aspiration will be bilateral, since the detection rate by bilateral aspiration as compared to unilateral is significantly higher, which in return results in a significant reduction of patients to be screened.

Explicit description of the handling of BM and blood samples is provided in the respective SOP attached in Supplements to this protocol. DTC in BM are detected by immunocytochemistry according to the SOP described in a Supplement to this protocol.

4.6.2.2 Derivation or calculation of outcome variable

A "DTC-positive" result is defined as the presence of immunocytochemically positive cells with tumour cell-like morphology or cells with morphological overlap between haematopoietic cells and tumour cells after exclusion of plasma cells (known to directly bind alkaline phosphatase of the reporting antibody labelling), obvious skin squamous epithelial cells and cellular or debris artefacts among a total analysed volume of 4 x 10⁶ mononucleated cells obtained from workup and immunostaining according to SOPs described in a Supplement to this protocol.

Of note, an increased cell volume is analysed in this trial in order to increase the likelihood of a higher median number of DTC, and hence facilitation of an improved reliable differentiation between numbers of DTC detected at randomisation and after 12 and 24 months of randomised treatment as compared to published data, with a median of 3 DTC detected per 2 x 106 mononucleated cells analysed (Braun et al, 2005b).

For an improved statistical discrimination of the number of DTC at randomisation and after 12 and 24 months, a total of 8 x 106 mononucleated cells will be analysed in all DTC-positive cases. Decision upon inclusion will be solely based on the enumeration of 4 x 106 mononucleated cells. As compared to the initial "DTC-positive" BM status, both a "DTC-negative" result and a "DTC-positive" result with more than 95% reduction in the number of DTC at the second / third BM aspiration are considered a significant reduction and a measurable therapeutic effect of the randomised treatment.

4.6.3 Clinical events

4.6.3.1 Methods of assessment

Clinical events are assessed by chest X-ray and liver ultrasound (alternatively by chest and abdominal CT scan) and whole body bone scan after 12 and 24 months of randomised treatment. Further tests rendering the diagnosis more precisely are at the individual investigator's discretion.

4.6.3.2 Derivation or calculation of outcome variable

Any reproducible radiographic / scintigraphic evidence of distant lymph node, bone and organ metastases, any histologic evidence of loco-regional recurrence and death after randomisation are considered as clinical event. The date of the onset of the clinical event should be recorded in the CRFs.

The date of recurrence is the date of the investigation/procedure (imaging, biopsy, etc) that led to the diagnosis of recurrence. If more than one investigation/procedure is performed, and assuming that more than one confirms recurrence, the date of recurrence is the date when the first investigation/procedure was performed. The date of the recurrence in the case of a biopsy refers to the date of the biopsy itself and not the date of the pathology report. In the case of more than one procedure, where the first one had unclear results that have been confirmed later, that date of recurrence is the date of the investigation/procedure with clear, definitive results. Recurrence should not be backdated to the earlier procedure. In the few cases where recurrence is based solely on clinical evidence, an explanation should be available in the medical notes at the hospital or clinic, and the date of recurrence, to the best knowledge of the physician, should be documented. If a patient dies prior to reporting recurrence, the date of recurrence will be considered as the date of death.

4.6.4 Molecular assessment of DTC, CTC and mDNA

4.6.4.1 Methods of assessment

The molecular assessment of DTC, CTC and methylated DNA (mDNA) is not part of the primary efficacy measurement but will serve for exploratory investigations. Sample handling and shipment is described in a Supplement to this protocol. Peripheral blood draws will be performed in exact parallel to all planned BM aspirations. Of note, recurrent and recurrence- free patients will have three peripheral blood draws per protocol for CTC and mDNA assessments.

In brief, for each patient, 2 PAXgene™ tubes (Qiagen, Hilden, Germany; #762125) each with 2.5 mL BM and 2 PAXgene™ tubes each with 2.5 niL peripheral blood and 1 EDTA tube with 5 mL peripheral blood will be sent to the Innsbruck laboratory.

PAXgene™ tubes are kept at room temperature (18-22°C) for 2-24 hrs (up to 96 hrs at room temperature are possible) and are then stored frozen at < -2O⁰C at Innsbruck Medical University, Department of Obstetrics and Gynecology, Laboratory for Biochemistry. RNA will be isolated according to the manufacturer's recommendations using the PAXgene™ Blood RNA Kit 50 (Qiagen, Hilden, Germany; #762134) for both BM and peripheral blood samples without further modifications (as described in a Supplement to this protocol). RT-PCR for the detection of DTC and CTC in BM and peripheral blood, respectively, are confidential intellectual property of Innsbruck Medical University, Department of Obstetrics and Gynecology, Laboratory for Biochemistry. Detection of mDNA will be performed as previously published (Muller et al, 2003; Widschwendter et al, 2004; Fiegl et al, 2005) and outlined in a Supplement to this protocol.

4.7 Safety measurements and variables 4.7.1 Adverse events Adverse event

An adverse event is the development of an undesirable medical condition or the deterioration of a pre-existing medical condition following or during exposure to a pharmaceutical product, whether or not considered causally related to the product. An undesirable medical condition can be symptoms (e.g., nausea, chest pain), signs (e.g., tachycardia, enlarged liver) or the abnormal results of an investigation (e.g., laboratory findings, electrocardiogram). In clinical studies, an AE can include an undesirable medical condition occurring at any time, including run-in or washout periods, even if no study treatment has been administered. Any events that are unequivocally because of progression of the disease under study must not be reported as an AE. Adverse events of bone pain that are not considered to be due to disease progression should be reported as an adverse event. Serious adverse event (SAE)

A SAE is an AE occurring during any study phase (i.e., run-in, treatment, washout, follow- up), and at any dose of the investigational product, comparator or placebo, that fulfils one or more of the following criteria:

• results in death

• is immediately life-threatening

• requires in-patient hospitalisation or prolongation of existing hospitalisation

• results in persistent or significant disability or incapacity

• is a congenital abnormality or birth defect

• is an important medical event that may jeopardise the patient or may require medical intervention to prevent one of the outcomes listed above.

The causality of SAEs (i.e., their relationship to study treatment) will be assessed by the investigator(s), who in completing the relevant case report form must answer "yes" or "no" to the question "Do you consider that there is a reasonable possibility that the event may have been caused by any of the following - study medication - other medication?". For further guidance on the definition of a SAE and a guide to the interpretation of the causality question (see Supplement A "Investigators and Study Administrative Structure"). Any serious events that are unequivocally because of progression of disease must not be reported as an SAE.

Note that SAEs that could be associated with any study procedure should also be reported. For such events the causal relationship is implied as "yes".

Other Significant Adverse Events (OAE)

OAEs will be identified by the Study Team Physician and, if applicable, by the Drug Safety Physician during the evaluation of safety data for the Clinical Study Report. Significant adverse events of particular clinical importance, other than SAEs and those AEs leading to discontinuation of the patient from study treatment, will be classified as OAEs. Examples of these are marked haematological and other laboratory abnormalities, and certain events that lead to intervention (other than those already classified as serious), dose reduction or significant additional treatment. For each OAE, a narrative may be written and included in the

Clinical Study Report.

4.7.2 Laboratory safety measurements and variables

4.7.2.1 Methods of assessment

The following tests will be performed prior to and on specified days during and following therapy (as indicated in section 3.1):

• Hematology:

• Neutrophils

• Platelets

• Clinical chemistry:

• Total bilirubin

• Alkaline Phosphatase (AP)

• AST, (SGOT)

• ALT, (SGPT)

• Creatinine

Abnormal laboratory values that are considered to be clinically significant by the investigator must be repeated as soon as possible after receiving the laboratory report to rule out laboratory error.

4.7.2.2 Derivation or calculation of outcome variables

Normal values for haematology and clinical chemistry are defined by the local laboratories. Abnormal values are considered values that are out of range as indicated below:

• Neutrophils < 1.5 x 109/1

• Platelets < 100 x 109/1

• Total bilirubin (except patients with confirmed Gilbert's syndrome) > Ix ULN,

• AST (SGOT) > 1.5x ULN, • ALT (SGPT) > 1.5x ULN,

• Alkaline phosphatases (AP) > Ix ULN.

• Creatinine > 1.1 x ULN

4.7.3 Vital signs, ECG and physical examination

The following tests will be performed prior to and on specified days during and following therapy (see also Table 1):

• Complete history of malignant and non-malignant diseases including known hypersensitivity reaction to anastrozole or fulvestrant.

• Full physical examination, vital signs (blood pressure, heart rate, temperature), height, weight, assessment of WHO performance status.

• Abnormal vital signs, ECG and physical examination that are considered to be clinically significant by the investigator must be reported as an AE and clarified by subsequent diagnostic tests as appropriate according to GCP guidelines.

4.8 Volume of bone marrow / blood sampling and handling of biological samples

The total volume of blood that will be drawn from each patient in this study is as follows: Table 2 — Volume of bone marrow / blood to be drawn from each patient

Assessment Sample volume (mL) No. of samples Total volume (mL)

Bone marrow, bilateral" 5-10 6 30-60

Peripheral venous blood

CTC^a 2.5 6 15 mDNA^a ( 10 3 15

Safety^b Clinical chemistry 5 10 50

Haematology 5 10 50 Total (bone marrow / blood) 30-60 / 130 a A total 3 assessments at Screening, after 12 and 24 months, respectively, b Clinical chemistry and haematology are controlled every 3 months. 4.8.1 Analysis of biological samples

Stability limits for BM and blood obtained for primary and exploratory efficacy analyses, respectively, defined by the Innsbruck Laboratory will be applied to all analyses performed. Innsbruck Laboratory will not analyse samples that fall outside these stability limits. Analytical data will not be reported if found to have been derived from a sample that fell outside these stability limits. The standards of procedure followed by Innsbruck Laboratory may be amended in accordance with its Standard Operating Procedures. Innsbruck Laboratory will inform ABCSG of the stability limits relevant to this study before the first patient gives informed consent to take part in the study.

Blood samples for safety are analysed locally in licensed laboratories of the respective study centres. ABCSG assures itself and provides assurance to AstraZeneca that the local laboratory will apply defined stability limits to all analyses performed on behalf of ABCSG. Samples falling outside these limits must not be analysed or data reported. The other laboratory will inform ABCSG of the stability limits relevant to this study before the first patient gives informed consent to take part in the study.

4.8.2 Stability limits of biological samples

Bone marrow samples in 50 mL tubes with EDTA are considered stable for a maximum of 72 hrs, however, best results are obtained after 24-48 hrs if the sample is kept at temperatures between +4°C and +22°C (i.e., room temperature 18-22°C is sufficient for transport). PAXgene tubes with either BM or peripheral blood for the detection of disseminated or circulating mRNA markers are kept at room temperature (18-22°C) for a minimum of 2 hrs, an optimum of 24 hrs and a maximum of 120 hrs.

EDTA tubes containing peripheral blood (5 mL) for analysis of circulating methylated DNA are shipped together with PAXgene tubes and EDTA-bone marrow, and will be further processed in the Innsbruck Laboratory.

6. STATISTICAL METHODS AND DETERMINATION OF SAMPLE SIZE

6.1 Statistical evaluation - general aspects

A comprehensive Statistical Analysis Plan (SAP) will be prepared before analysing the data. Each trial endpoint will be summarised / analysed at two time-points, using all data available up to and including the date at which the last scheduled patient receives the: • 12 month DTC assessment • 24 month DTC assessment.

In section 6.4, these two time-points will be referred to as "after 12 months" and "after 24 months".

6.2 Description of outcome variables in relation to objectives and hypotheses

Please refer to the Table 6 for a description of the relationship between specific study objectives and outcome variables.

Table 3 — Objectives and outcome variables

Objective/outcome variable type Outcome variable

Primary /efficacy DTC, clinical event after 12 months

Secondary/efficacy DTC, clinical event after 24 months

Secondary /safety Incidence, CTCAE grade and type of AEs, clinically significant laboratory abnormalities or changes in vital signs, and ECG changes 12 and 24 months

Exploratory/efficacy Disseminated ribonucleotides at 12 and 24 months

Circulating ribonucleotides 12 and 24 months Circulating tumour-cell derived methylated nucleotides 12 and 24 months Frequency of expression of hormone receptors, HER2 oncoprotein and broad stem cell characteristics of DTC at 12 and 24 months

6.3 Description of analysis sets

Efficacy data from this study will be analysed on an intention-to-treat basis using randomised treatment. The safety data for this study will be summarised using treatment received. The analysis population will consist of all randomised patients who received at least one dose of trial therapy.

Subgroups according to preceding chemotherapy; patterns of hormone-receptor expression; country of origin; lymph node metastasis; tumour size; and differentiation grade of the primary tumours are considered for exploratory analyses of efficacy. 6.4 Method of statistical analysis 6.4.1 Primary Objective - Efficacy 6.4.1.1 Recurrence and Presence/absence of DTC after 12 months

For the analysis of this endpoint, DTC negative patients who are alive with no clinical recurrence after 12 months will be considered to be "successes". Patients who are either:

• DTC positive with presence of clinical recurrence and/or death (from any cause),

• DTC positive with absence of clinical recurrence or death (from any cause),

• DTC negative with presence of clinical recurrence and/or death (from any cause)

will not be considered as "successes".

A logistic regression analysis, including treatment factor only, will be used to compare the proportion of "successes" between the two treatment groups. The results of the analysis will be presented in terms of odds ratios together with associated confidence intervals and 2-sided p-values. The estimate of the difference in the "success" rates and the corresponding 2-sided

95% confidence intervals will also be presented. It is assumed that censored patients will distribute equally to both treatment groups. This assumption will be checked in an exploratory manner.

A Cox's proportional hazards regression model will also be performed as a secondary analysis. The model will allow for the effect of treatment and will also include terms for preceding chemotherapy, hormone-receptor expression, country, axillary lymph-node metastases status, tumours <2 vs. >2cm in diameter and differentiation grade of primary tumours. The conclusion will be based on the unadjusted analysis, which is considered as primary. If the unadjusted analysis and the adjusted analysis yield different results, the consequences of the covariate adjustments will be explored.

A global test for the presence of the treatment by baseline covariate interactions will be performed at the 1% level of significance by including all the 2-way treatment by baseline covariate interactions in the model. The assumptions of proportionality will also be investigated with a time-dependent exploratory variable, which is defined as treatment- {log(time to event)}. If the p- value from the WaId Chi-squared statistic for this variable is less than 5% there is evidence of a departure from the adjusted model assumptions.

In this case, the reason will be explored and reported. 6.4.2 Secondary Objectives

6.4.2.1 Safety after 12 months and 24 months

Safety and tolerability data will be presented by treatment received. Appropriate summaries of these data will be presented. Safety and tolerability will be assessed in terms of AEs, laboratory data, and ECG changes, which will be collected for all patients. Data from all treatment periods will be combined in the presentation of safety data. AEs (both in terms of

MedDRA preferred terms and CTCAE grade), laboratory data and ECG data will be listed individually by patient and summarised by treatment received.

For patients who have a dose modification, all AE data (due to toxicity or otherwise) will be assigned to the initial treatment received group.

ECG changes will be summarized for each treatment group. Vital signs data will be listed for each patient and changes in vital signs will be summarized

6.4.2.2 Recurrence and Presence/absence of DTC after 24 months

For the analysis of this endpoint, DTC negative patients who are alive with no clinical recurrence after 24 months will be considered to be "successes". Patients who are either:

• DTC positive with presence of clinical recurrence and/or death (from any cause),

• DTC positive with absence of clinical recurrence or death (from any cause),

• DTC negative with presence of clinical recurrence and/or death (from any cause)

will not be considered as "successes".

A logistic regression analysis, including treatment factor only, will be used to compare the proportion of "successes" between the two treatment groups. The results of the analysis will be presented in terms of odds ratios together with associated confidence intervals and 2-sided p-values. The estimate of the difference in the "success" rates and the corresponding 2-sided

95% confidence intervals will also be presented. It is assumed that censored patients will distribute equally to both treatment groups. This assumption will be checked in an exploratory manner.

As secondary analyses, a Cox's proportional hazards regression model and a global test for the presence of the treatment by baseline covariate interactions will be performed as de-scribed in

Section 6.4.1.1. If it is the case that a high proportion of patients (i.e., beyond the expected percentage of dropouts) refuse bone marrow aspiration after 24 months this fact will be incorporated in a descriptive manner in the interpretation of the data. 6.4.3 Exploratory objectives

6.4.3.1 Outcome/survival and number of DTC after 12 months and 24 months

The numbers of DTC in patients at baseline after 12 months ±3 weeks and after 24 months ±3 weeks of randomised treatment will be summarised descriptively, by treatment group, using standard summary statistics (mean, minimum, maximum, sd and N).

In addition, in order to evaluate the degree of any DTC reduction in relation to occurrence of relapse, distant metastases, and deaths (overall and breast-cancer specific), separate summary tables, summarising the numbers of DTC, will be produced for each of these variables, split by treatment group.

6.4.3.2 Molecular DTC assessment after 12 months and 24 months

The association between the assessment of disseminated ribonucleotides using a multi-marker RT-PCR and the detection of DTC using standard immunocytochemical assay will be assessed by a linear regression model including also the treatment effect.

6.4.3.3 Molecular CTC assessment after 12 months and 24 months

The association between the assessment of circulating ribonucleotides using a multi-marker RT-PCR and the detection of DTC using standard immunocytochemical assay will be assessed by a linear regression model including also the treatment effect.

6.4.3.4 Molecular assessment of circulating methylated DNA after 12 months and 24 months

The association between the assessment of circulating tumour-cell derived methylated nucleotides using a molecular approach and the detection of DTC using standard immunocytochemical assay will be assessed by a linear regression model including also the treatment effect.

6.4.3.5 Assessment of DTC phenotype after 12 months and 24 months

The agreement of the expression of hormone receptors, HER2 oncoprotein and broad stem cell characteristics of DTC using a standard immunocytochemical assay will be measured and tested with the kappa coefficient. 6.5 Determination of sample size

Statistical calculations performed for the primary endpoint of the trial are as follows: Using 80% power, a 2-sided alpha = 0.05 and assuming that 50% of patients receiving anastrozole remain DTC-positive after 12 months of trial therapy, then 65 patients per group are required to demonstrate a halving or greater in the number of DTC-positive patients after 12 months of trial therapy (i.e.. number of DTC-positive patients in the anastrozole + fulvestrant treatment group is 25% or less). Thus 130 patients are required in total. Allowing for (up to approximately 26%) dropouts, approximately 176 patients are required. Assuming a 20% prevalence of DTC, 880 patients are required in total.

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Claims

Claims

1. A method of treatment of breast cancer which comprises administration of fulvestrant and simultaneous, separate or sequential treatment with an aromatase inhibitor to a patient in need thereof wherein the patient has hormone receptor positive early breast cancer.

2. A method according to claim 1 in which fulvestrant is administered as a 450-550 mg intramuscular injection monthly.

3. A method according to claim 2 wherein one additional 450-550 mg dose is administered at day 14 of the first month.

4. A method according to claim 2 or 3 in which the dose of fulvestrant is about 500mg.

5. A method according to any preceding claim in which the patient has hormone receptor positive early breast cancer and a biomarker indicative of a poor prognosis.

6. A method according to claim 5 in which the biomarker indicative of a poor prognosis is the presence of disseminated tumour cells in the bone marrow.

7. A method according to any preceding claim 1 in which the aromatase inhibitor is anastrozole, exemestane or letrozole.

8. A method according to claim 7 in which the aromatase inhibitor is anastrozole.

9. A method according to any preceding claim in which the patient is a postmenopausal female.

10. Fulvestrant for use in treatment of hormone receptor positive early breast cancer in a patient wherein the patient is receiving simultaneous, sequential or separate treatment with an aromatase inhibitor.

11. Fulvestrant in a package together with instructions for its use for treatment of hormone receptor positive early breast cancer with simultaneous, sequential or separate treatment with an aromatase inhibitor.