[18F]Fluoroestradiol-Positron Emission Tomography (PET)/CT Imaging of Invasive Lobular Carcinoma
Phase: PHASE2
Conditions: Invasive Lobular Breast Carcinoma
Sponsor: University of Utah

Current Version: 12-01-2023 
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Title: 
 
[18F]Fluoroestradiol-PET/CT Imaging of Invasive Lobular Carcinoma 
 
 
Matthew F. Covington, MD Principal Investigator 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Imaging radiopharmaceuticals to be used: 
 
            [18F]Fluoroestradiol ([18F]FES) 
                  IND # 151981 
 
2-deoxy-2-[18F]fluoro-D-glucose (FDG) 
 
       ANDA # 204498 
 
 
 
 
University of Utah IRB # 128055 
 
 
 
 
 
 
 
 

Current Version: 12-01-2023 
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1. OBJECTIVES 
5 
1.1. PRIMARY OBJECTIVE FOR PILOT PHASE (COMPLETED AS OF MARCH 2022 AMENDMENT) 
5 
1.2. SECONDARY OBJECTIVE FOR PILOT PHASE (COMPLETED AS OF MARCH 2022 AMENDMENT) 
5 
1.3. PRIMARY OBJECTIVE FOR EXPANSION PHASE (ADDED AS OF MARCH 2022 AMENDMENT) 
5 
1.4. SECONDARY OBJECTIVES FOR EXPANSION PHASE (ADDED AS OF MARCH 2022 AMENDMENT) 6 
1.5. EXPLORATORY OBJECTIVES FOR EXPANSION PHASE (ADDED AS OF MARCH 2022 
AMENDMENT) 
6 
2. BACKGROUND 
8 
2.1. INVASIVE LOBULAR CARCINOMA 
8 
2.2. IMAGING OF INVASIVE LOBULAR CANCER 
8 
2.3. OVERVIEW OF THE PET RADIOPHARMACEUTICAL [18F]FLUOROESTRADIOL 
9 
2.4 OVERVIEW OF THE FES/FDG SUVMAX RATIO 
10 
2.5 RESULTS FROM STUDY PILOT PHASE: 
11 
3.  PHARMACOLOGY, SAFETY AND RADIATION DOSIMETRY OF [18F]FES 
17 
3.1. PHARMACOLOGY AND SAFETY 
17 
3.2. HUMAN RADIATION DOSIMETRY 
21 
4. TRIAL DESIGN 
28 
4.1. PATIENT ELIGIBILITY 
28 
4.2. INCLUSION CRITERIA 
29 
4.3. EXCLUSION CRITERIA 
30 
4.4. PATIENT REGISTRATION 
31 
4.5. STUDY PROCEDURES, SCHEDULE OF EVENTS, TRACER ADMINISTRATION:  ROUTE AND DOSING
 
31 
4.6. DATA COLLECTION 
35 
5. METHODS FOR EVALUATION OF IMAGING STUDIES 
36 
5.1. [18F]FES-PET/CT IMAGING 
36 
5.2 [18F]FLUORODEOXYGLUCOSE PET IMAGING 
37 
6. DATA ANALYSIS AND STATISTICS 
37 
6.1. VALIDATION OF [18F]FLUOROESTRADIOL-PET/CT IMAGING TO ASSESS THE UPTAKE IN INVASIVE 
LOBULAR CARCINOMA 
37 
6.2 DATA ANALYSIS 
37 
6.3. JUSTIFICATION OF SAMPLE SIZE 
41 
7. REGULATORY AND REPORTING REQUIREMENTS 
41 
7.1. HUMAN SUBJECT PROTECTIONS 
42 
7.2 INSTITUTIONAL REVIEW 
42 
7.3 DATA AND SAFETY MONITORING PLAN 
42 
7.4 ADVERSE EVENTS / SERIOUS ADVERSE EVENTS 
43 
7.5 SAE REPORTING REQUIREMENTS 
45 
7.6 PROTOCOL AMENDMENTS 
46 
7.7 PROTOCOL DEVIATIONS 
47 
7.8 FDA ANNUAL REPORTING 
47 
7.9 CLINICAL TRIALS DATA BANK 
47 

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7.10 RECORD KEEPING 
47 
8. PET RADIOPHARMACEUTICAL PRODUCTION 
47 
8.1. [18F]FLUOROESTRADIOL 
47 
9. REFERENCES 
50 
10. APPENDICES 
56 
APPENDIX A: SCHEDULE OF EVENTS, REVISED FOR EXPANSION PHASE AS OF MARCH 2022 
AMENDMENT 
57 
APPENDIX B: [18F]FLUOROESTRADIOL INFUSION FLOW CHART FOR STAFF 
59 
APPENDIX C: [18F]FLUOROESTRADIOL PATIENT ADVERSE EVENT QUESTIONNAIRE 
60 
APPENDIX D: 5 YEAR FOLLOW-UP ASSESSMENT 
62 
APPENDIX E: NCI COMMON TOXICITY CRITERIA 
63 
 
 
 
 

Current Version: 12-01-2023 
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PROTOCOL SIGNATURE 
 
I confirm that I have read this protocol, and I will conduct the study as outlined herein 
and according to the ethical principles stated in the latest version of the Declaration of 
Helsinki, the applicable ICH guidelines for good clinical practice, and the applicable laws 
and regulations of the federal government. I will promptly submit the protocol to the IRB 
for review and approval. Once the protocol has been approved by the IRB, I understand 
that any modifications made during the course of the study must first be approved by 
the IRB prior to implementation except when such notification is made to remove an 
immediate hazard to the subject.   
 
I will provide copies of the protocol and all pertinent information to all individuals 
responsible to me who assist in the conduct of this study.  I will discuss this material 
with them to ensure that they are fully informed regarding the study treatment, the 
conduct of the study, and the obligations of confidentiality. 
 
Note: This document is signed electronically through submission and approval by the 
Principal Investigator in the University of Utah IRB Electronic Research Integrity and 
Compliance Administration (ERICA) system. 

Current Version: 12-01-2023 
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1. OBJECTIVES 
 
1.1. Primary Objective for Pilot Phase (Completed as of March 2022 Amendment) 
 
To assess the positive detection rate of invasive lobular carcinoma (ILC) on 18F]Fluoroestradiol-
PET/CT (FES-PET/CT).   
Primary endpoint:  
1) FES-PET/CT will detect at least 80% of histologically proven primary ILC estrogen receptor 
positive (ER+) tumors. Reference standard is histopathology from biopsy of a primary breast 
or metastatic lesion demonstrating ILC. The null hypothesis is that 60% of cases will 
demonstrate positive uptake on FES, equivalent to rates from pooled data for FDG-PET/CT 
[1-3]. A higher proportion of tumors are expected to demonstrate positive uptake by FES-
PET/CT.  
 
1.2. Secondary Objective for Pilot Phase (Completed as of March 2022 
Amendment) 
 
To assess FES-PET/CT concordance with ER status from biopsy and presence of inter-
tumoral ER heterogeneity.  
 
Secondary endpoints:  
1. Rate of estrogen receptor positive (ER+) ILC that does not demonstrate positive FES 
uptake, defined as focal uptake above background with SUV max of 1.5 or greater. 
2. Rate of estrogen receptor negative (ER-) ILC that does demonstrate positive FES 
uptake, defined as focal uptake above background with SUVmax of 1.5 or greater. 
3. Rate of same-patient (inter-tumoral) heterogeneous FES uptake defined as presence of 
FES uptake with SUVmax of 1.5 or greater in some but not all biopsy proven or 
suspected metastatic lesions. 
To assess for any differences between FDG- and FES-PET/CT uptake. 
 
Secondary endpoints:  
1. For cases with both FDG- and FES-PET/CT imaging, evaluate the rate of discordant 
uptake (FES positive/FDG negative or FES negative/FDG positive).  Discordant uptake 
will be evaluated for biopsy proven primary, any proven or suspected local nodal 
(axillary, intramammary, internal mammary, supraclavicular) and any proven or 
suspected distant metastatic lesions. Note that completion of an FDG-PET/CT study is 
not obligatory for study enrollment, but an optional FDG-PET/CT will be paid for by the 
study if the patient elects to complete this study, and if not already obtained for clinical 
evaluation.  
2. For cases with both FDG- and FES-PET/CT imaging, evaluate the correlation of lesion 
uptake between FES and FDG.   
 
1.3. Primary Objective for Expansion Phase (Added as of March 2022 
Amendment) 
 

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To assess change in staging of patients with newly diagnosed ILC (primary study arm, 
n=40) based on FES-PET/CT results per the American Joint Committee on Cancer 
(AJCC) TNM staging system. 
Primary endpoint: 
• 
Overall percentage of change of stage following research FES-PET/CT 
imaging compared to stage based on standard of care imaging.   
 
1.4. Secondary Objectives for Expansion Phase (Added as of March 2022 
Amendment) 
 
To assess whether quantity of methylated ctDNA at baseline (primary study arm, 
n=40) predicts patient stage at presentation per the American Joint Committee on 
Cancer (AJCC) TNM system.  
Secondary endpoint: 
• MethylPatch PCR ctDNA quantity at baseline as predictor of patient stage at 
baseline 
 
To assess whether quantity of methylated ctDNA at baseline (primary study arm, 
n=40) predicts survival at each follow-up interval starting at 6 months (follow-up 
performed at 6, 12, 18, 24, 36, 48 and 60 months).  
Secondary endpoint: 
• Correlation between quantity of methylated ctDNA with survival based on 
survival analysis at each follow-up period. 
 
To assess whether heterogeneous FES-PET/CT uptake at baseline (yes/no) (primary 
study arm, n=40), defined as abnormal FES uptake in some but not all proven or 
suspected ILC lesions, predicts survival at each follow-up interval starting at 6 
months (follow-up performed at 6, 12, 18, 24, 36, 48 and 60 months). 
Secondary endpoint: 
• Presence of heterogeneous FES-PET/CT uptake at baseline (binary 
variable/yes/no) as a predictor for survival as assessed by a survival analysis 
on follow-up assessments.  
 
1.5. Exploratory Objectives for Expansion Phase (Added as of March 2022 
Amendment) 
 
 
 
 

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Evaluate rate of complete ER blockade (yes/no) on optional post-therapy FES-
PET/CT study (Primary arm, n=10 patients) with ER+ ILC completing an optional FES-
PET/CT study 2-4 weeks after therapy onset. 
Secondary endpoint: 
• Rate of persistent abnormal FES uptake at sites of known or suspected ILC 
(yes/no).  
 
Evaluate rate of persistent FES uptake (yes/no) on optional FES-PET/CT 
(Exploratory Arm 1, n=10) for individuals with known metastatic initially ER positive ILC 
currently on hormonal therapy. 
Exploratory endpoint: 
• Rate of persistent FES uptake (yes/no) while on therapy 
 
Assess the negative predictive value of FES-PET/CT imaging (Exploratory Arm 2, 
n=5) for individuals with estrogen receptor (ER) negative ILC as determined by 
immunohistochemistry from core needle biopsy. 
Exploratory endpoint:  
• Negative predictive value for negative (no abnormal uptake) FES-PET/CT 
imaging 
 
Assess whether heterogeneous FES-PET/CT uptake at baseline (yes/no) for 
participants in Primary and Exploratory Arm 2, defined as abnormal FES uptake in 
some but not all proven or suspected ILC lesions, predicts development of recurrent 
disease (yes/no) for patients with no distant metastatic disease at baseline, during 
follow-up starting at 6 months (follow-up performed at 6, 12, 18, 24, 36, 48 and 60 
months). 
Exploratory endpoint: 
• Presence of heterogeneous FES-PET/CT uptake at baseline (binary 
variable/yes/no) and development of recurrent disease at follow-up (yes/no).  
 
To assess whether heterogeneous FES-PET/CT uptake at baseline (yes/no) for 
participants in all study arms, defined as abnormal FES uptake in some but not all 
proven or suspected ILC lesions, predicts development of new metastatic disease 
during follow-up (yes/no) for patients who have metastatic disease at baseline, during 
follow-up starting at 6 months (follow-up performed at 6, 12, 18, 24, 36, 48 and 60 
months). 
Exploratory endpoint: 
• Presence of heterogeneous FES-PET/CT uptake at baseline (binary 
variable/yes/no) and development of recurrent disease at follow-up (yes/no).  
 

Current Version: 12-01-2023 
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Assess whether quantity of methylated ctDNA at baseline for participants in Primary 
Arm and Exploratory Arm 2 predicts development of recurrent disease (yes/no) for 
patients with no distant metastatic disease at baseline, during follow-up starting at 6 
months (follow-up performed at 6, 12, 18, 24, 36, 48 and 60 months). 
Exploratory endpoint: 
• Quantity of methylated ctDNA at baseline as predictor of development of 
recurrent disease at follow-up (yes/no).  
 
To assess whether quantity of methylated ctDNA at baseline for participants in all 
study arms predicts development of new metastatic disease during follow-up 
(yes/no) for patients who have metastatic disease at baseline, during follow-up starting 
at 6 months (follow-up performed at 6, 12, 18, 24, 36, 48 and 60 months). 
Exploratory endpoint: 
Quantity of methylated ctDNA at baseline at baseline as predictor of development of 
new metastatic disease at follow-up (yes/no).  
 
 
2. BACKGROUND 
 
2.1. Invasive Lobular Carcinoma 
 
ILC is a distinct molecular and pathologic entity from invasive ductal carcinoma and is 
the second most common type of breast cancer next to invasive ductal carcinoma, 
comprising up to 15% of invasive breast cancer cases in the United States [1, 2, 4-6].  
 
2.2. Imaging of Invasive Lobular Cancer 
 
According to the National Comprehensive Cancer Network (NCCN) 2018 guidelines 
FDG-PET/CT may be performed as an alternative to a contrast-enhanced CT of the 
chest, abdomen and pelvis and Tc-99m MDP bone scan for evaluation of distant 
metastatic disease in newly diagnosed stage III breast cancer patients [7]. FDG-PET/CT 
is usually not obtained for stage I or stage II breast cancer patients as change in patient 
management is rare [7]. Prior studies have demonstrated FDG-PET/CT can identify 
sites of unsuspected metastatic disease in newly diagnosed breast cancer patients 
thereby altering treatment decisions given that palliative management is typical for 
stage IV disease, whereas neoadjuvant therapy followed by surgery and postoperative 
radiation may be considered for stage II and operable stage III disease [1, 7-11]. These 
guidelines consider invasive breast cancer as a single entity and do not consider 
whether tailoring imaging techniques for subtypes of breast cancer may be beneficial.  
However, prior research suggests that FDG-PET/CT may be more appropriate as an 
alternative to CT and bone scan for patients with invasive ductal carcinoma (IDC) rather 
than invasive lobular carcinoma (ILC) as FDG demonstrates comparatively reduced 
sensitivity for ILC metastases [1, 2, 6, 12, 13]. Compared to IDC, ILC is more often 
occult on mammography, ultrasound, and FDG-PET/CT [1-4, 6]; which is of importance 
for clinical management as ILC is more often multifocal and bilateral compared to IDC 

Current Version: 12-01-2023 
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[14].  Clinical breast examination also has lower sensitivity for detection of ILC 
compared to IDC, even for large tumors, as ILC may be indistinguishable from normal 
breast tissue on palpation [5, 15].  
 
A prior study evaluating systemic staging of newly diagnosed patients with stage I-III 
invasive breast cancer found that FDG-PET/CT is 1.98 times less likely to reveal 
unsuspected distant metastatic disease for women with ILC compared to IDC [1]. In this 
study, all IDC metastases demonstrated FDG avidity whereas 25% of ILC metastases 
(3 of 12) were not FDG avid [1]. Detection of local axillary metastatic disease on FDG-
PET/CT was also lower for ILC (0 of 146 patients) compared to IDC (7 of 89 patients)[1] 
despite data from the Surveillance, Epidemiology and End Results (SEER) database 
demonstrating similar rates for lymph node metastases between IDC and ILC [14]. 
Another study evaluating FDG-PET/CT for the diagnosis of primary breast cancer found 
that the false negative rate for detection of ILC by FDG was 65% (15 of 23 cases) 
compared to 23% for IDC (23 of 97 cases) when matching for tumors of the same size 
[3]. A final study reported a false negative rate of FDG for ILC detection of 13% (2 of 15 
patients) [2]. Mechanistically, ILC may not take up FDG as avidly as IDC due to lower 
tumor microvascularity, cellular density, proliferation rate, and number of GLUT 
transporters [2, 6, 12, 16-20].  ILC osseous metastatic disease is also more frequently 
occult on FDG-PET/CT compared to IDC [3, 12] as ILC osseous metastases are more 
frequently sclerotic, whereas FDG-PET/CT is more sensitive for lytic osseous 
metastases.  Sclerotic ILC osseous metastases also may be indistinguishable from 
benign bone islands on CT at initial staging, thereby necessitating biopsy or imaging 
follow-up for confirmation of osseous metastatic disease [3, 12].  Improved imaging 
strategies for primary and metastatic ILC are therefore warranted.     
 
Multiple studies have proven the efficacy of FES-PET/CT for imaging evaluation of ER+ 
invasive breast malignancy (evaluating both IDC and ILC together, with the large 
majority of cases comprising IDC) but, to our knowledge, no prior study has focused 
FES-PET/CT evaluation only to cases of ILC, nor have prior studies compared FES-
PET/CT directly with FDG-PET/CT for evaluation of newly diagnosed ILC. Given that all 
prior studies on FES-PET/CT have grouped a small number of ILC cases with a larger 
number of IDC cases, the imaging performance of FES-PET/CT specifically for ILC is 
unknown. ILC demonstrates higher rates of ER positivity than IDC with prior studies 
showing greater than 90% positivity for cases of ILC [1, 6].  Data from the SEER 
database also shows ILC demonstrates higher overall expression of ER than IDC (ILC 
95% positive for ER, n=17,503 vs IDC 74% positive for ER, n=172,379) [14]. FES-
PET/CT may therefore be suitable for imaging evaluation of a high proportion of patients 
with ILC.   
 
2.3. Overview of the PET Radiopharmaceutical [18F]Fluoroestradiol 
 
FES is an emerging PET radiopharmaceutical that utilizes binding of radioactive-labeled 
estradiol to estrogen receptors, thereby allowing direct imaging of estrogen receptor 
uptake within breast malignancy [21-47].  Prior studies have shown that FES binding to 
the ER is essentially equivalent to that of endogenous estradiol and FES uptake on PET 

Current Version: 12-01-2023 
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imaging has been shown to correlate with ER expression on in vitro assays such as 
radioligand binding or immunohistochemistry [43, 46].  
 
According to the Cancer Imaging Program of the National Cancer Institute (CEP-NCI) 
[18F]Fluoroestradiol Investigator Drug Brochure Version 5 (2017), over 1450 patients 
have received FES, either under the Radioactive Drug Research Committee program or 
under an Investigational New Drug (IND) application.  FES-PET/CT is increasing our 
understanding of the role of ER expression in invasive breast malignancies, both for 
diagnosis and staging of breast malignancy, in addition to informing the use of hormonal 
therapy versus chemotherapy or other therapies for patients with known breast cancer. 
In May 2020, FDA approved the use of FES manufactured by Zionexa for detection of 
estrogen receptor (ER)-positive lesions as an adjunct to biopsy in patients with recurrent 
or metastatic breast cancer. 
 
As a non-invasive agent, FES-PET/CT allows imaging of the entire body for abnormal 
estrogen receptor density without requiring a biopsy, thereby facilitating the in vivo 
assessment of estrogen receptors.  FES-PET/CT allows imaging of the entire volume of 
receptor status within every tumor, thereby avoiding potential sampling error from tissue 
sampling of only a portion of a tumor and providing a potential route to evaluate for 
estrogen receptor heterogeneity within a tumor or between sites of primary and 
metastatic malignancy.  Additionally, by providing an imaging biomarker for assessment 
of estradiol binding to the estrogen receptor, FES-PET/CT assesses the biological 
activity of the estrogen receptor, which is not possible by immunohistochemistry.   
 
Although beyond the scope of the current study, FES-PET/CT may guide therapeutic 
decision making by documenting the presence of estrogen receptor heterogeneity in 
primary and metastatic invasive breast cancer lesions which can be useful to guide 
therapeutic decision making between estrogen-targeting hormonal therapies versus 
chemotherapy.  Additionally, FES-PET uptake at sites devoid of native estrogen 
receptor expression, such as bone, may be highly specific for breast cancer 
metastases.  
 
According to the CEP-NCI [18F]FES Investigator Drug Brochure Version 5 (2017), more 
than 20 fluorinated estrogen derivatives have been evaluated for imaging, and FES 
remains the most promising to date.   
 
2.4 Overview of the FES/FDG SUVmax ratio 
Several recent prior studies have shown potential predictive utility for progression free 
survival using the FES/FDG SUVmax ratio [48-50]. Based on a traditional 
understanding of physiology, indolent tumors would be predicted to have a lower 
glycolytic rate and therefore show low FDG uptake.  Indolent tumors are also more likely 
to be well-differentiated and, for breast cancer, this would therefore suggest presence of 
functional ERs and high corresponding FES uptake.  The most indolent, well-
differentiated tumors may therefore show high FES uptake, low FDG uptake, and result 
in a high FES/FDG SUVmax ratio [28]. Additionally, as FES has been shown to be 
predictive of response to endocrine therapy, and FDG has been shown to predict 

Current Version: 12-01-2023 
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disease aggressiveness, it is possible that the combination of these factors will be 
predictive of progression free survival [28].   
 
However, Liu, et al. [48] found the opposite to be true in a recent study on a population 
of ER+ metastatic breast cancer patients (not specific to ILC with 82.8% IDC and 11.4% 
ILC) undergoing treatment with fulvestrant. In this study, lesions were classified as 
heterogeneous (patient with multiple tumors demonstrating variable FES-PET/CT 
uptake) or, if homogeneous, as low or high FES/FDG ratios with a cut-point of 0.96 
based on the median FES/FDG SUVmax ratio of the sample population.  Neither FES 
SUVmax nor FDG SUVmax predicted progression free survival, but the FES/FDG ratio 
did separate patients based on median progression free survival for low FES/FDG (29.4 
months) compared to high FES/FDG (14.7 months).  The heterogeneous group had the 
lowest median progression free survival (5.5 months) suggesting that imaging evidence 
of estrogen receptor dysfunction in some lesions is associated with shorter median 
progression free survival.  These three-way PET classifiers were shown to be 
independent, statistically significant prognostic factors for median progression free 
survival on fulvestrant therapy (p-value 0.006).   
 
The results of the study by Liu, et al. [48] are unique from what the study authors 
hypothesized in that patients with low FES/FDG ratios showed longer median 
progression free survival when the opposite may be predicted based on FES uptake 
correlating with well-differentiated tumors and FDG uptake as a marker of metabolic 
aggressiveness.  The study authors surmise that one reason may be 17B-estradiol 
activation of glucose uptake via ER-dependent PI3k/Akt activation in ER+ breast cancer 
cell lines [48].  
 
2.5 Results from Study Pilot Phase: 
The pilot phase of this study completed in 2021 by Dr. Covington and the Center for 
Quantitative Cancer Imaging at Huntsman Cancer Institute evaluated 17 patients 
(average age 58.4 years, all female sex) with ILC with FES-PET/CT and optional FDG-
PET/CT imaging.  The hypothesis of this trial was that FES-PET/CT would have 
superior detection of ILC compared to standard-of-care FDG-PET/CT, which, per a 
literature search, has a positive detection rate of 60%.  This study showed positive 
(abnormal) FES uptake within 22/25 lesions (88%, p-value = 0.0024) and within 14/17 
subjects (82.4%, p-value = 0.046).   
 
Heterogeneous FES uptake defined as uptake in some but not all suspected sites of 
metastatic disease based on biopsy and imaging in patients with more than 1 lesion 
was identified in 2/10 lesions (2.5 to 55.6% exact 95% binomial confidence interval).   
 
Discordant uptake was seen in 33/41 lesions and, for these lesions, there was 
significantly more FES+/FDG- than FES-/FDG+ cases (p = 0.016) using McNemar’s test 
for correlated proportions.  For cases with both FES and FDG imaging, Spearman 
correlation of lesion uptake based on maximal SUV uptake (SUVmax) showed a p-value 
of 0.00083 for biopsied lesions and a p-value of 0.0046 for all suspected abnormal 
lesions. 

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Interestingly, one patient with histologically proven ER- ILC from core needle biopsy 
was enrolled and did show abnormal FES uptake by qualitative and quantitative imaging 
assessment criteria.  It is unknown whether this result is secondary to disease 
heterogeneity with only an ER- portion of a tumor being sampled, with separate ER+ 
components being unsampled, or if this reflects a false negative abnormal finding on 
FES-PET/CT imaging.  
 
Of 17 cases of newly diagnosed ILC, 7 had histologically-proven nodal metastatic 
disease (42.8%) on core biopsy or sentinel lymph node biopsy and 3 of these (42.8%) 
were not detected on either standard of care ultrasound or MRI.  Diagnostic 
performance of study imaging for detection of axillary nodal metastatic disease is shown 
in Table 2.5.1. 
 
Table 2.5.1. Imaging performance for histologically proven axillary nodal metastatic 
disease.  
 
 
Sensitivity 
Specificity 
PPV 
NPV 
Accuracy 
N= 
Ultrasound 
29% 
90% 
66.7% 
64.3% 
64.7% 
17 
MRI 
57% 
90% 
80% 
75% 
76.5% 
17 
FDG-
PET/CT 
67% 
100% 
100% 
80% 
80% 
14 
FES-
PET/CT 
100% 
100% 
100% 
100% 
100% 
17 
Abbreviations: PPV: positive predictive value.  NPV: Negative predictive value. 
 
Six non-ILC malignant breast lesions were identified in our study population consisting 
of 2 IDC lesions and 4 lesions that were ductal carcinoma in situ (DCIS). Of these, FES-
PET/CT identified 5 of 6, FDG-PET/CT identified all 6, contrast-enhanced breast MRI 
detected 3 of 6, ultrasound detected 1 of 4 (2 cases had no ultrasound imaging), and 
mammography detected 1 of 6.  This suggests superiority of both FES- and FDG-
PET/CT for identification of additional lesions compared to standard of care contrast-
enhanced breast MRI, ultrasound, and mammography that will be further assessed in 
the present study.  
 
We calculated the mean SUVmax FES/FDG ratio for our cohort of ILC patients was 1.2 
compared to the mean ratio of 0.96 in the Liu, et al. study [48].  When assessing 
whether this ratio correlates with tumor grade, we found a p-value of 0.01 with R of -
.6042 showing a moderate negative correlation between FES/FDG ratios and tumor 
grade, suggesting that higher grade tumors have lower FES/FDG ratios. When grade 1 
or grade 2 disease as a function of the FES/FDG ratio being above or below the cut-
point of 1.2, this cut-point was deemed significant with a p-value of 0.04.   
 
When assessing whether lymph node metastatic disease varies as a function of 
FES/FDG ratios above or below a cut-point of 1.2 a p-value of 0.07 was found, and a 

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correlation coefficient R of 0.5004 with a p-value of 0.08, therefore suggesting a trend 
towards significance that will be further assessed in the present study.   
 
 
2.6 Overview of MethylPatch PCR Analysis for Breast Cancer Evaluation 
 
MethylPatch PCR analysis is a blood-based methylated circulating tumor DNA (ctDNA) 
assay that has been developed at the Huntsman Cancer Institute under the direction of 
K-T Varley, PhD. Many blood-based tumor marker tests have been developed to 
improve the detection of breast cancer [51, 52]. The most widely utilized is CA 15-3, but 
it is often detected in association with other non-malignant diseases and benign 
conditions, and has lower sensitivity for early stage disease and therefore cannot serve 
as a prognostic biomarker in clinical practice [51, 52]. There is a need for more sensitive 
and specific biomarkers for the detection of breast cancer. The discovery that tumor 
cells shed small amounts of DNA into the bloodstream, and that cancer-specific 
mutations can be detected by ultra-deep sequencing of DNA isolated from blood plasma 
has revolutionized the field of cancer diagnostics [53, 54]. Circulating tumor DNA 
(ctDNA) tests are a promising approach for detecting and monitoring breast cancer 
because a minimally invasive blood draw can be part of routine yearly wellness 
appointments [54].  
 
The  Methyl  Patch  PCR  assay  addresses several major  challenges  associated  with  
using  ctDNA  for  breast  cancer screening.  These include identifying markers present 
across patients and specific to cancer.  Most ctDNA tests rely on the detection of 
mutations in targeted panels of cancer genes. Dr. Varley’s lab analyzed tumor mutation 
data from The Cancer Genome Atlas (TCGA) and found that 18% (189/1066) of breast 
cancer tumors do not contain point mutations in 73 cancer genes covered by Guardant 
Health’s field-leading  ctDNA  assay (Guardant  360) [55]. This analysis indicates that 
even if the mutation-based ctDNA test had perfect analytical sensitivity, it would be 
unable detect cancer in 18% of patients with breast cancers. Single base substitutions 
in TP53 are the most common somatic mutation in breast cancer, and often the only 
cancer gene mutation found in patient tumors, but TP53 mutations are also found in 
blood plasma of healthy individuals due to clonal hematopoiesis of indeterminate 
potential [56]. These age-related false-positive TP53 mutations in healthy individuals 
diminish the specificity of mutation-based ctDNA screening tests.  
 
To address this challenge, Dr. Varley’s lab studied a different marker of ctDNA: DNA 
methylation.  Aberrant DNA methylation occurs early in tumor development at hundreds 
of specific genomic loci in each tumor.  Many cancer genomics research groups, 
including Dr. Varley’s, have identified hundreds of loci that exhibit cancer-specific 
methylation across cancer types [57-59]. While  some  patients’  tumors  contain  few,  
or  sometimes  zero, mutations  in  targeted  cancer  gene  panels,  every  tumor  will  
have  cancer-specific  DNA  methylation  at  the commonly methylated loci, ensuring 
specific detection of ctDNA in a larger portion of patients with cancer. The benefits of 
detecting methylated ctDNA were demonstrated in a recent ASCO presentation by the 
Circulating Cell-free Genome Atlas Study which reported that whole genome bisulfite 

Current Version: 12-01-2023 
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sequencing (a DNA methylation assay) had higher sensitivity than mutation-based 
assays [60]. 
 
Sensitive detection of early stage disease is also challenging using ctDNA assays.  
Early stage tumors produce lower amounts of cell-free DNA.  In three recent studies 
that evaluated ctDNA mutation assays for cancer detection, the frequency of ctDNA 
detection in Stage 1 patients was approximately 50% [53, 61, 62]. This suggests that 
tumor-specific mutations were present at a concentration of 0.5 molecules per blood 
specimen. One way to increase the probability of observing rare molecules is to 
increase the amount of blood collected from each patient, which is often prohibited by 
Institutional Review Boards due to patient safety. Alternatively, measuring multiple 
mutations increases the probability of observing rare ctDNA. If you could detect 6 
independent mutations in a patient, the probability of missing all 6 mutations due to 
undersampling is low, and 98% (1-0.5^6) of Stage I patients would have detectable 
ctDNA. Dr. Varley’s lab analyzed tumor mutation data from The Cancer Genome Atlas 
(TCGA) and found that less than 5% of breast cancers had 6 or more mutations in 
cancer genes. This result indicates that cancer gene panel assays are unlikely to be 
sensitive enough for detection of rare ctDNA molecules in early stage disease.  To 
increase the sensitivity and specificity of ctDNA mutation assays for detecting breast 
cancer, several recent studies have performed exome sequencing on patients’ tumors 
and developed custom assays to detect multiple mutations specific to each patient’s 
tumor.  In two recent studies using this approach, ctDNA was detected a median of 8.9 
and 10.7 months prior to clinically detectable disease recurrence, with sensitivities of 
89% and 96% [63, 64]. The success of these studies demonstrates that ctDNA assays 
can provide sensitive detection of breast cancer. Unfortunately, this strategy relies on 
sequencing a patient’s tumor to identify their unique mutation profile for assay 
development, and thus cannot be used for initial diagnosis. A  recent  study  that  
included  both  a  ‘cancer  gene’  mutation  panel  and  protein  markers  showed  
improved specificity for initial detection of breast cancer (99%), but the sensitivity for 
detecting breast cancer was only 33% [61]. Additionally, broader implementation of this 
type of multi-analyte test will inherently face challenges related to robustness and 
feasibility. Together these studies suggest that multiplexed biomarker tests are a 
promising direction to pursue, but a more universal, single-analyte approach is needed.  
 
An individual patient’s tumor contains many more methylation defects than mutations 
[65]. While it is unlikely that a patient’s tumor will contain 6 point mutations in a targeted 
cancer gene panel, the presence of 6 cancer-specific methylation events in a single 
patient is expected, because hundreds of loci are concurrently hyper-methylated in each 
tumor. A  multiplexed  ctDNA  test  designed  to  detect  cancer-specific  
hypermethylation  at  many  loci increases  the  probability  of  observing  rare  ctDNA  
molecules  in  the  blood  specimen.  This enables robust and sensitive detection of 
early stage tumors that release smaller quantities of DNA into the bloodstream.  
 
An additional drawback of using ctDNA mutation assays for early diagnosis is that many 
different tumor types contain the same mutations. The genes that are most frequently 
mutated in one cancer type (>20% of tumors) are also frequently mutated in other 

Current Version: 12-01-2023 
15 
 
cancer types (Figure 2.6.1A). If a ctDNA test detected a TP53 mutation during routine 
screening, it could indicate that the individual had cancer, but because TP53 mutations 
occur in all tumor types, it doesn’t predict the patient’s tumor type or location. This 
would lead to expensive follow-up imaging of multiple organs to identify the primary 
source of malignancy and determine which oncology subspecialist should manage the 
patient’s care. In contrast, different cancer types have distinct methylation profiles [66-
68] (Figure 2.6.1B). While most mutations are not unique to specific cancer types, if a 
breast cancer-specific DNA methylation profile is detected in the blood, it indicates a 
specific diagnosis of breast cancer leading to a more straightforward diagnostic breast 
imaging workup and referral to the 
appropriate breast oncology specialist.  
This study will utilize a new assay, called 
MethylPatch PCR, which is designed to 
provide deep targeted sequencing of 54 
regions of the genome that exhibit DNA   
methylation specifically in breast cancer. 
The multiplexed quantification of 
methylated ctDNA from many loci is a 
promising approach to achieve a more 
specific, sensitive and robust blood-based 
tumor marker of breast cancer. 
 
The MethylPatch PCR assay is designed 
to provide deep targeted sequencing of 
54 regions of the genome that exhibit 
DNA methylation in breast tumors. To 
determine if  this approach could be used 
to detect methylated ctDNA, Dr. Varley’s 
lab performed the MethylPatch PCR 
assay on DNA isolated from blood 
plasma collected from 15 breast cancer  
patients in  the  2-month  interval  
between  diagnosis  and  surgical  
resection  of  their tumors.  They also 
performed the assay on DNA isolated 
from blood plasma collected from 20 
healthy donors. They calculated the 
percentage of methylated UMIs 
(molecules) observed for each target 
region in each sample.  They then 
calculated normalized percent of methylated values for each target region in each 
sample by fitting the values to a standard curve using coefficients from a linear 
regression of the observed and expected values in a titration (Figure 2.6.2). In the 
healthy donor plasma samples, they observed fewer target regions with percent 
methylated values above 0.15 (LOD), and the mean of values in each of the healthy 
donor samples was lower than 0.15 (Figure 7). The breast cancer patient plasma 
A. 
 
B. 
 
 
 
Figure 2.6.1. A. The most common 
mutations in each cancer are found in 
other cancer types. B. Heatmap of 
genomic loci that exhibit cancer-type 
specific hypermethylation in TCGA data. 

Current Version: 12-01-2023 
16 
 
samples all had a larger number of target regions with percent methylated values above 
0.15 (LOD), and all produced mean normalized percent methylated values greater than 
0.15. The presence of higher quantities of methylated DNA in all of the breast cancer 
samples compared to all of the healthy donor samples confirms that our approach can 
detect low levels of methylated ctDNA in blood plasma samples from patients with 
breast cancer. Notably, four of the  
breast cancer patients were diagnosed with Stage I disease, demonstrating the ability to 
detect methylated ctDNA in patients with early 
stage disease and small tumors  
 (Figure 2.6.3). These results confirm that the 
MethylPatch PCR assay can detect and 
quantify methylated ctDNA specifically in breast 
cancer patients, and demonstrates that the 
highly multiplexed nature of this assay (54 
markers) provides more opportunities to 
sensitively and robustly detect rare ctDNA in 
each patient. 
 
Preliminary data from Dr. Varley’s lab has also 
identified genes in triple negative breast cancer 
that, when highly methylated, predict an early 
clinical relapse.  Additionally, early data 
suggests that if the methylation pattern 
identified in metastatic disease is seen when 
only primary breast malignancy but no 
metastatic disease is evident, this is also 
predictive of an early clinical relapse. Such 
cancer-specific DNA methylation changes on 
ctDNA in blood plasma may predate clinical 
relapse by approximately 10 months.  Ideally 
such markers should be monitored 
longitudinally throughout treatment and 
remission, and this study will provide this necessary and important information for 
patients with ILC.  
Pertinent to the current study, the number of circulating tumor cells are higher in ILC 
compared to IDC but the prognostic nature of having a higher circulating tumor cell 
burden was weaker compared to IDC suggesting that the circulating tumor cells in ILC 
are more dormant [69].  Additionally, there is a current challenge to differentiate 
between ILC tumors that have a worse prognosis given the near-uniformity in terms of 
ER and PR positivity, HER2 negative status, proliferation index, and grade [70].  This 
study will evaluate, in a longitudinal manner, whether MethylPatch PCR ctDNA analysis 
provides additional prognostic value beyond these standard-of-care metrics.   
 
 
 
 
Figure 2.6.2.  MethylPatch PCR 
can accurately quantify low levels of 
methylated DNA titrated into cell-
free DNA from healthy donors. 
Mean and 95% confidence intervals 
are depicted. 

Current Version: 12-01-2023 
17 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3.  PHARMACOLOGY, SAFETY and RADIATION DOSIMETRY of 
[18F]FES  
 
3.1. Pharmacology and Safety 
 
3.1.1. Pharmacology of [18F]FES in Humans 
 
The information provided herein is abstracted from the CEP-NCI [18F]FES Investigator 
Drug Brochure Version 5 (2017).  
 
The pharmacology of FES is best understood by analogy to estradiol, a naturally 
occurring product synthesized in the ovary in pre-menopausal women and by 
 
Figure 2.6.3.   Breast cancer patient plasma 
samples (N=15) contained methylated ctDNA 
from more target genomic regions (dots), higher 
percentage of methylated molecules from each 
region, and higher mean percentages of 
methylated molecules across regions 
(rectangles) compared to health donor plasma 
samples (green, N=20).   Stage I breast cancer 
patients are indicated by light pink dots. 

Current Version: 12-01-2023 
18 
 
conversion from adrenal steroids, largely through the action of aromatase enzymes. 
Pre-menopausal levels of estradiol vary widely during the menstrual cycle, reaching 
levels as high as 500 pg/ml (1.7 nM) mid-cycle. In post-menopausal women and in men, 
levels are generally less than 30 pg/ml (0.1 nM). Because FES-PET is not intended for 
therapeutic effect and is not used in sufficient concentration to elucidate a physiologic 
effect, mechanisms of action beyond metabolism and binding to ER have not been 
studied. 
Typical blood FES concentration after a 6 mCi injection is 1 µCi/ml (< 3 pmoles/ml) peak 
and by 60 minutes after injection is less than 150 fmoles/ml. For the limiting-case 
specific activity this corresponds to a peak blood FES level of 833 pg/ml and 42 pg/ml at 
one hour, comparable to pre-menopausal mid-cycle estradiol levels of 62 to 534 pg/ml 
and post-menopausal levels of 20 to 88 pg/ml. Males have levels similar to post-
menopausal women. Thus the short-term exposure to estrogen from an FES injection 
as part of an FES PET study transiently yields physiologic levels of the estrogenic 
steroid, decreasing to sub-physiologic levels after 60 minutes. 
 
The metabolism of estradiol has been well characterized. It occurs largely in the liver. 
The formation in the liver of sulfate conjugates at hydroxyl sites is an important route of 
metabolism. These conjugates are secreted into bile and have efficient enterohepatic 
circulation that serves as a reservoir for regulating estrogen levels. Glucuronides are 
also formed in the liver, to a lesser extent than sulfates, and their primary route of 
elimination is the urine. 
 
3.1.2. Toxicity of [18F]FES in Humans 
 
The information provided here is abstracted from the [18F]FES Investigator Drug 
Brochure, Version 5 (2017), with minor changes to reflect our local manufacturing 
process. The [18F]FES is a sterile, IV injectable solution in sodium chloride buffered by 
sodium ascorbate. The injected dose of [18F]FES is 6 mCi (185 MBq), with an allowable 
range of 3 to 6 mCi and a specific activity greater than 170 Ci/mmol at the time of 
injection. FES is the only active ingredient. There is no evidence that nonradioactive 
and radioactive FES molecules display different biochemical behavior. Approximately 
1500 patients are known to have received this drug, based upon published reports, 
without significant adverse effects. 
 
 [18F]FES is purified by HPLCand is formulated in a solution 0.9% (v/v) sodium 
phosphates for injection (USP), less than 10% ethanol (v/v) in normal saline mL with a 
maximum injection volume of 5 ml.. The concentration of ethanol in the final injectate is 
less than 32 mg/ml, or a maximum of 0.505 ml of ethanol. This is less than one-third of 
the amount of ethanol in one alcoholic drink and <0.05 ml/kg (< 0.04g/kg) for a standard 
56.8-kg woman. In RTECS, the LDLo is given as 1.4 g/kg orally for producing sleep, 
headache, nausea, and vomiting. Ethanol has also been administered intravenously to 
women experiencing premature labor (8 g/kg) without producing any lasting side effects. 
Based upon these reports and experience with over 100 patients over the past decade 

Current Version: 12-01-2023 
19 
 
receiving this amount of ethanol in injectates, we conclude that ethanol will not pose any 
danger of toxicity in this study. 
 
The other components of the final product solution; sterile water for injection, saline and 
sodium phosphates are all USP grade. These are all nontoxic for USP grade injectables 
at the concentrations that will be used. The final product is at pH 6.5 and the final 
injection volume is ≤ 5 ml. 
 
The potential contaminants in the final [18F]FES drug product are: acetone, acetonitrile, 
Kryptofix®, other reaction products. Residual solvents in the final product are limited to 
410 ppm of acetonitrile. Acetonitrile is used to dissolve the Kryptofix® [2.2.2] and is the 
solvent for the reaction. The permissible level of acetonitrile in the final product is ≤ 410 
ppm, the USP permissible level of acetonitrile in 2-[18F]FDG.   All of the residual solvent 
levels met our acceptance criteria in our initial three qualification syntheses. 
 
The toxicity for Kryptofix® has not been reported (RTECS Number Kryptofix® 222 
MP4750000). The MSDS for Kryptofix® lists the LD50 for intravenous administration in 
rats and mice as 35 and 32 mg/kg respectively. In light of these reported toxicities, the 
FDA has proposed a maximum permissible level of 50 µg/ml of Kryptofix® in 2-
[18F]FDG, as has the USPi; therefore, this maximum permissible level will also apply to 
the [18F]FES final product. All of the Kryptofix levels were < 50 µg/ml in our initial 
qualification syntheses (n=3). 
 
Although a relatively pure [18F]FES product is obtained, trace amounts of other reaction 
products may be found in the final product. For this reason, an upper limit of 5 µg per 
dose has been set for the total mass of any other materials in the final injectate. The 5 
µg is determined by assuming that the UV absorbing compounds at 280 nm have the 
same molar extinction coefficient as FES. 
 
Pregnant or nursing women: Reproductive toxicology studies have not been 
performed with [18F]FES. However, since [18F]FES is a radiopharmaceutical, it is 
assumed that fetal toxicity or embryo fetal toxicity may result if it is administered to a 
pregnant woman. Therefore, [18F]FES injection is not suitable for administration to 
pregnant or nursing women. 
 
Genotoxic and mutagenic potential: Carcinogenesis does not appear to be a risk of 
administration of [18F]FES as described in the Investigator’s Brochure based upon a 
large number of animal toxicity studies. Although estrogens, including the natural 
hormones estradiol and estrone, are carcinogenic in laboratory animals, synthetic 
estrogens such as 2-fluoroestradiol and 4-fluoroestradiol are poor carcinogens in the 
same animal model systems because the fluorine blocks metabolism when substituted 
in these positions. [18F]FES is labeled with fluorine at the 16 position of the estradiol. 
Administration of [18F]FES as described herein, for up to four PET scan procedures, 
results in intermittent and vastly reduced overall estrogenic exposure compared to 
regimens known to cause cancer in animals.  
 

Current Version: 12-01-2023 
20 
 
Findings in safety pharmacology and toxicology studies: Nonclinical toxicology and 
safety pharmacology studies with [18F]FES have not identified any specific target organs 
or adverse effects on nervous, respiratory, or cardiovascular systems. As such, no 
potential [18F]FES injection-related safety risks for humans have been identified on the 
basis of the nonclinical data. 
 
Expected risks to subjects participating in clinical studies: No serious adverse 
reactions have been reported from any published studies. [18F]FES could potentially 
exert toxic effects through 1 of 3 mechanisms: (1) radiation exposure to tissues from the 
radioactive label, (2) physiologic actions mediated through the ER, and (3) direct toxic 
or mutagenic effects of FES or metabolites. Radiation exposure from [18F]FES at 
injected activities used in PET (6 mCi, typical) is low, and is comparable to other nuclear 
medicine procedures. With respect to the other two mechanisms of toxicity, FES 
injected as a bolus for PET imaging (current mass dose limit of 5 micrograms) 
transiently reaches physiologic concentrations, but returns to sub-physiologic levels 
within an hour after injection. As such, toxic effects due to actions mediated through the 
ER and directly toxic effects of metabolites will be far less than those of natural ER 
ligands. All of the evidence supports the safety of [18F]FES-PET imaging. Intravenous 
injection and the use of an intravenous cannula are known to carry a small risk of 
infection and hematoma. The exposure to radiation will not exceed that which is 
considered acceptable in accordance with appropriate guidelines. 
 
Reports of intravenous administration of estrogens are rare; it is used in this form 
largely in the setting of acute dysfunctional uterine bleeding. Two studies have 
documented acute toxicities resulting from intravenous bolus doses. White and 
colleagues studied pharmacokinetics and tolerability of 17β-estradiol in eight 
postmenopausal women [71]. Estradiol was administered in doses of 25, 50, 100, or 
200 µg peripherally over a five second period. Peak serum levels were not reported; 
however, the authors did document approximate dosage proportionality with respect to 
serum area under the curve (AUC). An adverse event was reported in only one patient 
who experienced mild discomfort at the injection site immediately following her dose. 
This reaction lasted 3 – 4 seconds and did not recur. 
 
Intravenous administration of Premarin® 25 mg versus placebo in 34 patients with 
dysfunctional uterine bleeding resulted in mild adverse reactions reported for seven of 
18 treated patients (39%) versus two of 16 (13%) in the placebo group [72]. Adverse 
effects in the Premarin® treated patients included flushing, euphoria, dizziness, 
drowsiness, and taste disturbances. The mean changes from baseline following 
injection of Premarin® versus placebo for blood pressure, pulse and respiratory rate 
were not statistically significant. In another study, intravenous estradiol combined with 
oral estradiol was given in post-menopausal women with recurrent ischemia and history 
of unstable angina [73]. This study reported a low incidence of headache, edema, 
vaginal bleeding, and a 23% incidence of breast tenderness and mood changes. 
Results of these studies are shown in Table 3.1.  
 
Table 3.1. Adverse events from selected trials of intravenous estradiol 

C urre nt Versi o n: 1 2- 0 1- 2 0 2 3 
2 1 
 
N u m b er 
of 
P ati e nt s  
Dr u g  
D o s e  
D ur ati o n  
A d v er s e E v e nt s  
E str a di ol 
Bl o o d 
L e v el s  
S o ur c e  
8  
I V 
e str a di ol  
2 5 g  
5 0 g 
1 5 0 g 
3 0 0 g 
Si n gl e 
i nj e cti o n 
N o r e p ort e d A E  
B a s eli n e = 
4 2. 2 n g/ L  
6 0 mi n s 
p o st i nj = 
6 8 9. 6 n g/ L  
R o s a n o  
1 9 9 3 [ 7 4] 
1 8  
I V 
Pr e m ari n
® 
( C o nj u g at e d 
E q ui n e 
E str a di ol)  
2 5 m g  
D u al 
i nj e cti o n 
Fl u s hi n g, 
dr o w si n e s s, 
e u p h ori a, 
di z zi n e s s, 
n a u s e a  
N ot 
r e p ort e d  
D e v or e 
1 9 8 2 [ 7 2] 
1 0 0  
I V 
e str a di ol  
+ 
Or al 
e str a di ol  
1. 2 5 
m g  
+ 
1. 2 5 
m g/ Q D  
Si n gl e 
i nj e cti o n 
+ 
2 1 d a y s of 
or al E  
o  fl u s hi n g  
7 %  
o  h e a d a c h e     1 1 %  
o  e d e m a         1 1 %  
o  Va gi n al  
bl e e di n g      1 0 %  
o  br e a st 
t e n d er n e s s  2 3 % 
o  m o o d  
c h a n g e s        5 %  
N ot 
r e p ort e d 
S c h ul m a n [
7 3]  
 
T h e R o s a n o  et al st u d y i s m o st r el e v a nt t o t h e u s e of F E S -P E T [ 7 4]. F or t hi s st u d y u p t o 
3 0 0 µ g of e str a di ol w a s a d mi ni st er e d i ntr a v e n o u sl y. Bl o o d l e v el s of e str a di ol w er e u p t o 
6 9 0 n g/ L ( p g/ ml) at 6 0 mi n ut e s p o st i nj e cti o n, a n d t h er e w er e n o r e p ort e d a d v er s e 
e v e nt s. F or F E S u s e d f or P E T, t h e t y pi c al d o s e i s 1. 5 µ g or l e s s, wit h a m a xi m u m d o s e 
of 5 µ g, a n d f or t h e d o s e li mit i nj e cti o n t h e bl o o d l e v el at 6 0 mi n ut e s s h o ul d b e 4 2 p g/ ml. 
P E T st u di e s al s o i n di c at e t h at t h e ti s s u e di stri b uti o n a n d bl o o d cl e ar a n c e of [1 8 F ] F E S i s 
si mil ar t o I V e str a di ol.  
 
Wit h t h e e x c e pti o n of di s c o mf ort at t h e i nj e cti o n sit e, a d v er s e e v e nt s t o F E S I V i nj e cti o n 
at d o s e s u s e d f or P E T/ C T  h a v e n ot b e e n r e p ort e d  [ 7 5]. 
 
3. 2. H u m a n R a di ati o n D o si m etr y  
 
3. 2. 1. H u m a n R a di ati o n D o si m etr y of [ 1 8 F ] Fl u or o e str a di ol 
 
 
T h e a m o u nt of i nj e ct e d [1 8 F] F E S  a cti vit y u s e d i n t hi s pr ot o c ol will b e 2 2 2 M B q ( 6 m Ci). 
T h e d o si m etr y e sti m at e s b a s e d o n 4 9 p ati e nt s w a s pr e vi o u sl y p u bli s h e d a n d i s u s e d t o 
e sti m at e r a di ati o n d o s e a n d ri s k s i n t hi s pr ot o c ol  [ 7 6]. T h e gr e at e st or g a n a b s or b e d 
d o s e s f or a 2 2 2 M B q ( 6 m Ci) i nj e cti o n of [ 1 8 F] Fl u or o e str a di ol ar e t h e li v er ( 2 8 m G y) a n d 
g all bl a d d er ( 2 3 m G y), w hil e t h e eff e cti v e d o s e i s 4. 9 m S v ( T a bl e 3. 3).  
 

Current Version: 12-01-2023 
22 
 
 
3.2.2. Human Radiation Dosimetry of [18F]Fluorodeoxyglucose 
 
The amount of injected [18F]FDG activity used in this protocol will be 555 MBq (15.0 
mCi). This is the typical activity used for a clinical whole-body PET study. The 
radiation dosimetry estimates for [18F]FDG are based on Publication 106 issued by 
The International Commission on Radiation Protection (ICRP) [International 
Commission on Radiological Protection. Radiation dose to patients from 
radiopharmaceuticals. Addendum 4 to ICRP Publication 53. ICRP Publication 106. 
Feb 24, 2014.]. The greatest organ absorbed doses for a 555 MBq (15 mCi) injection 
of [18F]FDG are the bladder (72 mGy) and the heart (37 mGy), while the effective 
dose is 10.6 mSv (Table 3.4). 
 
 
3.2.2 Human Radiation Dosimetry for CT exams performed on the PET/CT 
Scanner 
The study will be performed on the research GE Discovery 710 PET/CT scanner in the 
Molecular Imaging Suite at HCI.  Each PET imaging study will require a helical CT scan 
from the top of the head through the knees with near diagnostic technique for both 
clinical interpretation and attenuation correction. The CT acquisition parameters for 
large patients receiving the greatest exposure will be 140 kVp, 0.5s rotation speed, 250 
mA tube current, 64 x 0.625 mm collimation, and a pitch of 1.35 for the helical scan. 
Note that most patients will utilize 120 kVp but dosimetry estimates are based on larger 
patients at 140 kVp. Automatic tube current modulation is used for all CT exams but for 
the purpose of estimating maximum risk, a maximum fixed tube current of 500 mA is 
used for all calculations in order to provide a conservative estimate of maximum risk. In 
order to estimate the absorbed doses of individual organs and the resulting effective 
dose, the ImPACT Scan CT Patient Dosimetry Calculator (Version 1.0.4) was used with 
the specific acquisition parameters used in this protocol and the NRPB monte carlo 
dose data sets for the GE Lightspeed VCT scanner produced in report SR250 dosimetry 
tables [3.) ImPACT, Imaging Performance Assessment of Computed Tomography 
Scanners (online). 2006, St. George's Hospital, ImPACT group: Tooting, 
London. http://www.impactscan.org/index.htm. 4.) Shrimpton, P.C., et al., Survey of CT 
Practice in the UK.  Part 2:  Dosimetric Aspects, NRBP-R249. 1991: London. 5.) Jones 
DG, Shrimpton PC. Survey of CT practice in the IK. Part 3: Normalized organ doses 
calculated using Monte Carlo techniques. Chilton, NRPB-SR250. 1991, (London, 
HMSO).]. A correction factor has been applied to account for the difference in the CT 
Dose index (CTDIvol) derived from ImPACT Calculator and that reported on the GE 
scanner. Note that the topogram contributes a negligible radiation exposure to the 
helical CT exam and was not included in the dose estimates. The greatest organ 
absorbed doses for a single helical body CT scan using the aforementioned acquisition 
parameters are bone surfaces (28 mGy) and thyroid (27 mGy), while the effective dose 
is 17 mSv (Table 3.3). 
 
 

Current Version: 12-01-2023 
23 
 
3.2.3. Cumulative Radiation Dosimetry for 18F]Fluoroestradiol-PET/CT only 
 
The cumulative radiation dose to a research subject participating in this clinical trial and 
receiving only the mandatory [18F]Fluoroestradiol-PET/CT has been compiled from the 
dosimetry estimates for a single [18F]Fluoroestradiol-PET study and the corresponding 
whole body CT scan that will be performed as part of the PET/CT procedure. For 
research subjects participating in the clinical trial and receiving only the single 
[18F]Fluoroestradiol-PET/CT study, the greatest cumulative organ absorbed doses for 
the study are the liver (44 mGy), and gallbladder (39 mGy) while the effective dose is 22 
mSv (Table 3.3). 
 
3.2.4. Cumulative Radiation Dosimetry for [18F]Fluoroestradiol-PET/CT and 
[18F]FDG-PET/CT 
 
For Pilot Phase Completed in 2021: 
 
The cumulative radiation dose to a research subject participating in this clinical trial and 
receiving both the mandatory [18F]Fluoroestradiol-PET/CT and the optional [18F]FDG-
PET/CT has been compiled from the dosimetry estimates for a single 
[18F]Fluoroestradiol PET study (Table 3.3), a single [18F]FDG-PET study, and the 2 
corresponding whole body CT scans that will be performed as part of the PET/CT 
procedures (Table 3.4). For research subjects participating in the clinical trial and 
receiving both the single [18F]Fluoroestradiol PET/CT study and the single [18F]FDG-
PET/CT study, the greatest cumulative organ absorbed doses for the study are the 
bladder (118 mGy, heart (78 mGy), and liver (72 mGy), while the effective dose is 49 
mSv (Table 3.4). 
 

Current Version: 12-01-2023 
24 
 
Table 3.3. Radiation dosimetry table for [18F]FES-PET/CT 
 
 
 

Current Version: 12-01-2023 
25 
 
Table 3.4. Radiation dosimetry table for [18F]FES-PET/CT + [18F]FDG-PET/CT 
 
For Expansion Phase Added in March 2022 Amendment: 
3.2.5. Cumulative Radiation Dosimetry for [18F]Fluoroestradiol-PET/CT and 
[18F]FDG-PET/CT 
 
The cumulative radiation dose to a research subject participating in this clinical trial and 
receiving both the mandatory [18F]Fluoroestradiol-PET/CT and the optional FDG-
PET/CT has been compiled from the dosimetry estimates for a single 
[18F]Fluoroestradiol-PET study, a single FDG-PET study, and the 2 corresponding 
wholebody CT scans (1 moderate dose and 1 diagnostic dose) that will be performed as 
part of the PET/CT procedures. For research subjects participating in the clinical trial 
and receiving both the single [18F]Fluoroestradiol PET/CT study and optional FDG-
PET/CT study, the greatest cumulative organ absorbed doses for the study are the 

Current Version: 12-01-2023 
26 
 
bladder (136 mGy), heart (96 mGy), and bone (94 mGy), while the effective dose is 66 
mSv (Table 3.5) 
 
Table 3.5. Radiation dosimetry table for [18F]FES-PET/CT + [18F]FDG-PET/CT 
 
 
 
 
3.2.6. Two [18F]FES-PET/CT at baseline and 2-4 weeks following therapy 
 
The cumulative radiation dose to a research subject participating in this clinical trial and 
receiving two [18F]Fluoroestradiol-PET/CT scans has been compiled from the dosimetry 
estimates for two [18F]Fluoroestradiol-PET study, and the 2 corresponding wholebody 
moderate dose CT scans that will be performed as part of the PET/CT procedures. For 
research subjects participating in the clinical trial and receiving both the baseline and 
post-therapy  [18F]Fluoroestradiol PET/CT studies, the greatest cumulative organ 
absorbed doses for the study are the liver (89 mGy), gall bladder (78 mGy), and bone 
(62 mGy), while the effective dose is 43 mSv (Table 3.6). 
 
Table 3.6. Radiation dosimetry table for 2 FES-PET/CT. 

Current Version: 12-01-2023 
27 
 
 
3.2.7. [18F]FES-PET/CT and [18F]FDG-PET/CT at baseline and [18F]FES-PET/CT 
2-4 weeks following therapy 
 
The cumulative radiation dose to a research subject participating in this clinical trial and 
receiving two [18F]Fluoroestradiol-PET/CT and the optional FDG-PET/CT has been 
compiled from the dosimetry estimates for a two [18F]Fluoroestradiol-PET studies, a 
single FDG-PET study, and the 3 corresponding wholebody CT scans (2 moderate dose 
and 1 diagnostic dose) that will be performed as part of the PET/CT procedures. For 
research subjects participating in the clinical trial and receiving both the 
two[18F]Fluoroestradiol PET/CT study and the single FDG-PET/CT study, the greatest 
cumulative organ absorbed doses for the study are the bladder (165 mGy), liver (133 
mGy), and bone (125 mGy), while the effective dose is 88 mSv (Table 3.7). 
 
 
 
 
Table 3.7. Radiation dosimetry table for 2 FES-PET/CT and 1 FDG-PET/CT. 
 

Current Version: 12-01-2023 
28 
 
 
 
4. TRIAL DESIGN 
 
4.1. Patient Eligibility 
 
For Pilot Phase Completed in 2021 
 
Study patients: Adult patients, (n = 24 tumors).  
 
The study duration is expected to be 1-2 years.  
 
For Expansion Phase Added in March 2022 Amendment:  
 
Additional patients to be enrolled per March  2022 study amendment:  
 
Study patients: Adult patients, (n = 55) to achieve at least 38 evaluable patients for the 
primary endpoint and up to 15 evaluable patients for exploratory objectives.   
 
 
 

Current Version: 12-01-2023 
29 
 
The study duration is expected to be 5 years.  
 
 
 
4.2. Inclusion Criteria 
 
For Pilot Phase Completed in 2021: 
 
• Adults aged 18 years or greater 
• All patients or legal guardians are willing and able to sign a written informed 
consent and HIPAA authorization in accordance with local and institutional 
guidelines. 
• Histologically confirmed invasive lobular carcinoma within the past 12 weeks 
confirmed from biopsy of primary tumor or metastasis. 
• Patient is willing to have their clinical records reviewed for at least 24 months 
after enrollment. 
For Expansion Phase Added in March 2022 Amendment: 
 
• Adults aged 18 years or greater 
• All patients or legal guardians are willing and able to sign a written informed 
consent and HIPAA authorization in accordance with local and institutional 
guidelines. 
• Patient must qualify for one of the following: 
o Primary endpoint analysis/Primary Arm: 
 Histologically confirmed ER+ invasive lobular carcinoma within the 
past 16 weeks confirmed from biopsy of primary tumor or 
metastasis (n=40). 
o Exploratory Arm 1: 
 Histologically confirmed ER+ invasive lobular carcinoma at any time 
in the past, confirmed from biopsy of primary tumor or metastasis, 
with confirmed or imaging suspected metastatic disease, currently 
on antihormonal therapy or chemotherapy (n=10). 
o Exploratory Arm 2: 
 Histologically confirmed ER- invasive lobular carcinoma (at any 
point) at any site with biopsy-proven or imaging suspected 
metastatic ILC (n=5). 
• Patient is willing to have their clinical records reviewed, and be contacted by 
phone during follow-up intervals specified, for approximately 60 months after 
enrollment. 
• Patient is willing to provide baseline blood specimens for ctDNA analysis.  
 
 

Current Version: 12-01-2023 
30 
 
4.3. Exclusion Criteria 
 
For Pilot Phase Completed in 2021: 
• Patients with known allergic or hypersensitivity reactions to previously 
administered radiopharmaceuticals. Patients with significant drug or other 
allergies or autoimmune diseases may be enrolled at the Investigator’s 
discretion. 
• Patients who require monitored anesthesia for PET/CT scanning. 
• Patients who are too claustrophobic to undergo PET/CT scanning. 
• Pregnancy or current breast feeding. 
• 
Any patient that is medically unstable defined as a patient requiring inpatient 
hospitalization or needing evaluation at an acute care or urgent care facility at time of 
imaging. 
• 
Patients undergoing treatment with estrogen receptor agonists (such as fulvestrant and 
tamoxifen) within 5 weeks of the FES-PET/CT scan. (Note that aromatase inhibitors and 
luteinizing hormone-releasing hormone agonists do not affect ER expression, or binding 
of FES to ER, and do not need to be discontinued or considered for inclusion or 
exclusion of patients).  
• Patient who have had the site(s) of biopsy proven invasive lobular carcinoma 
surgically resected.  
For Expansion Phase Added in March 2022 Amendment: 
 
• Patients with known allergic or hypersensitivity reactions to previously 
administered radiopharmaceuticals. Patients with significant drug or other 
allergies or autoimmune diseases may be enrolled at the Investigator’s 
discretion. 
• Patients who require monitored anesthesia for PET/CT scanning. 
• Patients who are too claustrophobic to undergo PET/CT scanning. 
• Pregnancy or current breast feeding. 
• Patient who have had the site(s) of biopsy proven invasive lobular carcinoma 
surgically resected. Note: This does not apply for participants being enrolled for 
Exploratory Arm 1.  
• Patients undergoing treatment with estrogen receptor agonists (such as 
fulvestrant and tamoxifen) within 5 weeks of the FES-PET/CT scan. (Note that 
aromatase inhibitors and luteinizing hormone-releasing hormone agonists do not 
affect ER expression, or binding of FES to ER, and do not need to be 
discontinued or considered for inclusion or exclusion of patients). Note: This does 
not apply for participants being enrolled for Exploratory Arm 1.  
 
 
 
 

Current Version: 12-01-2023 
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4.4. Patient Registration 
 
Participants must meet all of the eligibility requirements listed above and in Appendix A 
prior to registration.  New patient registrations will be submitted to the clinical trials office 
by the study coordinator by completing a Clinical Trials Office Patient Registration Form 
and sending to CTORegistrations@hci.utah.edu.  The study coordinator will also record 
study data on all patients entered into the study and complete subsequent forms. 
 
4.5. Study Procedures, Schedule of Events, Tracer Administration:  Route and 
Dosing 
 
 
 
 
Study Schema 
 
For Pilot Phase Completed in 2021: 
 
Study Schema 
 
 
*Note the optional [18F]FDG-PET/CT session may occur before or after the 
[18F]FES-PET/CT but may not occur on the same day 
 
 
 
 
 
For Expansion Phase Added in March 2022 Amendment: 
 
Screening
[18F]FES-
PET/CT 
Imaging 
Session
Optional 
[18F]FDG-
PET/CT 
Imaging 
Session*

Current Version: 12-01-2023 
32 
 
 
 
 
4.5.1. Initial Visits Prior to PET Imaging 
 
The patient will be evaluated by their treating oncologist or breast surgeon. The 
following additional patient data will be obtained: histological diagnosis (when available 
following biopsy or surgery), age at radiological diagnosis, gender, and other treatment 
modalities used. Clinical (non-research related) imaging studies, such as bone scan, 
contrast MRI, FDG-PET/CT, and/or standard clinical CT scans, will be collected 
according to standard of care.  Additionally, ER status of the primary tumor, ER status 
of metastases (if present and if ER status is known) will be documented.   
 
4.5.2. [18F]FES–PET/CT Exams 
 
Patient preparation prior to [18F]FES-PET/CT examinations includes a recommendation 
but not requirement for fasting with no food or drink except for plain water for 4 or more 
hours prior to imaging to reduce bowel uptake due to bile excretion.   
 
Patients who are not postmenopausal or surgically sterile must have a serum 
pregnancy test performed within 48 hours prior to each research PET imaging. 
Histologically 
confirmed invasive 
lobular carcinoma 
(n=55)
Primary Arm: Staging 
of ER+ ILC using FES-
PET/CT compared to 
standard of care 
(n=40)
Baseline FES-PET/CT, 
Optional FDG-PET/CT
ctDNA
Follow-up ctDNA 
(optional) and 
Survival Assessment 
per Schedule A
Optional post-therapy 
FES-PET/CT (n=10)
Exploratory Arm 1: 
FES-PET/CT for 
metastatic ILC on 
current therapy 
(n=10)
Baseline FES-PET/CT, 
Optional FDG-PET/CT
ctDNA
Follow-up ctDNA 
(optional) and 
Survival Assessment 
per Schedule A
Exploratory Arm 2: 
Evaluation of ER- ILC 
(n=5) 
Baseline FES-PET/CT, 
Optional FDG-PET/CT
ctDNA
Follow-up ctDNA 
(optional) and 
Survival Assessment 
per Schedule A

Current Version: 12-01-2023 
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4.5.3. Day of Research [18F]FES–PET/CT Scan (Required or Optional) 
 
The research subject will be queried regarding recommended but optional fasting and 
vital signs will be obtained.  Vital signs will include heart rate, blood pressure, and 
temperature. The patient will then have the appropriate IV access placed for radiotracer 
administration.  
 
Table 5.1. PET Radiopharmaceutical Administration: Route and Dosing 
 
Tracer 
Route 
Manner 
Injected Dose 
Scan Mode 
 
[18F]Fluoroestradiol 
 
IV 
 
~30s push 
 
Target injected 
activity is 
approximately 
6 mCi 
(allowable 
range of 
approximately 
3 to 6 mCi)  
 
Static 
 
The [18F]FES will be prepared by the PET radiochemists and radiopharmacists in the 
PET cyclotron facility at HCI on the day of the scanning session. The 
radiopharmaceutical will be administered to the patient by a physician, nuclear medicine 
technologist, or trained research personnel in the PET imaging suite. The [18F]FES will 
be pushed by hand as a manually timed infusion (~30s) as indicated by Table 5.1. The 
uptake time for the [18F]FES will be approximately 60 minutes +/- 10 minutes. The 
patient will be moved from the uptake room to the scanner with enough time to allow for 
a bathroom visit. The goal is to have the uptake time as close to 60 minutes as possible.  
A CT scan for PET attenuation correction and diagnostic interpretation will be acquired 
using x-ray CT (PET/CT scanner) from the level of the top of the head to the level of the 
knees.  PET images will then be acquired from the top of the head to the level of the 
knees. 
 
The infusion and imaging procedure will be terminated in any patient who exhibits 
anaphylaxis, significant hypotension (systolic blood pressure less than 80 mmHg, 
dyspnea, chest pain, grand mal seizure or an O2 saturation lower than 80%. 
 
Vital signs will be obtained again at the conclusion of the scan, as well as adverse 
events potentially related to the administration of [18F]FES.  Adverse events will also be 
established after approximately 24 hours but up to 72 hours. (Appendix B: Schedule of 
Events, Appendix C: [18F]FES Infusion Flow Chart, Appendix D: [18F]FES Patient 
Adverse Event Questionnaire). 
4.5.4. Optional [18F]Fluorodeoxyglucose–PET/CT Scan 
 

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If a clinical [18F]Fluorodeoxyglucose (FDG) scan has not been completed as part of 
standard clinical care, the patient may elect to complete an optional FDG-PET/CT, the 
costs of which will be covered by the study.   
 
If performed, the optional FDG-PET/CT must be completed within 4 weeks (before or 
after) of the [18F]FES-PET/CT study.  FDG-PET/CT would be performed according to 
standard departmental clinical protocol. CT imaging will be performed with a non-
contrast CT technique to avoid any potential for a contrast-reaction.  
 
4.5.5. MethylPatch ctDNA analysis 
 
After giving informed consent, subjects will undergo one phlebotomy procedure, at 
times specified Appenix A: Schedule of Events), to obtain at least 20 mL (two 10mL 
Streck BCT tubes) for methylated circulating tumor DNA (ctDNA) analysis using the 
MethylPatch assay. Blood specimens will be drawn by CQCI nuclear medicine research 
technologists and promptly delivered to HCI Biorepository and Molecular Pathology 
(BMP) Shared Resource for immediate isolation of plasma. Briefly, following blood 
collection the tube is centrifuged, and the plasma supernatant is collected by carefully 
excluding the buffy coat containing leukocytes. The plasma is centrifuged a second time 
to remove trace cells and macromolecules, and 4-5 mL of supernatant plasma is 
aliquoted into cryovials for long-term frozen storage.  Separate aliquots of buffy coat 
and plasma specimens will be encoded with a de-identified study identifier (Shadow ID 
& CC#) and stored in the BMP freezers for later disbursement. BMP will act as the 
honest broker to maintain the separation of PHI from the de-identified patient 
specimens.  This procedure will be repeated at the prescribed study intervals (described 
in Appendix A: Schedule of Events).   
  
The Varley Lab will submit disbursement requests to BMP for the de-identified plasma 
specimens at regular intervals for MethylPatch analysis. The first step of this analysis 
involves cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit. The quantity 
and quality of each cfDNA sample is assessed to ensure it meets critical metrics (> 40 
ng, >30% 150-250bp in size). If the cfDNA meets these criteria, then series of molecular 
biology reactions are performed to enrich for 54 biomarker regions of DNA and to 
convert unmethylated cytosines in the DNA sequence to uracil to reveal the methylation 
status of the DNA (enzymatic conversion). The DNA is then amplified by polymerase 
chain reaction and sequenced on an illumina Nova-seq instrument.  Bioinformatics 
analysis of the sequencing results is used to determine if the results meet critical quality 
metrics: 80% of the biomarkers have at least 1,000 molecules with >=5x read depth, 
and enzymatic conversion efficiency >98%.  Bioinformatics analysis is then performed 
on each sample that meets these criteria to quantify methylated ctDNA. The quantity of 
methylated ctDNA is reported for each de-identified sample as the percentage of 
molecules from each biomarker that were methylated, and the mean percentage of 
methylated molecules. The MethylPatch assay returns a positive result when at least 3 
methylated breast cancer biomarker regions are identified and the mean percent 
methylation across biomarkers is greater than 0.15%.   Statistical analysis will be used 
to evaluate the correlation between the quantity of methylated ctDNA detected in each 

Current Version: 12-01-2023 
35 
 
specimen and patient stage, tumor FES-PET/CT uptake, progression free survival, and 
overall survival. 
 
To support the exploratory objectives and correlative studies, blood samples will be 
collected at the time points indicated on the Schedule of Events. After completion of the 
MethylPatch PCR ctDNA assay, any remaining blood will be stored for future 
unspecified cancer research. With the participant’s approval, and as approved by the 
Institutional Review Board (IRB), de-identified biological samples will be stored at 
Huntsman Cancer Institute’s Biorepository. 
  
At the time of consent, subjects will be consented as part of enrollment to authorize the 
biobanking of any residual blood sample for use in future undisclosed cancer research 
that may include other types of ctDNA analysis. During the conduct of the study, an 
individual participant can choose to withdraw consent to have biological specimens 
stored for future research. However, the withdrawal of consent with regard to biosample 
storage may not be possible after the study is completed. 
  
Blood Samples: 
Up to 40 mLs of blood will be collected at the time-points indicated on the Schedule of 
Events. When samples are due to be collected on the same day as treatment 
administration, samples should be collected prior to study therapy.  
  
 
4.6. Data Collection 
 
4.6.1. Safety Data Recorded During [18F]Fluoroestradiol-PET/CT Scans 
 
Data collected on the visit for [18F]Fluoroestradiol-PET/CT scanning will be recorded on 
the [18F]Fluoroestradiol Protocol flow chart and will include: vital signs (including heart 
rate, blood pressure and temperature) recorded just prior to the [18F]Fluoroestradiol 
infusion and then after the completion of the [18F]Fluoroestradiol-PET/CT imaging study. 
In addition to monitoring the above-mentioned safety parameters, an emergency code 
cart is located in the PET suite and can be utilized by the PET imaging team, as well as 
accessed by the code team at the institution should an emergent situation arise. 
 
Additional information to be collected will include: 
• Questioning of patient regarding adverse events immediately after [18F]FES 
administration. 
• Patients may be discharged after the completion of the [18F]FES-PET/CT 
scan if stable and if no adverse events are noted on questioning. Patients will 
be evaluated for adverse events after the completion of the 
[18F]Fluoroestradiol-PET/CT scan and at approximately 24 hours 
 
4.6.2. Tissue Collection and Analysis 
 

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All patients enrolled on this study will have undergone a procedure (either biopsy or 
tumor resection) in order to obtain a histological diagnosis of their tumors as part of 
standard care. The surgical pathology report will be reviewed and the pathologic stage 
entered into the research database. Other pathologic assessment results including 
immunohistochemistry of receptor status will also be entered into the research 
database.   
 
5. METHODS FOR EVALUATION OF IMAGING STUDIES 
 
Each PET imaging study will be evaluated using qualitative visual assessment and 
semi-quantitative analysis techniques that have previously been used by us and other 
groups, and represent the standard and accepted means of evaluating static PET 
images. These assessments are described below.   
 
5.1. [18F]FES-PET/CT Imaging 
 
5.1.1. [18F]FES Qualitative Visual Assessment 
 
The uptake of [18F]Fluoroestradiol is mediated by binding of the [18F]Fluoroestradiol to 
the estrogen receptors which are found in approximately 70-75% of invasive breast 
cancers overall and greater than 90% of invasive lobular carcinomas [14, 34, 77]. The 
ability of [18F] Fluoroestradiol-PET to detect malignant lesions in humans has been 
assessed in studies by researchers at Washington University in St. Louis, University of 
Pennsylvania, and others [34, 38, 42-47, 76, 78-87].  
 
Visual assessment will be used to qualitatively assess for abnormal focal tracer uptake 
throughout the imaged body. Images will be reviewed in an unblinded manner to assess 
whether additional lesions are found when comparing with prior imaging.     
 
 
5.1.2. [18F]FES-PET Semi-Quantitative Assessment 
 
 [18F]FES uptake will be quantified by calculating body-weight-corrected Standardized 
Uptake Values (SUVmax) as follows: 
 
 
𝑆𝑆𝑆𝑆𝑆𝑆𝑚𝑚𝑚𝑚𝑚𝑚=
𝐴𝐴𝑚𝑚𝑚𝑚𝑚𝑚
𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (𝑚𝑚𝑚𝑚𝑚𝑚)
𝐵𝐵𝐵𝐵
ൗ
 
where  
𝐴𝐴𝑚𝑚𝑚𝑚𝑚𝑚= radioactive concentration (mCi/mL) 
 
 
𝐵𝐵𝐵𝐵= body weight BW (g) 
 
[88] 
 
The maximum SUV (SUVmax) and mean SUV (SUVmean) on the lesions identified on the 
qualitative visual analysis, with cutoff for abnormal FES accumulation of the body-
weight-corrected SUVmax greater or equal to 1.5, a cutoff which has been used in prior 
studies.  A region-of-interest (ROI) will be drawn over the lesions (on all image slices 

Current Version: 12-01-2023 
37 
 
with abnormal uptake). The slice values will then be averaged to obtain an SUVmean. 
Each lesional SUVmax and SUVmean value will then be averaged to obtain the average 
SUVmax and average SUVmean values on a per-person basis and also used for further 
analysis.   
 
 
5.2 [18F]Fluorodeoxyglucose PET Imaging 
 
5.2.1. [18F]Fluorodeoxyglucose Qualitative Visual Assessment 
 
Visual assessment will be used to qualitatively assess for abnormal focal tracer uptake 
above background for the same type of tissue throughout the imaged body. Images will 
be reviewed in an unblinded manner to assess whether additional lesions are found 
when comparing with prior imaging.     
 
5.2.2. [18F]Fluorodeoxyglucose Semi-Quantitative Assessment 
 
Semi-quantitative assessment of [18F]Fluorodeoxyglucose-PET uptake will be 
performed by calculating SUVmax and SUVmean for each lesion in the same manner as 
described for FES-PET in section 5.1.2. 
 
 
 
 
6. DATA ANALYSIS AND STATISTICS 
 
6.1. Validation of [18F]Fluoroestradiol-PET/CT Imaging to Assess the Uptake in 
Invasive Lobular Carcinoma 
 
For Pilot Phase Completed in 2021 
 
This study will provide preliminary exploratory data in the value [18F]FES to assess the 
avidity of invasive lobular carcinoma for both estrogen receptor positive and estrogen 
receptor negative cases.  
 
For Expansion Phase, as of March 2022 Amendment 
 
The study will provide exploratory data regarding use of [18F]FES-PET/CT for staging of 
patients with ILC, and for FES-PET/CT imaging in patients on endocrine therapies. The 
study will also provide exploratory data regarding use of MethylPatch PCR ctDNA assay 
for ILC staging and surveillance.   
 
6.2 Data Analysis 
 
For Pilot Phase Completed in 2021 
 

Current Version: 12-01-2023 
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Qualitative and Visual Assessment. The images for the [18F]Fluoroestradiol-PET/CT will 
be visually assessed for any artifacts. Images will then be assessed for presence of 
FES avidity in the biopsy proven invasive lobular carcinoma and in any other sites 
throughout the body.  Data will then be assessed and tabulated on a lesion by lesion 
basis as to the presence of abnormal FES-PET uptake.  
 
Semi- and Fully-Quantitative Assessments.  
 
Primary endpoint:  
1. The primary endpoint is the positive uptake rate of invasive lobular carcinoma on FES-
PET/CT (yes/no whether the tumor shows positive uptake on FES PET). The null 
hypothesis is that 60% will show positive uptake. The null hypothesis will be tested using 
a one-sided test of binomial proportions at Type I error (alpha) = 0.10. A 95% exact 
binomial confidence interval for positive uptake will also be reported.  
Secondary endpoints:  
1. Rate of estrogen receptor positive (ER+) ILC that does not demonstrate positive FES 
uptake, defined as focal uptake above background with SUV max of 1.5 or greater. The 
proportion of ER+ ILC that does not demonstrate positive FES uptake will be reported 
along with a 95% exact binomial confidence interval. 
 
2. Rate of estrogen receptor negative (ER-) ILC that does demonstrate positive FES 
uptake, defined as focal uptake above background with SUV max of 1.5 or greater. The 
proportion of ER- ILC that demonstrate positive FES uptake will be reported along with a 
95% exact binomial confidence interval. 
 
3. Rate of same-patient (inter-tumoral) heterogeneous FES uptake defined as presence of 
FES uptake in some but not all biopsy proven or suspected metastatic lesions. The 
proportion of patients with heterogeneous FES update will be reported along with a 95% 
exact binomial confidence interval. 
 
4. For cases with both FDG- and FES-PET/CT imaging, evaluate the rate of discordant 
uptake (FES positive/FDG negative or FES negative/FDG positive).  Discordant uptake 
will be tabulated and evaluated using McNemar’s test for correlated proportions.  
 
5. For cases with both [18F]Fluoroestradiol- PET/CT and [18F]FDG-PET/CT studies, 
Spearman correlation of lesion uptake on [18F]Fluoroestradiol- PET/CT and [18F]FDG-
PET/CT will be calculated. The number of lesions identified by [18F]Fluoroestradiol- 
PET/CT alone, and [18F]FDG-PET/CT alone, and both [18F]Fluoroestradiol- PET/CT and 
[18F]FDG-PET/CT will be tabulated.  
 
For Expansion Phase, as of March 2022 Amendment 
 
Patients in the Primary Arm (see Study Schema in Section 4.5) will contribute to the 
analysis of Primary and Secondary Endpoints.  
 

Current Version: 12-01-2023 
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Primary Endpoint: Overall percentage of change of stage (Yes/No) following research 
FES-PET/CT imaging compared to stage based on standard of care imaging.  
 
The primary endpoint will analyzed using a one-sided one-sample test of binomial 
proportions at one-sided alpha = 0.05. The null hypothesis (H0) is that the proportion of 
patients with a change in staging is 5% or less. The alternative hypothesis (H1) is that 
the proportion of patients with a change in staging is 20% or more. 
 
Secondary Endpoint 1: Correlation between methylated DNA and stage at 
presentation.  
 
Spearman correlation will be used to assess the correlation between ctDNA and stage.  
 
Secondary Endpoint 2: The relationship between the quantity of methylated ctDNA 
and overall survival.  
 
A proportional hazards model will be used to assess the relationship between 
methylated ctDNA and overall survival. To determine the relationship at various time 
points, the analysis will be performed with censoring at 6, 12, 18, 24, 36, 48 and 60 
months.    
 
Secondary Endpoint 3: Relationship between the presence of heterogeneous FES- 
PET/CT uptake at baseline (binary variable/yes/no) and overall survival.  
 
Kaplan-Meier methods and a log rank test will be used to assess the relationship 
between FES-PET/CT and overall survival.  
 
Exploratory Endpoint 1: The rate of complete ER blockade (yes/no) at 2-4 weeks 
post-therapy in ER+ ILC patients. This endpoint will be restricted to patients in the 
primary arm with ER+ ILC that complete an optional post-therapy FES-PET/CT.   
 
The number and proportion of sites with persistent abnormal FES uptake will be 
reported along with a 95% exact binomial confidence interval. Resampling methods 
may be used to report a confidence interval adjusted for possible correlation between 
multiple lesions in the same patient.  
 
Exploratory Endpoint 2: The rate of persistent abnormal FES uptake (yes/no) in 
metastatic patients on hormonal therapy with known ER+ ILC. This endpoint will be 
restricted to metastatic patients with ER+ ILC in Exploratory Arm 1 that complete an 
optional post-therapy FES-PET/CT.   
 
The number and proportion of sites with persistent abnormal FES uptake will be 
reported along with a 95% exact binomial confidence interval. Resampling methods 

Current Version: 12-01-2023 
40 
 
may be used to report a confidence interval adjusted for possible correlation between 
multiple lesions in the same patient.  
 
Exploratory Endpoint 3: The negative predictive value for negative (no abnormal 
uptake) FES-PET/CT imaging with estrogen receptor (ER) negative ILC as determined 
by immunohistochemistry from core needle biopsy. This endpoint will be restricted to 
patients in Exploratory Arm 2 with estrogen receptor (ER) negative ILC.  
 
The negative predictive value will be reported together with a 95% exact binomial 
confidence interval. Resampling methods may be used to report a confidence interval 
adjusted for possible correlation between multiple lesions in the same patient.  
 
Exploratory Endpoint 4: The relationship between heterogeneous FES-PET/CT 
uptake at baseline (yes/no) and development of metastatic disease during follow-up. 
This endpoint will be restricted to patients in the Primary Arm and Exploratory Arm 2 
who have no distant metastatic disease at baseline. 
 
Kaplan-Meier methods will be used to estimate the proportion of non-metastatic patients 
that develop metastatic disease at 6, 12, 18, 24, 36, 48 and 60 months. This will be 
done separately in patients with and without heterogeneous FES-PE/CT uptake. A log-
rank test will be used to compare the two groups.   
 
Exploratory Endpoint 5: The relationship between heterogeneous FES-PET/CT 
uptake at baseline (yes/no) and development of new metastatic disease during follow-
up. This endpoint will be restricted to patients in all study arms who have metastatic 
disease at baseline. 
 
Kaplan-Meier methods will be used to estimate the proportion of metastatic patients that 
develop new metastatic disease at 6, 12, 18, 24, 36, 48 and 60 months. This will be 
done separately in patients with and without heterogeneous FES-PE/CT uptake. A log-
rank test will be used to compare the two groups.   
 
Exploratory Endpoint 6: The relationship between the quantity of methylated ctDNA at 
baseline and development of recurrent disease (yes/no) for patients with no distant 
metastatic disease at baseline. This endpoint will be restricted to subjects in the Primary 
Arm and Exploratory Arm 2 with no distant metastatic disease. 
 
Logistic regression will be used to assess the relationship between methylated ctDNA 
and recurrent disease.  
 
Exploratory Endpoint 7: The relationship between the quantity of methylated ctDNA at 
baseline and development of new metastatic disease (yes/no) for patients with 
metastatic disease at baseline. This endpoint will be restricted to subjects in all arms 
with metastatic disease at baseline.  

Current Version: 12-01-2023 
41 
 
 
Logistic regression will be used to assess the relationship between methylated ctDNA 
and recurrent disease.  
 
 
6.3. Justification of Sample Size 
 
For Pilot Phase Completed in 2021 
 
The initial primary endpoint from the completed pilot study was to establish the positive 
uptake rate of invasive lobular carcinoma on FES-PET/CT (yes/no whether the tumor 
shows positive uptake on FES-PET). The null hypothesis is that 60% will show positive 
uptake, similar to FDG-PET/CT. A higher proportion of tumors are expected to have 
positive uptake by FES-PET/CT. The null hypothesis will be tested using a one-sided 
test of binomial proportions at Type I error (alpha) = 0.10. With data from 24 tumors 
there will 81% power provided the true proportion that show positive uptake is 80%. The 
null hypothesis will be rejected if at least 18/24 tumors show positive FES-PET/CT 
uptake.  
 
For Expansion Phase, as of March 2022 Amendment 
 
The revised primary endpoint is the change in tumor stage when staged with FES-
PET/CT compared to current standard of care imaging.  For the primary statistical 
analysis, change in staging with FES-PET/CT will be coded as a binary variable 
(changed, unchanged).  The null hypothesis (H0) is that the proportion of patients with a 
change in staging is 5% or less. The alternative hypothesis (H1) is that the proportion of 
patients with a change in staging is 20% or more. With these hypotheses, 38 evaluable 
patients will provided 90% power using a one sample exact binomial test at one-sided 
alpha = 0.05. H0 will be rejected if at least 5 patients have a change in staging. For the 
primary endpoint 40 patients will be recruited to allow 2 redundant patients to ensure 
that at least 38 evaluable patients will be available for statistical review.  An additional 
17 patients may be enrolled to achieve the exploratory aims of this study.  
 
 
 
7. REGULATORY AND REPORTING REQUIREMENTS 
 
The AE reporting time frame for this study’s investigational radiopharmaceutical is 24 
hours post injection.  Per the approval of IND #151981 for [18F]FES the following 
reporting of unexpected fatal or life threatening events, serious adverse events, and 
serious and unexpected adverse events will occur: (1) Reporting any unexpected fatal 
or life threatening adverse experience associated with the use of [18F]FES by telephone 
or fax no later than 7 calendar days after initial receipt of the information. (2) Reporting 
any adverse experience associated with the use of [18F]Fluoroestradiol, that is both 
serious (SAE) and unexpected in writing no later than 15 calendar days after initial 
receipt of the information. (3) Submitting annual reports. The reportable events will also 

Current Version: 12-01-2023 
42 
 
be submitted to the Institutional Review Board (IRB) using the University of Utah ERICA 
online system:  
https://erica.research.utah.edu/erica/Rooms/DisplayPages/LayoutInitial?Container=com
.webridge.entity.Entity%5BOID%5B5FD2DA60262617429607E459C0E09D92%5D%5D 
 
The descriptions and grading scales found in the revised NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 5.0 with subsequent modifications will be 
utilized for adverse event reporting (http://ctep.cancer.gov/reporting/index.html). 
 
All appropriate treatment areas will have access to a copy of the CTCAE version 5.0 
with modifications. A list of adverse events that have occurred or might occur (Reported 
Adverse Events and Potential Risks) can be found in Section 3 (Toxicity of [18F]FES in 
Humans) 
 
7.1. Human Subject Protections 
 
The study will be conducted in accordance with the appropriate FDA, IRB, ICH GCP, 
and other federal and local regulatory requirements, as applicable. Informed consent 
will be obtained from all research participants prior to performing any study procedures 
using the most recent IRB-approved version. All patients must be at least 18 years of 
age to participate. 
 
7.2 Institutional Review 
 
Before study initiation, the investigator must have written and dated approval/favorable 
opinion from the IRB/IEC for the protocol, consent form, subject recruitment materials 
(e.g., advertisements), and any other applicable patient-facing documents. The 
investigator should also provide the IRB/IEC with a copy of the Investigator Brochure or 
product labeling information. 
 
The investigator or designee should provide the IRB/IEC with reports, updates and other 
information (eg, expedited safety reports, amendments, and administrative letters) 
according to regulatory requirements or institution procedures. 
 
7.3 Data and Safety Monitoring Plan 
 
A Data and Safety Monitoring Committee (DSMC) is established at Huntsman Cancer 
Institute (HCI) and approved by the NCI to assure the well-being of patients enrolled on 
Investigator Initiated Trials that do not have an outside monitoring review.  Roles and 
responsibilities of the DSMC are set forth in the NCI approved plan.  The activities of 
this committee include review of adverse events including SAEs, important medical 
events, significant revisions or amendments to the protocol, and approval of cohort/dose 
escalations.  If the DSMC and/or the PI have concerns about unexpected safety issues, 
the study will be stopped and will not be resumed until the issues are resolved.  The 
DSMC also reviews and approves audit reports generated by the Research Compliance 
Office. 

Current Version: 12-01-2023 
43 
 
 
This study is classified as low risk per the NCI-approved DSM plan. Each low-risk study 
may be assigned a physician member of the DSMC as medical monitor, or in rare cases, 
an external medical monitor. The medical monitor will be notified of all serious adverse 
events (SAEs). SAEs occurring in patients treated at HCI or its affiliates will also be 
reviewed by the full DSMC monthly.  
Low-risk trials will be monitored by RCO personnel after the first patient is enrolled. 
Following the initial monitoring visit, the RCO will conduct audits for low-risk studies 
annually thereafter. Audits of low-risk studies may be conducted more frequently as 
requested by the DSMC, PRMC, IRB, RCO management, or the PI. Low-risk trials will be 
formally reviewed by the DSMC after the first patient is enrolled and then annually 
thereafter. 
 
 
7.3.1 Data Reporting 
 
This study will be monitored by the Principal Investigator using the ERICA system. In 
addition, the study will be monitored by the HCI Data and Safety Monitoring Committee. 
Cumulative data will be submitted electronically to ERICA, the HCI Data and Safety 
Monitoring Committee, and the IRB as required. 
 
7.4 Adverse Events / Serious Adverse Events 
 
This study will utilize the CTCAE (NCI Common Terminology Criteria for Adverse 
Events) version 5.0 for AE and SAE reporting. An electronic copy of the CTCAE version 
5.0 can be downloaded from: 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_50 
 
7.4.1 Adverse Events (AE) 
 
An adverse event is the appearance or worsening of any undesirable sign, symptom, or 
medical condition occurring after receiving the radioactive tracer(s) and 24 hours 
afterword even if the event is not considered to be related to the tracer.  For the 
purposes of this study, the terms toxicity and adverse event are used interchangeably.  
Medical conditions/diseases present before starting the study are only considered 
adverse events if they worsen after being injected with the radiopharmaceutical(s).  
Abnormal laboratory values or test results constitute adverse events only if they induce 
clinical signs or symptoms, are considered clinically significant, or require therapy. 
 
The collection of any adverse events will begin when a patient receives their first dose 
of [18F]FES and will end 24 hours after receiving that dose. Adverse event monitoring 
and reporting following administration of [18F]FDG is not necessary for this protocol as 
this is a clinically available, non-investigational imaging agent.  
 
Information about all adverse events, whether volunteered by the subject, discovered by 
the investigator questioning, or detected through physical examination, laboratory test 

Current Version: 12-01-2023 
44 
 
or other means, will be collected and recorded and followed as appropriate.  Those 
adverse events that are not associated with [18F]FES that do not require expedited 
reporting will be reported in the routine manner to the IRB. 
 
The occurrence of adverse events should be sought by non-directive questioning of the 
patient at each visit or phone contact during the study.  Adverse events also may be 
detected when they are volunteered by the patient during or between visits or through 
physical examination, laboratory tests, or other assessments.  As far as possible, each 
adverse event should be evaluated to determine: 
 
1. The severity grade based on CTCAE v.5 (Grade 1-5). 
2. Its relationship to the study radioactive tracer(s) (definite, probably, possible, 
unlikely, not related) 
3. Its duration (start and end dates or if continuing at final exam). 
4. Action taken (no action taken; study tracer dosage adjusted/temporarily 
interrupted; study tracer permanently discontinued due to this adverse event; 
concomitant medication taken; non-drug therapy given; hospitalization/prolonged 
hospitalization). 
5. Whether it constitutes an SAE. 
 
All adverse events will be treated appropriately. Once an adverse event is detected, it 
should be followed until its resolution, and assessment will include any changes in 
severity, the suspected relationship to the study tracer, the interventions required to 
treat it, and the outcome. 
 
Information about common side effects already known about the tracer is described in 
the Pharmacology and Safety of [18F]FES and Radiation Dosimetry of [18F]FES (Section 
3).  This information will be included in the patient informed consent and will be 
discussed with the patient during the study as needed. 
 
All adverse events will be immediately recorded in the patient research chart. 
 
7.4.2 Serious Adverse Event (SAE) 
 
Information about all serious adverse events will be collected and recorded during the 
24 hour reporting period.  A serious adverse event is an undesirable sign, symptom, or 
medical condition which: 
• Is fatal or life-threatening. 
• Results in persistent or significant disability/incapacity. 
• Is medically significant, i.e., defined as an event that jeopardizes the patient or 
may require medical or surgical intervention to prevent one of the outcomes 
listed above. 
• Causes congenital anomaly or birth defect. 
• Requires inpatient hospitalization or prolongation of existing hospitalization, 
unless hospitalization is for: 

Current Version: 12-01-2023 
45 
 
o Routine treatment or monitoring of the studies indication, not associated 
with any deterioration in condition (procedures such as central line 
placements, paracentesis, and pain control). 
o Elective or pre-planned treatment for a pre-existing condition that is 
unrelated to the indication under study and has not worsened since the 
start of study drug. 
o Treatment on an emergency outpatient basis for an event not fulfilling any 
of the definitions of a SAE given above and not resulting in hospital 
admission. 
o Social reasons and respite care in the absence of any deterioration in the 
patient’s general condition. 
 
Any death from any cause while a patient is receiving tracer on this protocol or up to 30 
days after the administration of a radioactive tracer(s), or any death which occurs more 
than 30 days after administration of a tracer(s) has ended but which is felt to be related 
to the tracer, must be reported. 
 
Note: All deaths on study will be reported 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 IRB within 24 hours of 
notification of the event, indicating that a full report will follow. Any unexpected fatal or 
life threatening adverse experience associated with the use of [18F]Fluoroestradiol will 
be reported to the FDA by telephone or fax no later than 7 calendar days after initial 
receipt of the information. All reportable adverse events will be submitted to the FDA & 
IRB within the required timeframe by as mandated by the FDA and IRB.  
 
Toxicities which fall within the definitions listed above must be reported as an SAE 
regardless if they are felt to be related to the radioactive tracer(s) or not.  Toxicities 
unrelated to the radioactive tracer(s) that do NOT fall within the definitions above, must 
simply be documented as AEs in the patient research chart. 
 
7.5 SAE Reporting Requirements 
 
SAEs must be reported to the DSMC, the FDA, and the IRB, according to the 
requirements described below: 
 
A MedWatch 3500 A form must be completed and submitted to 
compliance@hci.utah.edu as soon as possible, but no later than 10 days of first 
knowledge or notification of event (5 days for fatal or life threatening event). 
 
*MedWatch 3500A form can be found at: 
http://www.fda.gov/dowloads/Safety/MedWatch/HowToReport/DowloadForms/ucm0827
28.pdf 
 
DSMC Notifications: 

Current Version: 12-01-2023 
46 
 
• An HCI Research Compliance Officer (RCO) will process and submit the 
MedWatch form to the proper DSMC member as necessary for each individual 
study. 
• The RCO will summarize and present all reported SAEs according to the Data 
and Safety Monitoring Plan at the quarterly DSMC meeting. 
 
FDA Notifications: 
• Adverse events occurring during the course of a clinical study that meet the 
following criteria will be promptly reported to the FDA:  
o Serious 
o Unexpected 
o Definitely, Probably, or Possibly Related to the investigational drug 
o Fatal or life-threatening events that meet the criteria above will be reported 
within 7 calendar days after first knowledge of the event by the 
investigator; followed by as complete a report as possible within 8 
additional calendar days. 
o All other events that meet the criteria above will be reported within 15 
calendar days after first knowledge of the event by the investigator. 
o The RCO will review the MedWatch report for completeness, accuracy 
and applicability to the regulatory reporting requirements. 
o The RCO will ensure the complete, accurate and timely reporting of the 
event to the FDA. 
o The Regulatory Coordinator will submit the report as an amendment to the 
IND application. 
o All other adverse events and safety information not requiring expedited 
reporting that occur or are collected during the course of the study will be 
summarized and reported to the FDA through the IND Annual Report. 
 
IRB Notification: 
• Events meeting the University of Utah IRB reporting requirements 
(http://www.research.utah.edu/irb/) will be submitted through the IRB’s electronic 
reporting system within 10 working days. 
 
7.6 Protocol Amendments 
Any amendments or administrative changes in the research protocol during the period, 
for which the IRB approval has already been given, will not be initiated without 
submission of an amendment for IRB review and approval. 
 
These requirements for approval will in no way prevent any immediate action from being 
taken by the investigator in the interests of preserving the safety of all patients included 
in the trial. 
 
Any amendments to the protocol that significantly affect the safety of subjects, scope of 
the investigation, or the scientific quality of study are required to submit the amendment 
for FDA review. 
 

Current Version: 12-01-2023 
47 
 
7.7 Protocol Deviations 
A protocol deviation (or violation) is any departure from the defined procedures and 
treatment plans as outlined in the protocol version submitted and previously approved 
by the IRB.  Protocol deviations have the potential to place participants at risk and can 
also undermine the scientific integrity of the study thus jeopardizing the justification for 
the research.  Protocol deviations are unplanned and unintentional events. 
 
Because some protocol deviations pose no conceivable threat to participant safety or 
scientific integrity, reporting is left to the discretion of the PI within the context of the 
guidelines below.  The IRB requires the prompt reporting of protocol deviations which 
are: 
• Exceptions to eligibility criteria. 
• Intended to eliminate apparent immediate hazard to a research participant or 
• Harmful (caused harm to participants or others, or place them at increased risk of 
harm – including physical, psychological, economic, or social harm), or 
• Possible serious or continued noncompliance. 
 
7.8 FDA Annual Reporting 
An annual progress report will be submitted to the FDA within 60 days of the 
anniversary of the date that the IND went into effect. (21 CFR 312.22). 
 
7.9 Clinical Trials Data Bank 
The study will be registered on http://clinicaltrials.gov and the NCI CTRG (Clinical Trials 
Reporting Program) by the Clinical Trials Office. 
 
7.10 Record Keeping 
Per 21 CFR 312.57, Investigator records shall be maintained for a period of 2 years 
following the date a marketing application is approved; or, if no application is filed or the 
application is not approved, until 2 years after the investigation is discontinued and the 
FDA is notified. 
 
 
8. PET RADIOPHARMACEUTICAL PRODUCTION 
 
8.1. [18F]Fluoroestradiol 
 
8.1.1. Study Agent 
 
[18F]Fluoroestradiol is a fluorine-18 labelled PET radiopharmaceutical supplied as a 
ready-to-inject solution in either vials or syringes. The maximum dose volume is 5 ml. 
The drug substance is [18F]Fluoroestradiol. The formulation of [18F]FES drug product 
contains saline, 0.9% (v/v) sodium phosphates for injection and no more than 10% (v/v) 
ethanol. The pH of the drug product is 6-8. The radiochemical purity (RCP) is greater 
than or equal to 95% throughout the shelf-life (up to 6 hours). [18F]Fluoroestradiol 
injection is manufactured by automated radiosynthesis followed by formulation with 
buffer and aseptic dispensing in a remotely controlled system. Fluorine-18 decays by 

Current Version: 12-01-2023 
48 
 
positron emission (β+ decay, 96.7%) and orbital electron capture (3.3%) with a half-life 
of approximately 110 minutes (mins). The positron undergoes annihilation with an 
electron to produce two gamma photons each of energy 511 keV (193.4% emission).  
 
[18F]Fluoroestradiol injection is a sterile, aqueous solution of [18F]FES and excipients for 
intravenous administration. The product dose is supplied with a radioactive content of 
111-222 MBq/ml (3-6 mCi/ml) at the requested calibration time and time in an 
aseptically prepared syringe or a glass vial sealed with a synthetic rubber closure and 
aluminum overseal and then withdrawn into syringes at the clinical site. Each vial or 
syringe is transported in a lead or tungsten shield. The quality control (QC) analysis of a 
sample of the drug product as well as product release for human use is completed by a 
qualified quality assurance team before transportation of the drug product to the study 
site. The investigator (or nominated deputy) will receive release/reject information for 
the drug product. Only product for which confirmation of release has been received shall 
be used. Where the product is transported as a single patient dose, confirmation of the 
dose will be measured in a dose calibrator before administration. The calculation is 
based on the radioactive content, the half-life of fluorine-18 (109.8 mins), the reference 
date and time, the prescribed dose and the time of injection. Each patient dose will 
contain between 111-222 MBq (3.0-6.0 mCi) at the time of administration. The doses 
will contain no more than 5 μg [18F]FES and 5 ug related substances per injection. The 
maximum administered dose volume is 5 ml. 
 
8.1.2. Reported Adverse Events and Potential Risks 
 
No serious adverse reactions have been reported from any published studies in part 
3.1.2 of this document. The risks to subjects mainly relate to the intravenous injection 
and intravenous blood sampling procedures, and the radiation emitted by 
[18F]Fluoroestradiol. Intravenous injection and the use of an intravenous cannula are 
known to carry a small risk of infection and hematoma. The exposure to radiation will 
not exceed that which is considered acceptable in accordance with appropriate 
guidelines. 
8.1.3. Production of the Radiopharmaceutical 
 
The [18F]FES used in this study will be prepared locally by the PET Radiochemistry 
Group at the University of Utah. The precursors for the radiosynthesis include F-18 
prepared at the Huntsman Cancer Institute cyclotron from proton irradiation of [O-18] 
water and an organic precursor supplied along with other reagents are used on the 
TRACERlab FX-N synthesis module. [18F]Fluoroestradiol is manufactured by automated 
radiosynthesis on the TRACERlab FX-N followed by formulation with buffer and aseptic 
dispensing in a remotely controlled system. The formulation of [18F]FES drug product 
contains sodium phosphates buffer in saline. The pH of the drug product is 6-8. The 
radiochemical purity (RCP) is greater than or equal to 95% throughout the shelf-life (up 
to 6 hours). The radiopharmaceutical product is a clear and colorless liquid that is 
stored at room temperature in a sterile serum vial. The [18F]FES currently has an 
expiration time of 6 hours from the end of synthesis (EOS).   
 

Current Version: 12-01-2023 
49 
 
8.1.4. Agent Accountability 
 
[18F]Fluoroestradiol is a radiopharmaceutical produced in the cyclotron facility at the 
Huntsman Cancer Institute. The agent is investigational and approved by the FDA 
under IND#151981 (Hoffman).   
 
The shelf-life of [18F]FES is up to 6 hours from the end of synthesis and the product 
must not be used beyond this limit. [18F]Fluoroestradiol should be stored at room 
temperature (~15-25°C) in a shielded container. All non-radioactive containers 
(shielding, transport cans) must be returned to the manufacturing site. Containers that 
are radioactive or that contained radioactive products must be destroyed at either the 
study site or another designated facility, after the study and after overall drug 
accountability has been completed by the sponsor or its representative. Waste must be 
disposed of according to national regulations for radioactive material. Precautions for 
the safe handling of radioactive materials should be observed. 
 
Each radiosynthesis is done by University of Utah cyclotron and radiochemistry staff 
and the product [18F]Fluoroestradiol in a dose calibrated syringe will be released after 
passing all required quality control assays to the physician who will be responsible for 
administering the appropriate amount (John M. Hoffman, MD or his designee). The 
quality control tests that must be passed prior to release of the product [18F]FES for 
injection include the radioactive purity, the radiochemical purity, sterilizing filter integrity 
impurities tests, pyrogens and appearance. The [18F]FES dose is drawn into a syringe, 
assayed for mCi at the time of injection, and administered to the research subject. 
 
 
 

Current Version: 12-01-2023 
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56 
 
 
10. APPENDICES 
 
 
 
APPENDIX A 
 
 
 
Schedule of Events 
 
 
 
 
 
APPENDIX B 
 
 
 
[18F]Fluoroestradiol Infusion Flow Chart for Staff  
 
 
 
APPENDIX C 
 
 
 
[18F]Fluoroestradiol Patient Adverse Event Questionnaire  
 
APPENDIX D 
 
 
 
NCI Common Toxicity Criteria  
   
 
 

Current Version: 12-01-2023 
57 
 
 
APPENDIX A: Schedule of Events, Revised for Expansion Phase as of March 2022 
Amendment 
 
 
Screening 
Baseline 
FES-PET/CT 
Imaging 
Session  
Optional Baseline 
FDG-PET/CT Imaging 
Session 
Optional FES-PET/CT 
Imaging Session 4 
weeks following 
treatment start 
 
 
[18F]FES-
PET/CT 
[18F]FDG-PET/CT5 
[18F]FES-PET/CT 
Assessment/ 
Procedures 
 
 
 
 
Informed Consent 
X 
 
 
 
Inclusion/ Exclusion 
Criteria 
X 
 
 
 
Infusion of 
[18F]Fluoroestradiol 
 
X 
 
X 
[18F]Fluoroestradiol 
PET/CT Imaging 
 
X 
 
X 
Vital Signs1 
 
X1 
 
X1 
Initial AE 
Assessment2 
 
X 
 
X 
24-72 Hour AE 
Assessment2 
 
X 
 
X 
Pregnancy Test 
 
X3 
X3 
X3 
[18F]FDG-PET/CT 
Imaging 
 
 
X4 
 
MethylPatch ctDNA 
assay5 
 
X5 
 
X5 
 
 
 
 
(1) 
Includes: Heart Rate, Blood Pressure, and Temperature.  Vitals signs will be collected 
prior to infusion and again at the completion of the imaging study. Height and weight will 
be recorded at the beginning of an imaging session.  
(2) 
Adverse events collection will begin when a patient receives their dose of [18F]FES and 
continue until 24 hours post injection. The [18F]FES Patient Adverse Event Questionnaire 
(Appendix D) will be completed at the conclusion of the imaging study and again at 
approximately 24-72 hours post injection via telephone consultation. 
(3) 
Patient must be postmenopausal, surgically sterile, or confirmed not to be pregnant by 
serum pregnancy test performed within 48 hours prior to research PET imaging. 
(4) 
The optional [18F]FDG-PET/CT session may occur before or after the [18F]FES-PET/CT 
within 4 weeks before or after but may not occur on the same day. 
(5) 
Requires one phlebotomy procedure and delivery of sample to HCI Biorepository and 
Molecular Pathology Shared Resource and subsequent MethylPatch PCR analysis by the 
HCI Varley Lab 
 
Appendix A.2 
 
MethylPatch ctDNA Survival Assessment Schedule 

Current Version: 12-01-2023 
58 
 
 
Assessment/ 
Procedures 
Informed 
Consent 
Inclusion/Exc
lusion 
Criteria 
Optional MethylPatch 
ctDNA Assay 
Assessment of 
Recurrence and 
Survival  
 
 
 
 
 
Screening 
X 
X 
 
 
6-month follow-up 
(+/- 1 month) 
 
 
X1 
X2 
12-month follow-up 
(+/- 1 month) 
 
 
X1 
X2 
18-month follow-up 
(+/- 2 months) 
 
 
X1 
X2 
24-month follow-up 
(+/- 2 months) 
 
 
X1 
X2 
36-month follow-up 
(+/- 2 months) 
 
 
X1 
X2 
48-month follow-up 
(+/- 2 months) 
 
 
X1 
X2 
60-month follow-up 
(+/- 2 months) 
 
 
X1 
X2 
 
 
 
 
1. Requires one phlebotomy procedure and delivery of sample to HCI Biorepository and 
Molecular Pathology Shared Resource and subsequent MethylPatch PCR analysis by the 
HCI Varley Lab 
2. Survival assessment as based on electronic medical record clinical visit follow-up and 
participant phone call over a total 5-year time-to-event endpoint.  Recurrence evaluation will 
include assessment of recurrent and/or metastatic disease in participants without evidence 
of distant metastatic disease at baseline assessment.  
 
 

Current Version: 12-01-2023 
59 
 
APPENDIX B: [18F]Fluoroestradiol Infusion Flow Chart for Staff 
 
 
Subject Name and MRN: _____________________________________________________ 
 
 
Subject Study ID (if enrolled): ______________________________  
                    
 
 
Projected Study Start Date: __________________    Referring MD: ______________________ 
  
                                                                                               
 
 
 
 
 
 
Administered dose of [18F]FES   ___________ mCi 
 
 
Specific Activity of [18F]FES   _____________Ci/mmol 
 
                                                                                              
Initials of Person 
Making Assessment 
Time 
Study 
Temp 
BP 
HR 
Weight 
Height 
 
 
Baseline 
 
 
 
 
 
 
 
End of 
Study 
 
 
 
NA 
NA 
 
 
 
  Completed by: ______________________________________________ Date: _________________________  
The infusion and imaging procedure will be terminated in any patient who exhibits anaphylaxis, significant hypotension 
(systolic blood pressure less than 80 mmHg, dyspnea, chest pain, grand mal seizure or an O2 saturation lower than 80%. 

Current Version: 12-01-2023 
60 
 
APPENDIX C: [18F]Fluoroestradiol Patient Adverse Event Questionnaire  
Subject Name and MRN: _____________________________________________  
 
Subject Study ID (if enrolled): _____________________________  
                    
Projected Study Start Date: _________________    Referring MD: _______________________________ 
 
Day of Imaging Session  
Possible Adverse Event 
Yes 
No 
Comment on possible AE 
 
Body as a Whole: 
 
 
 
Pain (Abdominal) 
 
 
 
Pain (Chest/Breast) 
 
 
 
Pain (Other site) 
 
 
 
 
 
 
 
Fever 
 
 
 
Injection site reaction 
 
 
 
 
 
 
 
Cardiovascular System: 
 
 
 
Vasodilation (flushing) 
 
 
 
Tachycardia (fast heart rate) 
 
 
 
 
 
 
 
Digestive System: 
 
 
 
Nausea 
 
 
 
Diarrhea 
 
 
 
Vomiting 
 
 
 
 
 
 
 
Respiratory System: 
 
 
 
Dyspnea (Shortness of breath) 
 
 
 
 
 
 
 
Skin and Appendages: 
 
 
 
Rash 
 
 
 
Pruritus (Itching) 
 
 
 
Urticaria (Hives) 
 
 
 
Sweating  
 
 
 
Cyanosis (discoloration of 
fingers/toes) 
 
 
 
 
 
 
 
Central Nervous System 
 
 
 
Visual disturbances 
 
 
 
Numbness of feet 
 
 
 
Numbness of fingers/hands 
 
 
 
Weakness of feet 
 
 
 
Weakness of fingers 
 
 
 
Burning sensation in feet 
 
 
 
Burning sensation of fingers 
 
 
 
 
Performed by: _____________________________  Date: _______________Time: ________ 
 
PI Oversight: _________________________________  Date: _______________ 
24-72 hour post-injection assessment 

Current Version: 12-01-2023 
61 
 
Possible Adverse Event 
Yes 
No 
Comment on possible AE 
 
Body as a Whole: 
 
 
 
Pain (Abdominal) 
 
 
 
Pain (Chest/Breast) 
 
 
 
Pain (Other site) 
 
 
 
 
 
 
 
Fever 
 
 
 
Injection site reaction 
 
 
 
 
 
 
 
Cardiovascular System: 
 
 
 
Vasodilation (flushing) 
 
 
 
Tachycardia (fast heart rate) 
 
 
 
 
 
 
 
Digestive System: 
 
 
 
Nausea 
 
 
 
Diarrhea 
 
 
 
Vomiting 
 
 
 
 
 
 
 
Respiratory System: 
 
 
 
Dyspnea (Shortness of breath) 
 
 
 
 
 
 
 
Skin and Appendages: 
 
 
 
Rash 
 
 
 
Pruritus (Itching) 
 
 
 
Urticaria (Hives) 
 
 
 
Sweating  
 
 
 
Cyanosis (discoloration of 
fingers/toes) 
 
 
 
 
 
 
 
Central Nervous System 
 
 
 
Visual disturbances 
 
 
 
Numbness of feet 
 
 
 
Numbness of fingers/hands 
 
 
 
Weakness of feet 
 
 
 
Weakness of fingers 
 
 
 
Burning sensation in feet 
 
 
 
Burning sensation of fingers 
 
 
 
 
Performed by: _____________________________  Date: _______________Time: ________ 
 
 
PI Oversight: _________________________________  Date: _______________ 

Current Version: 12-01-2023 
 
Page 62 of 63 
 
APPENDIX D: 5 Year Follow-up Assessment  
 
 
 
Subject Name and MRN: _____________________________________________  
 
Subject Study ID (if enrolled): _____________________________  
 
Assessment 
Timepoint 
Is the 
participant 
alive?  
Imaging 
performed at 
our institution 
since prior 
assessment? 
Imaging 
performed at 
outside 
institution 
since prior 
assessment? 
Clinical 
evaluation at 
our institution 
since prior 
assessment? 
Clinical 
evaluation at 
outside 
institution 
since prior 
assessment? 
Is there 
documented 
recurrent or 
new metastatic 
disease based 
on these 
assessments? 
6-month 
follow-up 
(+/- 1 
month) 
 
 
 
 
 
 
12-month 
follow-up 
(+/- 1 
month) 
 
 
 
 
 
 
18-month 
follow-up 
(+/- 2 
months) 
 
 
 
 
 
 
24-month 
follow-up 
(+/- 2 
months) 
 
 
 
 
 
 
36-month 
follow-up 
(+/- 2 
months) 
 
 
 
 
 
 
48-month 
follow-up 
(+/- 2 
months) 
 
 
 
 
 
 
60-month 
follow-up 
(+/- 2 
months) 
 
 
 
 
 
 
 
Instructions: Mark yes, no, unable to confirm for each of the questions above. Any 
imaging results or pertinent clinical records from the clinical providers monitoring the 
patient’s breast cancer therapy (as determined by the study team/PI) will be obtained 
and uploaded into the research record.   
 
 
 

Current Version: 12-01-2023 
 
Page 63 of 63 
 
 
APPENDIX E: NCI Common Toxicity Criteria 
 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/
docs/CTCAE_v5_Quick_Reference_5x7.pdf 
 
 
  
 
 
 
 
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