A Phase II Trial of Neoadjuvant PD 0332991, a Cyclin-Dependent Kinase (Cdk) 4/6 Inhibitor, in Combination With Anastrozole in Women With Clinical Stage 2 or 3 Estrogen Receptor Positive and HER2 Negative Breast Cancer
Phase: PHASE2
Conditions: Breast Neoplasms
Sponsor: Washington University School of Medicine

 
Protocol Version: 06/29/2022 
 
Page 1 of 109 
 
 
A Phase II Trial of Neoadjuvant PD 0332991, a Cyclin-Dependent Kinase (Cdk) 4/6 
inhibitor, in Combination with Anastrozole in Women with Clinical Stage 2 or 3 Estrogen 
Receptor Positive and HER2 Negative Breast Cancer 
 
Washington University School of Medicine, Division of Oncology 
660 South Euclid Avenue, Campus Box 8056, St. Louis, MO  63110 
 
Protocol#:  201301106 
Version Date: 06/29/2022 
 
Coordinating Center: 
Washington University School of Medicine 
 
 
 
Principal Investigator:  
Cynthia Ma, M.D., Ph.D. 
Washington University School of Medicine 
MSC 8076-0041-03 
660 South Euclid Avenue  
St. Louis MO  63110 
Telephone: 314-362-9383 
Fax: 314-362-7086 
Email: cynthiaxma@wustl.edu 
 
Co-Investigators 
Institution 
Modality 
Matthew J. C. Ellis, M.B., Ph.D. 
Baylor College of Medicine 
Medical Oncology 
Julie A. Margenthaler, M.D. 
Washington University 
Surgical Oncology 
Mark Watson, M.D. 
Washington University 
Pathology and Immunology 
Souzan Sanati, M.D. 
Washington University 
Anatomic and Molecular Pathology 
Hussam Al-Kateb, M.Sc., Ph.D.,  
Washington University 
Genomics and Pathology Services  
Matthew Goetz, M.D. 
Mayo Clinic – Rochester 
Medical Oncology 
Tina Hieken, M.D. 
Mayo Clinic – Rochester 
Surgical Oncology 
Donald W. Northfelt, M.D. 
Mayo Clinic – Scottsdale 
Medical Oncology 
Andres Forero, M.D. 
University of Alabama 
Medical Oncology  
 
 
Feng Gao, M.D., Ph.D. 
Washington University 
Biostatistics 
 
Study Team Contact:  
Caroline Bumb 
Phone: (314) 362-7249 
Email: cbumb@wustl.edu 
 
Study Drug:  
PD 0332991 (palbociclib) 
IND#:  
 
117008 
Clinical Trials.gov#: NCT01723774 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to 
obtain informed consent, may not be disclosed to another party unless law or regulations require such 
disclosure.  Persons to whom the information is disclosed must be informed that the information is 
confidential and may not be further disclosed by them 
 

 
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A Phase II Trial of Neoadjuvant PD 0332991, a Cyclin-Dependent Kinase (Cdk) 4/6 
inhibitor, in Combination with Anastrozole in Women with Clinical Stage 2 or 3 Estrogen 
Receptor Positive and HER2 Negative Breast Cancer 
 
Protocol Revision History 
 
Initial Approval Version 
 
 
 
 
 
 
01/15/2013 
Amendment #1 Version 
 
 
 
 
 
 
03/01/2013 
Amendment #2 Version 
 
 
 
 
 
 
10/11/2013 
Amendment #3 Version 
 
 
 
 
 
 
01/29/2014 
Amendment #4 Version 
 
 
 
 
 
 
04/24/2014 
Amendment #5 Version 
 
 
 
 
 
 
09/29/2014 
Amendment #6 Version 
 
 
 
 
 
 
02/06/2015 
Amendment #7 Version 
 
 
 
 
 
 
11/25/2015 
Amendment #8 Version 
 
 
 
 
 
 
09/14/2016 
Amendment #9 Version 
 
 
 
 
 
 
06/29/2022 
 
 
 

 
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A Phase II Trial of Neoadjuvant PD 0332991, a Cyclin-Dependent Kinase (Cdk) 4/6 
inhibitor, in Combination with Anastrozole in Women with Clinical Stage 2 or 3 Estrogen 
Receptor Positive and HER2 Negative Breast Cancer 
 
Principal Investigator Signature Page 
 
 
Principal Investigator: 
 
Cynthia X. Ma, M.D., Ph.D. 
 
 
 
 
 
Signature of Investigator 
 
Date 
 
 
 
Printed Name of Investigator 
 
 
 
By my signature, I agree to personally supervise the 
conduct of this study and to ensure its conduct in 
compliance with the protocol, informed consent, 
IRB/HRPO procedures, the Declaration of Helsinki, ICH 
Good Clinical Practices guidelines, and the applicable 
parts of the United States Code of Federal Regulations or 
local regulations governing the conduct of clinical 
studies. 
 
 
 
 
 
 
 
Contact Information 
 
Principal Investigator:  
 
Cynthia X. Ma, M.D., Ph.D. 
 
 
 
 
 
Washington University School of Medicine 
 
 
 
 
 
Division of Oncology 
 
 
 
 
 
660 South Euclid Avenue, Campus Box 8076 
 
 
 
 
 
St. Louis, MO  63110 
 
 
 
 
 
Phone: (314) 362-8903 
 
 
 
 
 
Fax:  (314) 747-9320 
 
 
 
 
 
E-mail:  cynthiaxma@wustl.edu 
 
 
 

 
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SCHEMA 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1Cycle 0 starts while waiting for the result of PIK3CA analysis          
2Cycle length=28 Days 
3Biopsy prior to PD0332991 is not required in the endocrine resistant cohort if the patient had a research tumor sample collected 
at the time of Ki67 analysis while on endocrine therapy  
4Optional biopsy on Day 15 of subsequent cycles is recommended if C1D15 Ki67 was indeterminate or if not performed. 
5Cycle 5 duration is 10-12 days  and administered in patients whose absolute neutrophil is recovered to > 1.5k/mcL and platelet > 
100k /mcL and treatment related AEs to grade 1 or less within 3 weeks after completion of cycle 4.  Patients who do  not 
meet this criteria may proceed to surgery in 3-5 weeks post cycle 4 day 21 PD0332991.  
6Anastrozole is continued until the day of surgery.  If premenopausal continue goserelin 3.6 mg SC monthly throughout the 
neoadjuvant duration.  PD0332991 is also continued until the day of surgery in patients who started cycle 5 therapy. 
7 See Postoperative Therapy and Follow-up Schema  
*NCI9170: A phase II trial of neoadjuvant MK-2206 in combination with anastrozole for PIK3CA mutant clinical stage 2 or 3 
ER+ and HER2- breast cancer 
†If the NCI9170 trial is not open or was open but is now closed at the participating institution 
 
Mutation Negative 
Ki67 > 10% 
Registration to  
the PIK3CA Wild Type Cohort 
Post Operative Therapy 
and follow up 7 
Registration to NCI 9170* or 
the PIK3CA Mutant Cohort† 
Cycle 1 Day 15 (or within the 3rd week): 
Required Tumor biopsy for Ki67 and research assay post PD 0332991 
Ki67 ≤ 10% or indeterminate4 
Continue therapy for a total of 4 cycles (Cycles 1 to 4)  
PD 0332991 PO daily 3 weeks on and 1 week off 
(Days 1-21 on a 28-day cycle) 
Anastrozole5 1 mg PO daily (Days 1 – 28 each cycle) 
If premenopausal add: Goserelin 3.6mg SC monthly (Day 1 each cycle) 
Neoadjuvant chemotherapy per 
physician decision 
Follow up path report 
Surgery6  
Evaluation for pathologic response 
Tumor sampling for research 
PIK3CA Wild Type or Mutant Cohorts (Closed as of Amendment #7) 
Eligible patients: clinical stage II or III ER+ HER2- breast cancer  
with complete resection of tumor as a goal 
 
Had Pre-registered to NCI 9170 trial* or this trial# and started the following procedure: 
 
#1:  Tumor biopsy for PIK3CA mutation testing and research 
#2  Cycle 0 treatment1:  Anastrozole 1 mg PO daily Cycle 0 Days 1-28 
If premenopausal add: Goserelin 3.6mg SC on Day 1 
Cycle 1 Day 12 
Required tumor biopsy for research assay before starting PD 03329913 
Start PD 0332991 orally daily on Days 1-21 each cycle  
Continue Anastrozole 1 mg PO daily  
Mutation Positive 
No surgery  
Disease recurrence  
Patient refusal for followup 
Endocrine Resistant Cohort 
Clinical stage II or III ER+ 
HER2- breast cancer 
 
Ki67 > 10% on neoadjuvant 
endocrine therapy  
5Cycle 5 (10-12 days) 
(PD 0332991 daily + 6 anastrozole daily for 10-12 days) 
Off Study 

 
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POSTOPERATIVE THERAPY AND FOLLOW-UP SCHEMA 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
# Except those who refuse for followup or did not undergo surgery 
* Follow-up for patients who do not receive adjuvant PD 0332991 will take place annually for 5 years post-surgery 
or until recurrence (whichever comes first).  Follow-up for patients who do receive adjuvant PD 0332991 will be tied 
to treatment, not to date of surgery.  These patients will be followed for a total of 5 years (inclusive of the up to 2 
years of adjuvant PD 0332991 treatment), with follow-up after discontinuation of PD 0332991 taking place annually 
after the last dose or until recurrence (whichever comes first).  Patients who discontinue adjuvant PD 0332991 early 
(do not complete the full 23 cycles of treatment) will be followed annually following the last dose of PD 0332991 to 
get as close as possible to 5 years of post-surgery follow-up or until recurrence (whichever comes first). 
1 Endocrine therapy that is not Anastrozole can be used if patient cannot tolerate Anastrozole (See Section 5.3) 
 
 
 
PIK3CA Mutant or WT Cohort 
Endocrine Resistant Cohort 
C1D1 Ki67 > 2.7%  
C1D15 Ki67 < 2.7% 
# All others 
# All others 
C1D15 Ki67 < 10% 
Adjuvant PD 0332991 + Anastrozole1 x 2 years 
followed by standard hormonal therapy to complete at 
least 5 years of adjuvant hormonal therapy 
Neoadjuvant 
therapy then 
curative surgery 
Adjuvant chemotherapy and radiation if indicated 
Follow up annually for 5 years post surgery  
Research blood yearly for 5 years post surgery* 
Treatment per 
physician choice  
Treatment per 
physician choice  
Neoadjuvant 
therapy then 
curative surgery  

 
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Glossary of Abbreviations  
 
AE 
Adverse event 
ALT (SGPT) 
Alanine transaminase (serum glutamate pyruvic transaminase) 
ANC 
Absolute neutrophil count 
AST (SGOT) 
Aspartate transaminase (serum glutamic oxaloacetic transaminase) 
AUC 
Area under the curve 
BCS 
Breast conserving surgery 
B-HCG 
Beta human chorionic gonadotropin  
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
Cdk 
Cyclin dependent kinase 
CFR 
Code of Federal Regulations 
CNS 
Central nervous system 
CR 
Complete response 
CRF 
Case report form 
CT 
Computed tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
DCIS 
Ductal carcinoma in situ 
DNA 
deoxyribonucleic acid 
DOB 
Date of birth 
DSM 
Data and Safety Monitoring  
DSMC 
Data Safety Monitoring Committee 
ECG (or EKG) 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
EDTA 
ethylenediaminetetraacetic acid 
ER 
Estrogen receptor 
FDA 
Food and Drug Administration 
FFPE 
Formalin fixed paraffin embedded 
FISH 
fluorescent in situ hybridization 
FSH 
Follicle-stimulating hormone 
FWA 
Federal wide assurance 
GCP 
Good Clinical Practice 
GnRH 
Gonadotropin-releasing hormone 
HER2 
Human epidermal growth factor receptor 2 
HHS 
Department of Health and Human Services’ 
HIV 
Human Immunodeficiency Virus 
HRPO 
Human Research Protection Office (IRB) 
IHC 
Immunohistochemistry 
IND 
Investigational New Drug 

 
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IRB 
Institutional Review Board 
IV 
Intravenous 
LD 
Longest diameter 
LDH 
Lactate dehydrogenase 
LH 
Luteinizing hormone 
LHRH 
Luteinizing hormone-releasing hormone 
MRI 
Magnetic resonance imaging 
MTD 
Maximum tolerated dose 
NC 
No change 
NCCN 
National Cancer Center Network 
NCI 
National Cancer Institute 
OHRP 
Office of Human Research Protections  
OS 
Overall survival 
pCR 
Pathologic complete response 
PD 
Progressive disease 
PEPI 
Preoperative Endocrine Prognostic Index 
PI 
Principal investigator 
PK 
Pharmacokinetic 
PO 
Per os (by mouth) 
PR 
Partial response 
QASMC 
Quality Assurance and Safety Monitoring Committee 
QD 
Quaque die (one a day) 
RECIST 
Response Evaluation Criteria in Solid Tumors (Committee) 
RFS 
Relapse-free survival 
RR 
Response rate 
SAE 
Serious adverse event 
SCC 
Siteman Cancer Center 
TSH 
Thyroid stimulating hormone 
TTP 
Time to progression 
UPN 
Unique patient number 
US 
Ultrasound 
WBC 
White blood cell (count) 
WHO 
World Health Organization 
WUSM 
Washington University School of Medicine 
 
 

 
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Table of Contents 
SCHEMA ........................................................................................................................................ 4 
POSTOPERATIVE THERAPY AND FOLLOW-UP SCHEMA .................................................. 5 
1.0 
BACKGROUND AND RATIONALE .............................................................................. 10 
1.1 
ER Positive and HER2 Negative Breast Cancer and Study Overview .......................... 10 
1.2 
Neoadjuvant Endocrine Therapy in ER+ Breast Cancer ................................................ 11 
1.3 
Cyclin D/Cdk4/6 Pathway in ER+ Breast Cancer .......................................................... 13 
1.4 
PD 0332991 .................................................................................................................... 15 
1.5 
Anastrozole..................................................................................................................... 19 
1.6 
Goserelin ........................................................................................................................ 21 
1.7 
Rationale to Investigate PD 0332991 in Combination with Anastrozole ...................... 22 
1.8 
Rationale for Adding Adjuvant PD 0332991 in Combination with Anastrozole in 
Patients who Derived benefit from the Addition of PD 0332991 during Neoadjuvant Therapy 
(Amendment #7) ....................................................................................................................... 25 
1.9 
Correlative Studies Background..................................................................................... 26 
2.0 
OBJECTIVES .................................................................................................................... 29 
2.1 
Primary Objective .......................................................................................................... 30 
2.2 
Secondary Objectives ..................................................................................................... 30 
2.3 
Exploratory Objectives ................................................................................................... 31 
3.0 
PATIENT SELECTION .................................................................................................... 32 
3.1 
Pre-registration Eligibility Criteria for the PIK3CA Mutant Cohort ............................. 32 
3.2 
Registration Eligibility Criteria for the PIK3CA Mutant Cohort ................................... 34 
3.3 
Eligibility Criteria for the PIK3CA Wild Type Cohort.................................................. 35 
3.4 
Eligibility Criteria for the Endocrine Resistant Cohort .................................................. 38 
3.5 
Eligibility Criteria for the Adjuvant Cohort ................................................................... 40 
3.6 
Inclusion of Women and Minorities............................................................................... 43 
4.0 
PRE-REGISTRATION AND REGISTRATION PROCEDURES ................................... 43 
4.1 
Pre-Registration .............................................................................................................. 43 
4.2 
Registration .................................................................................................................... 44 
5.0 
TREATMENT PLAN ........................................................................................................ 46 
5.1 
Neoadjuvant Treatment .................................................................................................. 46 
5.2 
Surgery ........................................................................................................................... 47 
5.3 
Post Surgery Therapy ..................................................................................................... 47 
5.4 
General Concomitant Medication and Supportive Care Guidelines .............................. 48 
5.5 
Women of Childbearing Potential .................................................................................. 48 
5.6 
Duration of Therapy ....................................................................................................... 48 
5.7 
Treatment/Follow-up Decision Tree .............................................................................. 49 
5.8 
Duration of Follow-up .................................................................................................... 51 
6.0 
DOSE DELAYS/DOSE MODIFICATIONS .................................................................... 51 
6.1 
Dose Modifications for Anastrozole .............................................................................. 51 
6.2 
Dose Modifications for PD 0332991 ............................................................................. 51 
6.3 
Re-Treatment Criteria for Neoadjuvant Cycles 1-4 and Adjuvant Therapy .................. 53 
6.4 
Re-Treatment Criteria for Cycle 5 ................................................................................. 53 
7.0 
REGULATORY AND REPORTING REQUIREMENTS ............................................... 54 
7.2 
Reporting to the Human Research Protection Office (HRPO) at Washington University
 
56 

 
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7.3 
Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at 
Washington University .............................................................................................................. 57 
7.4 
Reporting Requirements for Secondary Sites ................................................................ 57 
7.5 
Reporting to Secondary Sites ......................................................................................... 57 
7.6 
Reporting to the FDA ..................................................................................................... 57 
7.7 
Reporting to Pfizer ......................................................................................................... 58 
7.8 
Timeframe for Reporting Required Events .................................................................... 58 
8.0 
PHARMACEUTICAL INFORMATION .......................................................................... 59 
8.1 
Study Agent (PD 0332991) ............................................................................................ 59 
8.2 
Anastrozole..................................................................................................................... 64 
8.3 
Goserelin ........................................................................................................................ 65 
9.0 
CORRELATIVE STUDIES .............................................................................................. 66 
9.1 
Sample Collection, Processing, and Shipment ............................................................... 66 
9.2 
Real Time Integral Biomarker Studies ........................................................................... 71 
9.3 
Tumor Ki67 Assessment on Cycle 1 Day 1 (Pre-PD 0332991) and Cycle 1 Day 15 .... 72 
9.4 
Laboratory Correlative Studies ...................................................................................... 73 
10.0 STUDY CALENDARS ..................................................................................................... 76 
10.1 Pre-Registration and Cycle 0 Calendar .......................................................................... 76 
10.2 Neoadjuvant Study Treatment Calendar (ALL Cohorts) ............................................... 77 
10.3 Post Surgery Treatment Calendar .................................................................................. 78 
11.0 DATA SUBMISSION SCHEDULE ................................................................................. 79 
12.0 MEASUREMENT OF EFFECT........................................................................................ 80 
12.1 Neoadjuvant Treatment .................................................................................................. 80 
12.2 Treatment Resistance ..................................................................................................... 80 
12.3 Surgery ........................................................................................................................... 81 
12.4 Post-surgery .................................................................................................................... 81 
13.0 DATA AND SAFETY MONITORING ............................................................................ 81 
14.0 AUDITING ........................................................................................................................ 82 
15.0 STATISTICAL CONSIDERATIONS............................................................................... 83 
15.1 Purpose ........................................................................................................................... 83 
15.2 Primary Endpoint ........................................................................................................... 83 
15.3 Trial Design .................................................................................................................... 83 
15.4 Sample Size and Trial Duration ..................................................................................... 85 
15.5 Data Analysis ................................................................................................................. 85 
15.6 Correlative Studies ......................................................................................................... 86 
16.0 MULTICENTER REGULATORY REQUIREMENTS ................................................... 87 
17.0 REFERENCES .................................................................................................................. 89 
APPENDIX A: ECOG Performance Status Scale ........................................................................ 95 
APPENDIX B: Neoadjuvant PD 0332991: Registration Worksheet ........................................... 96 
APPENDIX C: Adjuvant PD 0332991 Registration Worksheet .................................................. 99 
APPENDIX D: Medication Diary – PD 0332991 ...................................................................... 102 
APPENDIX E: Medication Diary – Endocrine Therapy ............................................................ 103 
APPENDIX F: Pfizer Reportable Event Cover Sheet ................................................................ 105 
APPENDIX H: Strong CYP3A4 Inhibitors or Inducers ............................................................. 107 
 
 
 

 
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1.0 
BACKGROUND AND RATIONALE 
 
1.1 
ER Positive and HER2 Negative Breast Cancer and Study Overview 
 
Estrogen receptor positive (ER+) and Human Epidermal Growth Factor Receptor 2 
negative (HER2-) breast cancer represents approximately 70% of breast cancer cases[1].  
Although most patients are diagnosed at an early stage and treated with curative intent, 
there is a persistent risk of relapse over decades[2].  As a result, approximately one-third 
of patients diagnosed with early stage ER+ breast cancer eventually experience disease 
recurrence, indicating the failure of current treatments regimens to completely eradicate 
cancer cells from these patients.  
 
Conventional chemotherapy has been extensively examined in this setting but the impact 
of these agents is modest in addition to resulting in significant toxicity with little benefit 
for most patients. In the recent Early Breast Cancer Trialists’ Collaborative Group 
(EBCTCG) overview analysis of randomized trials, the 5-year gain in reducing recurrence 
is about twice as great for ER poor disease as for ER+ disease[2].  Consistent with this 
finding is the low rate of pathologic complete response (pCR) for ER+ disease in response 
to neoadjuvant chemotherapy [3-9].   The pCR rate in the ER+ HER2- subgroup is 
particularly low, ranging 1.8% to 6%, compared to the over 20% pCR rate in the ER- or 
HER+ populations, following treatment with neoadjuvant anthracycline/taxane-containing 
regimens [3, 4, 6].   
 
Endocrine therapy is the most effective systemic adjuvant treatment for ER+ HER2- breast 
cancer; however a protracted course of therapy is necessary to prevent recurrence, resulting 
in toxicity and economic burden. Unfortunately, many patients still suffer disease relapse 
despite long-term endocrine therapy. The therapeutic effects of endocrine treatments are 
thought to depend on an inhibitory or “cytostatic” effect on the tumor cell cycle [10, 11].  
Unlike cytotoxic chemotherapy, it has never been clearly demonstrated that endocrine 
interventions promote cell death through apoptosis, perhaps explaining why maintenance 
therapy is necessary and there is a very high frequency of delayed recurrence [12].  In three 
relatively large studies with aromatase inhibitors, the pCR rate was no more than 1% [13-
15].   
 
Recent studies indicated that cyclin D is required for tumor maintenance in breast cancer 
[16], and inhibition of Cdk 4/6 was able to induce senescence, a permanent form of cell 
cycle arrest, in preclinical studies [16-20].  The high frequency of cyclin D/Cdk4/6 pathway 
alteration as a result of cyclin D amplification or loss of negative regulators in ER+ HER2- 
breast cancer [21] provides a strong scientific rationale for the investigation of Cdk4/6 
inhibitors in this subtype.  This is supported by the clinical efficacy observed in the 
randomized phase II trial of the Cdk4/6 inhibitor PD 0332991 in combination with letrozole 
versus letrozole alone as first line therapy for metastatic ER+ HER2- breast cancer, which 
demonstrated that the combination therapy was associated with a significant improvement 
in progression free survival (PFS) from 7.5 to 26.1 months (HR 0.37, 95% CI 0.21 – 0.63, 

 
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p<0.001) [22].  This has led to the FDA fasttrack listing of this agent to accelerate the 
review and approval process.   
 
The goal of this neoadjuvant trial is to investigate whether the combination of the 
aromatase inhibitor anastrozole and the Cdk4/6 inhibitor PD 0332991 induces cell cycle 
arrest more effectively than anastrozole alone and whether there is induction of senescence 
with short-term neoadjuvant treatment. In addition, serial tumor samples are collected for 
investigation of predictive and pharmacodynamics markers of response to treatment.  The 
results of this trial will provide essential efficacy and tolerability information of this 
combination as well as biomarkers for patient selection for future adjuvant clinical trials.   
 
1.2 
 Neoadjuvant Endocrine Therapy in ER+ Breast Cancer  
 
1.2.1 Neoadjuvant Aromatase Inhibitor in Postmenopausal Women  
 
Neoadjuvant endocrine therapy with an aromatase inhibitor is now considered a 
standard treatment option in postmenopausal women with estrogen dependent 
tumors that are 2 cm or greater (NCCN Guidelines http://www.nccn.org). The 
primary clinical goal in this setting is reduction in the size of the tumor to allow 
breast conservation [23].  Preoperative therapy has several additional advantages.  
From the standpoint of clinical practice, tumor response to neoadjuvant endocrine 
therapy provides an in vivo assessment of its endocrine sensitivity and predicts long 
term outcome.  Most recently a prognostic model has been developed that integrates 
post-neoadjuvant endocrine therapy pathological stage, ER status and Ki67 level (a 
marker of cell proliferation) into a Preoperative Endocrine Prognostic Index (PEPI) 
[11, 24].  In addition, the level of Ki67 following short-term (2-4 weeks) of 
neoadjuvant endocrine therapy and degree of Ki67 suppression compared to 
baseline correlated with relapse and survival [12, 25, 26].  From a research 
standpoint, the neoadjuvant setting provides a fertile environment for using 
biological endpoints such as Ki67 based biomarkers to efficiently test new 
therapeutic strategies and to study mechanisms of endocrine therapy resistance 
[27].   
 
P024 Trial 
The P024 trial compared letrozole to tamoxifen in postmenopausal ER+ breast 
cancer.  Letrozole proved superior to tamoxifen in the primary endpoints of clinical 
response rate, radiographic response rate, and the incidence of breast conservation 
[14].  Despite a response rate of 60% to letrozole in the 124 confirmed ER+ patients, 
only one patient achieved a pathologic complete response (pCR)[14].  While the 
low pCR rate can be an argument against neoadjuvant endocrine therapy, in fact a 
low rate of pCR is also seen with neoadjuvant chemotherapy regimens in ER+ 
disease [6].  Thus, ER+ and HER2- breast cancer must be relatively resistant to 
cancer therapy induced cell death, regardless of the treatment modality.   
 
In the P024 study, assessment of the proliferative marker Ki67 was performed at 
baseline and at the time of definitive surgery. Interestingly pretreatment Ki67 score 

 
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did not correlate with relapse free survival in the P024 study.  However, subsequent 
analyses showed that a model (PEPI score) incorporating post treatment Ki67 score, 
ER status and pathologic stage predicted relapse free survival and breast cancer 
specific survival [11].  
 
IMPACT Trial 
The IMPACT (Immediate Preoperative Anastrozole, Tamoxifen or Combined with 
Tamoxifen) trial randomized 330 postmenopausal patients to anastrozole, 
tamoxifen or the combination for 12 weeks prior to definitive surgery.  The overall 
clinical response rate was not significantly different between the three arms; 
however, significantly more patients classified as requiring mastectomy were 
eligible for Breast Conservation Surgery (BCS) on the anastrozole treatment arm.  
Analysis of tumor cell proliferation after 2 weeks of therapy showed significantly 
lower Ki67 values with anastrozole alone than with either tamoxifen or the 
combination[25, 26]. Consistent with results obtained in the P024 study, post 
treatment Ki67 score was a more powerful predictor of recurrence free survival 
than baseline Ki67 [25].   
 
Surprisingly, neither tamoxifen nor anastrozole treatment in the IMPACT trial 
activated classical apoptotic cell death in breast tumors.  In fact, anastrozole 
treatment resulted in a significant decrease in TUNEL staining in ER+ breast 
tumors [26].  This conclusion underscores the postulate that ER+ disease tends to 
be quite resistant to stimuli that can cause apoptosis in normal hormone regulated 
tissues.  An important implication of these studies is that endocrine therapy 
primarily controls disease through cytostasis, but may not kill disseminated tumor 
cells.  Consequently, when patients stop adjuvant endocrine therapy the relapse rate 
increases.   
 
ACOSOG Z1031  
The ACOSOG Z1031 trial is a randomized phase II study of postmenopausal 
women with clinical stage II or III ER positive breast cancer to receive anastrozole, 
exemestane, or letrozole 16-18 weeks prior to surgery.  A total of 374 patients were 
enrolled between January 2006 and January 2009.   Clinical response rates were 
similar among the three treatment arms: 69.1% (95% CI: 60.1-77.1%) for 
anastrozole, 62.9% (95% CI: 53.8-71.4%) for exemestane and 74.8% (95% CI: 
66.3-82.15%) for letrozole.  In addition, changes in Ki67 after treatment showed no 
difference between treatment arms (p=0.45). Geometric mean percentage change 
in Ki67 was 78% with anastrozole, 81.2% with exemestane and 87.1% with 
letrozole [28].  In this trial, the pCR rate was approximately 1% (data from Dr. 
Matthew Ellis). 
 
1.2.2 Neoadjuvant Aromatase Inhibitor in Combination with Ovarian 
Suppression in Premenopausal Women 
 
Ovarian suppression with GnRH agonist such as goserelin is effective in preventing 
relapse in premenopausal women with early stage ER+ breast cancer[29].  

 
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Goserelin in combination with tamoxifen is more efficacious than tamoxifen alone 
in TTP, RR and OS in premenopausal women with advanced disease[30].  
Goserelin in combination with letrozole in premenopausal women has similar 
efficacy as compared to letrozole alone in postmenopausal women as the first line 
hormonal therapy in the metastatic setting[31].  Since aromatase inhibitors are more 
effective than tamoxifen as adjuvant hormonal therapy for postmenopausal women, 
there has been considerable interest in testing the combination of goserelin and an 
aromatase inhibitor in the early stage setting for premenopausal women with ER+ 
breast cancer. 
 
The comparison of goserelin in combination with either anastrozole or tamoxifen 
as neoadjuvant therapy for premenopausal women with ER+ HER2- operable 
breast cancer from the randomized, double-blind, multicenter phase III study of 
tamoxifen or anastrozole in combination with goserelin (STAGE) was recently 
reported in abstract forms[32, 33].  Anastrozole and goserelin combination was 
found to be more effective than the tamoxifen combination in inducing higher 
response rate by caliper (70.4% vs 50.0%, p=0.004) as well as by ultrasound (58.2% 
vs 42.4%, p=0.027) and MRI (64.3% vs 37.4%, p<0.001)[32]. A greater reduction 
in Ki67 was also observed after 24 weeks of anastrozole plus goserelin (n=96, 
21.9% to 2.9%) when compared to that of tamoxifen plus goserelin (n=96, 21.6% 
to 8.0%)[33]. In the same study, the levels of E1 and E2 reached steady state level 
at 1 month following goserelin.  Therefore, we have chosen the combination of 
goserelin and anastrozole for this study in those who are premenopausal at study 
entry. 
 
1.3 
Cyclin D/Cdk4/6 Pathway in ER+ Breast Cancer 
 
1.3.1 Interaction of Estrogens and Cyclin-Dependent Kinases in Breast 
Cancer Cells 
 
Studies of ER-positive breast cancer cell lines indicate that estrogens[34] and 
antiestrogens[35] act on sensitive populations of cells in early to mid-G1 phase. G1/S 
transition is under the control of cyclin-dependent kinases (Cdks) activated by 
specific complex formation with regulatory cyclins. Cdk4 and Cdk6 are activated 
by binding to D-type cyclins and act early in G1 phase[36-39]. A primary target of 
Cdk action in G1 phase is the retinoblastoma susceptibility gene product (pRb), 
which mediates G1 arrest through sequestration of transcriptional factors of the 
E2F-DP family. Phosphorylation of pRb and other members of the pocket protein 
family (p107 and p130) by active cyclin-Cdk complexes leads to release of E2F 
and DP transcription factors and transcription of requisite genes for S-phase 
entry[39].  D-type cyclins play an essential role in recognition of extracellular 
growth stimuli and initiation of G1 transit[40, 41], and several lines of evidence have 
linked estrogen regulation of cellular proliferation to cyclin D1 expression. 
Estrogen-induced proliferation of normal uterine and breast epithelium in vivo is 
associated with increased expression of cyclin D1 mRNA and protein[42-45]. 
Expression of cyclin D1 in breast tumor isolates correlates with ER-positive 

 
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status[46-48]. MCF-7 breast cancer cells treated with estrogen exhibit increased 
expression of cyclin D1 mRNA and protein, formation of active cyclin D1-Cdk4 
complexes, and phosphorylation of pRb leading to G1/S transition[49-52]. 
Estrogen-induced S-phase entry in these cells is inhibited by microinjection of 
antibodies to cyclin D1[53]. Antiestrogen-induced growth arrest of ER-positive 
breast cancer cells is associated with decreased cyclin D1 expression[54]. 
Collectively, these studies are consistent with a model of estrogen action in which 
receptor activation induces increased cyclin D1 expression, Cdk4 activation, and 
cell cycle progression. An upstream role for cyclin D1 has been suggested by recent 
reports describing direct physical interactions between cyclin D1 and the ER, 
leading to recruitment of steroid receptor coactivators and activation of ER-
dependent transcription. This occurs in the absence of hormone and is independent 
of D cyclin association with Cdk4 [55-58]. 
 
Constraint upon Cdk activity and G1 progression is provided by the universal Cdk 
inhibitors of the Cip-Kip family, including p21Cip1 and p27Kip1, and the specific 
Cdk4 and Cdk6 inhibitors of the INK4 family, typified by p16INK4a[41, 59-62]. The 
p16INK4a gene product inhibits formation of active D cyclin-Cdk complexes through 
specific binding interactions with Cdk4 or Cdk6 that prevent D cyclin-Cdk 
association[63-65]. Overexpression of p16INK4a in cells with functional pRb results 
in inhibition of both Cdk4-and Cdk6-associated kinase activity and pRb 
phosphorylation, with subsequent cell cycle arrest[63, 64]. In addition, inhibition 
of D cyclin-Cdk4 complex formation by p16INK4a prevents sequestration of p21Cip1 
and p27Kip1 by these complexes in early G1, leading to suppression of cyclin E-Cdk2 
activity[66-68]. Adenoviral transduction of p16INK4a into MCF-7 cells leads to G1 
arrest associated with inhibited Cdk activity[69, 70]. Cell cycle progression induced 
by estradiol requires action of the steroid through mid-G1, well beyond the point of 
cyclin D1-Cdk4 activation[50]. Functional association of cyclin D1-Cdk4 is 
required for estrogen-induced Cdk2 activation and G1/S transition and estrogen 
regulates expression of p21Cip1, p27Kip1, and Cdc25A independent of D cyclin-Cdk4 
function[71]. 
 
1.3.2 Deregulation of Cell Cycle Related Genes and Proteins in Breast 
Cancer 
 
Cell cycle related genes and proteins are frequently deregulated in breast cancer.  
Approximately 15-20% of human breast cancers exhibit amplification of D1 
(CCND1) gene[72-74], while the majority of human mammary carcinomas 
overexpress CCND1 protein[75-77]. Overexpression of CCND1 is seen early in 
breast cancer, and it is maintained at all stages of breast cancer progression, 
including metastatic lesions[75, 78]. There is a mounting body of evidence linking 
a specific CCND1 polymorphism (G/A870) to increased risk of cancer and outcome 
in a variety of tumor types including breast cancer. This polymorphism results in a 
splice variant, altered protein structure and enhanced oncogenic activity in 
experimental models[79]. The continued presence of Cdk4-associated kinase 
activity is actually required to maintain breast tumorigenesis[80]. Direct analyses 

 
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of primary tumors have revealed loss of Rb expression in 20-35% of tumors, and 
loss of heterozygosity or other alterations of the Rb locus in 7–37% of tumors[81-
84]. In preclinical models, Rb depletion appears to be associated with resistance to 
antiestrogen therapy[85]. 
 
Finally, virtually all ER-positive cell lines harbor loss of 16ink4a[86, 87], and low 
expression of Cdk inhibitors p21 and p27 and high expression level of cyclin E and 
D1 have all been associated with resistance to anti-estrogen therapy. 
 
Data from the Cancer Genome Atlas project confirmed that ER+ breast cancer is 
enriched for CCND1 (Cyclin D1) amplification (luminal B: 58%; luminal A: 29%), 
gain of CDK4 (25% in luminal B versus 14% in luminal A), and loss of negative 
regulators including CDKN2A (p16) and CDKN2C (p18) [21].  In contrast to basal-
like breast cancers, Rb is intact in most luminal breast cancer.  Since a functional 
Rb is a pre-requisite for the efficacy of CDK4/6 inhibitors, luminal B breast cancers 
are ideal candidate for these agents and early success has been observed in clinical 
trial of CDK4/6 inhibitors.   
 
1.4 
PD 0332991 
 
1.4.1 Mechanism of Action   
 
PD 0332991 is a highly selective inhibitor of Cdk4/cyclinD1 kinase activity. PD 
0332991 has selectivity for Cdk4/6, with little or no activity against a large panel 
of 34 other protein kinases including other Cdks and a wide variety of tyrosine and 
serine/threonine kinases. Cdk6, another enzyme that also complexes with cyclin-D 
subunits, is also commonly expressed in mammalian cells and tumors. Cdk6 is 
highly homologous to Cdk4 and can perform the same function by phosphorylating 
Rb, thus potentially creating a redundant mechanism to promote cell cycle 
progression.  Consequently, inhibition of both enzymes is necessary to ensure 
complete suppression of Rb phosphorylation and the greatest possible spectrum of 
antitumor activity. Results indicate that PD 0332991 inhibits Cdk6 with equivalent 
potency to Cdk4.  
 
1.4.2 Nonclinical Studies 
 
In vitro single-agent activity of PD 0332991 
The only known natural substrate for Cdk4/cyclinD1 is the retinoblastoma gene 
product (Rb).  Specific Cdk4 phosphorylation sites on Rb include serine-780 and 
serine-795. Therefore, the phosphorylation status of Rb at these specific sites in 
treated tumors can serve as an appropriate biomarker for target modulation by PD 
0332991. The IC50 for reduction of Rb phosphorylation at serine-780 in the MDA-
MB-435 breast carcinoma cell line was 0.066 μM. PD 0332991 was equally 
effective at reducing Rb phosphorylation at serine-795 in this tumor cell line with 
an IC50 of 0.063 μM. Similar effects on serine-780 and serine-795 Phosphorylation 
were obtained in the Colo-205 colon carcinoma cell line. 
 

 
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PD 0332991 inhibits cellular proliferation and prevents cellular DNA synthesis by 
preventing cells from entering S phase of the cell cycle. PD 0332991 inhibited 
thymidine incorporation into the DNA of a panel of Rb-positive human breast, 
colon, and lung carcinomas, with IC50 values ranging from 0.040 to 0.17 μM. PD 
0332991 was also effective in preventing cell cycle progression in human 
leukemias and in non-transformed human epithelial cells and fibroblasts and was 
equally effective in suppressing cell division in human tumor cell lines. A selective 
Cdk4/cyclin D inhibitor should cause a specific accumulation of cells in G1, but 
have no effect on other phases of the cell cycle, in which cells should continue to 
progress and eventually decline in number. MDA-MB-453 breast carcinoma cells 
that were exposed to various concentrations of PD 0332991 for 24 hours show a 
significant increase in the percentage of cells in G1 in the presence of as little as 
0.04 μM PD 0332991 with a concomitant decline in other phases of the cell cycle.  
 
Finally, to provide further evidence of the selectivity of PD 0332991, the compound 
was tested against Rb-negative tumor cells, which should not be sensitive to a 
specific Cdk4 inhibitor. PD 0332991 was tested against the MDA-MB-468 human 
breast carcinoma and the H2009 human non-small cell lung carcinoma, both of 
which have deleted Rb. The compound had no anti-proliferative activity on these 
cells when assayed at 3 μM (highest concentration tested), which is 1 to 2 orders of 
magnitude higher than the concentration necessary to inhibit Rb-positive tumor 
cells. 
 
In Vivo Activity Studies 
PD 0332991-0002 (hydrochloride salt) was used in all in vivo tumor models. 
Additionally, PD 0332991-0054 (isethionate salt) was used in the MDA-MB-435 
breast carcinoma model, and had comparable efficacy to the hydrochloride salt. 
 
The MTD in SCID mice was 150 mg/kg/day when administered orally, once a day, 
for 14 days. The MTD was defined as the highest dose that was nonlethal (<LD10). 
At the MTD on this regimen, PD 0332991 has significant antitumor efficacy against 
multiple human tumor xenograft models. The Colo-205 model is exquisitely 
sensitive to PD 0332991. At doses as low as 12.5 mg/kg, a 13-day growth delay 
was obtained, indicating a 90% inhibition of tumor growth rate. PD 0332991 was 
inactive against the H23 lung and the SW-620 colon carcinomas. The lack of 
response may be associated with the presence of oncogenic K-ras mutations in SW-
620 and H23; none of the xenografts sensitive to PD 0332991 had such mutations.  
 
Further evidence that the anti-tumor activity observed in Rb-positive tumors is due 
to inhibition of Cdk4/Cdk6 protein kinase activity was obtained by testing PD 
0332991 in the MDA-MB-468 breast carcinoma and the DU-145 prostate tumor 
models. These are Rb negative tumors; neither of which responded to this 
compound. The lack of efficacy in Rb-negative tumors is consistent with the lack 
of anti-proliferative activity observed in vitro. Taken together, these results support 
the proposed mechanism of PD 0332991 (inhibition of Cdk4/6-mediated Rb 
phosphorylation) and the specificity of the compound demonstrated in enzyme 

 
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activity tests. 
 
Further studies investigated whether continuous daily dosing of PD 0332991 was 
needed for optimal efficacy. Four dosing schedules were employed against the 
MDA-MB-435 breast carcinoma model over 14 days of treatment, including 
continuous daily, every other day, every third day, and 3 courses of 3 days dosing 
followed by 4-day drug holidays. The design of this experiment was such that the 
total compound administered over the 2-week period was identical for each 
treatment schedule. The results show that a similar degree of efficacy was attained 
with all schedules, implying that an intermittent regimen is feasible without 
compromising activity.  Similar experiments were conducted against the Colo-205 
colon carcinoma model. Again, intermittent schedules were as efficacious as daily 
dosing, with tumor regressions occurring during all dosing regimens. 
 
During the 14-day treatment period employed for most of the efficacy experiments, 
no cures were documented, and the tumors grew back after therapy. It is possible 
that a tumor variant had selectively grown back and acquired resistance to the 
compound. To address this possibility, Colo-205 colon tumors that had initially 
significantly regressed in response to treatment with PD 0332991 were harvested 
and reimplanted into naive mice. After the tumors grew to 100 to 150 mg, these 
tumor-bearing mice were treated with PD 0332991 with a dose and dosing schedule 
identical to the original experiment. The tumors responded with equal sensitivity to 
the drug and fully regressed, indicating that no resistance had developed during the 
initial treatment. A similar result was observed with retreated MDAMB-435 
tumors. 
 
Pharmacokinetics  
The single-dose pharmacokinetics of PD 0332991 following IV or PO routes of 
administration were investigated in Sprague-Dawley rats and Beagle dogs 
(toxicology species), and in cynomolgus monkeys. PD 0332991 was administered 
intravenously to determine elimination kinetics and absolute bioavailability from 
the PO route. Following IV administration, mean plasma clearance values of PD 
0332991 in all species were low to moderate and were all lower than the 
corresponding hepatic blood flow. The mean apparent volumes of distribution at 
steady state were approximately 10-fold greater than total body water. Mean 
absolute oral bioavailability of PD 0332991 was moderate in all species tested.  In 
rats on Day 1 of repeat dose studies, mean PD 0332991 Cmax and AUC values 
increased in a dose-related manner up to 300 mg/kg. In dogs, mean PD 0332991 
Cmax and AUC values increased in a dose-related manner up to 20 mg/kg on Day 
1, and did not increase between 20 and 40 mg/kg. Mean PD 0332991 Cmax and 
AUC values in female rats were less than in male rats (up to one ninth and up to 
one sixteenth the values for male rats, respectively). There was no observed sex 
difference in systemic exposure in dogs. Mean PD 0332991 Cmax and AUC values 
following 3 weeks of dosing indicate up to 3-fold accumulation upon multiple 
dosing in both rats and dogs. 
 

 
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1.4.3 Clinical Development of PD 0332991 
 
Currently, fourteen studies evaluating the safety, efficacy, pharmacodynamics and 
PK of PD 0332991 as single agent or in combination have started. Results from 
earlier phase I trials have been reported and are discussed below. 
 
1.4.4 Clinical Pharmacokinetics 
 
To date pharmacokinetic data is available from four studies (see Section 8.1.3). The 
exposure (AUC(0-10) and Cmax) increased in a dose-proportional manner over the 
dose range of 25-225 mg QD following PD 0332991 administration on Days 1 and 
8 of Cycle 1.  At a steady state (Day 14 or Day 21), PD 0332991 was absorbed with 
a median Tmax of ~4 hours. PD 0332991 extensively penetrates into peripheral 
tissues, and was eliminated slowly; the mean elimination half-life (t1/2) was 26.5 
hours. 
 
The preliminary results from the recently performed food-effect study (“A5481021, 
a Phase 1, open-label 4 sequence 4 period crossover study of palbociclib (PD-
0332991) in healthy volunteers to estimate the effect of food on the bioavailability 
of palbociclib”) has provided evidence that when a single 125 mg dose of 
palbociclib was administered under fed conditions (including high fat or low fat 
meal given together with palbociclib, or moderate fat meal given 1 hour before and 
2 hours after palbociclib) as a freebase formulation the palbociclib exposure levels 
were more uniform across the population than when taken in the fasting condition. 
 
1.4.5 Clinical Toxicology  
 
Since protocols A5481001 and A5481002 both tested PD 0332991 as a single agent 
in advanced cancers, the relevant safety data have been combined (see Section 
8.1.11 Table 16). The most frequently reported AEs were predominantly 
considered treatment-related. These treatment-related events included fatigue, 
neutropenia, diarrhea, nausea, anemia, and thrombocytopenia. The most common 
Grade 4 adverse events were neutropenia and thrombocytopenia 
 
QT Interval Effects 
Data from non-clinical (in vitro and in vivo) studies indicated that PD 0332991 has 
the potential to delay cardiac repolarization as measured by prolongation of the QT 
interval on the ECG. Prolongation of QTcF (maximum increase of < 30 msec from 
baseline) was observed in a majority of patients in two Phase I trials with PD 
0332991 as a single agent.  No patient had a maximum QTcF value of > 500 msec 
during treatment.  Notably, one female patient receiving PD 0332991 at 75 mg QD 
on Schedule 3/1 had a maximum QTcF increase of 67 msec from baseline to Cycle 
1.  Additionally, QTcF increases ranging from 39 to 51 msec compared to baseline 
persisted throughout her ECG collection period of 5 subsequent cycles.  No 
significant changes in blood pressure, pulse rate and body weight have been 
observed in the two completed Phase 1 clinical studies in advanced cancers.  

 
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The patients enrolled in clinical studies should be closely monitored for potential 
cardiovascular symptoms. Appropriate monitoring should include clinical 
examinations, vital signs, routine ECGs, and AEs monitoring.  
 
1.4.6 Developmental/Reproductive Toxicity 
 
Fertility and teratology studies with PD 0332991 have not been conducted. Women 
of childbearing potential must have a negative pregnancy test prior to treatment 
with PD 0332991. Female patients must be surgically sterile or be postmenopausal, 
or must agree to use effective contraceptive during the period of the trial and for at 
least 90 days after completion of treatment.  
 
1.5 
Anastrozole 
 
1.5.1 Mechanism of Action 
 
Anastrozole is an FDA-approved agent used in the management of hormone 
receptor positive breast cancers.  In postmenopausal women, the principal source 
of circulating estrogen (primarily estradiol) is conversion of adrenally-generated 
androstenedione to estrone by aromatase in peripheral tissues, such as adipose 
tissue, with further conversion of estrone to estradiol. Many breast cancers also 
contain aromatase; the importance of tumor-generated estrogens is uncertain. 
Treatment of breast cancer has included efforts to decrease estrogen levels, by 
ovariectomy premenopausally and by use of anti-estrogens and progestational 
agents both pre- and post-menopausally; and these interventions lead to decreased 
tumor mass or delayed progression of tumor growth in some women.  Anastrozole 
is a potent and selective non-steroidal aromatase inhibitor. It significantly lowers 
serum estradiol concentrations and has no detectable effect on formation of adrenal 
corticosteroids or aldosterone [AstraZeneca, Package Insert]. 
 
1.5.2 Pharmacodynamics/kinetics 
 
Inhibition of aromatase activity is primarily due to anastrozole, the parent drug. 
Studies with radiolabeled drug have demonstrated that orally administered 
anastrozole is well absorbed into the systemic circulation with 83 to 85% of the 
radiolabel recovered in urine and feces. Food does not affect the extent of 
absorption. Elimination of anastrozole is primarily via hepatic metabolism 
(approximately 85%) and to a lesser extent, renal excretion (approximately 11%), 
and anastrozole has a mean terminal elimination half-life of approximately 50 hours 
in postmenopausal women.  The major circulating metabolite of anastrozole, 
triazole, lacks pharmacologic activity. The pharmacokinetic parameters are similar 
in patients and in healthy postmenopausal volunteers. The pharmacokinetics of 
anastrozole are linear over the dose range of 1 to 20 mg and do not change with 
repeated dosing. Consistent with the approximately 2-day terminal elimination 
half-life, plasma concentrations approach steady-state levels at about 7 days of once 

 
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daily dosing and steady-state levels are approximately three- to four-fold higher 
than levels observed after a single dose of anastrozole. Anastrozole is 40% bound 
to plasma proteins in the therapeutic range [AstraZeneca, Package Insert]. 
 
Effect on Estradiol: Mean serum concentrations of estradiol were evaluated in 
multiple daily dosing trials with 0.5, 1, 3, 5, and 10 mg of anastrozole in 
postmenopausal women with advanced breast cancer. Clinically significant 
suppression of serum estradiol was seen with all doses. Doses of 1 mg and higher 
resulted in suppression of mean serum concentrations of estradiol to the lower limit 
of detection (3.7 pmol/L). The recommended daily dose, anastrozole 1 mg, reduced 
estradiol by approximately 70% within 24 hours and by approximately 80% after 
14 days of daily dosing. Suppression of serum estradiol was maintained for up to 6 
days after cessation of daily dosing with anastrozole 1 mg.  
 
Effect on corticosteroids: In multiple daily dosing trials with 3, 5, and 10 mg, the 
selectivity of anastrozole was assessed by examining effects on corticosteroid 
synthesis. For all doses, anastrozole did not affect cortisol or aldosterone secretion 
at baseline or in response to ACTH. No glucocorticoid or mineralocorticoid 
replacement therapy is necessary with anastrozole.  
 
Other Endocrine Effects: In multiple daily dosing trials with 5 and 10 mg, thyroid 
stimulating hormone (TSH) was measured; there was no increase in TSH during 
the administration of anastrozole. Anastrozole does not possess direct 
progestogenic, androgenic, or estrogenic activity in animals, but does perturb the 
circulating levels of progesterone, androgens, and estrogens [AstraZeneca, Package 
Insert]. 
 
Studies in postmenopausal women demonstrated that anastrozole is extensively 
metabolized with about 10% of the dose excreted in the urine as unchanged drug 
within 72 hours of dosing, and the remainder (about 60% of the dose) is excreted 
in urine as metabolites. Metabolism of anastrozole occurs by N-dealkylation, 
hydroxylation and glucuronidation. Three metabolites of anastrozole have been 
identified in human plasma and urine. The known metabolites are triazole, a 
glucuronide conjugate of hydroxy-anastrozole, and a glucuronide of anastrozole 
itself. Several minor (less than 5% of the radioactive dose) metabolites have not 
been identified. Because renal elimination is not a significant pathway of 
elimination, total body clearance of anastrozole is unchanged even in severe 
(creatinine clearance less than 30 mL/min/1.73m2) renal impairment, dosing 
adjustment in patients with renal dysfunction is not necessary. Dosage adjustment 
is also unnecessary in patients with stable hepatic cirrhosis [AstraZeneca, Package 
Insert]. 
 
 

 
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1.6 
Goserelin 
 
1.6.1 Mechanism of Action 
 
Goserelin is a synthetic analog of endogenous gonadotropin-releasing hormone 
(GnRH) also known as luteinizing hormone releasing hormone (LHRH) agonist. 
LHRH regulates follicle-stimulating hormone (FSH) and luteinizing hormone 
synthesis and secretion by the anterior pituitary gland, which in turn stimulates the 
production of sex hormones estrogen and testosterone by the ovary and testis 
respectively. In response to LHRH, FSH and LH synthesis initially increases, 
causing a transient increase in circulating levels of sex hormones.  These hormones 
are however, regulated by feedback loops, so further hormone release is suppressed. 
Chronic administration of goserelin leads to sustained suppression of pituitary 
gonadotropins. With continued administration for more than 1-3 weeks, the 
pituitary gland down-regulates and desensitizes LHRH receptors, reducing FSH 
and LH secretion. Although the physiologic effects are complicated, the end result 
of continuous goserelin administration is an effective chemical castration. In 
women the estradiol levels transiently increases and later falls to postmenopausal 
levels by three weeks of continuous therapy.  Normal pituitary and gonadal 
functions typically returns within three months of discontinuing goserelin.   
 
1.6.2 Pharmacodynamics/kinetics 
 
In females, a down-regulation of the pituitary gland by chronic exposure to 
goserelin leads to suppression of gonadotropin secretion, a decrease in serum 
estradiol to levels consistent with the postmenopausal state, and would be expected 
to lead to a reduction of ovarian size and function, reduction in the size of the uterus 
and mammary gland, as well as a regression of sex hormone-responsive tumors, if 
present. Serum estradiol is suppressed to levels similar to those observed in 
postmenopausal women within 3 weeks following initial administration; however, 
after suppression was attained, isolated elevations of estradiol were seen in 10% of 
the patients enrolled in clinical trials. Serum LH and FSH are suppressed to 
follicular phase levels within four weeks after initial administration of drug and are 
usually maintained at that range with continued use of goserelin. In 5% or less of 
women treated with goserelin, FSH and LH levels may not be suppressed to 
follicular phase levels on day 28 post treatment with use of a single 3.6 mg depot 
injection. In certain individuals, suppression of any of these hormones to such 
levels may not be achieved with goserelin. Estradiol, LH and FSH levels return to 
pretreatment values within 12 weeks following the last implant administration in 
all but rare cases [AstraZeneca, Package Insert]. 
 
The pharmacokinetics of goserelin have been determined in both male and female 
healthy volunteers and patients. In these studies, goserelin was administered as a 
single 250 µg (aqueous solution) dose and as a single or multiple 3.6 mg depot dose 
by subcutaneous route. The absorption of radiolabeled drug was rapid, and the peak 

 
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blood radioactivity levels occurred between 0.5 and 1.0 hour after dosing.  
 
Goserelin is released from the depot at a much slower rate initially for the first 8 
days, and then there is more rapid and continuous release for the remainder of the 
28-day dosing period. Despite the change in the releasing rate of goserelin, 
administration of goserelin every 28 days resulted in testosterone levels that were 
suppressed to and maintained in the range normally seen in surgically castrated 
men. When goserelin 3.6 mg depot was used for treating male and female patients 
with normal renal and hepatic function, there was no significant evidence of drug 
accumulation. However, in clinical trials the minimum serum levels of a few 
patients were increased. These levels can be attributed to interpatient variation. The 
apparent volumes of distribution determined after subcutaneous administration of 
250 µg aqueous solution of goserelin were 44.1 and 20.3 liters for males and 
females, respectively. The plasma protein binding of goserelin obtained from one 
sample was found to be 27.3%. Metabolism of goserelin, by hydrolysis of the C-
terminal amino acids, is the major clearance mechanism. The metabolism of 
goserelin in humans yields a similar but narrow profile of metabolites to that found 
in other species. All metabolites found in humans have also been found in 
toxicology species. Clearance of goserelin following subcutaneous administration 
of the solution formulation of goserelin is very rapid and occurs via a combination 
of hepatic metabolism and urinary excretion. More than 90% of a subcutaneous 
radiolabeled solution formulation dose of goserelin is excreted in urine. 
Approximately 20% of the dose in urine is accounted for by unchanged goserelin. 
The total body clearance of goserelin (administered subcutaneously as a 3.6 mg 
depot) was significantly (p<0.05) greater (163.9 versus 110.5 L/min) in females 
compared to males. [AstraZeneca, Package Insert]. 
 
1.7 
Rationale to Investigate PD 0332991 in Combination with Anastrozole 
 
In preclinical studies, endocrine resistance was associated with persistent cyclin D 
expression and RB phosphorylation despite efficient blockade of ER and the poor 
prognosis luminal B breast cancers were associated with a gene expression signature of 
RB-dysfunction [20].  PD 0332991 was shown to have anti-tumor activity for multiple 
tumor types including breast cancer in both in vitro and in vivo studies [88, 89]. In 
preclinical studies, PD 0332991 was particularly effective in inhibiting cell growth of 
luminal breast cancer subtype [88]. A synergistic anti-tumor effect was also observed when 
combined with tamoxifen in tamoxifen-sensitive and resistant cell lines [88]. Importantly, 
treatment with PD 0332991 effectively suppressed proliferation of ER+ breast cancer cell 
lines resistant to anti-estrogen and led to irreversible cell cycle arrest and features of 
cellular senescence [20]. These studies provided a preclinical rationale to combine 
endocrine therapy and a Cdk4/6 inhibitor to improve tumor control for ER+ breast cancer.   
 
The combination of PD 0332991 and endocrine therapy has shown highly promising results 
in patients with advanced ER+ HER2- breast cancer.  In the randomized phase 2 study of 
letrozole with or without PD 0332991 as first line therapy for metastatic ER+, HER2- 
breast cancer, the PFS was significantly better in the combination arm (26.1 months vs 7.5 

 
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months (HR 0.37, 95% CI 0.21 – 0.63, p<0.001) [22]. The response rate for the 
combination arm (n = 84) was 31% vs. 26% for the letrozole arm (n = 81) and the clinical 
benefit rate was 68% vs. 44%, respectively. Toxicities were tolerable.  The most commonly 
reported treatment-related AEs in the combination arm were neutropenia (grade 1/2: 23%; 
grade 3: 55%, grade 4: 6%),  leukopenia (grade 1/2: 29%; grade 3: 17%, grade 4: 0%), 
anemia  (grade 1/2: 24%; grade 3: 5%, grade 4: 1%), and fatigue (grade 1/2: 35%; grade 3: 
2%, grade 4: 2%) and nausea  (grade 1/2: 23%; grade 3:2%, grade 4: 0%). The promising 
clinical data led to the FDA fast-track listing of PD 0332991.  
 
In the Ki67 analysis of the serial tumor biopsy samples collected from the first 7 patients 
enrolled in this trial, comparing data for C1D15 (2 weeks on PD 0332991 and 6 weeks on 
anastrozole) and C1D1 (4 weeks on single agent anastrozole), the addition of PD 0332991 
to anastrozole led to further reduction in Ki67 (Figure 1) in 4 patients.  In these responsive 
tumors, Ki67 was reduced to <1% after 2 weeks of combination therapy, indicating a 
profound anti-proliferative effect of PD 0332991.  In contrast, Ki67 was not suppressed by 
either anastrozole or the combination in 3 other patients, indicating treatment resistance. 
Interestingly, one patient (PD001) had a tumor Ki67 >10% on C1D1, but achieved 
complete cell cycle arrest, Ki67 at 0.5% on C1D15 after combination therapy, indicating 
that endocrine resistant tumors could be responsive to the combination therapy with PD 
0332991. 
 
Based on these preliminary data and the accumulating evidence that PD 0332991 exerts 
anti-tumor effects through inhibition of cell cycle, inducing senescence, without apoptosis, 
we have elected to modify the primary endpoint of the study from pathologic complete 
response at surgery to complete cell cycle arrest on treatment.  We propose to investigate 
whether the combination of PD 0332991 and anastrozole is able to improve the rate of 
complete cell cycle arrest when compared to the historical control of single agent aromatase 
inhibitors and also adding an endocrine therapy resistant cohort and the PIK3CA mutant 
cohort. 
 

 
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Since PD 0332991 is commonly associated with myelosuppression, surgery is 
scheduled in 3-5 weeks following the last dose of PD 0332991 in this trial.  As a 
result, patients are off PD 0332991 at the time of surgery.  Therefore the effect of 
combination therapy on cell cycle arrest will be evaluated at an earlier time point, 
Cycle 1 Day 15, when patients are taking both PD 0332991 and anastrozole.   
 
In this trial, we define complete cell cycle arrest as tumor Ki67 <2.7% since this is 
the Ki67 cutpoint developed for PEPI score calculation in the PO24 trial [11] and 
validated in the our Ki67 scoring SOP [90].   
 
We will use data from the ACOSOG Z1031 trial, a large neoadjuvant aromatase 
inhibitor trial in ER+ HER2- clinical stage II or III breast cancer, as the historical 
control as it enrolled the same patient population [28].  In the neoadjuvant 
ACOSOG Z1031 trial, 95 of 215 (44%) patients had Ki67 <2.7% on 2-4 week 
tumor biopsy (Personal Communication with Matthew Ellis). 
 
1.7.1 PIK3CA mutation in ER-positive breast cancer and rationale to 
investigate PD 0332991 in both PIK3CA wild type and mutant 
populations 
 
PIK3CA, the alpha catalytic subunit of PI3K, is the most common significantly 
mutated genes identified in luminal breast cancer, occurring at a frequency of 45% 
and 29% in luminal A and luminal B, respectively [21, 91-93].  Up to 80% of the 
mutations are restricted to “hotspots” within the helical (HD) and the kinase 
domains (KD) encoded by exons 9 and 20, respectively [94].  The three hot spot 
mutations include E542K, E545K and H1047R [94]. Consistent with potentially 
Figure 1  Tumor Ki67 response (preliminary data from the first 7 patients enrolled in the 
current trial).  
 
 
Ongoing Ki67 data from current trial 
ID# 
Baseline  
C1D1 
C1D15 
PD 001 
22% 
14% 
0.5% 
PD 002 
27% 
17% 
80% 
PD 003 
14% 
4% 
1% 
PD 004 
26% 
3% 
0% 
PD 005 
39% 
10% 
33% 
PD 006 
65% 
37% 
48% 
PD 007 
11% 
2% 
< 1% 
Note that 4 patients had Ki67 <1% on C1D15, 
indicating profound inhibition of cell proliferation 
by PD 0332991. The 3 patients with Ki67 >10% 
on C1D15 were considered resistant to therapy 
and have been taken off study per protocol. 
Baseline (pre-registration): prior to anastrozole 
C1D1: post 1 month of single agent anstrozole 
C1D15: post 2 weeks after the addition of PD 0332991 

 
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important roles of these mutations in the process of tumorigenesis, the majority of 
these mutations are missense mutations that occur in evolutionarily conserved 
regions [94].  Many of the PIK3CA mutations, including the 3 hot spot mutations, 
have been shown to increase levels of phosphorylated AKT (pAKT) and induce 
cellular transformation and invasion in vitro and tumor formation in vivo when 
introduced into human mammary epithelial cells [95, 96].  Tumor cells with 
mutations in PIK3CA have shown to be highly dependent on p110 alpha for cell 
survival [97].  
 
The relationship between PIK3CA mutation and endocrine therapy responsiveness 
in ER+ breast cancer was investigated retrospectively in several neoadjuvant 
endocrine trials [93, 98].   No interaction was between PIK3CA mutation and 
response to endocrine therapy was identified in these studies [93, 98].  
 
The relationship between PIK3CA mutation and responsiveness of ER+ breast 
cancers to CDK4/6 inhibitors has not been investigated.  In preclinical studies, 
PD0332991 was found to be effective in a subpopulation of both PIK3CA mutant 
and wild type cancer [88].  The most promising predictors of response identified in 
preclinical studies have been the presence of intact Rb or down regulation of p16; 
however, this has not been confirmed in the recently reported clinical trial of 
letrozole in combination with PD 0332991 as first line therapy for metastatic ER+ 
breast cancer [22].  Since PIK3CA mutation is an important driver event in ER+ 
breast cancer, and direct inhibitors of PI3K, including those that target specifically 
the alpha catalytic subunit, are being developed to treat tumors with PIK3CA 
mutation, it is important to investigate the anti-tumor effect and predictors of 
response to PD 0332991 in PIK3CA wild type and mutant population separately.   
 
The study was designed initially to focus on the population of ER+ breast cancer 
without PIK3CA hotspot mutations since trials of inhibitors against PI3K pathway, 
such as the NCI9170, were ongoing the same time for the population with PIK3CA 
hotspot mutations. As the NCI9170 trial that enrolls patients with PIK3CA mutant 
tumors is approaching its enrollment target as of November 2013, we plan to open 
the PIK3CA mutant cohort for this trial, starting in institutions that do not have 
NCI9170 open. 
 
1.8 
Rationale for Adding Adjuvant PD 0332991 in Combination with Anastrozole 
in Patients who Derived benefit from the Addition of PD 0332991 during 
Neoadjuvant Therapy (Amendment #7) 
 
Between 4/23/2013 and 4/24/2015, the study enrolled 50 patients with clinical stage II or 
III ER+ HER2- breast cancer to the PIK3CA mutant and PIK3CA wild type (WT) cohorts.  
Of these 50 patients,  45 (PIK3CA WT, n=28; PIK3CA mutant, n=15; PIK3CA unknown, 
n=2) were evaluable for Ki67 analysis at C1D15 after completion of 6 weeks of anastrozole 
and 2 weeks of PD 0332991 (for the primary endpoint).  The data was presented at 2015 
SABCS {Ma, 2015 #3841}.  Complete cell cycle arrest (CCCA) was achieved in 39 of the 
45 (87%, 90% CI: 75-94%) evaluable patients, 22 of 28 (79%, 90% CI: 62-90%) patients 

 
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in the PIK3CA WT cohort and all 15 (100%, 90% CI: 82-100%) patients in the PIK3CA 
mutant cohort. The study met its primary endpoint of achieving a CCCA rate of 66% in 
C1D15.  In addition, 26 patients who did not achieve CCCA with anastrozole alone on 
C1D1 achieved complete CCCA in C1D15 post 2 weeks of combination therapy.  These 
data justify the evaluation of PD 0332991 in the adjuvant setting.  The international 
PALLAS (PALbociclib CoLlaborative Adjuvant Study) trial is currently enrolling patients 
with high risk ER+ HER2- breast cancer to receive 2 years of palbociclib (PD 0332991) in 
the adjuvant setting.  However, patients who received neoadjuvant PD 0332991 
(palbociclib) are not eligible for the adjuvant PALLAS trial. We therefore propose to 
include in this trial the option of adjuvant PD 0332991 with anastrozole for 23 cycles after 
chemotherapy and radiation therapy for those who derived benefit from PD 0332991 in the 
neoadjuvant setting in this trial.  This includes: 
• The 26 patients who achieved complete cell cycle arrest only after the addition of 
PD 0332991 (C1D1 Ki67 >2.7% and C1D15 Ki67 ≤ 2.7%) (in the PIK3CA WT, 
mutant, or unknown cohorts).  
• Patients who have a Ki67 ≤ 10% on C1D15 biopsy in the endocrine resistant cohort 
(including patients enrolled prior to activation of Amendment #7).   
 
All patients (with the exception of those who withdraw consent for follow-up and those 
who did not undergo surgery) will be followed for invasive disease recurrence and overall 
survival for 5 years in the adjuvant setting or until recurrence (whichever comes first).  
Blood will be drawn for serum, plasma, and circulating cell-free DNA (cfDNA) yearly.  
   
1.9 
Correlative Studies Background 
 
1.9.1 Early Tumor Ki67 Assessment during Neoadjuvant Endocrine 
Therapy to Determine Treatment Response  
 
In this trial, we plan to perform tumor biopsy to assess Ki67 following one month 
of endocrine therapy (pre-PD 0332991) and 2 weeks on combination of PD 
0332991 and anastrozole (Cycle 1 Day 15).  This is based on data from previous 
neoadjuvant endocrine trials which indicated that 2-4 week tumor Ki67 expression 
on neoadjuvant endocrine therapy is predictive of individual patient outcome long 
term[11, 25, 26].  In the IMPACT trial, 2-week Ki67 was a significant independent 
predictor of RFS (HR = 1.95; 95% CI = 1.23–3.07; P = .004) [25]. The 5-year RFS 
rates were 85%, 75%, and 60% for the lowest, middle, and highest values of 2-
week Ki67 expression, respectively[25]. In the P024 trial, while baseline Ki67 was 
not associated with relapse, post 16-week treatment Ki67 levels had a robust 
association with RFS (HR = 1.4, CI = 1.2–1.6 per natural log unit increase; 
P < .001), and breast cancer–specific survival (HR = 1.4, CI = 1.1–1.7; P = .009) 
[11].  
 
To further investigate these findings, Ellis et al examined the interaction between 
Ki67 levels and a PAM50-based definition of luminal A breast cancer versus 
luminal B breast cancer. By using ROC methodology, a cut point of Ki67 10% 
served as the best surrogate for the LumA versus LumB distinction [99]. The 10% 

 
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Ki67 cut point was then applied to the baseline and early on-treatment data in two 
data sets (Table 2), Preoperative Letrozole study (POL) [13] and IMPACT trial 
[25]. At baseline the dichotomized Ki67 definition was not significantly predictive 
for surgical Ki67 level, PEPI score or RFS in these modest sized sample sets. In 
contrast, high levels of Ki67 on the one month POL samples predicted a higher 
level of Ki67 in the surgical samples at four months after treatment initiation 
(P=.01), a poorer PEPI score (P=0.01), a smaller number of patients in the PEPI-0 
group (P=0.08) and worse RFS (P=0.003). IMPACT data confirmed that a 2-week 
Ki67 >10% predicted higher Ki67 in the surgical specimen (P=0.001), a poorer 
PEPI score (P=0.001), smaller numbers of patients in the PEPI-0 group (P= 0.004) 
and worse RFS (P=0.008) (Table 2.3). 
 
Table 2 Early Ki67 Assessments and Outcome in IMPACT and POL Trials 
POL 4W Ki67 
% PEPI 0 
RFS (events) 
>10% 
1/19 (5%) 
5/21 (23%) 
≤10% 
10/36 (28%) 
1/41 (2.4%) 
P Value 
P=0.08 (Fisher) 
P=0.003 (log rank) 
 
IMPACT 2W Ki67 
% PEPI 0 
RFS (events) 
>10% 
0/32 (0%) 
9/35 (26%) 
≤10% 
21/101 (21%) 
13/118 (11%) 
P Value 
P=0.004 (Fisher) 
P=0.008 (log rank) 
 
In this trial, to avoid futile therapy, we plan to obtain tumor biopsy post 2 weeks 
of PD 0332991 in combination with anastrozole.   Patients whose Day 15 
Ki67>10% will be considered resistant to the combination therapy.  These patients 
will be recommended alternative therapies such as immediate surgery or 
neoadjuvant chemotherapy.  The pre-PD 0332991 Ki67 will not be used for clinical 
decision making since PD 0332991 will be added to the treatment regimen.  It is, 
however, important to obtain the pre-PD 0332991 Ki67 since this allows a 
comparison between anastrozole alone and the combination of anastrozole and PD 
0332991 on the degree of Ki67 suppression.  
 
1.9.2 To Assess Tumor Cell Apoptosis on Biopsies Taken Pre-PD 0332991 
and Cycle 1 Day 15 
 
We will investigate whether there is an increase in tumor cell apoptosis with the 
combination of PD 0332991 and anastrozole (or anastrozole in combination with 
goserelin).  Anastrozole 1mg daily dosing reduced estradiol level by 70% by 24 
hours and by approximately 80% after 14 days of daily dosing [AstraZeneca, 
Package Insert], therefore, full estrogen deprivation is expected by 28 days of 
anastrozole administration.  In the case of goserelin, serum estradiol is suppressed 
to levels similar to those observed in postmenopausal women within 3 weeks 

 
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following administration.  In addition, only 5% or less of women treated with 
goserelin, FSH and LH may not be suppressed to follicular phase levels on day 28 
post treatment with a single 3.6mg depot injection [AstraZeneca, Package Insert].   
 
1.9.3 To Assess the Degree of Ki67 Suppression before (Pre-PD 0332991) and 
after PD 0332991 (Cycle 1 Day 15) 
 
The degree of Ki67 suppression following 2-4 weeks of therapy has been associated 
with the effectiveness of endocrine agents or the combination of endocrine agent 
with an mTOR inhibitor.   In the IMPACT trial, suppression of Ki67 after 2 and 12 
weeks was significantly greater with anastrozole than with tamoxifen (P = 0.004) 
which mirrored the result based on RFS in the ATAC trial [100]. These data 
indicate that Ki67 response early on therapy could potentially be employed as a 
measurement of treatment efficacy. 
 
1.9.4 To Assess Effect of PD 0332991 on Markers of Tumor Cell Senescence  
 
Studies in preclinical models of ER+ breast cancer indicated that PD 0332991 
induced stable cell cycle arrest that was fundamentally distinct from those elicited 
by ER antagonists and was capable of inducing aspects of cellular senescence, as 
measured by expression of senescence-associated b-galactosidase [20]. In addition, 
candidate proteins important for senescence including p16, IL6, cyclin D1, 
activated mTOR (such as pS6), FOXM1 phosphorylation, will be examined [19, 
101]. These markers will be assayed on tumors collected at all time points.  Since 
surgery occurs after at least 3 weeks after the last dose of PD 0332991, a 
comparison of these markers as well as Ki67 level between surgery and C1D15 
sample will be informative in determining whether 4 months of combination 
therapy induced irreversible cell cycle arrest and cells have entered senescence.   
 
1.9.5 Evaluation of Target Inhibition on the Cdk4/6-Rb Pathway 
 
The pharmacodynamic effect of PD 0332991 in combination with anastrozole on 
the Cdk4/6-Rb pathway activities will be assessed by phosphoroproteomics and 
immunohistochemistry analysis on serial tumor biopsies.  Since PD 0332991 
inhibits Cdk4/6 phosphorylation of Rb at Ser 801/811 and Ser780, the levels of total 
and phosphorylated RB will be examined at baseline, post 1 cycle of single agent 
anastrozole, then post 2 weeks of PD 0332991 in combination with anastrozole.  
 
1.9.6 Preoperative Endocrine Prognostic Index (PEPI)  
 
In a multivariable analysis conducted on the P024 trial, three other post-
neoadjuvant endocrine therapy tumor factors were determined to have independent 
prognostic value for relapse and death after relapse in addition to Ki67 [11]. These 
included pathological tumor size (T1/2 versus T3/4), pathological node status 
(positive or negative), the natural logarithm of the Ki67 value and the ER status of 
the tumor. A prognostic score, the preoperative endocrine prognostic index (PEPI), 

 
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was developed, which weighs each of these factors according to their associated 
hazard ratios. PEPI was then validated in an independent data set from the IMPACT 
trial [11]. No relapses were recorded in either trial in patients with T1, N0 tumors 
with a PEPI score of 0 (residual tumor with Ki67 index of 2.7% - natural logarithm 
of 1- or less with maintained ER expression) or in the rare patient with a pCR. PEPI 
has also recently been validated in the POL Trial (PreOperative Letrozole trial: A 
multicenter phase II trial of letrozole in postmenopausal women with clinical stage 
II or III hormone receptor positive breast cancer) [13]. In the combined analysis of 
P024 trial/POL trial, no relapse was observed with a median follow up of 61.3 
months in the 24 patients in the PEPI 0 category.  PEPI 0 as a prognostic marker 
on therapy therapy is being validated prospectively in the A011106 trial.  An 
improvement in the rate of PEPI 0 following neoadjuvant PD 0332991 and 
anastrozole compared to the historical control would be of great interest for the 
design of adjuvant trials of PD 0332991. 
 
1.9.7 Assessment of Serum Estradiol Level Before and Following PD 
0332991 
 
The goal of this study is to ensure that PD 0332991 does not interact with 
anastrozole in reducing serum estradiol levels in addition to monitor changes in 
estradiol in premenopausal women being treated with goserelin, anastrozole before 
and after PD 0332991 during the course of therapy.  Since there are no obvious 
theoretical pharmacokinetic interactions between the two agents, we do not 
anticipate a change in serum estradiol levels following PD 0332991 administration.  
Blood will be collected from premenopausal women for sensitive estradiol 
measurement, to be done at the clinical laboratory, at baseline, end of 4 weeks of 
anastrozole (or in combination with goserelin), Cycle 3 Day 1, and at time of 
surgery.  In patients on goserelin because of initial premenopausal status prior to 
pre-registration, estradiol level must be in the postmenopausal range per 
institutional standard after Cycle 0 therapy to be eligible for the study drug therapy 
on this trial.  In addition, these patients should go off study drug therapy and be 
considered inevaluable for the primary endpoint if the estradiol level rose to 
premenopausal range on Cycle 3 Day 1 or at Surgery or any time during Cycles 1-
4 of treatment. 
 
 
2.0 
OBJECTIVES 
 
Since surgery occurs 3 to 5 weeks following the last dose of PD 0332991 in order to avoid 
complications related to PD 0332991-induced myelosuppression, we propose to assess the primary 
endpoint of complete cell cycle arrest (Ki67 ≤ 2.7%) on C1D15 while patients are receiving PD 
0332991.  The value of Ki67 on C1D15 and at surgery will provide data on the potential 
reversibility of Ki67 decrease when off PD 0332991 for at least 3 weeks. These data may help to 
hypothesize regarding the duration of the biologic effect of PD 0332991. 
 
 

 
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Amendment #5: 
Please note that as of Amendment #5, a fifth cycle of daily PD 0332991 (consisting of 10-12 days 
of treatment, plus continued daily anastrozole) is added prior to surgery based on preliminary 
analysis of the first 18 patients showing an increase in tumor Ki67 at the time of surgery which 
was performed 3 to 5 weeks after the last dose of PD 0332991 (Cycle 4 Day 21).  This data 
indicates that PD 0332991 has a reversible effect on cell cycle arrest. However, alternative 
explanations exist—for example, the emergence of resistance clones.  To invesitigate these 
possibilities, we propose to re-initiate PD 0332991 (and continue anastrozole) for patients whose 
blood counts and other AEs recover sufficiently after 4 complete neoadjuvant cycles of PD 
0332991 + anastrozole, and treat for an additional 10 to 12 days, with the last dose of PD 0332991 
and anastrozole being administered on the day before surgery.  Based on previous phase I studies, 
the mean ANC within 10-12 days of treatment is between 2.2 k-4.2 k/mcL (personal 
communication with Pfizer), therefore we expect majority of our patients will be able to complete 
Cycle 5.  In fact, a neoadjuvant study consisting of 14 days of aromatase inhibitor +/- PD 0332991 
before surgery is ongoing, and none of the first 9 patients randomized to receive PD 0332991 had 
ANC < 1,000 on Day 10 (personal communication with Pfizer). 
 
2.1 
Primary Objective 
 
1. To determine the rate of complete cell cycle arrest, defined by Ki67 <2.7%,  following 
2 weeks (C1D15) of neoadjuvant PD 0332991 in combination with anastrozole in 
women with clinical stage II or III ER+/HER2- breast cancer without PIK3CA hot spot 
mutation (PIK3CA Wild Type Cohort).  
 
2. To determine the rate of complete cell cycle arrest, defined by Ki67 <2.7%,  following 
2 weeks (C1D15) of neoadjuvant PD 0332991 in combination with anastrozole in 
women with clinical stage II or III ER+/HER2- endocrine-resistant breast cancer.  
 
2.2 
 Secondary Objectives 
 
1. To determine the rate of complete cell cycle arrest, defined by Ki67 <2.7%,  following 
2 weeks (C1D15) of neoadjuvant PD 0332991 in combination with anastrozole in 
women with clinical stage II or III ER+/HER2- breast cancer with PIK3CA hot spot 
mutation.  
 
2. 
To determine the rate of complete cell cycle arrest, defined by Ki67 <2.7%,  following 
2 weeks (C1D15) of neoadjuvant PD 0332991 in combination with anastrozole in 
women with clinical stage II or III ER+/HER2- endocrine-resistant breast cancer.  
 
The following objectives will be assessed in PIK3CA mutant cohort and in PIK3CA 
Wild Type (WT) cohort combined or separately, and assessed separately in the 
endocrine resistant cohort. 
 
3. 
To compare the rate of complete cell cycle arrest between C1D1 and C1D15 
 

 
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4. 
To assess Ki67 level on serially collected tumor specimens (baseline, C1D1, C1D15, 
and surgery) 
 
5. 
To determine the rate of PEPI 0 score with the study drug therapy 
 
6. 
To determine the pathologic complete response (pCR) rate of study drug therapy 
 
7. 
To assess the rate of clinical response and radiologic response in the neoadjuvant 
setting 
 
8. 
To determine the safety profile (on therapy and post 30 days of therapy completion) 
of the study therapy during 4 months of neoadjuvant therapy. 
 
9. 
To determine the safety profile of the study therapy during 2 years of adjuvant therapy. 
 
10. To assess the long term outcomes of patients treated in this trial. 
 
2.3 
Exploratory Objectives  
 
The following objectives will be assessed in PIK3CA mutant cohort and in PIK3CA 
WT cohort combined or separately, and assessed separately in the endocrine resistant 
cohort. 
 
1. 
To assess tumor cell apoptosis index and senescence markers on serially collected 
tumor specimens  
 
2. 
To examine the pharmacodynamic effect of PD 0332991 in combination with 
anastrozole using serially collected tumor specimens (baseline, C1D1, 2 weeks on 
combination therapy, and surgery)  
 
3. 
To explore molecular mechanisms which could affect tumor response to the 
combination PD 0332991 and anastrozole by tumor genomic, transcriptomic, and 
proteomic analysis 
 
4. 
To document pathologic staging (tumor size, lymph node status) following 
neoadjuvant chemotherapy in patients who had tumor Ki67>10% on C1D15 and 
elected to receive neoadjuvant chemotherapy 
 
5. 
To explore circulating markers predictive of cancer recurrence.  
 
6. 
To assess the concentrations of anastrozole prior to and 90 minutes following 
anastrozole (without PD 0332991) on Cycle 1 Day 1 and to compare these 
concentrations to the concentrations of anastrozole prior to and 90 minutes 
following both anastrozole and PD 0332991 on Cycle 1 Day 15. 
 
7. 
To assess the concentration of PD 0332991 prior to and 90 minutes following both 

 
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anastrozole and PD 0332991 on Cycle 1 Day 15. 
 
 
3.0 
PATIENT SELECTION 
 
There are three cohorts of patients for this trial: 1) PIK3CA Wild Type Cohort, 2) PIK3CA Mutant 
Cohort, and 3) Endocrine Resistant Cohort. As of Amendment #7, enrollment to the PIK3CA 
Wild Type and Mutant Cohorts is closed; patients may enroll to the Endocrine Resistant 
Cohort for neoadjuvant treatment and to the Adjuvant Cohort for adjuvant treatment.   
 
Applicable to the PIK3CA Wild Type and Mutant Cohort:  
Patients who were pre-registered to NCI 9170 trial (Phase II Trial of Neoadjuvant MK-2206 in 
Combination with either Anastrozole if Postmenopausal or Anastrozole and Goserelin if 
Premenopausal in Women with Clinical Stage 2 or 3 PIK3CA Mutant Estrogen Receptor Positive 
and HER2 Negative Invasive Breast Cancer), started anastrozole (or anastrozole plus goserelin if 
premenopausal) ≤ 6 weeks, and were found negative for PIK3CA hotspot mutations are eligible to 
be screened for the wild type cohort. 
 
In institutions without NCI9170 open, or after completion of enrollment to NCI9170 in institutions 
where it is open, patients will be pre-registered to this trial and those with PIK3CA mutations will 
be enrolled to the PIK3CA mutant cohort. 
 
Applicable to the Endocrine Resistant Cohort: 
Pre-registration is not required for patients to be enrolled in the endocrine resistant cohort, as 
PIK3CA mutation status will not be assessed for those patients. 
 
Patients who are potentially eligible to receive adjuvant treatment with PD 0332991 are 
required to be screened using the eligibility criteria in Section 3.5. 
 
3.1 
Pre-registration Eligibility Criteria for the PIK3CA Mutant Cohort 
 
Note: enrollment to this cohort is CLOSED as of Amendment #7. 
 
3.1.1 Inclusion Criteria 
 
1. Clinical T2-T4c, any N, M0 invasive ER+ (Allred Score of 6-8) and HER2 
negative (0 or 1+ by IHC or FISH negative for amplification) breast cancer, by 
AJCC 7th edition clinical staging, with the goal being surgery to completely 
excise the tumor in the breast and the lymph node. 
 
Note: Patients with invasive ER+ (Allred Score of 6-8) HER2- breast cancer or 
DCIS in the contralateral breast the patient are eligible  
 
2. Female >18 years of age.   
 
3. ECOG performance status of 0, 1 or 2 (Appendix A).   
 

 
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4. Life expectancy > 4 months. 
 
5. If premenopausal, patient must be willing to comply with pregnancy 
requirements laid out in Section 5.5. 
 
6. Adequate organ and marrow function as defined below: 
a. leukocytes > 3,000/mcL 
b. absolute neutrophil count > 1,500/mcL 
c. platelets  > 100,000/mcL 
d. total bilirubin ≤ upper normal institutional limits 
e. AST(SGOT)/ and ALT(SGPT) < 2.5 X institutional upper limit 
normal  
 
 
f. Creatinine ≤ upper normal institutional limits 
 
7. Able to understand and willing to sign an IRB-approved written informed 
consent document. 
 
3.1.2 Exclusion Criteria 
 
1. Prior treatment of this cancer including: 
a. Surgery,  
b. Radiation therapy,  
c. Chemotherapy,  
d. Biotherapy,  
e. Hormonal therapy  
f. Investigational agent prior to study entry. 
 
2. Receiving any other investigational agents. 
 
3. Prior therapy with any Cdk4 inhibitor. 
 
4. Any of the following in the previous 6 months:  
a. myocardial infarction 
b. severe/unstable angina 
c. coronary/peripheral artery bypass graft 
d. symptomatic congestive heart failure 
e. cerebrovascular accident 
f. transient ischemic attack   
g. symptomatic pulmonary embolism. 
 
5. Uncontrolled intercurrent illness including, but not limited to: 
a. ongoing or active infection 
b. symptomatic congestive heart failure 
c. unstable angina pectoris 
d. uncontrolled symptomatic cardiac arrhythmia, 
e. psychiatric illness/social situations that would limit compliance with study 

 
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requirements.  
 
6. Pregnant/nursing. 
 
7. Unwilling to employ adequate contraception. 
 
8. Known HIV-positive on combination antiretroviral therapy. 
 
NOTE: HIV-positive patients on combination antiretroviral therapy are 
ineligible because of the potential for pharmacokinetic interactions with PD 
0332991.  In addition, these patients are at increased risk of lethal infections 
when treated with marrow-suppressive therapy. 
 
9. Evidence of inflammatory cancer (clinical presentation of skin erythema 
involving more than one third of the breast or pathological evidence of dermal 
lymphatic involvement) 
 
10. Known metastatic disease. 
 
11. Current use of anticoagulation therapy.   
 
12. Previous excisional biopsy of the breast cancer or sentinel lymph node biopsy. 
 
13. Any condition that impairs patient’s ability to swallow PD 0332991 tablets 
(e.g., gastrointestinal tract disease resulting in an inability to take oral 
medication or a requirement for IV alimentation, prior surgical procedures 
affecting absorption) 
 
14. History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to PD 0332991 or other agents used in the study. 
 
15. Corrected QT (QTc) interval >470 msec. 
 
3.2 
Registration Eligibility Criteria for the PIK3CA Mutant Cohort 
 
Note: enrollment to this cohort is CLOSED as of Amendment #7. 
 
3.2.1 Inclusion Criteria 
 
The criteria below must be met for registration onto the study in addition to the pre-
registration criteria, except treatment with endocrine therapy for this cancer is 
allowed prior to registration. 
 
1. For the PIK3CA mutant cohort:  tumor PIK3CA mutation present 
 
2. In premenopausal women, serum estradiol level in postmenopausal range ≤ 7 

 
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days prior to registration. 
 
3.2.2 Exclusion Criteria 
 
The criteria below must be met for registration onto the study in addition to the pre-
registration criteria. 
 
 
1. Current use or anticipated need for food or drugs that are known strong 
CYP3A4 inhibitors (i.e. grapefruit juice, verapamil, ketoconazole, miconazole, 
itraconazole, posaconazole, erythromycin, clarithromycin, telithromycin, 
indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, amprenavir, 
fosamprenavir, nefazodone, diltiazem, and delavirdine) or inducers (i.e. 
dexamethasone, glucocorticoids, progesterone, rifampin, phenobarbital, St. 
John’s wort). 
 
 
3.3 
Eligibility Criteria for the PIK3CA Wild Type Cohort 
 
Note: enrollment to this cohort is CLOSED as of Amendment #7. 
 
3.3.1 Inclusion Criteria 
 
1. Clinical T2-T4c, any N, M0 invasive ER+ (Allred Score of 6-8) and HER2 
negative (0 or 1+ by IHC or FISH negative for amplification) breast cancer, by 
AJCC 7th edition clinical staging, with the goal being surgery to completely 
excise the tumor in the breast and the lymph node. 
 
Note: Patients with invasive ER+ (Allred Score of 6-8) HER2- breast cancer or 
DCIS in the contralateral breast the patient are eligible  
 
2. For the PIK3CA wild type cohort: tumor PIK3CA mutation absent 
 
Note that if a patient did not have sufficient research tissue for PIK3CA 
sequencing at pre-registration or if PIK3CA sequencing result is delayed, she 
could be registered and enrolled on the PD991 trial without assigning to a 
particular cohort at the time of enrollment.  PIK3CA sequencing will be 
performed in the future on tumors collected at subsequent time points to assign 
the treatment cohort or when the PIK3CA sequencing data is available. 
 
3. For the endocrine resistant cohort: Ki67 > 10% by central testing at Washington 
University AMP laboratory from a tumor biopsy performed after at least 2 
weeks on neoadjuvant endocrine therapy. 
a. Note that prior neoadjuvant endocrine therapy could include any endocrine 
therapy (including aromatase inhibitor, tamoxifen, fulvestrant) alone or in 
combination, or endocrine therapy in combination with any investigational 

 
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agent that is not a Cdk 4/6 inhibitor. 
b. Patients who had a Day 17 Ki67 > 10% from the NCI9170 trial are eligible 
for the endocrine resistant cohort. 
c. Note that enrollment to the endocrine resistant cohort will depend on the 
funding availability.  Please contact the study chair before enrolling patients 
to this cohort.  
 
4. Female >18 years of age.   
 
5. ECOG performance status of 0, 1 or 2 (Appendix A).   
 
6. Life expectancy > 4 months. 
 
7. If premenopausal, patient must be willing to comply with pregnancy 
requirements laid out in Section 5.5. 
 
8. Adequate organ and marrow function as defined below: 
a. Leukocytes > 3,000/mcL 
b. absolute neutrophil count > 1,500/mcL 
c. platelets > 100,000/mcL 
d. total bilirubin ≤ upper normal institutional limits 
e. AST(SGOT)/ and ALT(SGPT) < 2.5 X institutional upper limit 
normal  
 
 
f. Creatinine ≤ upper normal institutional limits 
 
9. In premenopausal women, serum estradiol level in postmenopausal range ≤ 7 
days prior to registration. 
 
10. Able to understand and willing to sign an IRB-approved written informed 
consent document. 
 
3.3.2 Exclusion Criteria 
 
1. Prior treatment of this cancer including: 
a. Surgery,  
b. Radiation therapy,  
c. Chemotherapy,  
d. Biotherapy,  
e. Hormonal therapy  
f. Investigational agent prior to study entry. 
 
2. Receiving any other investigational agents. 
 
3. Prior therapy with any Cdk4 inhibitor. 
 
4. Any of the following in the previous 6 months:  

 
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a. myocardial infarction 
b. severe/unstable angina 
c. coronary/peripheral artery bypass graft 
d. symptomatic congestive heart failure 
e. cerebrovascular accident 
f. transient ischemic attack   
g. symptomatic pulmonary embolism. 
 
5. Uncontrolled intercurrent illness including, but not limited to: 
a. ongoing or active infection 
b. symptomatic congestive heart failure 
c. unstable angina pectoris 
d. uncontrolled symptomatic cardiac arrhythmia, 
e. psychiatric illness/social situations that would limit compliance with study 
requirements.  
 
6. Pregnant/nursing. 
 
7. Unwilling to employ adequate contraception. 
 
8. Known HIV-positive on combination antiretroviral therapy. 
 
NOTE: HIV-positive patients on combination antiretroviral therapy are 
ineligible because of the potential for pharmacokinetic interactions with PD 
0332991.  In addition, these patients are at increased risk of lethal infections 
when treated with marrow-suppressive therapy. 
 
9. Evidence of inflammatory cancer (clinical presentation of skin erythema 
involving more than one third of the breast or pathological evidence of dermal 
lymphatic involvement) 
 
10. Known metastatic disease. 
 
11. Current use of anticoagulation therapy.   
 
12. Previous excisional biopsy of the breast cancer or sentinel lymph node biopsy. 
 
13. Any condition that impairs patient’s ability to swallow PD 0332991 tablets 
(e.g., gastrointestinal tract disease resulting in an inability to take oral 
medication or a requirement for IV alimentation, prior surgical procedures 
affecting absorption) 
 
14. History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to PD 0332991 or other agents used in the study. 
 
15. Corrected QT (QTc) interval >470 msec. 

 
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16. Current use or anticipated need for food or drugs that are known strong 
CYP3A4 inhibitors (i.e. grapefruit juice, verapamil, ketoconazole, miconazole, 
itraconazole, posaconazole, erythromycin, clarithromycin, telithromycin, 
indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, amprenavir, 
fosamprenavir, nefazodone, diltiazem, and delavirdine) or inducers (i.e. 
dexamethasone, glucocorticoids, progesterone, rifampin, phenobarbital, St. 
John’s wort). 
 
 
3.4 
Eligibility Criteria for the Endocrine Resistant Cohort 
 
3.4.1 Inclusion Criteria 
 
1. Clinical T2-T4c at diagnosis or screening, any N, M0 invasive ER+ (Allred 
Score at least 3 or > 1% ER positivity) and HER2 negative (0 or 1+ by IHC or 
FISH negative or equivocal) breast cancer, by AJCC 7th edition clinical staging, 
with the goal being surgery to completely excise the tumor in the breast and the 
lymph node. 
 
Note: Patients with invasive breast cancer that is ER pos, HER2 neg or 
equivocal or DCIS in the contralateral breast are eligible; multi-focal diseases 
are not excluded.  The dominant lesion will be followed per protocol. 
 
2. Ki67 > 10% by central testing at Washington University AMP laboratory from 
a tumor biopsy performed after at least 2 weeks on neoadjuvant endocrine 
therapy. 
 
If Ki67 is > 10% by local testing, the Ki67 slide and H&E slide need to be 
reviewed by the study pathologist to confirm eligibility (discuss with Study 
Chair).  For patients external to Washington University, please contact the 
Washington University coordinator by email so that a screening ID# can be 
assigned prior to shipment of the slides. 
 
Note that prior neoadjuvant endocrine therapy could include any endocrine 
therapy (including aromatase inhibitor, tamoxifen, fulvestrant) alone or in 
combination, or endocrine therapy in combination with any investigational 
agent that is not a Cdk 4/6 inhibitor. 
 
3. Female >18 years of age.   
 
4. ECOG performance status of 0, 1 or 2 (Appendix A).   
 
5. Pre- or post-menopausal women are eligible 
 

 
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If premenopausal, patient must be willing to comply with pregnancy 
requirements laid out in Section 5.5 and agrees with GnRH agonist therapy for 
ovarian suppression during the study. 
 
6. Adequate organ and marrow function as defined below: 
a. Leukocytes > 3,000/mcL 
b. Absolute neutrophil count > 1,500/mcL 
c. Platelets > 100,000/mcL 
d. Total bilirubin ≤ upper normal institutional limits 
e. AST(SGOT)/ and ALT(SGPT) < 2.5 X institutional upper limit 
normal  
 
 
f. Creatinine ≤ upper normal institutional limits 
 
7. Able to understand and willing to sign an IRB-approved written informed 
consent document. 
 
3.4.2 Exclusion Criteria 
 
1. Prior treatment of this cancer including: 
a. Surgery  
b. Radiation therapy  
c. Chemotherapy  
 
2. Receiving any other investigational agents. 
 
3. Prior therapy with any Cdk4 inhibitor. 
 
4. Any of the following in the previous 6 months:  
a. myocardial infarction 
b. severe/unstable angina 
c. coronary/peripheral artery bypass graft 
d. symptomatic congestive heart failure 
e. cerebrovascular accident 
f. transient ischemic attack   
g. symptomatic pulmonary embolism. 
 
5. Uncontrolled intercurrent illness including, but not limited to: 
a. ongoing or active infection 
b. symptomatic congestive heart failure 
c. unstable angina pectoris 
d. uncontrolled symptomatic cardiac arrhythmia, 
e. psychiatric illness/social situations that would limit compliance with study 
requirements.  
 
6. Pregnant/nursing. 
 

 
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7. Unwilling to employ adequate contraception. 
 
8. Known HIV-positive on combination antiretroviral therapy. 
 
NOTE: HIV-positive patients on combination antiretroviral therapy are 
ineligible because of the potential for pharmacokinetic interactions with PD 
0332991.  In addition, these patients are at increased risk of lethal infections 
when treated with marrow-suppressive therapy. 
 
9. Known metastatic disease. 
 
10. Current use of anticoagulation therapy.   
 
11. Previous excisional biopsy of the breast cancer or sentinel lymph node biopsy. 
 
12. Any condition that impairs patient’s ability to swallow PD 0332991 tablets 
(e.g., gastrointestinal tract disease resulting in an inability to take oral 
medication or a requirement for IV alimentation, prior surgical procedures 
affecting absorption) 
 
13. History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to PD 0332991 or other agents used in the study. 
 
14. Corrected QT (QTc) interval >470 msec. 
 
 
15. Current use or anticipated need for food or drugs that are known strong 
CYP3A4 inhibitors (i.e. grapefruit juice, verapamil, ketoconazole, miconazole, 
itraconazole, posaconazole, erythromycin, clarithromycin, telithromycin, 
indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, amprenavir, 
fosamprenavir, nefazodone, diltiazem, and delavirdine) or inducers (i.e. 
dexamethasone, glucocorticoids, progesterone, rifampin, phenobarbital, St. 
John’s wort). See Appendix H 
 
 
3.5 
Eligibility Criteria for the Adjuvant Cohort 
 
3.5.1 Inclusion Criteria 
 
1. Derived benefit from PD 0332991 in the neoadjuvant setting in this trial.  This 
includes the 26 patients who achieved complete cell cycle arrest only after the 
addition of PD 0332991 (C1D1 Ki67 >2.7% and C1D15 Ki67 ≤ 2.7%) from the 
main study (PIK3CA WT, mutant, or unknown cohorts) as well as any patients 
who have a Ki67 ≤ 10% on C1D15 biopsy in the endocrine resistant cohort.   
 
2. ECOG performance status of 0, 1 or 2 (Appendix A).   

 
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3. If premenopausal, patient must be willing to comply with pregnancy 
requirements laid out in Section 5.5. 
 
4. Adequate organ and marrow function as defined below: 
a. leukocytes ≥ 3,000/mcL 
b. absolute neutrophil count ≥ 1,500/mcL 
c. platelets ≥ 100,000/mcL 
d. total bilirubin ≤ upper normal institutional limits 
e. AST(SGOT)/ and ALT(SGPT) ≤ 2.5 X institutional upper limit normal 
 
f. Creatinine ≤ upper normal institutional limits 
 
5. Underwent surgery of the breast and axilla for curative intent.   
 
6. At least 4 weeks post completion of adjuvant chemotherapy and radiation 
therapy if indicated.   
 
7. Patients who already started on adjuvant hormonal therapy are eligible under 
the following conditions: 
a. For the 26 patients who enrolled in the initial cohorts and derived benefit  
from neoadjuvant PD 0332991 (C1D1 Ki67 >2.7% and C1D15 Ki67 ≤ 
2.7%), adjuvant PD 0332991 should be initiated as soon as possible if 
adjuvant hormonal therapy has been initiated and the patient has completed 
radiation if indicated.  
b. For patients who enrolled in the endocrine resistant cohort and derived 
benefit from neoadjuvant PD 0332991 (C1D15 Ki67 ≤ 10%), adjuvant PD 
0332991 should be initiated within 6 months or sooner after initation of 
adjuvant hormonal therapy. 
 
8. Able to understand and willing to sign an IRB-approved written informed 
consent document. 
 
3.5.2 Exclusion Criteria 
 
1. Any of the following in the previous 6 months:  
a. myocardial infarction 
b. severe/unstable angina 
c. coronary/peripheral artery bypass graft 
d. symptomatic congestive heart failure 
e. cerebrovascular accident 
f. transient ischemic attack   
g. symptomatic pulmonary embolism. 
 
2. Uncontrolled intercurrent illness including, but not limited to: 
a. ongoing or active infection 
b. symptomatic congestive heart failure 

 
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c. unstable angina pectoris 
d. uncontrolled symptomatic cardiac arrhythmia 
e. Psychiatric illness/social situations that would limit compliance with study 
requirements.  
 
3. Pregnant/nursing. 
 
4. Unwilling to employ adequate contraception. 
 
5. Known HIV-positive on combination antiretroviral therapy. 
 
NOTE: HIV-positive patients on combination antiretroviral therapy are 
ineligible because of the potential for pharmacokinetic interactions with PD 
0332991.  In addition, these patients are at increased risk of lethal infections 
when treated with marrow-suppressive therapy. 
 
6. Known metastatic disease. 
 
7. Any condition that impairs patient’s ability to swallow PD 0332991 tablets 
(e.g., gastrointestinal tract disease resulting in an inability to take oral 
medication or a requirement for IV alimentation, prior surgical procedures 
affecting absorption) 
 
8. History of allergic reactions attributed to compounds of similar chemical or 
biologic composition to PD 0332991 or other agents used in the study. 
 
9. Corrected QT (QTc) interval >470 msec. 
 
 
10. Current use or anticipated need for food or drugs that are known strong 
CYP3A4 inhibitors (i.e. grapefruit juice, verapamil, ketoconazole, miconazole, 
itraconazole, posaconazole, erythromycin, clarithromycin, telithromycin, 
indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, amprenavir, 
fosamprenavir, nefazodone, diltiazem, and delavirdine) or inducers (i.e. 
dexamethasone, glucocorticoids, progesterone, rifampin, phenobarbital, St. 
John’s wort). See Appendix H 
 
 
 

 
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3.6 
Inclusion of Women and Minorities 
 
Women and members of all races and ethnic groups are eligible for this trial.   
 
 
4.0 
PRE-REGISTRATION AND REGISTRATION PROCEDURES 
 
4.1 
Pre-Registration 
 
This subsection applies only to patients enrolling to the PIK3CA Mutant and WT 
cohorts.  Enrollment to those cohorts is closed as of Amendment #7. 
 
Patients will be pre-registered to the NCI 9170 trial if it is active for patient 
enrollment at the participating institution unless patient is not an appropriate 
candidate for the NCI9170 trial per treating physician.  Otherwise, patients will be 
pre-registered through this trial using the following procedures for PIK3CA 
sequencing. No pre-registration is required for the Endocrine Resistant Cohort. 
 
Patients must not start any protocol intervention prior to pre-registration through 
the Siteman Cancer Center. All eligible and consenting patients will be pre-registered to 
this protocol for the purposes of PIK3CA sequencing; if mutation is identified, the patient 
will then be registered to the PIK3CA Mutant Cohort if eligible for study treatment.  If 
mutation is not identified, the patient will then be registered to the PIK3CA Wild Type 
Cohort if eligible for study treatment.  Pre-registration is not required for patients to be 
enrolled in the endocrine resistant cohort, as PIK3CA mutation status will not be assessed. 
 
The following steps must be taken: 
 
1. Confirmation of patient pre-registration eligibility by Washington University 
2. Pre-Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN)  
 
Once the patient has been entered in the Siteman Cancer Center database, the WUSM 
coordinator will forward verification of enrollment and the UPN via email. 
 
4.1.1 Confirmation of Patient Eligibility for Pre-Registration 
 
Confirm patient eligibility by scanning or faxing the information listed below to the 
research coordinator listed in the Siteman Cancer Center Clinical Trials Core 
Protocol Procedures for Secondary Sites packet and Caroline Bumb 
(cbumb@dom.wustl.edu) at least one business day prior to registering patient: 
 
1. Your name and contact information (telephone number, fax number, and 
email address) 
2. Your site PI’s name, the registering MD’s name, and your institution name 

 
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3. Patient’s race, sex, and DOB 
4. Three letters (or two letters and a dash) for the patient’s initials 
5. Currently approved protocol version date 
6. Copy of signed consent form (patient name may be blacked out) 
7. Planned date of sample shipment 
8. Completed pre-registration eligibility checklist, signed and dated by a 
member of the study team 
9. Copy of appropriate source documentation confirming patient eligibility 
 
4.1.2 Patient Pre-Registration in the Siteman Cancer Center Database  
 
Registrations may be submitted Monday through Friday between 8am and 5pm CT. 
Urgent late afternoon or early morning enrollments should be planned in advance 
and coordinated with the Washington University research coordinator.  Registration 
will be confirmed by the research coordinator or his/her delegate by email or fax 
within one business day. Verification of eligibility and pre-registration should be 
kept in the patient chart. 
 
Patients at all sites must be pre-registered through the Siteman Cancer Center 
database at Washington University. 
 
4.1.3 Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  
Patients will also be identified by first, middle, and last initials.  If the patient has 
no middle initial, a dash will be used on the case report forms (CRFs).  All data will 
be recorded with this identification number on the appropriate CRFs. 
 
4.1.4 Additional Notes 
 
• Pre-registration tests/procedures (see Section 10.0) must be completed within 
the guidelines specified on the test schedule. 
• At the time of IRB submission, site should request several biopsy kits by 
contacting the Alliance Central Specimen Bank (address and phone numbers 
listed in Section 9.1). 
• Following pre-registration and research tumor biopsy, patient may start Cycle 
0 anastrozole (and goserelin if premenopausal) according to the instructions in 
Section 5.1. 
 
4.2 
Registration 
 
The information in this section applies to patients enrolling to ALL cohorts. 
 
Patients must not start any protocol intervention prior to registration through the 
Siteman Cancer Center. 
 

 
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The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility by Washington University 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) if not pre-registered through this trial 
 
Once the patient has been entered in the Siteman Cancer Center database, the WUSM 
coordinator will forward verification of enrollment via email. 
 
4.2.1 Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below and scanning and 
emailing to the research coordinator listed in the Siteman Cancer Center Clinical 
Trials Core Protocol Procedures for Secondary Sites packet and Caroline Bumb at 
cbumb@ wustl.edu at least one business day prior to registering patient: 
 
1. Your name and contact information (telephone number, fax number, and email 
address) 
2. Your site PI’s name, the registering MD’s name, and your institution name 
3. Patient’s race, sex, and DOB 
4. Three letters (or two letters and a dash) for the patient’s initials 
5. Currently approved protocol version date 
6. Copy of signed consent form (patient name may be blacked out) 
7. Planned date of enrollment 
8. Completed eligibility checklist (Appendix B), signed and dated by a member of 
the study team 
9. Copy of appropriate source documentation confirming patient eligibility 
 
4.2.2 Patient Registration in the Siteman Cancer Center Database 
 
Registrations may be submitted Monday through Friday between 8am and 5pm CT. 
Urgent late afternoon or early morning enrollments should be planned in advance 
and coordinated with the Washington University research coordinator.  Registration 
will be confirmed by the research coordinator or his/her delegate by email within 
one business day. Verification of eligibility and registration should be kept in the 
patient chart. 
 
All patients at all sites must be registered through the Siteman Cancer Center 
database at Washington University. 
 
4.2.3 Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  
Patients will also be identified by first, middle, and last initials.  If the patient has 
no middle initial, a dash will be used on the case report forms (CRFs).  All data will 
be recorded with this identification number on the appropriate CRFs. 

 
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5.0 
TREATMENT PLAN 
 
5.1 
Neoadjuvant Treatment 
 
Treatment will be administered on an outpatient basis.  Patients will be instructed to make 
use of medication diaries (Appendices D and E) to act as a records for administration of 
PD 0332991 and anastrozole. Appropriate dose modifications are described in Section 6.0.  
No investigational or commercial agents or therapies other than those described below may 
be administered with the intent to treat the patient’s malignancy. 
 
Cycle 0 (Only applicable for PIK3CA Mutant and WT cohorts) 
Before Cycle 1 Day 1 starts, patients who enrolled in the PIK3CA mutant or WT cohort 
should have completed Cycle 0.  Cycle 0 therapy is 28 days of anastrozole (1mg PO daily) 
and, if premenopausal, goserelin 3.6mg SC every 28 days.  
  
Cycles 1-5 
Treatment with PD 0332991 combined with anastrozole (and goserelin if premenopausal) 
is to be 4 28-day cycles followed by a fifth cycle of 10-12 days duration consisting of daily 
PD 0332991 and anastrozole, with the last dose of both drugs to be given the day before 
surgery. 
 
REGIMEN DESCRIPTION 
Agent 
Dose 
Route 
Schedule 
Cycle Length 
PD 0332991 
125 mg 
PO 
Daily 
(Days 1-21) 
 Cycles 1-4: 28 days 
Cycle 5: 10-12 days 
Adjuvant treatment: 28 
days 
Anastrozole 
1 mg 
PO 
Daily 
(Days 1-28) 
Goserelin* 
3.6 mg 
SC 
Q28 days 
* only if premenopausal; although it is preferred to be given on Day 1 of each cycle, it may be 
administered any day of the cycle to accommodate the Q28-day cycle for goserelin.  
  
PD 0332991 should be taken with food.  Anastrozole should be taken daily with or without 
food.   
 
For both PD 0332991 and anastrozole: 
• If a patient misses a day’s dose entirely, she must be instructed not to make it up the 
next day but just take her regular dose the following day. 
• If a patient vomits anytime after taking a dose, she must be instructed not to make it up 
but to resume subsequent does the next day as prescribed. 
• If a patient inadvertently takes an extra dose during a day, she must be instructed to not 
take the next day’s dose. 
   
Special Considerations for Cycle 5 
Cycle 5 Day 1 of PD 0332991 may be delayed by up to 3 weeks to allow  ANC to recover 
to ≥ 1500/mcL and platelets to recover to ≥ 100,000/mcL and other treatment related AEs 

 
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to recover to ≤ grade 1 as well as to accommodate surgery scheduling. Anastrozole is 
continued in all patients during the neoadjuvant period. Patients who receive goserelin 
should also continue goserelin every 28 days during the neoadjuvant period.  
 
CBC+D is to be performed 3 days (±1 day) prior to surgery in patients who receive Cycle 
5 therapy.  Treatment with PD 0332991 should be interrupted if ANC < 1,000/mcL, 
platelets < 100,000/mcL, or patients present with any ≥ grade 2 treatment-related toxicities.  
If a patient needs to stop treatment prematurely in Cycle 5, she will have surgery on the 
scheduled date unless contraindicated, as the recovery of neutropenia is usually prompt 
after stopping PD 0332991 due to its mechanisms of action (investigator brochure). 
However, surgery may be rescheduled at the discretion of the treating physician and the 
study chair.  Patients should continue with anastrozole until the day before surgery 
regardless of whether they complete Cycle 5 of PD 0332991. 
 
Patients whose ANC and/or platelets do not recover within 3 weeks following the completion of 
Cycle 4 will proceed to surgery as planned (3 to 5 weeks following the last dose of PD 0332991 
(Cycle 4 Day 21)).  For these patients, anastrozole is to be continued until the day of surgery, 
with the last dose of anastrozole given the day before surgery.  
 
5.2 
Surgery 
 
Standard surgery (breast and axillary lymph node surgery) will be performed per 
institutional standards 2-4 weeks following the completion (Day 28) of Cycle 4 in those 
who did not receive Cycle 5.  In patients who receive Cycle 5, surgery occurs on Day 11, 
12, or 13 of Cycle 5.   
 
Note that tissue collection is mandatory at the time of surgery for all patients.  
 
5.3 
Post Surgery Therapy 
 
Standard adjuvant treatment, including chemotherapy, hormonal therapy, and radiation if 
needed, are recommended after surgery at the discretion of the treating physician. 
 
Patients who derived benefit from the combination therapy have the option of taking PD 
0332991 in combination with anastrozole for 23 cycles after surgery and adjuvant 
chemotherapy and radiation if indicated.  Patients are determined to have derived benefit 
if their Ki67 is ≤ 10% on C1D15 tumor biopsy if they are enrolled to the endocrine resistant 
cohort (including patients who enrolled prior to activation of Amendment #7) OR if they 
are among the 26 patients who enrolled initially to the main trial who required PD 0332991 
to achieve complete cell cycle arrest. 
 
PD 0332991 should be re-started at least 4 weeks after the completion of chemotherapy 
and radiation therapy if these treatments were planned.  Hormonal therapy may be started 
earlier (during radiation therapy) if desired. 
   
Anastrozole is preferred in this trial to combine with PD 0332991 in the adjuvant setting 

 
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as this is the same regimen used in the neoadjvuant setting, but alternative hormonal 
therapy such as another aromatase inhibitor or tamoxifen is allowed if patient could not 
tolerate adjuvant anastrozole.  Goserelin should be administered if aromatase inhibitor is 
used in patients who are pre-menopausal determined by institutional standard.  
 
After completion of adjuvant PD 0332991, patients should continue standard of care 
hormonal therapy (for example, anastrozole) to complete at least 5 years of adjuvant 
hormonal therapy.  Further therapy after that is at the discretion of the treating physician.   
 
5.4 
General Concomitant Medication and Supportive Care Guidelines 
 
While taking PD 0332991, patients should be instructed to avoid food or drugs that are 
known strong CYP3A4 inhibitors or inducers.  Please refer to Appendix H for a list of 
prohibited medications.  
 
 
No specific antidotes exist for the treatment of PD 0332991 overdose. Since renal excretion 
of PD 0332991 is minimal, the benefit of hemodialysis in the treatment of a PD 0332991 
overdose is probably negligible. The treatment of overdose of PD 0332991 should consist 
of general supportive measures. If indicated, elimination of unabsorbed drug should be 
achieved by emesis or gastric lavage. 
 
No prophylactic medications are required prior to administration of anastrozole or 
goserelin. 
 
5.5 
Women of Childbearing Potential 
 
Women of childbearing potential (defined as women with regular menses, women with 
amenorrhea, women with irregular cycles, women using a contraceptive method that 
precludes withdrawal bleeding, and women who have had a tubal ligation) are required to 
have a negative serum pregnancy test within 7 days prior to the first dose of the study agent.   
 
Women of childbearing potential are required to use two forms of acceptable contraception, 
including one barrier method, during participation in the study and for 90 days following 
the last dose of PD 0332991.  
 
If a patient is suspected to be pregnant, all study drugs should be immediately discontinued.  
In addition a positive urine test must be confirmed by a serum pregnancy test.  If it is 
confirmed that the patient is not pregnant, she may resume dosing. 
 
5.6 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 

 
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In the absence of treatment delays due to adverse events, neoadjuvant treatment may 
continue for 4 cycles plus the fifth short cycle or one of the following criteria applies: 
• Documented and confirmed disease progression or Ki67>10 % on C1D15 
• Death 
• Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
• General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
• Suspected pregnancy 
• Serious non-compliance with the study protocol 
• Lost to follow-up 
• Patient withdraws consent 
• Investigator removes the patient from study 
• The Siteman Cancer Center decides to close the study 
 
Adjuvant treatment (in patients eligible to receive it) may continue for up to 23 cycles or 
until one of the following criteria applies: 
• Documented and confirmed disease progression 
• Death 
• Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
• General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
• Suspected pregnancy 
• Serious non-compliance with the study protocol (missing 50% dosing in any cycle 
due to non-compliance or missing study required procedures due to non-
compliance determined by site investigator) 
• Lost to follow-up 
• Patient withdraws consent 
• Investigator removes the patient from study 
• The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.7 
Treatment/Follow-up Decision Tree 
 
Any patient who registers on study and then withdraws her consent prior to receiving any 
PD 0332991 will be considered a cancellation.  No further follow-up is required. Future 
treatment is at the discretion of their treating physician. 
 
If a patient is found not to have fulfilled all the eligibility requirements after starting PD 
0332991, she will be removed from study treatment. All study forms up to the point of 
study treatment discontinuation are to be submitted.  These patients will be observed for 

 
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AEs for 30 days following the last dose of PD 0332991 or the resolution of AEs at least 
possibly related to PD 0332991, whichever comes later. Future treatment is at the discretion 
of the treating physician. No further follow-up is required. 
   
All patients except for those who withdrew consent for follow up or who refused to undergo 
surgery will be followed for 30 days after surgery, then yearly to document recurrence and 
survival status for 5 years or until recurrence whichever comes first (see Section 10.3).   
 
Patients who received adjuvant PD 0332991 will be followed as described in Section 10.3 
during adjuvant PD 0332991 therapy, then yearly following completion of PD 0332991 to 
complete a total of 5 years of follow-up post surgery.  Should a patient discontinue adjuvant 
PD 0332991 early, she would be followed annually following the completion of PD 
0332991 to get as close as possible to 5 years of post-surgery follow-up.   
 
If protocol therapy was discontinued due to Ki67 > 10% on C1D15 or estradiol level in the 
premenopausal range in patients on goserelin, or discontinued PD 0332991 prior to 
completion of planned duration of therapy, the patient will be observed for AEs for 30 days 
following the last dose of PD 0332991 or the resolution of AEs at least possibly related to 
PD 0332991, then yearly to complete 5 years of follow up after surgery or until recurrence 
(whichever comes first).  Neoadjuvant chemotherapy per physician choice is recommended 
before surgery.  Pathologic response will be documented.  
 
If there is physical evidence for clinical progression with bi-dimensional tape, ruler, or 
caliper tumor measurements of the primary tumor, a mammogram and/or ultrasound of the 
breast should be done to confirm/rule out progressive disease.  If there is physical evidence 
for clinical progression with clinical assessment of the lymph node mass, an ultrasound of 
the axilla should be done to confirm/rule out progressive disease.  
   
If progression is confirmed by ultrasound OR mammographic imaging, discontinue the 
study drug and operate as soon as possible or begin other anti-neoplastic approaches such 
as chemotherapy or radiation therapy at the treating physician’s discretion. NOTE: Other 
anti-neoplastic approaches such as chemotherapy or radiation must not be administered 
while the patient is taking study drug. 
 
For these patients:  
• Obtain the serum and plasma samples (can be obtained at surgery) 
• Obtain core biopsies (two frozen in OCT and two formalin-fixed) before the patient 
receives non-protocol chemotherapy (could be obtained at surgery) 
• Complete case report form indicating whether the patient will go to immediate 
surgery or begin other anti-neoplastic approaches such as chemotherapy or 
radiation therapy.  If patient goes to immediate surgery, complete surgery and 
pathology case report forms.  
• Follow-up for 30 days following the last dose of PD 0332991 or the resolution of 
AEs at least possibly related to PD 0332991, then yearly to complete 5 years of 
followup after surgery or until recurrence (whichever comes first). 
 

 
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If disease progression is not confirmed by ultrasound or mammographic imaging, study 
drug may be continued at the investigator’s discretion, and the protocol followed as 
described.  These patients are not eligible for adjuvant PD 0332991 therapy. 
 
Clinical progression outside the primary site (i.e., the development of a new breast mass or 
the development of clinical suspicion for advanced disease) should lead to further imaging 
evaluation and if confirmed, study drug will be discontinued.  Optional tumor biopsy and 
blood will be obtained prior to the subsequent neoadjuvant therapy or at the time of surgery 
is surgery is the next step.  Subsequent management is at the investigator’s discretion.  The 
patient will go off study. 
 
Patients in whom surgery is not performed will be observed for 30-60 days after last dose 
of study drug and will not undergo annual follow-up.  
 
If a patient is found to be pregnant, the study drug will be discontinued immediately. The 
patient will be followed for adverse events through the end of this pregnancy. Future 
treatment decisions are at the discretion of the patient’s treating physician.   
 
If a patient withdraws consent at any point in this study, all study data should be submitted 
up to the point of consent withdrawal.  
 
5.8 
Duration of Follow-up 
 
Patients who receive the study therapy will be observed post-surgery for 30-60 days or 
resolution of AEs, whichever comes later, then yearly for recurrence and survival status 
for 5 years. Patients who received adjuvant PD 0332991 will be followed as described in 
Section 10.3 during adjuvant PD 0332991 therapy, then yearly following completion of 
PD 0332991 to complete a total of 5 years of follow-up post-surgery.   
 
 
6.0 
DOSE DELAYS/DOSE MODIFICATIONS  
 
Table 3. Dose Modification Table. 
Dose Level 
PD 0332991 Days 1-21 of each 28-day 
cycle 
Anastrozole (daily) 
1 (starting dose) 
125 mg/day 
1 mg/day 
-1 
100 mg/day 
1 mg/day 
-2 
75 mg/day 
1 mg/day 
Note that PD 0332991 dose reduction below 75 mg/day is not allowed 
 
6.1 
Dose Modifications for Anastrozole 
 
No dose adjustment is permitted for anastrozole but interruptions are allowed per physician 
discretion.  During adjuvant phase, switiching to another hormonal therapy is allowed.  
 
6.2 
Dose Modifications for PD 0332991 

 
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Patients will be monitored for toxicity and the dose of PD 0332991 may be adjusted as 
indicated in Table 3.  Dose reduction by 1, and if needed, 2 dose levels will be allowed 
depending on the type and severity of toxicity encountered (Table 4).  Patients requiring 
more than 2 dose reductions will be discontinued from the study. 
 
Recommended dose reductions for PD 0332991 are detailed in Table 4. Doses may be held 
as needed for toxicity resolution during a cycle. Doses omitted for toxicity are not replaced 
or restored within the same cycle (meaning that the cycle remains 28 days regardless of the 
number of doses of taken). 
 
Treatment with PD 0332991 should be permanently discontinued if toxicity has not 
recovered to grade ≤ 2 within two weeks (including the scheduled 1-week off treatment 
period within a cycle).  
 
Table 4. PD 0332991 Dose Modifications Based on Worst Treatment-Related Toxicity in 
the Previous Cycle 
 
Worst Toxicity During Previous 
Cycle  
Action 
New Dose Level  
Grade 4 neutropenia  
Hold until ANC > 1000/mm3 
Decrease by one dose level  
Grade 4 thrombocytopenia  
Hold until platelets > 50,000 
Decrease by one dose level  
Grade 3 neutropenia associated with 
a documented infection or fever ≥ 
38.5 ºC  
Hold until ANC > 1000/mm3 
without fever 
Decrease by one dose level  
Grade 3 neutropenia without fever 
Hold until ANC > 1000/mm3 
1st occurrence: resume at the same dose 
2nd occurrence: decrease by one dose 
level 
3rd occurrence: decrease by one dose 
level 
Grade ≥ 3 non-hematologic toxicity 
(includes nausea, vomiting, 
diarrhea, and hypertension only if 
persisting despite maximal medical 
treatment)  
Hold until recover to < grade 1 
(or < grade 2 if investigator does 
not consider the AE a safety 
risk) 
Decrease by one dose level  
Inability to deliver at least 80% of 
the planned dose of PD 0332991 or 
anastrozole due to adverse events 
possibly related to study treatment  
 
Decrease by one dose level 
Grade > 2 non-hematologic toxicity 
(except alopecia) that persists 
longer than 4 weeks despite 
maximal supportive care and is 
unacceptable to patient and/or 
investigator 
 
Decrease by one dose level 
 
 
6.2.1 Dose Adjustments Due to QTc Prolongation 

 
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Any patients who develops new grade 2 or greater ECG QT corrected interval 
prolonged at any time during the study will need to have the ECG repeated 
immediately for confirmation. 
 
Grade 2: no adjustments; continue at same dose level 
Grade 3 (reversible cause identified and corrected): withhold treatment until QTc 
≤ 470 msec, then resume treatment at the same dose level 
Grade 3 (no reversible cause identified): withhold treatment until QTc ≤ 470 msec, 
then decrease PD 0332991 by one dose level 
Grade 4: permanently discontinue PD 0332991 
 
6.3 
Re-Treatment Criteria for Neoadjuvant Cycles 1-4 and Adjuvant Therapy 
 
A new cycle of treatment with PD 0332991 may begin only if: 
• ANC ≥1,000/mcL. 
• Platelet count ≥50,000/mcL. 
• Non-hematologic toxicities have returned to baseline or Grade ≤1 severity (or, at 
the Investigator’s discretion, Grade ≤2 if not considered a safety risk for the 
patient). 
 
Criteria for dose interruption within cycle: 
• ANC <1,000/mcL. 
• Platelet count <50,000/mcL. 
 
Re-treatment within the cycle may only be started when ANC ≥1,000/mcL and platelet 
count ≥50,000/mcL. 
 
Doses omitted for toxicity within a cycle are not replaced or restored within the same cycle 
(meaning that the cycle remains 28 days regardless of the number of doses of taken). 
 
If these conditions are not met, anastrozole treatment may be continued but treatment with 
PD 0332991 must be delayed by one week. If, after a one-week delay, all toxicities have 
recovered within the limits described above, treatment with PD 0332991 can be resumed. 
 
If the patient has not recovered after 2 weeks (including the scheduled 1-week off treatment 
period within a cycle) despite dose reduction to the lowest dose level, treatment with PD 
0332991 will be permanently discontinued.  
 
Adjuvant PD 0332991 may be interrupted for a maximum of 4 weeks due to intercurrent 
illness or surgery (discuss with study chair).  
 
Anastrozole is administered in a continuous regimen and therefore no re-treatment criteria 
apply. 
 
6.4 
Re-Treatment Criteria for Cycle 5 

 
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Cycle 5 Day 1 with PD 0332991 may begin only if: 
• ANC ≥1,500/mcL 
• Platelet count ≥100,000/mcL 
• Non-hematologic toxicities have returned to baseline or Grade ≤1 severity (or, at 
the Investigator’s discretion, Grade ≤2 if not considered a safety risk for the 
patient). 
 
If these conditions are not met, anastrozole treatment will be continued but treatment with 
PD 0332991 will be delayed by one to three weeks. If all toxicities have recovered within 
the limits described above within 3 weeks, treatment with PD 0332991 for Cycle 5 will be 
initiated.  If the patient has not recovered after a 3 week delay, treatment with PD 0332991 
will be permanently discontinued and patients should proceed to surgery within 3 to 5 
weeks after the last dose of PD0332991 (Cycle 4 Day 21) if possible.  
 
Anastrozole is administered in a continuous regimen and therefore no re-treatment criteria 
apply. 
 
 
7.0 
REGULATORY AND REPORTING REQUIREMENTS 
 
The entities providing oversight of safety and compliance with the protocol require reporting as 
outline below. 
 
The Washington University Human Research Protection Office (HRPO) requires that all events 
meeting the definition of unanticipated problem or serious noncompliance be reported as outlined 
in Section 7.2. 
 
The FDA requires that all serious and unexpected adverse events be reported as outlined in Section 
7.4.  In addition, any fatal or life-threatening adverse experiences where there is a reasonable 
possibility of relationship to study intervention must be reported. 
 
Pfizer requires that all events be reported as outlined in Section 7.7. 
 
7.1 Definitions 
 
7.1.1 Adverse Events (AEs) 
 
Definition: any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading: the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for 
all toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from 
the CTEP website. 
 

 
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Attribution (relatedness), Expectedness, and Seriousness: the definitions for the 
terms listed that should be used are those provided by the Department of Health 
and Human Services’ Office for Human Research Protections (OHRP).  A copy of 
this guidance can be found on OHRP’s website: 
http://www.hhs.gov/ohrp/policy/advevntguid.html 
 
7.1.2 Serious Adverse Event (SAE) 
 
Definition:  any adverse drug experience occurring at any dose that results in any 
of the following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
 
All unexpected SAEs must be reported to the FDA. 
 
7.1.3 Unexpected Adverse Experience 
 
Definition: any adverse drug experience, the specificity or severity of which is not 
consistent with the current investigator brochure (or risk information, if an IB is not 
required or available). 
 
Events that are both serious AND unexpected must be reported to the FDA.  
 
7.1.4 Life-Threatening Adverse Experience  
 
Definition: any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 
Life-threatening adverse experiences must be reported to the FDA. 
 
 
7.1.5 Unanticipated Problems 
 
Definition: 
• unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB-approved research protocol and informed consent document; and (b) 

 
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the characteristics of the subject population being studied; 
• related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and 
• suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was 
previously known or recognized. 
 
7.1.6 Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that 
govern human subjects research or failure to follow the determinations of the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate 
choice to ignore regulations, institutional policies, or determinations of the IRB. 
 
7.1.7 Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial 
harm to subjects or others, or that materially compromises the rights or welfare of 
participants. 
 
7.1.8 Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the 
research team’s control. Exceptions apply only to a single participant or a singular 
situation. 
 
Local IRB Pre-approval of all protocol exceptions must be obtained prior to the 
event.  For secondary sites, the Washington University PI will issue approval of the 
exception, but it must also be submitted to the local IRB with documentation of 
approval forwarded to Washington University.  HRPO approval is not required for 
protocol exceptions occurring at secondary sites. 
 
7.2 
Reporting to the Human Research Protection Office (HRPO) at Washington 
University 
 
The PI is required to promptly notify the IRB of the following events: 
 
• Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
• Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
• Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 

 
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These events must be reported to the IRB within 10 working days of the occurrence 
of the event or notification to the PI of the event.  The death of a research participant 
that qualifies as a reportable event should be reported within 1 working day of the 
occurrence of the event or notification to the PI of the event. 
 
7.3 
Reporting to the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University 
 
The PI is required to notify the QASMC of any unanticipated problem occurring at WU or 
any BJH or SLCH institution that has been reported to and acknowledged by HRPO as 
reportable.  (Unanticipated problems reported to HRPO and withdrawn during the review 
process need not be reported to QASMC.) 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via email to 
a QASMC auditor. 
 
7.4 
Reporting Requirements for Secondary Sites 
 
The research team at each secondary site is required to promptly notify the Washington 
University PI and research coordinator of all reportable events (as described in Section 7.6) 
within 1 working day of the occurrence of the event or notification of the secondary site’s 
PI of the event.  This notification may take place via email if there is not yet enough 
information for a formal written report (using an FDA MedWatch form).  A formal written 
report must be sent to the Washington University PI and research coordinator within 10 
working days of the occurrence of the event or notification of the secondary site’s PI of 
the event.  The death of a research participant that qualifies as a reportable event should be 
reported within 1 working day of the occurrence of the event or notification of the 
secondary site’s PI of the event. 
 
The research team at a secondary site is responsible for following its site’s guidelines for 
reporting applicable events to its site’s IRB according to its own institutional guidelines. 
The research team at Washington University is responsible for reporting all applicable 
events to the FDA. 
 
7.5 
Reporting to Secondary Sites 
 
The Washington University PI (or designee) will notify the research team at each secondary 
site of all reportable events that have occurred at other sites within 10 working days of the 
occurrence of the event or notification of the PI of the event.  This includes events that take 
place both at Washington University and at other secondary sites, if applicable. 
 
7.6 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 

 
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FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the Washington University principal investigator to report any 
unanticipated problem to the FDA as follows: 
 
• Report any unexpected fatal or life-threatening adverse experiences (Section 7.1.4) 
associated with use of the drug by telephone or fax no later than 7 calendar days 
after initial receipt of the information.   
• Report any serious, unexpected adverse experiences (Section 7.1.2), as well as 
results from animal studies that suggest significant clinical risk within 15 calendar 
days after initial receipt of this information.   
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
Secondary sites must submit a completed MedWatch form to the Washington University 
PI and research coordinator within 4 calendar days (for fatal or life-threatening adverse 
experiences) or 11 calendar days (for serious, unexpected adverse experiences).  The 
Washington University PI will be responsible for submitting all MedWatch forms from 
secondary sites to the FDA within the timeframes specified above. 
 
7.7 
Reporting to Pfizer 
 
Within 24 hours of first awareness of the event (immediately if the event is fatal or life-
threatening), the PI or designee will report to Pfizer by facsimile any serious adverse drug 
experience (as defined in Section 7.9) that occurs during the SAE reporting period (as 
defined in Section 7.11) in a study subject assigned to receive PD 0332991.  Such SAEs 
will be reported using MedWatch form and the Pfizer Reportable Event Fax Cover Sheet 
(Appendix G) should also be included.  SAEs should be reported as soon as they are 
determined to meet the definition, even if complete information is not yet available. 
 
Even though there may not be an associated SAE, exposure to PD 0332991 during 
pregnancy or lactation is reportable. 
 
Secondary sites must submit a completed MedWatch form to the Washington University 
PI and research coordinator within the specified time frame.  The Washington University 
PI will be responsible for submitting all MedWatch forms from secondary sites to Pfizer. 
 
7.8 
Timeframe for Reporting Required Events  
 

 
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Adverse events will be tracked for 30 days following the last day of study treatment. 
 
8.0 
PHARMACEUTICAL INFORMATION 
 
8.1 
Study Agent (PD 0332991)   
 
8.1.1 PD 0332991 Description 
 
Laboratory Code: PD 0332991-0000 
Molecular Weight: 447.5 
Molecular Formula: C24H29H7O2 
 
Formulation: Capsules that use common compendial excipients (corn starch, 
microcrystalline cellulose, sodium starch glycolate, magnesium stearate 
(nonbovine)) will be used in clinical programs.  The capsule shells are 
manufactured from gelatin NF. 
 
8.1.2 Clinical Pharmacology 
 
PD 0332991 is a highly selective inhibitor of Cdk4/cyclinD1 kinase activity (IC50 = 
11 nM; Ki = 2 nM). PD 0332991 has selectivity for Cdk4/6, with little or no activity 
against a large panel of 34 other protein kinases including other Cdks and a wide 
variety of tyrosine and serine/threonine kinases. Cdk6, another enzyme that also 
complexes with cyclin-D subunits, is also commonly expressed in mammalian cells 
and tumors. Cdk6 is highly homologous to Cdk4 and can perform the same function 
by phosphorylating Rb, thus potentially creating a redundant mechanism to 
promote cell cycle progression. Consequently, inhibition of both enzymes is 
necessary to ensure complete suppression of Rb phosphorylation and the greatest 
possible spectrum of antitumor activity. Results indicate that PD 0332991 inhibits 
Cdk6 with equivalent potency to Cdk4. 
 
8.1.3 Pharmacokinetics and Drug Metabolism 
 
To date pharmacokinetic data have been reported for four studies (A5481001, 
A5481002, A5481003 and A5481004). Final PK data are available from studies 
A5481001 and A5481002.  Pharmacokinetic parameters are available from all 74 
patients enrolled in Protocol A5481001 following a single-dose (Day 1 of Cycle 1), 
and from 51 patients following multiple-dose administration (Day 8 of Cycle 1) of 
daily doses ranging from 25 to 225 mg of PD 0332991 (Table 4.). On Day 1, all 
patients had detectable plasma concentrations of PD 0332991 at the first measured 
time point (1 hour) following oral administration. The exposure (AUC(0-10) and 
Cmax) increased in a dose-proportional manner over the dose range of 25-225 mg 
QD following PD 0332991 administration on Days 1 and 8 of Cycle 1, although 
some variability (low to moderate) around these doses was observed particularly at 
the 150 mg QD dose level (Table 5). 
 
 

 
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Table 5 
 
 
Steady-state PK parameters are available (Table 6) for nine patients on Day 14 of 
Cycle 1 (receiving 200 mg SC 0332991 QD for 2 weeks) and four patients on Day 
21 of Cycle 1 (receiving 125 mg QD for 3 weeks).  PD 0332991 was absorbed with 
a median Tmax of ~4 hours. The mean PD 0332991 Vz/F was 3103 L, which is 
significantly greater than total body water (42 L), indicating that PD 0332991 
extensively penetrates into peripheral tissues. PD 0332991 was eliminated slowly; 
the mean elimination half-life (t½) was 26.5 hours and the mean CL/F was 86.1 
L/hour. PD 0332991 accumulated following repeated dosing with a median Rac of 
2.4, which is consistent with the elimination half-life.  
 
 

 
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Table 6 
 
 
Renal excretion of PD 0332991 was a minor route of elimination with ~1.7% of the 
drug excreted unchanged in urine over the 10-hour collection period in the 125 mg 
and 200 mg dose group, combined. The mean renal clearance (CLR) was 6.59 
L/hour.  
 
An exploratory evaluation of the circulating metabolites for PD 0332991 was 
conducted in plasma samples obtained from patients treated with PD 0332991 200 
mg QD. Preliminary assessment of the pooled plasma samples on Day 14 of Cycle 
1 indicated that the glucuronide conjugate of PD 0332991 and the lactam of 
PD 0332991 were the main metabolites present in plasma. Other metabolites 
observed were the glucuronide conjugates of hydroxylated PD 0332991 and the 
glucuronide conjugate of reduced PD 0332991. 
  
The preliminary results from the recently performed food-effect study (“A5481021, 
a Phase 1, open-label 4 sequence 4 period crossover study of palbociclib (PD-
0332991) in healthy volunteers to estimate the effect of food on the bioavailability 
of palbociclib”) has provided evidence that when a single 125 mg dose of 
palbociclib was administered under fed conditions (including high fat or low fat 
meal given together with palbociclib, or moderate fat meal given 1 hour before and 
2 hours after palbociclib) as a freebase formulation the palbociclib exposure levels 
were more uniform across the population than when taken in the fasting condition. 
 
 
 
Drug-drug interaction between PD 0332991 and letrozole was evaluated during the 
Phase 1 portion of a breast cancer study (A5481003). The preliminary data indicate 
a lack of a potential for drug-drug interaction between PD 0332991 and letrozole 
when administered in combination. 
 
 

 
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8.1.4 Supplier(s) 
 
Pfizer will supply the study agent.  The study agent will be free of charge to the 
patient.  
 
8.1.5 Dosage Form and Preparation 
 
Medication will be provided in non-patient specific bottles containing either 125  
mg, 100 mg, or 75 mg capsules. The patient number and the protocol number should 
be recorded on the bottle label in the spaces provided. Site personnel must ensure 
that patients clearly understand the directions for self-medication. Patients should 
be given a sufficient supply to last until their next study visit. Unused drug and/or 
empty bottles should be returned to the site at the next study visit. PD 0332991 is 
an agent that must be handled and administered with care. Patients should be 
instructed to keep their medication in the bottles provided and not transfer it to any 
other container. Due to possible unknown hazards associated with topical and 
environmental exposure to experimental agents, capsules must not be opened 
and/or emptied into any vehicle for oral ingestion; capsules must be swallowed 
intact. 
 
8.1.6 Storage and Stability 
 
Please store PD 0332991 capsules according to storage conditions on the label. 
Medication should be kept in a secured locked area at the study site in accordance 
with applicable regulatory requirements. Returned medication should be stored 
separately from medication that needs to be dispensed.  
 
To ensure adequate records, PD 0332991 capsules will be accounted for as 
instructed by Pfizer. Unless otherwise authorized by Pfizer, at the end of the clinical 
trial all drug supplies unallocated or unused by the subjects must be returned to 
Pfizer or its designee. All containers of PD 0332991 that were sent to the 
investigator throughout the study must be returned to the sponsor or designee, 
whether they are used or unused, and whether they are empty or contain capsules. 
 
8.1.7 Administration 
 
Patients should be instructed to swallow PD 0332991 capsules whole and not to 
chew them prior to swallowing. No capsule should be ingested if it is broken, 
cracked, or otherwise not intact. Patients should take PD 0332991 with food and 
should be encouraged to take their dose at approximately the same time each day. 
 
8.1.8 Special Handling Instructions 
 
Females of childbearing potential should not handle or administer the study agent 
unless they are wearing gloves. 

 
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8.1.9 Pregnancy 
 
Fertility and teratology studies with PD 0332991 have not been conducted; 
therefore, safety for pregnant women of childbearing capacity and for the fetus 
cannot be implied from the existing data. If the drug is used during pregnancy, or 
if the patient becomes pregnant while receiving this drug, the patient should be 
apprised of the potential hazard to the fetus.  
 
PD 0332991 caused testicular degeneration in rats and dogs. The incidence and 
severity was dose related and correlated with decreases in testicular weight in the 
rat. Testicular degeneration was not reversed after cessation of treatment and 
progressed in severity in both species. Testicular degeneration produced by PD 
0332991 is consistent with Cdk inhibition and alterations in cell cycle kinetics. 
 
Women of childbearing potential must have a negative pregnancy test prior to 
treatment with PD 0332991. Female patients must be surgically sterile or be 
postmenopausal, or must agree to use effective contraceptive during the period of 
the trial and for at least 90 days after completion of treatment. The decision of 
effective contraception will be based on the judgment of the principal investigator 
or a designated associate. 
 
8.1.10 QT Interval 
 
The patients enrolled in clinical studies should be closely monitored for potential 
cardiovascular symptoms. Appropriate monitoring should include clinical 
examinations, vital signs, routine ECGs, and AEs monitoring. In case of QTc 
prolongation, concomitant conditions such as electrolyte unbalances or use of 
medications affecting the QT interval should be ruled out or corrected. In case of 
clinically significant toxicities, PD 0332991 administration should be interrupted 
and the dose reduced as indicated in clinical protocols. 
 
In Study A5481001 using QTcF, 46 of 73 patients had a maximum increase from 
baseline of <30 msec and no patient had a maximum on treatment value of  ≥500 
msec. Notably, one female patient who had received PD 0332991 at 75 mg QD on 
Schedule 3/1, had a maximum QTcF increase of 67 msec from baseline to Cycle 1. 
Additionally, QTcF increases ranging from 39 to 51 msec compared to baseline 
persisted throughout her ECG collection period of 5 subsequent cycles. After 7 
cycles, the dose was increased to 100 mg QD. The patient remained on treatment 
for a total of 39 cycles with no cardiac related adverse events. QT data analysis for 
study A5481002 indicated no clinically significant mean changes with ECGs. 
Using Fridericia's correction in the A5481002 study, all 17 subjects in the analysis 
had a maximum increase from baseline of <30 msec and a maximum post-baseline 
value for QTc of <500 msec. 
 
 

 
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8.2 
Anastrozole 
 
8.2.1 Anastrozole Description 
 
Anastrozole is a nonsteroidal aromatase inhibitor. 
 
Chemical Name or Amino Acid Sequence: 1,3-Benzenediacetonitrile, a, a, a', a'-
tetramethyl-5-(1H-1,2,4-triazol-1-ylmethyl) 
Other Names: Arimidex 
Classification: Aromatase inhibitor 
Molecular Formula: C17H19N5 
 
M.W.:  293.4 
Approximate Solubility:  Anastrozole has moderate aqueous solubility (0.5 mg/mL 
at 25°C); solubility is independent of pH in the physiological range. Anastrozole is 
freely soluble in methanol, acetone, ethanol, and tetrahydrofuran, and very soluble 
in acetonitrile. 
 
8.2.2 Mode of Action 
 
Anastrozole is a potent and selective non-steroidal aromatase inhibitor. It 
significantly lowers serum estradiol concentrations and has no detectable effect on 
formation of adrenal corticosteroids or aldosterone [AstraZeneca, Package Insert]. 
 
8.2.3 How Supplied 
 
Anastrozole tablets are manufactured by AstraZeneca. Anastrozole tablets for oral 
administration contain 1 mg of anastrozole. 
 
Anastrozole is commercially available and will be billed to the patient or her 
insurance. 
 
8.2.4 Dosage Form and Preparation 
 
Anastrozole is an off-white powder. Each tablet contains as inactive ingredients: 
lactose, magnesium stearate hydroxypropylmethylcellulose, polyethylene glycol, 
povidone, sodium starch glycolate, and titanium dioxide [AstraZeneca, Package 
Insert]. 
 
8.2.5 Storage 
 
Store at controlled room temperature at 20-25°C. 
 
8.2.6 Method of Administration 
 
Patients should be instructed to take anastrozole tablets by mouth with or without 
food. 

 
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8.2.7 Potential Drug Interactions 
 
Anastrozole is generally safe to administer with other medicines. However, 
concomitant use of agents and herbal products that alter ER function are 
specifically not allowed. 
 
For further information, please refer to the FDA-approved package insert for   
anastrozole. 
 
8.3 
Goserelin 
 
8.3.1 Goserelin Description 
 
Synthetic decapeptide analogue of GnRH. 
 
Chemical Name or Amino Acid Sequence: [D-Ser(But)6,Azgly10].  Its chemical 
structure 
is 
pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 
acetate. 
Other Names: Zoladex 
Classification: GnRH agonist 
Molecular Formula: [C59H84N18O14•(C2H4O2)x where x = 1 to 2.4] 
 
M.W.:  1269  
Approximate Solubility:  Goserelin is freely soluble in glacial acetic acid. It is 
soluble in water, 0.1M hydrochloric acid, 0.1M sodium hydroxide, 
dimethylformamide and dimethyl sulfoxide. Goserelin acetate is practically 
insoluble in acetone, chloroform and ether [AstraZeneca, Package Insert]. 
 
8.3.2 Mode of Action 
 
Goserelin has actions similar to those of naturally occurring GnRH (also known as 
LHRH). Normally, GnRH is released in a pulsatile manner to maintain levels of 
gonadotropins. Goserelin, in contrast, is continuously administered, which leads to 
down-regulation of the GnRH receptor on the pituitary gland and ultimately 
decreased production of FSH and LH. 
 
8.3.3 How Supplied 
 
Goserelin is supplied as a sterile, biodegradable product containing goserelin 
acetate equivalent to 3.6 mg of goserelin. Goserelin is designed for subcutaneous 
injection with continuous release over a 28-day period. Goserelin acetate is 
dispersed in a matrix of D,L-lactic and glycolic acids copolymer (13.3-14.3 
mg/dose) containing less than 2.5% acetic acid and up to 12% goserelin-related 
substances and presented as a sterile, white to cream colored 1-mm diameter 
cylinder, preloaded in a special single use syringe with a 16-gauge x 36 +/- 0.5 mm 
siliconized needle with protective needle sleeve (SafeSystem™ Syringe) in a 

 
Protocol Version: 06/29/2022 
 
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sealed, light and moisture proof, aluminum foil laminate pouch containing a 
desiccant capsule. Studies of the D,L-lactic and glycolic acids copolymer have 
indicated that it is completely biodegradable and has no demonstrable antigenic 
potential [AstraZeneca, Package Insert]. 
 
Goserelin is commercially available and will be billed to the patient or her insurance. 
 
8.3.4 Storage 
 
Store at controlled room temperature (do not exceed 25°C). 
 
8.3.5 Method of Administration 
 
Goserelin should be administered subcutaneously every 28 days into the anterior 
abdominal wall below the navel line using an aseptic technique under the 
supervision of a physician. 
 
8.3.6 Potential Drug Interactions 
 
Goserelin is generally safe to administer with other medicines.  
 
For further information, please refer to the FDA-approved package insert for 
goserelin. 
 
 
9.0 
CORRELATIVE STUDIES 
 
9.1 
Sample Collection, Processing, and Shipment 
 
9.1.1 Sample Collection/Shipment Kit for Neoadjuvant Tumor and Blood 
Collection 
 
 
The kit is a two-chamber kit in which it is possible to send both frozen and ambient 
specimens.  The kit is stocked with all the necessary items needed to draw blood 
and obtain tissue specimens.  It also contains the necessary materials used to 
process and prepare specimens for shipment in accordance to IATA regulations.  
Finally the kit contains all required documentation and labels required to return the 
kit to the Washington University Tissue Procurement Core Facility (address listed 
below).  
 
It is advised that the baseline samples are harvested during ultrasound guided clip 
placement to optimize tissue accrual. 
 
Sample collection/shipment kits are available for institutions by sending the request 
to Washington University Tissue Procurement Facility below: 
 
 

 
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Wash U Alliance/ACOSOG-CBS/TPS  
425 S. Euclid Ave, Room 5120 
St. Louis, MO  63110-1005 
Phone: 314-454-7615 
Email: tbank@wudosis.wustl.edu 
 
9.1.2 Sample Collection and Schedule in the Neoadjuvant Setting (PIK3CA 
Mutant and WT Cohorts) 
 
All sample collection is mandatory. 
Correlative 
Study 
Blood /  
Tumor  
Type of Tube 
Volume to 
Collect 
Time point 
Process at 
Site? 
Temperature Conditions for 
Storage / Shipping 
Tumor for 
PIK3CA testing 
Core 
biopsies 
2 cores in OCT 
2 cores in 10% 
formalin 
14-G core 
needle 
Pre-anastrozole 
No 
Cores in OCT should be 
immediately frozen; cores in 
formalin should be stored / 
shipped at ambient temperature 
Tumor for 
research♣ 
Core 
biopsies 
2 cores in OCT 
2 cores in 10% 
formalin 
14-G core 
needle 
Pre-PD0332991,  
C1D15, Surgery* 
No 
Cores in OCT should be 
immediately frozen; cores in 
formalin should be stored / 
shipped at ambient temperature 
Plasma for 
research**  
Whole 
blood 
EDTA (purple) 
10 mL 
Pre-anastrozole†, 
Pre-PD0332991, 
C1D15, Surgery 
Yes 
-80ºC 
Plasma for PK 
studies 
Whole 
blood 
K2EDTA (purple) 
10 mL 
Pre-PD0332991#, 
C1D15# 
Yes 
-80ºC (or -20ºC) 
Serum for 
research** 
Whole 
blood 
Clot-tube (red) 
10 mL 
Pre-anastrozole†, 
Pre-PD0332991, 
C1D15, Surgery 
Yes 
-80ºC 
Germline DNA 
for research 
Whole 
blood 
EDTA (purple) 
10 mL 
Pre-anastrozole†, 
Pre-PD0332991 
No 
Ambient 
Whole blood for 
plasma 
circulating DNA 
Whole 
blood 
Cell-free DNA 
BCT 
10 mL 
Pre-anastrozole, 
Pre-PD0332991, 
C1D15, Surgery 
No 
Room temperature, same-day 
shipment (avoid Friday blood 
draw) 
Pre-anastrozole: C0D1;  Pre-PD0332991: C1D1 
* Additional surgical tumor specimens are required; please refer to instruction ‘g’ below 
** Blood could be collected the same time as the PK samples (either prior to or after drug administration) on Pre-
PD0332991 and C1D15. 
† For patients who pre-registered to this protocol only.  
# Prior to and 90 minutes following drug administration (see Section 9.4.7). 
♣ Tumor collection prior to PD0332991 is not required for the endocrine resistant cohort if the patient had undergone 
research tumor collection as part of the tumor biopsy for the Ki67 analysis prior to registration to this trial. 
 
Instructions for sample collection and processing: 
Note that sample collection/shipment kits are available for the study. 
 
a:  For biopsies at each time point: 4 cores (14 G core needle) will be taken at each time point, 
with 2 in 10% formalin and 2 freshly frozen in OCT.  Care needs to be taken to reduce the 
ischemic time to as much as possible to less than 30 min. 
b:  A repeat biopsy may be needed in consented patients if the first biopsy did not yield enough 
tumor cells for Ki67. 
c:  Samples can be obtained at the time of surgery. Care needs to be taken to reduce the 
ischemic time to as much as possible to less than 30 minutes. 

 
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d: Plasma processing: Upon collection of the blood PK samples, keep the samples on wet 
ice at all times prior to processing to plasma.  The blood samples must be processed to 
plasma and placed in the freezer at -20°C within one hour of collection.  To process the 
blood samples to plasma, centrifuge the blood samples at approximately 4°C at 1700xg for 
approximately 10 minutes.  Using a separate pipette for each time point, transfer the plasma 
samples into pre-labeled amber polypropylene cryovials and store at approximately -20°C 
until shipment.  As much as practical, keep the blood and plasma samples away from direct 
sunlight and unfiltered lab light.  Ship the samples on dry ice to the analytical labs.  
e: Germ line DNA processing: The EDTA tube should be mixed several times and labeled 
with the patient’s study number, date of birth, and collection date and time. Whole blood 
specimens are shipped in the specimen kit and must be received by the Washington 
University Tissue Procurement Facility within 48 hours of the time of collection. Do not 
freeze whole blood.  
f:   Whole blood for plasma circulating DNA: The cell free DNA collection BCT tube 
(provided with the kits) should be mixed several times and labeled with the patient’s study 
number, date of birth, and collection date and time. Whole blood specimens are shipped in 
the specimen kit and must be received by the Washington University Tissue Procurement 
Facility within 48 hours of the time of collection. Do not freeze whole blood. 
g:  Slide submission for the surgical specimen is required for all patients for research, in 
addition to the biopsies described.  The request of these additional surgical materials is due 
to the concern that the biopsy samples from the surgical specimens may not contain 
sufficient tumor for correlative studies of residual tumors. 
h:  When the local pathological analysis of tumor samples from the definitive surgical 
procedure is complete, 10 unstained Superfrost Plus slides and the corresponding 
pathology report should be submitted to the Washington University Tissue Procurement 
Facility.  Alternatively, a tissue block containing the residual cancer may be submitted and 
stored at the Washington University Tissue Procurement Facility unless a request is 
received to return the block (at which point 10 sections and 4 1-mm TMA cores will be 
taken, and then the block will be returned).  Further fixed material from the diagnostic 
biopsy or surgical specimen may be requested by the Washington University Tissue 
Procurement Facility at a later date to complete sample pairs if insufficient tumor is present 
in the specimens that were previously provided.  Samples should be shipped as soon as 
possible after surgery.   
 
 

 
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9.1.3 Sample Collection and Schedule in the Neoadjuvant Setting (Endocrine 
Resistant Cohort) 
 
All sample collection is mandatory. 
Correlative 
Study 
Blood /  
Tumor  
Type of Tube 
Volume to 
Collect 
Time point 
Process at 
Site? 
Temperature Conditions for 
Storage / Shipping 
Tumor for 
research♣ 
Core 
biopsies 
2 cores in OCT 
2 cores in 10% 
formalin 
14-G core 
needle 
Pre-PD0332991,  
C1D15, Surgery* 
No 
Cores in OCT should be 
immediately frozen; cores in 
formalin should be stored / 
shipped at ambient temperature 
Plasma for 
research  
Whole 
blood 
EDTA (purple) 
10 mL 
Pre-PD0332991, 
C1D15, Surgery 
Yes 
-80ºC 
Serum for 
research 
Whole 
blood 
Clot-tube (red) 
10 mL 
Pre-PD0332991, 
C1D15, Surgery 
Yes 
-80ºC 
Germline DNA 
for research 
Whole 
blood 
EDTA (purple) 
10 mL 
Pre-PD0332991 
No 
Ambient, same-day shipment 
(avoid Friday blood draw) 
Whole blood for 
plasma 
circulating DNA 
Whole 
blood 
Cell-free DNA 
BCT 
10 mL x2 
Pre-PD0332991, 
C1D15, Surgery 
No 
Ambient, same-day shipment 
(avoid Friday blood draw) 
Pre-PD0332991: C1D1 
♣ Tumor collection prior to PD0332991 is not required for the endocrine resistant cohort if the patient had undergone 
research tumor collection as part of the tumor biopsy for the Ki67 analysis prior to registration to this trial. Consent 
to research of archival tumor specimens collected prior to and during this trial is required for all patients.    
* Additional surgical tumor specimens are required; please refer to instruction ‘g’ below 
 
Instructions for sample collection and processing: 
Note that sample collection/shipment kits are available for the study. 
 
a:  For biopsies at each time point: 4 cores (14 G core needle) will be taken at each time point, 
with 2 in 10% formalin and 2 freshly frozen in OCT.  Care needs to be taken to reduce the 
ischemic time to as much as possible to less than 30 min. 
b:  A repeat biopsy may be needed in consented patients if the first biopsy did not yield enough 
tumor cells for Ki67. 
c:  Samples can be obtained at the time of surgery. Care needs to be taken to reduce the 
ischemic time to as much as possible to less than 30 minutes. 
d: Serum and plasma processing: Keep the samples on wet ice at all times prior to 
processing to plasma.  The blood samples must be processed to plasma and placed in the 
freezer at -20°C within one hour of collection.  To process the blood samples to plasma, 
centrifuge the blood samples at approximately 4°C at 1700xg for approximately 10 
minutes.  Using a separate pipette for each time point, transfer the plasma samples into pre-
labeled amber polypropylene cryovials and store at approximately -20°C until shipment.  
As much as practical, keep the blood and plasma samples away from direct sunlight and 
unfiltered lab light.  Ship the samples on dry ice to the analytical labs.  
e: Germ line DNA processing: The EDTA tube should be mixed several times and labeled 
with the patient’s study number, date of birth, and collection date and time. Whole blood 
specimens are shipped in the specimen kit and must be received by the Washington 
University Tissue Procurement Facility within 48 hours of the time of collection. Do not 
freeze whole blood.  
f:   Whole blood for plasma circulating DNA: The cell free DNA collection BCT tube 
(provided with the kits) should be mixed several times and labeled with the patient’s study 
number, date of birth, and collection date and time. Whole blood specimens are shipped in 

 
Protocol Version: 06/29/2022 
 
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the specimen kit and must be received by the Washington University Tissue Procurement 
Facility within 48 hours of the time of collection. Do not freeze whole blood. 
g:  Slide submission for the surgical specimen is required for all patients for research, in 
addition to the biopsies described.  The request of these additional surgical materials is due 
to the concern that the biopsy samples from the surgical specimens may not contain 
sufficient tumor for correlative studies of residual tumors. 
h:  When the local pathological analysis of tumor samples from the definitive surgical 
procedure is complete, 10 unstained Superfrost Plus slides and the corresponding 
pathology report should be submitted to the Washington University Tissue Procurement 
Facility.  Alternatively, a tissue block containing the residual cancer may be submitted and 
stored at the Washington University Tissue Procurement Facility unless a request is 
received to return the block (at which point 10 sections and 4 1-mm TMA cores will be 
taken, and then the block will be returned).  Further fixed material from the diagnostic 
biopsy or surgical specimen may be requested by the Washington University Tissue 
Procurement Facility at a later date to complete sample pairs if insufficient tumor is present 
in the specimens that were previously provided.  Samples should be shipped as soon as 
possible after surgery.   
 
9.1.4 Sample Collection and Schedule during Follow-up  
 
Patients who received adjuvant PD 0332991 
Research blood includes the collection of plasma, serum, and whole blood for 
cfDNA.  Research blood will be collected on Day 1 +/- 3 days of adjuvant Cycles 
1 and 12 and at the end of treatment (either end of Cycle 23 or end of the last cycle 
received if the patient ends treatment early).  Additional research draws will take 
place annually for 3 years following the last dose of PD 0332991 (+/- 8 weeks). 
 
All other patients 
Research blood includes the collection of plasma, serum, and whole blood for 
cfDNA.  Research blood will be collected 1 year post surgery, then annually for a 
total of 5 years following surgery or until recurrence (whichever comes first). 
 
Blood for all patients will be collected as follows: 
• 10 mL in an EDTA (purple top) tube for plasma to be stored at -80°C; to 
process the blood samples to plasma, centrifuge the blood samples at 
approximately 4°C at 1700xg for approximately 10 minutes. 
• 10 mL in a clot (red top) tube for serum to be stored at -80°C 
• 10 mL each in 2 cell-free DNA BCT tubes to be stored at room temperature 
and shipped same day to the Washington University Tissue Procurement 
Core Facility; the cell free DNA collection BCT tube (provided) should be 
mixed several times and labeled with the patient’s study number, date of 
birth, and collection date and time. Whole blood specimens are shipped and 
must be received by the Washington University Tissue Procurement 
Facility within 48 hours of the time of collection. 
 
9.1.5 Shipment of Samples 
 
All samples should be labeled with institutional surgical pathology number (tumor 

 
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samples), study number, patient ID number patient initials, sample collection date 
and time and be accompanied by the completed specimen submission forms 
(Appendix F/K/L (as appropriate)).  
 
All samples should be shipped to the Wash U Tissue Procurement Core Facility.  
 
Specimens may be sent to the Wash U Tissue Procurement Facility on Monday 
through Thursday for next day delivery. The Bank cannot receive specimens on 
Saturdays, Sundays, or holidays. Do not send specimens on Friday, Saturday, 
or the day before a holiday. 
 
The institution is expected to pay the cost of mailing specimens and will be 
reimbursed through capitation fees set for each individual study. 
 
Arrange for Federal Express pick-up through your usual institutional procedure. 
Ship specimens to the address below: 
 
Wash U Alliance/ACOSOG-CBS/TPS  
425 S. Euclid Ave, Room 5120 
St. Louis, MO 63110-1005 
Phone: (314) 454-7615 
E-mail:  tbank@wudosis.wustl.edu 
 
On the day that specimens are sent to the specimen bank, please contact the  
bank by phone, fax, or e-mail to notify what is being sent and when the 
shipment is expected to arrive.  
 
9.2 
Real Time Integral Biomarker Studies 
 
Note that the PIK3CA (Gene ID: 5290) hot spot sequencing assay that covers exons 1, 4, 
7, 9 and 20, is done while the patient was pre-registered to the NCI 9170 trial or through 
this trial.  Briefly, sequencing of these 5 exons and exon-intron splice junctions should be 
performed using the established protocol at the CLIA certified GPS@WU, a CAP-
accredited (#27556-03) and CLIA-licensed (#26D0698685) laboratory environment under 
the unified supervision by faculty in the Departments of Genetics and Pathology and 
Immunology at Washington University.   
 
Tumor biopsy will be sectioned and H&E stained for evaluation of tumor cellularity by a 
pathologist and processed to tumor DNA and RAN extraction at Washiongton University 
once it arrives at the Alliance Central Specimen Bank.  The first of the two formalin fixed 
and paraffin embedded (FFPE) tumor cores will be sectioned and stained by H&E to assess 
for tumor cellularity.  If the total normal of tumor nuclei is greater than 75% in the biopsy, 
serial sections will be immediately prepared for DNA and RNA extraction.  If tumor 
cellularity is less than 75%, the second frozen core will be evaluated.  In cases where both 
FFPE tumor biopsies contain less than 75% neoplastic cellularity, macro- or micro-
dissection of tumor rich area will be performed if there is sufficient tumor.  If neither 

 
Protocol Version: 06/29/2022 
 
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baseline sampling core contains sufficient tumor cellularity, the frozen tissue will be 
examined.  If needed a second biopsy may be required if the patient consents. 
 
High quality DNA sequence reads that are concordant on both strands will be analyzed 
from each amplicon.  Variant calls (as compared to reference sequence) will be reported as 
a qualitative / non-ordered categorical result.  The position (nucleotide, exon, amino acid, 
and protein domain), sequence variant, and predicted change (if any) in corresponding 
amino acid sequence will be reported.  If a non-synonymous variant identified has never 
been reported to be a somatic mutation, germ-line DNA will be sequenced from the same 
patient to determine whether this is a somatic mutation or a polymorphism.  The result will 
be further classified as:  1) Previously described somatic mutation; 2) Novel somatic 
mutation, not a germ-line polymorphism. Results will be emailed to the study coordinator 
at the participating site.  Contact cbumb@ wustl.edu for questions related to the sequencing 
result. 
 
Patients with or without a somatic mutation in PIK3CA hotspot region are eligible for the 
trial. 
 
9.3 
Tumor Ki67 Assessment on Cycle 1 Day 1 (Pre-PD 0332991) and Cycle 1 Day 15 
 
9.3.1 Rationale 
 
If Ki67 is over 10% on Cycle 1 Day 15, patient will be taken off the study drug 
therapy and recommended to either immediate surgery or neoadjuvant 
chemotherapy.  If Ki67 is 10% or less, the patient will continue therapy for a total 
of 5 cycles of combination PD 0332991 and endocrine therapy. 
 
9.3.2 Tissue Processing and Ki67 Analysis 
 
Upon receipt of the specimen, the 2 fixed biopsy specimens (A & B) at each time 
point will be further processed for tumor Ki67 analysis at the CLIA certified 
Anatomic and Molecular Pathology Core Labs at Barnes Jewish Hospital at St. 
Louis (CLIA number 26D2013203). After embedding in paraffin, one section from 
the tumor block will be stained with H&E to assess biopsy adequacy. Another 
section from the block will be incubated with antibody against Ki67 (clone 30-9) 
and then assayed using the Ventana Benchmark platform. The block and any 
remaining sections will be returned to the Tissue Procurement Core Facility at the 
completion of testing. Both cores will be reviewed and will be taken into account 
for the scoring.  Ki67 scoring is reported as a quantitative/continuously distributed 
value.  
 
9.3.3 Ki67 Scoring and Reporting 
 
 
Ki67 scoring will be performed using a standard SOP established at the CLIA 
laboratory, with the result expressed as immunoreactive over total numbers of cells. 
The Ki67 data for C1D15 biopsy is available real time to the treating physician and 

 
Protocol Version: 06/29/2022 
 
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the patients. Patients will be continued on protocol therapy while awaiting analysis 
results. Results of tumor Ki67 will be reported within 10 working days upon receipt 
of the samples. Wash U Path Coordinator will email and fax the results to both the 
CRA listed on Specimen Submission CRF that is sent with the tissue samples and 
Wash U CRA. Contact cbumb@wustl.edu for questions related to the Ki67 result. 
 
Note: If the biopsy yielded no tumor cells, the patient may continue on study drug 
therapy or proceed with a second biopsy for Ki67 determination.  
 
9.4 
Laboratory Correlative Studies 
 
Laboratory correlative studies are performed on leftover tumor specimens following the 
integral biomarker analysis (Section 9.2) from baseline, on Cycle 1 Day 1 (pre-PD 
0332991), Cycle 1 Day 15, and on surgical specimens.   
 
9.4.1 To assess tumor cell apoptosis on serial tumor specimens 
 
Methods:  FFPE tumor blocks obtained from biopsies at baseline and post therapy 
will be sectioned at 5 micron sections for immunohistochemistry analysis of 
cleaved caspase 3 or TUNEL staining with established method at the Study PI and 
Co-PI’s laboratory.  Apoptotic index will be calculated as the percentage of tumor 
cells staining positive for apoptotic markers (either cleaved caspase 3 or TUNEL 
staining). 
 
9.4.2 To assess Ki67 level on serial tumor biopsies 
 
Methods:  Ki67 at baseline, Cycle 1 Day 1 (pre-PD 0332991), Cycle 1 Day 15, and 
surgery will be performed at the CLIA certified Anatomic and Molecular Pathology 
(AMP) facility at Washington University on FFPE sections of serially collected 
tumor biopsies.  Ki67 index will be calculated as the percentage of tumor cells 
stained positive at each time point.   
 
9.4.3 To assess effect of PD 0332991 on markers of tumor cell senescence  
 
Methods: Candidate proteins important for senescence including p16, cyclin D1, 
activated mTOR (such as pS6), IL6, senescence associated beta-galactosidase, 
FOXM1 phosphorylation, will be examined by IHC.   
 
9.4.4 To examine the pharmacodynamic effect of PD 0332991 in combination 
with endocrine therapy on Cdk4/6 Rb signaling activities 
 
Methods:  The pharmacodynamic effect of PD 0332991 in combination with 
anastrozole on Cdk4/6-Rb signaling will be assessed by phosphoroproteomics and 
immunohistochemistry analysis of pRB and other candidate molecules on serial 
tumor biopsies. 
   

 
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9.4.5 Assessment of serum estradiol level before and following PD 0332991   
 
Sensitive estradiol level in premenopausal women will be tested in the clinical 
laboratory at the end of 4 weeks of anastrozole (or in combination with goserelin) 
(at baseline), Cycle 1 Day 15, and surgery. 
 
9.4.6 Explore potential markers of sensitivity and resistance 
 
In preclinical studies, RB proficient cell lines with low p16 expression were found 
to be most sensitive to PD 0332991 in ovarian cancer. In addition, copy number 
variations of CDKN2A, RB, CCNE1, CCND1 were associated with response[102].   
Therefore, tumor specimens collected on Cycle 1 day 1 will be analyzed for the 
protein expression of these genes by immunohistochemistry.  Gene copy number 
changes will be assessed by aCGH method.  These studies will be performed in the 
study PI and Co-PI’s laboratory.  Other studies include hypothesis generating 
exploratory DNA and RNA studies as well as proteomic analysis.  
 
9.4.7 Pharmacokinetics 
 
Ten mL of blood will be drawn into each of 2 K2EDTA tubes at the following time 
points to evaluate the concentrations of PD 0332991 and the effect of PD 0332991 
on the concentrations of anastrozole: 
• Prior to initiation of PD 0332991 but after patients have taken anastrozole 
alone or in combination with goserelin for at least 2 weeks (this could be 
done on Cycle 1 Day 1): 
o pre-dose of anastrozole 
o 90 minutes following this same dose of anastrozole 
• Cycle 1 Day 15 (may take place up to Day 21 if samples are missed) 
o prior to both anastrozole and PD 0332991 
o 90 minutes following administration of both drugs  
 
Process blood to plasma within 30-45 minutes by centrifuging in a refrigerated 
centrifuge (1700 x g for about 10 minutes at 4ºC) within 1 hour of collection.  The 
collection tube containing PD 0332991 should be covered with aluminum foil to 
protect from light.  Transfer plasma into pre-labeled amber polypropylene storage 
cryovials and store at -20ºC.  Ship the samples on dry ice to the following address: 
 
Wash U Alliance/ACOSOG-CBS/TPS 
425 S. Euclid Ave., Room 5120 
St. Louis, MO 63110-1005 
Phone: (314) 454-7615 // E-mail:  tbank@wudosis.wustl.edu 
 
At the completion of the 1st stage and the 2nd stage of the study, batched samples 
will be sent by Washington University CRA to the following addresses for analysis: 
 
For PD 0332991 analysis: 

 
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Maria Edwards c/o PPD 
2244 Dabney Road 
Richmond, VA 23230 
Phone: (804) 977-8430 // E-mail: maria.edwards@ppdi.com 
 
For anastrozole analysis: 
Robert Twieg c/o Covance Laboratories, Inc. 
8211 SciCor Drive, Suite B 
Indianapolis, IN 46214 
Phone: (317) 715-3964 // E-mail: Robert.Twieg@covance.com 
 
 

 
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10.0 
STUDY CALENDARS 
 
10.1 
Pre-Registration and Cycle 0 Calendar 
 
This applies only to the PIK3CA WT and Mutant Cohorts (closed as of Amendment #7). 
 
 
≤ 14 days 
prior to 
pre-registration 
Pre-Registration 
≤ 14 days 
Pre- 
Anastrozole 
Cycle 0 
Day 1 A 
(≤ 7 days post 
pre-registration) 
TREATMENT 
 
Anastrozole  
 
 
Days 1-28B 
GoserelinC  
 
 
X 
CLINICAL ASSESSMENT 
 
H&P, PS 
X 
X 
 
Clinical measurements of breast lesionsD 
X 
X 
 
Adverse event assessment 
 
 
 
CBC and differential 
X 
X 
 
Serum chemistryE 
X 
X 
 
Serum pregnancy testC 
X 
 
 
Serum estradiol 
X 
 
 
EKG (12 lead)  
X 
 
 
RADIOLOGY 
 
Mammogram and ultrasound of breast and 
axillary massesF 
XF 
 
 
SAMPLE COLLECTION 
 
Tumor biopsies R 
 
XG 
 
Research blood samples R 
 
XH 
 
A. Cycle 0 Day 1 is to start < 7 days post pre-registration.  Tumor biopsy and blood collections must be done 
prior to the start of anastrozole.  
B.   All effort is to be made to keep Cycle 0 length at 28 days, but up to 42 days of Cycle 0 therapy is allowed 
prior to the start of Cycle 1 Day 1 due to delays in PIK3CA sequencing result or scheduling difficulties. In 
this situation, goserelin should be administered every 28 days throughout the study regardless of cycle days. 
C.  In premenopausal women only   
D. Using a standard cm calibrated caliper, tape or ruler, the longest axis and the perpendicular axis of the tumor 
are to be measured and recorded in metric notation. In patients with synchronous lesions, it is recommended 
that both lesions be measured and biopsied the same if they are both >1cm with at least one dimension.  
E.  Serum chemistry is albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, 
creatinine, glucose, potassium, total protein, SGOT/AST, SGPT/ALT, sodium 
F. Mammogram and ultrasound of the diseased breast must be completed within 42 days of pre-registration. 
Imaging studies must include bidimensional breast tumor measurements, although retrospective 
measurement of the imaging study is allowed after patient pre-registered on the study.  
G. Pre-anastrozole core biopsies for correlative studies are required prior to the initiation of anastrozole 
therapy.  Samples obtained at the time of diagnosis before registration to this trial (for other studies) may 
be submitted if they were collected according to the tissue acquisition instructions in Section 9. It is advised 
that the baseline samples are harvested during ultrasound guided clip placement to optimize tissue accrual. 
H. For patients pre-registered to this trial only.   
R. Research funded 

 
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10.2 
Neoadjuvant Study Treatment Calendar (ALL Cohorts) 
A.  C1D1 office visit,  labs, EKG,as well as pre-PD 0332991tumor biopsies may be done within 14 days prior to C1D1. 
B.   Please refer to section 5.1. Surgery occurs between Cycle 5 day 11 and day 13 in patients who received Cycle 5 treatment.  In patients who did not 
receive Cycle 5 treatment, surgery occurs 3 to 5 weeks following the last dose of PD 0332991. 
C. 30-60 days post-surgery, then yearly to document recurrence and survival for 5 years. 
E.  Anastrozole (and goserelin if premenopausal) should have been started at least 28 days prior to C1D1 **(only applies to PIK3CA Mutant and Wild 
Type cohorts). 
F.  Patients will keep a pill diary (Appendix D). 
G.  If C1D15 Ki67 is > 10%, patient will discontinue PD 0332991 and be observed for AEs 30-60 days following the last dose, then followed per 
protocol. Further treatment is at the discretion of the treating physician. 
H. Prior to start of PD 0332991 (within 12 weeks prior to registration) and at the end of cycle 4 before surgery. 
J.  2D measurements of the breast lesion should be performed. Using a standard cm calibrated caliper, tape or ruler, the longest axis and the perpendicular 
axis of the tumor are to be measured and recorded in metric notation. In patients with synchronous lesions, it is recommended that both lesions be 
measured and biopsied the same if they are both >1cm with at least one dimension; otherwise, the dominant mass is followed clinically and by Ki67 
K.  Serum chemistry is albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, potassium, total protein, 
SGOT/AST, SGPT/ALT, sodium 
L.  In premenopausal women only 
N.  EKG will be performed at baseline, pre-surgery, and as needed.  Surgery may be delayed if QTc prolongation has occurred. 
O.  Mammogram and ultrasound of the diseased breast must be completed within 12 weeks prior to registration. Imaging studies must include 
bidimensional breast tumor measurements, but retrospective measurement is allowed in the study. 
P. 
C1D15 biopsy and blood draw may be done up to C1D20 for scheduling convenience, although C1D15 is strongly preferred.   
Q.   Optional biopsy on C2D15, C3D15, or C4D15 in patients with indeterminate Ki67 on C1D15 biopsy or who missed the C1D15 biopsy.   
R.  Research funded.  Research blood samples are mandatory except where indicated. 
S.  Optional 
T. 
If counts recover within 3 weeks of the end of C4, re-start PD 0332991 for 10 to 12 doses (C5) (last dose to be the day before surgery). 
U. PKs to be drawn on a day prior to initiation of PD 0332991 (may be C1D1) pre-anastrozole and again 90 minutes post-anastrozole. Other research 
blood include serum, plasma, whole blood for germline DNA and whole blood for circulating tumor DNA is also collected on C1D1.  Details are 
described in Section 9.1.2.  PK samples are not required in the endocrine resistant cohort. 
V. PKs to be drawn pre-dose of both drugs and again 90 minutes post-dose of both drugs. PK samples are not required in the endocrine resistant cohort. 
W. Not required in endocrine resistant cohort if a research biopsy was performed prior to registration or an archival tumor block is available 
Y.    Only required in patients who are in Cycle 5. 
 
 
≤ 14 days 
prior to 
registration 
C1D1A 
C1D15 
±1 day 
C2-5   
D1±3  
days 
3 (+/-1) days 
prior to 
surgery 
Off Study -  
Progression 
SurgeryB 
Post-
surgeryC 
TREATMENT 
 
AnastrozoleE 
 
Daily until the day of surgery (if no progression) 
 
GoserelinE 
 
Every 28 days (if no progression) 
 
PD 0332991F 
 
3 weeks on and 1 week off G,T 
D/C 
 
 
CLINICAL ASSESSMENT 
 
H&P, PS  
X 
X 
 
X 
 
X 
 
X 
Clinical meas. of breast lesionsJ 
X 
X 
 
X 
 
X 
 
Exam 
Adverse event assessment 
X 
X 
 
X 
 
X 
 
X 
CBC + differential 
X 
 
 
X 
XY 
 
 
X 
Serum chemistryK 
X 
 
 
X 
 
 
 
X 
Serum pregnancy testL 
 
X 
 
 
X 
X 
 
 
Drug compliance assessment 
 
 
 
X 
 
 
 
 
EKG (lead II) N 
X 
 
 
 
 
 
X 
 
RADIOLOGY 
 
Mammogram and US of breast 
and axillary massesO 
XH 
 
 
XH 
 
X 
 
 
SAMPLE COLLECTION 
 
Tumor biopsies R 
 
XW 
XP 
XQ 
 
XS 
X  
 
Research blood R 
 
XU 
XP,V 
 
 
XS 
X 
 

 
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10.3 
Post Surgery Treatment Calendar 
 
Patients who had a Ki67<10% on the neoadjuvant C1D15 tumor biopsy in the endocrine 
resistant cohort (before or after activation of Amendment #7) and the 26 patients who 
enrolled initially to the main trial (PIK3CA WT or mutant cohort) who required PD 
0332991 to achieve complete cell cycle arrest have the option of restarting PD 0332991 in 
combination with anastrozole for adjuvant treatment (23 cycles after surgery).  These 
patients will need to be consented and screened for the adjuvant portion of the study, and 
may only begin treatment with adjuvant PD 0332991 if they meet the eligibility criteria in 
Section 3.4 and if they have provided informed consent. 
 
PD 0332991 should be re-started at least 4 weeks after the completion of chemotherapy 
and radiation therapy if these treatments were planned.  Anastrozole may be started earlier 
while the patient receives radiation therapy if desired.  After completion of PD 0332991, 
patients should continue standard of care hormonal therapy (for example anastrozole) to 
complete at least 5 years of therapy.  Further therapy after that is at the discretion of treating 
physician. 
 
 
Screening 
Adjuvant  
Cycles 1-2 
Adjuvant  
Cycles 3 to 23 
End of C23 
or early 
termination  
Follow up 
Yearly post the last 
dose of palbociclib to 
complete 5 years of 
follow up post surgery 
or recurrenceF 
Day of cycle 
-30 to 0 
D1  
+/- 3 days 
D14 
+/- 3 days 
D1 +/- 7 days 
(C3, 6, 9, 12, 
15, 18, 21) 
4 weeks 
+/- 8 weeks 
TREATMENT 
 
Endocrine Therapy A 
 
Daily  
PD 0332991 
 
Days 1-21 each cycle  
 
 
CLINICAL ASSESSMENT 
 
Informed consent 
X 
 
 
 
 
 
Concomitant medication  
X 
X 
 
X 
X 
 
H&P, PS  
X 
X 
 
X 
X 
 
Adverse event assessment 
X 
X 
X 
X 
X 
X 
ECG 
X 
 
Disease monitoringB  
X 
Per standard of care 
CBC+ differential 
X 
X 
X 
X 
X 
X 
Serum chemistry C 
X 
X 
 
X 
X 
X 
Serum/urine pregnancy testG 
X 
X 
 
X 
X 
 
Drug compliance 
assessmentD 
 
X 
 
X 
X 
 
Research bloodE 
 
X 
 
X (C12 only) 
X 
X 
A. Premenopausal women requires goserelin  
B. 
Disease monitoring is per standard of care.  
C. Serum chemistry is albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, potassium, total 
protein, SGOT/AST, SGPT/ALT, sodium.   
D. Patients will keep a pill diary (Appendix D). 
E. 
Research blood includes the collection of plasma, serum and whole blood for cfDNA.   
F. 
For patients who did not receive adjuvant PD 0332991 the follow up is yearly for 5 years post surgery  or until recurrence whichever comes 
first.   
G.  In premenopausal women only. 

 
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11.0 
DATA SUBMISSION SCHEDULE 
 
Case report forms with appropriate source documentation will be completed according to the 
schedule listed in this section.  Electronic data management systems will be used in this trial in 
collaboration with the Center for Biomedical Informatics core at Washington University.   
 
ClinPortal is a web-based clinical studies data management system that will be used for capture of 
clinical data from this trial.  The case report forms developed for this trial will be transformed to 
electronic format. An electronic study calendar will drive the study’s data collection workflow.  An 
Oracle database securely stores PHI in compliance with HIPAA and IRB regulations. 
 
Identical study databases will be created in ClinPortal for each participating center; each center 
has access only to data from its own participants.  Washington University, as the data coordinating 
center, has access to data from all sites. 
 
Training in entering data in ClinPortal is required before a participating center will be given access 
to its site’s database.  The Center for Biomedical Informatics at Washington University offers 
monthly web-based training for external users; the schedule may be found at the following URL: 
http://cbmi.wustl.edu/?q=clinportal-training-details.  Users must RSVP at least 3 days prior to the 
training sessions. 
 
In addition, a participating center must have IRB approval of this protocol prior to initiation of 
ClinPortal data entry training, which must be completed prior to site study activation. 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to (pre-) registration 
Pre-registration Form 
Registration Form 
Eligibility Form 
Pre-Study Form 
Prior to starting treatment 
Treatment Form 
Labs Form 
Every cycle 
Adjuvant Form 
Every cycle of adjuvant therapy 
Toxicity Form 
Continuous 
Treatment Summary Form 
Completion of treatment 
Disease-Related Assessment Form 
Day 1 of each cycle and end of Cycle 4 
MedWatch Form 
See Section 7.0 for reporting requirements 
Surgery Form 
1 month post surgery 
Correlative Studies Form 
Refer to Section 9.0 
Follow-Up Form 
Annually for 5 years after surgery 
 
Any queries generated by Washington University must be responded to within 28 days of receipt 
by the participating site.  The Washington University research team will conduct a regular review 
of data status at all secondary sites, with appropriate corrective action to be requested as needed. 

 
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12.0 
MEASUREMENT OF EFFECT 
 
12.1 
Neoadjuvant Treatment 
 
  
Complete cell cycle arrest:  This is defined as Ki67 ≤ 2.7%, which will be assessed at 
C1D1, C1D15, and surgery.  In patients without C1D15 Ki67, Ki67 on D15 of subsequent 
cycles could be used instead. 
 
Clinical Evaluation: Prior to Cycle 0 (or performed during pre-registration for study NCI 
9170),  and at the end of each neoadjuvant treatment cycle cycles (that is, at the end of 
Cycles 1-4) the longest axis and the perpendicular axis of the measurable lesion should be 
measured and recorded in metric notation by tape, ruler or caliper technique on the case 
report forms.    
 
Radiologic evaluation of tumor size:  Mammogram and ultrasound imaging will be 
performed prior to Cycle 0 (or performed during pre-registration for study NCI 9170) and 
at the end of Cycle 4 combination therapy for bidimensional measurement of the tumor.  
 
WHO criteria will be used to assess clinical and radiologic responses. 
 
Complete Response (CR) is defined as the disappearance of all known disease based on 
a comparison between the pre-treatment measurements and the measurements taken at the 
completion of neo-adjuvant therapy (that is, at the end of cycle 4 neo-adjuvant combination 
therapy).  In addition there is no appearance of new lesions.  
 
Partial Response (PR) is defined as a 50% or greater decrease in the product of the bi-
dimensional measurements of the lesion (total tumor size) between the pre-treatment 
measurements and the measurements taken at the completion of neo-adjuvant therapy (that 
is, at the end of cycle 4 neo-adjuvant combination therapy).  In addition there can be no 
appearance of new lesions or progression of any lesion. 
 
No Change (NC): a 50% decrease in total tumor size cannot be established nor has a 25% 
increase in the size of the lesion been demonstrated. 
 
Progressive Disease (PD): A 25% or greater increase in the total tumor size of the lesion 
from its pretreatment measurements or the appearance of new lesions.  
 
12.2 
Treatment Resistance 
 
A patient is said to have resistance disease if her Ki67 is > 10 % on biopsy taken on Cycle 
1 Day 15 or if progressive disease is documented any time during neoadjuvant endocrine 
therapy. 
 
 

 
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12.3 
Surgery 
 
A pathologic complete response (pCR) is defined as no histology evidence of invasive 
tumor cells in the surgical breast specimen and sentinel or axillary lymph nodes.  
 
All eligible women who have been begun treatment with combination therapy and have 
tumor Ki67 determined on C1D15 (or a subsequent cycle) are included in the analysis of 
complete cell cycle arrest.  Women with tumor Ki67>2.7% on therapy are considered to 
have a non-complete cell cycle arrest.   
 
12.4 
Post-surgery 
 
Local recurrence is defined as histologic evidence of ductal carcinoma in situ or invasive 
breast cancer in the ipsilateral breast or chest wall.  
 
Regional recurrence is defined as the cytologic or histologic evidence of disease in the 
ipsilateral internal mammary, ipsilateral supraclavicular, ipsilateral infraclavicular and/or 
ipsilateral axillary nodes or soft tissue of the ipsilateral axilla. 
 
Distant recurrence is defined as the cytologic, histologic, and/or radiographic evidence 
of disease in the skin, subcutaneous tissue, lymph nodes (other than local or regional 
metastasis), lung, bone narrow, central nervous system or histologic and/or radiographic 
evidence of skeletal or liver metastasis.  
 
Second primary breast cancer is defined histologic evidence of ductal carcinoma in situ 
or invasive breast cancer in the contralateral breast or chest wall.  
 
Second primary cancer (non-breast) is defined as any non-breast second primary cancer 
other than squamous or basal cell carcinoma of the skin, melanoma in situ, or carcinoma 
in situ of the cervix is to be reported and should be confirmed histologically whenever 
possible. 
 
 
13.0 
DATA AND SAFETY MONITORING 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Data and Safety Monitoring Committee (DSMC) will meet to review toxicity data at least every 6 
months following the activation of the first secondary site.  The report will be prepared by the 
statistician with assistance from the study team and will be submitted to the Quality Assurance and 
Safety Monitoring Committee (QASMC).  This report will include: 
 
• HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
• Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 

 
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• History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
• Study-wide target accrual and study-wide actual accrual including numbers from 
participating sites 
• Protocol activation date at each participating site 
• Average rate of accrual observed in year 1, year 2, and subsequent years at each 
participating site 
• Expected accrual end date and accrual by site.  
• Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
• Measures of efficacy 
• Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
• Summary of toxicities at all participating sites 
• Abstract submissions/publications 
• Summary of any recent literature that may affect the safety or ethics of the study 
 
Until such a time as the first secondary site activates this protocol, a semi-annual DSM report to 
be prepared by the study team will be submitted to the QASM Committee beginning 6 months 
after study activation at Washington University. 
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 
A DSMC will consist of no fewer than 3 members including 2 clinical investigators and a 
biostatistician.  Like investigators, DSMC members are subject to the Washington University 
School of Medicine policies regarding standards of conduct. Individuals invited to serve on the 
DSMC will disclose any potential conflicts of interest to the trial principal investigator and/or 
appropriate university officials, in accordance with institution policies. Potential conflicts that 
develop during a trial or a member’s tenure on a DSMC must also be disclosed.  
 
Refer to the Washington University Quality Assurance and Data Safety Monitoring Committee 
Policies and Procedures for full details on the responsibilities of the DSMC at 
http://www.siteman.wustl.edu/uploadedFiles/Research_Programs/Clinical_Research_Resources/
Protocol_Review_and_Monitoring_Committee/QASMCQualityAssurcance.pdf 
 
 
14.0 
AUDITING 
 
Since Washington University is the coordinating center, each site will be audited annually by 
Siteman Cancer Center personnel (QASMC) unless the outside institution has an auditing 
mechanism in place and can provide a report. The outside sites will be asked to send copies of all 
audit materials, including source documentation.  The audit notification will be sent to the 

 
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Washington University Research Patient Coordinator, who will obtain the audit materials from the 
participating institution. 
 
Notification of an upcoming audit will be sent to the research team one month ahead of the audit. 
Once accrual numbers are confirmed, and approximately 30 days prior to the audit, a list of the 
cases selected for review (up to 10 for each site) will be sent to the research team. However, if 
during the audit the need arises to review cases not initially selected, the research team will be 
asked to provide the additional charts within two working days. 
 
Additional details regarding the auditing policies and procedures can be found at 
https://siteman.wustl.edu/wp-content/uploads/2015/10/QASMC-Policies-and-Procedures-
03.31.2015.pdf   
 
 
15.0 
STATISTICAL CONSIDERATIONS 
 
15.1 
Purpose 
 
This is an open label phase II trial consisting of three independent cohorts that are run 
separately—the PIK3CA wild type cohort, the PIK3CA mutant cohort, and the endocrine 
resistant cohort—in women with clinical stage 2 or 3 estrogen receptor positive and HER2 
negative invasive breast cancer to assess the anti-tumor activity (in terms of rate of 
complete cell cycle arrest) and safety profile of neoadjuvant PD 0332991 in combination 
with anastrozole for postmenopausal women or with anastrozole and goserelin for 
premenopausal women.  
 
15.2 
Primary Endpoint 
 
The primary endpoint of this trial is complete cell cycle arrest. All eligible women who 
begin combination treatment and have an evaluable tumor Ki67 on Cycle 1 Day 15 will be 
included in the analyses of the primary study endpoint.    
 
A patient with tumor Ki67 value > 2.7% on Cycle 1 Day 15 of combination treatment is 
considered to not have had a complete cell cycle arrest.   
 
15.3 
Trial Design 
 
The expectation for complete cell cycle arrest, defined as < 2.7%, at 2-4 weeks post 
neoadjuvant aromatase inhibitor alone, is 44% based on data from Z1031 Cohort B.  Since 
PIK3CA mutation status does not affect neoadjuvant Ki67 response, the expected rate of 
complete cell cycle arrest for the PIK3CA mutant and wild type cohorts for single agent 
anastrozole is expected to be 44%.  
 
The study is designed to ensure the sample size for the PIK3CA wild type cohort for the 
primary endpoint analysis. For the PIK3CA wild type cohort, thirty-three eligible patients 
will be enrolled. The sample size was chosen using Fleming's single-stage phase II  design 

 
Protocol Version: 06/29/2022 
 
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to test the hypothesis that adding PD 0332991 would result in at least 50% improvement 
(44% versus 66%) in the complete cell cycle arrest rate, with 80% power and at 1-sided 
0.05 significance level. If at least 20 patients in the cohort meet the criteria for complete 
cell cycle arrest, this regimen would be considered for future randomized trials. 
 
Patients pre-registered to this trial and found to have PIK3CA mutation in the tumor are 
eligible to receive therapy in the PIK3CA mutant cohort.  However, the enrollment to both 
cohorts will stop when 33 patients with PIK3CA wild type tumors have been enrolled and 
analyzed for the primary endpoint.  
 
For the PIK3CA mutant cohort, depending on the frequency of mutation, 14 to 17 patients 
will be enrolled. The sample size for mutant cohort is determined primarily based on 
clinical feasibility rather than statistical power. However, according to the general 
guidelines regarding the sample size for translational studies[103], a sample size of 10-20 
patients would provide reasonable precision for the estimation of pilot information 
regarding efficacy. If 10 complete cell-cycle arrest are observed out of 15 patients, for 
example, we will have 80% confidence that the “true” rate will fall between is ranged 47% 
and 83%. 
 
For the endocrine resistant cohort, a Simon optimal two stage phase II clinical trial design 
will be used to assess whether the complete cell cycle arrest rate is at most 5% against the 
alternative that the complete cell cycle arrest rate is at least 20%.  The study design 
proposed below yields a 90% chance of detecting a complete cell cycle arrest rate of at 
least 20% at an alpha level of 0.1. 
 
Stage 1: Enroll 12 patients.  If at least one complete cell cycle arrest is documented 
among these 12 patients, continue to Stage 2.  Otherwise terminate enrollment for 
this patient cohort and declare that this regimen has insufficient anti-tumor activity 
in the neoadjuvant setting to recommend it for further testing in this patient 
population. 
 
Stage 2: Enroll an additional 25 patients.  If at most 3 complete cell cycle arrests 
are documented among the 37 total patients enrolled, this regimen will be 
considered to have insufficient anti-tumor activity in the neoadjuvant setting to 
recommend it for further testing in this patient population.  Otherwise, this regimen 
may be recommended for further testing in the neoadjuvant setting for this patient 
population.  For such a design, we will only have 7% chance to erroneously stop 
the trial at the 1st-stage if the true the complete cell cycle arrest rate is at least 20%.  
Conversely,  there will be 54% chance to stop the trial at the 1st-stage if the true 
the complete cell cycle arrest rate is 5% or less.    
 
Adverse events, the pace of accrual, other scientific discoveries, or changes in standard of 
care will be taken into account in any decision to terminate this trial earlier than designed. 
 
A 90% confidence interval for the true complete cell cycle arrest rate will be calculated 
using the Duffy-Santner approach. 

 
Protocol Version: 06/29/2022 
 
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15.4 
Sample Size and Trial Duration 
 
We anticipate that 4-6 patients per month with clinical stage 2 or 3 estrogen receptor 
positive and HER2 negative invasive breast cancer will be screened for the PIK3CA.  
Approximately 40% of the patients have breast cancer with PIK3CA hotspot mutations. 
Thus, approximately 55 women with clinical stage 2 or 3 estrogen receptor positive and 
HER2 negative invasive breast cancer will be screened to enroll 33 evaluable patients to 
the PIK3CA wild type cohort.  
 
The period encompassing enrollment, study treatment, and surgery will be about 20 
months. 
 
For the endocrine resistant cohort, we anticipate enrolling about one patient per month, 
completing Stage 1 in the approximately one year.  If Stage 1 is successful, we will consider 
adding more sites to the study. 
  
15.5 
Data Analysis  
 
All data analyses will be performed separately for each cohort unless otherwise specified. 
 
Demographic and clinical characteristics of the sample, as well as response, toxicity by 
grade, and loss to follow-up will be summarized using descriptive statistics. The rate of 
complete cell cycle arrest will be calculated and the corresponding 90% confidence interval 
(CI) will be provided. The rates for pCR and PEPI 0 and their 90% CIs will also be 
calculated.  The differences in overall survival (OS) and relapse-free survival (RFS) for 
patients with or without complete cell cycle arrest on C1D1 will be described using Kaplan-
Meier product limit estimator and compared by log-rank tests. Similar analysis will be 
performed for complete cell cycle arrest on C1D15 or patients with or without PEPI 0, with 
the two cohorts combined or separately.  A trajectory analysis (see below) will also be 
performed for this comparison.  
 
Analysis of other secondary endpoints: 
                           
Clinical response rate: The clinical response rate will be estimated by the number of 
patients whose disease meets the WHO criteria of complete or partial response prior to 
surgery divided by the total number of eligible patients who began combination 
neoadjuvant treatment. A 90% confidence interval for the true clinical response rate will 
be calculated using the Duffy-Santner approach for the endocrine resistant cohort (2-stage 
design).  The complete cell cycle arrest rates and the corresponding 90% confidence 
intervals  will also be calculated for PIK3CA wild type and mutant cohorts separately and 
combined. 
 
Radiological response rate: The radiological response rate will be estimated by the number 
of patients whose disease meets with WHO criteria for complete or partial response at the 
evaluation prior to surgery divided by the total number of eligible patients who began 

 
Protocol Version: 06/29/2022 
 
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combination neo-adjuvant therapy. A 90% confidence interval for the true radiographic 
response rate will be calculated using the Duffy-Santner approach.  The analysis will be 
performed with the two cohorts separately and combined. 
 
Adverse Events: All eligible patients that have intitiated treatment will be considered 
evaluable for assessing adverse event rate(s).  The maximum grade for each type of adverse 
event will be recorded for each patient using the NCI-CTCAE v4.0, and frequency tables 
will be reviewed to determine patterns.  Additionally, the relationship of the adverse 
event(s) to the study treatment will be taken into consideration. 
 
Ki67 suppression on Cycle 1 Day 1, Cycle 1 Day 15 and surgery: Following the approach 
of Dowsett et al. [26], the percent change in the Ki67 level from baseline will be determined 
on the log scale. A 95% t-confidence interval for the mean percent change in the Ki67 level 
from baseline will be constructed (if appropriate). The rate of complete cell cycle arrest on 
C1D1, C1D15, and surgery will be described by contingency tables compared by 
McNemar tests. Using trajectory analysis, we will also assess the association between 
distinct patterns of Ki67 change over time and clinical outcomes. Specifically, trajectory 
groups will be formulated to describe clusters of individuals with similar temporal patterns 
of Ki67 over time using finite mixture models with SAS procedure TRAJ.  This method 
enables identification of the number of discrete trajectory types, and characterization of the 
shape and estimation of the prevalence of each type.  We will choose the best models based 
on statistical tests (including Bayesian and Akaike information criterion, each of which test 
goodness-of-fit) and clinical judgment.  
 
To assess the long term outcomes of patients treated in this trial: The long-term outcomes 
(i.e., pathologic complete response (pCr), local and distant recurrence, etc, as defined in 
section 12.4) will also be assessed in the PIK3CA wild and mutnt cohorts (separately and 
combined) and the endocrine resistant cohort, respectively. The categorical outcome will 
be summarized using contingency tables and compared by Fisher’s exact test, while the 
time-to-event outcomes will be described using Kaplan-Meier product limit estimators and 
compared by log-rank test. 
 
15.6 
Correlative Studies  
 
Tumor cell apoptotic index: Apoptosis studies based on the TUNEL assay conducted in 
the context of the IMPACT study showed that the rate of apoptosis actually declined after 
two weeks of anastrozole treatment when the percentage TUNEL positive cells was less 
than one percent, with an SD of less than one percent [12].  Given the very low background 
of TUNEL positivity, a modest sized study is sufficient to demonstrate an increase in cell 
death. We will use the TUNEL assay to assess apoptosis at baseline and in the sample taken 
on PD 0332991. The TUNEL-positive cells will be counted using the point counting 
approach used for Ki67 and the apoptosis index will be calculated as the number of positive 
cells divided by the number of all cells and multiplied by 100. 
 
Steady state levels of estradiol: Steady state levels of estradiol on serially collected serum 
samples will be examined in premenopausal women to confirm the efficacy of the 

 
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anastrozole goserelin therapy in suppressing both ovarian and peripheal estradiol 
production. The change of estradiol level over time will be described by summary statistics 
and also compared using 2-way ANOVA for repeated measurement data 
 
Pharmacodynamic markers and markers of tumor cell senescence: The 
pharmacodynamic effect of PD 0332991 in combination with anastrozole on RB 
phosphorylation and markers of senescence will be assessed by phosphoroproteomics and 
immunohistochemistry analysis on serial tumor biopsies. The change of RB 
phosphorylation and senescence markers over time will be described by summary statistics 
and also compared using 2-way ANOVA for repeated measurement data.   
 
Explore potential markers of sensitivity and resistance: In preclinical studies, RB 
proficient cell lines with low p16 expression were found to be most sensitive to PD 
0332991 in ovarian cancer. In addition, copy number variations of CDKN2A, RB, CCNE1, 
CCND1 were associated with response [102].   Therefore, tumor specimens collected on 
Cycle 1 day 1 will be analyzed for the protein expression of these genes by 
immunohistochemistry.  Gene copy number changes will be assessed by circular binary 
segment (CBS) analysis using Signal Map Software (Nimblegen, Madison, WI, USA) and 
its association with clinical outcomes will also be assessed using contingency tables and 
fisher’s exact test. The gene expression levels between responses will also be summarized 
using means, standard deviations, medians, and compared by t-test or Mann-Whitney rank-
sum test as appropriate.  
 
To assess the concentrations of anastrozole and PD 0332991: The concentrations of 
anastrozole prior to and 90 minutes following anastrozole (without PD 0332991) on Cycle 
1 Day 1, as well as the concentrations of anastrozole prior to and 90 minutes following 
both anastrozole and PD 0332991 on Cycle 1 Day 15 will be summarized using means and 
standard deviations, and the differneces will be compared using two-way ANOVA for 
repeated measurement data. Data transformation will also be performed as necessary to 
better satisfy normality assumption. Similar analysis will be performed for the 
concentration of of PD 0332991. 
 
 
16.0 
MULTICENTER REGULATORY REQUIREMENTS 
 
Washington University requires that each participating site sends its informed consent document 
to be reviewed and approved by the Washington University Regulatory Coordinator (or designee) 
prior to IRB/IEC submission.    
 
Site activation is defined as when the secondary site has received official written documentation 
from the coordinating center that the site has been approved to begin enrollmkent.  At a minimum, 
each participating institution must have the following documents on file at Washington University 
prior to study activation: 
 
• Documentation of IRB approval of the study in the form of a letter or other official 
document from the participating institution’s IRB.  This documentation must show 

 
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which version of the protocol was approved by the IRB. 
• Documentation of IRB approval of an informed consent form. The consent must 
include a statement that data will be shared with Washington University, including the 
Quality Assurance and Safety Monitoring Committee (QASMC), the DSMC (if 
applicable), and the Washington University study team. 
• Documentation of FWA, signed FDA Form 1572 (if applicable), and the CVs of all 
participating investigators. 
• Protocol signature page signed and dated by the investigator at each participating site. 
 
The coordinating center Principal Investigator (or designee) is responsible for disseminating to the 
participating sites all study updates, amendments, reportable adverse events, etc.  Protocol/consent 
modifications and IB updates will be forwarded electronically to the secondary sites within 4 
weeks of obtaining Washington University IRB approval.  Activated secondary sites are expected 
to submit protocol/consent/IB modifications to their local IRBs within 4 weeks of receipt unless 
otherwise noted.  Upon the secondary sites obtaining local IRB approval, documentation of such 
shall be sent to the Washington University study team within 2 weeks of receipt of approval. 
 
Documentation of participating sites’ IRB approval of annual continuing reviews, protocol 
amendments or revisions, all SAE reports, and all protocol violations/deviations/exceptions must 
be kept on file at Washington University. 
 
The investigator or a designee from each institution must participate in a regular conference call 
to update and inform regarding the progress of the trial. 
 
 

 
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17.0 
REFERENCES 
 
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APPENDIX A: ECOG Performance Status Scale 
 
 
Grade 
 
 
Description 
0 
Normal activity.  Fully active, able to carry on all pre-disease 
performance without restriction. 
1 
Symptoms, but ambulatory.  Restricted in physically strenuous 
activity, but ambulatory and able to carry out work of a light or 
sedentary nature (e.g., light housework, office work). 
2 
In bed <50% of the time.  Ambulatory and capable of all self-care, but 
unable to carry out any work activities.  Up and about more than 50% 
of waking hours. 
3 
 
In bed >50% of the time.  Capable of only limited self-care, confined 
to bed or chair more than 50% of waking hours. 
 
4 
100% bedridden.  Completely disabled.  Cannot carry on any self-care.  
Totally confined to bed or chair. 
5 
 
Dead. 
 
 
 
 

 
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APPENDIX B: Neoadjuvant PD 0332991: Registration Worksheet 
 
Protocol #: 
Neoadjuvant PD 0332991 (HRPO# 
201301106) 
Patient Initials 
________-_________-______ 
PI Name:  
 
Institution Name: 
 
Registering MD 
name: 
 
Site Number: 
 
Study Coordinator 
Name and Email: 
 
 
Site Telephone # 
 
Please ensure the following are included with this form: (Please ensure all patient identification 
information is removed) 
 
 Corresponding Source Documentation for Inclusion/Exclusion 
 
 
 
Menopausal Status 
 Pre/Peri- menopausal 
  Postmenopausal 
DOB:  
_____ / _____ / _____ 
Zip Code: 
____ ____ ____ ____ ____ 
Date endocrine therapy initiated: 
_____ / _____ / _____ 
Local Ki67 after at least 2 weeks of endocrine 
therapy: 
________________ 
Planned Date Investigational Treatment 
Initiation: 
_____ / _____ / _____ 
Date of signed informed consent: 
_____ / _____ / _____ 

 
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REGISTRATION INCLUSION CRITERIA 
 
1. Clinical T2-T4c at diagnosis or screening, any N, M0 invasive ER+ (Allred Score at least 3 or 
> 1% ER positivity) and HER2 negative (0 or 1+ by IHC or FISH negative equivocal) breast 
cancer, by AJCC 7th edition clinical staging, with the goal being surgery to completely excise 
the tumor in the breast and the lymph node. 
 
Note: If the patient has invasive breast cancer that is ER pos, HER2 neg or equivocal, or 
DCIS in the contralateral breast, the patient is eligible.  Multifocal diseases are not excluded.  
 
2. Ki 67 > 10% by central testing after at least 2 weeks on neoadjuvant endocrine therapy. 
 
3. Female >18 years of age.   
 
4. ECOG performance status of 0, 1 or 2 
ECOG: _________ 
Date Assessed: ____________ 
 
5. If premenopausal, patient must be willing to comply with pregnancy requirements laid out in 
Section 5.5. 
6. Laboratory Values (Within 14 days of Registration) 
 
Leukocytes > 3,000/mcL 
 
Screening result:____________ 
 
Absolute Neutrophil Count (ANC) ≥ 1,500/mcL  
Screening result:____________ 
 
 
Platelets (Plts) > 100,000/mcL 
Screening result:____________ 
 
 
Total bilirubin ≤ upper normal institutional limits 
 
Screening result:____________ 
ULN:____________ 
 
Alanine aminotransferase (ALT) and aspartate 
aminotransferase (AST) ≤ 2.5 x upper limit of normal (ULN) 
 
ALT screening result:__________ 
ULN:___________ 
AST screening result:_________ 
ULN:____________ 
 
Serum creatinine ≤ upper normal institutional limits 
Screening result:____________ 
ULN:____________ 
 
7. Able to understand and willing to sign an IRB-approved written informed consent document  
 
 
 

 
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REGISTRATION EXCLUSION CRITERIA 
 
1. Prior treatment of this cancer including: surgery, radiation therapy, or chemotherapy. 
 
2. Receiving any other investigational agents. 
 
3. Prior therapy with any Cdk4 inhibitor 
 
4. Any of the following in the previous 6 months: myocardial infarction, severe/unstable angina, 
coronary/peripheral artery bypass graft, symptomatic congestive heart failure, cerebrovascular 
accident, transient ischemic attack,  symptomatic pulmonary embolism 
 
5. Uncontrolled intercurrent illness including, but not limited to ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, uncontrolled symptomatic 
cardiac arrhythmia, psychiatric illness/social situations that would limit compliance with study 
requirements 
 
6. Pregnant/nursing. 
 
7. Unwilling to employ adequate contraception. 
 
8. Known HIV-positive on combination antiretroviral therapy. 
 
9. Known metastatic disease 
 
10. Current use of anticoagulation therapy 
 
11. Previous excisional biopsy of the breast cancer or sentinel lymph node biopsy 
 
12. Any condition that impairs patient’s ability to swallow PD 0332991 tablets (e.g., gastrointestinal 
tract disease resulting in an inability to take oral medication or a requirement for IV alimentation, 
prior surgical procedures affecting absorption) 
 
13. History of allergic reactions attributed to compounds of similar chemical or biologic composition 
to PD 0332991 or other agents used in the study 
 
14. Correct QT (QTc) interval > 470 msec 
QTc: ______________ 
Date:______________ 
 
15.  
 
16. Current use or anticipated need for food or drugs that are known strong CYP3A4 inhibitors (i.e. 
grapefruit 
juice, 
verapamil, 
ketoconazole, 
miconazole, 
itraconazole, 
posaconazole, 
erythromycin, clarithromycin, telithromycin, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, 
atazanavir, amprenavir, fosamprenavir, nefazodone, diltiazem, and delavirdine) or inducers (i.e. 
dexamethasone, glucocorticoids, progesterone, rifampin, phenobarbital, St. John’s wort). 
 
17.  
 
Completed by: ___________________________ 
 
Date: _____ / _____ / _____ 
Investigator Signature: _________________________ 
Date: _____ / _____ / _____ 
 
For Wash U Use Only 
 
 
 
 
Screen Failure 
 
Subject Identifier: 
 Yes                  No 
 
 __ __ - __ __ __ 
 
Signature (Wash U designee): 
___________________________ 
Date: _____ / _____ / _____ 

 
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APPENDIX C: Adjuvant PD 0332991 Registration Worksheet 
 
 
Protocol #: 
Neoadjuvant PD 0332991 (HRPO# 
201301106) 
Patient Initials 
________-_________-______ 
PI Name:  
 
Institution Name: 
 
Registering MD 
name: 
 
Site Number: 
 
Study Coordinator 
Name and Email: 
 
 
Site Telephone # 
 
Please ensure the following are included with this form: (Please ensure all patient identification 
information is removed) 
 
 Corresponding Source Documentation for Inclusion/Exclusion 
 
 
 
Menopausal Status 
 Pre/Peri- menopausal 
  Postmenopausal 
DOB:  
_____ / _____ / _____ 
Zip Code: 
____ ____ ____ ____ ____ 
Date endocrine therapy initiated: 
_____ / _____ / _____ 
Planned Date Investigational Treatment 
Initiation: 
_____ / _____ / _____ 
Date of signed informed consent: 
_____ / _____ / _____ 
Patient ID for Neoadjuvant Trial: 
 

 
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REGISTRATION INCLUSION CRITERIA 
 
1. Derived benefit from PD 0332991 in the neoadjuvant setting in this trial. 
 
2. ECOG performance status of 0, 1 or 2    
ECOG: _________ 
Date Assessed:______________ 
 
3. If premenopausal, patient must be willing to comply with pregnancy requirements laid out in 
Section 5.5. 
4. Laboratory Values (Within 14 days of Registration) 
 
Leukocytes > 3,000/mcL 
 
Screening result:____________ 
 
 
Absolute Neutrophil Count (ANC) ≥ 1,500/mcL  
Screening result:____________ 
 
 
Platelets (Plts) > 100,000/mcL 
Screening result:____________ 
 
 
Total bilirubin ≤ upper normal institutional limits 
 
Screening result:____________ 
ULN:____________ 
 
Alanine aminotransferase (ALT) and aspartate 
aminotransferase (AST) ≤ 2.5 x upper limit of normal (ULN) 
 
ALT screening result:__________ 
ULN:___________ 
AST screening result:_________ 
ULN:____________ 
 
Serum creatinine ≤ upper normal institutional limits 
Screening result:____________ 
ULN:____________ 
 
5. Underwent surgery of the breast and axilla for curative intent.     Date of Surgery: _________ 
 
6. At least 4 weeks post-completion of adjuvant chemotherapy and radiation therapy if indicated. 
 
7. Patients who already started on adjuvant hormonal therapy are eligible under the following 
conditions: 
a. For the 26 patients who enrolled in the initial cohorts and derived benefit  from neoadjuvant 
PD 0332991 (C1D1 Ki67 >2.7% and C1D15 Ki67 ≤ 2.7%), adjuvant PD 0332991 should 
be initiated as soon as possible if adjuvant hormonal therapy has been initiated and the 
patient has completed radiation if indicated.  
b. For patients who enrolled in the endocrine resistant cohort and derived benefit from 
neoadjuvant PD 0332991 (C1D15 Ki67 ≤ 10%), adjuvant PD 0332991 should be initiated 
within 6 months or sooner after initation of adjuvant hormonal therapy. 
 
8. Able to understand and willing to sign an IRB-approved written informed consent document 
 
 
 

 
Protocol Version: 06/29/2022 
 
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REGISTRATION EXCLUSION CRITERIA 
 
1. Any of the following in the previous 6 months: myocardial infarction, severe/unstable angina, 
coronary/peripheral artery bypass graft, symptomatic congestive heart failure, cerebrovascular 
accident, transient ischemic attack,  symptomatic pulmonary embolism 
 
2. Uncontrolled intercurrent illness including, but not limited to ongoing or active infection, 
symptomatic congestive heart failure, unstable angina pectoris, uncontrolled symptomatic 
cardiac arrhythmia, psychiatric illness/social situations that would limit compliance with study 
requirements 
 
3. Pregnant/nursing. 
 
4. Unwilling to employ adequate contraception. 
 
5. Known HIV-positive on combination antiretroviral therapy. 
 
6. Known metastatic disease 
 
7. Any condition that impairs patient’s ability to swallow PD 0332991 tablets (e.g., gastrointestinal 
tract disease resulting in an inability to take oral medication or a requirement for IV alimentation, 
prior surgical procedures affecting absorption) 
 
8. History of allergic reactions attributed to compounds of similar chemical or biologic composition 
to PD 0332991 or other agents used in the study 
 
9. Correct QT (QTc) interval > 470 msec 
QTc:______________ 
Date: _____________ 
 
10.  
 
11. Current use or anticipated need for food or drugs that are known strong CYP3A4 inhibitors (i.e. 
grapefruit 
juice, 
verapamil, 
ketoconazole, 
miconazole, 
itraconazole, 
posaconazole, 
erythromycin, clarithromycin, telithromycin, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, 
atazanavir, amprenavir, fosamprenavir, nefazodone, diltiazem, and delavirdine) or inducers (i.e. 
dexamethasone, glucocorticoids, progesterone, rifampin, phenobarbital, St. John’s wort). 
 
12.  
 
Completed by: ___________________________ 
 
Date: _____ / _____ / _____ 
Investigator Signature: _________________________ 
Date: _____ / _____ / _____ 
 
For Wash U Use Only 
 
 
 
 
Screen Failure 
 
Subject Identifier: 
 Yes                  No 
 
 __ __ - __ __ __ 
 
Signature (Wash U designee): 
___________________________ 
Date: _____ / _____ / _____ 

 
Protocol Version: 06/29/2022 
 
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APPENDIX D: Medication Diary – PD 0332991 
Today’s Date:    
 
 
 
 
 
 
Agent:    
 
 
 
 
Cycle:    
 
 
 
 
 
 
 
 
 
Patient Name:    
 
 
 
 
 
 
Study ID#:   
 
 
 
INSTRUCTIONS TO THE PATIENT: 
1. Take 1 _____mg pill one time daily for 21 days, followed by 7 days of rest (no pills).  
2. Take PD 0332991 at approximately the same time each day with a meal. Swallow the tablets whole and do not 
chew them. 
3. If you forget to take your dose before 6:00PM, then do not take a dose that day.  Restart taking it the next day. 
4. Avoid St. John’s Wort, Seville oranges, grapefruit, grapefruit juice, grapefruit hybrids, pummelos, and exotic 
citrus fruits from 7 days before you start taking PD 0332991 and throughout the entire study.   
5. Complete this drug diary form for each 28-day cycle. 
6. Record the date, time (include AM or PM), and the number of tablets taken (for each strength). 
7. If you have any questions or notice any side effects, please record them in the comments section.  Record the 
time if you should vomit. 
8. If you have any questions, please call the study coordinator. 
9. Please return the forms to your physician or your study coordinator when you go to your next appointment. 
10. Please bring your unused study medications and/or empty bottles with you to each clinic visit so that a pill count 
can be done. 
 
Day 
Date 
What time was 
dose taken? 
# of 100 mg 
tablets taken 
# of 25 mg 
tablets taken 
Comments 
1 
 
 
 
 
 
2 
 
 
 
 
 
3 
 
 
 
 
 
4 
 
 
 
 
 
5 
 
 
 
 
 
6 
 
 
 
 
 
7 
 
 
 
 
 
8 
 
 
 
 
 
9 
 
 
 
 
 
10 
 
 
 
 
 
11 
 
 
 
 
 
12 
 
 
 
 
 
13 
 
 
 
 
 
14 
 
 
 
 
 
15 
 
 
 
 
 
16 
 
 
 
 
 
17 
 
 
 
 
 
18 
 
 
 
 
 
19 
 
 
 
 
 
20 
 
 
 
 
 
21 
 
 
 
 
 
  
 

 
Protocol Version: 06/29/2022 
 
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APPENDIX E: Medication Diary – Endocrine Therapy 
Today’s Date:    
 
 
 
 
 
 
Agent:    
 
 
 
 
Cycle:    
 
 
 
 
 
 
 
 
 
Patient Name:    
 
 
 
 
 
 
Study ID#:   
 
 
 
INSTRUCTIONS TO THE PATIENT: 
1. Complete one form for each month.  Take _____mg ( ___tabets) of anastrozole at approximately the same time 
each day. Swallow the tablets whole and do not chew them. 
2. Record the date, the number of tablets taken, and when you took them. 
3. If you forget to take your dose before 6:00PM, then do not take a dose that day.  Restart taking it the next day. 
4. If you have any questions or notice any side effects, please record them in the comments section.  Record the 
time if you should vomit. 
5. Please return the forms to your physician or your study coordinator when you go to your next appointment.  
Please bring your unused study medications and/or empty bottles with you to each clinic visit so that a pill count 
can be done. 
Day 
Date 
What time was 
dose taken? 
# of tablets 
taken 
Comments 
1 
 
 
 
 
2 
 
 
 
 
3 
 
 
 
 
4 
 
 
 
 
5 
 
 
 
 
6 
 
 
 
 
7 
 
 
 
 
8 
 
 
 
 
9 
 
 
 
 
10 
 
 
 
 
11 
 
 
 
 
12 
 
 
 
 
13 
 
 
 
 
14 
 
 
 
 
15 
 
 
 
 
16 
 
 
 
 
17 
 
 
 
 
18 
 
 
 
 
19 
 
 
 
 
20 
 
 
 
 
21 
 
 
 
 
22 
 
 
 
 
23 
 
 
 
 
24 
 
 
 
 
25 
 
 
 
 
26 
 
 
 
 
27 
 
 
 
 
28 
 
 
 
 
 

 
 
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APPENDIX F: 
Pfizer Reportable 
Event Cover Sheet 
 
 

 
 
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APPENDIX H: Strong CYP3A4 Inhibitors or Inducers 
(http://medicine.iupui.edu/clinpharm/ddis/clinical-table/) 
 
Inhibitors 
Indinavir 
Nelfinavir 
Ritonavir 
Clarithromycin 
Itraconazole 
Ketoconazole 
Nefazodone 
 
Inducers 
Carbamazepine 
Efavirenz 
Nevirapine 
Phenobarbital 
Phenytoin 
Pioglitazone 
Rifabutin 
Rifampin 
St. John’s Wort 
Troglitazone 
 
 

 
 
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