Clinical trial NCT01216176
Conditions: Breast Cancer
Keywords: Breast Cancer Postmenopausal Metastatic Phase I ONLY AZD0530 (saracatinib) Anastrozole Phase I for metastatic disease Phase II for newly diagnosed breast cancer Pharmacokinetic PK Hormone Receptor Estrogen Receptor Progesterone Receptor ER+ PR+ HER negative Aromatase Inhibitors
Sponsor: Joyce Marie Slingerland, MD
Start date: 2008-10-21; Completion date: 2018-02-07
Enrollment: 71 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The investigators propose to conduct a Phase I/randomized Phase II study design in order to test the tolerability and efficacy of AZD0530 (also called saracatinib) when used together with anastrozole in therapy for ER+ and/or PR+, postmenopausal breast cancer. The Phase I pharmacokinetic (PK) cohort of the study (cohort A) in postmenopausal women with metastatic breast cancer 2008-2009 showed initial safety,tolerability and good bioavailability of both drugs and determined the doses for use in the ongoing Phase II trial. In the randomized Phase II cohort of the study (cohort B), postmenopausal women with newly diagnosed, previously untreated ER+, HER2 negative breast cancer that is at least 2 cm or more in diameter by clinical exam or radiology will be randomized to either neoadjuvant treatment with anastrozole plus placebo, or anastrozole in combination with AZD0530 (saracatinib). The Phase II cohort will permit extended assays of tolerability, initial estimates of efficacy, and the investigation of molecular predictors of drug efficacy.

Detailed description:

Arms: arm 1: Dual treatment with 1 mg anastrozole orally once daily together with AZD0530 (saracatinib) 175 mg orally once daily, or as specified per protocol, until disease progression for treatment of metastatic breast cancer arm 2: Dual treatment with 1 mg anastrozole orally once daily together with AZD0530 (saracatinib) 175 mg orally once daily, or as specified per protocol, until disease progression or 4-6 months of treatment completed. arm 3: Dual treatment with 1 mg anastrozole orally once daily together with Placebo orally once daily, or as specified per protocol, until disease progression or4-6 months of treatment completed.
Interventions: intervention 1: Anastrozole intervention 2: AZD0530 (saracatinib) intervention 3: Placebo
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: None

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Revised December 5,  2016   
 
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EPROST # 20080325 
SCCC # 2008002 
 
IND # 102,186 
 
A Phase I Pharmacokinetic and Randomized Phase II Trial of Neoadjuvant Treatment with 
Anastrozole plus AZD0530 in Postmenopausal Patients with Hormone Receptor Positive 
Breast Cancer 
 
 
Principal Investigator 
Joyce M. Slingerland, MD, PhD 
Braman Family Breast Cancer Institute 
Division of Hematology Oncology 
University of Miami Sylvester Comprehensive Cancer Center 
 
 
Sub-Investigators 
J. Hurley; S. Richman; R. Mahtani; M. Lippman; M. Jahanzeb; C. Calfa; A. Tobon Perez; C. 
Welsh; K. Hernandez 
 
Collaborator 
Mark Pegram 
 
Statistician 
Isildinha M Reis, PhD 
 
 
Study Sites 
University of Miami and Stanford University 
 
 
 
 
REVISED December 5, 2016 


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Table of Contents 
1.0 OBJECTIVES ............................................................................................................................ 6 
1.1 Primary Objectives .................................................................................................................... 6 
Cohort A (Phase I PK cohort in MBC patients): ........................................................................... 6 
Cohort B (randomized phase II neoadjuvant cohort) .................................................................. 6 
1.2 Secondary Objectives ............................................................................................................... 6 
Cohort A: .......................................................................................................................................... 6 
Cohort B: .......................................................................................................................................... 6 
1.3 Study Schema ........................................................................................................................... 8 
2.0 BACKGROUND ......................................................................................................................... 9 
 
2.1 Aromatase Inhibitors as Endocrine Therapy for Breast Cancer .......................................... 9 
Targeting ER + breast cancer by inhibition of estradiol biosynthesis: aromatase inhibition . 9 
The aromatase inhibitor anastrozole ............................................................................................ 9 
Superiority of anastrozole over tamoxifen in metastatic breast cancer .................................... 9 
Primary adjuvant AI treatment of early breast cancer ............................................................... 10 
Anastrozole metabolism and drug interactions ......................................................................... 11 
2.2 Neoadjuvant Endocrine Therapy ........................................................................................... 11 
Neoadjuvant therapy of early breast cancer .............................................................................. 11 
Neoadjuvant endocrine therapy for breast cancer .................................................................... 12 
Tamoxifen as neoadjuvant therapy for breast cancer ............................................................... 13 
AIs versus tamoxifen as neoadjuvant therapy ........................................................................... 13 
2.3 Activation of the Src oncogene: a mediator of antiestrogen therapy resistance ............. 14 
Src activation is common in human breast cancer ................................................................... 14 
The cell cycle inhibitor p27 is a key regulator of cell proliferation .......................................... 15 
p27 is required for growth arrest by tamoxifen and by estrogen deprivation ........................ 15 
Src phosphorylates p27 to promote p27 degradation ............................................................... 15 
2.4 Pre-clinical Data Using Src Inhibitor AZD0530 with Anastrozole ....................................... 16 
Src inhibitorAZDO530 cooperates with anastrozole to inhibit breast cancer growth in vitro 16 
AZD0530 augments antitumor effects of anastrozole in breast xenografts in vivo................ 16 
2.5 Clinical Development of the Src Inhibitor AZD0530 ............................................................ 17 
2.6 Summary of scientific rationale for targeting Src in combination with aromatase 
inhibition in ER+ postmenopausal breast cancer ...................................................................... 18 
2.7 Scientific Rationale for the Study .......................................................................................... 19 
2.8 Rationale for AZD0530 Dosing and Scheduling ................................................................... 19 
2.9 Rationale for Molecular Investigations ................................................................................. 20 
2.10 Rationale for Imaging Studies ............................................................................................. 21 
3.0 PATIENT SELECTION ............................................................................................................ 24 
3.1 Inclusion Criteria ..................................................................................................................... 24 


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      Inclusion Criteria (phase I PK cohort A) ............................................................................... 24 
      Inclusion Criteria (randomized, phase II neoadjuvant cohort B) ........................................ 25 
    3.2 Exclusion Criteria.................................................................................................................... 26 
            Exclusion Criteria (cohort A) ................................................................................................. 26 
            Exclusion Criteria (cohort B) ................................................................................................. 27 
3.3 Enrollment procedures ........................................................................................................... 29 
Enrollment ............................................................................................................................... 29 
Cancellation Guidelines.......................................................................................................... 30 
Randomization and Blinding in Cohort B ............................................................................. 30 
Additional procedures ............................................................................................................ 31 
3.4 Regulatory Authority Approval .............................................................................................. 31 
3.5 Ethical Conduct of the Study ................................................................................................. 32 
3.6 Institutional Review Board/Ethics Committee ...................................................................... 32 
4.0 TREATMENT PLAN ................................................................................................................ 32 
4.1 Cohort A (Phase I PK cohort) Study Treatment ................................................................... 32 
      AZD0530 dose finding for combined treatment with anastrozole. ..................................... 32 
Pharmacokinetic evaluation ................................................................................................... 34 
Summary of Phase I Data ....................................................................................................... 34 
 
4.2 Treatment Plan Phase II Cohort B (randomized neoadjuvant cohort) ............................... 35 
Pharmacokinetic evaluation ................................................................................................... 36 
4.3 Supportive Care Guidelines ................................................................................................... 36 
Use of Hematopoietic Growth Factors .................................................................................. 36 
 
G-CSF .............................................................................................................................. 37 
 
Erythropoietic agents .................................................................................................... 37 
 
     Prophylactic Antibiotics ................................................................................................ 37 
4.4 Duration of Therapy ................................................................................................................ 37 
4.5 Dose Delays/Dose Modifications ........................................................................................... 38 
Dose Delays for Neutropenia ................................................................................................. 38 
Dose Reductions for Febrile Neutropenia ............................................................................ 38 
Dose Reductions for Infection ............................................................................................... 39 
Thrombocytopenia .................................................................................................................. 39 
Anemia ..................................................................................................................................... 39 
Other Toxicities ....................................................................................................................... 39 
Discontinuation of AZD0530 Alone ....................................................................................... 40 
Dose Reductions and Delays for Anastrozole ...................................................................... 40 
Concomitant Treatment .......................................................................................................... 40 
5.0 CLINICAL AND LABORATORY EVALUATIONS .................................................................. 41 
5.1 Baseline/Pretreatment Assessments .................................................................................... 41 
5.2 Assessments during AZD0530 treatment ............................................................................. 42 
5.3 Cohort B Assessments at End of Anastrozole + AZD0530/Presurgical Visit .................... 43 
5.4 Assessments at Post-Surgical Visit ...................................................................................... 44 
6.0 AGENT FORMULATION AND PROCUREMENT ................................................................... 45 


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6.1 Anastrozole .............................................................................................................................. 45 
6.2 AZD0530 ................................................................................................................................... 45 
6.3 Study Drug Storage ................................................................................................................ 45 
7.0 CORRELATIVE/SPECIAL STUDIES ...................................................................................... 45 
7.1 Tissue Collection and Molecular Studies ............................................................................. 45 
Core Biopsies (Baseline) ........................................................................................................ 46 
Definitive Surgery ................................................................................................................... 46 
8.0 MEASUREMENT OF EFFECT ................................................................................................ 47 
8.1 Functional Imaging Studies ................................................................................................... 47 
8.2 Clinical and Molecular Efficacy Assessment ....................................................................... 48 
8.3 Clinical Response Assessment ............................................................................................. 48 
Assessment of Response by Physical Examination and MRI Evaluation ......................... 48 
Assessment of Pathologic Response ................................................................................... 48 
Breast-conserving Surgery .................................................................................................... 48 
9.0 SAFETY ................................................................................................................................... 49 
9.1 Definition of an Adverse Event (AE) ...................................................................................... 49 
9.2 Reporting of Adverse Events ................................................................................................. 50 
9.3 Grading of Adverse Events .................................................................................................... 50 
9.4 Monitoring of Adverse Events ............................................................................................... 50 
9.5 Serious Adverse Events ......................................................................................................... 51 
9.6 Reporting of Serious Adverse Events ................................................................................... 51 
9.7 Reporting for Investigational Agents .................................................................................... 53 
9.8 Reports of Patient Death ........................................................................................................ 53 
9.9 Period of Observation ............................................................................................................. 53 
10.0 DATA REPORTING ............................................................................................................... 54 
10.1 Study Monitoring ................................................................................................................... 54 
  Data and Safety Monitoring Plan ......................................................................................... 55 
10.2 Auditing .................................................................................................................................. 55 
10.3 Modification of the Protocol ................................................................................................. 55 
10.4 Information Disclosure and Publications ........................................................................... 56 
11.0 CRITERIA FOR DISCONTINUATION OF THERAPY ........................................................... 56 
12.0 STATISTICAL METHODS AND CONSIDERATIONS .......................................................... 56 
12.1 Phase I PK cohort: Statistical Considerations ................................................................... 56 
12.2 Randomized double-blind phase II cohort: Statistical Considerations ............................ 57 
12.3 Correlative Molecular Studies: ............................................................................................ 58 
12.4 Randomization and Blinding in Cohort B ........................................................................... 59 


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12.5 Safety Evaluation .................................................................................................................. 59 
   Interim monitoring of phase II component of study ......................................................... 59 
   Guidelines for early stopping due to toxicity .................................................................... 60 
   Guideline for early stopping for futility .............................................................................. 61 
13.0 REFERENCES ...................................................................................................................... 63 
APPENDIX I Schedule of Events Phase II Study ....................................................................... 72 
APPENDIX II Tumor Tissue Collection and Preparation .......................................................... 74 
APPENDIX III Web Address NCI Common Terminology Criteria for Adverse Events ........... 77 
APPENDIX IV Data and Safety Monitoring Plan ........................................................................ 78 
APPENDIX V ECOG Performance Scale .................................................................................... 79 
APPENDIX VI RECIST Criteria .................................................................................................... 79 
APPENDIX VII Reverse Phase Protein Lysate Arrays (RPPA) ................................................. 83 
APPENDIX VIII Gene Expression Analysis by Microarray ........................................................ 83 
APPENDIX IX Pharmacokinetic Evaluation ............................................................................... 87 
APPENDIX X Summary of Strong CYP3A Inducers and Inhibitors Prohibited on Study
 
……………………………………………………………………………………………………..88 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1.0 OBJECTIVES 
We propose to conduct a Phase I/randomized double-blind Phase II study design in order to test the 
tolerability and efficacy of AZD0530 when used together with anastrozole in therapy of ER+ and/or 
PR+ postmenopausal breast cancer. The Phase I pharmacokinetic (PK) cohort of the study (cohort 
A) will be conducted in postmenopausal women with metastatic disease and will ascertain safety 
and toxicity. Patients in the randomized Phase II cohort of the study (cohort B) will consist of 
postmenopausal women with newly diagnosed ER+ and/or PgR+, Her2- breast cancer who are 
randomized to either neoadjuvant treatment with anastrozole plus placebo, or anastrozole in 
combination with AZD0530. The Phase II cohort will permit extended assays of tolerability, initial 
estimates of efficacy, and the investigation of molecular predictors of drug efficacy.  
 
1.1 Primary Objectives 
 
Cohort A (Phase I PK cohort in MBC patients): 
The primary objective of this open label phase I study will be to determine if a well-tolerated dose 
of AZD0530 can be used in combination with anastrozole for post-menopausal women with ER+ 
and/or PR+ metastatic breast cancer. 
 
Cohort B (randomized phase II neoadjuvant cohort): 
The primary objective will be to compare treatment groups (AZD0530 and anastrozole versus 
anastrozole with placebo) with respect to clinical response, defined as percentage 
change/reduction in tumor size, by investigator measurements at diagnosis and on completion of 
neoadjuvant treatment prior to surgery.  
 
1.2 Secondary Objectives   
 
Cohort A: 
The secondary objective will be to test effects of giving the two drugs together on pharmacokinetics 
of both drugs. 
 
Cohort B: 
Secondary objectives will include evaluation of a) clinical response, defined as percentage 
change/reduction in tumor size, as measured by dynamic contrast-enhanced magnetic resonance 
imaging (MRI) measurements of tumor volume at diagnosis and on completion of neoadjuvant 
treatment prior to surgery b)pathological complete response (pCR)  c) clinical response (complete 


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(CR), or partial (PR) responses) and clinical benefit (CR, PR, or stable disease (SD)), as measured 
by physician measurement of tumor size and by MRI measurements of tumor volume at diagnosis 
and prior to surgery; d) the qualitative and quantitative toxicities; e) to identify molecular/biologic 
correlates as indicators of treatment response 
 
. Other 
biomarkers may be considered pending initial IHC results. 
 
 
 
 


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1.3 STUDY SCHEMA  
 
 
 
220,000 new cases of invasive breast cancer are diagnosed per year in the United States.  About 
two thirds of these express the estrogen receptor (ER). ER protein, assayed in newly diagnosed 
breast cancers, is a weak prognostic factor and predicts tamoxifen response. Over 120,000 new 
patients per year will start adjuvant therapy with an aromatase inhibitor or tamoxifen to prevent 
disease recurrence. In the USA alone, approximately one million breast cancer patients are on 
tamoxifen therapy and over 6 million are on this treatment worldwide. Aromatase inhibitors (AI), 
both reversible, non-steroidal (anastrozole and letrozole) and irreversible steroidal (exemestane) 
block peripheral conversion of adrenal androstenedione to estradiol and effectively cut off 
mitogenic estrogen to breast cancer in post-menopausal patients. AIs are now considered to be 
first line agents in both the metastatic and adjuvant settings for in post-menopausal women (Wong 
and Ellis, 2004). 
 
 


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2.0 BACKGROUND 
 
2.1 Aromatase Inhibitors as Endocrine Therapy for Breast Cancer 
 
Targeting ER + breast cancer by inhibition of estradiol biosynthesis: aromatase inhibition 
Aromatase, product of the CYP19 gene, is an enzyme of the cytochrome P-450 super family. It is 
expressed in several tissues, including subcutaneous fat, liver, muscle, brain, normal breast 
tissues, and mammary adenocarcinoma (Nelson and Bulun, 2001). Aromatase is responsible for 
the conversion of the adrenal androgen substrate androstenedione to estrogen in peripheral (non-
ovarian) tissues (Evans et al., 1986), the predominant source of estrogen in postmenopausal 
women. Aromatase inhibitors (AIs) can reduce estrogen production by more than 90% (Geisler et 
al., 1998). Because AIs do not fully inhibit ovarian production of estrogen, only women without 
functioning ovaries are currently treated with AIs (Altundag and Ibrahim, 2006). 
 
The aromatase inhibitor anastrozole 
Anastrozole is a potent and highly selective non-steroidal aromatase inhibitor. In postmenopausal 
women, estradiol is produced primarily from the conversion of androstenedione to estrone through 
the aromatase enzyme complex in peripheral tissues. Estrone is subsequently converted to 
estradiol. Reducing circulating estradiol levels has been shown to produce a beneficial effect in 
women with breast cancer. In postmenopausal women, anastrozole at a daily dose of 1 mg 
produced estradiol suppression of greater than 80% using a highly sensitive assay. Anastrozole 
does not possess progestogenic, androgenic or estrogenic activity. Daily doses of anastrozole up 
to 10 mg do not have any effect on cortisol or aldosterone secretion, measured before or after 
standard ACTH challenge testing. Corticoid supplements are therefore not needed. 
 
An extensive phase III clinical study program showed that anastrozole is an effective treatment of 
early breast cancer and advanced breast cancer in postmenopausal women suitable for endocrine 
therapy. 
 
Superiority of anastrozole over tamoxifen in metastatic breast cancer 
Several phase III studies have compared the efficacy of the AIs with that of tamoxifen as first-line 
therapy for metastatic breast cancer (MBC) (Bonneterre et al., 2001a); (Mouridsen et al., 2003); 
(Paridaens, 2004); (Bonneterre et al., 2000); (Nabholtz et al., 2000). The Tamoxifen and 
Anastrozole Randomized Group Efficacy and Tolerability (TARGET) trial was a multicenter 


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randomized trial conducted simultaneously in Europe and in North America (Paridaens, 2004); 
(Bonneterre et al., 2000)comparing anastrozole with tamoxifen as first-line therapy for MBC. While 
the n=282 European cohort did not show a significant difference between the two drugs, the322 
patient cohort of the North American TARGET showed a significantly higher clinical benefit rate 
with anastrozole (59.1%) than with tamoxifen (45.6%; p = .0098). The TTP was 11.1 months in the 
anastrozole group and 5.6 months in the tamoxifen group (p = 0.005). On the basis of these two 
trials, anastrozole was approved as first-line therapy for MBC (Bonneterre et al., 2000); (Nabholtz 
et al., 2000).  Analysis of the combined patient population in both trials (n = 1,021; median follow-
up, 18.2 months) showed that in the subset of patients whose tumors were positive for ER and/or 
PgR (excluding those with unknown receptor status) anastrozole was superior to tamoxifen with 
respect to TTP (median, 10.7 months for anastrozole and 6.4 months for tamoxifen; p = .022) 
(Altundag and Ibrahim, 2006). 
 
Primary adjuvant AI treatment of early breast cancer 
Several trials have shown superiority of AIs over tamoxifen in the adjuvant therapy of breast 
cancer. The ATAC trial, (Buzdar, 2003) demonstrated superiority of adjuvant anastrozole over 
tamoxifen to prevent disease recurrence, with reduced time to progression and contralateral 
disease, and improved survival. Overall, anastrozole was well tolerated. Patients receiving 
anastrozole had a decrease in hot flushes, vaginal bleeding, vaginal discharge, endometrial 
cancer, venous thromboembolic events and ischemic cerebrovascular events compared with 
patients receiving tamoxifen. Patients receiving anastrozole had an increase in joint disorders 
(including arthritis, arthrosis and arthralgia) and fractures compared with patients receiving 
tamoxifen. A fracture rate of 22 per 1000 patient years was observed on anastrozole and 15 per 
1000 patient years with the tamoxifen group with a median follow up of 68 months. The fracture 
rate for anastrozole falls within the broad range of the fracture rates reported in an age matched 
postmenopausal population. 
 
The Breast International Group (BIG) 1-98 trial also indicate superiority of adjuvant letrozole over 
tamoxifen; indeed the survival benefit appeared to be modestly higher for letrozole over that 
observed with anastrozole (Thurlimann et al., 2005). The increased risks of thrombosis and 
endometrial cancer observed with long term tamoxifen are not seen with AIs. AIs are generally 
well tolerated, with 10-20% patients experiencing hot flushes, and up to 20% with arthralgias and 
increased menopause related osteopenia. Thus, AI-treated patients should be treated with vitamin 
D, calcium and if indicated, bisphosphonates to reduce risk of accelerating osteopenia. 


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In a phase III trial (ABCSG 8) conducted in 2579 postmenopausal women with hormone receptor 
positive early breast cancer being treated with adjuvant tamoxifen, patients had a superior 
disease-free survival when switched to anastrozole compared with those continuing on tamoxifen. 
Two further similar trials (GABG/ARNO 95 and ITA) with anastrozole, as well as a combined 
analysis of ABCSG 8 and GABG/ARNO 95, supported these results. The anastrozole safety profile 
in these 3 studies was consistent with the known safety profile established in post-menopausal 
women with hormone-receptor positive early breast cancer. 
 
Anastrozole metabolism and drug interactions  
Anastrozole is eliminated slowly with a plasma elimination half-life of 40 to 50 hours. Food slightly 
decreases the rate but not the extent of absorption. The small change in the rate of absorption is 
not expected to result in a clinically significant effect on steady-state plasma concentrations during 
once daily dosing of anastrozole tablets. Approximately 90 to 95% of plasma anastrozole steady-
state concentrations are attained after 7 daily doses. There is no evidence of time or dose 
dependency of anastrozole pharmacokinetic parameters. Anastrozole is extensively metabolized 
by postmenopausal women with less than 10% of the dose excreted in the urine unchanged within 
72 hours of dosing. Metabolism of anastrozole occurs by N dealkylation, hydroxylation and 
glucuronidation. The metabolites are excreted primarily via the urine. 
 
Antipyrine and cimetidine clinical interaction studies indicate that the co-administration of 
anastrozole with other drugs is unlikely to result in clinically significant drug interactions mediated 
by cytochrome P450. A review of the clinical trial safety database did not reveal evidence of 
clinically significant interaction in patients treated with anastrozole who also received other 
commonly prescribed drugs. There were no clinically significant interactions with bisphosphonates. 
Tamoxifen and/or other therapies containing estrogen should not be co-administered with 
anastrozole as they may diminish its pharmacological action. 
 
2.2 Neoadjuvant Endocrine Therapy 
 
Neoadjuvant therapy of early breast cancer 
A number of observations support the use of neoadjuvant therapy in early breast cancer. First, 
neoadjuvant therapy provides the earliest possible systemic treatment, potentially eradicating 
micrometastases prior to surgery through changes in tumor cell growth kinetics or alterations in the 


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balance of circulating pro- and anti-angiogenic factors (Fisher et al., 1989; Fidler and Ellis, 1994).  
In addition, neoadjuvant therapy permits an in vivo assessment of drug efficacy (clinical response), 
which may guide the choice of subsequent treatments. Finally, the likelihood that breast-
conserving surgery can be performed is increased with the use of neoadjuvant therapy (Ellis and 
Smith, 1996). 
 
Several large randomized trials have compared neoadjuvant therapeutic strategies (with cytotoxic 
chemotherapy regimens) to standard adjuvant treatments (Powles et al., 1995); (Scholl et al., 
1994); (Semiglazov et al., 1994); (Mauriac et al., 1991); (Fisher et al., 1997).  By far the largest 
phase III study is the NSABP B-18 trial, which randomized 1,523 patients with operable breast 
cancer to preoperative or postoperative doxorubicin-cyclophosphamide (AC) chemotherapy 
(Fisher et al., 1997). There was no significant difference in disease-free or overall survival between 
the groups and more patients receiving neoadjuvant chemotherapy underwent breast-conserving 
surgery (68% vs 60%).  In light of these data, the concept of neoadjuvant therapy is now well 
established, with this approach being considered a standard treatment option for patients with 
early breast cancer (Macaskill et al., 2006). 
 
Neoadjuvant endocrine therapy for breast cancer 
Neoadjuvant antiestrogen therapy for hormone receptor positive early stage breast cancer in 
postmenopausal women is attractive for a number of reasons. Patients receive a form of systemic 
therapy that in the adjuvant setting is as good as chemotherapy in providing long term freedom 
from relapse or death (Abe et al., 2005). The improved potential for breast conserving therapy with 
neoadjuvant endocrine therapy is as good as with neoadjuvant chemotherapy (Ma and Ellis, 
2006a) and there is no indication that deferring surgery during neoadjuvant endocrine treatment is 
detrimental. Hormonal therapy has a well-tolerated toxicity profile compared to chemotherapy. 
Neoadjuvant therapy allows immediate assessment of short term efficacy of therapy. Finally, in 
this context, serial tumor samples can be obtained for ascertainment of biologic endpoints or 
markers of disease response. Biomarker studies can be done to help define the subgroup of 
tumors that are likely to respond to therapy. Although pathologic complete remission (pCR) tend to 
be low in neoadjuvant hormonal therapy studies, clinical response rates of 37-55% are reported for 
AIs and can be corroborated by imaging studies and molecular markers of cell proliferation and 
death (Ma and Ellis, 2006b). In addition to providing information about short term efficacy of 
therapy and identifying predictive biomarkers, neoadjuvant therapy also has the potential to predict 
efficacy of endocrine treatment in the adjuvant setting. Powerful results obtained with new targeted 


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therapies and the identification of predictive biomarkers in the neoadjuvant setting would provide 
strong impetus to initiate more cost and labor intensive phase II-III clinical trials in the adjuvant 
setting.  
 
      Tamoxifen as neoadjuvant therapy for breast cancer 
Tamoxifen is a competitive antagonist of estradiol binding to the estrogen receptor. Tamoxifen has 
been used in lieu of surgery in frail and elderly patients, with response rates of 49-68% and stable 
disease in up to 30% of patients. Median time to maximal tamoxifen response is reported as 14 
weeks (Akhtar et al., 1991). Hormonal therapy has been used in postmenopausal patients with 
locally advanced breast cancer to reduce tumor bulk to allow breast conserving surgery. It is 
noteworthy that though pathologic complete response rates are low (<10%), lower pCR rates in 
ER-positive postmenopausal disease are also reported after neoadjuvant chemotherapy. 
Unfortunately, tamoxifen increases the risk for endometrial cancer by 2.4 times and the risk for 
thromboembolic disease by 1.9 times (Cuzick et al., 2003). Furthermore, despite 5 years adjuvant 
tamoxifen relapses continue even beyond 15 years after therapy (Fisher et al., 2001). Therefore, 
an alternative or additional hormonal therapy may help reduce breast cancer mortality more than 
tamoxifen does and have a more favorable toxicity profile (Altundag and Ibrahim, 2006). 
 
AIs versus tamoxifen as neoadjuvant therapy 
Several studies showed efficacy of AIs as neoadjuvant therapy for hormone receptor-positive 
breast cancers. In a phase II trial, 112 postmenopausal women with locally advanced ER-positive 
breast cancer received neoadjuvant anastrozole (Milla-Santos et al., 2004). Fifty-five percent of 
the patients had complete clinical responses, and 29% had partial clinical responses. However, an 
impressive 23% of the patients had complete pathologic responses. In a phase III randomized trial, 
324 postmenopausal patients with stage II or III hormone receptor-positive breast cancer were 
treated with letrozole or tamoxifen as neoadjuvant therapy (Eiermann et al., 2001). The clinical 
response rate to letrozole was significantly higher than to tamoxifen (55% vs. 36%; p < .001). 
Patients receiving letrozole also had a higher incidence of breast-conserving surgery (Ellis et al., 
2001). 
 
The randomized, double-blind phase III Preoperative Anastrozole Compared with Tamoxifen 
(PROACT) trial compared 12 weeks of anastrozole versus tamoxifen as neoadjuvant therapy in 
451 postmenopausal women with hormone receptor-positive breast tumors (Cataliotti et al., 
2006b). Additional preoperative chemotherapy was optional and was determined before 


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randomization. In the subset of patients who received only hormonal therapy (n = 314), 43% of 
those treated with anastrozole had improved surgical status in favor of breast-conserving surgery 
compared with 31% of those treated with tamoxifen (p = .04) (Cataliotti et al., 2006c). 
 
In the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) 
trial, 330 postmenopausal women with ER-positive, invasive, operable breast cancer were 
randomized in a double-blinded fashion to 3 months of preoperative treatment with anastrozole, 
tamoxifen, or both. The ORR was 37.2% in the anastrozole group, 36.1% in the tamoxifen group, 
and 39.4% in the combination group. In patients who were assessed as requiring mastectomy at 
baseline (n = 124), 44% received breast-conserving surgery after anastrozole compared with 31% 
of those given tamoxifen (p = .23); this difference became significant for patients who were 
deemed candidates for breast-conserving surgery by their surgeons (46% vs. 22%, respectively; p 
= .03). All treatments were well tolerated [43] (Altundag and Ibrahim, 2006). 
 
To date only one trial has compared neoadjuvant chemotherapy with AIs (Semiglazov, 2007). 
Semiglazov et al randomized 121 patients to four cycles of doxorubicin 60mg/m2 with paclitaxel 
200 mg/m2 versus exemestane or anastrozole for 3 months. Clinical response rates were 
encouraging and did not differ significantly (76% vs 82% vs 76%) nor did the pCR rates (7.4% vs 
6.8% vs 4%). While this data suggest potential equivalency in response to neoadjuvant chemo or 
AI therapy, this small single institution study has not been repeated. 
 
2.3 Activation of the Src oncogene: a mediator of antiestrogen therapy resistance 
 
      Src activation is common in human breast cancer 
cSrc is over-expressed in up to 70% of primary human breast cancers. Up to 70% of primary 
human breast cancers show Src activation, with 40% at high levels. Src is activated by the IGF-
1R, EGFR and HER2 and further phosphorylates and activates these receptor tyrosine kinases to 
stimulate cell proliferation, motility and survival. Estrogen bound ER recruits and activates Src, 
leading to Shc, and MAPK activation. Recent gene expression profiling has defined a gene 
signature of Src activation that is observed in up to 40% of human breast cancers (Nevins ref). 
This Src signature was a powerful predictor of antiproliferative responsiveness to a Src inhibitor 
drug in vitro. Overexpression of HER2 or Src in ER positive breast cancer cells can cause 
resistance to antiestrogen therapies by misregulating the cell cycle inhibitor, p27 (see below).  
 


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The cell cycle inhibitor p27 is a key regulator of cell proliferation 
Since this protocol investigates how a Src inhibitor drug may increase responsiveness anastrozole 
by activating p27, the cell cycle will be briefly reviewed. Cell cycle progression is governed by cyclin 
dependent kinases (Cdks) (Reed et al., 1994) that coordinate cell cycle transitions.  The Cdks are 
activated by cyclin binding and regulated by the cdk inhibitors, including p27. p27 is a key regulator 
of proliferation in mammary epithelial cells p27 binds and inhibits cyclin E-Cdk2, an activator of DNA 
replication and cell proliferation p27 levels are high in growth arrested cells and p27 levels must fall 
to allow cells to proliferate p27 null animals exhibit hyperproliferation of many organs and p27+/- 
heterozygous animals are more tumor prone. Oncogenic activation of signaling pathways leads 
almost invariably to loss of p27 function in human cancers, most commonly through accelerated p27 
proteolysis. We and others have shown that p27 levels are frequently reduced in up to 60% of 
primary human breast cancers (Catzavelos et al., 1997). 
 
p27 is required for growth arrest by tamoxifen and by estrogen deprivation 
Estrogen stimulates breast cancer proliferation by causing p27 degradation and cyclin E-Cdk2 
activation. Antisense inhibition of p27 expression caused cells arrested by tamoxifen, fulvestrant or 
estrogen deprivation to re-enter cell cycle, thus, p27 is required for the therapeutic antiproliferative 
effect of tamoxifen and AIs (Cariou et al., 2000). Others have made similar findings (Carol ref). 
 
Src phosphorylates p27 to promote p27 degradation  
Src plays a critical role to trigger the degradation of p27 (Chu et al., 2007). Since we had shown 
that estrogen-driven breast cell proliferation requires rapid p27 proteolysis (Cariou et al., 2000) and 
since Src was known to be activated in breast cancer by growth factors receptors and by estrogen, 
we investigated whether Src could contribute to p27 proteolysis. We showed that Src 
phosphorylates p27 and reduces its inhibitory function toward cyclin E-Cdk2, thereby facilitating 
subsequent p27 degradation (Chu et al., 2007). Data from human cancers also supports a 
relationship between Src activation and p27 loss. The levels of p27 and of activated cSrc 
(detected with an anti-pY416-Src antibody) were assayed by immunohistochemistry in 482 primary 
human breast cancers. Reduced nuclear p27 was seen in 37% of these tumors. Little or no cSrc 
activation was detected in a minority (23%) and 39% of the breast cancers showed strong cSrc 
activation. Of 392 ER positive cancers, 127/339 (37%) showed strong Src activation. This 
proportion is similar to the fraction of ER positive breast cancers that manifest de novo 


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antiestrogen resistance. Reduced p27 was strongly associated with Src activation (p=0.02) (Chu 
et al., 2007).   
 
2.4 Pre-clinical Data Using Src Inhibitor AZD0530 with Anastrozole 
 
Src inhibitorAZDO530 cooperates with anastrozole to inhibit breast cancer growth in vitro 
Preclinical data indicates that the Src inhibitor drug, AZD0530 can cooperate with anastrozole to 
inhibit breast cancer cell growth. To make a breast cancer line that could be treated with aromatase, 
the aromatase gene was introduced into MCF-7 cells The resulting MCF-AROM5 line is estrogen 
dependent and arrests with a low % of cells S phase (%S) when deprived of estrogen, but 
resumes proliferation in the presence of androstenedione, reflecting the intracellular conversion of 
androstenedione to estrone by the aromatase enzyme. Cells were treated with anastrozole, or Src 
inhibitor (AZD0530) or both. While each drug alone had a partial effect, the two used together 
more effectively stopped cell proliferation (See Table 1). 
  
Table 1. Effects of Anastrozole, Src inhibitor or both together on cell cycle 
estrogen 
+ 
- 
- 
- 
- 
- 
no estrogen 
- 
+ 
+ 
+ 
+ 
+ 
androstenedione 
- 
- 
25nM 
25nM 
25nM 
25nM 
anastrozole 
- 
- 
- 
- 
100M  
100
M 
Src inhibitor 
- 
- 
- 
1M 
- 
1M 
S% 
43 
16 
53 
48 
27 
14 
 
Furthermore, when these cells were treated with a dose of AZD0530 that on its own had no effect, 
the dose of anastrozole needed to arrest cell growth was tenfold lower than when anastrozole was 
used alone. These data indicate synergy between AZD0530 and anastrozole in vitro. 
 
AZD0530 augments antitumor effects of anastrozole in breast xenografts in vivo 
MCFAROM5 was growth as xenograft tumors in nude mice. While AZD0530 alone had little effect 
on breast cancer xenograft tumor growth, and anastrozole partially inhibited tumor growth, growth 
inhibition by anastrozole was augmented by concurrent administration of AZD0530 (Fig 1). These 
data support clinical trials of Src inhibitors to prevent or delay the development of anastrozole 
resistance in ER positive breast cancer.  


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0
2
4
6
8
10
12
14
0
4
7
14
21
25
28
32
35
39
relative tumor volum
without androstenedione
androstenedione s.c. 5mg/kg
anaastrozole s.c. 0.25mg/kg
Src inhibitor i.g. 50mg/kg
anastrozole+Src inhibitor
 
 
Fig. 1 Anastrozole and Src inhibitor effects on in vivo growth of breast cancer xenograft 
tumors. All of the groups except that marked without androstenedione received androstenedione 
s.c. 5mg/kg/d. 
 
2.5 Clinical Development of the Src Inhibitor AZD0530 
AZD0530 is a potent, orally available dual inhibitor of Abl and Src family kinases (Ple et al., 2004). 
It showed potent (<uM IC50) antiproliferative activity in 17 human cancer cell lines and inhibited 
growth of 6/13 xenograft tumor models. AZD0530 inhibits cell motility and invasion in vitro (Hiscox 
et al., 2005) and inhibits metastasis in animal models in vivo T. Green, ASCO Abstract , 2005). 
 
Four Phase I studies in 163 healthy subjects have been completed and dosing is complete in a 
Phase I study in 237 patients with advanced solid malignancies (as of March 2008), providing data 
on pharmacokinetics, pharmacodynamics, bioavailability, safety and tolerability of AZD0530. 
AZD0530 is relatively slowly absorbed (median tmax of 5 hours) and is cleared moderately slowly 
(t1/2 of approximately 40 hours). AZD0530 is moderate inhibitor of CYP3A4. At steady state, the 
amount recovered in urine, (AZD0530 plus the N desmethyl metabolite) ranged from 10% to 18% 
indicating the relative importance of renal clearance. Food had no effect on the bioavailability of 
the AZD0530 Phase II formulation. 
 
In a single ascending dose Phase I study, doses between 2.5 and 500 mg were well tolerated by 
healthy subjects and 1000 mg was declared as the maximum tolerated single dose (as a result of 
grade 3 diarrhea in 2 of 5 subjects). Multiply dosed healthy subjects showed a dose-related effect 
of AZD0530 on neutrophil and platelet counts. Neutrophil nadirs were above 1.0 x 109/L in all 
cases occurring between 10-14 days. For all individuals the counts returned to normal within 4 
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days of the nadir. A decrease in platelet count was seen in 1 of 9 subjects treated with 125 mg 
AZD0530 with nadir 118 x 109/L on Day 12, with recovery to within normal range during ongoing 
treatment. A dose-dependent rise in serum creatinine was noted which did not exceed the upper 
limit of normal and returned to pre-dose levels within 6 days after the final dose. In total, 163 
volunteers and 237 patients have been exposed to AZD0530 as of March 2008. In heavily pre-
treated cancer patients (mean # prior systemic therapies = 5; range 1-13) with advanced solid 
malignancies and high metastatic burden, events of ≥2 CTCAE Grade 2 occurring in greater than 
10% of the study population included asthenia, anorexia, anemia, diarrhea, dyspnea, neutropenia, 
fatigue and anorexia. Many of these events are within the realm of what would be expected in this 
patient population. 
 
 
 
 
 
 
 
 
   
 
Preliminary efficacy analysis showed 10 patients had stable disease for longer than 21 days and 6 
patients had stable disease for ≥60 days. These data indicate that Src inhibition in vivo by 
AZD0530 in human subjects is clinically feasible with oral bioavailability, predictable 
pharmacokinetics, and manageable toxicities. Please refer to the AZD0530 Investigator Brochure 
for descriptions of completed Phase I and ongoing phase II trials. 
 
2.6 Summary of scientific rationale for targeting Src in combination with aromatase 
inhibition in ER+ postmenopausal breast cancer 
Aromatase inhibition is a highly effective antiestrogen therapy for ER+ post-menopausal breast 
cancers and is more effective than tamoxifen for metastatic and adjuvant therapy (Wong and Ellis, 
2004) . In the aromatase transfected cell line MCF-AROM5, we found that AZD0530 showed a 
synergistic inhibition of proliferation when added to anastrozole. Anastrozole together with AZD0530 
caused a greater rise in the p27 level, a greater increase in p27 binding to its target cyclin E-Cdk2 
and a greater proliferative arrest than did either drug alone (data not shown). Our xenograft data 
also indicate that AZD0530 could cooperate with anastrozole to arrest the growth of orthotopic MCF-


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AROM5 tumors in nude mice (Fig 1). Taken together, These data, along with Phase I data indicating 
safety and tolerability of bio-effective doses of AZD0530 from Phase I trials in humans, provide a 
strong rationale for the proposed Phase I-randomized Phase II trials to test safety of co-
administration of these drugs and then test their short term efficacy in a context in which molecular 
endpoints of drug effect can be ascertained in serial tissue samples from treated tumors.  
 
2.7 Scientific Rationale for the Study 
We postulated that Src inhibition would augment p27 to promote the anti-proliferative effects of 
anastrozole in ER positive breast cancer cells, leading to a greater reduction in tumor volume, and 
increase in pCR rate. Moreover, it is postulated that responsive cancers can be distinguished from 
poorly or non-responsive cancers by expression of a Src activated protein or gene expression 
profiles in the primary tumors and/or by changes in these parameters during treatment. We also 
postulate that changes in the viability of tumor initiating cells (cancer stem cells) may be critically 
linked to both immediate responsiveness to neoadjuvant therapy and to long term outcome. We will 
investigate the hypothesis that Src inhibition will augment p27 to promote the anti-proliferative 
effects of anastrozole in ER positive breast cancer, leading to a greater reduction in tumor volume, 
and increase in pCR rate. Moreover, we postulate that responsive cancers can be distinguished 
from poorly or non-responsive cancers by expression of a Src activated protein or gene expression 
profile in the primary tumors and/or by changes in these parameters during treatment. We also 
postulate that changes in the tumor initiating cells (cancer stem cells) may be critically linked to 
both immediate responsiveness to neoadjuvant therapy and to long term outcome. 
 
2.8 Rationale for AZD0530 Dosing and Scheduling 
As noted above, four Phase I clinical trials of AZD0530 have been completed, three in healthy 
volunteers and one in patients with advanced solid tumors. From the cancer patient study, an MTD 
of AZD0530 of 175mg po od has been established. At this dose no SAEs were experienced and few 
DLTs. Hemopoetic toxicity, with transient grade 3 neutropenia, was observed in <10% of study 
patients within the first 10-15 days of continuous oral therapy, returning to baseline with continued 
drug treatment. A non-physiologically relevant rise in serum creatinine (Cr) (mean 10%, maximum 
25%) was not accompanied by a reduction in GFR tested pre- and post-treatment and returned to 
baseline within 6 days of stopping the drug. Among the 237 cancer patients and 163 healthy 
volunteers studied in Phase I trials and who have been exposed to AZD0530 prior to March 2008, 
no permanent change in Cr was noted. The 175 mg dose caused no significant, non-hematologic 


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toxicities. Diarrhea, significant only above 500 mg, was not above that in placebo treated controls at 
the 175 mg dose. A papular rash, noted at higher doses, was not observed at 175 mg. 
  
As noted in the background section, AZD0530 is metabolized predominantly via the cytochrome 
P450 CYP3A4 enzyme. Phase I AZD0530 studies with concurrent medazolam administration 
indicate that AZD0530 administered concurrently with anastrozole may increase modestly the 
bioavailability of anastrozole.  Anastrozole is well tolerated, with the most common adverse effect, 
hot flushes, seen in up to 20% patients, but diminishing over the first three months of continuous 
therapy. Other ADE includes joint pains and stiffness and symptoms related to reduce estrogen: 
vaginal dryness and hair thinning. Rare effects include rash, GI symptoms (nausea and diarrhea), 
carpal tunnel syndrome, in <0.1-1%. Earlier studies in women with metastatic breast cancer showed 
that both 1 mg and 10 mg daily oral dosing levels of anastrozole were effective and non-toxic 
(Buzdar et al., 1996). Thus, concurrent AZD0530 and anastrozole administration may modestly 
increase the anastrozole bioavailability but not to levels associated with adverse effects. Moreover, 
co-administration of anastrozole with AZD0530 is not anticipated to reduce bioavailability of 
AZD0530, nor is it expected to increase AZD0530 bioavailability, leading to increased drug toxicity. 
In vitro pharmacokinetic studies have shown that anastrozole can modestly impair CYP3A4 activity, 
but only when administered at 30 fold higher doses than those planned in the present study (Grimm 
and Dyroff, 1997). Thus the serum levels of AZD0530 are not expected to be decreased or 
augmented by concurrent therapy with anastrozole and vice versa.  
 
The Phase I trial will test the safety, tolerability, and bioavailability of anastrozole given with 
AZD0530. Although such studies usually test PK on the new drug starting at 0.5X, then 0.75X and 
only later 1X the MTD of the new biologic agent, because the MDT of 175 mg AZD0530 has minimal 
ADE and because pharmacokinetic studies of both drugs indicate that neither should interfere with 
bioavailability nor augment toxicity of the other, we will start the Phase I study using the MTD of both 
drugs. Daily oral AZD0530 therapy will be added to anastrozole in patients with ER positive MBC 
whose disease has progressed after any hormonal therapy and have been on anastrozole for at 
least one week. Toxicity will be closely monitored and both AZD0530 and anastrozole levels will be 
measured before and at intervals after the addition of AZD0530. 
 
2.9 Rationale for Molecular Investigations 
In addition to assessing the safety of the anastrozole + AZD0530 regimen, another principal aim of 
this study is to investigate whether changes in gene expression, or the expression of specific 


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biomarkers, are either predictive of response to AZD0530 or indicative of response. Given the 
sample size, the molecular data obtained in this trial will be considered hypothesis-generating, with 
the ultimate goal being to develop additional trials which will identify which patients have a greater 
likelihood of benefiting from anastrozole + AZD0530 therapy. The ability to identify markers that 
can predict response, or are indicative of response, will allow more rational use of AZD0530 in 
breast cancer (and potentially other solid tumors). 
 
We propose to assay molecular targets and upstream regulators of Src in diagnostic samples and 
resected cancers at mastectomy or lumpectomy after 4-6 months of treatment with anastrozole 
alone or anastrozole and AZD0530
 
 
 in an 
effort to define molecular endpoints of treatment response that could be used to guide patient 
selection and monitor disease response in subsequent trials. 
 
Pre-clinical studies in cell lines and xenograft studies have suggested that tumor cells with the 
highest Src activation have the greatest anti-proliferative response to AZD0530. These data 
suggest that cancer cells may manifest “Src addiction”’ when this pathway is oncogenically 
activated and not tolerate its inhibition. Since Src activation is at least one of the most probable 
determinants of drug efficacy, we will test whether combined anastrozole/AZD0530 may be most 
effective in Src activated cancers. We will focus the molecular and cellular correlative assays to 
accompany this trial on pathways indicative of Src activation. 
 
 Actual protocols 
used will be updated at the time of molecular analysis on completion of study accrual. 
 
      2.10 Rationale for Imaging Studies 
Dynamic Contrast Enhanced MRI 
A considerable literature indicates that breast MRI may provide important advantages in 
documenting tumor response. It is possible using MRI to calculate tumor volume and gain graphic 
information about tumor blood flow (Tao et al., 2005) and (Hylton, 2006a). Recent MRI studies 
indicate that AIs may reduce tumor blood flow (Tao et al., 2005).  MRI may provide a greater 
accuracy in clinical response measurement than ultrasound, that is highly operator dependent, 
than mammography, since some tumors are not mammographically detected. Dynamic contrast-
enhanced MRI was shown to predict neoadjuvant chemotherapy responses (Martincich et al., 


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2004c)and not only affords 3-dimensional tumor characterization, but has revealed distinct tumor 
imaging patterns that may predict treatment response (Esserman et al., 2001a), (Chou et al., 
2007), (Yankeelov et al., 2007).. While MRI-measured tumor volume after two cycles of treatment 
was predictive of neoadjuvant chemotherapy response (Martincich et al., 2004b), (Hylton, 2006b), 
to date no studies have ascertained the predictive value of MRI after short term neoadjuvant 
endocrine therapy. 
 
Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) performed at high temporal 
resolution following the administration of gadolinium (Gd)-chelated contrast medium enables non-
invasive evaluation of tumor and normal tissue vasculature. DCE-MRI studies of the breast can be 
performed to be sensitive to the vascular phase of contrast medium delivery which reflect on tissue 
perfusion and blood volume or sensitive to the presence of contrast medium in the extravascular 
extracellular space (EES) which reflect on microvessel permeability and extracellular leakage 
space in addition to perfusion (Padhani, 2003). 
 
A number of studies have attempted to correlate the morphological findings on breast MRI with 
key histological features (Knopp et al., 1999); (Buadu et al., 1996); (Matsubayashi et al., 2000); 
(Stomper et al., 1997). For example, early rim enhancement appears to correlate with high 
microvessel density particularly in the tumor periphery, increased expression of VEGF, relatively 
small amounts of fibrosis and small cancer cell nests. Other studies have attempted to correlate 
the kinetic DCE-MRI parameters with immunohistochemical staining in breast cancer. Broad 
correlations between MRI parameters and MVD have been shown by some studies (Buckley et al., 
1997); (Hulka et al., 1997) while others have found no correlation (Su et al., 2003); (Morgan, 
2003). It appears that factors other than MVD are important in determining the degree of tissue 
enhancement (Knopp et al., 1999). For example, Knopp et al used high temporal resolution 
imaging to quantify contrast enhancement, and were able to calculate the intensity of 
enhancement (amplitude), redistribution rate constant (kep) and the elimination rate constant (kel), 
in a study comparing the vasculature of benign and malignant breast lesions (Knopp et al., 1999). 
As a correlate, tissue vascular density was assessed using CD31 and expression of VEGF 
determined using immunohistochemistry. The results suggested there were significantly faster 
exchange rates in malignant lesions compared to benign breast tissue, distinct differences 
between histologic subtypes and a significant dependence of enhancement kinetics on VEGF 
expression. However, some authors have not found a correlation between VEGF expression and 
the degree of enhancement in breast cancer (Su et al., 2003). 


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A number of clinical studies have reported on the usage of DCE-MRI in phase 1 trials of 
antiangiogenesis cancer drugs (Morgan, 2003); (Jayson et al., 2002); (Galbraith et al., 2002); 
(Yung, 2003); (Galbraith et al., 2003). The use of DCE-MRI was to provide biological evidence of 
drug action and to impart confidence and thus accelerate the development of a particular drug or 
approach (go-no-go decisions). Good examples of the use of DCE-MRI in this way can be found in 
studies performed by Jayson et al, Morgan et al. and Galbraith et al (Morgan, 2003); (Jayson et 
al., 2002); (Galbraith et al., 2002); (Knopp, 2007). The value of DCE-MRI as an early predictor of 
the efficacy of neoadjuvant chemotherapy in patients with breast cancer has not been fully 
assessed. Both Knopp and Hayes et al. have shown that successful treatment causes decreases 
in the rate and magnitude of enhancement and that poor response results in persistent abnormal 
enhancement (Hayes et al., 2002); (Ah-See, 2004). DCE-MRI changes become pronounced after 
2 cycles of therapy and DCE-MRI at that stage can be used to identify potential non-responders 
(Martincich et al., 2004a); (Leach et al., 2003). A small neoadjuvant trial in patients with 
inflammatory breast cancer utilized DCE-MRI to assess the effect of bevacizumab on the tumor 
before and after combining with the preoperative chemotherapy (Wedam SB, 2004). The results 
demonstrated marked changes in the permeability as measured by the K21 as well as other 
measures of tumor blood flow. 
 
The goal of functional imaging in this study is to establish a correlation between DCE-MRI 
parameters (as outlined above) with clinical/molecular parameters, and to identify whether DCE-
MRI can be used as an early predictor of response to endocrine therapy with or without Src 
perturbation. Thus, in detail the aims are: 
1. To assess whether parameters derived from the DCE-MRI study correlate with molecular 
parameters that reflect the underlying tumor response. 
2. To assess whether changes in MRI kinetic parameters following the treatment anastrozole 
vs. anastrozole + AZD0530 enabling assessments of drug effect. 
3. To determine if changes in DCE-MRI parameters after 10 weeks (+/- 5 business days) of 
combination therapy correlate with or predict final pathological outcome following 
anastrozole alone or anastrozole plus AZD0530. 
4. To determine which MRI parameter is best in being able to predict pathological 
responsiveness/non-responsiveness to combination therapy. 
 


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3.0 PATIENT SELECTION 
Cohort A (PK cohort): The target population consists of postmenopausal patients with hormone 
receptor positive relapsed or metastatic breast cancer who are candidates for therapy with an 
aromatase inhibitor. 
 
Cohort B (randomized double-blind phase II neoadjuvant cohort): The target population includes 
female patients with invasive, > 2 cm, HER2-negative breast cancer who have not received prior 
therapy. Patients must be postmenopausal and tumor must demonstrate expression of the 
estrogen receptor and/or progesterone receptor. Patients with distant metastatic disease are 
excluded from study. Patients with no distant metastasis but with local metastatic extension of 
tumor to skin will not be excluded. Patients may not have inflammatory breast cancer. Bilateral or 
multifocal invasive breast cancer is not an exclusion criteria. The patient may have concurrent 
DCIS but the DCIS will not be measured as part of the study endpoints. 
 
3.1 Inclusion Criteria 
 
Inclusion Criteria (phase I PK cohort A) 
1. Female patient > 18 years. 
2. Patient must be postmenopausal, verified by 1 of the following:  
a. Bilateral surgical oophorectomy 
b. No spontaneous menses > 1 year  
c. No menses for < 1 year with FSH and estradiol levels in postmenopausal range 
If a study subject under the age of 60 reports prior surgery in which the ovaries were removed 
and if the operative report cannot be obtained to confirm bilateral salpingo-oophorectomy, the 
subject will have serum estradiol, LH and FSH drawn to confirm menopausal status prior to 
study entry. 
3. Postmenopausal women with primary invasive breast cancer, histologically confirmed by core 
needle (or incisional biopsy), whose tumors are estrogen (ER) and/or progesterone (PgR) 
positive. Estrogen- and/or progesterone-receptor positive disease based on 10% or more 
nuclear staining of the invasive component of the tumor. 
4. Stage IV disease (as defined by the AJCC Staging Manual, 6th Edition, 2002); or locally 
relapsed, unresectable disease. 
5. Measurable or evaluable disease according to RECIST criteria (see appendix VI). 


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6. Both HER2-positive and HER2-negative disease (as defined by IHC or by fluorescence in situ 
hybridization [FISH]). HER2+ must have had prior treatment with trastuzumab and/or lapatinib. 
7. ECOG performance status 0-2 (see appendix V). 
8. Patients are suitable candidates for treatment with anastrozole (patients may have had any 
prior endocrine therapy or prior chemotherapy for treatment of their disease, either as adjuvant 
therapy, or as treatment for advanced disease). There is no restriction on the number of prior 
regimens in the phase I cohort A. 
9. Patient is accessible and willing to comply with treatment and follow-up. 
10. Patient is willing to provide written informed consent prior to the performance of any study-
related procedures. 
11. Required laboratory values 
a. Absolute neutrophil count > 1.5 x 109/L 
b. Hemoglobin > 9.0 g/dL 
c. Platelet count > 100 x 109/L 
d. Creatinine < 1.5 mg/dLTotal bilirubin < 1.0 x upper limit of normal (ULN) 
e. Alkaline phosphatase and AST/ALT within the following parameters. In determining 
eligibility, the more abnormal of the two values (AST or ALT) should be used. 
. 
TABLE 2 
AST or ALT: 
ALK PHOS: 
 ULN 
>1x but 1.5x  >1.5x but 2.5x  >2.5x ULN 
 ULN 
Eligible 
Eligible 
Eligible 
Ineligible 
>1x but 2.5x  
Eligible 
Eligible 
Ineligible 
Ineligible 
>2.5x but 5x  
Eligible 
Ineligible 
Ineligible 
Ineligible 
>5x ULN 
Ineligible 
Ineligible 
Ineligible 
Ineligible 
 
Inclusion Criteria (randomized, phase II neoadjuvant cohort B) 
1. Female patient > 18 years 
2. Patient must be postmenopausal, verified by 1 of the following:  
a. Bilateral surgical oophorectomy 
b. No spontaneous menses > 1 year  
c. No menses for < 1 year with FSH and estradiol levels in postmenopausal range 
If a study subject under the age of 60 reports prior surgery in which the ovaries were removed 
and if the operative report cannot be obtained to confirm bilateral salpingo-oophorectomy, the 


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subject will have serum estradiol, LH and FSH drawn to confirm menopausal status prior to 
study entry. 
3. Postmenopausal women with primary invasive breast cancer, histologically confirmed by core 
needle (or incisional biopsy), whose tumors are estrogen (ER) and/or progesterone (PgR) 
positive. Estrogen- and/or progesterone-receptor positive disease based on 10% or more 
nuclear staining of the invasive component of the tumor. Bilateral or multifocal invasive breast 
cancer is not an exclusion criteria. The patient may have concurrent DCIS in either breast but 
the DCIS will not be measured as part of the study endpoints. 
4. Tumor size > 2 cm  
5. Tumor measurable either by clinical examination, mammography, MRI, or ultrasound. 
6. HER2-negative disease (as defined by fluorescence in situ hybridization [FISH] or by IHC) 
7. ECOG performance status 0-1 (see Appendix V) 
8. Patient is accessible and willing to comply with treatment and follow-up. 
9. Patient is willing to provide written informed consent prior to the performance of any study-
related procedures. 
10. Required laboratory values 
a. Absolute neutrophil count > 1.5 x 109/L 
b. Hemoglobin > 9.0 g/dL 
c. Platelet count > 70 x 109/L 
d. Creatinine < 1.5 mg/dL 
e. Total bilirubin < 1.5 x upper limit of normal (ULN)  
f. 
Alkaline phosphatase and AST/ALT < 1.5 x upper limit of normal (ULN). 
 
3.2 Exclusion Criteria 
 
 
Exclusion Criteria (cohort A) 
1. Concurrent therapy with any other non-protocol anti-cancer therapy. 
2. Any agent with estrogenic or putatively estrogenic properties, including herbal preparations, 
must be stopped at least one week prior to registration. 
3. Ongoing, chronic administration of bisphosphonate therapy is allowed so long as such 
treatment was ongoing at the time of study entry.   
4. Current therapy with hormone replacement therapy, or any hormonal agent such as raloxifene, 
tamoxifen, or other selective estrogen receptor modulators (agents must be stopped prior to 
randomization). 


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5. Presence of neuropathy > grade 2 (NCI-CTC version 3.0) at baseline. 
6. History of any other malignancy within the past 5 years, with the exception of non-melanoma 
skin cancer or carcinoma-in-situ of the cervix. 
7. Clinically significant cardiovascular disease (e.g., hypertension [BP > 150/100], history of 
myocardial infarction or stroke within 6 months, unstable angina), New York Heart Association 
(NYHA) Grade II or greater congestive heart failure, or serious cardiac arrhythmia requiring 
medication. 
8. Active, uncontrolled infection requiring parenteral antimicrobials. 
9. A history of a severe hypersensitivity reaction to anastrozole, or AZD0530 or their excipients. 
10. Evidence of bleeding diathesis or coagulopathy. 
11. Resting EKG with measurable QTc interval of >480 msec at 2 or more time points within a 24 
hr. period. 
12. Since AZD0530 is a substrate and inhibitor of CYP3A4, patients requiring medication with 
drugs listed in Appendix X should be excluded from study.  
13. Any evidence of severe or uncontrolled systemic medical or psychiatric conditions (e.g. Severe 
hepatic impairment, interstitial lung disease [bilateral, diffuse, parenchymal lung disease]) or 
current unstable or uncompensated respiratory or cardiac conditions which make it undesirable 
for the patient to participate in the study or which could jeopardize compliance with the 
protocol. 
14. Evidence of underlying pulmonary dysfunction as evidenced by oxygen saturation <90% by 
pulse oximetry, interstitial pulmonary infiltrates on high resolution CT scan prior to study entry 
and/or symptomatic pulmonary (pleural or parenchymal) metastasis. 
 
Exclusion Criteria (cohort B) 
1. Prior chemotherapy, endocrine therapy, or radiotherapy for the presenting breast cancer. Prior 
incidence and treatment of contralateral invasive or non-invasive breast cancer is not an 
exclusion criterion. 
2. Inflammatory BC, clinically defined as the presence of erythema or induration involving one-
third or more of the breast, or pathologically defined as dermal lymphatic invasion. 
3. Prior excisional biopsy or complete resection of the primary invasive tumor (prior sentinel node 
biopsy allowed). 
4. Prior ipsilateral radiation therapy for invasive or non-invasive breast cancer. 
5. Distant metastatic disease is an exclusion criterion – Isolated ipsilateral supraclavicular node 
involvement and/or direct invasion of the primary tumor into skin is allowed. 


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6. Concurrent therapy with any other non-protocol anti-cancer therapy. 
7. Any agent with estrogenic or putatively estrogenic properties, including herbal preparations, 
must be stopped at least one week prior to registration . 
8. Current therapy with hormone replacement therapy, or any hormonal agent such as raloxifene, 
tamoxifen, or other selective estrogen receptor modulators (agents must be stopped at least 
one week prior to randomization). 
9. Presence of neuropathy > grade 2 (NCI-CTC AE version 3.0) at baseline. 
10. History of any other malignancy within the past 5 years, with the exception of non-melanoma 
skin cancer or carcinoma-in-situ of the cervix. 
11. Clinically significant cardiovascular disease (e.g., history of myocardial infarction or stroke 
within 6 months, unstable angina), New York Heart Association (NYHA) Grade II or greater 
congestive heart failure, or serious cardiac arrhythmia requiring medication. 
12. Active, uncontrolled infection requiring parenteral antimicrobials. 
13. A history of a severe hypersensitivity reaction to anastrozole, or AZD0530 or their excipients. 
14. Evidence of bleeding diathesis or coagulopathy. 
15. Resting EKG with measurable QTc interval of >480 msec at 2 or more time points within a 24 
hr. period. 
16. AZD0530 is a substrate and inhibitor of CYP3A4. Since concurrent administration of AZD0530 
with other CYP3A4 substrates has been shown to be well tolerated, continuation or initiation of 
medically indicated drugs that are substrates of CYP3A4 is permitted at MD discretion. Drugs 
listed in Appendix X that are known to strongly induce or inhibit CYP3A4 activity should be 
discontinued prior to study entry and should not be initiated during protocol treatment.   
17. Any evidence of severe or uncontrolled systemic psychiatric or medical conditions (e.g. Severe 
hepatic impairment, interstitial lung disease [bilateral, diffuse, parenchymal lung disease]) or 
current unstable or uncompensated respiratory or cardiac conditions which make it undesirable 
for the patient to participate in the study or which could jeopardize compliance with the 
protocol. 
18.  Evidence of underlying pulmonary dysfunction as evidenced by oxygen saturation <90% by 
pulse oximetry prior to study entry and/or symptomatic pulmonary (pleural or parenchymal) 
disease. 
19. Subjects unwilling or unable to undergo breast MRI as required by protocol will be excluded 
from study. 
 


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3.3 Enrollment procedures 
A signed, written informed consent form must be obtained prior to initiation of any study-specific 
procedures. The screening for metastatic disease is part of routine patient work up and should 
proceed whether or not the patient has signed consent. Staging work-up is not paid for by the 
study. 
 
Prior to the performance of any study-specific procedures or assessments, each subject must sign 
an IRB approved informed consent form. The investigator or member of the research team must 
explain to each subject the nature of the study, its purposes, the procedures involved, the 
expected duration, potential risks, and benefits, and any discomfort it may entail. Each subject 
must be informed that participation is voluntary, and that she may withdraw from the study at any 
time without prejudicing further medical treatment or her relationship with the treating physician. 
 
The person obtaining consent, and the subject, must sign and date the informed consent form. 
The subject must receive a copy of the signed and dated consent form, and the original must be 
retained in the site’s records. 
 
The informed consent is considered part of the study protocol, and any changes to the informed 
consent must be approved by the IRB. 
 
      Enrollment  
All eligible patients must be enrolled prior to the start of the study. 
 
The Phase II portion of this study will be opened at both U of Miami (JMH and Sylvester 
Comprehensive Cancer Center) and at Stanford University (Dr. Slingerland PI at UM and Dr. 
Pegram as PI at Stanford). Enrollment procedures below are detailed for both sites (UM and 
Stanford).  
 
To enter a patient, the investigator or study team member will contact the assigned study 
coordinator at the respective study site. All eligibility requirements must be reviewed by the study 
coordinator and the eligibility checklist must be completed and signed by the Principal Investigator 
or designated Sub-Investigator prior to initiating registration to enter the patient on study. All 
eligibility documents for Stanford patients must be reviewed and approved by the PI (J. 


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Slingerland) or her designees at UM prior to patient enrollment and randomization. The following 
information will be retained in the patient’s study chart:  
 
1) Completed and signed protocol-specific eligibility checklist; 
2) All pages of the original signed informed consent forms (ICFs), including HIPAA Form B. 
3) Relevant source documents such as: subject medical history and physical exam, admission or 
discharge notes, diagnostic reports, pathologic confirmation of diagnosis, and relevant subject-
specific written communication. 
 
The study coordinator will enter a patient on this study once eligibility requirements have been 
verified. 
 
Cancellation Guidelines 
If a patient does not receive protocol therapy, the patient may withdraw. Contact the assigned 
study coordinator at the respective study site or e-mail the information including the reasons for 
withdrawal within 10 working days. Patients who are enrolled on study but not treated will be 
excluded from all analyses. 
 
Randomization and Blinding in Cohort B 
For cohort B, after a signed informed consent has been obtained and eligibility has been 
established, patients will be randomized using a stratified permuted block design to ensure 
allocation 2:1 ratio between combined treatment of AZD0530 plus anastrozole (40 patients) or 
anastrozole plus placebo (20 patients). Patients will be stratified by study site (UM/SCCC or 
Stanford Cancer Institute), and baseline tumor size (≥ 2 and < 6 cm; ≥ 6 cm).  
 
In order to preserve the double-blind nature of this study, the investigators, study team, and 
patients will be blinded to assignment of AZD0530 or placebo.  The UM/SCCC CRS office will be 
responsible for centralized treatment assignment for all registered study patients. Patients will not 
be unblinded until accrual of all patients is completed. However, if a patient should experience a 
serious adverse event that is unexpected and possibly or likely drug related, unblinding will be 
permitted to assess the study drug related SAE as medically indicated. The investigator or sub-
investigator will contact the sponsor who will arrange for unblinding of the individual. 
 


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Randomization lists for each site will be prepared by SCCC Biostatistics and provided to the 
SCCC CRS-Informatics office, where they will be maintained independent of the study team and 
implemented by CRS-Informatics at the UM site. Apart from designated unblinded research 
Pharmacists and monitors at each study site, members of the study team, including those 
responsible for patient enrollment, will not have access to the randomization lists.  
 
After all eligibility requirements are met and signed off, UM CRS study coordinators will register 
the patient by entering the required information into an informatics database. The CRS 
coordinators at each site will not directly receive the designated study arm information. The 
automated patient registration and randomization software used for this study will automatically 
determine the patient’s treatment assignment, and this information will be sent by e-mail to the 
“unblinded” Research Pharmacist at the respective site, in charge of providing study drug to 
patients at the particular site (UM/SCCC and Stanford Cancer Institute). After randomization is 
completed the CRS coordinator will provide a printed copy of the randomization confirmation to the 
Research Pharmacist at the particular site (UM/SCCC and Stanford Cancer Institute). All 
registration documents and randomization confirmations will be printed by the study coordinator 
and placed in the patient’s research chart at each study site.  
 
Additional procedures 
All study procedures/treatments must occur within the following time periods (also refer to the 
Study Schema): 
 
No more than 28 days must elapse between registration and the start of protocol treatment. 
All screening studies and procedures must be performed in this window. 
 
In cohort B, repeat dynamic MRI should be performed on week 10 (+/- 5 business days) 
after the start of AZD0530. The final pre-surgical MRI study must be performed prior to 
definitive surgery.  
 
For cohort B, definitive surgery, should occur within 28 days after the final dose of 
AZD0530. 
 
The post-surgery visit should occur between 4-6 weeks after surgery. 
 
3.4 Regulatory Authority Approval 
The principal investigator will be responsible for obtaining approval, in accordance with all specific 
regulatory requirements, to conduct the study under appropriate regulatory agencies. 
 


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3.5 Ethical Conduct of the Study 
This study will be conducted in accordance with Good Clinical Practice (GCP) and ICH guidelines, 
the Declaration of Helsinki (Edinburgh, Scotland Amendment, 2000), and all applicable local 
regulatory requirements. 
 
3.6 Institutional Review Board/Ethics Committee 
The protocol, informed consent form, and all other required documents will be reviewed and 
approved by a properly constituted Institutional Review Board (IRB). A signed and dated statement 
that the IRB has approved the study must be forwarded to the sponsor prior to the initiation of the 
trial. Any amendments to the protocol must also be approved by the IRB, according to applicable 
regulations. 
 
The Investigator is responsible for filing required progress reports with the IRB and with reporting 
serious adverse events, life-threatening events, and deaths. The Investigator is also responsible 
for filing with the IRB reports of serious adverse events occurring with study treatment in other 
trials, as reported by the sponsor. The Investigator is responsible for informing the IRB of the 
termination of the study. 
 
4.0 TREATMENT PLAN 
 
4.1 Cohort A (Phase I PK cohort) Study Treatment 
 
AZD0530 Dose Finding for Combined Treatment with Anastrozole 
We will begin by testing the combination of AZD0530 at 175mg with anastrozole 1 mg (both drugs 
administered once daily P.O.). Since the steady state anastrozole is reached in one week, study 
patients will be treated with anastrozole alone for one week, followed by three weeks of combined 
daily treatment of AZD0530 plus anastrozole. When given alone, steady state AZD0530 
concentrations are reached in 11 days. Thus, we propose to evaluate dose limiting toxicity (DLT) 
during the first 21 days of combined treatment.  
 
In this study, DLT is defined as any grade 3 or higher non-hematologic toxicity or a grade 4 
hematologic toxicity as per NCI CTCAE v3.0, that is of clinical significance (as defined by the 
investigator as an event that results in an alteration to the planned routine management which would 
not normally be necessary), and occurs within the first 21 days of combined treatment and is 


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considered to be related to the combination of AZD0530 and anastrozole. Combined treatment will 
be continued for as long as the disease is stable or responding to treatment, provided the patient 
tolerates the treatment.  
 
We will use the standard design (cohorts of 3-6 patients), testing initial dose of AZD530 at 175 
mg/day (weeks 2-4) in combination with fixed 1 mg/day anastrozole (in weeks 1-4). In this study, 
simultaneous enrollment is only permitted under the following circumstances: if the first patient to 
receive a dose level does not experience DLT within the first 21 days of combined treatment, 
patients 2 and 3 may be enrolled simultaneously at the same dose level; if no DLTs occur in the first 
3 patients evaluated for minimum 21 days of combined treatment, patients 4 and 5 may be enrolled 
simultaneously at the same dose level; and if no DLTs in first 4 patients, patients 5 and 6 may be 
enrolled simultaneously at the same dose level. This procedure ensures no more than two patients 
will experience DLTs among the first 6 patients treated. 
 
Dose de-escalation of AZD0530: If one patient with DLT is observed in the first cohort of 3 patients, 
additional 1-3 patients will be enrolled at the 175 mg AZD0530 dose. If 2 out of 2-6 patients 
experience a DLT, we will dose de-escalate to accrue a further 2-6 patients at the dose of 125mg 
AZD0530 together with anastrozole 1 mg daily. (AZD0530 is available in pills of 50 mg and 125 mg). 
If 2 out of 2-6 of this de-escalated cohort have DLTs at 125mg AZD0530 and 1 mg anastrozole, this 
will lead to further dose reduction of AZD0530 at 100 mg, which will be tested in the same manner in 
cohorts of 2-6 patients.  
 
 
Table 3 
AZD0530 Dose Reduction Guidelines 
 
Drug 
Starting dose 
Dose Level -1 
Dose Level -2 
Anastrozole 
1 mg 
1 mg 
1 mg 
AZD0530 
175 mg 
125 mg 
100 mg 
              
 
No dose re-escalations will be permitted. 
 
The MTD (maximum tolerated dose) of AZD0530 combined with fixed anastrozole 1 mg is defined 
as the highest dose of AZD0530 for which the incidence of DLT is less than 33% in 6 patients. We 
will enroll additional patients at the determined MTD of AZD0530 (in combination with anastrozole 


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1mg daily), to evaluate pharmacokinetics in a total of 12 patients. The determined MTD of AZD0530 
will be then be used in the phase II portion of study.  
 
Pharmacokinetic evaluation 
At intervals after initiation of AZD0530 on day one at 6, 12, 24, 48, and 72 hours after the first 
dose of AZD0530; and on days 8, 15 and 22 blood will be drawn and sent to AstraZeneca for 
pharmacokinetic measurement of AZD0530 and anastrozole. Because of potential inter-patient 
variability in drug clearance, once drug doses that allow effective bioavailability, without undue 
toxicity are determined, we will accrue a total of twelve patients at those doses for complete PK 
determinations. Samples will be taken before the patients ingested the drug doses on the day of 
the sample to make sure the concentrations were actually at a minimum. Anastrozole 
concentrations during AZD0530 treatment would be compared with those measured prior to 
initiating AZD0530; AZD0530 concentrations would be compared with data on file at AstraZeneca.  
 
      Summary of Phase I Data 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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4.2 Treatment Plan Phase II Cohort B (randomized neoadjuvant cohort)  
 
The phase I cohort of the study established a dose of AZD0530 that can be used together with 
anastrozole that is safe and potentially efficacious for subsequent Phase II and III studies in 
patients with locally advanced or metastatic breast cancer. The neoadjuvant context offers a 
number of advantages for Phase II drug development: 1. it allows a rapid in vivo response 
assessment; 2 the disease context allows comparison of diagnostic biopsy and surgical specimen 
to determine biologic indicators of treatment response; 3. promising new adjuvant endocrine 
strategies can be tested short term against standard treatment; 4. greater response rates, surgical 
outcomes and evidence of enhanced efficacy at the cellular level provide a sound basis for taking 
this therapy forward and committing the resources required for Phase III trials.  
 
When a combination of non-toxic doses of both drugs is found that allows good bioavailability of 
both, we will proceed to a randomized Phase II trial of anastrozole and AZD0530 for neoadjuvant 
first line treatment of postmenopausal patients with ER positive with newly diagnosed disease given 
for 4-6 months prior to surgery. There is considerable prior data on anastrozole given alone as 
neoadjuvant endocrine therapy (Smith et al., 2005; Cataliotti et al., 2006a) . We postulate that 
AZD0530 may reverse or delay development of resistance to AIs such as anastrozole and more 
effectively treat early breast cancers. Patients will be accrued in a 2:1 AZD0530 plus anastrozole vs. 
anastrozole plus placebo randomization protocol. The randomization modestly reduces our power to 
detect differences, but will give the greatest information about the potential safety and efficacy of the 
new drug combination. Patients will be treated with anastrozole and AZD0530 or placebo starting on 
day 1.Treatment efficacy will be indicated by clinical measurement and magnetic resonance imaging 
(MRI) of the primary tumor and the rate of pathologic complete response observed at the time of 
definitive surgery (lumpectomy or mastectomy). The randomization not only permits an indication of 
early efficacy, it will allow the cleanest ascertainment of molecular and cellular endpoints specific to 
the combined therapy that are not observed with anastrozole alone.  
 
 
Patients with newly diagnosed ER and/or PR positive breast cancer (tumors ≥2 cm) will be 
randomized to receive anastrozole (1 mg po/day) plus placebo; or combined anastrozole 1 mg 
po/day and the dose of AZD0530 determined in the phase I cohort of study above (175mg), for up 
to 6 months prior to definitive lumpectomy or mastectomy. Informed consent must be signed 
before randomization and study drugs will be initiated within 10 business days of randomization. 
For study subjects at Stanford University, randomization will be done at U of Miami. Response to 


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AIs is typically slower than with neoadjuvant chemotherapy. Moreover clinical response with 
neoadjuvant AI at 4 months has been reported as greater than that at 3 months, suggesting that 
some may benefit from a greater duration of pre-operative therapy (Ma and Ellis, 2006). 
Responding patients therefore will be treated for up to 6 months. Progression on neoadjuvant 
anastrozole alone has been reported in as few as <11% (Smith, et al., 2005). Patients with 
primarily non-responsive disease or a clinically palpable tumor that responds initially then shows 
stability over the three subsequent 4-week cycles will have their preoperative MRI and proceed to 
surgery directly.  
 
Inclusion criteria include a tumor that may be measurable either by clinical examination, 
mammography, MRI, or ultrasound at diagnosis.  
 
For non-palpable tumors: If a tumor is not clinically palpable at diagnosis and is measurable only 
by radiologic means (MRI, mammography or ultrasound), treatment response will be evaluated by 
both physical exam and radiologic tumor evaluation. The first on treatment radiologic evaluation 
will be the planned MRI at 10 weeks. If the week 10 MRI shows the tumor size unchanged 
compared to pre-treatment, further radiologic evaluation will be performed 4 weeks later (14 
weeks) (See Appendix I). Radiologic imaging must show decrease in tumor size within 14 weeks. 
If the tumor shows no size reduction at that time, the patient will be sent to surgery. Non-palpable 
tumors that increase in size radiologically will be sent for surgery. If the week 10 MRI shows size 
reduction, the next planned radiologic evaluation will be at 18 weeks for subjects completing 20-24 
weeks or at end of study for subjects who stop therapy after 4 cycles. Responding tumors that 
decrease or remain stable on imaging between 10 and 18 weeks will complete planned therapy 
provided there is no sign of tumor progression on physical exam. 
 
Pharmacokinetic evaluation 
The day on which study drugs are first taken for Phase II Cohort B will be designated day zero. To 
extend PK data obtained from Cohort A, Cohort B patients will have PK draws on days 28 and at 
the follow-up visit of the next 2 cycles thereafter (days 56 & 84+/- 3 business days)). 
 
4.3 Supportive Care Guidelines 
 
Use of Hematopoietic Growth Factors 


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i.G-CSF 
The use of G-CSF as primary prophylaxis is not permitted. G-CSF is permitted at the 
discretion of the treating investigator for patients with febrile neutropenia, or grade 4 
neutropenia lasting >7 days. Patients receiving Neupogen® or Neulasta® should receive the 
following doses and schedules: 
 
 
Neupogen®: 
300 ug or 480 ug subcutaneously for 7-10 days  
 
 
Neulasta®: 
6 mg subcutaneously  
 
ii.Erythropoietic agents 
Erythropoietic agents may be used at the discretion of the treating investigator for patients with 
an Hgb < 11 g/dL. Epoietin-alpha, epoietin-beta, or darbepoietin may be used, and each 
should be administered according to standard clinical practice guidelines for the Phase I trial 
only. 
 
iii.Prophylactic Antibiotics 
 
The use of prophylactic antibiotics is not permitted. 
 
4.4 Duration of Therapy 
Protocol treatment will continue until one of the following criteria is met: 
 
Completion of all prescribed protocol therapy. 
 
Disease progression (Phase I and II) and in the Phase II trial (Cohort B patients) disease 
stability over three successive 4-week cycles. In the Phase II trial, patients with palpable 
tumor size unchanged after 3 successive 4-week cycles will be sent for surgery. See 
criteria for evaluation of non-palpable tumors on page 36. 
 
Unacceptable toxicity (defined as toxicity necessitating the discontinuation of study drug, 
as outlined in Section 4.5 below).  
 
Withdrawal of patient consent.    
 
Continuation would, in the judgment of the investigator, not be in the best interests of the 
subject.  
 
Clinical review for toxicity will be performed weekly for four weeks at the time of PK draws, and 
tumor assessments by physical examinations will then be monthly until evidence of disease 


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progression or removal from protocol for Cohort A. Patients in Cohort B will be assessed at 
baseline and then every 4 weeks (+/- 3 business days). Imaging studies will also be obtained 
serially as described in Section 5.2 below. 
 
4.5 DOSE DELAYS/DOSE MODIFICATIONS 
In the event of severe toxicities or failure of hematologic recovery, AZD0530 may be delayed up to 
3 weeks. Any toxicity necessitating a treatment delay greater than 3 weeks will result in the 
discontinuation of study drug treatment. Patients will remain on study (but off AZD0530) during the 
period required to assure resolution of possible or actual AZD0530 related AEs. Since anastrozole 
has been in use for decades and is very well tolerated on its own, and since anastrozole is of 
known significant efficacy in the neoadjuvant setting for patients being treated with curative intent, 
patients are to remain on study-supplied anastrozole during evaluation of potential AZD0530 
toxicity. 
 
 
Dose reductions should be made according to the organ system demonstrating the greatest 
degree of toxicity. Dose reductions should be made according to Table 4.  
 
 
 
No dose re-escalations will be permitted. 
 
Dose Delays for Neutropenia 
 
The following modifications should be instituted for neutropenia: 
 
 
Table 4 Delayed ANC Recovery Guidelines 
Neutrophil count (x 109/L) 
Action to be taken 
> 1.5 
Treat on time 
< 1.5 
Delay AZD0530 for 1 week and repeat CBC 
1. If ANC > 1.5, proceed with full-dose AZD0530 
 
2. If ANC < 1.5, hold AZD0530 for one additional week 
If no recovery by day 14, patient should discontinue AZD0530 
 
 
Dose Reductions for Febrile Neutropenia 
Febrile neutropenia is defined as a fever > 38.5 in the presence of neutropenia (ANC < 1.0 x 
109/L). 


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Febrile neutropenia should be immediately treated according to institutional guidelines. Such 
treatment should include the use of empiric antibiotics and hospital admission if indicated. 
 
 
In-hospital care for neutropenic fever will be per standard of care. 
 
After a first episode of febrile neutropenia, AZD0530 should be reduced one dose level per table 4, 
and use of g-CSF considered. 
 
Patients who develop two episodes of febrile neutropenia will not receive further study drug 
treatment and will be discontinued from the study. 
 
Dose Reductions for Infection 
In the event a patient develops grade 3 or grade 4 infection during treatment with AZD0530, the 
drug will be reduced one dose level. 
 
Patients who develop two episodes of grade 3 or grade 4 infection despite G-CSF treatment will 
not receive further study drug treatment. 
 
Thrombocytopenia 
 
The following dose modifications will occur for thrombocytopenia, based on the platelet count: 
 
 
Table 5 Dose Modifications for Thrombocytopenia 
Platelet Count (x 109/L) 
Dose Modification 
> 50 
No dose modification. Treat as planned. 
< 50 
Hold AZD0530 and repeat platelet count weekly. 
If count > 50 after delay, reduce AZD0530 one level for current 
and all subsequent cycles. 
If count < 50 after 2 weeks, discontinue study drug treatment. 
 
Anemia 
The use of erythropoietic agents is permitted in all patients who develop an Hgb < 11 g/dL while 
receiving AZD0530 in the Phase I study. Erythropoietic agents should be administered according 
to institutional guidelines. Blood transfusions and/or an erythropoietic agent should be 
administered for all patients developing grade 3 or greater anemia. For patients developing grade 
3 or grade 4 anemia, AZD0530 should be reduced one dose level and the patient should be 
considered for treatment with an erythropoietic agent (Phase I only). 
 
Other Toxicities 
All toxicities should be treated symptomatically whenever possible. 
 


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For grade 3 toxicities, AZD0530 will be held for a maximum of 3 weeks, until resolution to < grade 
1, and then reinstituted if medically appropriate. AZD0530 should be reduced one dose level for all 
subsequent cycles. If the toxicity does not resolve to < grade 1, study drug treatment will be 
discontinued. 
 
The Phase I part of this study will exclude patients with interstitial lung disease as diagnosed by 
baseline high resolution CT scan. Further evaluation for development of interstitial pneumonitis will 
include a second and third high resolution CT scan, at 8 and 16 weeks respectively, during the 
Phase I study only. Decreased O2 saturation by pulse oximetry, or new pulmonary symptoms 
(dyspnea and/or cough) shall prompt further pulmonary studies using high resolution CT to look for 
evidence of pulmonary interstitial pneumonitis. If interstitial lung disease (bilateral, diffuse, 
parenchymal lung disease) is observed during these evaluations AZD0530 therapy should be 
permanently discontinued and appropriate management should be initiated as per local practice. 
 
Study drug treatment will be discontinued for all grade 4 non-hematologic toxicities. 
 
Discontinuation of AZD0530 Alone 
Patients requiring discontinuation of AZD0530 for AZD0530-specific toxicity will be removed from the 
study after the appropriate follow-up to monitor toxicity resolution (see section 9.8). 
 
Dose Reductions and Delays for Anastrozole 
No dose reductions will be permitted for anastrozole. 
 
Concomitant Treatment 
Patients should be maintained on the same medications throughout the study, as medically feasible. 
All concomitant medications will be recorded in the CRF with indication, dose information, and dates 
of administration up until the date of surgery or end of study biopsy for the Phase II Cohort B patients. 
Please refer to the “Appendix X List of medications which strongly inhibit or induce the CYP3A 
metabolic pathway”. Since concurrent administration of AZD0530 with other CYP3A4 substrates has 
been shown to be well tolerated, continuation of medically indicated drugs that are substrates of 
CYP3A4 is permitted at MD discretion. AZD0530 administered concurrently with CYP3A4 substrates 
may increase modestly drug bioavailability and careful review of concurrent medications and AEs is 
required at each follow up visit while Phase II Cohort B patients are on therapy. 
 


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The following will not be permitted: 
 
Prophylactic antibiotics 
 
Any other investigational agent. 
 
5.0 CLINICAL AND LABORATORY EVALUATIONS 
5.1 Baseline/Pretreatment Assessments 
The following will be obtained at screening: 
Table 6 Screening Assessments (both Phase I and II) 
Assessment/Procedure 
Explanation 
Timing 
Informed consent 
Signed 
Prior to registration 
Medical history 
General medical history including prior and 
concomitant conditions and medications 
(phase II only); tumor history including date 
of diagnosis, histology, stage, all prior 
therapy, hormone receptor status, and 
HER2 status by FISH or IHC; history of all 
baseline toxicities and ECOG performance 
status 
Within 28 days prior to 
start 
of 
protocol 
treatment 
Physical examination 
Physical exam performed by an ARNP or 
physician, including vital signs and clinical 
tumor status by physical examination (bi-
dimensional) if palpable  
Within 28 days prior to 
start 
of 
protocol 
treatment  
Electrocardiogram 
12-lead ECG 
Within 28 days prior to 
start 
of 
protocol 
treatment  
Dynamic MRI (cohort B 
only) 
MRI of breasts 
Within 28 days prior to 
start 
of 
protocol 
treatment. 
Hematology 
Complete blood count including differential 
Within 28 days prior to 
start 
of 
protocol 
treatment 
Standard 
chemistry 
evaluation 
Sodium, potassium, chloride, bicarbonate, 
creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase 
Within 28 days prior to 
start 
of 
protocol 
treatment 
HER2 status 
By FISH or IHC 
 
Prior to start of protocol 
treatment 
Hormone 
Receptor 
Status 
ER and PgR status by IHC  
Prior to study enrollment  
Tumor status 
Documentation of measurable and non-
measurable disease for Phase I. For Phase 
II: 
clinical 
tumor 
status 
by 
physical 
examination (bi-dimensional measurements) 
at time of entry or by radiologic imaging if 
non-palpable.  
Within 28 days prior to 
study entry 
Radiologic 
assessment 
to exclude metastases in 
Will include bone scan and CT abdomen 
with and without contrast and a CT scan of 
Prior to study entry 


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cohort B 
lungs that may be done without contrast.  
Tumor tissue collection  
(snap 
frozen 
and 
paraffin-embedded) 
See Appendix II for collection requirements  
Prior to start of protocol 
therapy 
O2 saturation 
Pulse oximetry (room air) 
 
Within 28 days prior to 
start of therapy 
 
 
5.2 Assessments during AZD0530 treatment 
 
Table 7A Phase I Cohort A 
Assessment/ 
Procedure 
Explanation 
Timing 
Toxicity Evaluation 
 
History of all toxicities or adverse events 
(AE)  and ECOG performance status 
Every 
28 
days 
± 
3 
business days  
 
Physical examination 
 
Physical exam, including vital signs, and bi-
dimensional 
tumor 
measurement 
if 
measurable clinically 
Every 
28 
days 
± 
3 
business days  
 
Hematology 
 
Complete blood count including differential 
Every 
28 
days 
± 
3 
business days  
Standard 
chemistry 
evaluation 
 
Sodium, potassium, chloride, bicarbonate, 
creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase 
Every 
28 
days 
± 
3 
business days  
 
Tumor status 
Documentation of clinical tumor status by 
physical examination if palpable and by 
radiologic investigations CT scans of chest, 
abdomen, and pelvis (and bone scan, CT 
brain scan and/or PET scan if clinically 
indicated) 
Every 
8 
weeks 
± 
3 
business days 
High resolution lung 
CT scan 
 
 
 
 
 
  
Week 8 and 16 if still on 
protocol  ±2 weeks 
 


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Table 7B Phase II Cohort B  
Assessment/ 
Procedure 
Explanation 
Timing 
Toxicity Evaluation 
History 
of 
all 
toxicities 
or 
AE 
and 
concomitant 
medication 
and 
ECOG 
performance status 
Every 
28 
days 
± 
3 
business days  
 
Physical examination 
 
Physical exam, including vital signs, and bi-
dimensional breast tumor measurement* 
Every 
28 
days 
± 
3 
business days  
 
Hematology 
 
Complete blood count including differential 
Every 
28 
days 
± 
3 
business days  
Standard 
chemistry 
evaluation 
 
Sodium, potassium, chloride, bicarbonate, 
creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase 
Every 
28 
days 
± 
3 
business days  
 
Dynamic breast MRI  
 
To monitor tumor size 
Week 10  5 business 
days, 
and 
prior 
to 
definitive surgery (within 4 
weeks ± 3 business days 
of stopping study drug) 
** For non-palpable tumors: If a tumor is not clinically palpable at diagnosis and is measurable only 
radiologically (MRI, mammography or ultrasound), treatment response will be evaluated by both 
physical exam and radiologic tumor evaluation. The first on treatment radiologic evaluation will be the 
planned MRI at 10 weeks. If the week 10 MRI shows the tumor size unchanged compared to pre-
treatment, further radiologic evaluation will be performed 4 weeks later (14 weeks). If tumor size is not 
reduced at that time, the patient will be sent to surgery. Non-palpable tumors that increase in size 
radiologically will be sent for surgery. If the week 10 MRI shows size reduction, the next planned 
radiologic evaluation will be at 18 weeks for subjects completing 20-24 weeks or at end of study for 
subjects who stop therapy after 4  cycles. Responding patients whose tumors decrease or remain 
stable on imaging between 10 and 18 weeks will complete planned therapy provided there is no sign 
of tumor progression on physical exam. 
 
5.3 Cohort B Assessments at End of Anastrozole + AZD0530/Presurgical Visit 
 
The following assessments and procedures will be performed at the conclusion of neoadjuvant 
therapy, prior to definitive surgery (if surgery is to be performed) or radiotherapy: 
 
Table 8 Assessments at End of Neoadjuvant Therapy (Pre-surgical Visit or Early Study 
Withdrawal) 
Assessment/Procedure 
Explanation 
Timing 
Toxicity evaluation 
History since prior exam, including 
history of all toxicities or AE, concomitant 
medication 
and 
ECOG 
performance 
status 
Within 4 weeks prior to 
surgery or at withdrawal 
from study 
Physical examination 
Physical exam, including vital signs, 
weight  
Within 4 weeks prior to 
surgery or at withdrawal 
from study 


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Hematology 
Complete 
blood 
count 
including 
differential 
Within 4 weeks prior to 
surgery or at withdrawal 
from study 
Standard 
chemistry 
evaluation 
Sodium, 
potassium, 
chloride, 
bicarbonate, creatinine, total bilirubin, 
AST, ALT, alkaline phosphatase 
Within 4 weeks prior to 
surgery or at withdrawal 
from study 
Tumor status 
Documentation of bi-dimensional clinical 
tumor size by physical examination  
Within 4 weeks prior to 
surgery or at withdrawal 
from study 
Tumor 
tissue 
(snap 
frozen)* 
See 
Appendix 
II 
for 
collection 
requirements 
At the time of definitive 
surgery or at withdrawal 
from study* 
Pathologic assessment 
Evaluation of tumor specimen in patients 
undergoing 
mastectomy 
or 
breast-
conserving 
procedure 
to 
determine 
response (pCR).  
After definitive surgery or 
at withdrawal from study 
Dynamic breast MRI 
To monitor tumor size 
Prior 
to 
surgery 
(if 
operable) and within 4 
weeks of stopping study 
drugs 
*Per study amendment June 2014, patients that withdraw from study for any reason will have an end 
of study MRI within 4 weeks of stopping study drug and every effort will be made to obtain an end of 
study biopsy or tissue sample at the time of surgical tumor removal. For patients withdrawing from 
study due to SAE, it will not be considered a study violation if they fail to have a final study biopsy. For 
patients whose tumors remain inoperable, repeat core biopsies or an incisional biopsy may be 
obtained after the completion of neoadjuvant study therapy, prior to the administration of additional 
therapy. 
 
 
5.4 Assessments at Post-Surgical Visit 
The following assessments will be performed at a mandatory follow-up visit 4-6 weeks after 
surgery (if surgery is performed). 
 
Table 9 Assessments at Post-Surgical Visit 
Assessment/Procedure 
Explanation 
Toxicity evaluation 
History since prior exam, including history of all toxicities or AEs 
and ECOG performance status 
Physical examination 
Physical exam, including vital signs, weight, wound assessment.  
Hematology 
Complete blood count only as clinically indicated 
Standard 
chemistry 
evaluation 
Sodium, potassium, chloride, bicarbonate, creatinine, total 
bilirubin, AST, ALT, alkaline phosphatase only if abnormal pre-
operatively or if clinically indicated 


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Pathology report 
Document pathologic tumor size and lymph node involvement 
 
6.0 AGENT FORMULATION AND PROCUREMENT 
 
6.1 Anastrozole 
Anastrozole will be obtained from AstraZeneca, Inc., for the purposes of this study. 
 
The drug should be stored, prepared, and administered according to the manufacturer’s guidelines 
and institutional practice. 
 
The dose administered will be 1 mg PO daily.   
 
6.2 AZD0530 
AZD0530 will be obtained from AstraZeneca, Inc. for the purposes of this study. 
 
The drug should be stored, prepared, and administered according to the manufacturer’s 
guidelines. 
 
The dose administered will be 175 mg PO daily or as otherwise specified in Section 4 above. 
Further details on the administration of AZD0530 are found in Section 4 of this protocol. 
 
6.3 Study Drug Storage 
Upon receipt of AZD0530 or placebo, study drug is to be stored according to the manufacturer’s 
specifications. 
 
7.0 CORRELATIVE/SPECIAL STUDIES 
 
7.1 Tissue Collection and Molecular Studies 
 
A principal aim of this study is to explore whether changes in gene expression, or the expression 
of specific biomarkers, are either predictive of response to AZD0530 or indicative of response. 
Given the sample size, the molecular data obtained in this trial will be considered hypothesis-
generating, with the ultimate goal being to develop additional trials that will identify which patients 
have a greater likelihood of benefiting from AZD0530 therapy. 


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To perform the molecular studies on tumor cells, tumor tissue will be obtained serially at the 
following time points: 
 
Core Biopsies (Baseline) 
Tumor samples must be collected at the time of the initial diagnosis (or in a subsequent 
procedure) either by core needle or incisional biopsy. Excisional biopsy will not be allowed. 
Adequate core biopsies, (or the equivalent amount of tissue with an incisional biopsy), are required 
to perform the molecular analyses identified below.  
 
Once the core biopsies have been removed, a minimum of one sample must be immediately snap-
frozen using the procedure outlined in Appendix II or an equivalent technique, then stored in liquid 
nitrogen or at -70 to -80 °C on site. A minimum of one biopsy sample should undergo formalin 
fixation and embedding in paraffin according to standard institutional guidelines.   
 
Definitive Surgery 
A second tumor sample must be collected by biopsy before surgery using the same SOP for pre-
treatment sample if tumor size permits or at the time of the patient’s definitive surgery in the same 
manner as above. Please refer to Appendix II for further details on the procedure. 
 
In patients whose tumors remain inoperable after the completion of chemotherapy, repeat core 
biopsies or an incisional biopsy will be obtained and prepared as described in Appendix II or using 
an equivalent technique. 
 
In the event that a sentinel node biopsy procedure is performed, the dye can be injected into the 
tumor bed after tumor removal. The dye should not be injected into the tumor itself as this may 
alter the tissue and affect the molecular analyses. In the event of tumor response with no evidence 
of tumor at surgery, tissue surrounding the marker clip will be recovered for study evaluations, if 
feasible. 
 


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8.0 MEASUREMENT OF EFFECT 
 
8.1 Functional Imaging Studies 
All patients will have a dynamic contrast-enhanced MRI obtained at the study center at baseline, at 
10 weeks ± 5 business days after initiating study drugs, and prior to surgery. The radiologist 
carrying out the DCE-MRI will be blinded to the patient treatment arm. Contrast enhanced MRI will 
be carried our according to institutional protocol. Patients will be imaged in the prone position with 
IV access established prior to onset of imaging. High resolution 3-dimensional, fat-suppressing 
images will be performed immediately before, after, and at a delayed time point following injection 
of gadolinium-DTPA. Gd-DTPA will be given at a dose of 0.1 mmol/kg body weight over 15 sec, 
followed by a saline flush over 15 sec. Pulse sequence parameters will be as described 
(Esserman et al., 2001b). One three-dimensional acquisition of 64 slices, covering the entire tumor 
affected breast will be acquired in a scan time of 5 minutes. Tumor diameter, blood flow and 
volumes will a calculated using computer assisted diagnostic software as described (Hylton, 
2006c).  Statistical considerations for tumor size measurement will be as above. 
 
We anticipate that MRI evaluation will not only provide a more rigorous evaluation of tumor size 
(diameter), but will also enhance the accuracy of volumetric calculations and provide data on 
effects of hormonal therapy on tumor blood flow that have not been systematically studied 
previously in the context of endocrine therapy. The blood flow information may be of biologic 
interest and may stimulate further analysis of angiogenic factors that may be revealed in the 
molecular endpoint studies. Response to neoadjuvant chemotherapy can be predicted for some 
regimens by MRI response after two treatment cycles. It will be of interest to ascertain if MRI 
response is predictive of clinical and pathologic response to an AI and the novel biologic agent 
AZD0530 in this study. 
 
The following 4 dynamic MRI modeling parameters will result from the dynamic MRI analyses: 
 
 
Transfer constant (Ktrans) 
 
Tissue leakage space (ve) 
 
Rate constant (kep) 
 
Area under the Gd-DTPA curve at 60-seconds (AUGC60) 
 
All imaging studies will be reviewed and scored by radiologists blinded to treatment assignment or 


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outcome.  
8.2 Clinical and Molecular Efficacy Assessment 
In patients with assessable disease, the response to treatment will be determined by evaluating 
both the clinical objective response rate and, in those who undergo mastectomy or a breast-
conserving procedure, the pathologic complete response rate. Efficacy will also be evaluated by 
determining the percentage of patients in each group who undergo breast-conserving surgery. 
 
8.3 Clinical Response Assessment 
 
Assessment of Response by Physical Examination and MRI Evaluation 
The primary objective of the phase II part of the study will be to compare treatment groups 
(AZD0530 and anastrozole versus anastrozole alone) with respect to relative change in tumor size, 
as documented by the greatest tumor diameter on bi-dimensional measurement as measured by 
MD. A secondary objective is to assess size change by MRI volumetric assays at diagnosis and after 
neoadjuvant treatment (prior to surgery). Since clinical improvement is anticipated in both treatment 
groups, we expect to compare the two treatment groups with respect to relative reduction in tumor 
size. Tumors will be measured by the attending physician each 4 week cycle and an interim MRI 
evaluation is planned at 10 weeks after treatment initiation and within 4 weeks after stopping study 
drug. We chose relative reduction/change in tumor size as the primary endpoint of the phase II part 
of our study, because relative reduction/change in tumor size leads to a more statistically sensitive 
comparison of treatments groups compared to RECIST categories. Detailed methodology is 
discussed in the statistical section of the protocol (cf. below). 
  
 
Assessment of Pathologic Response 
A pathologic complete response will be defined as the absence of viable tumor cells in the 
resected specimen, as determined by standard histologic examination. All specimens will be 
reviewed by a central pathologist to determine pathologic response. 
 
Breast-conserving Surgery 
Efficacy will also be evaluated by determining the percentage of patients in each group who 
undergo breast-conserving surgery. 
 


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9.0 SAFETY 
The investigator is responsible for the detection and documentation of events meeting the criteria 
and definitions of an Adverse Event (AE) or Serious Adverse Event (SAE). 
9.1 Definition of an Adverse Event (AE) 
An AE is any undesirable sign, symptom, or medical condition occurring after starting study 
treatment, even if the event is not considered to be related to study treatment. This can include 
any physical or clinical change experienced by the patient whether or not considered related to the 
use of the study drug. An adverse event can therefore be any unfavorable or unintended sign 
(including an abnormal laboratory finding, for example), symptom or disease (including the onset 
of new illness and the exacerbation of pre-existing conditions) temporally associated with the use 
of the study treatment. In this protocol, study treatment refers to AZD0530 (or placebo) or 
anastrozole plus AZD0530 (or placebo). 
 
Examples of an AE include: 
 
Exacerbation of a chronic or intermittent pre-existing condition, including either an increase 
in frequency and/or intensity of the condition. 
 
 
New conditions diagnosed or detected after study treatment administration, even though it 
may have been present prior to the start of the study. 
 
 
Signs, symptoms, or the clinical sequelae of a suspected interaction. 
 
 
Signs, symptoms, or the clinical sequelae of a suspected overdose. 
 
Examples of an AE do not include: 
 
medical or surgical procedures (the condition that leads to the procedure is an AE). 
 
 
Anticipated day-to-day fluctuations of pre-existing diseases or conditions present or 
detected at the start of the study that do not worsen. 
 
 
Progression of the disease/disorder being studied, unless more severe than expected for 
the subject’s condition. 
 


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9.2 Reporting of Adverse Events 
All adverse events must be recorded in the patient's medical records and on the Case Report 
Form. The onset and end dates, severity, duration, effect on study drug administration (e.g., 
discontinuation), relationship to study drug, and administration of any other drug(s) to treat this 
event will be recorded for each AE. The severity of the AE and relationship to study drug will be 
assessed according to specific guidelines. Patients will be questioned and/or examined by the 
Investigator or his/her designee for evidence of AEs. The presence or absence of specific AEs 
may also be solicited from patients by review of systems, as clinically indicated. 
 
9.3 Grading of Adverse Events 
Adverse events will be graded according to the National Cancer Institute Common Terminology 
Criteria for Adverse Events (NCI CTCAE), version 3.0 (Appendix III). 
 
For AEs which are not covered by the NCI CTCAE grading system, the following terminology will 
be used: 
 
 
Mild – awareness of sign, symptom, or event, but easily tolerated. 
 
 
Moderate – discomfort severe enough to cause interference with usual activity and which 
may warrant intervention. 
 
 
Severe - incapacitating with inability to do usual activities or significantly affects clinical 
status, and warrants intervention such as hospitalization. 
 
 
Life-threatening – immediate risk of death. 
 
9.4 MONITORING OF ADVERSE EVENTS 
Patients having adverse events will be monitored with relevant clinical assessments and laboratory 
tests as determined by the Investigator. All adverse events must be followed to satisfactory 
resolution or stabilization of the event(s). Any actions taken and follow-up results must be recorded 
on the appropriate page of the Case Report Form, as well as in the patient’s source 
documentation. Follow-up laboratory results should be filed with the patient's source 
documentation. 
 


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9.5 Serious Adverse Events 
In the event of a serious adverse event (SAE) the first concern will be for the safety of the subject.   
 
An SAE or STEAE is any sign, symptom or medical condition that emerges during study drug 
treatment or during a post-treatment follow-up period that (1) was not present at the start of study 
drug treatment and it is not a chronic condition that is part of the patient’s medical history, OR (2) 
was present at the start of study drug treatment or as part of the patient’s medical history but 
worsened in severity and/or frequency during therapy, AND that meets any of the following 
regulatory serious criteria: 
 
Results in death. 
 
Is life-threatening (subject was at immediate risk of death at the time of the event). 
 
Requires in-patient hospitalization or prolongation of an existing hospitalization. 
 
Results in persistent or significant disability/incapacity. 
 
Constitutes a congenital anomaly or birth defect. 
 
Is medically significant, in that is may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed above. 
 
In the event of an SAE that is felt to be possibly or probably linked to study drug, the subject may 
be unblinded if it is felt to be in the medical interest of the subject to do so, that is, if knowledge of 
use of placebo or saracatinib might assist in medical management of the SAE. This will be done at 
the discretion of the PI in consultation with the Sponsor-Investigator, Dr. Slingerland. The PI 
involved will obtain study drug allocation information from the Research Pharmacist and may 
disclose this to the patient and relevant medical personnel involved in this patient’s management. 
The date, circumstances and the reason for unblinding of the subject will be documented in the 
patient’s study chart in a note to file by the CRC and by the Investigator or Sub-Investigator in the 
physician clinic note. The Research Pharmacist involved will also record the study subject’s 
unblinding in a note to file in the Pharmacy records. 
 
9.6 Reporting of Serious Adverse Events 
Reports of all serious adverse events, as well as all Alert Safety Reports sent by Astra Zeneca, 
Inc. to the Investigators (an IND Safety Report is an adverse event determined by the sponsor to 
require immediate distribution to the Investigators and appropriate Health Regulatory Authorities), 
must be reported to the appropriate Institutional Review Board/Ethics Committee and/or reported 


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in accordance with local law/regulations. The Investigator must keep documentation of such 
notifications at the trial site.   
 
 
The causality of SAEs (their relationship to study treatment) will be assessed by the 
investigator(s).All serious adverse events, whether or not deemed drug-related or expected, must 
be reported by the Investigator or sub-Investigator immediately or within 24 hours (one working 
day) of when the investigator is notified of the event by telephone to the Sponsor. A written report 
to the IRB must follow within 14 days, which includes a full description of the event and any 
sequelae. This includes serious adverse events that occur anytime while enrolled in the study or 
30 days past the end of therapy or withdrawal visit. 
 
All SAE’s must be recorded on a MedWatch 3500 form and faxed or emailed to the Sponsor.  The 
Sponsor will submit to the IRB.  Participating sites should follow their IRB policies and guidelines. 
 
Investigators and other site personnel must inform the FDA, via a Medwatch form, of any 
unexpected and possibly study drug related SAE that occurs according to the FDA reporting 
requirement timelines.  A copy of the MedWatch report must be faxed to AstraZeneca at the time 
the event is reported to the FDA.  It is the responsibility of the investigator to compile all necessary 
information and ensure that the FDA receives a report according to the FDA reporting requirement 
timelines and to ensure that these reports are also submitted to AstraZeneca at the same time. 
 
A cover page should accompany the Medwatch form indicating the following: 
 
 
Arimidex/AZD0530 Investigator Sponsored Study (ISS) 
 
The investigator IND number assigned by the FDA 
 
The investigator’s name and address 
 
The trial name and AstraZeneca D5390L00104 
 
Send by way of fax to: 
 
 
If a non-serious AE becomes serious, this and other relevant follow-up information must also be 
provided to AstraZeneca and the FDA. 
 


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9.7 Reporting for Investigational Agents 
 
The 
FDA 
MedWatch 
3500 
Reporting 
Form 
that 
can 
be 
obtained 
electronically 
at 
http://www.fda.gov/downloads/AboutFDA/ReportsManualsForms/Forms/UCM163919.pdf for FDA IND 
agents. 
Expedited reporting requirements for adverse events experienced by patients on study arm(s) who have received at least one dose of an 
investigational agent 
Attribution 
Grade 2 
Grade3 
Grade 4 
Grade 5b 
Protocol-
specific 
Require-
ments/ 
Exceptions 
Unexpecte
d 
Unexpecte
d 
Expected 
Unexpecte
d 
Expected 
Unexpecte
d 
Expected 
See footnote 
(c) for special 
requirements. 
See footnote 
(d) for special 
exceptions. 
Unrelated 
or 
Unlikely 
 
MedWatch 
3500 
 
if 
Hospitalize
d 
MedWatch 
3500 
if 
Hospitalize
d 
MedWatch 
3500   
MedWatch 
3500   
MedWatch 
3500   
MedWatch 
3500   
Possible, 
Probable, 
or 
Definite 
MedWatch 
3500  a 
MedWatch 
3500  a 
MedWatch 
3500 
if 
Hospitalize
d 
a 
MedWatch 3500A reporting is only required if the event is related to the investigational agent(s); it is not required if the event is 
related only to the commercial agent(s) included in the protocol treatment. 
b 
This includes all deaths within 30 days of the last dose of treatment with an investigational agent(s), regardless of attribution. Any 
death that occurs more than 30 days after the last dose of treatment with an investigational agent(s) and is attributed (possibly, probably, 
or definitely) to the agent(s) and is not due to cancer recurrence must be reported via MedWatch 3500A. 
c 
Protocol-specific expedited reporting requirements: The adverse events listed below also require expedited monitoring for 
this trial: 
Hospitalization:  
Any grade 1 or 2 adverse event which precipitates a hospitalization lasting ≥ 24 hours or prolongs 
hospitalization must be submitted. 
d 
The adverse events listed below do not require reporting: 
 
Grade 4 myelosuppression 
 
 
9.8 Reports of Patient Death 
The death of any patient during the study or follow-up phase, regardless of the cause, must be 
reported within 24 hours by telephone to the principal investigator and to the IRB in accordance 
with local law/regulations. The Investigator must keep documentation of such notifications at the 
trial site. A written report must follow as soon as possible. If an autopsy is performed, the report 
must be provided.  
 
9.9 Period of Observation 
The period of observation for collection and reporting of adverse events for this study is the 
interval between when the subject starts study treatment and  the date of surgical tumor removal 
or end-of-study biopsy, within 28 days after discontinuing study drug. During the Phase II trial, if a 
patient has an AE or SAE that precipitates study withdrawal, the patient should remain on study 
until complete resolution of the AE. Thus, the saracatinib (AZD0530) may be withheld for 
maximum 3 weeks, before the decision is made to stop saracatinib entirely. After this 3 weeks in 


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which saracatinib is stopped, the patient will be monitored for at least an additional for 30 days, or 
until full resolution of the SAE. During the time of monitoring for resolution of the adverse event, 
the patient should continue to take anastrozole (provided by the study) unless this exceeds the six 
4 week cycles of treatment stipulated by protocol. The patient will be considered off study and no 
longer monitored once the AE is resolved. 
 
If an SAE occurs after the period of observation expires, the Investigator should be notified to 
determine how the SAE should be reported. In general, late fatal complications or conditions of 
medical importance should be documented and reported as an SAE. 
 
10.0 DATA REPORTING 
Data will be collected using designated Case Report Forms. Subject data necessary for analysis 
and reporting the results of the study will be entered into a validated, secure database. Clinical 
data management and data validation will be performed in accordance with defined procedures. 
Access to the data will be strictly controlled. 
 
The investigator must complete the Case Report Forms and transmit the data as instructed, and 
must store copies of the Case Report Forms with other study documents in a secure location in 
accordance with all regulatory requirements. All entries to the Case Report Forms must be made 
as described in the completion guidelines. 
 
Essential documents must be retained by the investigator for as long as necessary to comply with 
national and international regulations. Essential documents must be retained for 15 years. 
Essential documents include: 
 
IRB/EC approvals for the protocol, informed consent, and all amendments 
 
All source documents and laboratory records 
 
CRF paper copies 
 
Informed consent forms 
 
FDA Form 1572 (as required) 
 
Any other pertinent study document 
 
10.1 STUDY MONITORING  
The study will be monitored by the UM Data Safety and Monitoring Committee. The extent, nature, 
and frequency of review will be based on enrollment rate and other relevant considerations.   


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Data and Safety Monitoring Plan 
The Research Team will continuously monitor study accruals, toxicities, and response to treatment 
at both study sites. Patients will be monitored closely throughout the study for any toxicity or 
adverse reaction to treatment. All toxicities, regardless their grade, will be recorded in the patient 
case report form using NCI/CTEP Common Terminology Criteria for Adverse Events (CTCAE) 
version 3.0 (Appendix A). Dose escalation/de-escalation and stopping rules described in section 
5.0 will be followed.   
 
The UM/Sylvester Comprehensive Cancer Center’s Data and Safety Monitoring Committee 
(DSMC) will monitor all patients entered on study at all sites for this protocol according to the 
Cancer Center’s DSM Plan (Appendix IV). Data and Safety monitoring for Stanford study site 
patients will be monitored by the UM DSMC. In its oversight capacity, the DSMC bears 
responsibility for suspending or terminating this study. In the event of a major AE resulting in 
death, the Sponsor will be informed, make the decision to unblind the study case affected and 
make further decisions in consultation with the UM DSMC. 
 
DSMC oversight of the conduct of this trial includes ongoing review of accrual and adverse event 
data. In addition, the DSMC will review reports from all audits or study reviews pertaining to this 
clinical trial and take appropriate action. 
 
10.2 Auditing 
In addition to routine monitoring procedures, the IRB, principal investigator and/or the industry 
collaborators may conduct audit for the purposes of quality assurance, and to ensure compliance 
with GCP, ICH, and other applicable guidelines. Regulatory authorities may also conduct audits at 
any time during or after the completion of the study.  
 
10.3 Modification of the Protocol 
Any substantive modifications of the protocol that may affect the conduct of the study, patient 
safety, or scientific validity of the study require a protocol amendment. Such amendments must be 
approved by the IRB/EC prior to implementation at a site. 
 


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10.4 Information Disclosure and Publications 
All information provided by and/or generated relating to this study or the investigational agent, and 
not previously published, is considered confidential and proprietary. This confidential information 
shall not be disclosed or used (except in the performance of this study) without prior written 
authorization. 
 
No publication, abstract, or presentation of any aspect of the study shall be made without the 
approval of the principal investigators and Astra Zeneca, Inc. A pre-specified procedure will be 
used to determine the authorship list of any publication or presentation, taking into account 
participation in the leadership, design, and conduct of the trial as well as patient accrual. 
 
11.0 CRITERIA FOR DISCONTINUATION OF THERAPY 
Criteria for removal from the study include the following: 
 
 
Completion of all prescribed protocol therapy. 
 
Disease progression.  
 
Unacceptable toxicity (defined as toxicity necessitating the discontinuation of study drug, 
as outlined in Section 4).  
 
Withdrawal of patient consent   . 
 
Continuation would, in the judgment of the investigator, not be in the best interests of the 
subject. 
 
12.0 STATISTICAL METHODS AND CONSIDERATIONS 
 
12.1 Phase I PK cohort: Statistical Considerations  
Study patient demographics (age and race) and disease characteristics will be recorded, including 
previous therapy. The safety data, including toxicities, number of treatment cycles initiated and 
completed, reasons for study withdrawal, laboratory data, and concomitant medications, will be 
summarized descriptively. Toxicities will be tabulated by type, grade, duration, attribution to 
treatment, and administered dose of study drug in accordance with Data Safety Monitoring 
Committee guidelines. 
 
 
 


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The effect of AZD0530 on pharmacokinetics of anastrozole will be summarized as geometric 
means and standard deviations for peak concentration (PC) of study drug in plasma and 
corresponding areas under the curve (AUC).  
   12.2 Randomized double-blind phase II cohort: Statistical Considerations 
Study size of 60 patients is based on patient availability and the known efficacy of anastrozole as a 
single agent. Subject accrual will continue until sixty subjects have completed at least 4 of the planned 
6 months of therapy. For data analysis, subjects treated for at least 4 months will be considered to 
have completed therapy. Patients removed from treatment due to disease progression have 
completed treatment, but disease progression will be recorded in the CRF. Patients will be 
randomized in a 2:1 ratio; 40 patients will receive combined treatment of AZD0530 and anastrozole 
and 20 patients will receive anastrozole alone. Patients will be stratified by study site (UM/SCCC or 
Stanford Cancer Institute), and baseline tumor size (≥2 and <6 cm; ≥6 cm). In order to prevent 
selection bias and protect the assignment sequence, the investigators, study team, and patients will 
be blinded to assignment of AZD0530 or placebo. This study size will allow an ongoing evaluation of 
toxicity and initial assessment of efficacy. The primary objective of the phase II part of the study will be 
to compare treatment groups (AZD0530 and anastrozole versus anastrozole alone) with respect to 
relative change in tumor size, as documented by the greatest tumor diameter on bi-dimensional 
measurement. Since clinical improvement is anticipated in both treatment groups, we expect to 
compare the two treatment groups with respect to relative reduction in tumor size. Tumors will be 
measured each 4 week cycle. We chose relative reduction/change in tumor size as the primary 
endpoint of the phase II part of our study, because relative reduction/change in tumor size leads to a 
more statistically sensitive comparison of treatments groups compared to RECIST categories.  
 
Secondary endpoints include tumor size evaluation by MRI, pathologic complete response (pCR), 
clinical response (complete (CR), or partial (PR) responses), and clinical benefit (CR, PR, or stable 
disease (SD)) by RECIST criteria; toxicities; and identification of biologic correlates as indicators of 
treatment response. Because tumors are assayed by MRI at fewer time points (pre-treatment, 10 
weeks and at end of neoadjuvant treatment), the MRI-based tumor size will be analyzed only at the 
end of the study and will not be part of the interim analysis (See Section 12.4.1.2). 
 
We will use clinical improvement as the main study endpoint, defined as relative reduction 
(percentage change) in tumor size = 100(ts1 – ts2)/ (ts1), where ts1 and ts2 are respectively the 
tumor sizes (largest diameter measured by MD) at baseline and after completing neoadjuvant 
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With 60 patients allocated 2:1 to the combined treatment, our study has 83.8% power to detect an 
absolute mean difference of 20%,  corresponding to an anticipated mean relative reduction in tumor 
size of 60% in the combined AZD0530 + anastrozole group (40 patients) and 40% in patients 
receiving anastrozole alone. Furthermore, a smaller absolute difference of 15% between treatment 
arms, i.e. 55% vs. 40%, can be detected with 59.4% power. Power calculations assume common 
standard deviation of 24% and comparison based on a two-sided t-test with 0.045 significance level at 
final analysis, accommodating an interim analysis at 0.005 significance. (See section 12.4.1.2)  Note 
that assuming a slightly smaller standard deviation of 20% increases power to 94.3% to detect a 
difference 60% vs. 40%, and to 75.4% to detect a difference 55% vs. 40%. 
 
Analysis of Phase II data will include a description of baseline characteristics and summary of safety 
data by treatment arm. We will characterize tumor size (measured by MD and by MRI) in study 
patients at baseline and post neoadjuvant treatment (prior to surgery) by descriptive statistics 
(minimum, maximum, median, average, standard deviation). A similar description will be given for 
relative reduction in tumor size = 100(ts1 – ts2)/ (ts1), where ts1 and ts2 are respectively the tumor 
sizes at baseline and post neoadjuvant treatment. The mean relative reduction in tumor size between 
the two treatments will be compared by a two-sample t-test. The pathologic complete response rate 
(pCR) and overall clinical response rate (CR or PR), clinical benefit (CR, PR, or SD), and individual 
response categories (CR, PR, and SD), will be estimated by the exact binomial method.  
 
12.3 Correlative Molecular Studies  
For the purposes of this protocol, tumor tissue samples will be considered evaluable if: 
 
Patient meets eligibility criteria. 
 
Patient has received AZD0530 or placebo, together with anastrozole. 
 
Frozen and paraffin-embedded tumor tissues are collected before and after protocol 
treatment as specified in Appendix II and are obtained by the principal investigator 
in the UM Sylvester research laboratory. 
 
Tumor tissue specimens are adequate for molecular assessment. The adequacy 
will be evaluated by the UM Sylvester laboratory according to the following criteria: 
 
Sufficient tissue to perform immunohistochemistry. 
 
RNA available for microarrays. 
 
Acceptable RNA quality confirmed by Oncogenomics Core, with a rRNA ratio 
(28S/18S) of 1.8-2.4. 


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Given these assumptions, it is expected that approximately 40-50 matched specimens (pre- and 
post-treatment) will be obtained for molecular analysis.   
 
12.4 Randomization and Blinding in Cohort B 
After a signed informed consent has been obtained and eligibility has been established, patients 
will be randomized using a stratified permuted block design to ensure allocation 2:1 ratio between 
combined treatment of AZD0530 + anastrozole (40 patients) and anastrozole alone (20 patients).  
In order to prevent selection bias and protect the assignment sequence, the investigators, study 
team, and patients will be blinded to assignment of AZD0530 or placebo.  See more details in 
section 3.3.  
 
12.5 Safety Evaluation 
The safety analysis will be conducted on all patients who received all or any portion of one pill of 
any study drug. 
 
Descriptive statistics will be used to summarize the number and types of adverse events, serious 
adverse events, abnormal laboratory data, and number of patients in whom study treatment had to 
be stopped, dose-reduced, or delayed. In the phase II part of the trial (Cohort B), adverse events 
in the two study arms will be compared using two-tailed χ² tests or, when expected counts are low, 
Fisher's exact test.  
 
Interim monitoring of phase II component of study 
The Research Team will continuously monitor study accruals, toxicities, and clinical outcome. The 
Sylvester Comprehensive Cancer Center’s Data and Safety Monitoring Committee (DSMC) will 
monitor this protocol according to the Cancer Center’s DSM Plan. DSMC oversight of the conduct 
of this trial includes ongoing review of adverse event data and periodic review of trial outcomes. 
The DSMC also reviews reports from internal monitoring of protocol compliance and data integrity 
conducted by the University of Miami, Office of Clinical Research Operations, and Regulatory 
Support. Additionally, the Sylvester Protocol Review Committee will monitor study progress with 
respect to patient accrual.  
 
In order to preserve the blinding of the phase II part of the study, all analysis needed for 
DMSC review will be done by an independent biostatistician and reported directly to the 


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Sylvester DSMC. These reports will not be provided to the PI or any study team member 
until the trial is completed. 
 
Guidelines for early stopping due to toxicity  
We propose the following guidelines for the DSMC in its review of accumulating data on toxicity. 
The proposed guidelines were developed using Bayesian methods, which can be applied at any 
stage of enrollment without advance specification of the number of interim analyses to be 
performed, or the number of patients evaluable for toxicity, at the time such assessments are 
made (Spiegelhalter, et al., 1994; Fayers, et. al, 1997). 
 
Under the Bayesian method, we assign a prior probability (level of belief at the start of the trial) to 
a range of possible values for the true toxicity rate. As data on treated patients become available, 
the prior probability distribution is revised and the resulting posterior probability becomes the basis 
for recommending either early termination or continuation of the study. Specific stopping 
guidelines based on posterior probabilities for both acute and late occurring toxicity are given 
below along with underlying assumptions for the prior distributions.  
 
Toxicity will be tabulated by arm and if the stopping guideline is exceeded in either arm, the DSMC 
will consider study termination.  
 
If a treatment-related (possible, probable, or definite) death occurs, enrollment will be suspended 
and continuation of the study will be reassessed by the DSMC. 
 
For the purposes of safety monitoring, we define unacceptable toxicity to be any of the following 
treatment-related (possible, probable, or definite) adverse events: 
 
Grade 3 or higher non-hematologic toxicity, excluding rash and fatigue, 
 
Grade 4 or higher hematologic toxicity, excluding lymphopenia, leucopenia, and neutropenia 
lasting ≤ 7 days, and anemia. 
 
Unacceptable toxicity is expected to occur in no more than 10% of patients in either treatment arm 
during the phase II component of study.  If there is evidence that the true rate of this toxicity 
exceeds 20% in either arm, then the study should be suspended or possibly terminated early. 
Specifically, we suggest as a guideline for early termination a posterior probability of 90% or higher 


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that the true rate exceeds 20%. Table 10 shows specific instances where this guideline is met, 
suggesting early termination due to evidence of excessive toxicity.  
 
 
 
 
 
 
Posterior 
probabilities used to derive guidelines for toxicity were calculated under a prior beta distribution 
with parameters 1 = 0.2 and 2 = 1.8, which corresponds to an expected rate of 10% based on 
prior information roughly equivalent to having studied 2 patients. Furthermore, this prior distribution 
assigns a small a priori chance (17.5%) to the possibility that the true rate of unacceptable toxicity 
is 20% or greater.  
 
Guideline for early stopping for futility  
One interim analysis will be conducted during Phase II when 20 patients in the 
anastrozole/AZD00530 arm and 10 patients in the anastrozole only arm have been evaluated for 
the primary efficacy endpoint, ‘relative reduction in tumor size’ as defined in the second paragraph 
of section 17. The interim statistical review of disease response/toxicity requested by the DSMC will 
use clinical size determination alone.  A two-sample t-test will be used to compare treatment arms 
with respect to the mean relative reduction in tumor size from baseline to post neoadjuvant 
therapy. The interim analysis will use a two-sided significance level of 0.005, and the final analysis 
will be done at the 0.045 significance level.  
 
Early termination for futility will be based on the significance of the t-test and the direction of the 
difference as follows. If p≤0.005 and the mean relative reduction in tumor size is greater for 
anastrozole alone than for anastrozole/AZD0530, the DSMC should consider stopping the study 
Table 
10. 
Early 
Stopping 
Guidelines 
due 
to 
toxicity  
to be applied to each arm in phase II. 
Patients 
with  
unacceptable toxicity 
Patients 
evaluated 
 for toxicity 
Observed  
toxicity rate ≥ 
3 
3 to 5 
60% 
4 
6 to 9 
44% 
5 
10 to 12 
42% 
6 
13 to 16 
38% 
7 
17 to 20 
35% 
8 
21 to 24 
33% 
9 
25 to 28 
32% 
10 
29 to 32 
31% 
11 
33 to 36 
31% 
12 
37 to 40 
30% 
Unacceptable toxicity: any of the following treatment-related (possible, 
probable, or definite) adverse events: 
Grade 3 or higher non-hematologic toxicity, excluding rash and fatigue. 
Grade 4 or higher hematologic toxicity, excluding lymphopenia, leucopenia, and 
neutropenia lasting ≤ 7 days, and anemia. 


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early. However, if p≤0.005 and the mean relative reduction is greater for anastrozole/AZD0530 
than for anastrozole alone, the study will continue to the full planned accrual of 60 patients to 
permit the analysis of molecular endpoints of the study and the evaluation of MRI measurement as 
surrogate for MD measurement as planned. Continuation to full accrual if the direction of 
difference favors combined treatment will allow confirmation of the interim finding and better 
precision for estimating the tumor size reduction and other secondary endpoints in both arms. 
 
Since reduction of tumor size is expected with anastrozole alone, accrual should not be 
suspended during the interim analysis.  


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Padhani,A.R. (2003). MRI for assessing antivascular cancer treatments. Br J Radiol 76, S60-S80. 
Paridaens,R. (2004). First line hormonal treatment (HT) for metastatic breast cancer (MBC) with 
exemestane (E) or tamoxifen (T) in postmenopausal patients (pts) - A randomized phase III trial of the 
EORTC Breast Group. Proc Am Soc Clin Oncol 23. 
 
Ple,P.A., Green,T.P., Hennequin,L.F., Curwen,J., Fennell,M., Allen,J., Lambert-Van Der Brempt,C., 
and Costello,G. (2004). Discovery of a new class of anilinoquinazoline inhibitors with high affinity and 
specificity for the tyrosine kinase domain of c-Src. J. Med. Chem. 47, 871-887. 
 
Powles,T.J., Hickish,T.F., Makris,A., Ashley,S.E., O'Brien,M.E., Tidy,V.A., Casey,S., Nash,A.G., 
Sacks,N., Cosgrove,D., and . (1995). Randomized trial of chemoendocrine therapy started before or 
after surgery for treatment of primary breast cancer. J Clin Oncol 13, 547-552. 
 
Reed,S.I., Bailly,E., Dulic,V., Hengst,L., Resnitzky,D., and Slingerland,J. (1994). G1 control in 
mammalian cells. J Cell Sci Suppl 18, 69-73. 
 
Scholl,S.M., Fourquet,A., Asselain,B., Pierga,J.Y., Vilcoq,J.R., Durand,J.C., Dorval,T., Palangie,T., 
Jouve,M., Beuzeboc,P., and . (1994). Neoadjuvant versus adjuvant chemotherapy in premenopausal 
patients with tumours considered too large for breast conserving surgery: preliminary results of a 
randomised trial: S6. Eur. J Cancer 30A, 645-652. 
 
Semiglazov,V.F., Topuzov,E.E., Bavli,J.L., Moiseyenko,V.M., Ivanova,O.A., Seleznev,I.K., Orlov,A.A., 
Barash,N.Y., Golubeva,O.M., and Chepic,O.F. (1994). Primary (neoadjuvant) chemotherapy and 
radiotherapy compared with primary radiotherapy alone in stage IIb-IIIa breast cancer. Ann. Oncol 5, 
591-595. 
 


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Semiglazov, VF. Phase 2 randomized trial of primary endocrine therapy versus chemotherapy in 
postmenopausal patients with estrogen receptor-positive breast cancer. Cancer 110[2], 244-254. 
2007.  
 
Smith,I.E., Dowsett,M., Ebbs,S.R., Dixon,J.M., Skene,A., Blohmer,J.U., Ashley,S.E., Francis,S., 
Boeddinghaus,I., and Walsh,G. (2005). Neoadjuvant treatment of postmenopausal breast cancer with 
anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, 
or Combined with Tamoxifen (IMPACT) multicenter double-blind randomized trial. J Clin Oncol. 23, 
5108-5116. 
 
Stomper,P.C., Winston,J.S., Herman,S., Klippenstein,D.L., Arredondo,M.A., and Blumenson,L.E. 
(1997). Angiogenesis and dynamic MR imaging gadolinium enhancement of malignant and benign 
breast lesions. Breast Cancer Res Treat 45, 39-46. 
 
Su,M.Y., Cheung,Y.C., Fruehauf,J.P., Yu,H., Nalcioglu,O., Mechetner,E., Kyshtoobayeva,A., 
Chen,S.C., Hsueh,S., McLaren,C.E., and Wan,Y.L. (2003). Correlation of dynamic contrast 
enhancement MRI parameters with microvessel density and VEGF for assessment of angiogenesis in 
breast cancer. J Magn Reson. Imaging 18, 467-477. 
 
Tao,Y., Klause,A., Vickers,A., Bae,K., and Ellis,M. (2005). Clinical and biomarker endpoint analysis in 
neoadjuvant endocrine therapy trials. J Steroid Biochem. Mol. Biol. 95, 91-95. 
 
Thurlimann,B., Keshaviah,A., Coates,A.S., Mouridsen,H., Mauriac,L., Forbes,J.F., Paridaens,R., 
Castiglione-Gertsch,M., Gelber,R.D., Rabaglio,M., Smith,I., Wardley,A., Price,K.N., and Goldhirsch,A. 
(2005). A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer. 
N. Engl. J. Med. 353, 2747-2757. 
 
Wedam SB. A pilot study to evaluate response and angiogenesis after treatment with bevacizumab in 
patients with inflammatory breast cancer. Low JA and Yang X. Proc Am Soc Clin Oncol 22. 2004.  
 
Wong,Z.W. and Ellis,M.J. (2004). First-line endocrine treatment of breast cancer: aromatase inhibitor 
or antioestrogen? Br. J. Cancer 90, 20-25. 
 


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Yankeelov,T.E., Lepage,M., Chakravarthy,A., Broome,E.E., Niermann,K.J., Kelley,M.C., Meszoely,I., 
Mayer,I.A., Herman,C.R., McManus,K., Price,R.R., and Gore,J.C. (2007). Integration of quantitative 
DCE-MRI and ADC mapping to monitor treatment response in human breast cancer: initial results. 
Magn Reson. Imaging 25, 1-13. 
 
Yung, W. K. A phase I trial of single-agent PTK 787/ZK 222584 (PTK/ZK), an oral VEGFR tyrosine 
kinase inhibitor, in patients with recurrent glioblastoma multiforme. Proc Am Soc Clin Oncol 22, 99. 
2003. Abstract 
 
Spiegelhalter DJ, Freedman LS, Parmar MK.  Bayesian approaches to randomized trials (with 
discussion). J Royal Stat Soc Ser A 1994; 157:357-416. 
 
Fayers PM, Ashby D, Parmar MKB. Tutorial in biostatistics: Bayesian data monitoring in clinical trials. 
Stat Med  1997;16:1413 - 30. 
 
 
 


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APPENDIX I   
SCHEDULE OF EVENTS PHASE II STUDY
 
 
Parameter 
 
Screening/ 
Baseline 
 
On 
study 
evaluations 
(q 28 days)  
 
End 
of 
AZD0530/ 
Presurg Visite 
 
Post-Surgical 
Visit 
 
Informed Consent 
x 
 
 
 
 
Medical History – up to 28 days 
x 
x 
x 
x 
 
Physical examination – up to 
28 days prior to study entry 
x 
x 
x 
x 
 
Performance Status 
x 
x 
x 
 
 
Toxicity Assessment 
 
x 
x 
 
 
Clinical tumor status – up to 28 
days prior to study entry 
x 
X** 
x 
 
 
Electrocardiogram (ECG) 
x 
 
 
 
 
Dynamic MRIa 
x 
xf 
xf 
 
 
CBC, differential, platelet count 
x 
x 
x 
xg 
 
Serum 
Chemistries 
(electrolytes, creatinine, total 
bilirubin, alkaline phosphatase, 
AST, ALT) 
 
x 
 
x 
 
x 
 
xh 
 
HER2 status (by FISH or IHC) 
x 
 
 
 
 
Hormone Receptor status (IHC; 
local lab) 
x 
 
 
 
 
Radiologic 
Assessment 
to 
exclude distant metastasesb 
x 
 
 
 
 
PK Drawsc 
 
x c 
 
 
 
O2 
saturation 
by 
pulse 
oximetry 
x 
 
 
 
 
Core 
biopsy/tumor 
tissue 
collection (snap frozen and 
paraffin-embedded) 
x 
 
x d 
 
 
Adverse Events 
 
x 
x 
x 
 


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a The baseline Dynamic MRI must be within 28 days of study entry and should, if possible, precede core biopsy 
collection.   
b Are mandatory and will include bone scan and CT abdomen with and without contrast and a CT scan of lungs 
that may be done without contrast.  
c Blood will be collected for PK evaluation of drug levels at day 28 and every 28 days for the next two cycles 
(+/- 3 business days).  
d If surgery is to be performed, tumor tissue collection (See Appendix II) will occur at the time of definitive 
surgery. For patients whose tumors remain inoperable after neoadjuvant therapy, repeat core biopsies or an 
incisional biopsy may be obtained after the completion of study drugs and prior to the administration of 
additional therapy. Patients who withdraw from study due to toxicity will be asked but are not required to have 
repeat study biopsy. 
e Procedures/studies do not need to be performed if previously performed within 28 days. 
f MRI scan at week 10 ± 5 business days and prior to surgery within 4 weeks of stopping study drug 
g,h Only if clinically indicated or abnormal pre-operatively 
**In the event that a tumor is not clinically palpable at diagnosis and is measurable only by radiologic means 
(MRI, mammography or ultrasound), serial radiologic evaluations will include the week 10 MRI and evaluation 
at week 18 if subject is responding, or at end of study if therapy is stopped after cycle 4, see page 43. 


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APPENDIX II TUMOR TISSUE COLLECTION AND PREPARATION 
AZD0530/ANASTROZOLE CLINICAL TRIAL TISSUE COLLECTION 
 
STANDARD OPERATING PROCEDURE 
 
PURPOSE OF STUDY RELATED CORE BIOPSY: 
Patients will usually have been identified as study candidates based on imaging and 
page or on a prior diagnostic biopsy which identifies them as having invasive ER+ and/or 
PR+ (HR+), HER2 negative breast cancer in the post-menopausal setting. If the patient 
has not had a prior diagnostic biopsy, the patient will be consented for the Tumor Bank 
Core Facility and during the diagnostic biopsy, sufficient material will be banked to 
permit retrieval for study related molecular assays, should be patient subsequently 
consent to trial. If the patient has already had a diagnostic biopsy either at UM or JMH, 
but no sample was banked, and the patient subsequently consents to the 
AZD0530/anastrozole Neoadjuvant trial (SCCC20080325), a study related biopsy will be 
performed to permit molecular correlative studies on the pre-treatment specimen 
associated with this clinical trial. Verification of the receptor positive and HER2 negative 
status must be done before the patient is formally entered on the trial.  
 
In this Neoadjuvant clinical trial of anastrozole +/- AZD0530, patients will receive daily 
oral therapy with anastrozole 1 mg po, together with either placebo or AZD0530 175 mg 
po for up to 6 months prior to definitive surgery. All patients must have an adequate core 
biopsy prior to study entry for the purpose of acquisition of samples for molecular 
correlative studies per the protocol. The procedure will consist of an image guided cores 
biopsies taken for: 
 
1. 
At least one but if possible two cores (depending on the tumor size) will be snap 
frozen in OCT per protocol below and stored at -70-800C), in a manner that permits 
subsequent histopathologic verification of the presence of tumor in acquired specimen 
prior to RNA extraction for gene expression profiling. The SOP detailed below provides 
the methodology for acquisition of biopsy samples for subsequent RNA extraction. 
2. 
One core will be placed in a cryo-vial without OCT and immediately frozen in 
liquid nitrogen for subsequent RPPA proteomics analysis 
3. 
Cores will be formalin fixed (two if possible, but at least one) and embedded in 
paraffin for study related IHC analysis. Biopsy samples will be immediately placed in 
formalin per clinical pathology SOP for subsequent embedding and slide preparation. 
 
PRINCIPLE: 
The ability to obtain high quality RNA from fresh tissue is highly dependent on the tissue 
procurement. Delays in tissue processing usually lead to RNA degradation. Inadequate 
freezing can be a major source of pre-analytic variance in downstream applications. In 
addition, estimating the amount of tissue needed to generate a sufficient quantity of RNA 
can be difficult. The following procedure addresses these issues. 
 
SPECIMEN: 
Fresh, unfixed clinical tissue.  
 
 


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MATERIALS: 
Dry Ice 
Disposable Gloves 
Nunc cryovial for the RPPA specimen 
Liquid Nitrogen dewar with sufficient liquid nitrogen to cover samples for RPPA 
Formalin obtained from the pathology department for tissue fixation for IHC studies 
Cryomold© Embedding Molds (VWR, Cat# 25608-924, 1-800-932-5000) 
Cold Plate, 4x4 Stainless Steel sheet (VWR, Cat# 25608-942, 1-800-932-5000) 
OCT Embedding Compound (VWR, Cat#25608-930, 1-800-932-5000) 
BD 10mL Syringe (VWR, Cat# BD309604, 1-800-932-5000)  
Forceps (VWR, Cat#82027-386, 1-800-932-5000) 
Reclosable Zip Bag (VWR, Cat#89005-302, 1-800-932-5000) 
 
STORAGE REQUIREMENTS:   
Tissue samples for RNA extraction must be kept frozen on dry ice or at -80 C. 
The RPPA sample will be kept in Liquid Nitrogen after placement in a cryovial 
 
PROCEDURE: 
1. Obtain dry ice.  
2. Place the dry ice into the styrofoam container; fill 1/3 with dry ice and replace lid.   
3. (3) Cryomolds are provided for potential cores and labeled with the study 
participant’s unique ID number using a permanent marker or barcode label.   
4. Place the cold plate on the dry ice so that it is level and will be cold prior to the 
actual removal of tissue from the individual. 
5. Upon arriving at the site, place the Cryomold containers onto the cold plate. 
6. Using the syringe of OCT embedding compound, fill the depth of each Cryomold 
½ - ¾ full with OCT. The OCT should freeze within minutes. Do NOT transport 
the syringe of OCT or OCT bottle on dry ice. These are to be carried separately 
from the rest of the kit. 
7. If diagnostic tissue is being obtained for the clinical management of the patient, 
the attending Pathologist or Pathology designee must release the tissue for 
research. This can only be done if sufficient tumor is obtained for diagnosis.  
8. If diagnosis has previously been established, the tissue is released for research. 
9. Place the provided tissue immediately onto the frozen layer of OCT using 
forceps.   
10. With its Cryomold on the cold plate once again, place a top layer of OCT on the 
specimen. For optimal quality, the sample should be acquired and frozen within 5 
minutes.   
11. Replace the lid on the container so that the top layer of OCT will freeze. 
12. After the samples are frozen, place them into the provided plastic bag and seal it. 
13. Samples will be transported in Liquid Nitrogen or in dry ice to the Slingerland Lab 
for storage to maintain T of - 70°C -80°C until samples are batched for RNA 
extraction and gene expression analysis.  
14. RNA extraction will be carried out after for frozen section analysis verifies the 
presence of tumor in the cryopreserved tissue.  
15. For patients on clinical trial the submitting physician, phone, pager, email, time, 
date, trial ID number,
 
 
 


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APPENDIX III    WEB ADDRESS NCI COMMON TERMINOLOGY CRITERIA FOR 
ADVERSE EVENTS 
 
The NCI Common Terminology Criteria for Adverse Events, version 3.0, can be 
accessed at the following website: 
 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf 


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APPENDIX IV DATA AND SAFETY MONITORING PLAN 
The Sylvester Comprehensive Cancer Center (SCCC) Data and Safety Monitoring 
Committee (DSMC) will monitor this clinical trial according to the Cancer Center’s DSM 
Plan. In its oversight capacity, the DSMC bears responsibility for suspending or 
terminating this study. 
 
DSMC oversight of the conduct of this trial includes ongoing review of accrual and 
adverse event data, and periodic review of response. The guidelines appearing in 
Section 11 are offered for DSMC consideration in assessing adverse events and 
response to study treatment. In addition, the DSMC will review reports from all audits, 
site visits, or study reviews pertaining to this clinical trial and take appropriate action. 


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APPENDIX V ECOG PERFORMANCE SCALE 
 
 
Point 
 
Description 
 
0 
 
Normal activity 
 
1 
 
Symptoms of disease, but ambulatory and able to carry out 
activities of daily living 
 
2 
 
Out of bed more than 50% of the time; occasionally needs 
assistance 
 
3 
 
In bed more than 50% of the time; needs nursing care 
 
4 
 
Bedridden; may need hospitalization 
 


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APPENDIX VI RECIST CRITERIA 
Definitions 
 
 
Measurable disease – the presence of at least one measurable lesion. 
 
Measurable lesions – lesions that can be accurately measured in at least 
one dimension with longest diameter >20 mm using conventional techniques or >10 mm 
using spiral CT. 
 
Non-measurable lesions – all other lesions, including small lesions (longest 
diameter <20 mm with conventional techniques or <10 mm with spiral CT scan), bone 
lesions, leptomeningeal disease, ascites, pleural/pericardial effusion, inflammatory 
breast disease, lymphangitis cutis/pulmonis, cystic lesions, and abdominal masses that 
are not confirmed and followed by imaging techniques. 
 
Baseline Documentation of Target and Non-Target Lesions 
 
All measurable lesions, up to a maximum of 5 lesions per organ and 10 lesions in total, 
representative of all involved organs, should be identified as Target Lesions and 
recorded and measured at baseline. 
 
Target lesions should be selected on the basis of their size (lesions with the longest 
diameter) and their suitability for accurate repeated measurements (either by imaging 
techniques or clinically). 
 
A sum of the longest diameter (LD) for all target lesions will be calculated and reported 
as the baseline sum LD. The baseline sum LD will be used as a reference by which to 
characterize the objective tumor. 
 
All other lesions (or sites of disease) should be identified as Non-Target Lesions and 
should also be recorded at baseline. Measurements of these lesions are not required, 
but the presence or absence or worsening of each should be noted throughout follow-up. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Response Criteria 
 
Tables 1 and 2 summarize the criteria to be used to evaluate response. 
 
Table 1 Response Criteria for Target Lesions 
 
Response Criteria 
Evaluation of Target Lesions 
Complete 
response 
(CR) 
Disappearance of all target lesions 
 
Partial response (PR) 
At least a 30% decrease in the sum of the LD of target lesions,  
taking as reference the baseline sum LD 
 
Progressive 
disease 
(PD) 
At least a 20% increase in the sum of the LD of target lesions,  
taking as reference the smallest sum LD recorded since the treatment 
started, or the appearance of one or more new lesions 
 
Stable disease (SD) 
Neither sufficient shrinkage to qualify for PR nor sufficient increase  
to qualify for PD, taking as reference the smallest sum LD recorded  
since the treatment started 
 
 
 
Table 2 Response Criteria for Non-Target Lesions 
 
Response Criteria 
Evaluation of Non-Target Lesions 
Complete response (CR) 
Disappearance of all non-target lesions and normalization of tumor  
marker  level 
 
Incomplete response or  
stable disease (SD) 
Persistence of one or more non-target lesions and/or maintenance 
of tumor marker level above normal limits 
 
Progressive disease (PD) Appearance of one or more new lesions and/or unequivocal 
progression  
of non-target lesions 
 
Unknown (UNK) 
Progression has not been documented and one or more non-target  
lesions have not been assessed or have been assessed using a less  
sensitive technique than at baseline 
 
Table 3 summarizes the assessment of overall response, which is a composite of target 
lesion response, non-target lesion response, and presence of new lesions. 
 
 
 
 
 
 
 
 
 


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Table 3 Evaluation of Overall Response 
 
Target lesions 
Non-target lesions 
New 
lesions 
Overall response 
CR 
CR 
No 
CR 
CR 
Incomplete 
response/SD 
No 
PR 
CR, PR, SD 
UNK 
No 
UNK 
PR 
Non-PD and not UNK No 
PR 
SD 
Non-PD and not UNK No 
SD 
UNK 
Non-PD and/or UNK 
No 
UNK 
PD 
Any 
Yes or No 
PD 
Any 
PD 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
Note: 
Subjects with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
classified as having “symptomatic deterioration”.  Every effort should be made to 
document objective progression after discontinuation of treatment. 
 
In some circumstances, it may be difficult to distinguish residual disease from normal 
tissue.  When the evaluation of complete response depends on this determination, it is 
recommended that the residual lesion be investigated (fine needle aspirate/biopsy) to 
confirm the complete response status. 


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APPENDIX VII REVERSE PHASE PROTEIN LYSATE ARRAYS (RPPA) 
 
Combinatorial effects of the multiple genomic aberrations in breast cancer may be most 
clearly manifest at the proteomic level. Thus, functional proteomic analysis has the 
potential to assist in identification of pathways whose activation may account for 
heterogeneity in response to AZD0530.  
 
Reverse phase protein lysate arrays (RPPA) is a novel, high throughput, quantitative 
and cost effective antibody-based approach to the study of functional proteomic patterns 
in tumors. RPPA offers an approach to quantitative profiling of the levels and activation 
of multiple proteins in breast cancer patient samples. It is a moderate throughput, 
quantitative, inexpensive, multiplexed ELISA with the potential to map protein levels as 
well as function in different intracellular pathways in a comprehensive, convenient, and 
sensitive manner. Thus, RPPA provides a practical approach to identification and 
validation of biomarkers for prognosis and prediction of response to Anastrozole 
/AZD0530. Although RPPA is fully validated for analysis of cell lines1-9, additional 
questions remain for its efficient application to breast cancer patient samples and 
eventually to patient management. 
 
 
RPPA allows high throughput quantitation (up to 1000 samples concurrently), low cost 
(~$0.50 per sample/antibody), sensitivity (fg of target) and amount of sample material 
required (ng of lysate). RPPA currently costs about 20 times less than ELISA-based 
approaches and about 10 times less than Luminex (BD). As tiny amounts of protein are 
required, RPPA is applicable to needle biopsies. These characteristics allow the 
simultaneous quantitative assessment of multiple different proteins in a single sample 
giving a comprehensive picture of the activation status of the tumor. The major 
disadvantage of RPPA is that it does not provide spatial organization, but this is resolved 
with complementary IHC. 
 
 
  
 
 
 
 


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APPENDIX VIII GENE EXPRESSION ANALYSIS BY MICROARRAY 
 
Matched pre- and post-treatment tumor samples will be collected from up to 60 patients. 
A direct comparison of pre and post-treatment samples will be done to characterize the 
molecular response to AZD0530 or placebo.  
details will be updated at the time 
of molecular analysis at trial completion. The following provides a procedural guideline 
only. Fluor reversals will be done on each sample pair to minimize the effects of dye 
bias, and each fluor reversal pair will be combined to create an experiment.  In addition, 
a reference sample design will be used to compare each pretreatment sample to a 
reference sample.  Additional microarray experiments will be performed on those 
patients from whom tumor tissue (and usable RNA) can be obtained at the time of 
definitive surgery.  
 
. 
  
 
 
 
 
 
 
 
. 
 
A significant gene expression change will be defined as a 1.50-fold change with a p-
value of 0.01. Histograms of the frequency of gene changes per patient will be 
generated.  The number of groups defined will depend on the distribution of the samples 
across the sum of gene changes, with a minimum of two and a maximum of three 
groups defined. 
 
In addition to treatment response, analysis will also be performed on tumors grouped on 
the basis of the other molecular markers being measured. 
 
Data Normalization 
Normalization for dye bias and background on a single array will be calculated using 
appropriate software.  This software flags outliers, computes statistics on inlier pixels 
and local background, does a background subtraction on features, performs dye 
normalization, and calculates a p-value that is a confidence measure of gene differential 
expression.  Dye and background normalization are performed on a channel basis rather 
than on intensity ratios, so the corrected intensities for the channel corresponding to the 
tumor sample can be used to compare tumor samples across arrays.  For instance, a 
direct comparison between pre- and post-treatment could be made by extracting the 
normalized intensity value for the appropriate channel in each array. 
 
 
 
 


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Supervised Classification Analysis 
Linear discrimination analysis and nearest-neighbor classifiers will be used on filtered 
gene lists to predict tumor response.  Cross validation procedures will be used to assess 
the repeatability of the results on new data. 
 
Selecting Significant Genes 
Both clustering and supervised classification algorithms are sensitive to the genes 
selected for analysis.  We propose to use several selection methods to determine 
significant genes, and then run the analysis on each of these gene sets. 
 
 
 
 
 
 
   
 
 
 
 
 
 
 
Detecting a gene expression signature of Src activation 
 
 
 
 
 
 
 


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APPENDIX IX PHARMACOKINETIC EVALUATION – PHASE I ONLY 
 
Drug 
substance 
6 hours 
12 
hours 
24 
hours 
48 
hours 
72 
hours 
Day 8 
Day 
15 
Day 22 
anastrozole x 
x 
x 
x 
x 
x 
x 
X 
AZD0530 
x 
x 
x 
x 
x 
x 
x 
x 
At intervals after initiation of AZD0530 at 6, 12, 24, 48, and 72 hours; and on days 8, 15 
and 22 blood will be drawn and sent to AstraZeneca for pharmacokinetic measurement 
of AZD0530 and anastrozole. 


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APPENDIX X SUMMARY OF STRONG CYP3A INDUCERS AND INHIBITORS 
PROHIBITED ON STUDY 
The listed strong CYP3A4 inducers and inhibitors should not be used while on protocol 
medications. They are prohibited. 
CYP3A4 inhibitors including: 
 
Antibiotics (clarithromycin, telithromycin) 
 
Antifungals (ketoconazole, itraconazole, voriconazole) 
 
Antiretroviral protease inhibitors (atazanavir, indinavir, nelfinavir) 
 
Grapefruit 
 
CYP3A4 inducers including: 
 
Rifampin, rifabutin, rifapentin 
 
Phenytoin, barbiturates (Phenobarbital) 
 
Carbamazepine 
 
Other (dexamethasone, St. John’s Wort) 
 
Since concurrent administration of AZD0530 with other CYP3A4 substrates that are not 
strong inducers or inhibitors of CYP3A4 is well tolerated, continuation of medically 
indicated CYP3A4 substrate drugs is permitted at physician discretion. AZD0530 
administered concurrently with CYP3A4 substrates may increase modestly drug 
bioavailability and careful review of concurrent medications and AEs is required at each 
follow up visit while Phase II Cohort B patients are on therapy. Consult 
http://www.fda.gov/drugs/developmentapprovalprocess/developmentresources/druginter
actionslabeling/ucm093664.htm for list of known CYP3A4 substrates.