Clinical trial NCT02913430
Conditions: Metastatic Breast Cancer
Keywords: ESR1 mutation
Sponsor: Shannon Puhalla
Start date: 2018-04-24; Completion date: 2024-04-30
Enrollment: 7 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
To assess longitudinal changes in allele frequency of ESR1 mutation in plasma in patients treated with Fulvestrant plus palbociclib compared to tamoxifen plus palbociclib

Detailed description:
The primary objectives are to assess longitudinal changes in allele frequency of ESR1 mutation in plasma in patients treated with Fulvestrant plus palbociclib compared to tamoxifen plus palbociclib.

Patients with ER+ breast cancer who had 1 to 3 prior lines of endocrine therapy and up to one line of chemotherapy for MBC, excluding fulvestrant and tamoxifen, will be randomized in a 1:1 ratio to receive fulvestrant 500mg IM Q28 days with one extra dose on Day15 of the first cycle (as a loading dose) plus palbociclib 125mg/day PO on a 21 days on/7 days off schedule or tamoxifen 20mg PO daily plus palbociclib 125mg/day PO on a 21 days on/7 days off schedule.

Arms: arm 1: fulvestrant administered 500mg IM Q28 days plus palbociclib125mg/day PO on a 21 days on/7 days off schedule arm 2: Tamoxifen is administered orally, at a dose of20mg PO Qdaily plus palbociclib125mg/day PO on a 21 days on/7 days off schedule
Interventions: intervention 1: Fulvestrant intervention 2: Tamoxifen intervention 3: Palbociclib
Intervention descriptions: intervention 1: 500mg IM Q28 days intervention 2: 20mg PO Qdaily intervention 3: Tamoxifen or Fulvestrant plus palbociclib125mg/day PO on a 21 days on/7 days off schedule

Protocol documents:
Prot_SAP_000.pdf: 
Clinical Study Protocol Synopsis 
Drug Substance Fulvestrant 
Study Code Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 1  
 
 
Drug Substance 
Fulvestrant 
Study Number 
Pending 
Version Number 
12 
Date 
7-16-2020 
Treatment of Metastatic Breast Cancer with Fulvestrant plus Palbociclib or 
Tamoxifen plus Palbociclib: A Randomized Pilot Trial with ESR1 mutation 
tested in Circulating Tumor DNA. 
 
 
 
 
 
 
PI: Shannon L. Huggins-Puhalla, MD 
University of Pittsburgh  
Magee Womens Cancer Center 
300 Halket St. Suite 4628 
Pittsburgh, PA 15213 
 
Phone: 412-641-6500/ Fax: 412-641-6461 
 
puhallasl@upmc.edu 
 
 
 
 
 
 
 
 


Clinical Study Protocol Synopsis 
Drug Substance Fulvestrant 
Study Code Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 2  
 
 
 
Additional Study Sites in UPMC Network: 
 
 
Arnold Palmer Pavilion at Mountain View 
Medical Park 
Arnold Palmer Pavilion, Mount Pleasant 
Arnold Palmer Pavilion at Oakbrook 
Commons 
UPMC Hillman Cancer Center 
Tony Teramana Cancer Center, 
Steubenville 
UPMC CancerCenter at UPMC 
McKeesport 
UPMC CancerCenter, Beaver 
UPMC CancerCenter, Jefferson 
UPMC CancerCenter at UPMC East 
(Monroeville) 
*UPMC CancerCenter, New Castle 
UPMC CancerCenter at UPMC Passavant 
(OHA) 
UPMC CancerCenter at UPMC Passavant 
(HOA) 
UPMC CancerCenter at UPMC St. 
Margaret 
*UPMC CancerCenter, Uniontown 
UPMC CancerCenter, Upper St. Clair 
UPMC CancerCenter, Greenville 
UPMC CancerCenter, Horizon 
UPMC CancerCenter, Northwest 
UPMC CancerCenter, Indiana 
UPMC CancerCenter, Murtha 
UPMC CancerCenter, Washington 
Butler Health System Medical Oncology 
UPMC Pinnacle Health System 


Clinical Study Protocol Synopsis 
Drug Substance Fulvestrant 
Study Code Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 3  
 
 
 
Additional study sites: 
 
Case Western Comprehensive Cancer Center 
Local PI: Shaveta Vinayak, MD, MS 
Case  Comprehensive Cancer Center 
Case Western Reserve University School of Medicine 
Division of Hematology and Oncology 
11100 Euclid Avenue/ LKS 5079 
Cleveland, OH 44106 
Email: shaveta.vinayak@uhhospitals.org 
 
Cleveland Clinic 
 
Local PI: G. Thomas Budd, MD 
 
Department of Hematology/Medical Oncology, Desk R35 
The Cleveland Clinic Foundation 
9500 Euclid Avenue 
Cleveland, OH 44195 
Email: buddg@ccf.org 
 


Clinical Study Protocol Synopsis 
Drug Substance Fulvestrant 
Study Code Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 4  
 
 
 
 
 
Patient Population (see section 4 for details) 
• Second to third line in the metastatic setting 
• Pre-menopausal (must receive chemical ovarian ablation) and post-menopausal 
• All patients must have prior exposure to an AI in the adjuvant, neoadjuvant or metastatic 
setting 
• Prior Palbociclib required 
• Prior fulvestrant not allowed. 
• Prior tamoxifen allowed in adjuvant setting only if followed by AI for ≥ 2years. 
• Prior everolimus or other biologic agents are allowed. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 5  
 
 
 
TABLE OF CONTENTS 
PAGE 
 
TITLE PAGE ............................................................................................................................ 1 
TABLE OF CONTENTS ......................................................................................................... 5 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................................. 8 
1. INTRODUCTION .............................................................................................................. 10 
1.1 Background ....................................................................................................................... 10 
1.2 Research hypothesis ......................................................................................................... 15 
1.3 Rationale for conducting this study .................................................................................. 15 
1.4 Benefit/risk and ethical assessment .................................................................................. 16 
2. STUDY OBJECTIVES ...................................................................................................... 17 
2.1 Primary objective .............................................................................................................. 17 
2.2 Secondary objectives ........................................................................................................ 17 
2.3 Exploratory objectives ...................................................................................................... 17 
3. STUDY PLAN AND PROCEDURES ............................................................................... 17 
3.1 Overall study design and flow chart ................................................................................. 17 
3.2 Rationale for study design, doses and control groups ...................................................... 18 
4. SUBJECT SELECTION CRITERIA ................................................................................. 19 
4.1 Inclusion criteria ............................................................................................................... 19 
4.2 Exclusion criteria .............................................................................................................. 20 
5. STUDY CONDUCT .......................................................................................................... 21 
5.1 Randomization Proceedures ............................................................................................. 21 
5.1.1 Regulatory Requirements .............................................................................................. 21 
5.1.2 Patient Randomization ................................................................................................... 21 
5.2 Treatments ........................................................................................................................ 22 
5.2.1 Identity of investigational products ............................................................................... 22 
5.2.2 Doses and treatment regimens ....................................................................................... 27 
5.3 Restrictions and concomitant therapies during the study ................................................. 28 
5.4 Treatment adherence ........................................................................................................ 29 
5.5 Discontinuation of treament ............................................................................................. 29 
5.6 Procedures for discontinuation of a subject from treatment ............................................. 29 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 6  
 
5.7 Training of study site personnel ....................................................................................... 29 
5.8 Withdrawal from the study ............................................................................................... 27 
6. COLLECTION OF STUDY VARIABLES ....................................................................... 29 
6.1 Recording of data.............................................................................................................. 29 
6.2 Data collection at enrollment and follow-up .................................................................... 30 
6.3 Efficacy ............................................................................................................................. 31 
6.3.1 Measurable systemic disease ......................................................................................... 31 
6.3.2 Non-measurable disease ................................................................................................ 30 
6.3.3 Specifications by methods of measurements ................................................................. 32 
6.3.4 Response Assessment .................................................................................................... 33 
6.3.5 Correlative Studies ........................................................................................................ 34 
6.4 Safety ................................................................................................................................ 38 
6.4.1 Definition of adverse events .......................................................................................... 38 
6.4.2 Definitions of serious adverse event (SAE) .................................................................. 39 
6.4.4 Reporting of serious adverse events .............................................................................. 41 
6.4.5 Causality of adverse event by study compound. .......................................................... 42 
6.4.6 Time period for collection of adverse events ................................................................ 42 
7. BIOLOGICAL SAMPLING PROCEDURES ................................................................... 43 
7.1 Solid Tumor Biopsy Samples ........................................................................................... 43 
7.2 Blood Samples .................................................................................................................. 43 
7.3 Leftover samples. ............................................................................................................. 44 
7.4 Handling, storage and destruction of biological samples ................................................. 44 
7.5 Withdrawal of informed consent for donated biological samples .................................... 44 
8. ETHICAL AND REGULATORY REQUIREMENTS ..................................................... 44 
8.1 Ethical conduct of the study ............................................................................................. 44 
8.2 Ethics and regulatory review ............................................................................................ 46 
8.3 Informed consent .............................................................................................................. 45 
8.4 Changes to the protocol and informed consent form ........................................................ 46 
8.5 Audits and inspections ...................................................................................................... 47 
9. STUDY MANAGEMENT ................................................................................................. 47 
9.1 TABLE 4. Study timetable and end of study.................................................................... 47 
9.2 Study Management. .......................................................................................................... 48 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 7  
 
10. DATA MANAGEMENT ................................................................................................. 52 
11. EVALUATION AND CALCULATION OF VARIABLES ........................................... 53 
11.1 Calculation or derivation of efficacy variable(s) ............................................................ 53 
11.2 Calculation or derivation of safety variable(s) ............................................................... 53 
12 STATISTICAL METHODS AND SAMPLE SIZE DETERMINATION........................ 54 
12.1 Statistical Analysis Plan  ................................................................................................ 54 
12.2 Determination of sample size ......................................................................................... 54 
13. LIST OF REFERENCES ................................................................................................. 58 
 
 
 
 
 
LIST OF TABLES 
TABLE 1. Abbreviations 
 
TABLE 2. Time to response definition 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 8  
 
TABLE 3. MammoSeq gene list 
 
TABLE 4. Study timetable and end of study 
 
TABLE 5. Probability of detecting a "promising" effect 
 
LIST OF FIGURES 
FIGURE 1. ESR1 Mutations are associated with poor outcome 
 
FIGURE 2. Drug Response in ESR1 wildtype and mutant cells, and context dependent 
gain-of-function of mutant ER in genome-edited cells. 
 
FIGURE 3. Detection of ESR1 mutation in liver metastasis and plasma 
FIGURE 4. Study Schema 
 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
The following abbreviations and special terms are used in this study Clinical Study Protocol. 
TABLE 1. List of abbreviations 
Abbreviation or 
special term 
Explanation 
AE 
Adverse events 
AI 
Aromatase Inhibitor 
CR 
Complete response 
CRS 
Clinical research services 
CTCAE 
Common terminology criteria for adverse events 
ctDNA 
Circulating Tumor DNA 
ddPCR 
Droplet digital PCR 
DMC 
Data monitoring committee 
DSMC 
Data safety monitoring committee 
eCFR 
Electronic case report forms 
ER+ 
Estrogen Receptor Positive 
ESR1 
Estrogen Receptor 1 
ESR1-mt 
Estrogen Receptor 1 mutant 
ESR1-wt 
Estrogen Receptor 1 wild type 
FDA 
Food and Drug Administration 
GDP 
Good clinical practice 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 9  
 
 
Abbreviation or 
special term 
Explanation 
ICH 
International clinical research 
IRB 
Institutional review board 
ISS 
Investigator sponsored study 
LBD 
Ligand binding-domain 
MBC 
Metastatic Breast Cancer 
OS 
Overall Survival 
PD 
Progressive disease 
PFS 
Progression Free Survival 
PR 
Partial response 
PR 
Progesterone receptor 
RR 
Response rate 
SAE 
Serious adverse events 
SD 
Stable disease 
SRM 
Study reference manual 
TAM 
Tamoxifen 
TTP 
Time to progression 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 10  
 
1. INTRODUCTION 
1.1 Background 
A major challenge is to identify the drivers of progression from primary to metastatic breast 
cancer, and identify new targets for treatment. Mutations in the estrogen receptor (ESR1) are 
a potential resistance mechanism to endocrine therapy (1-9). In cell based-models, the 
presence of a mutation in ESR1 confers constitutive and ligand independent activity.(3) 
Although these mutations are extremely rare in previously untreated patients with early 
breast cancer, the development of these mutations over time after exposure to hormonal 
therapy with tamoxifen or AIs has been recently demonstrated to occur in as many as 14-54% 
of patients with estrogen receptor- positive (ER+) metastatic breast cancer (MBC).(7) 
The mutations primarily occur in the ligand binding domain (LBD) of ESR-1 and there have 
been at least 6 mutations described. A recent study by Chandarlapathy identified additional 
(albeit very rare) ESR1 mutations ( 10) Patients whose tumors harbor ESR1 mutations have 
poor overall survival.  In a recent large meta-analysis including data from1540 patients, 28% 
had ESR mutations and had significantly worse PFS. The presence of ESR1 mutation, 
however, did not predict outcome on fulvestrant based therapy. (11) Even using a small 
cohort of patients, we reported shorter progression free survival (PFS) in patients with 
endocrine-resistant ER+ breast cancer receiving palbociclib who had ESR1 mutations 
detected in cfDNA (12)   
 Together these observations support the tenable hypothesis that these mutations are 
clinically significant and the tumors that harbor them are strongly dependent upon ESR1 
signaling. 
Interestingly, in vitro, both tamoxifen and fulvestrant appear to have activity in the setting of 
ESR1 mutations; although different ESR1 mutations appear to have varying thresholds for 
effective dosing of both agents that define their level of partial resistance (3).  Our group has 
recently performed detailed analysis of drug response in genome-edited T47D and MCF-7 
cells, and these data were published Bahreini et al (13)Briefly, we introduced the most 
frequent ESR1 hotspot mutations D538G and 
Y537S, and observed not only ligand-
independent activity of the mutant ER in growth 
and transcriptional activity assays, but also 
observed antiestrogen resistance: cells with 
mutant ER had higher IC50 for the SERMs 
4OHT and raloxifene, and the SERDs 
fulvestrant and AZD9496 compared to WT 
(Figure 2A).  We did observe differences 
between the mutants, with Y537S displaying 
increased resistance compared to D538G.   
Figure 2:  A) ESR1 mutant-cells display resistance 
against selective estrogen receptor modulators 
(SERMs) and selective estrogen receptor degraders 
(SERDs). Graphical (a) and tabular (b) presentation of half 
maximal inhibitory concentration (IC50) values that were 
determined in dose–response curves in wild-type (WT), 
 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 11  
 
Y537S and D538G cells treated with 20 pM estradiol (E2) plus varying doses of 4OHT, raloxifene (Ral), 
fulvestrant (Ful), and AZD9496 in T47D and MCF7 cell lines. One-way analysis of variance was 
performed to compare the IC50 values of mutants to WT within each cell line and drug (*p < 0.05, **p < 
0.01). (13). 
 
Of note, our recent unpublished data shows metastatic properties of the D538G mutant 
receptor which might explain high MAF of this hotspot mutation despite decreased 
resistance compared to Y537S. We hypothesize that these novel gain-of-function activities 
are related to target genes specifically regulated by mutant ER (Figure 2B). 
 
 
 
 
 
 
 
 
 
 
Figure 2B: Genome-wide transcriptomic analysis reveals regulation of ligand-independent 
estrogen receptor (ER) targets, and of novel target genes by ERα mutants in T47D and MC7 
cells. T47D and MCF7 cell lines were hormone-deprived for 3 days, treated with vehicle (veh) or 1 
nM of estradiol (E2) for 24 h, RNA was isolated and RNA sequencing analysis was performed. The 
heat map shows normalized log2 fold change (FC) of genes differentially regulated in mutants vs 
wild-type (WT) in the absence of ligand (FC >2, p value <0.005). The genes are sorted based on E2 
regulation in WT (red arrow ligand-independent E2 activated genes, blue arrow ligand-independent 
E2 downregulated genes, green circle ligand-independent non-E2 regulated genes, i.e. “novel target 
genes”). (13). 
 
As our studies indicate, the effect of different ESR1 mutations and their response to therapy 
are cell and context dependent. Thus it is important to determine the clinical relevance of 
these mutations and the efficacy of tamoxifen and fulvestrant in the setting of AI resistance 
especially since these two drugs possess distinct modes-of-action. 
Specifically, as mutations in ESR1 appear to constitutively activate the receptor, the intrinsic 
ability of fulvestrant (in contrast to tamoxifen) to facilitate ER degradation may provide a 
mechanism to circumvent AI resistance with a more durable response. Our lab has recently 
published two publications on this important topic (17, 18).  
Both fulvestrant and tamoxifen are standard hormonal therapies in patients with AI resistance. 
In a prospective setting, the efficacy and potential resistance of these agents in ESR1-mt 
tumors is unknown. There is some evidence that Fulvestrant treatment has efficacy in the 
tumors with ESR1 mutations, but this is less known for Tamoxifen.  The prevalence of ESR1-


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 12  
 
mt in the metastatic breast cancer population has been reported to be 15-50%. Some of these 
patients were heavily pre-treated endocrine- refractory patients. There is some but limited 
information on the prevalence of ESR1 mutations after AI failure and the incidence of ESR1 
mutation as an effect of subsequent endocrine therapies. It is important to ascertain the 
prevalence of ESR1-mutations in patients earlier in their metastatic disease course, which is 
the time in which agents such as tamoxifen and fulvestrant are typically used. It is critical, 
therefore, to identify biomarkers in patient subsets that are predictive of rationale 
combinations of hormonal therapies as strategies to combat endocrine resistance from the 
outset.  
 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 13  
 
Many patients now receive palbociclib with an AI in the first-line metastatic setting. ER- 
positive luminal breast cancer is particularly dependent on the interaction of cyclin Ds with 
the protein complexes CDK4 and CDK6 to drive cellular proliferation(18, 19). Together, 
they facilitate phosphorylation/inactivation of the Retinoblastoma tumor suppressor protein 
(RB), and this leads to progression through the G1 restriction point to the S phase of the cell 
cycle and, thus, cellular proliferation. Loss of RB is a potential mechanism of resistance to 
CDK4/6 inhibitors in vitro, but this needs to be validated in patients. Palbociclib (Ibrance, 
Pfizer) is an oral small molecule inhibitor of CDK4 and CDK6. Palbociclib received 
accelerated FDA approval based on the results of PALOMA-1, a randomized Phase 2 study 
comparing palbociclib plus letrozole versus letrozole alone in women with ER-positive 
HER-2-negative advanced breast cancer. PALOMA-1 showed significantly longer PFS with 
palbociclib plus letrozole (20.2 months) than with letrozole alone (10.2 months) (20) 
The PALOMA-2 trial was designed as a confirmatory study and was a double-blind, 
randomized, Phase 3 2:1 comparison of palbociclib plus letrozole versus placebo plus 
letrozole. The median progression free survival was 24.8 months with the combination 
compared to 14.5 months in the placebo-letrozole group. The most common grade 3 or 4 
adverse events were neutropenia, leukopenia, and fatigue (21). 
The PALOMA-3 trial was a randomized, double-blind, placebo-controlled phase 3 trial 
comparing pelvis occluded in combination with fulvestrant compared with placebo plus 
fulvestrant in women with hormone receptor positive HER-2 negative metastatic breast cancer 
after progression on prior endocrine therapy. The median PFS on palbociclib plus fulvestrant 
was about 11 months, and it was not different in patients with greater than or equal to grade 3 
neutropenia versus those who did not (22). 
 
However, there is significant need for predictive biomarker development to guide therapy in 
patients who are resistant to AIs and/or have received prior palbociclib. Loss of RB is one 
potential biomarker of CDK4/6 resistance, but it is actually not commonly described in ER- 
positive breast cancer. Other potential biomarkers in include CCnE1, amplification of E2F, 
cyclin E levels, or loss of CDKN1A, that has been linked to tamoxifen resistance.


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 14  
 
In addition, there is currently no data to support CDK4/6 beyond progression in patients with 
AI-resistant disease. It is unknown whether these agents can prevent emergence of resistance 
in patients formerly treated with an AI/CDK4/6 combination. It is also unknown whether 
they could circumvent the ligand-independent activation of ER signaling that occurs in the 
setting of an ESR1mutation. Limited ongoing studies seek to address this question. The 
BioPER trial (NCT013184090) is an open label non-controlled phase II trial assessing 
Palbociclib in combination with endocrine therapy of investigators choice after prior 
successful treatment with a Palbociclib containing regimen, looking for molecular profiles 
identifying patient who may benefit from continued Palbocicilib in this setting. This trial 
focuses on Rb status and related signaling as potential biomarkers. 
 
A second study of Palbociclib after CDK and endocrine therapy (PACE) (NCT03147287) is 
examining Fulvestrant, Palbociclib and Avelumab for patients with metastatic disease. This 
trial is not estimated to be completed until 2024. A third ongoing trial (NCT02738866), phase 
II trial of Palbociclib and Fulvestrant in patients with HR+ MBC who have progressed on 
treatment with Palbociclib and an AI. There are currently no trials examining the 
combination of Palbciclib with Tamoxifen vs Palbociclib with Fulvestrant in patients 
previously exposed to a CDK4/6 inhibitor. 
 
The focus of this study will be examination of ESR1 mutations as a biomarker. Both 
tamoxifen and fulvestrant are possible standard-of-care therapies in this clinical setting. We 
will assess the detection of ESR1 mutations in plasma over time on both therapies.. 
Additionally, since testing for ESR1 is not fully standardized and because it is a clinically 
relevant question, we will collect response data as measured by PFS  for fulvestrant plus 
palbociclib vs tamoxifen plus palbociclibfor treatment of MBC, unselected by ESR1. 
 
While previous studies have predominately evaluated for ESR1-mt in tumor tissues, use of 
novel technology to assess for ESR1-mt in plasma makes it clinically feasible to assess the 
variability of this marker over time in response to treatment. A PCR Sanger sequencing-based 
assay for detection of the five ESR1 mutations is available for solid tumor detection. A new 
next generation sequencing assay, MammaSeqSM, which measures all reported ESR1 
mutations, and mutations and copy number alterations in another 77 genes commonly mutated 
in breast cancer, is also under development in the UPMC Molecular Pathology laboratory and 
will be incorporated into this protocol. We have recently published on the generation and use 
of MammaSeqSM (20)  For detection of ESR1 mutations in plasma, a droplet digital PCR 
(ddPCR) assay is performed in the research laboratory of Drs. Adrian V. Lee PhD and Steffi 
Oesterreich. Using this assay, we have identified ESR1 mutations in 5/30 (16.7%) of patients 
with metastatic breast cancer, and when only considering those with an ER-positive primary 
breast cancer, we find ESR1 mutations in 5/20 (25%) of patients. The MammaSeqSM 
capabilities would allow us to more comprehensively assess the initial presence and also the 
acquisition of mutations during treatment with tamoxifen plus palbociclib and fulvestrant plus 
palbociclib. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 15  
 
This may provide an additional means to discern differences between these two 
mechanistically distinct treatments and also enable us to target our analysis to more 
specifically determine the significance of ESR1 mutations per se as prognostic and predictive 
indicators. 
1.2 Research hypothesis 
1. In the 2nd to 3rd line of AI resistant WT ESR1 MBC, Fulvestrant plus palbociclib 
treatment will result in earlier detection of ESR1 mutations compared to treatment with 
tamoxifen plus palbociclib  
 
2. In the 2nd to 3rd line of AI resistant mutant ESR1 MBC, Fulvestrant plus palbociclib will 
result in increased PFS compared with tamoxifen plus palbociclib 
 
3. Change of allele frequency of ESR1 mutation is associated with PFS in all groups 
 
 
1.2 Rationale for conducting this study 
We propose a pilot clinical trial comparing the standard doses of fulvestrant plus palbociclib 
to tamoxifen plus palbociclib while evaluating subjects for the presence of ESR1 mutation. 
The goal is to obtain plasma, and tumor samples if available, that will look at the changes in 
time in the detection of ESR mutations while on non-AI therapy but with continued exposure 
to palbociclib. As both tamoxifen or faslodex would be standard next line options for patients 
progressing on AI, we would like to look at these two populations, This project will provide 
needed information regarding the prevalence of ESR1-mt in patients with ER+ MBC in the 
2nd -3rd line setting when hormonal therapies are predominantly used. We will determine the 
presence of ESR1-mt by analyzing tumor biopsies and by making use of the novel technology 
of detection of ESR1-mt from circulating tumor DNA (ctDNA) by digital droplet PCR 
(ddPCR) in plasma. This technology allows the non-invasive detection and measurement of 
the level of circulating ESR1-mt over time and in response to treatment. Measuring ESR1 
both in tissue and plasma will allow for determination of concordance between both sites, and 
may remove any bias that metastatic tumor heterogeneity could contribute. The tissues 
collected will also be assessed for other alterations in ESR1 such as amplifications or fusions, 
as well as assessment of ESR1 abnormalities on ER binding sites and whether this is altered 
after fulvestrant treatment. Our group has been able to demonstrate congruence between an 
ESR1 mutation found on liver biopsy and in plasma using ddPCR in a patient with ER+ 
MBC. (figure 4, unpublished and reference 23). 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 16  
 
FIGURE 3. Detection of ESR1 mutation in liver metastasis and plasma 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
This proposal will provide a prospective pilot study of the effect of the presence or absence of 
ESR1-mt on response to a commonly used therapy in the metastatic setting. This study will 
provide important data regarding a potentially very common and clinically relevant mutation 
that likely effects response to frequently used treatments.  The results obtained can then be 
used as springboard to assess other potential therapies in this setting and to further study the 
biologic relevance of the mutation. 
1.3 Benefit/risk and ethical assessment 
This study will be conducted in accordance with the ethical principles that have their origin in 
the current Declaration of Helsinki and will be consistent with applicable US regulatory 
requirements and International Conference on Harmonization Good Clinical Practice (ICH 
GCP). 
The study will be conducted in compliance with the protocol. The protocol and any 
amendments and the patient informed consent will receive Institutional Review Board (IRB) 
approval prior to initiation of the study. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 17  
 
Study personnel involved in conducting this trial will be qualified by education, training, and 
experience to perform their respective task(s). This trial will not use the services of 
investigators or study personnel where sanctions have been invoked or where there has been 
scientific misconduct or fraud (e.g., loss of medical licensure, debarment). Investigators are 
responsible for the conduct of the study at their study site. 
 
2. STUDY OBJECTIVES 
 
2.1 Primary objective 
1. Assessment of longitudinal changes in allele frequency of ESR1 mutation in 
plasma in patients treated with Fulvestrant plus palbociclib compared to 
tamoxifen plus palbociclib  
 
2. Assessment of genomic alterations (using MammaSeq) associated with 
wildtype or mutant ESR1 
 
3. Assessment of the functionality of ESR1 in tumor biopsies collected prior to and 
following treatment with fulvestrant plus palbociclib, or tamoxifen plus palbociclib, as 
assessed by RNAseq 
 
4. Safety and toxicity assessment 
 
2.2 Secondary objectives 
1. Collection of clinical outcome data in this limited patient population 
a. Progression free survival (PFS), Objective response rate (ORR) and 
Clinical benefit rate (defined as CR+PR+ stable disease at any tumor 
assessment) and clinical benefit rate at 6 months (CBR6) (defined as 
CR+PR+ stable disease for at least 6 months) in the entire cohort, and the 
ESR1-mt cohorts 
 
2. Correlation between longitudinal changes in circulating levels of ESR1 
mutations and clinical outcome 
3. Assessment of concordance of ESR1 status in plasma and biopsy samples 
4. Use of cfRNA to determine gene expression in AI resistant mutant ESR1 MBC 
 
 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 18  
 
 
3. STUDY PLAN AND PROCEDURES 
3.1 Overall study design and flow chart 
Patients with ER+ breast cancer who had 1 to 3 prior lines of endocrine therapy and up to one 
line of chemotherapy for MBC, excluding fulvestrant and tamoxifen, will be randomized in a 
1:1 ratio to receive fulvestrant 500mg IM Q28 days with one extra dose on D15 of the first 
cycle (as a loading dose) plus palbociclib 125mg/day PO on a 21 days on/7 days off schedule 
or tamoxifen 20mg PO daily plus palbociclib 125mg/day PO on a 21 days on/7 days off 
schedule. Patient accrual will be a goal of 40 evaluable at up to 4 sites. At randomization, 
plasma will 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 19  
 
be obtained for evaluation of ESR1-mt and a biopsy will be obtained when possible. During 
treatment, all patients will undergo ct-DNA ESR1 mutation evaluation every 28 days. 
Treatment will continue until disease progression or unacceptable toxicity. At the time of 
disease progression, a second biopsy will be obtained when possible and the last blood drawn 
for evaluation of ct-DNA ESR1-mt will occur. All tissue obtained will also undergo molecular 
analysis to determine the activity of mutant ESR1. 
 
 
 
FIGURE 5. Study Schema 
 
 
3.2 Rationale for study design, doses and control groups 
Prior data supports the use of fulvestrant 500mg monthly with one extra dose 14 days after the 
first dose in metastatic breast cancer that is ER+. Two phase III trials comparing fulvestrant 
250mg monthly to anastrozole published in 2002 and showing similar efficacy were the basis 
of approval by the FDA. (9) In 2004, Howell et al published the first trial comparing 
fulvestrant with tamoxifen in previously untreated MBC that was ER+ in postmenopausal 
woman. This study used the approved dose of fulvestrant in 2004 of 250mg IM monthly 
compared with tamoxifen 20mg PO daily and included 563 patients. Not all patients had 
known ER+ in the study and the primary outcome of time-to-progression (TTP) was analyzed 
both in the intention-to-treat (ITT) population and in the subgroup of known ER+ patients 
which corresponded to 78% of the initial cohort. Time to progression was similar in both 
analyses. Amongst all patients the TTP was 6.8 months for fulvestrant and 8.3 months for 
tamoxifen (p=0.08). Amongst ER+ patient the TTP was 8.2 months and 8.3 months, 
respectively (p=0.39). Both regimens were well tolerated and fulvestrant resulted in similar 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 20  
 
incidence of nausea, injection site pain or inflammation and thromboembolic events (3.3% vs. 
5.8%). Fulvestrant was associated with lower incidence of hot flashes (17.7% vs. 24.7%, 
p=0.05). (10) 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 21  
 
The FIRST study evaluated fulvestrant 500mg monthly compared with anastrozole in the first 
line setting of ER+ MBC. This was a phase II study with 205 patients that used clinical benefit 
rate (CBR) as the primary endpoint. CBR was similar between both drugs, but time to 
progression, which was a secondary end point, was superior to fulvestrant 500mg monthly. 
(11) More recently, an update of the FIRST trial reported improved OS of fulvestrant 500mg 
monthly. (12) Based on this data, the FALCON study which is a phase III clinical trial 
comparing fulvestrant 500mg monthly with anastrozole and using PFS as the primary  
outcome was initiated. This study was recently reported and showed superiority of fulvestrant 
in the 1st line setting over anastrozole with median PFS in the fulvestrant arm of 16.6 months 
and 13.8 months with anastrozole (HR 0.797, 95% CT 0.637-0.999).   (13) In 2014 the 
CONFIRM trial comparing fulvestrant 250 mg monthly with fulvestrant 500mg monthly 
reported improved OS for the higher dose. (14) Therefore, fulvestrant 500mg monthly is the 
current standard dose. Based on the data above and our own unpublished preclinical data 
showing a dose-response relationship of fulvestrant and inhibition of breast cancer cells, we 
hypothesized that fulvestrant 500mg IM monthly could result in longer PFS compared with 
tamoxifen. We also hypothesize that this effect could be more pronounced in endocrine 
resistant breast cancer cells through ESR1 mutation as shown above. Because of that, we 
propose a phase II trial comparing the PFS of fulvestrant 500mg IM monthly with that of 
tamoxifen in the 2nd to 3rd line setting of ER+ MBC and with the exploratory evaluation of 
ESR1 mutation from solid tumor biopsies and ctDNA. The randomized study will be open 
label (unblinded) because the therapies are different modalities (injection, oral) and blinding 
would therefore be an onerous burden to the patients. However, we emphasize that clinic visits 
and disease assessments are on the same schedule for both treatment arms. 
4. SUBJECT SELECTION CRITERIA 
4.1 Inclusion criteria 
For inclusion in the study subjects should fulfill the following criteria: 
1. Signed informed consent 
2. Patients must have histologically or cytologically confirmed invasive breast cancer that is 
ER+ (>1% staining) with radiographical or clinical evidence of metastatic disease 
a. Measurable and/or non-measurable disease 
3. Prior therapies: 
a. Patients must have previously received an aromatase inhibitor in the adjuvant, 
neo-adjuvant or metastatic setting. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 22  
 
b. Patients must have previously received palbociclib in the adjuvant, neo- 
adjuvant or metastatic setting. If patient is currently taking palbociclib at time 
of screening for the trial they may continue taking palbociclib. 
 
c. The minimum duration of AI in the adjuvant setting is 2 years. 
d. There is no minimum duration of AI in the metastatic setting or neoadjuvant 
setting. 
e. Patients may have been previously treated with an mTOR inhibitor or other 
investigational agent in addition to an aromatase inhibitor. 
f. Prior treatment with tamoxifen is allowed in the adjuvant setting provided that 
it was followed by a minimum of 2 years of an AI. 
4. Brain metastasis is allowed if previously treated, stable and off steroids for a minimum of 
56 days 
5. Age > 18 years 
6. Male or female breast cancer is allowed 
7. Patients may be pre- or post-menopausal; pre-menopausal patients must be on ovarian 
suppression and must be adequately suppressed on LHRH agonists with estradiol levels in 
the post-menopausal range 
a. Premenopausal patients cannot be pregnant and must agree to adequate birth 
control in addition to ovarian suppression. Agreement by the patient and/or 
partner to use highly effective, nonhormonal form of contraception or two 
effective forms of non-hormonal contraception. Contraception use should 
continue during the duration of study treatment and for at least 6 months after 
the last dose of study treatment. 
8. ECOG performance status 0-2 
9. Adequate bone marrow function as indicated by the following, within 14 days of 
enrollment: 
a. ANC ≥ 1500 cells/mm3 
b. Platelets ≥ 100,000 cells/ mm3 
c. Hemoglobin ≥ 9 g/dL 
10. Adequate liver function, as indicated by the following, within 14 days of enrollment. 
a. Total bilirubin 1.5 upper limit of normal (ULN) 
b. AST  1.5  ULN 
c. ALT ≤ 2.5  ULN 
d. Alkaline phosphatase ≤ 2.5  ULN with the following exception; ALP ≤ 5× 
ULN in patients with bone metastases. 
11. Adequate hemostatic function as determined by PT, INR and aPTT < 1.5× ULN (unless on 
therapeutic coagulation, in which case the adequate level of anticoagulation will be 
determined by the investigator). 
12. Adequate renal function, as indicated by creatinine ≤ 1.5  ULN. 
4.2 Exclusion criteria 
Subjects should not enter the study if any of the following exclusion criteria are fulfilled 
1. Prior therapy exclusions: 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 23  
 
a. Prior therapy with fulvestrant 
b. Prior therapy with tamoxifen in the metastatic setting 
c. More than 3 prior lines of endocrine therapy in the metastatic setting 
d. More than one prior line of chemotherapy in the metastatic setting 
2. Washout of 2 weeks is required for aromatase inhibitors; washout of 4 weeks is required 
for, everolimus or other biological agents with the exception of Palbociclib. 
3. Patients must not be receiving any other investigational agent. 
4. Patients with symptomatic, untreated CNS metastases are not eligible. 
5. Patients may not have significant concurrent illness, infection, pregnancy or lactation 
6. Patients must not have a different active malignancy, except for skin basal cell carcinoma, 
skin squamous cell carcinoma and cervical intraepithelial neoplasia. 
 
5. STUDY CONDUCT 
5.1 Randomization Procedures 
 
5.1.1 Regulatory Requirements 
Before a site may enter patients, protocol-specific regulatory and other documents must be 
submitted to the Coordinating Center at the University of Pittsburgh Cancer Institute (UPCI), 
as noted in study materials. Detailed information regarding document submission and control 
is provided to each site in separate study materials. 
Once all required documents are received, reviewed and approved by the Coordinating 
Center’s designated representative(s), a patient may be enrolled. 
 
5.1.2 Patient Randomization 
A member of the study team will confirm eligibility criteria and complete the protocol - 
specific eligibility checklist. Registration must occur prior to the initiation of therapy. Any 
participant not registered to the protocol before treatment begins will be considered ineligible 
and registration will be denied. 
The Coordinating Center (UPMC Hillman Cancer Center) will review the submitted 
documents in order to verify eligibility and consent. To complete the registration process, the 
Coordinating Center will: 
1. Register the participant on the study with the UPMC Clinical Research Services 
Coordinating Center (CRSCC). 
 
2. Upon confirmation of registration, the Coordinating Center will will provide the study 
specific participant case number, and assigned treatment arm to the participating 
institution. 
 
Following registration, participants may begin protocol treatment and must begin treatment 
within 5 to 7 days. Issues that would cause treatment delays should be discussed with the 
Principal Investigator. If a participant does not receive protocol therapy 5 to 7 days following 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 24  
 
registration, the participant’s protocol status must be changed and the occurrence must be 
communicated to the UPMC CRSCC of participant status changes as soon as possible. 
5.2 Treatments 
 
5.2.1 Identity of investigational products (note commercial supply will be used for both 
drugs. Palbociclib will be provided Pfizer.) 
 
Treatment 
Dosage form and strength 
Manufacturer 
Fulvestrant 
500mg IM Q28 days 
AstraZeneca 
Tamoxifen 
Palbociclib 
20mg PO Qdaily 
125mg PO D1-21 Q28 days 
AstraZeneca 
Pfizer 
 
 
5.2.1.1 Fulvestrant 
Please refer to the package insert for complete prescribing and toxicity information. 
 
1. 
Other Names 
ICI-182, 780; Faslodex 
2. 
Classification 
Fulvestrant is an estrogen receptor down regulator. One of its main differences from 
tamoxifen and the other SERMs is its lack of agonistic activity. It represents a class of drug 
that binds, blocks and degrades the ER. 
3. 
Mode of Action 
Estrogen Receptor Antagonist. 
4. 
Storage and Stability 
Refrigerate, 2°-8°C (36°-46°F). To protect from light, store in the original carton until 
time of use. 
5. 
Administration 
Fulvestrant 500mg should be administered intramuscularly into the buttocks slowly (1- 
2 minutes per injection) as two 5mL injections, one in each buttock. 
6. 
Supply 
Fulvestrant is supplied as 50mg/ml injection in 5-mL syringes for intramuscular 
administration. 
7. 
Incompatibilities 
No known drug-drug interactions. 
 
8. 
Adverse Effects 
10% incidence reported: 
a. Endocrine & metabolic: Hot flushes (7% to 13%) 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 25  
 
b. Hepatic: Alkaline phosphatase increased (>15%; grades 3/4: 1% to 2%), 
transaminases increased (>15%; grades 3/4: 1% to 2%) 
c. Local: Injection site pain (12% to 14%) 
d. Neuromuscular & skeletal: Joint disorders (14% to 19%) 
1% to 10% incidence reported: 
a. Cardiovascular: Ischemic disorder (1%) 
b. Central nervous system: Fatigue (8%), headache (8%) 
c. Gastrointestinal: Nausea (10%), anorexia (6%), vomiting (6%), constipation (5%), 
weight gain (≤1%) 
d. Genitourinary: Urinary tract infection (2% to 4%) 
e. Neuromuscular & skeletal: Bone pain (9%), arthralgia (8%), back pain (8%), 
extremity pain (7%), musculoskeletal pain (6%), weakness (6%) 
f. Respiratory: Cough (5%), dyspnea (4%) 
<1% incidence reported: (Limited to important or life-threatening; reported with 250 
mg or 500 mg dose): Angioedema, hepatitis, hypersensitivity reactions, leukopenia, liver 
failure, osteoporosis, thrombosis, vaginal bleeding 
5.2.1.2 Tamoxifen 
Note: Please refer to package insert for complete prescribing and toxicity information. 
1. 
Other Names 
Soltamox, Nolvadex 
2. 
Classification 
Tamoxifen is a selective estrogen receptor modulator that has antagonistic and 
agonistic effect on the estrogen receptor, depending on the target tissue. 
3. 
Mode of Action 
Selective Estrogen Receptor Modulator. 
4. 
Storage and Stability 
Store at controlled room temperature, 20-25°C (68-77°F). Dispense in a well-closed, 
light-resistant container. 
5. 
Administration 
20 mg orally daily. 
6. 
Supply 
Tamoxifen is supplied in 20mg tablets in bottles containing 90 tablets. 
7. 
Incompatibilities 
Please see the Tamoxifen Package Insert for more details on the known medication 
interactions. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 26  
 
8. 
Adverse Effects 
10% incidence reported: 
 
a. Cardiovascular: Vasodilation (41%), flushing (33%), hypertension (11%), 
peripheral edema (11%) 
b. Central nervous system: Mood changes (12% to 18%), pain (3% to 16%), 
depression (2% to 12%) 
c. Dermatologic: Skin changes (6% to 19%), rash (13%) 
d. Endocrine & metabolic: Hot flashes (3% to 80%), fluid retention (32%), altered 
menses (13% to 25%), amenorrhea (16%) 
e. Gastrointestinal: Nausea (5% to 26%), weight loss (23%), vomiting (12%) 
f. Genitourinary: Vaginal discharge (13% to 55%), vaginal bleeding (2% to 23%) 
g. Neuromuscular & skeletal: Weakness (18%), arthritis (14%), arthralgia (11%) 
h. Respiratory: Pharyngitis (14%) 
i. Miscellaneous: Lymphedema (11%) 
1% to 10% incidence reported: 
a. Cardiovascular: Chest pain (5%), venous thrombotic events (5%), edema (4%), 
cardiovascular ischemia (3%), angina (2%), deep venous thrombus (≤2%), MI (1%) 
b. Central nervous system: Insomnia (9%), dizziness (8%), headache (8%), anxiety 
(6%), fatigue (4%) 
c. Dermatologic: Alopecia (≤5%) 
d. Endocrine & metabolic: Oligomenorrhea (9%), breast pain (6%), menstrual 
disorder (6%), breast neoplasm (5%), hypercholesterolemia (4%) 
e. Gastrointestinal: Abdominal pain (9%), weight gain (9%), constipation (4% to 8%), 
diarrhea (7%), dyspepsia (6%), throat irritation (oral solution 5%), abdominal 
cramps (1%), anorexia (1%) 
f. 
Genitourinary: Urinary tract infection (10%), leukorrhea (9%), vaginal hemorrhage 
(6%), vaginitis (5%), vulvovaginitis (5%), ovarian cyst (3%) 
g. Hematologic: Thrombocytopenia (≤10%), anemia (5%) 
h. Hepatic: AST increased (5%), serum bilirubin increased (2%) 
i. 
Neuromuscular & skeletal: Back pain (10%), bone pain (6% to 10%), osteoporosis 
(7%), fracture (7%), arthrosis (5%), joint disorder (5%), myalgia (5%), paresthesia 
(5%), musculoskeletal pain (3%) 
j. 
Ocular: Cataract (7%) 
k. Renal: Serum creatinine increased (≤2%) 
l. 
Respiratory: Cough (4% to 9%), dyspnea (8%), bronchitis (5%), sinusitis (5%) 
Less than 1% or frequency not defined: Angioedema, bullous pemphigoid, cholestasis, 
corneal changes, endometrial cancer, endometrial hyperplasia, endometrial polyps, 
endometriosis, erythema multiforme, fatty liver, hepatic necrosis, hepatitis, hypercalcemia, 
hyperlipidemia, hypersensitivity reactions, hypertriglyceridemia, impotence (males), 
interstitial pneumonitis, loss of libido (males), pancreatitis, phlebitis, pruritus vulvae, 
pulmonary embolism, retinal vein thrombosis, retinopathy, second primary tumors, Stevens- 
Johnson syndrome, stroke; tumor pain and local disease flare (including increase in lesion size 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 27  
 
and erythema) during treatment of metastatic breast cancer (generally resolves with 
continuation); uterine fibroids, vaginal dryness, visual color perception changes 
 
 
5.2.1.2 Palbociclib 
Note: Please refer to package insert for complete prescribing and toxicity information. 
9. 
Other Names 
10. 
Classification 
CDK4/6 inhibitor. 
11. 
Mode of Action 
CDK4/6 inhibitor 
 
12. 
Storage and Stability 
Palbociclib capsules and blisters should be stored at controlled and monitored room 
temperature specified on the product labels. Further storage and stability conditions are stated 
in the palbociclib IB. Investigators and site staff are requested to check storage temperatures 
daily (i.e. manually or by using alarm systems to alert of any excursions) and ensure that 
thermometers are working correctly as required for proper storage of investigational products. 
 
Deviations from the storage requirements, including any actions taken, must be documented 
and reported to AFT. Once a deviation is identified, palbociclib must be quarantined and not 
used until receipt of documentation of permission to use the investigational product. 
 
Medication should be kept in a secured locked area at the study site in accordance with 
applicable regulatory requirements. Medication which has been returned by the patients 
should be stored separately from medication that needs to be dispensed. 
 
13. 
Administration 
125 mg orally daily, day 1-21 of a 28 day cycle. 
Palbociclib should be taken orally, once per day with food around the same time, starting at 
the 125-mg dose. Treatment is continuous daily for 21 days, followed by 7 days off, to 
complete a 28-day cycle. 
 
Expected toxicities and potential risks as well as allowable dose modifications are described in 
section 9. No investigational or commercial agents or therapies other than those described 
below may be administered with the intent to treat the participant’s malignancy within the 
Treatment Phase. 
 
Administration is performed on an outpatient, self-administration basis. On days when a 
patient is scheduled for a clinic visit (dispensing visit), the patient should take the scheduled 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 28  
 
palbociclib dose once all visit assessments have been performed and are within acceptable 
range unless otherwise indicated. 
 
Missed doses of palbociclib (meaning doses that are missed for one day) should not be made 
up. For example, if a dose is entirely missed for one day, dose should be skipped and NOT 
retaken the next day; patients should resume regular dosing as prescribed the following day. If 
a dose is vomited at any time after taking palbociclib, a replacement dose should NOT be 
taken. Patients who inadvertently take 1 extra dose during a day must skip the next day’s dose 
and notify his or her treating physician. If a patient takes more than two doses of palbociclib 
in a day, the patient should bring this to the attention of his or her treating physician. 
 
Patients should be instructed to record daily administration of the study drugs in a drug or 
medication diary. 
 
14. 
Supply 
 
Palbociclib will be manufactured by Pfizer and provided by the study Sponsor as capsules 
containing 75 mg, 100 mg, or 125 mg equivalents of palbociclib free base. Palbociclib will be 
supplied to sites in High Density Polyethylene (HDPE) bottles containing 75 mg, 100 mg, or 
125 mg capsules and are labeled for clinical use. 
15. 
Incompatibilities 
Please see the Palbociclib Package Insert for more details on the known medication 
interactions. 
16. 
Adverse Effects 
Likely (greater than or equal to 50% chance that this will happen): 
• Neutropenia. A condition in which the number of white bloods cells called neutrophils is 
abnormally low. This increases the risk of infection, which may be serious or life threatening. 
You will be monitored closely for this risk. 
Frequent (between a 10 to 50% chance that this will happen): 
• Fatigue. Participants experiencing fatigue while taking palbociclib should exercise caution 
when driving or operating machinery. 
• Weakness 
• Low number of red blood cells that can causes tiredness and shortness of breath. May require 
a blood transfusion. (Anemia) 
• Diarrhea 
• Nausea 
• Low number of platelets, which may cause bleeding and bruising. Bleeding may be serious 
or life threatening and may require a blood transfusion. 
• Decreased appetite 
• Constipation 
• Mouth blisters/sores 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 29  
 
• Infection of the sinus or lung 
• Vomiting 
• Loss of touch or sensation of pins and needles or numbness on the skin 
 
Occasional (Between a 1 to 10% chance that this will happen): 
• Rash 
• Bloating 
• Temporary hair loss 
• Swelling in extremities (hands and feet) 
• Headache 
• Nosebleed 
• Muscle spasm 
• Inflammation of the mucous membranes 
• Fever 
• Dry mouth 
• Fever with dangerously low white blood cell count 
• Abnormal taste 
 
Rare (Less than a 1 % chance that this will happen): 
• Abnormal electrical conduction within the heart which may lead to arrhythmias or irregular 
heartbeat 
• A blood clot that causes a sudden blockage in a lung blood vessel, usually due to a 
blood clot that traveled to the lung from the leg. A pulmonary embolism is a serious condition 
that can cause: permanent damage to part of your lung from lack of blood flow to lung tissue; 
low oxygen levels in your blood; damage to other organs in your body from not getting 
enough oxygen. If a clot is large, or if there are many clots, a pulmonary embolism can cause 
death. 
 
5.2.2 Doses and treatment regimens 
Fulvestrant Arm 
1. Loading dose: Patients will receive a loading dose of 500mg of fulvestrant 
administered as two 250 mg intramuscular (IM) injections, one into each buttock, on 
day 1 and day 15. 
2. Maintenance Dose: Patients will receive fulvestrant 500mg IM administered as two 
250mg intramuscular (IM) injections, one into each buttock, once every 4 weeks 
starting on day 29. Each cycle will consist of 28 days during which fulvestrant will be 
administered on day 1. 
3. Palbociclib dose: Patients will receive 125mg PO days 1-21 of a 28 day cycle. 
 
Tamoxifen Arm 
 
1. Patients will receive tamoxifen 20mg PO daily 
2. Palbociclib dose: Patients will receive 125mg PO days 1-21 of a 28 day cycle. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 30  
 
5.2.3 Dose adjustment/modifications 
Typically both tamoxifen and fulvestrant are well tolerated standard agents. There are not any 
a priori dose modifications for toxicity, treatment should continue until progression occurs. 
In unusual circumstances where fulvestrant or tamoxifen must be discontinued prior to disease 
progression due to intolerable fulvestrant-associated toxicity, the UPMC Hillman Cancer 
Center study PI should be contacted. Tamoxifen can be continued at investigator’s discretion 
if thrombosis occurs and if adequately anti-coagulated. 
Investigators can hold either agent for up to 3 weeks at their discretion (patient travel, clinic 
holiday, etc.). If the delivery of any study drug is delayed for more than 3 weeks due to 
toxicity, that drug should be permanently discontinued. 
If an overdose occurs in the course of the study, then investigators or other site personnel 
inform appropriate AstraZeneca and Pfizer representatives within one day, i.e., immediately 
but no later than the end of the next business day of when the PI becomes aware of it. The 
designated AstraZeneca and/or Pfizer representative works with the investigator to ensure that 
all relevant information is provided to the AstraZeneca and Pfizer Patient Safety data entry 
sites. 
Palbociclib dose modifications should be performed according to prescribing information. 
 
5.3 Restrictions and concomitant therapies during the study 
The following restrictions apply during the entire duration of the study: 
1. No other investigational therapy is allowed during the study period. If an investigational 
therapy was used prior to study enrollment, a washout period of 28 days is required 
before enrollment. 
2. No anticancer agents other than the study medications may be given to patients during 
the study period, with the following exceptions: 
1. Bisphosphonates, Zometa (zoledronic acid), or Xgeva (Denosumab) 
2. GnRH analog agents. 
3. If any other anticancer agents are required for a patient then the patient must first be 
withdrawn from the study. 
4. Concurrent radiation therapy is prohibited, however palliative radiotherapy may be 
considered on a case by case basis after discussion with the Study Chair. 
5. Grapefruit and other citrus juices affect P450 and PgP activity. Concomitant use will be 
avoided. All CYP3A4 inducers and inhibitors should be avoided. 
6. Medications that inhibit or induce CYP2D6, , CYP2C8/9 and CYP2D6 will be avoided 
and used at the discretion of the principal investigator or treating physician on a case by 
case basis. Please see appendix A for a list of these medications. 
7. Anticoagulant medications can be used during the study period. The appropriate PT, 
INR and PTT levels will be determined at the discretion of the treating physician. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 31  
 
5.4 Treatment adherence 
The PI will determine when lack of adherence to the study protocol should lead to removal 
from study. The patient removed for lack of adherence will still be included in the ITT 
analysis. 
5.5 Discontinuation of treatment 
Discontinuation of the fulvestrant plus Palbociclibor tamoxifen plus Palbociclib will occur in 
the case of unacceptable toxicity, disease progression, patient's own request, treating 
physician's discretion or if the study is closed. 
5.6 Procedures for discontinuation of a subject from treatment 
All patients who initiate protocol treatment will be included in the overall evaluation of 
response as part of the ITT analysis. All reasons for discontinuation of treatment should be 
documented clearly in the study record. In case of treatment discontinuation due to 
unacceptable toxicity as defined in section 6.4, the patients will still be included in the overall 
evaluation of response by ITT. If the study medication is discontinued at the patient’s own 
request, the reason for discontinuation from the study must be documented and the patient 
followed for outcomes by ITT analysis. 
5.7 Training of study site personnel 
The participating sites will provide experienced staff, and adequate equipment and facilities to 
support this clinical trial. The participating sites will also be responsible for research staff 
training in computer applications, human subject research, and HIPAA compliance, as well as 
the continuing education in these areas as required by local institutional standards. 
 
 
5.8 Withdrawal from study 
Withdrawal from study may occur at patient’s own request. The reason for discontinuation 
from the study must be documented. The patients will be included in the overall evaluation of 
response by ITT. The patient’s permission for active and passive follow-up will be sought, 
with response censored at the time of study withdrawal only if the patient withdraws consent 
for any follow-up. 
 
6. COLLECTION OF STUDY VARIABLES 
6.1 Recording of data 
All information will be collected on study-specific case report forms by the study staff at each 
institution. The necessary forms will be provided to each site by the UPMC Hillman Cancer 
Center CRSCC. The 
Coordinating Center staff will enter the data required by the protocol within 10 days into the 
Electronic Case Report Forms (eCRF). The Principal Investigator is responsible for assuring 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 32  
 
that the data entered into eCRF is complete, accurate, and that entry and updates are 
performed in a timely manner. 
6.2 Data collection at enrollment and follow-up 
The completed forms will be forwarded vie email or fax to the Coordinating Center for central 
review and inclusion in the study dataset with relevant source documentation as outlined in 
the case report forms. The data submission schedule is as follows: 
1. At the time of registration: 
a. Registration Forms 
b. Informed Consent Form (signed by the subject and approved investigator) 
c. Eligibility Checklist 
d. Source documents related to eligibility and randomization 
e. Research tissue samples with documentation, if baseline tumor biopsy is 
collected. 
 
2. At the start of each new cycle: 
a. Adverse event forms 
b. Drug diary (both arms) 
c. Study blood along with documentation (sent on same day of draw) 
d. Pertinent medication list 
 
3. Final Assessment (off-treatment): 
a. AE form 
b. Pertinent source documents 
c. Research tissue/blood samples with documentation 
 
4. Follow-up (disease status, survival every 3 months) 
 
All study data will be reviewed for completeness and accuracy by the Protocol Chair or 
his/her designee. The Principal Investigator (or his/her designee) at each respective institution 
is responsible for review, and ensuring the completeness and accuracy, of the data generated 
by his/her institution. 
Peripheral blood collected during the study will be sent to UPMC Magee-Womens Research 
Institute for analysis of ESR1 mutation. Solid tumor biopsies will be sent to the Magee- 
Womens Hospital of UPMC Department of Pathology for analysis. When solid tumor 
biopsies are obtained, one core will be sent for MammoSeq testing, which will include ESR 
assessment as well as other genes described above. This test will be performed at the Division 
of Molecular Genomic Pathology of UPMC Magee-Womens Hospital. The other core will be 
used for the NanoString studies. A 3rd core, if obtained, will be banked as fresh frozen tissue 
to be used in the event that the two (2) other core biopsy samples do not contain sufficient 
usable tissue for either analysis of ESR1 mutation and/or MammoSeq testing . 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 33  
 
6.3 Efficacy 
Response and progression will be evaluated in this study using the international criteria 
proposed by the revised Response Evaluation Criteria in Solid Tumours (RECIST) Committee 
(version 1.1). Changes in only the largest diameter (unidimensional measurement) of the 
tumor lesions are used in the RECIST criteria. Patients will be evaluated for response after 
every 2 cycles of therapy. In the case of non-measurable disease (as defined in 6.3.2), 
progression of disease will be determined by the appearance of new metastatic lesions or 
clinical worsening secondary to the non-measurable metastatic site at the treating physician's 
discretion. 
 
6.3.1 Measurable systemic disease 
Tumor lesions: Must be accurately measured in at least one dimension (longest diameter in the 
plane of measurement is to be recorded) with a minimum size of: 
• 
10mm by CT scan (CT scan slice thickness no greater than 5 mm). 
• 
10mm calliper measurement by clinical exam (lesions which cannot be accurately 
measured with callipers should be recorded as non-measurable). 
• 
20mm by chest X-ray. 
Malignant lymph nodes: To be considered pathologically enlarged and measurable, a lymph 
node must be ≥15mm in short axis when assessed by CT scan (CT scan slice thickness 
recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis 
will be measured and followed. 
 
6.3.2 Non-measurable disease 
All other lesions, including small lesions (longest diameter <10mm or pathological lymph 
nodes with ≥10 to <15mm short axis) as well as truly non-measurable lesions. Lesions 
considered truly non-measurable include: leptomeningeal disease, ascites, pleural or 
pericardial effusion, inflammatory breast disease, lymphangitic involvement of skin or lung, 
abdominal masses/abdominal organomegaly identified by physical exam that is not 
measurable by reproducible imaging techniques. 
 
Special considerations regarding lesion measurability 
Bone lesions, cystic lesions, and lesions previously treated with local therapy require 
particular comment: 
Bone lesions: 
• 
Bone scan, PET scan or plain films are not considered adequate imaging techniques to 
measure bone lesions. However, these techniques can be used to confirm the presence 
or disappearance of bone lesions. 
• 
Lytic bone lesions or mixed lytic-blastic lesions, with identifiable soft tissue 
components, that can be evaluated by cross-sectional imaging techniques such as CT 
or MRI can be considered as measurable lesions if the soft tissue component meets the 
definition of measurability described above. 
• 
Blastic bone lesions are non-measurable. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 34  
 
Cystic lesions: 
• 
Lesions that meet the criteria for radiographically defined simple cysts should not be 
considered as malignant lesions (neither measurable nor non-measurable) since they 
are, by definition, simple cysts. 
• 
‘Cystic lesions’ thought to represent cystic metastases can be considered as 
measurable lesions, if they meet the definition of measurability described above. 
However, if non-cystic lesions are present in the same patient, these are preferred for 
selection as target lesions. 
Lesions with prior local treatment: 
• Tumor lesions situated in a previously irradiated area, or in an area subjected to other 
loco-regional therapy, are usually not considered measurable unless there has been 
demonstrated progression after local treatment. 
 
6.3.3 Specifications by methods of measurements 
Measurement of lesions 
All measurements should be recorded in metric notation, using callipers if clinically assessed. 
All baseline evaluations should be performed as close as possible to the treatment start and 
never more than 28 days before the beginning of the treatment. 
Method of assessment 
The same method of assessment and the same technique should be used to characterise each 
identified and reported lesion at baseline and during follow-up. Imaging based evaluation 
should always be done rather than clinical examination unless the lesion(s) being followed 
cannot be imaged but are assessable by clinical exam. 
Clinical lesions: Clinical lesions will only be considered measurable when they are superficial 
and ≥10mm diameter as assessed using callipers (e.g., skin nodules). For the case of skin 
lesions, documentation by color photography including a ruler to estimate the size of the 
lesion is suggested. When lesions can be evaluated by both clinical exam and imaging, 
imaging evaluation should be undertaken since it is more objective and may also be reviewed 
at the end of the study. 
Chest X-ray: Chest CT is preferred over chest X-ray, particularly when progression is an 
important endpoint, since CT is more sensitive than X-ray, particularly in identifying new 
lesions. However, lesions on chest X-ray may be considered measurable if they are clearly 
defined and surrounded by aerated lung. 
CT, MRI: CT is the best currently available and reproducible method to measure lesions 
selected for response assessment. Guidelines have defined measurability of lesions on CT scan 
based on the assumption that CT slice thickness is 5mm or less. When CT scans have slice 
thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the 
slice thickness. 
MRI is also acceptable in certain situations (e.g. for body scans). 
Ultrasound: Ultrasound is not useful in assessment of lesion size and should not be used as a 
method of measurement. Ultrasound examinations cannot be reproduced in their entirety for 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 35  
 
independent review at a later date and, because they are operator dependent, it cannot be 
guaranteed that the same technique and measurements will be taken from one assessment to 
the next. If new lesions are identified by ultrasound in the course of the study, confirmation by 
CT or MRI is advised. If there is concern about radiation exposure at CT, MRI may be used 
instead of CT in selected instances. 
Endoscopy, laparoscopy: The utilisation of these techniques for objective tumor evaluation is 
not advised. However, they can be useful to confirm complete pathological response when 
biopsies are obtained or to determine relapse in trials where recurrence following complete 
response or surgical resection is an endpoint. 
Tumor markers: Tumor markers alone cannot be used to assess objective tumor response. If 
markers are initially above the upper normal limit, however, they must normalise for a patient 
to be considered in complete response. 
Cytology, histology: These techniques can be used to differentiate between PR and CR in rare 
cases if required by protocol (for example, residual lesions in tumor types such as germ cell 
tumors, where known residual benign tumors can remain). When effusions are known to be a 
potential adverse effect of treatment (e.g. with certain taxane compounds or angiogenesis 
inhibitors), the cytological confirmation of the neoplastic origin of any effusion that appears 
or worsens during treatment can be considered if the measurable tumor has met criteria for 
response or stable disease in order to differentiate between response (or stable disease) and 
progressive disease. 
 
6.3.4 Response Assessment 
Target Lesions 
When more than one measurable lesion is present at baseline all lesions up to a maximum 
of five lesions total (and a maximum of two lesions per organ) representative of all involved 
organs should be identified as target lesions and will be recorded and measured at baseline 
(this means in instances where patients have only one or two organ sites involved a 
maximum of two and four lesions respectively will be recorded). 
Evaluation of Target Lesions 
 
• Complete Response (CR): Disappearance of all target lesions. Any pathological lymph 
nodes (whether target or non-target) must have reduction in short axis to <10 mm. 
 
• Partial Response (PR): At least a 30% decrease in the sum of diameters of target 
lesions, taking as reference the baseline sum diameters. 
 
• Progressive Disease (PD): At least a 20% increase in the sum of diameters of target 
lesions, taking as reference the smallest sum on study (this includes the baseline sum 
if that is the smallest on study). In addition to the relative increase of 20%, the sum 
must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of 
one or more new lesions is also considered progression). 
 
• Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 36  
 
increase to qualify for PD, taking as reference the smallest sum of diameters while on 
study. 
 
Non-target lesions 
All other lesions (or sites of disease) including pathological lymph nodes should be 
identified as non-target lesions and should also be recorded at baseline. Measurements are 
not required and these lesions should be followed as ‘present’, ‘absent’, or in rare cases 
‘unequivocal progression’. In exceptional circumstances, unequivocal progression in non- 
target lesions may be accepted as disease progression. In addition, it is possible to record 
multiple non-target lesions involving the same organ as a single item on the case record 
form (e.g. ‘multiple enlarged pelvic lymph nodes’ or ‘multiple liver metastases’). 
When the patient only has non-measurable disease, the increase in overall disease burden 
should be comparable in magnitude to the increase that would be required to declare PD for 
measurable disease: i.e., an increase in tumor burden from “trace” to “large”, an increase in 
nodal disease from “localized” to “widespread”, or an increase sufficient to require a change 
in therapy. 
Evaluation of Non-Target Lesions 
 
• Complete Response (CR): Disappearance of all non-target lesions and normalisation 
of tumor marker level. All lymph nodes must be non-pathological in size (<10mm 
short axis). 
 
• Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance 
of tumor marker level above the normal limits. 
 
• Progressive Disease (PD): Unequivocal progression of existing non-target lesions. 
(Note: the appearance of one or more new lesions is also considered progression). 
 
 
Evaluation of Best Overall Response 
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started). The patient’s best overall response 
assignment will depend on the findings of both target and non-target disease and will also 
take into consideration the appearance of new lesions. 
TABLE 2. Time to response definition 
 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 37  
 
 
CR 
CR 
No 
CR 
CR 
Non CR/Non 
PD 
No 
PR 
CR 
Not 
evaluated 
No 
PR 
PR 
Non PD or 
not all 
evaluated 
No 
PR 
SD 
Non PD or 
not all 
evaluated 
No 
SD 
Not all 
evaluated 
Non PD 
No 
NE 
PD 
Any 
Yes or no 
PD 
Any 
PD 
Yes or no 
PD 
Any 
Any 
Yes 
PD 
CR= complete response, PR= partial response, PD= progressive disease, SD= stable disease, 
NE= not evaluable. 
 
Note: Patients with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be reported 
as “symptomatic deterioration”. Every effort should be made to document the objective 
progression even after discontinuation of treatment. 
 
Progression Free Survival 
PFS is defined as the duration of time from time of randomization to time of progression or 
death, whichever occurs first. PFS for a subject without an event will be censored on the date 
of last tumor assessment. If an interval of 6 months passes without a tumor assessment, PFS 
will be censored at the time of the earlier tumor assessment, even if an event (progressive 
disease or death) is later observed. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 38  
 
6.3.5 Correlative Studies 
We will determine the prevalence of ESR1 mutations in plasma and metastatic biopsies of 
patients with metastatic breast cancer. We have an ongoing program for Sanger sequencing 
based detection of ESR1-mut in tumor specimens in the Division of Molecular Genomic 
Pathology of UPMC Magee-Womens Hospital . Detection of ESR1-mut in ctDNA will be 
performed at the UPMC Pathology laboratories. Patients in the trial will have blood assessed 
serially for the presence or absence of ESR1-mt to determine the mutational prevalence in a 
2nd -3rd line ER+ MBC population with prior tamoxifen/AI exposure. We will also be able to 
follow the ctDNA levels over time in response to treatment. Patients with disease amenable to 
biopsy will be offered the option and encouraged to have a metastatic site biopsy performed 
prior to study treatment and again at the time of progression. The hypothesis is that circulating 
levels of ESR-mut will be detectable in 25% of patients in this setting (consistent with our 
pilot study) and will decrease in response to fulvestrant. Correlation of plasma results and 
tumor results will help to determine concordance between these two measurements as well as 
to assess for potential heterogeneity between plasma and a specific metastatic tumor site. For 
instance, while ESR1-mut have been previously identified in a number of metastatic sites 
including lymph node and liver metastases, there has not been identification of these 
mutations in bone specimens. Whether this is an effect of the tissue processing or whether 
there is truly biologic relevance to this remains to be seen. We may find that in plasma the 
detection of ESR1-mut may be much higher than the prevalence seen in metastatic biopsies 
for that reason (i.e. bone only ER+MBC). We will also perform ddPCR and NGS in ctDNA 
and tumor biopsies in conjunction with AstraZeneca scientists, as an additional exploratory 
objective for ESR and other co-existent mutations of interest. . 
These tumor biopsies will be assessed for the most commonly described ESR1 mutations 
using Sanger sequencing of the three ESR1 exons, which will assess for 23 different ESR1 
mutations (including the 6 most commonly described: S463P, L536Q, Y537S, Y537C, 
Y537N, and D538G)). These biopsies will also undergo next generation sequencing for other 
mutations that may be associated with ESR1-mut and could affect response to fulvestrant. 
This will be done using the MammoSeq assay developed at the UPMC Hillman Cancer 
Center, which includes genes commonly mutated in breast cancer, common actionable 
mutations in non-breast cancers, rare but actionable mutations such as ErbB2, as well as copy 
number amplifications and deletions. This will allow for determination of other mutations, 
such as PIK3CA, that are frequently encountered in ER+ MBC and could either be passenger 
mutations with the ESR1-mut or driver mutations that will provide greater influence over 
response to therapy. (see Table 3) Assessment of these mutations may also help to identify 
subsequent therapies for patients. As there is not any prospective data that currently uses 
molecularly targeted therapies towards other cancer genes (with exception of ER and HER-2) 
to guide therapies as a standard of care, we do not feel this should influence subsequent 
enrollment onto this trial of a standardly used agent. The hypothesis is that while 25% of ER+ 
MBC patients will have the most common ESR1-mut present in tumor specimens; there may 
be additional patients who will have other ESR1 alterations that will affect ESR1 activity and 
response to treatment. We will ask for archived tumor samples as well so that we can 
determine if the ESR1 mutation was acquired as a result of previous endocrine therapy or 
present at the time of diagnosis. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 39  
 
Metastatic biopsies obtained in the clinical trial will also undergo molecular analysis to 
determine the activity of mutant ESR1. Although tumors harboring ESR1 mutations appear to 
be acquired as a result of estrogen deprivation therapy (1-6), and patients with these particular 
tumors have a much worse overall survival (7), relatively little is known, mechanistically, 
about the role these mutants have in metastatic disease progression and tropism. We 
hypothesize that ESR1 mutants confer a selective advantage due not only to their intrinsic 
constitutive activity in the relative absence of estrogen, but also to their propensity to 
preferentially transactivate specific target genes, in response to microenvironment cues, that 
are required for tumor cell survival, colonization, and metastatic outgrowth. Thus, we 
anticipate that ER target gene expression profiles obtained from primary tumors and their 
matched ESR1-expressing metastatic counterparts will display distinct qualitative and/or 
quantitative differences. Furthermore, we hypothesize that a subset of genes from these 
derived differential gene signatures will be shared in metastases from the same distant sites 
among different patients harboring one of several ESR1 point mutations or gene fusions that 
also confer constitutive activity. This convergent mutant ER gene signature could then provide 
mechanistic insights into ESR1 metastatic dependencies and thus serve as a prognostic 
indicator of metastatic progression (and tropism) and as a predictive marker of therapeutic 
response to HD fulvestrant and next generation mutant ER-directed SERDs, SERMs, and 
allosteric antagonists. Most importantly, this approach offers the opportunity to correlate 
specific target gene expression with fulvestrant dose and clinical outcome and thereby 
elucidate the molecular basis for ESR1 conferred partial resistance to fulvestrant. Identifying 
these mutant ER-dependent essential genes could then define particular targeted combination 
strategies to address this resistance. 
Our laboratories have developed and implemented methods for determining gene expression 
profiles from formalin-fixed paraffin embedded (FFPE) solid tumor biopsies that will be 
collected. We will use NanoString nCounter technology, which enables a highly multiplexed 
direct profiling of individual RNA molecules without amplification and is ideally suited for 
degraded RNA from small biopsy material (requiring only 100ng of RNA). We previously 
used nCounter to measure expression of genes (n=158) in the five major breast cancer 
molecular tests (PAM50, Oncotype Dx, BCI, MammaPrint and EndoPredict) and in the 78 
cases of primary breast cancer measured we found the platform to be robust and highly 
reproducible. For this proposal, we have developed a wild type ERa target gene expression 
profile (n=208 genes) which is robustly increased or decreased by estrogen stimulation of 
ERa. Initially, to help validate this approach for discerning differences in ER-target gene 
expression profiles between wildtype and mutant ER at different estrogen and fulvestrant 
concentrations, we used CRISPR/cas9 targeted genome editing to generate isogenic pairs of 
T47D-derived cell lines expressing wildtype ER and its mutant (D538G)-expressing 
counterpart. Since it is quite possible that mutant ER can have specific target genes distinct 
from those of wild type ER (and therefore not be represented in our current probe kit), we will 
also conduct a gene expression microarray analysis using the isogenic cell line pair to identify 
potential mutant ER-specific target genes and accordingly expand our NanoString-based 
probe kit. The effect of estrogen and fulvestrant on gene expression profiles will be measured 
in vitro to produce a pharmacodynamic profile of receptor activity 
TABLE 3. MammoSeq gene list: 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 40  
 
 
ABL1 
CDK4* 
FGFR4* 
KIT 
NOTCH1 
AKT1 
CDKN1B 
FOXA1 
KRAS 
NRAS 
AKT3* 
CDKN2A 
GATA3 
MAP2K4 
PAK1* 
ALK 
CTNNB1 
GRB7* 
MAP3K1 
PDGFRA 
AR* 
DNAH14 HIST2H2BE MAP3K4 
PIK3CA* 
ARID1A 
EGFR* 
HLA-A 
MDM2* 
PIK3R1 
ATM 
ERBB2* 
HRAS 
MDM4* 
PTCH1 
AURKA* 
ERBB3* 
IDH1 
MET* 
PTEN 
AURKB* 
ERBB4 
IGF1R* 
MLL3 
RB1 
BRAF 
ESR1* 
INPP4B* 
MTOR 
RET 
BRCA1 
EZH2 
INSR* 
MYC* 
RPTOR 
BRCA2 
FGF19** 
JAK2* 
NCOA3* 
RUNX1 
CCND1* 
FGFR1* 
JAK3* 
NCOR1 
SMO 
CCNE1* 
FGFR2* 
JUN 
NCOR2 
STK11 
CDH1 
FGFR3 
KDR 
NF1 
TP53 
6.4 Safety 
6.4.1 Definition of adverse events 
An adverse event is the development of an undesirable medical condition or the deterioration 
of a pre-existing medical condition following or during exposure to a pharmaceutical product, 
whether or not considered causally related to the product. An undesirable medical condition 
can be symptoms (e.g., nausea, chest pain), signs (e.g., tachycardia, enlarged liver) or the 
abnormal results of an investigation (e.g., laboratory findings, electrocardiogram). In clinical 
studies, an AE can include an undesirable medical condition occurring at any time, including 
run-in or washout periods, even if no study treatment has been administered. The term AE is 
used to include both serious and non-serious AEs. Definition of AE will be in accordance to 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 41  
 
the CTCAE version 4.03. All grades of AE's will be captured, greater than grade 1. AE's will 
start to be captured on day 1 of study drug administration. 
6.4.2 Definitions of serious adverse event (SAE) 
A serious adverse event is an AE occurring during any study phase (i.e., run-in, treatment, 
washout, follow-up), that fulfils one or more of the following criteria: 
• Results in death 
• Is immediately life-threatening 
• Requires in-patient hospitalization or prolongation of existing hospitalization 
• Results in persistent or significant disability/incapacity or substantial disruption of 
the ability to conduct normal life functions 
• Results in a congenital abnormality or birth defect 
• Is an important medical event that may jeopardize the subject or may require medical 
intervention to prevent one of the outcomes listed above. 
 
6.4.3 Recording of Adverse Events 
General: All adverse events will be captured on the appropriate study-specific case 
report forms (CRFs). 
 
Serious Adverse Events: All serious adverse events, regardless of causality to study 
drug, will be reported to the Principal Investigator and/or the Study Coordinator 
at each institution, and also to the Coordinating Center. 
 
All serious adverse events must be reported to the Coordinating Center within 1 business day 
after the investigator becomes aware of the event. Events should be reported using a 
MedWatch form (3500) as available on the FDA website 
(http://www.fda.gov/Safety/MedWatch). Follow-up information must also be reported within 
1 business day of receipt of the information by the investigator. 
The Coordinating Center will disseminate information regarding serious adverse events to the 
participating sites within 5 days of review of the information by the Protocol Chair (or her 
designee in the event of extended absence) only in the case that the event(s) is believed to be 
related (i.e., possibly, probably, or definitely) to the study medication. 
The following variables will be collected for each AE: 
• AE (verbatim) 
• The date when the AE started and stopped 
• CTCAE grade 
• Whether the AE is serious or not 
• Investigator causality rating against the Investigational Product (yes or no) 
• Action taken with regard to investigational product 
• AE caused subject’s withdrawal from study (yes or no) 
• Outcome. 
 
In addition, the following variables will be collected for SAEs: 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 42  
 
• Date AE met criteria for serious AE 
• Date Investigator became aware of serious AE 
• AE is serious due to 
• Date of hospitalization 
• Date of discharge 
• Probable cause of death 
• Date of death 
• Autopsy performed 
• Causality assessment in relation to Study procedure(s) 
• Causality assessment in relation to Other medication 
• Causality assessment in relation to Additional Study Drug 
• Description of AE. 
 
It is important to distinguish between serious and severe AEs. Severity is a measure of 
intensity whereas seriousness is defined. An AE of severe intensity need not necessarily be 
considered serious. For example, nausea that persists for several hours may be considered 
severe nausea, but not a SAE. On the other hand, a stroke that results in only a limited degree 
of disability may be considered a mild stroke but would be a SAE. 
Adverse Events based on signs and symptoms 
When collecting AEs, the recording of diagnoses is preferred (when possible) to recording a 
list of signs and symptoms. However, if a diagnosis is known and there are other signs or 
symptoms that are not generally part of the diagnosis, the diagnosis and each sign or symptom 
will be recorded separately. 
Adverse Events based on examinations and tests 
If deterioration in a laboratory value/vital sign is associated with clinical signs and symptoms, 
the sign or symptom will be reported as an AE and the associated laboratory result/vital sign 
will be considered as additional information. Wherever possible the reporting investigator 
uses the clinical, rather than the laboratory term (e.g., anemia versus low hemoglobin value). 
In the absence of clinical signs or symptoms, clinically relevant deteriorations in non- 
mandated parameters should be reported as AE(s s). 
Deterioration of a laboratory value, which is unequivocally due to disease progression, should 
not be reported as an AE/SAE. 
Any new or aggravated clinically relevant abnormal medical finding at a physical examination 
as compared with the baseline assessment will be reported as an AE. 
Disease progression 
Disease progression can be considered as a worsening of a subject’s condition attributable to 
the disease for which the investigational product is being studied. It may be an increase in the 
severity of the disease under study and/or increases in the symptoms of the disease. The 
development of new, or progression of disease, or death attributable to breast cancer should be 
considered as disease progression and not an AE. Events, which are unequivocally due to 
disease, should not be reported as an AE. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 43  
 
6.4.4 Reporting of serious adverse events 
Investigators and other site personnel must inform the FDA, via a MedWatch form, of any 
serious or unexpected adverse events that occur in accordance with the reporting obligations 
of 21 CFR 312.32, and will concurrently forward all such reports to AstraZeneca and Pfizer. 
A copy of the MedWatch report must be faxed to AstraZeneca and Pfizer at the time the event 
is reported to the FDA. It is the responsibility of the investigator to compile all necessary 
information and ensure that the FDA receives a report according to the FDA reporting 
requirement timelines and to ensure that these reports are also submitted to AstraZeneca and 
Pfizer at the same time. 
When reporting to AstraZeneca and Pfizer, a cover page should accompany the MedWatch 
form indicating the following: 
• Investigator Sponsored Study (ISS) 
• The investigator IND number assigned by the FDA 
• The investigator’s name and address 
• The trial name/title and AstraZeneca ISS reference number 
Investigative site must also indicate, either in the SAE report or the cover page, the causality 
of events in relation to all study medications and if the SAE is related to disease progression, 
as determined by the principal investigator. 
Send SAE report and accompanying cover page to AstraZeneca by email to AE Mailbox 
Clinical Trial (TCS) <AEMailboxClinicalTrialTCS@astrazeneca.com> or by fax to 1-302- 
886-4114 (US Fax number). Email is the preferred method. Send SAE report and 
accompanying cover page to Pfizer by email to AE Mailbox Clinical Trial (TCS) <XXX> or 
by fax to 1-XXXXXX (US Fax number). Email is the preferred method. 
Serious adverse events that do not require expedited reporting to the FDA need to be 
reported to AstraZeneca and Pfizer preferably using the MedDRA coding language for 
serious adverse events. 
All SAEs have to be reported to AstraZeneca and Pfizer, whether or not considered causally 
related to the investigational product. All SAEs will be documented. The investigator is 
responsible for informing the IRB and/or the Regulatory Authority of the SAE as per local 
requirements. 
All SAE’s should be reported to AstraZeneca and Pfizer within 5 days. 
Institutional Review Board 
 
All adverse events (AEs) and serious adverse events (SAEs) will be reported to the IRB per 
current institutional standards. If an adverse event requires modification of the informed 
consent, these modifications will be provided to the IRB with the report of the adverse event. 
If an adverse event requires modification to the study protocol, these modifications will be 
provided to the IRB as soon as is possible. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 44  
 
6.4.5 Causality of Adverse Event by Study Compound 
The causality of SAEs (their relationship to all study treatment/procedures) will be assessed 
by the investigator(s) and communicated to AstraZeneca. 
• 
Definitely: An adverse event which has a timely relationship to the administration 
of the investigational drug/agent, follows a known pattern of response, for which 
no alternative cause is present. 
• 
Probably: An adverse event, which has a timely relationship to the administration 
of the investigational drug/agent, follows a known pattern of response, but for 
which a potential alternative cause may be present. 
 
• 
Possibly: An adverse event, which has a timely relationship to the administration 
of the investigational drug/agent, follows no known pattern of response, but a 
potential alternative cause does not exist. 
• 
Unlikely: An adverse event which does not have a timely relationship to the 
administration of the investigational drug/agent, follows no known pattern of 
response, does not reappear or worsen after re-administration of the 
investigational drug/agent (if applicable), and for which there is evidence that it is 
related to a cause other than the investigational drug/agent. 
 
• 
Unrelated: An adverse event, for which there is evidence that it is definitely 
related to a cause other than the investigational drug/agent. In general, there is no 
timely relationship to the administration of the investigational drug/agent, or if 
there is a timely relationship, the event does not follow a known pattern of 
response, and there is an alternative cause. 
6.4.6 Time period for collection of adverse events 
All serious adverse events must be reported to the Coordinating Center within 1 business day 
after the investigator becomes aware of the event. 
Follow-up of unresolved adverse events 
Follow-up information is sent to the same contact(s) to whom the original SAE Report Form 
was sent, using a new SAE Report Form stating that this is a follow-up to the previously 
reported SAE and giving the date of the original report. Each re-occurrence, complication, or 
progression of the original event should be reported as a follow-up to that event regardless of 
when it occurs. The follow-up information should describe whether the event has resolved or 
continues, if and how it was treated, and whether the patient continued or withdrew from 
study participation. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 45  
 
7. BIOLOGICAL SAMPLING PROCEDURES 
7.1 Solid Tumor Biopsy Samples 
Patients who opt to undergo the optional biopsy and have tumors amenable to safely consider 
research biopsy, as determined by interventional radiology, will undergo a biopsy after 
signing consent and before initiation of study drug. 
Patients who opt for the 2nd optional biopsy at the end of study participation will undergo the 
2nd biopsy upon progression of disease or discontinuation of study drug due to any other 
reason (e.g. unacceptable toxicity, discontinuation of study drug at physician discretion, 
discontinuation of study drug at patient request). 
Patients may opt to have the optional biopsy at the beginning of the study only, at the end of 
the study only, or both. Therefore, patients who decided against the optional biopsy at the 
beginning of the study may still opt to have the 2nd biopsy at the end of the study only, and 
should be encouraged by the local PI to do so. 
When patients opt to have the optional biopsy, there will be 2-3 core biopsies obtained. Two 
of these cores will be paraffin embedded. One core will be sent for MammoSeq testing, which 
will include ESR assessment as well as other genes described above. 
The other core will be used for the NanoString studies. A 3rd core, if obtained, will be banked 
as fresh frozen to be used in the event that the 2 other core biopsy samples do not contain 
enough usable tissue. 
The core for MammoSeq will be performed as specified in the Laboratory Manual. 
Leftover Samples 
 
Any leftover study blood and tissue samples will be stored at Magee-Womens Hospital 
Tissue Bank or the Magee-Womens Research Institute. Samples will be de-identified and 
stored indefinitely. At enrollment, patients will have the option to allow that these samples be 
used in future research, given that consent for such is signed. 
The study coordinator will keep a tumor sample log that includes the study number, a 
specimen serial number, the patient’s name, time point in therapy, and the date and time that 
the sample was drawn. The sample will be labeled with a serial number only. The laboratory 
technician will keep a log with the specimen number, conditions, processing and storage 
information. 
Note: The correlative sample collection schedules outlined above are based on an ideal 
subject. The sample schedule should be followed as closely as is realistically possible; 
however, the schedule may be modified due to problems such as scheduling delays or 
conflicts (e.g., clinic closure, poor weather conditions, vacations, etc). 
7.2 Blood Samples 
Peripheral blood will be collected for 2 purposes: laboratory monitoring of adverse event, and 
analysis of ESR1 mutation presence and frequency. The volume of blood required for each 
collection is 10mls, and 40mls respectively. The blood sample for monitoring adverse events 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 46  
 
will be sent to the center's laboratory. Blood specimens for determination of the presence of 
ESR1 mutation in circulating tumor DNA by ddPCR will be drawn into 4- 10ml collection 
tubes, and store at ambient temperature. They will be delivered to the laboratory as specified 
in the Laboratory Manual within 3 days. In the laboratory, ctDNA and genomic DNA will be 
isolated and stored at –20°C, as described in the Laboratory Manual. These samples will be 
analyzed for ESR ctDNA internally and for dd PCR and NGS for other genes of potential 
interest in collaboration with AstraZeneca. 
The study coordinator will keep a blood sample collection log that includes the study number, 
a specimen serial number, the patient’s name, time point in therapy, and the date and time that 
the sample was drawn. The sample will be labeled with a serial number only. The laboratory 
technician will keep a log with the specimen number, conditions, processing and storage 
information. 
The correlative sample collection schedules outlined above are based on an ideal subject. The 
sample schedule should be followed as closely as is realistically possible; however, the 
schedule may be modified due to problems such as scheduling delays or conflicts such as 
clinic closure, poor weather conditions, vacations, etc. 
7.3 Leftover Samples 
Any leftover study blood and tissue samples will be stored at Magee-Womens Hospital 
Tissue Bank or the Magee-Womens Research Institute. Samples will be de-identified and 
stored indefinitely. At enrollment, patients will have the option to allow that these samples be 
used in future research given that consent for such is signed. 
 
7.4 Handling, storage and destruction of biological samples 
Peripheral blood obtained for study monitoring of side effects and for analysis of ctDNA will 
be sent to the center's laboratory as described in section 7.2 and the Laboratory Manual. 
Remaining samples after analysis will be stored under the supervision of Dr. Oesterreich if the 
patient has agreed and signed informed consent allowing tissue storage. 
7.5 Withdrawal of informed consent for donated biological samples 
If any subject withdraws consent for donated biological samples, no further biological samples 
will be obtained. Of note, the case will still be included in the intention-to-treat analysis given 
that the subject has received at least one dose of the study compound. The obtained tissue will 
be analyzed, unless the patient has requested during study consent that all samples are 
destroyed. 
 
 
8. ETHICAL AND REGULATORY REQUIREMENTS 
8.1 Ethical conduct of the study 
This study must be carried out in compliance with the protocol and Good Clinical Practice, as 
described in: 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 47  
 
1. 
ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996. 
 
2. 
US 21 Code of Federal Regulations dealing with clinical studies (including parts 50 
and 56 concerning informed consent and IRB regulations). 
 
3. 
Declaration of Helsinki, concerning medical research in humans (Recommendations 
Guiding Physicians in Biomedical Research Involving Human Subjects, Helsinki 1964, 
amended Tokyo 1975, Venice 1983, Hong Kong 1989, Somerset West 1996). 
 
The investigator agrees to adhere to the instructions and procedures described in it and thereby 
to adhere to the principles of Good Clinical Practice that it conforms to. 
 
8.2 Ethics and regulatory review 
• Institutional Review Board: Information regarding study conduct and progress will be 
reported to the Institutional Review Board (IRB) per the current institutional standards 
of each participating center. 
• Food and Drug Administration (FDA): This trial does not involve an Investigational 
New Drug (IND) application and no reporting is required with regards to the clinical 
trial outlined at this time. 
 
8.3 Informed consent 
An investigator will explain to each subject the nature of the study, its purpose, procedures 
involved, expected duration, potential risks and benefits. Each subject will be informed that 
participation in the study is voluntary and that she/he may withdraw from the study at any 
time, and that withdrawal of consent will not affect his/her subsequent medical treatment. This 
informed consent will be given by means of a standard written statement and will be 
submitted for IRB approval prior to use. No patient will enter the study before informed 
consent has been obtained. In accordance with the Health Information Portability and 
Accountability Act (HIPAA), the written informed consent document (or a separate document 
to be given in conjunction with the consent document) will include a subject authorization to 
release medical information to the study sponsor and supporting agencies and/or allow these 
bodies, a regulatory authority, or Institutional Review Board access to subjects’ medical 
information that includes all hospital records relevant to the study, including subjects’ medical 
history. 
 
 
8.4 Changes to the protocol and informed consent form 
Any change or addition to the protocol can only be made in a written protocol amendment that 
must be approved by AstraZeneca, Health Authorities where required, and the IRB/IEC/REB. 
Only amendments that are required for patient safety may be implemented prior to 
IRB/IEC/REB approval. Notwithstanding the need for approval of formal protocol 
amendments, the investigator is expected to take any immediate action required for the safety 
of any patient included in this study, even if this action represents a deviation from the 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 48  
 
protocol. In such cases, AstraZeneca and IRB/IEC at the study site should be notified of this 
action in accordance with local IRB requirements. 
8.5 Audits and inspections 
Source data/documents must be available to inspections by AstraZeneca designee or 
regulatory health authorities. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 49 
 
 
9. STUDY MANAGEMENT 
9.1 TABLE 4. Study timetable and end of study 
 
Procedure 
Screeninga 
Visit 1 
Every 
4 weeks 
Every 
8 weeks 
Off Treatment Follow Up 
INFORMED CONSENT 
X 
CLINICAL ASSESSMENTS 
Physical Exam 
X 
X 
Xb 
X 
Xc 
Medical History 
X 
X 
Xc 
Vital Signs, Height, Weight 
X 
Xd 
X 
X 
(X) 
ECOG Performance Status 
X 
X 
X 
X 
LABORATORY TESTS: 
Hematology 
(CBC with diff, platelets) 
X 
Xe 
Xf 
(X) 
Chemistries and liver function tests g 
X 
Xe 
Xf 
(X) 
PT, INR, PTTq 
          X 
Estradiol ͪ 
X 
Pregnancy Test ͥ 
X 
X 
IMAGING: 
CT Chest/Abd/Pelvisᶨ 
Xj 
X 
(X) 
(X) 
Brain MRI 
Xk 
(X) 
(X) 
TREATMENT: 
Tamoxifen 
XL 
Fulvestrant 
X 
Xᴸ 
Palbociclib 
XP 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 49 
 
 
 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 49 
 
 
OTHER ASSESSMENTS: 
Concomitant Medication Review 
X 
X 
X 
X 
Symptom/Toxicity Assessment 
X 
X 
X 
Xm 
Follow-up/Survival 
Xn 
Xc 
CORRELATIVE STUDIES 
Blood collection for ESR1-mt 
analyzed by ct-DNA 
X 
X 
X 
Archived tissue sample (from breast 
primary) 
X 
Optional Solid Tumor Biopsy 
Xo 
Xo 
a) Screening assessments are required within 28 days of initiation of treatment unless otherwise noted. 
b) Required ≤ 1 day prior to each specified treatment (exceptions may be made to extend this window, with approval by the Protocol Chair/designee). 
c) To continue until patient death 
d) Height at baseline only 
e) Required ≤ 14 days prior to first day of treatment; baseline labs may be used if within window. 
f) Required ≤ 1 day prior to each specified treatment (exceptions may be made to extend this window, with approval by the Protocol Chair/designee). 
g) Chemistries to include measurement of sodium, potassium, chloride, CO2, BUN, creatinine, glucose, total bilirubin, calcium, total protein, albumin, 
AST, ALT, and alkaline phosphatase. 
h) Estradiol if required to determine post-menopausal status can be performed any time prior to Visit 1 treatment 
i) Serum Pregnancy test is required for women of childbearing potential only. Required ≤ 7 days prior to first treatment (i.e., day 1) unless otherwise 
noted. All outcomes of pregnancy should be reported to the PI and AstraZeneca. 
j) PET/CT is allowed and the choice between using a CT or PET with CT will belong to the treating physician. If a PET is chosen, the CT is mandatory 
and must be of diagnostic quality. 
k) Only if known CNS metastasis 
L) Fulvestrant will be dosed as follows: 2x250 mg injections on days 1, 15, 29, then next in 4 weeks (day 57) and continuing every 4 weeks thereafter. 
Tamoxifen will be dosed as follows: Tamoxifen 20mg PO Daily. 
m) All subjects must have a final toxicity assessment at least 30 days following the last dose of study drug 
n) Subjects will be followed approximately every 3 months after completion of study treatment for disease status and survival until death through clinic 
visit, phone call or record review. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 49 
 
 
o) Optional tumor biopsy will be performed ≤ 30 days prior to first day of treatment and within 30 days of treatment discontinuation. 
p) Palbociclib will be dosed 125mg PO QD days1-21 of 28 day cycle 
q) PT, INR, PTT only needs to be repeated for optional biopsies if the patient on anticoagulants 
(X) = OPTIONAL 
Note: Additional tests may be performed at the discretion of the treating investigator as clinically indicated. The sample collection schedules outlined 
above are based on an ideal subject. The sample schedule should be followed as closely as is realistically possible; however, the schedule may be modified 
due to problems such as scheduling delays or conflicts (e.g., clinic closure, poor weather conditions, vacations, etc.). 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 51  
 
9.2 Study Management 
 
Principal Investigator 
The Principal Investigator is responsible for performing the following tasks: 
1. Coordinating, developing, submitting, and obtaining approval for the protocol as well 
as its subsequent amendments. 
2. Assuring that all participating institutions are using the correct version of the protocol. 
3. Taking responsibility for the overall conduct of the study at all participating 
institutions and for monitoring the progress of the study. 
4. Reviewing and ensuring reporting of Serious Adverse Events (SAE). 
5. Reviewing data from all sites. 
 
Coordinating Center 
The UPMC Hillman Cancer Center Coordinating Center is responsible for performing the 
following tasks: 
1. Ensuring that IRB approval has been obtained at each participating site prior to the 
first patient registration at that site, and maintaining copies of IRB approvals from each 
site. 
2. Managing central patient registration. 
3. Collecting and compiling data from each site. 
4. Establishing procedures for documentation, reporting, and submitting of AE’s and 
SAE’s to the Protocol Chair, and all applicable parties. 
5. Facilitating audits by securing selected source documents and research records from 
participating sites for audit. 
 
Participating Sites 
We currently estimate participation of 3 sites. Participating sites are responsible for 
performing the following tasks: 
1. Following the protocol as written, and the guidelines of Good Clinical Practice (GCP). 
2. Submitting data to the Coordinating Center. 
3. Registering all patients with the Coordinating Center by submitting patient registration 
form, signed informed consent, and all required documentation promptly 
4. Providing sufficient, experienced clinical and administrative staff and adequate 
facilities and equipment to conduct a collaborative trial according to the protocol. 
5. Maintaining regulatory binders on site and providing copies of all required documents 
to the Coordinating Center. 
6. Collecting and submitting data within 10 days. 
 
Staffing 
UPMC Hillman Cancer Center Coordinating Site - Staffing will include a Clinical Research 
Manager, Clinical Research Coordinator and Research Associate. The participating sites will 
provide experienced staff, and adequate equipment and facilities to support this clinical trial. 
The participating sites will also be responsible for research staff training in computer 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 52  
applications, human subject research, and HIPAA compliance, as well as the continuing 
education in these areas as required by local institutional standards. 
Documentation 
Each participating site is responsible for submitting copies of all relevant regulatory 
documentation to the Coordinating Center. The required documents include, but are not 
limited to the following: local IRB approvals (i.e., protocol, consent form, amendments, 
patient brochures and recruitment material, etc.), IRB membership rosters, summary of 
unanticipated problems or protocol deviations, and documentation of expertise of the 
investigators. The Coordinating Center will provide each participating site with a 
comprehensive list of the necessary documents. It is the responsibility of the participating sites 
to maintain copies of all documentation submitted to the Coordinating Center. 
Confidentiality 
All unpublished information that the Coordinating Center gives to the investigator shall be 
kept confidential and shall not be published or disclosed to a third party without the prior 
written consent of the Protocol Chair (or her designee). 
Record Retention 
Following closure of the study, each participating center will maintain a copy of all site study 
records in a safe and secure location. Investigator records should be maintained for a 
minimum of 7 years after completion or discontinuation of the study or for longer if required 
by applicable local regulations. 
Additional Information 
Each participating site is responsible for submitting additional information as requested by the 
Protocol Chair (or her designee). The Coordinating Center may terminate the study at a 
participating site in the event that these conditions are not followed. 
 
10. DATA MANAGEMENT 
Data will be scanned to Coordinating site. Site personnel will review and issue queries as 
needed. Electronic data queries stating the nature of the problem and requesting clarification 
will be created for discrepancies and missing values. Designated investigator site staff are 
required to respond promptly to queries and to make any necessary changes to the data. 
Concomitant treatments and prior medications entered into the database will be coded using 
the WHO Drug Reference List, which employs the Anatomical Therapeutic Chemical 
classification system. Medical history/current medical conditions and adverse events will be 
coded using the Medical dictionary for regulatory activities (MedDRA) terminology. 
Laboratory samples for hematology, biochemistry, liver function tests, urinalysis, and 
coagulation will be performed locally. The occurrence of any protocol violations will be 
determined. After these actions have been completed and the data has been verified to be 
complete and accurate, the database will be declared locked 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 53  
11. EVALUATION AND CALCULATION OF VARIABLES 
11.1 Calculation or derivation of efficacy variable(s) 
The primary objectives of the study are descriptive assessments of  ESR1 mutation detection 
in plasma. One of the secondary endpoints of the study is PFS. PFS is defined as the duration 
of time from time of randomization to time of progression or death, whichever occurs first. 
PFS for a subject without an event will be censored on the date of last tumor assessment. If an 
interval of 6 months passes without a tumor assessment, PFS will be censored at the time of 
the earlier tumor assessment, even if an event (progressive disease or death) is later 
observed.If a patient has not had an event, PFS will be censored at the date of the last 
adequate tumor evaluation. PFS will also be censored at the date of tumor evaluation when 
the next tumor evaluation (or death) is >6 months after the previous evaluation without an 
event. PFS will be assessed via local radiology assessment according to RECIST 1.1. 
Although central review may occur as part of site calibration or an audit, the local assessment 
will be considered as the basis for the primary efficacy analysis. In the absence of measurable 
disease at baseline, the following criteria (per RECIST 1.1) will be used to assess disease 
progression among the patients with nonmeasurable lytic or mixed (lytic + blastic) bone 
lesions: 1) The appearance of one or more new lesions or 2) Unequivocal progression of 
existing non-measurable lesions. 
Additional secondary endpoints include response rate, clinical benefit rate, (defined as 
CR+PR+ SD at any tumor assessment), clinical benefit rate at 6 months (CBR6), in the entire 
cohort, and the ESR1-mut cohorts.  Response rate will be reported from best overall response 
according to RECIST 1.1 and as described in section 6.3. The response rate will then be 
compared amongst the tamoxifen plus palbociclib and fulvestrant plus palbociclib arms in the 
entire cohort and in the subgroup of ESR1-mt patients. 
ESR1 mutation will be analyzed both from tumor biopsies and peripheral blood circulating 
tumor DNA. These tumor biopsies and peripheral blood circulating tumor DNA will be 
assessed for the following most commonly described ESR1 mutations using Sanger 
sequencing of the three ESR1 exons which will assess for 23 different ESR1 mutations 
including the 6 most commonly described (S463P, L536Q, Y537S, Y537C, Y537N, and 
D538G). Next generation sequencing (NGS) and/or digital droplet PCR (ddPCR) of blood 
and tumor specimens will also be performed to look at co-existent mutations. It will then be 
reported as the frequency of occurrence of ESR1 mutation in the cohort of enrolled patients as 
well as in the cohort of patients treated with fulvestrant plus palbociclib and tamoxifen plus 
palbociclib. 
 
11.2 Calculation or derivation of safety variable(s) 
All adverse events will be captured on the appropriate study-specific case report forms (CRFs) 
and will be calculated as a frequency of occurrence according to the CTCAE v4.03. The 
frequency of adverse events will categorized in accordance to the grading system present in 
CTCAE v4.03, using the highest grade recorded for each AE for each patient. AE rates will be 
calculated from the tamoxifen plus palbociclib and fulvestrant plus palbociclib cohorts and 
compared using the mid-p correction to Fisher’s exact test. 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 54  
12 . STATISTICAL METHODS AND SAMPLE SIZE DETERMINATION 
 
 
 
12.1 Statistical analysis plan 
The primary analysis will be conducted at least 6 months after the last patient is enrolled. 
Cox proportional hazards regression will be used to estimate hazard ratios comparing PFS for 
tamoxifen plus palbociclib and fulvestrant plus palbociclib. Fulvestrant will be considered 
promising for the unselected population if the fitted hazard ratio is < 0.8, and for the ESR1- 
mut subpopulation if the hazard ratio is < 0.65. 
Response rate will be compared between treatment arms using the mid-P correction to 
Fisher’s exact test for univariate comparisons, and logistic regression for exploratory 
multivariable models. Cox proportional hazards regression will be used for analyses with 
overall survival as the endpoint. 
Adverse events will be reported for all patients in the safety cohort defined above, 
summarized by treatment group. Adverse events and serious adverse events will be tabulated 
in order of prevalence, with the highest grade reported by each patient. 
Concordance of ESR1 status in plasma and biopsy samples will be assessed primarily by 
agreement regarding presence/absence of mutations for tissue and blood samples collected no 
more than 30 days apart and without intervening therapy. Kappa statistics adjusted for 
clustering (both pre-therapy and post-therapy time points for some subjects) will describe 
basic agreement. Additionally, between-subject and within-subject variation in mutant allele 
frequency will be compared using linear mixed models and the concordance correlation 
coefficient. This approach (complementary analysis of categorical and continuous versions of 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 55  
ESR1 mutation status) may also be applied to assessment of within-patient longitudinal 
change. The primary analysis for these correlative studies will examine presence and allele 
frequency of any ESR1 mutation.  Secondary analyses will examine and compare tissue- 
blood concordance and longitudinal change in liquid biopsy measures for specific ESR1 
mutations. Level and change in circulating levels of ESR1-mt will be assessed as predictors of 
PFS using Cox proportional hazards regression with time-varying covariates. All hypothesis 
tests will be two-sided. 
 
12.2 Sample size justification 
 
The study sample size is restricted by external constraints: a planned study with a PFS 
endpoint powered to direct future therapy choices failed to accrue, in part because of the ever-
shifting treatment landscape as described above. A study with n=40, 20 each receiving  
fulvestrant plus palbociclib or tamoxifen plus palbociclib, will allow sufficient exploration of 
a variety of study aims, in order to prioritize and inform the aims of future studies.  If a 
sufficient number of accrued patients have ESR1-mt disease, it may be feasible to evaluate 
the association between mutational burden   and response to continued  endocrine+palbociclib 
therapy.  Alternatively, there may be a strong signal for evolution of mutational frequency as 
different for patients receiving fulvestrant+palbociclib versus tamoxifen+palbociclib.   
Similarly, there may be a signal suggesting that continued endocrine+palbociclib therapy is 
associated with clinical benefit  only in patients with ESR1-mt tumors who receive 
fulvestrant+palbociclib


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 56  
13. LIST OF REFERENCES 
1. Li et al, Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic 
Characterization of Breast-Cancer-Derived Xenografts. Cell Reports 2013 
2. Robinson et al, Activating ESR1 mutations in hormone-resistant metastatic breast cancer. 
Nature Genetics 2013 
3. Toy et al, ESR1 ligand-binding domain mutations in hormone resistant breast cancer. 
Nature Genetics 2013 
4. Merenbakh-Lamin et al, D538G Mutation in ERa: A Novel Mechanism for Acquired 
Endocrine Resistance in Breast Cancer. Cancer Research 2013 
5. Oesterreich and Davidson, The search for ESR1 mutations in breast cancer. Nature 
Genetics 2013 
6. Jeselsohn et al, Emergence of Constitutively Active ERa Mutations in Pretreated Advanced 
ER+ Breast Cancer. Clinical Cancer Res 2014 
7. Jeselsohn et al. ESR1 mutations - a mechanism for acquired endocrine resistance in breast 
cancer. Nat. Rev. Clin. Oncol. 2015. 
8. Andre Fabrice et al. Genomic and immune characterization of metastatic breast cancer: An 
ancillary study of the SAFIRO1 and MOSCATO trials. Annals of Oncology (2014) 25 
(Suppl_4). 
9. Schiavon G, Hrebien S, Garcia-Murillas I, Cutts RJ, Pearson A, Tarazona N, Fenwick K, 
Kozarewa I, Lopez-Knowles E, Ribas R, Nerurkar A, Osin P, Chandarlapaty S, Martin LA, 
Dowsett M, Smith IE, Turner NC. Analysis of ESR1 mutation in circulating tumor DNA 
demonstrates evolution during therapy for metastatic breast cancer. Sci Transl Med. 
2015;7(313):313ra182. doi: 10.1126/scitranslmed.aac7551. PubMed PMID: 26560360. 
10. 
Chandarlapaty S, Chen D, He W, Sung P, Samoila A, You D, Bhatt T, Patel P, Voi 
M, Gnant M, Hortobagyi G, Baselga J, Moynahan ME. Prevalence of ESR1 Mutations in 
Cell-Free DNA and Outcomes in Metastatic Breast Cancer: A Secondary Analysis of the 
BOLERO-2 Clinical Trial. JAMA Oncol. 2016;2(10):1310-5. doi: 
10.1001/jamaoncol.2016.1279. PubMed PMID: 27532364; PMCID: PMC5063698. 
11. Zhang K, Hong R, Xu F, eta l. Clinical value of circulating ESR1 mutations for patients 
with metastatic breast cancer: a meta-analysis. Cancer Manag Res 2018 
12. Gyanchandani R, Kota K, Jonnalagadda, et al. Detection of ESR1 mutations in circulating 
cell-free DNA from patients with metastatic breast cancer treated with palbociclib and 
letrozole. Oncotarget 2017.  
 
13. 
Bahreini et al. Mutation site and context dependent effects of ESR1 mutation in genome- 
edited breast cancer cell models. Breast Cancer Research (2017) 
 
14. 
Wang et al. Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary 
Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients. Clin Cancer Res. 
2015 
 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 57  
15. 
Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev 
Cancer. 2009;9(3):153-66. 
 
16. 
Musgrove EA et al. Cyclin D as a therapeutic target in cancer. Nat Rev Cancer. 
2011;11(8):558-72. 
 
 
17. 
Finn, R.S., et al., The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination 
with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, 
HER2negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 
study. Lancet Oncol, 2015. 16(1): p. 25-35. 29. 
 
18. 
Finn, R.S., et al., Palbociclib and Letrozole in Advanced Breast Cancer. NEJM, 2016. 
Nov 17; 375 (20):1925-36. 
 
19. 
Cristofanilli, M., et al., Fulvestrant plus palbociclib versus fulvestrant plus placebo for 
treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that 
progressed on previous endocrine therapy (PALOMA-3): final analysis of the 
multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol, 2016. 
17(4): p. 425-39 
 
20. 
Smith NG, Gyanchandani R, Shah OS, et al. Targeted mutation detection in breast 
cancer using MammaSeq™. Breast Cancer Res 2019 
 
21. 
Howell et al. Comparison of Fulvestrant Versus Tamoxifen for the Treatment of 
Advanced Breast Cancer in Postmenopausal Women Previously Untreated with 
Endocrine Therapy: A Multinational, Double-Blind, Randomized Trial. J Clin Oncol, 
2004 
 
22. 
Robertson JFR, Llombart-Cussac A, Feltl D, et al. Activity of Fulvestrant 500 mg 
versus anastrozole as first-line treatment for advanced breast cancer: Results of the 
FIRST Study. J Clin Oncol 2009 
 
23. 
Robertson JFR, Llombart-Cussac A, Feltl D, et al. Fulvestrant 500 mg versus 
anastrozole as first-line treatment for advanced breast cancer: Overall survival from the 
phase II “first” study. 2014 San Antonio Breast Cancer Symposium 
 
24. 
Robertson JFR, Bondarenko IM, Trishkina E, et al. Fulvestrant 500mg versus 
anastrozole 1 mg for hormone receptor-positive advanced breast cancer (FALCON): an 
international, randomized, double-blind, phase 3 trial. Lancet 2016; 388: 2997-3005 
 
25. 
Leo AD et al. Final Overall Survival: Fulvestrant 500 mg vs. 250 mg in the 
Randomized CONFIRM Trial. JNCJ J Natl Cancer Inst 2014. 
 
 
 
 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 58  
 
 
 
 
 
 
 
 
 
 
 
 
 
APPENDIX A 
 
List of medications that interact with CYP2D6, CYP3A4/5, CYP2C8/9 and CYP2D6 and 
should be avoided: 
 
 
alfentanil 
alprazolam 
alprenolol 
amitriptyline 
amlodipine 
amodiaquine2 
amphetamine 
aprepitant 
aripiprazole 
astemizole 
 
atomoxetine 
atorvastatin 
boceprevir 
bufuralol1 
buspirone 
cafergot 
carbamazepine 
carvedilol 
celecoxib 
cerivastatin 
 
cerivastatin 
chlorpheniramine 
chlorpromazine 
cilostazol 
cisapride 
clarithromycin 
clomipramine 
clonidine 
cyclosporine 
dapsone 


Clinical Study Protocol 
Drug Substance Fulvestrant 
Study Number Pending 
Edition Number 12 
Date 7-16-2020 
 
Page 59  
desipramine 
dexamethasone 
dexfenfluramine 
dextromethorphan1 
dextromethorphan2 
diclofenac1 
diltiazem 
docetaxel 
domperidone 
donepezil 
duloxetine 
encainide 
eplerenone 
estradiol 
felodipine 
fentanyl 
finasteride 
flecainide 
fluoxetine 
fluvastatin 
fluvoxamine 
gleevec 
glibenclamide 
glimepiride 
glipizide 
glyburide 
haloperidol 
hydrocortisone 
ibuprofen 
imipramine 
indinavir 
irbesartan 
irinotecan 
lercanidipine 
lidocaine 
lornoxicam 
losartan 
lovastatin 
meloxicam 
methadone 
methoxyamphetamine 
metoclopramide 
mexiletine 
midazolam1 
minaprine 
nateglinide 
nebivolol 
nelfinavir 
nevirapine 
nifedipine2 
nisoldipine 
nitrendipine 
nortriptyline 
ondansetron 
oxycodone 
paclitaxel 
paroxetine 
perhexiline 
perphenazine 
phenacetin 
phenformin 
pimozide 
piroxicam 
progesterone 
promethazine 
propafenone 
propranolol 
quetiapine 
quinine 
repaglinide 
risperidone 
ritonavir 
romidepsin 
rosiglitazone 
salmeterol 
saquinavir 
sildenafil 
simvastatin 
sirolimus 
S-metoprolol 
sorafenib 
sparteine 
sunitinib 
suprofen 
tacrolimus (FK506) 
telaprevir 
telithromycin 
terfenadine 
thioridazine 
timolol 
tolbutamide 
tolbutamide1 
torisel 
torsemide 
tramadol 
trazodone 
triazolam2 
valproic acid 
vemurafenib 
venlafaxine 
verapamil 
vincristine 
zakirlukast 
zaleplon 
ziprasidone 
zolpidem 
zuclopenthixol