Serial [F-18] Fluoroestradiol (FES) PET Imaging to Evaluate Endocrine-Targeted Therapy
Phase: NA
Conditions: Estrogen Receptor Positive, Recurrent Breast Carcinoma, Stage IV Breast Cancer AJCC v6 and v7
Sponsor: University of Washington

Local Protocol 7184 IN D#101203 [18F]fluoroestradiol 
6.0 Study Protocol  
RO RECC FEB 28 2019 
Local Protocol #: 7184 
TITLE: Serial IF-181 fluoroestradiol (FES) PET Imaging to Evaluate 
Endocrine-Targeted Therapy 
Principal Investigator: • 
Co-Investigators: 
Hannah Linden, MD 
University of Washington School of Medicine 
Department of Medicine / Division of Medical Oncology 
825 Eastlake Avenue E, G3-200 
Seattle, Washington 98109 
Telephone: (206) 606-6710 
Fax: (206) 606-2054 
hmlindenu.washinoton.edu 
 
Janet F. Eary, MD 
Department of Radiology 
University of Alabama 
JT N412 
619 19th St South 
Birmingham, AL 35249-6830 
Telephone: (205) 934-3138 
jeary@uabmc.edu  
Andrew Shields, MD 
University of Washington Medical Center 
1959 NE Pacific St 
Box 356113 
Seattle, WA 98195-6004 
Telephone: (206) 559-6856 
Fax: (206) 598-4192 
andrewsu.washinoton.edu  
Jean Lee, MD 
University of Washington Medical Center 
1959 NE Pacific St, BB308 
Box 356113 
Seattle, WA 98195-6004 
Telephone: (206) 598-0024 
Fax: (206) 598-0252 
jeanhleee.u.washington.edu  
David Mankoff, MD, PhD 
University of Pennsylvania 
116 Donner Building 
FHCRC IRB Approval 
MAR 2 0 2019 
Document Released Date 
Page 1 of 35  
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Local Protocol 7184 I NC:4101203 ["F]fluoroestradiol 
Hospital of the University of Pennsylvania 
3400 Spruce Street 
Philadelphia, PA 19104-4283 
Telephone: (215) 615-3687 
Fax: (215) 349-5843 
david.mankoff@uphs.upenn.edu  
Kenneth A. Krohn, PhD 
University of Washington Medical Center 
1959 NE Pacific St., Rm NW041 
Box 356004 
Seattle, WA 98195-6004 
Telephone: (206) 598-6245 
Fax: (206) 598-4192 
kkrohnu.washinciton.edu 
 
Dr. Krohn is a radiochemist and is not involved in patient care. 
Jeanne M. Link, PhD 
Oregon Health Sciences and University 
3181 SW Sam Jackson Park Road 
Mail Code L340 
Portland, OR 97239 
Telephone: (206) 406-4509 
Fax: (503) 494-4982 
Iiieaohsu.edu 
 
Dr. Link is a radiochemist and is not involved in patient care. 
Steven C. Shoner, PhD 
University of Washington Medical Center 
Box 356004 
Seattle, WA 98195-6004 
Telephone: (206) 598-6711 
Fax: (206) 598-4192 
scshoneru.washionqton.edu  
Dr. Shoner is a radiochemist and is not involved in patient care. 
Elahe A. Mostaghel, MD, PhD 
Fred Hutchinson Cancer Research Center 
1100 Fairview Ave North, Mailstop D4-100 
Seattle, WA 981 09-1 024 
Telephone: (206) 667-3506 
emostaqhfhcrc.orq 
Dr. Mostaghel conducts histopathological analysis of biopsy samples 
and is not involved in patient care. 
Peggy Porter, MD 
Fred Hutchinson Cancer Research Center 
1100 Fairview Ave North, Mailstop C1-015 
Seattle, WA 981 09-1 024 
Tel: 206-667-3751 
pporter@fhcrc.org  
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Local Protocol 7184 I N D# 101203 [18F]fluoroestradiol 
Dr. Porter conducts histopathological analysis of biopsy samples 
and is not involved in patient care. 
Carolyn L. Wang, MD 
University of Washington Medical Center 
1959 NE Pacific St, BB308 
Box 357115 
Seattle, WA 98195-6004 
Telephone: (206) 598-0024 
Fax: (206) 598-0252 
wanqclu.washington.edu 
 
David H. Lewis, MD 
University of Washington School of Medicine 
Department of Radiology / Nuclear Medicine Division 
325 Ninth Ave, Box 359728 
Seattle, WA 98104-2499 
Telephone: (206) 744-3471 
Fax: (206) 744-8560 
lewis@u.washington.edu  
Geraldine Liao, MD 
University of Washington School of Medicine 
Department of Radiology / Division of Breast Imaging 
825 Eastlake Ave E 
Seattle, WA 98109 
Telephone: (206) 606-7446 
liao@u.washington.edu  
Statisticians: 
Brenda F. Kurland, PhD 
Biostatistics Univ. of Pittsburgh & 
Univ. of Pittsburgh Cancer Institute 
Suite 325 Sterling Plaza, 201 North Craig St 
Pittsburgh, PA 15213 
phone: (412) 383-1128 
BFK10@pitt.edu  
Qian Vicky Wu, PhD 
Fred Hutchinson Cancer Research Center 
1100 Fairview Ave North, Mailstop M2-B823 
Seattle, WA 981 09-1 024 
Telephone: (206) 667-3358 
owufredhutch.orq 
Study Coordinator / 
Data Manager: 
• 
Alena Novakova 
825 Eastlake Avenue East 
Mail Box 358081 
Mailstop LV200 
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Local Protocol 7184 IND#101203 [18F]fluoroestradiol 
Seattle, WA 98109 
Telephone: (206) 606-6498 
Fax: (206) 606-7543 
alenanuw.edu 
 
Study Coordinator / 
Research Scientist: 
LaneII Peterson 
825 Eastlake Avenue East 
Mail Box 358081 
Mailstop LV200 
Seattle, WA 98109 
Telephone: (206) 606-1050 
Fax: (206) 606-7543 
lanell@u.washington.edu  
Current Protocol Version Date: October 23, 2017 
Previous Protocol Version Date: August 16, 2017 
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Local Protocol 7184 IND#101203 [189fluoroestradiol 
Table of Contents  
Page 
6.1. Schema  
6-07 
6.2. Objectives  
6-10 
6.2.1. Primary Objective  
6-10 
6.2.2. Secondary Objectives  
6-10 
6.3. Background  
6-10 
6.3.1. Study Disease  
6-10 
6.3.2. Investigational Agent  
6-12 
6.3.3. Rationale  
6-13 
6.4. Patient Selection  
6-15 
6.4.1. Inclusion Criteria  
6-15 
6.4.2. Exclusion Criteria  
6-15 
6.4.3. Inclusion of Women and Minorities  
6-16 
6.5. Registration Procedures  
6-16 
6.6. Treatment Plan  
6-16 
6.6.1. [18F]FES Administration  
6-16 
6.6.2. Study Procedures and Schedule of Events  
6-17 
6.6.3. General Concomitant Medication and Supportive Care Guidelines 6-19 
6.6.4. Duration of Therapy  
6-19 
6.6.5. Duration of Follow Up  
6-19 
6.7. Dosing Delays / Dose Modifications  
6-19 
6.8. Adverse Events: List and Reporting Guideline  
6-19 
6.8.1. Adverse Events and Potential Risks List (CAEPR)  
6-19 
6.8.2. Adverse Events characterization  
6-20 
6.8.3. Expedited Adverse Event Reporting  
6-21 
6.9. Pharmaceutical Information  
6-22 
6.9.1. Production of the Radiopharmaceutical  
6-22 
6.9.2 Reported Adverse Events and Potential Risks  
6-22 
6.9.3 Agent Accountability  
6-22 
6.10. Correlative / Special Studies  
6-23 
6.10.1. FDG-PET Imaging  
6-23 
6.10.2. [18F]FES-PET Image Assessment  
6-24 
6.10.3. Biologic Correlates  
6-26 
6.11. Study Calendar  
6-27 
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Local Protocol 7184 I N D# 101203 [18F]fluoroestradiol 
Table of Contents (cont.) 
Page 
6.12. Measurement of Effect 
6-29 
6.12.1  Time to Progression 
6-29 
6.13.  Data Reporting / Regulatory Considerations 
6-29 
6.13.1. Data Reporting 
6-29 
6.14. Statistical Considerations 
6-29 
6.14.1. Analysis Plan 
6-29 
6.14.2. Sample Size / Composition / Accrual Rate 
6-30 
6.14.3. Stratification Factors 
6-31 
6.14.4. Reporting and Exclusions 
6-31 
6.15. Literature Cited 
6-32 
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Local Protocol 7184 I N D# 101203 [I'Flfluoroestradiol 
6.1 SCHEMA 
This clinical study will investigate the utility of [18F]FES in patients with estrogen 
receptor (ER) positive, primary, recurrent or metastatic, breast carcinoma. [18F]FES 
PET has been validated as a measure of estrogen receptor expression. This trial is 
also designed to test the safety of [189FES. In addition, this study will obtain data on 
the value of [18F]FES to predict and evaluate response to endocrine-targeted therapies 
as a measure of ER expression in this patient population. If promising, these data will 
be used to design a phase II multi-center clinical trial. 
We anticipate that the imaging measure of receptor density will predict the likelihood 
that a patient will respond to hormone targeted therapy, evaluate subsequent changes 
in this measure as the patient undergoes therapy and that eventually [18F]FES imaging 
will help us design therapeutic trials for better treatment outcomes. We also expect 
that repeat [18F]FES PET imaging shortly after the start of endocrine-targeted therapy 
will reflect the pharmacodynamics of endocrine targeted therapy. 
This protocol is outlined in the following schematic. Briefly, patients with primary, 
recurrent or metastatic breast cancer from an ER+ primary tumor who will be 
undergoing endocrine-targeted therapy will be imaged with [F-18]-16a-fluoroestradiol. 
All patients will undergo a pre-therapy FES PET or FES PET/CT scan (hereafter FES 
PET will be used to denote both FES PET and FES PET/CT unless specifically noted). 
In some patients receiving non-interfering medications such as aromatase inhibitors, 
scans may be performed with patients already on a treatment who are to start an 
alternative or additional endocrine targeted treatment. Patients will undergo a repeat 
FES PET and FDG PET on study after starting the endocrine-targeted therapy. The 
exact timing of the repeat scans may vary depending on the treatment regimen. In the 
repeat studies, we will be testing the ability of FES PET and FDG PET to measure the 
pharmacologic effect of endocrine-targeted therapy and early measures that may help 
predict response to such targeted agents. In patients on selective estrogen receptor 
degrader (SERD) therapies, where repeat FES PET is used primarily to evaluate 
residual ER availability rather than tumor response, repeat FDG PET may be omitted, 
as determined by requirements of the co-enrolling companion therapeutic protocol. A 
subset of patients may undergo a third FES PET at a later time point determined by the 
treatment regimen. 
FDG PET and CT will be used as staging tests for this study and FDG will also serve 
as a comparator for tumor localization with the FES imaging. Patients will have a 
clinical FDG PET scan prior to the start of treatment as part of their standard care; their 
treating physician will order this. In rare cases where an FDG PET has recently been 
performed at an outside medical facility but the scan has (1) inadequate image quality 
or (2) insufficient quantitative information, or for patients who would not be ordered as a 
standard part of the patient's care, a baseline FDG scan will be done as part of this 
protocol. A second FDG PET scan will be done as a study procedure concurrent with 
the FES PET timing stipulated by treatment regimen (this may vary from 1 to 12 weeks 
post treatment start depending on regimen). In the subset of patients undergoing a third 
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Local Protocol 7184 I N D# 101203 [18F]flu oroestradiol 
FES PET scan there may also be a research FDG PET done at the same time point if 
determined by the treatment regimen. 
After the FES and FDG baseline imaging (see schematic below), the patients will start 
or change therapy, typically within 30 days, and be followed clinically for up to 20 years. 
Lesion location will be identified using clinical FDG scans with reference to clinical CT 
for metastatic disease or ultrasound, mammography, and/or breast MRI for primary 
tumors in patients presenting with metastatic or recurrent disease that still have a 
primary breast lesion. FES and FDG images will be spatially co-registered and tumor 
regions will be drawn on the FDG images and transferred to the FES images for 
quantitative analysis of FES uptake in both ER positive and ER negative tumors. For 
FDG studies, SUVs will be generated. 
The FES uptake from images will be analyzed qualitatively by visual assessment and 
quantitatively using a standard uptake value (SUV). Serial measurements of hormone 
levels in peripheral blood may be used with some treatment regimens to allow 
determination of efficacy of endocrine therapy. ER, PR and HER2 assays will allow 
determination of hormone receptor status and whether HER2 over-expression indicates 
estrogen-independent growth in some treatment studies, however, there may not be 
pathologic correlates in all studies. 
We emphasize that this is an observational study in that [18F]FES PET will not be 
used to direct therapy. Treatment regimens are chosen by the referring oncologist 
prior to the [18F]FES PET study on the basis of clinical criteria or participation in 
experimental treatment protocols. While patients and referring physicians will not be 
blinded to [18F]FES PET results, treatment choices will be made prior to the entry of the 
patient into the study and will not be altered because of the FES PET results. 
Patients with metastatic or recurrent disease may consent to optional research biopsies 
performed prior to start of the selected endocrine-targeted therapy, and after 1-12 
weeks on therapy. Obtained tissues will be analyzed for expression of prognostic 
markers and/or for intra-tumor steroid hormone concentrations to evaluate correlations 
between results of imaging and histo-pathological studies and to enhance 
understanding of mechanisms of response and resistance in the context of tumor 
microenvironment. 
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Local Protocol 7184 I ND#101203 r 8F1fluoroestradiol 
Primary or Metastatic Breast Cancer Patients (with history of ER+ Primary) 
Planning a Course of Endocrine-Targeted Therapy 
a) Diagnostic FDG PET or PET/CT 
FES PET or PET/CT 
(in a small number of patients FES PET may precede the FDG PET or PET/CT) 
b) Obtain metastatic or recurrent tumor tissue from an FDG and/or FES avid site 
(optional biopsy — baseline) 
Begin endocrine targeted therapy 
Repeat a) c) FDG and FES PET 
(timing of repeat imaging may vary from 1 week to 12 weeks post treatment start 
denendina on the treatment reaimenl 
Obtain metastatic or recurrent tumor tissue (optional biopsy — early response) 
(Timing may vary from Ito 12 weeks post treatment start; a sample should be 
obtained after the completion of imaging, from the same site as the baseline 
sample whenever possible) 
N't  
Repeat a) FDG and FES PET 
(if indicated by treatment protocol time points or to evaluate ER status at time of 
progression) 
Evaluation of disease-free/progression-free survival (for up to 20 years post-Rx) 
Figure 6.1 Outline (Schema) of the [18F]FES Study Protocol. a) FDG PET is not 
required for primary breast cancer patients unless required for localization or 
requested by treating physician. b) Where available and medically appropriate, a 
clinical or research biopsy that involved sampling of a relevant lesion within 6 
months prior to the enrollment may be utilized in place of the baseline optional 
biopsy. C) Repeat FDG PET may be omitted in patients on SERD therapies. 
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Local Protocol 7184 IND#101203 [18F]fluoroestradiol 
6. 2 OBJECTIVES 
6.2.1 Primary Objective 
Measure the effect of endocrine targeted therapy on ER expression and 
estradiol binding to the receptor using serial FES PET and FDG PET. 
6.2.2 Secondary Objectives 
Document the safety profile of FES PET in patients with breast cancer. 
Examine associations between FES PET results and serial 
measurements of hormone or other levels in peripheral blood, as related 
to efficacy of endocrine-targeted therapy. Correlate FES PET uptake 
measures with histopathological assays and tumor microenvironment 
studies on biopsy specimens, if relevant to specific treatment regimen. 
6.3 BACKGROUND 
6.3.1 Study Disease 
6.3.1.a Invasive breast carcinoma: In 2007, breast cancer will be diagnosed in 
over 180,000 women and account for over 40,000 deaths in the United 
States (Jemal, 2007b). The incidence of hormone receptor positive 
disease appears to be increasing (Li, 2003). Advances in diagnosis and 
therapy have resulted in increased survival for early breast cancer 
(Jemal, 2007a; Piccart-Gebbhart, 2005; Romond, 2005; Winer, 2005; 
Jemal, 2006). This increase in breast cancer survival may result in more 
patients diagnosed with advanced or metastatic breast cancer. Improved 
and targeted treatment of advanced breast cancer has also resulted in 
improved outcomes (Chia, 2003). However, better tailoring of therapy to 
patients is likely to result in higher quality of life (Greenberg, 1996). 
6.3.1.b Invasive breast carcinoma and hormone receptors: Nearly two-thirds 
of breast cancer patients have hormone receptor bearing (HR+) tumors 
(Carlson, 2002). Treatment options for HR+ patients have expanded to 
more potent and less toxic therapies, inviting clinicians to offer endocrine 
therapy to more patients (Carlson, 2002; Lonning, 2001). Nonetheless, 
chemotherapy is frequently offered to patients without proof of superior 
outcomes. Measurement of tumor hormone receptor expression, both 
estrogen receptor (ER) and progesterone receptor (PgR) at the time of 
primary diagnosis is now the standard of clinical care (Carlson, 2004). 
Knowledge of hormone receptor expression is essential for selection of 
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Local Protocol 7184 IND#101203 [189fluoroestradiol 
appropriate therapy but biopsies and serum assays may not provide 
sufficiently complete information (Anonymous, 1998; Osborne, 1998). 
6.3.1.c Treatment of breast carcinoma: Treatment selection for breast cancer 
poses several challenges. Heterogeneity is a challenge in management of 
breast cancer, especially for locally advanced or metastatic disease. In 
cases where tissue sampling is feasible, the expression of ER may be 
heterogeneous, and ER expression at one site does not guarantee 
expression at all sites. This issue presents significant practical challenges 
for assessing ER expression in breast cancer. The potential for 
misidentification of patients appropriate for endocrine therapy is significant. 
While ER expression predicts measurable tumor regression (objective 
response) in 30% to 77% of patients with a new diagnosis of breast 
cancer, response rates are more typically 7% to 21% in patients with 
recurrent disease and prior breast cancer treatment (Lonning, 2001). A 
predictive assay of hormone sensitivity capable of assessing ER 
expression at all sites of disease would be highly valuable for selection of 
which patients are more likely to benefit from endocrine therapy. 
6.3.1.d Recent advances in endocrine therapy: A significant advance in the 
management of ER+ metastatic breast cancer is the introduction of 
aromatase inhibitors (Al) (Winer, 2005; Mouridsen, 2001; Nabholz, 2000). 
These agents are more effective and result in a longer time to 
progression than previous therapies, and have therefore displaced 
tamoxifen as first-line treatment for metastases and adjuvant therapy 
(Winer, 2005; Mouridsen, 2001; Nabholz, 2000, Carlson 2005). However, 
several endocrine therapies are active agents for the treatment of 
hormone responsive disease: fulvestrant, a selective estrogen receptor 
demodulator agent or SERD; tamoxifen, a selective ER blocking drug, or 
SERM; as well as megestrol, testosterone, and diethylstilbesterol (DES) 
(Lonning, 2001; Carlson, 2005; Osborne, 2002; Buckner, 1989). 
Increasing use of Als in the adjuvant setting will lead to increased use of 
alternative endocrine treatment strategies in the metastatic setting. 
6.3.1.e Endocrine therapy for breast carcinoma: The optimal management of 
patients who experience progression of disease while on adjuvant 
aromatase inhibitor treatment, and of premenopausal patients, remains 
under investigation. There is evidence to support the usefulness of 
irreversible steroidal Als (exemestane) for treating patients who progress 
on nonsteroidal Als (Lonning, 2000). Clinical experience suggests a 
primary role for this strategy in patients who have a significant disease-
free interval on a hormonal adjuvant therapy. Currently active 
investigational protocols will determine the optimal management of 
premenopausal women with hormonal therapy. However, tamoxifen 
remains standard management and tamoxifen therapy in pre-menopausal 
patients may be complicated by high estradiol levels due to tamoxifen 
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Local Protocol 7184 I ND#101203 [18F]fluoroestradiol 
stimulation of the ovary (Jordan, 1991; Sunderland, 1991). Ovarian 
suppression alone or with tamoxifen or an Al is also an effective strategy 
(Carlson, 2004; Davidson, 2000; Klijn, 2000; Klijn, 2001; Milla Santos, 
2002), but the optimal sequence of endocrine treatment in 
premenopausal patients is not yet clear (Prowell, 2004). Our study will 
test the value of FES PET for predicting the effectiveness of endocrine 
therapy, with the eventual goal of selecting patients with primary or 
metastatic disease most likely to benefit from this expanding choice of 
agents for endocrine therapy. In addition, our hypothesis is that FES PET 
will be useful as a predictive assay for those patients who have failed one 
endocrine therapy agent and are being considered for an alternative 
endocrine treatment. 
6.3.1.f Imaging of ER with [189FES: PET ER imaging using [18F]-fluoroestradiol 
(FES) poses an attractive possibility to meet this clinical need. Previous 
work evaluating the ER binding, radiation dosimetry, blood clearance and 
protein interactions of FES in women with hormone receptor positive 
breast cancer has shown that uptake correlates with in vitro assay of 
expression in breast cancer tissue samples (Mankoff, 2001; Mintun, 
1988) . Furthermore FES positron emission tomography (with either PET 
or PET/CT) measures the heterogeneity of ER expression and function in 
metastatic disease (Mankoff, 2002; Dehdashti, 1995). Prior studies have 
shown that the level of FES uptake predicts the response of advanced 
breast cancer to hormonal therapy; these therapies were predominantly 
tamoxifen (Mortimer, 2001). We also showed that the level of FES uptake 
as an in vivo indicator of ER function predicted response to endocrine 
therapy (Linden, 2006). We measured FES uptake in patients with 
recurrent or metastatic breast cancer treated largely with aromatase 
inhibitors. In this study we will test FES predictive capability in patients 
undergoing endocrine therapy of breast cancer, and also to test the effect 
of ER-directed breast cancer treatments on ER expression and/or ER 
blockade. 
6.3.2 Investigational Agent 
6.3.2.a 16a-[189-fluoro-170-estradiol (FES) is a radiolabeled imaging agent that 
has been used for investigating tumor estrogen receptor activity with 
positron emission tomography (PET). It was developed in a collaboration 
of the Welch and Katzenellenbogen laboratories (Kiesewetter, 1984) as a 
positron-emitting radiopharmaceutical for imaging ER expression. 
Studies have shown that the strength ( KD) of FES binding to the ER is 
nearly identical to estradiol (Kiesewetter, 1984). FES binding to sex 
steroid transport protein (SBP or SHBG) is also nearly identical to 
estradiol (Kiesewetter, 1984; Tewson, 1999). Fluorine-18 labeled FES 
can be synthesized with high specific activity so the quantity of estrogenic 
material injected with the radiopharmaceutical is <5 pg (Kiesewetter, 
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Local Protocol 7184 IND#101203 [189fluoroestradiol 
1984; Lim, 1996). The typical peak molar concentrations of [19F]FES 
from a [18F]FES injection is 1 pmol/mL, a value comparable to circulating 
estrogen levels. This value decreases by 60 minutes post-injection to 
0.05 pmol/mL. [18F]FES can generate high quality PET images of regional 
estradiol binding using a 6 mCi injection, with acceptable radiation dose 
to the patient (Mankoff, 2001). In breast cancer, the uptake of FES 
measured by PET has been shown to correlate with ER expression in 
biopsy material assayed by in vitro radioligand binding (Mintun, 1988) or 
by immunohistochemistry (Peterson, 2008). As has been found for in vitro 
assays of ER expression, the level of FES uptake in breast cancer is 
predictive of response to endocrine therapy in advanced tumors treated 
with primary tamoxifen therapy (Mortimer, 2001) and metastatic cancer 
treated by salvage hormonal therapy, mostly in the form of aromatase 
inhibitors (Linden, 2006). This protocol will evaluate FES as an in vivo 
predictive assay for endocrine therapy of newly diagnosed metastatic 
breast cancer. 
6.3.3 Rationale 
6.3.3.a The primary hypothesis to be tested is that pre-therapy and post-therapy 
[18F]FES uptake will measure the effect of endocrine-targeted therapy on 
ER expression and/or estradiol binding to the ER. 
6.3.3.b This single-site study will determine the ability of [18F]FES to quantify 
changes in ER expression and estradiol binding in vivo in patients with 
breast cancer for whom endocrine-targeted therapy has been chosen 
based on clinical presentation or as part of therapeutic clinical trial. The 
results of this trial will help evaluate the effect of ER-directed therapies on 
ER expression and estrogen binding to ER, and secondarily whether the 
FES PET measures of estrogen receptor function, and changes with 
treatment, predict response to endocrine targeted therapy in patients with 
known ER+ primary or metastatic breast cancer as measured by 
immunohistochemistry (IFIC) of biopsy material. The primary goal is to 
gain insight into the efficacy of ER-targeted therapies, especially novel 
therapies, on ER expression and estrogen binding to ER. For example, 
serial FES PET imaging could be used to test the efficacy of an ER 
antagonist in blocking estradiol binding, or the ability of an agent directed 
at altering ER expression to change ER expression levels in tumors. 
Such in vivo measures of the pharmacodynamics of ER-directed therapy 
have traditionally required serial biopsy and have been quite challenging. 
FES PET presents a non-invasive alternative to measure the effects of 
ER-directed therapy. 
6.3.3.c We have examined the role of FES in 47 heavily pretreated patients 
(Linden, 2006). Initial FES uptake was measured and correlated with 
subsequent tumor response to 6 months of hormonal treatment. In this 
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Local Protocol 7184 IND#101203 renfluoroestradiol 
study, 11/47 (23%) patients had an objective response. Quantitative FES 
uptake and response were significantly associated; 0/15 patients with 
initial FES SUV<1.5 responded to hormonal therapy, compared to 11/32 
(34%) with FES SUV *1.5 (p < .01). In the subset of patients whose 
tumors did not over-express HER2/neu, 11/24 (46%) of patients with SUV 
1.5 responded. These pilot results support the further evaluation of the 
predictive capability of FES PET. 
6.3.3.d FES uptake predicted a greater likelihood of response to endocrine 
therapy. Patients with low or absent FES uptake were unlikely to manifest 
a response to treatment, yet FES did not help to distinguish which 
patients would experience a clinical benefit, defined as stable disease. 
Several factors may have contributed to this finding. Stable disease over 
6 months included patients with disease that may have progressed 
slowly, independent of treatment, as well as patients with rapidly 
progressive disease in whom therapy slowed but did not abrogate tumor 
growth. The study was not designed to distinguish between these two 
outcomes. In addition, we identified PgR (progesterone receptor) 
expression as contributing to likelihood of response, and noted that 
elevated estradiol levels and HER2 expression were associated with non-
response. In this new study we propose to document tumor 
characteristics including quantitative ER mRNA levels measured by PCR 
and semi-quantitative levels of ER, PgR, AR and HER2 by IHC . 
Gathering these indices as part of the proposed study may help to 
enhance the predictive value of PET FES, allow understanding of 
mechanism of resistance to endocrine therapy, and potentially determine 
which patients are likely to appreciate clinical benefit as a result of 
endocrine therapy, even in the absence of an objective response. 
6.3.3.e In prior observational imaging trials (unpublished data) we have measured 
regional estrogen-ER binding using FES PET before and after treatment 
with estrogen depleting therapies, Aromatase inhibitors (Al), and estrogen 
blocking therapies, Tamoxifen (TAM), or fulvestrant (FUL) in 30 
metastatic breast cancer patients. As expected, tumor FES uptake 
declined markedly on ER blockers TAM and FUL (average 54% decline), 
but less than 15% on average on estrogen-depleting Als (p<0.001). All 
5/5 patients taking TAM showed complete tumor blockade (FES SUV 5 
1.5 after treatment), compared to 4/11 of FUL patients (p=0.009). We 
concluded that Molecular imaging can assess the in vivo 
pharmacodynamics of targeted agents, and may give insight into the 
activity of established therapeutic agents. 
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Local Protocol 7184 !NEVI 01203 [18F]fluoroestradiol 
6.4 PATIENT SELECTION 
6.4.1 Inclusion Criteria: 
6.4.1.a Adult, non-pregnant patients with biopsy-proven or clinically obvious 
primary, recurrent or metastatic breast cancer 
6.4.1.b Breast cancer from ER+ primary that is seen on other imaging tests. 
Tumor ER expression must have been confirmed by 
immunohistocytochemistry of primary tumor or recurrent disease. 
6.4.1.c At least one site of disease 1.5 cm or greater is needed to meet the 
spatial resolution limits of PET imaging. 
6.4.1.d Patients must have been off tamoxifen or other estrogen receptor 
blocking agents for at least 6 weeks and off chemotherapy for 3 weeks for 
the initial baseline FES. 
6.4.1.e Patients must be selected for an endocrine targeted therapy regimen for 
treatment of their breast cancer by the referring oncologist. Selected 
treatments may be part of experimental treatment protocols for which the 
patient would be separately consented. 
6.4.1.f Patients must be willing to undergo serial imaging procedures. 
6.4.1.g Patients must agree to allow access to clinical records regarding 
response to treatment and long term follow up. 
6.4.2 Exclusion Criteria: 
6.4.2.a An inability to lie still for the tests 
6.4.2.b Individuals weighing more than 300 lb. (this is the weight limit of the 
scanner table) 
6.4.2.c Pregnant or lactating. Women of childbearing potential with either a 
positive or no pregnancy test at baseline are excluded. 
6.4.2.d Any other life-threatening illness (e.g. serious, uncontrolled concurrent 
infection or clinically significant cardiac disease — congestive heart failure, 
symptomatic coronary artery disease, cardiac arrhythmia not well 
controlled with medication). 
6.4.2.e Use of tamoxifen, faslodex, DES or any other ER blocking agent < 6 
weeks or chemotherapy < 3 weeks prior to imaging scan. 
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Local Protocol 7184 I N I1# 1 0 1 203 [189fluoroestradiol 
6.4.2.f Unwillingness or inability to give informed consent. 
6.4.2.g Uncontrolled diabetes mellitus (fasting glucose > 200 mg/dL) 
6.4.2.h Adult patients who require monitored anesthesia for PET scanning. 
6.4.3 Inclusion of Women and Minorities: 
Refer to Section 6.14.4 
6.5 REGISTRATION PROCEDURES 
Not applicable; this is a single-institution trial 
6.6 TREATMENT PLAN 
Treatment will be administered on an outpatient basis. Reported adverse 
events and potential risks are described in Section 6.8.1. There will be no 
dose modifications for FES administration. No investigational or commercial 
agents or therapies other than those described below will be administered 
with the intent to treat the patient's malignancy. 
6.6.1 [18F1FES Administration: 
FES will be administered in the PET imaging suite at the University of 
Washington Medical Center or at the Seattle Cancer Care Alliance (SCCA). 
The [18F]FES for injection will be prepared as described in section 6.6.2, 
Study Procedures and Schedule of Events. Upon arrival, the patient will have 
an opportunity to have questions answered regarding the procedure. The 
patient will have an intravenous line placed prior to [18F]FES administration. 
A dose of nominally 220 MBq (6 mCi with a range of 110 to 220 MBq = 3 to 
6 mCi) of [18F]FES will be administered intravenously by a physician. The 
injection will be infused over approximately 2 minutes and followed by a 
saline flush. The infusion and imaging procedure will be terminated in any 
patient who exhibits anaphylaxis, physical signs of significant hypotension, 
significant dyspnea or chest pain. We do not expect to observe any of these 
effects as no events have been reported in more than 300 scans performed 
with this compound at our institution to this date. The patient will then be 
allowed to rest comfortably in a prep room prior to being positioned in the 
PET scanner to commence the image acquisition at 60 +/- 10 minutes post 
FES injection. 
Page 16 of 35  
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Local Protocol 7184 IND#101203 ['8F]fluoroestradiol 
Heart rate and blood pressure will be recorded pre- and post-FES dosing, 
and will continue to be monitored after injection until baseline is achieved. 
Patients will be monitored for adverse events, potentially related to the 
administration of [18F]FES, from the time of the initial injection to 10 hours 
post injection. Any adverse events will be recorded and reported according 
to FDA requirements (see section 6.8 Adverse Events). 
6.6.2 Study Procedures and Schedule of Events 
6.6.2.a Initial visits prior to [18F]FES PET: Patients who are identified as 
potential candidates for the [18F]FES PET study will be approached to 
explain the nature of the study and to obtain their written informed 
consent to be enrolled in the protocol. Heart rate and blood pressure will 
be recorded at the time of clinic visit where study is considered or at the 
time of consent. The patient will have a clinical FDG PET/CT scan if a 
recent FDG scan has not already been obtained. The FDG PET/CT scan 
should be obtained close to the time of FES PET/CT, typically within 14 
days of the [18F]FES PET/CT scan. An additional FDG PET/CT scan may 
be done as a research scan if the patient has undergone a recent PET 
scan at an outside institution or if they are unable to obtain a clinical PET 
scan as part of their clinical care. The research FDG PET/CT scan in this 
instance will be identical in procedure to a clinical FDG scan. The 
following additional patient data will be obtained: histological diagnosis, 
age at radiologic diagnosis, weight, gender, ECOG score and other 
treatment modalities used. Correlative radiology including CT, MRI, or 
bone scan, and laboratory tests measuring renal, blood and liver function 
will also be collected, typically for values within 30 days of the FES 
PET/CT scan. For patients referred from outside the UW health system, 
patient records, and biopsy material and imaging as necessary, will be 
reviewed to determine eligibility for the study. 
6.6.2.b Day of [18F]FES PET/CT Scan: After the FES injection and rest period 
(section 6.6.1), the patient will be positioned supine in the PET/CT 
scanner. A low dose CT scan (60 mA) will provide attenuation correction 
data using non-contrast CT. The PET image acquisition sequence will be 
the same as that used for clinical FDG scans. This consists of a torso 
survey series of five-minute images acquired over 15 cm survey fields of 
view (F0V5) extending from the base of the skull to the mid thigh (5 to 7 
FOVs in total). The images will be corrected for radioactive decay of the 
tracer and normalized to the injected dose and body weight. This results 
in regional standardized uptake values (SUVs) whole-body image sets. 
Prior to injection, a whole blood sample may be taken for determination of 
albumin, estradiol and testosterone levels, heart rate and blood pressure 
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Local Protocol 7184 IND/0101203 [189fluoroestradiol 
will be recorded. A known quantity of nonradioactive FES will be added 
to plasma (from blood collected prior to injection) to determine binding to 
sex steroid binding protein (SBP). For patients in whom a second venous 
access line is feasible and has been placed, venous blood samples may 
be collected at 5, 20 and 60 minutes after injection of the [18F]FES for 
metabolite analysis in selected patients. Heart rate and blood pressure 
will be measured after the FES injection. 
6.6.2.c After the [18F]FES PET Scan: A follow up phone call will be made by 
study staff within 24 hours after the FES PET scan to ask about any signs 
of adverse effects. Patients will typically start the selected hormone 
targeted therapy within 30 days of the FES PET scan. Patients who have 
started aromatase inhibitors prior to [18F]FES PET will be permitted to 
enter the study, if an additional endocrine targeted treatment is planned. 
6.6.2.d In patients who consent to optional biopsies, a baseline tumor sample will 
be obtained after completion of the baseline PET imaging but prior to 
starting the selected endocrine targeted therapy. Where available and 
medically appropriate, a clinical or research biopsy that involved sampling 
of a relevant lesion within 6 months prior to the enrollment may be utilized 
in place of the baseline optional biopsy. 
In some patients undergoing biopsies, a whole blood sample will be 
collected prior'to starting the therapy, to allow correlation of steroid 
hormone levels in serum and biopsy tissue. 
6.6.2.e Repeat FES PET scan and FDG PET scan: Patients will undergo a 
second FES PET and FDG PET scan after starting endocrine targeted 
therapy to assess the effect of therapy on ER expression and estradiol 
binding to the receptor. Exact timing of the second FES and FDG scans, 
will depend upon the endocrine targeted regimen and dosing, but typically 
will be 1 — 12 weeks after starting therapy. 
The repeat FDG PET may be omitted in patients on SERD therapies, 
where repeat FES PET is used primarily to evaluate residual ER 
availability rather than tumor response. This will be determined by 
requirements of the co-enrolling companion therapeutic protocol. 
6.6.2.f In patients who consent to optional biopsies, an early response tumor 
sample will be obtained 1-12 weeks after starting the selected therapy. 
This biopsy should take place after completion of the repeat PET imaging 
and should target the same tumor site as the baseline biopsy. A whole 
blood sample may be drawn for correlation of steroid hormone levels in 
serum and biopsy tissue. 
6.6.2.g Week 1 to 20 years after [18F]FES PET Scan: Clinical follow-up will 
continue for up to 20 years, time of progression and date of death will be 
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Local Protocol 7184 IND#1 01 203 [18F]fluoroestradiol 
recorded. For some patients, especially those receiving two endocrine-
directed treatments, a third FES PET scan and additional research FDG 
PET scan may also be obtained 1-12 weeks after the second FES PET 
scan. For each patient, a maximum of 3 FES PET and 3 FDG PET scans 
would be done. 
6.6.3 General Concomitant Medication and Supportive Care Guidelines 
Because there is unlikely to be interaction of < 5 pg of [18F]FES with other 
concomitantly administered drugs through the cytochrome P450 system, 
this section is not applicable. 
The infusion and imaging procedure will be terminated in any patient who 
exhibits adverse reactions as described in section 6.6.1 and 6.8.1. 
6.6.4 Duration of Therapy 
As this is an observational study, the results of [18F]FES PETs and repeat 
FDG PETs are not used to direct therapy, and the duration of therapy is 
determined on the basis of clinical grounds by the referring physician, and 
is not influenced by the FES study. 
6.6.5 Duration of Follow Up 
Patients will be followed up to 20 years after [18F]FES PET. Patients 
removed from study for unacceptable adverse events will be followed until 
resolution or stabilization of the adverse event. 
6.7 DOSING DELAYS/DOSE MODIFICATIONS 
As this is a trial of a diagnostic imaging agent to be administered before 
endocrine therapy, dosing delays and dose modifications are not relevant to this 
protocol. 
6.8 ADVERSE EVENTS: LIST AND REPORTING GUIDELINE 
6.8.1 Adverse Events and Potential Risks 
No adverse events have been reported for diagnostic [18FJFES 
administration at the strength described for this study in 267 patients studied 
at the University of Washington. Thus no adverse effects are expected as a 
result of the administration of [18F]FES. The [18F]FES dose is less than 
0.002 of the recommended safe oral dose assuming an oral conjugated 
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Local Protocol 7184 IND#101203 C8F]fluoroestradiol 
estrogen dose of 2.5 mg in one day. Unexpected but potential adverse 
effects are listed below. 
Anaphylaxis, Hypotension, Dyspnea, Chest pain, Flushing, Euphoria 
Infection at the injection site or systemic infection 
Extravasation of the dose 
6.8.2 Adverse Events Characterization 
6.8.2.1 the general definitions for adverse events are: 
Adverse Event: Any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated 
with the use of a medical treatment or procedure regardless of whether it 
is considered related to the medical treatment or procedure (attribution of 
unrelated, unlikely, possible, probable, or definite). Each AE is a unique 
representation of a specific event used for medical documentation and 
scientific analysis. 
Serious Adverse Event (SAE): An SAE is defined in the FDA CFR 312 as 
any adverse drug experience occurring at any dose that results in any of 
the following outcomes: death, a life-threatening adverse drug 
experience, inpatient hospitalization, or prolongation of existing 
hospitalization, a persistent or significant disability/incapacity, or a 
congenital anomaly/birth defect. Important medical events that may not 
result in death, be life threatening, or require hospitalization may be 
considered a serious adverse drug experience when, based upon medical 
judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in 
this definition. We will report serious AEs via expedited reporting to FDA 
and to our local IRB. 
Life-Threatening Adverse Event: Any adverse drug experience that 
places the patient or subject, in the view of the investigator, at immediate 
risk of death from the reaction as it occurred, i.e., it does not include a 
reaction that, had it occurred in a more severe form, might have caused 
death. AEs can be `Unexpected' or 'Expected' for expedited reporting 
purposes only. A list of adverse events that have occurred or might occur 
(Reported Adverse Events and Potential Risks) can be found in Section 
6.8.1. 
In addition to oversight by the Pis, this study will be monitored by the 
UW/FHCRC Cancer Consortium Institutional Review Board and the 
Radiation Safety Committee. 
Page 20 of 35  
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Local Protocol 7184 IND#101203 ("F]fluoroestradiol 
6.8.3 Expedited Adverse Event Reporting 
Expedited AE reporting for this study will be done through the Cancer 
Consortium IRB and FDA and as required by FDA MedWatch. These 
requirements are briefly outlined in the table below. 
All life threatening adverse reactions reports are submitted to the FDA, our 
local IRB and to all investigators. A copy of the report is kept on file. 
Table 6.iii.2 Reportina Re uirements 
Unexpected 
Expected 
Adverse Reaction 
(known or suspected attributable to 
the use of C8F1FES) 
AE not 
attributable to 
[189FES 
AE, AR 
Serious including 
life-threatening (or 
death) 
Nonserious 
Life-Threatening or 
serious or not 
serious 
Not 
applicable to 
[18FIFES 
Reporting 
Time 
Requirement 
to the FDA 
Report to FDA 
ASAP and within 7 
days of discovery 
of event (section 
6.iii.8.7) 
Annual 
Continuation 
Review 
submission 
Annual Continuation 
Review submission 
Not 
applicable to 
[189FES 
Reporting 
Form for the 
FDA 
IND Safety report of 
potentially serious 
risk 
Annual Reports / 
Case reports 
Annual Reports / 
Case reports 
Not 
applicable to 
[18FjFES 
Reporting 
Time 
Requirement 
to the local 
IRB 
Report to IRB 
ASAP within 10 
days of discovery 
of event (suspected 
is defined as 50% 
probability 
attributable to 
[18F]FES study) this 
also includes any 
increased risks with 
the study even 
without an AE 
At continuation 
review time 
At continuation 
review time 
Not 
applicable to 
[18F]FES 
Reporing 
form for the 
IRB 
Expedited Reporting 
Form for 
Unanticipated 
Problems or 
Noncompliance and 
Adverse Event 
Reporting Form 
Form for 
Unanticipated 
Problems or 
Noncompliance, 
Case reports on 
continuation 
form, Data Saety 
Monoring 
Reports 
Form for 
Unanticipated 
Problems or 
Noncompliance, 
Case reports on 
continuation form, 
Data Safety 
Monitoring Reports 
Not 
applicable 
to [18F]FES 
Page 21 of 35  
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Local Protocol 7184 IND#101203 [189fluoroestradiol 
6.8.3.a Expedited Reporting Guidelines 
All deaths for subjects while on study will be reported to FDA using expedited 
reporting regardless of causality. Attribution to treatment or other cause 
will be provided. 
Fatal and life-threatening events will be reported to the University of Washington 
IRB within 24 hours of notification of the event, indicating that a full report 
will follow. All adverse events will be submitted to the IRB within10 days of 
notification of the event. , 
Fatal and life-threatening events will also be reported within 24 hours to 
the FDA following the MedWatch guidelines set by the FDA in compliance 
with the 21 CFR 312.32. 
Life-threatening (or fatal) adverse reactions must be reported within 7 
days to the FDA. The FDA should be notified as soon as the adverse 
reaction is discovered by telephone or fax or email. The instructions and 
forms are available at 
http://www.fda.gov/Safety/MedWatch/HowToReport/default.htm The 
report should be sent ASAP by mail and followed with a follow-up report. 
Individual IND safety reports to FDA are submitted on the Medwatch FDA 
Form 3500A as an "IND Safety Report". The form should be sent to The 
Director, Office of Generic Drugs in the Center for Drug Evaluation and 
Research at FDA. The address and phone numbers are available at: 
http://www.fda.qov/AboutFDA/CentersOffices/CDER/ucm119100.htm.  
6.9 PHARMACEUTICAL INFORMATION 
6.9.1 Production of the Radiopharmaceutical 
[189FES will be produced at the University of Washington according to the 
methods described in Section 7 of this IND application, Chemistry, 
Manufacturing and Controls. 
6.9.2 Reported Adverse Events and Potential Risks: 
A list of the adverse events and potential risks associated with [18F]FES was 
provided in Section 6.8.1. 
6.9.3 Agent Accountability: 
The chemical precursor for [189FES is provided in house or by ABX in 
single-use, 2 mg vials that are delivered to the custody of Dr. Steven 
Shoner, the lead radiochemist for this project. They are stored in a 
controlled temperature freezer in a locked and secure room and they are 
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Local Protocol 7184 IND#101203 [18F]fluoroestradiol 
inventoried with a chain of custody maintained from the time of receipt. 
Each radiosynthesis is done by Dr. Shoner or his designee and, after 
passing all required quality control assays, the product [18F]FES dose is 
drawn up under the supervision of the nuclear medicine physician 
investigators, Jean Lee, MD, Andrew Shields, MD or David Lewis, MD. The 
quality control tests that must be passed prior to release of the product 
[18F]FES for injection include the pH, radioactive purity, radiochemical purity, 
specific activity, sterilizing filter integrity, tests for Kryptofix [2.2.2], 
acetonitrile, endotoxins and particulates. Acceptance criteria are detailed in 
the CMC document. The [18F]FES dose is drawn up into a syringe, assayed 
for mCi at time of injection, labeled and administered to the research 
subject. 
6.10 CORRELATIVE/SPECIAL STUDIES 
6.10.1 FDG-PET Imaging 
In most cases, a standard clinical diagnostic FDG PET or PET/CT scan 
will be obtained prior to the [18F]FES PET scan. The FDG PET or PET/CT 
scan serves to help identify active sites of breast cancer and is used to 
help determine sites on the FES PET scan where estradiol binding would 
be expected. At least one site of measurable disease outside of the liver, 
where quantification of FES uptake can be difficult because of normal 
biodistribution, must be evident on the FDG PET scan and CT or MRI. 
FDG PET imaging will be completed close to the time of the FES PET 
scan. In select cases where the baseline FDG PET is being repeated at 
our institution for the purposes of this study it may be completed after the 
FES PET. The FDG PET will be a clinical 5 axial field-of-view (AFOV) 
scan from skull base to mid-thigh. Maximum SUV of identified lesions will 
be calculated and recorded as per standard clinical procedure. 
For FDG PET or PET/CT, all patients will fast for at least 4 hours before 
PET scanning. All patients must have a medical history negative for 
diabetes and/or a pre-scan serum glucose level less than 200 mg/dL. A 
dose of 260-370 MBq (7-10 mCi) of FDG will be injected into a peripheral 
intravenous or central venous catheter. Patients will rest comfortably in a 
supine position prior to scanning. Imaging will be performed with a 
commercially made PET/CT scanner operating in the high sensitivity 
mode. Imaging will consist of a torso survey covering five adjacent 15-cm 
axial fields of view (F0Vs). For the PET/CT device, a low dose CT scan 
beginning at approximately 60 minutes after FDG injection provides 
attenuation data for attenuation correction using a non-contrast CT. 
Emission data will be collected for all five axial FOVs, typically five 
minutes per FOV.. 
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Local Protocol 7184 IND#101203 [18F]fluoroestradiol 
Emission data will be reconstructed according to standard protocols. 
Images will be corrected for radioactive decay of the tracer and 
normalized to the injected dose and body weight, which results in regional 
standardized uptake values (SUVs): as defined in section 6.10.3. 
We emphasize that the baseline FDG PET or PET/CT is part of standard 
clinical care of metastatic and recurrent breast cancer and will be 
performed as part of routine clinical care in the vast majority of patients 
participating in the study. Methodology for FDG PET is included in this 
section for reference purposes. In some cases, FDG PET or PET/CT will 
have already been performed at an outside center at the time of 
enrollment. On rare occasions when patients have been referred to the 
study with a recent FDG PET scan already completed at an outside site 
that is unavailable or of insufficient detail for our study, or the patient is 
unable to obtain a PET scan as part of standard clinical management, we 
will repeat the FDG PET at the University of Washington as a research 
scan, however, the imaging procedure will not vary from that of a 
standard clinical scan. 
Patients will undergo a second (and potentially a third in a subset of 
patients) FDG PET following the same imaging procedure as part of the 
research study. This second scan will be performed at a time point 
stipulated by the treatment regimen and may vary from 1 to 12 weeks post 
therapy start. 
6.10.2 [189FES-PET Image Assessment 
6.10.2.a Summary of Assessment 
The FES PET scan will be assessed in multiple ways. 
Qualitative FES uptake: all known disease sites positive vs. 
heterogeneous or absent FES uptake. 
Quantitative FES uptake: SUVs in all lesions measured at baseline and 
repeat scan (and at third scan if indicated) 
Quantitative change in FES uptake: percentage and SUV unit change in 
SUV between baseline and repeat FES PET scan 
6.10.2.b Qualitative Visual Assessment 
Qualitative (visual) assessment of level of uptake in sites of recurrent 
breast cancer is performed for all studies. In this analysis, trained 
observers determine whether FES uptake is present above background at 
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Local Protocol 7184 I ND#101203 riFyluoroestradiol 
sites of breast cancer. Active disease sites are identified by the FDG 
PET scan performed close to the time of FES PET, in conjunction with 
conventional imaging such as CT, MRI, and bone scan. Active sites of 
disease are defined as sites with (1) abnormal uptake on FDG PET and 
(2) either biopsy confirmation or unequivocal correlative findings on at 
least one other modality. For each site of active disease, two trained 
observers blinded to the clinical data, but with any available FDG PET 
and correlative imaging, will determine whether FES above background 
levels is present or absent. Differences between observers will be 
resolved by consensus. 
6.10.2.c Quantitative Assessment 
FES static survey images will undergo region of interest (ROI) analysis as 
follows: Lesion location will be identified using FES scans along with 
FDG scans with reference to CT or ultrasound. All patients will have 
undergone clinically motivated FDG PET scans for staging prior to FES 
imaging. CT, MRI, ultrasound or bone scan will be available on all 
patients with suspected metastatic disease. Although FES and FDG 
imaging will be performed on separate days, studies will be performed 
with the patient in the same position and images will be co-registered 
using common anatomic landmarks such as the lung outline, cardiac 
blood pool, liver contour, and body outline. In this way, regions can be 
drawn on the FDG images and transferred to the FES images for 
quantitative analysis which is important in identifying tumor locations, 
especially in ER-negative tumors which are not visualized on FES 
imaging. For each disease site, a set of 1.5 cm diameter regions on three 
adjacent planes with the highest lesion FES uptake will be drawn to 
determine maximal FES uptake. Up to the 10 largest sites seen on the 
static torso survey will be quantified. Partial-volume correction will not be 
used because only sites 1.5 cm or greater diameter will be included in 
quantitative analysis. 
FES uptake will be quantified using the standard uptake value (SUV, 
defined below). For each site, we will record the average and maximum 
SUV within the ROI set for each site of disease. 
For each subject, we will calculate the average and range of FES uptake 
values for active disease sites. The FES uptake will be compared with 
subsequent response to endocrine therapy and UP. 
FDG studies will also be quantified for uptake in a similar fashion. On 
FDG studies, simple SUVs will be generated using the average tissue 
activity over the imaging period, which are typically 45 and 75 minutes 
after injection. We will record the maximum FDG SUV for each site of 
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Local Protocol 7184 I N D# 101203 [18F]fluo roestrad iol 
disease. Uptake at tumor sites using the standardized uptake value 
(SUV) is calculated as: 
SUV- ID/wt 
where Ct is the average tumor uptake after injection for the period of the 
static imaging for torso surveys (MBq/mL), ID is the injected dose (GBq) 
and wt is the patient's weight (kg). Note that either standard or metric 
units can be used to calculate SUV as long as only one type of unit is 
used for each calculation. 
6.10.3 Biologic Correlates 
Optional blood sample may be collected at the time of the FES PET scan 
procedure for determination of serum hormone levels (estradiol, estrone, 
follicle-stimulating hormone, sex hormone binding globulin, testosterone, 
free testosterone). 
Patients participating in this study will have undergone clinical biopsy of the 
primary tumor and/or metastasis for which ER, PgR, and HER2 expression 
will be determined by IHC. Clinical records of these assays will be 
reviewed, and the assays themselves may be reviewed as needed. 
On patients for whom archival biopsy material can be obtained, additional 
assays may be performed, and may include repeat and standardized ER, 
PR, and HER2 assays. 
Patients with metastatic or recurrent disease may consent to optional 
research biopsies of the tumor performed at the baseline, i.e. prior to start 
of the selected endocrine targeted therapy, and after 1-12 weeks on 
therapy. Obtained tissues will be analyzed for a baseline expression and 
post-therapy changes in ER, PgR, HER2, AR, EGFR and VEGF using IHC 
methods. Some samples will be assayed for intra-tumor steroid hormone 
levels (estradiol, estrone, testosterone, dihydrotestosterone) using IHC 
and/or mass spectrometry techniques. 
In some patients undergoing biopsy, a whole blood sample will be drawn 
during the same time point to allow correlation of steroid hormone levels in 
serum with levels in biopsy tissue. 
Assays for ER and PgR will be read using the Allred scoring system, 
which provides a semi-quantitative and qualitative assessment of tumor 
expression levels (Harvey, 1999). IHC is the primary assay methodology 
for HER2. Scoring of HER2 is done by assessing intensity of the 
membranous stain (0, 1+, 2+, 3+). Scores of 0 and 1+ are considered 
negative and score of 3+ is considered positive. Score of 2+ is considered 
indeterminate and will be validated by fluorescence in situ hybridization 
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Local Protocol 7184 I N D# 101203 [1 8F]fluo roestradiol 
(FISH) (HER-2 DNA probe kit; Abbott Molecular Inc., Des Plaines, 
IL)(Yaziji, 2004). Analysis of HER2 will be done using information from the 
IHC assays and the FISH confirmation when required. 
Measurement of these tumor-associated indices may enhance the 
predictive value of PET FES, provide information to better understand 
mechanisms of resistance to endocrine therapy, and eventually determine 
which patients are likely to appreciate clinical benefit as a result of 
endocrine therapy, even in the absence of an objective response. 
6.11 STUDY CALENDAR 
Baseline evaluations are to be conducted prior to administration of protocol therapy, 
typically within 30 days. Baseline FDG PET/CT scan will be obtained near the time 
of the [18F]FES-PET/CT scan, typically within 14 days. Procedures are outlined 
below in Table 6.2. 
Table 6.2. Study Calendar. 
Pre- 
Study 
Imaging 
1 
Rx 
1 
Imaging 
25  
Rx 
2 
Imaging 
36 
Follow up 
Informed Consent 
X 
Demographics 
X 
Pregnancy Test' 
X 
Vital Signs2  
X 
X 
X 
Research lab tests3  
X 
X 
X 
ER, PgR, HER24  
X 
FDG PET/CT5'6'7  
X 
X 
X 
[18F]FES injection 
and PET5'7  
X 
X 
X 
Collect serum 
hormone levels8  
X 
X 
X 
Adverse Event 
Evaluation 
X (0- 
24hr) 
X (0- 
24hr) 
X (0 - 
24hr) 
Optional tumor 
tissue biopsy and 
blood draw9  
X 
X 
1If patient is pre-menopausal, and birth control use is not confirmed 
2 Vital signs include heart rate and blood pressure 
3Additional blood tests to measure hormone levels (e.g. estradiol, estrone, testosterone, DHT, 
SHBG) or other hormone related measures might be performed in some patients depending on 
the treatment regimen. 
4 Pathology results from primary or metastatic biopsy will be reviewed and additional pathology 
tests may be performed on some patients depending on the treatment regimen. 
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Local Protocol 7184 IND#101203 [18F]fluoroestradiol 
5 FES PET/CT and FDG PET/CT will be done at 1-12 weeks after the Imaging 1 time point 
depending on the treatment regimen. 
6 Repeat FDG PET may be omitted in patients on SERD therapies. 
7  FES PET/CT and FDG PET/CT may be done at 1-12 weeks after the Imaging 2 time point 
depending on the treatment regimen. 
8 Optional blood sample for estradiol, estrone, follicle-stimulating hormone, sex hormone 
binding globulin, testosterone and free testosterone serum levels. 
8 In patients consented to optional biopsies, obtain metastatic or recurrent tumor tissue from an 
FDG and/or FES avid site after completion of PET imaging but prior to start of the endocrine 
targeted therapy. Repeat the biopsy from the same site after 1-12 weeks on treatment. (Where 
available and medically appropriate, a clinical or research biopsy that involved sampling of a 
relevant lesion within 6 months prior to the enrollment may be utilized in place of the baseline 
optional biopsy.) A blood draw done during the same time point will accompany some biopsies. 
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Local Protocol 7184 I ND#101203 [18F]fluoroestradiol 
6.12 MEASUREMENT OF EFFECT 
For the purposes of this study, the outcome measured will be the change in FES 
and FDG PET measures between the serial sets of scans. Response evaluations 
will not be evaluated as part of this study, though the information may be recorded if 
it is collected for treatment studies the patient may be participating. Patients may 
also be followed for up to 20 years for disease progression and overall survival. 
6.12.1 Time to Progression 
Time to progression (and progression-free survival) will be measured as the 
time from the start of endocrine therapy to the time the patient is first 
recorded as having disease progression. If a patient never progresses 
while being followed, the patient will be censored at the time he/she 
terminates follow-up or by date of disease progression or death. 
6.13 DATA REPORTING/ REGULATORY CONSIDERATIONS 
Adverse event lists, guidelines, and instructions for AE reporting are described in 
Section 6.8. 
6.13.1 Data Reporting 
The Principal Investigator is responsible for maintaining complete and 
timely, HIPAA-compliant electronic records of clinical and image datasets. 
6.14 STATISTICAL CONSIDERATIONS 
6.14.1 Analysis Plan 
The primary application will be pilot studies with enrollment of about 20 
patients, in which the in vivo effects of endocrine-targeted therapy are 
measured by serial FES PET. The primary endpoints will be quantitative and 
qualitative measures of FES uptake, and measures of change in FES uptake 
(Section 6.10.2). Additionally, patients will be followed for disease 
'progression. Associations among FES and FDG PET measures, 
progression, and biological correlates (Section 6.10.3) may be examined in 
descriptive analyses. 
An expected primary analysis is to examine whether FES uptake changed on 
average between serial assessments. This may be assessed by a one-
sample test of the percent change in FES SUV. For example, a one-sided 
test at the 0.05 level of significance could evaluate whether the average 
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Local Protocol 7184 I N D# 1 01203 [18F]fluoroestradiol 
percent change in FES SUV was greater than 20%, considered a biologically 
significant change. In serial FES SUV measurements of heavily-pretreated 
breast cancer patients, change in FES SUV was approximately normally 
distributed with a standard deviation of about 25 percentage points, for both 
patients treated with blocking therapy (tamoxifen or fulvestrant) or non-
blocking therapy (aromatase inhibitor). Using this standard deviation and 
n=20 to test whether the average change was greater than 20%, the power 
would be 0.97 if the true change is 40%, and 0.85 if the true change is 35%. 
To conclude only that the average percentage change was different from 
zero (using a two-sided test at the 0.05 level of significance), power would be 
0.95 to detect a true average change of 20% (and 0.85 if the true standard 
deviation were 30 percentage points). 
Other analyses for pilot studies could examine what proportion of patients 
experienced a threshold in percentage change, or surpassed a targeted 
follow-up FES SUV value. These percentages will be reported using a 90% 
Wilson score binomial confidence interval (Agresti, 1998). Width of these 
confidence intervals for pilot studies of n=20, n=25, and n=30 are given in 
Table 6.3 for a range of observed percentages. 
Table 6.3: 90% Wilson score binomial confidence intervals (Cis) by percent 
observed and sample size. 
observed 
percentage (Cl) 
for n=20 
observed 
percentage (Cl) 
for n=25 
observed 
percentage (Cl) 
for n=30 
0.1 (0.03, 0.26) 
0.12 (0.05, 0.27) 0.1 (0.04, 0.23) 
0.2 (0.09, 0.38) 
0.20 (0.10, 0.36) 0.2 (0.11, 0.34) 
0.3 (0.16, 0.48) 
0.32 (0.19, 0.48) 0.3 (0.18, 0.45) 
0.4 (0.24, 0.58) 
0.40 (0.26, 0.56) 0.4 (0.27, 0.55) 
0.5 (0.33, 0.67) 
0.48 (0.33, 0.64) 0.5 (0.36, 0.64) 
6.14.2 Expected Gender, Ethnic and Racial Composition 
Breast cancer is a disease of adults with low frequency of incidence in 
males. We have not excluded males from participation in our studies and 
will continue this policy but anticipate that none will enroll due to the rarity 
of presentation. 
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Local Protocol 7184 1ND#101203 rnfluoroestradiol 
Table 6.4. Planned Enrollment. 
TARGETED/PLANNED ENROLLMENT: 
no range 
Number of Subjects (must provide actual numbers. I.e. 
_ 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
3 
0 
3 
Not Hispanic or Latino 
47 
0 
47 
Ethnic Category Total of All Subjects* 50 
0 
50 
Racial Categories 
American Indian/Alaska Native 
1 
0 
1 
Asian 
8 
0 
8 
Native Hawaiian or Other Pacific 
Islander 
1 
0 
1 
Black or African American 
7 
0 
7 
White 
33 
0 
33 
Racial Categories: Total of All 
Subjects * 
50 
0 
50 
Males will not be excluded; however, breast cancer is rare in males. 
6.14.3 Stratification Factors 
, 
Not Applicable 
6.14.4 Reporting and Exclusions 
6.14.4.a Evaluation of Toxicity: All patients will be evaluable for toxicity for 24 
hours after each [18FJFES PET. 
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