Phase 1 Feasibility Trial: Improved Staging of Lobular Breast Cancer With Novel Amino Acid Metabolic and Tumor Neovasculature Receptor Imaging
Phase: PHASE1
Conditions: Anatomic Stage IV Breast Cancer AJCC v8, Invasive Breast Lobular Carcinoma, Metastatic Breast Lobular Carcinoma, Prognostic Stage IV Breast Cancer AJCC v8
Sponsor: Emory University

 
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Winship Protocol: X
Improved staging of lobular breast cancer with novel amino acid 
metabolic and tumor neovasculature receptor imaging
PI: David Schuster, MD. Professor and Director, 
Division of Nuclear Medicine and Molecular Imaging
Department of Radiology and Imaging Sciences
Emory University Hospital, Room E152
1364 Clifton Road, Atlanta, GA 30322
404-712-4859; dschust@emory.edu
Co-Investigators:
Sarah Friend, MD
Assistant Professor, 
Department of Hematology and Medical Oncology
Emory University; sarah.c.friend@emory.edu
Manali Bhave, MD 
Assistant Professor,
Department of Hematology and Medical Oncology
Emory University; manali.ajay.bhave@emory.edu
Mylin A Torres, MD
Associate Professor
Louisa and Rand Glenn Family Chair in Breast Cancer Research
Department of Radiation Oncology; Emory University; matorre@emory.edu
Toncred Marya Styblo, MD, MS, FACS
Associate Professor, 
Chair, Emory Integrated Cancer Network
Division of Surgical Oncology, Department of Surgery
Emory University; tstyblo@emory.edu
Saima Muzahir, MD
Assistant Professor
Division of Nuclear Medicine and Molecular Imaging
Department of Radiology and Imaging Sciences
Emory University; saima.muzahir@emory.edu
Anna Holbrook, MD
Assistant Professor
Division of Breast Imaging
Department of Radiology and Imaging Sciences
Emory University;anna.holbrook@emory.edu
Jeffrey M. Switchenko, Ph.D. 
Research Assistant Professor, 
Department of Biostatistics & Bioinformatics, Rollins School of Public Health
Biostatistics & Bioinformatics Shared Resource, Winship Cancer Institute; jswitch@emory.edu
Funding Source: NIH. Applying for R21. Currently internal. 

 
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Table of Contents
1.1 Synopsis .............................................................................................................3
1.2 Schema ...............................................................................................................4
1.3 Schedule of Activities........................................................................................6
2.  Introduction .................................................................................................................7
2.1 Study Rationale....................................................................................................7
2.2 Background..........................................................................................................8
2.3 Potential Risks and Benefits ..............................................................................10
3. Objectives and Endpoints.........................................................................................12
3.1 Study Objectives: ...............................................................................................12
3.2 Study Endpoints:................................................................................................12
4. Study Design ..............................................................................................................13
4.1 Overall Design....................................................................................................13
4.2 Scientific Rationale for Study Design.................................................................14
4.3 Justification for Dose..........................................................................................14
4.4 End of Study Definition.......................................................................................14
5. Study Population........................................................................................................15
5.1 Inclusion Criteria ................................................................................................15
5.2 Exclusion Criteria ...............................................................................................15
6.  Registration Procedures ..........................................................................................16
7.  Study Intervention.....................................................................................................16
7.1 Study Specific Experimental Procedures...........................................................17
8. Study assessments and Procedures. ......................................................................18
8.1 Schedule of Procedures.....................................................................................18
9. Measurement of Effect...............................................................................................19
10. Statistical considerations.........................................................................................20
11. Adverse Events:  List and Reporting Requirements..............................................20
12. Data Reporting / Regulatory Requirements............................................................23
13. Ethics and Protection Of Human Subjects .............................................................23
14. References .................................................................................................................25
APPENDIX A   Abbreviations and definition of terms ........................................27

 
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1.1 Synopsis
Invasive lobular breast cancer (ILC) accounts for approximately 10-15% of breast malignancies [1]. ILC 
has a distinct histology and discohesive growth which makes it difficult to visualize with current imaging 
modalities such as mammography, ultrasound and breast and whole body MR. Suboptimal imaging 
adversely impacts accurate staging upon which therapeutic decisions are based [2]. The metastatic 
pattern of ILC also differs from that of invasive ductal carcinoma (IDC) with a tendency to involve the 
gastrointestinal tract, genitourinary tract, peritoneum, retroperitoneum and leptomeninges. While 
fluorodeoxyglucose (FDG) PET has been shown to clinically upstage patients with locally advanced IDC, 
this is not so with ILC, which has been described as “initially indolent but slowly progressive”. There are 
currently no accurate imaging techniques for staging ILC. Our goal is to address an unmet public 
health need by improved staging of ILC, specifically detection of metastatic disease. 
Amino acid transporters are upregulated in breast cancer cells [3]. We and others have 
successfully imaged breast cancer with fluciclovine (18F) PET, an FDA approved synthetic amino acid 
radiotracer originally developed at Emory for imaging of cerebral glioma and prostate cancer. In 
exploratory studies conducted at Emory and Memorial Sloan Kettering in 39 women with breast cancer 
(13 patients with ILC), fluciclovine PET demonstrated promising results in detection of ILC in primary 
tumors and locoregional metastases [4-6]. Distant disease was not studied. Post-hoc RNA sequencing 
of the Emory cohort reveals that numerous molecular signaling pathways correlate with fluciclovine 
uptake including PI3K/Akt signalling, which mediates a range of biological endpoints necessary for 
cancer growth including cell survival, apoptosis, autophagy, cell cycle progression and angiogenesis. 
Since the pro-angiogenic factor VEGF contributes to Akt activation to improve oxygenation and 
nutrient supply to areas of poor vascularization, attention has turned to neoangiogenesis as an imaging 
biomarker for breast cancer. A different PET radiotracer in clinical trials for prostate cancer, prostate 
specific membrane antigen (PSMA) may also detect breast cancer due to the upregulation of PSMA 
receptors in tumor angiogenesis. Prostate-specific is actually a misnomer as PSMA receptors are 
upregulated in tumor neovasculature in many cancers including breast cancer. Evolving data suggests 
PSMA radiotracers may also have utility in breast cancer, including ILC [7-11].  
Our preliminary data support a key scientific premise that metabolic imaging with fluciclovine (18F) 
PET has shown promise in the detection of ILC. There is also emerging data that tumor neovasculature 
imaging with PSMA PET may improve detection of ILC. We hypothesize that amino acid transport 
metabolic imaging with fluciclovine (18F) PET will improve staging of ILC, particularly for distant 
metastases, compared to conventional imaging. We also hypothesize that receptor directed 
PSMA imaging of tumor associated neovasculature in ILC will reveal unique information to 
complement metabolic interrogation with fluciclovine PET. Improved staging will facilitate more 
appropriate management decisions.
To test these hypotheses with the highest scientific rigor, we propose an early phase feasibility 
trial with fluciclovine and PSMA PET strategies centered on detection of metastasis in patients with 
advanced ILC using histology as the gold standard. As an exploratory aim, we will also correlate the 
occurrence of circulating tumor DNA (ctDNA) directed against ESR1 and PI3K with presence of 
metastasis and tumor burden. We will explore if a multiparametric strategy with ctDNA and imaging will 
help define which patients should undergo molecular imaging and also identify those who may forego 
biopsy if distant lesions are detected on imaging. We expect this pilot trial will generate sufficient 
preliminary data to determine feasibility for a definitive NIH sponsored trial developing more accurate 
staging techniques to help modify current practice for imaging of ILC. 
Specific Aim 1: Improve detection of metastasis with fluciclovine and PSMA PET versus best 
standard of care conventional imaging, as confirmed with histology. We will image 20 patients with 
ILC who have either: a) clinical or imaging suspicion of metastatic disease; or b) proven metastatic 
disease but in whom there is suspicion of an even greater tumor burden that could change therapy 
approach. Abnormal foci which correlate to anatomic lesions previously unsuspected on conventional 
imaging will undergo optional biopsy as clinically indicated in the safest manner possible (if not already 
done) to determine the verified detection rate of metastatic disease.
Specific Aim 2: Determine concordance and discordance of ILC detection with PSMA versus 
fluciclovine PET, as confirmed with histology. We will compare verified detection rates for 
metastasis between fluciclovine and PSMA PET modalities. This may necessitate a second biopsy of a 

 
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distant lesion discordant between the 2 PET techniques. We will also analyze images for biodistribution 
in the primary lesion(s) for both radiotracers.
Exploratory Aim 3: Establish the role of molecular detection by ctDNA mutations including 
PIK3CA, ESR1, HER2, and AKT1 in characterizing the degree of tumor burden as identified by 
metabolic amino acid transport and/or tumor neovasculature receptor imaging [12]. We will collect 
peripheral blood for ctDNA in all patients and determine if ctDNA correlates with presence or absence of 
metastases and tumor burden as identified by fluciclovine (metabolic) and/or PSMA (angiogenesis) PET. 
1.2 Schema
We will undertake a single arm feasibility study with n=20 patients with treatment naïve biopsy proven 
ILC who have either: a) clinical or imaging suspicion of metastatic disease; b) biopsy proven metastatic 
disease but in whom there is suspicion of an even greater tumor burden that could change therapy 
approach. All patients will be recruited from the Winship Glenn Family Breast Cancer Program at any 
Emory facility and they will have already undergone best standard of care conventional imaging (CT or 
MR and possibly bone scan depending on clinical presentation). Standard of care conventional 
imaging typically does not include FDG PET, since FDG PET is not recommended for lobular 
cancer due to low sensitivity. We will then complete both fluciclovine PET and Ga PSMA PET scans 
on the same patient on separate days separated by at least 10 half-lives to allow for radioactive decay. 
There is no need for randomization since each patient will undergo both imaging techniques, and the 
results will be interpreted blindly. Thus, radiopharmacy scheduling will dictate the order of the radiotracers 
to ensure the patient will undergo both imaging modalities in the shortest time frame. Sometimes this will 
mean PSMA is completed first, followed by fluciclovine, and other times the opposite. After interpretation 
of both studies, careful correlation to already obtained conventional imaging will then be done to 
determine if a previously unsuspected anatomic correlate can be identified which would impact therapy. 
One or two metastases (depending on scan findings) will then undergo biopsy in the safest manner 
possible as standard of care (if not already done) to determine the verified detection rate of metastatic 
disease. Uncommonly, definitive imaging such as MR for skeletal or liver lesions may be accepted in lieu 
of biopsy, if biopsy is not feasible. We will then compare verified detection rate of each PET study to 
conventional imaging and to each other. In addition, we will collect peripheral blood for ctDNA in all 
patients and determine if ctDNA correlates with presence or absence of metastases and tumor burden 
as identified by conventional imaging, fluciclovine and/or PSMA PET. Study Specific Experimental 
Procedures will only consist of both PET studies and ctDNA analysis. All other procedures will be 
conducted per standard of care. See Figure 1.

 
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Figure 1: Trial Schema, Flow diagram 
 
Total 20:  Consent/ Screening and Enrollment.
Collect 20 cc peripheral venous blood
Pregnancy test as appropriate
Complete PSMA or fluciclovine (PET 1)
Follow-up assessments of study endpoints and safety
Phone call from study nurse (5-10 business days > PET)
Correlate PET scans to truth standard and conventional imaging.
Pregnancy test as appropriate
 Complete PSMA or fluciclovine (PET 2)
(i.e. If PSMA completed, patient then undergoes fluciclovine and vis versa)
Final Analysis
Compare verified detection rate of both PET scans to 
conventional imaging and to each other (Aims 1 and 2).
Correlate ctDNA with PET results and CI (Aim 3). 
Visit 1; Day 1
PET 1 
1-30 days >
Visit 2; Day 2
PET 2 
1-15 days > 

 
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1.3 Schedule of Activities
a Study intervention is either the PSMA or fluciclovine PET
b Pregnancy test within 24 hours prior to  each PET as applicable
c Uncommonly, definitive imaging such as MR for skeletal or liver lesions may be accepted in lieu of biopsy, if biopsy is not 
feasible.  
d Blood collection may also occur at other visit 
e There will be no further study specific visits beyond visit 2 (except for followup phone call from study nurse at 5-10 business 
days inquiring as to AE which does not require a “visit”). All further followup will be via medical record of subsequent history, 
biopsy/histology, imaging, etc. 
Procedures
Enrollment/
Screening (-60 days)
Study Visit 1
PET 1
(within 30 days of 
enrollment) 
Study Visit 2
PET2
(within 15 days of 
Visit 1)
Follow up e
(Up to 5 years)
Informed consent
X
Demographics
X
Medical history
X
X
Administer study intervention
X
X
Pregnancy test b
X
X
AE Assessment
X
X
Optional biopsy c
X
X
X
Follow medical history
X
Peripheral blood sample (ctDNA)d
X
Complete Case Report Forms 
(CRFs)
X
X
X
X

 
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2.  Introduction 
2.1 Study Rationale 
Lobular breast cancer is a lethal disease. It is difficult to detect and accurately stage, especially distant 
metastases. This difficulty is in part secondary to the discohesive growth pattern due to loss of E-cadherin 
which results in diffuse spread of cancer cells in atypical and sometimes unexpected patterns which may 
resemble inflammation. ILC may not demonstrate significant FDG uptake and also may be present in 
difficult to image locations such as peritoneum and central nervous system [2]. Thus, with ILC there may 
be significant understaging leading to inappropriate therapeutic decisions. Finally, since lobular breast 
cancer is not mass forming as is IDC, response to therapy may be difficult to evaluate. For these same 
reasons recurrent disease is difficult to detect with ILC, thus potentially limiting salvage therapy options. 
Therefore, the scientific justification for improved detection of metastatic disease is compelling.
Fluciclovine
Tumor glucose metabolism, specifically the shift away from oxidative phosphorylation to aerobic 
glycolysis (Warburg Effect), has formed the basis of advanced molecular imaging of breast cancer. Other 
metabolic activity upregulated in breast cancer includes use of amino acids. The amino acid transporters, 
specifically LAT1, ASCT2, ATB0,+ SNAT1 and XCT, are overexpressed in breast cancer and reported to 
be associated with tumor growth, metastasis and hormone receptor status [13-16]. Shennan and 
coauthors demonstrated that certain human breast cancer cell lines, MDA-MB-231 and MCF-7, express 
system L (large neutral amino acid) transporters [17, 18]. In fact, Shennan has also demonstrated that 
growth of cultured human breast cancer cells may be decreased via inhibition of system L transporters 
[14]. Fluciclovine is a synthetic amino acid analog PET radiotracer transported via system L (LAT1) and 
ASCT (ASCT2) and has shown promise in the imaging of prostate and other cancers [19]. Fluciclovine 
uptake has been shown in vitro in breast carcinoma cell lines as well as in animal models of breast cancer 
[16]. This has led to the study of amino acid transport as a basis for superior detection of breast cancer 
by our group. As noted in the preliminary data section of this proposal, we unexpectedly discovered that 
ILC was better detected with amino acid transport-based PET with fluciclovine compared to glycolytic 
based FDG PET. Preliminary studies understandably focused on the detection of primary and 
locoregional disease. We propose to expand this concept to also include detection of distant disease 
which could alter the stage of the patient and thus therapeutic approach. Improved initial staging and 
treatment selection could impact the survival of nearly 40,000 women with ILC per year, and potentially 
enhance the evaluation of therapy response in women living with metastatic ILC.
PSMA
PSMA receptors are upregulated in many cancers besides prostate cancer since tumor associated 
neovasculature has upregulated PSMA expression [8]. It is likely that PSMA cleavage of vitamin B9 (folic 
acid) stimulates oncogenic signaling through glutamate receptors with downstream activation of the PI3K-
Akt-mTOR signaling pathway [20]. Utilizing PSMA as a biologic marker of tumor neovasculature thus has 
a solid scientific underpinning and may yield significant biologic information in reference to lobular cancer. 
Many non-prostate 68Ga-PSMA avid malignancies have been reported in the literature including breast 
cancer, yet there is little information specifically on ILC [10]. Furthermore, if we can demonstrate that ILC 
is sufficiently PSMA avid, there is a possibility that a theranostic approach with Lu-177 PSMA 
radiotherapy may be investigated as is currently occurring with metastatic prostate cancer [21]. In fact, 
there has been one case report of metastatic IDC triple negative breast cancer treated with Lu-177 PSMA 
[11]. While 68Ga-PSMA is FDA approved for prostate cancer, it is not FDA approved for breast cancer. 
Ga-PSMA is currently produced at the Emory Center for Systems Imaging for an ongoing prostate cancer 
trial, and Dr. Schuster holds an IND allowing research applications.
ctDNA
The role of ctDNA specifically in regard to ILC has been little studied, especially the ability of ctDNA in 
predicting distant disease. The premise in this exploratory aim is specifically studying the association of 
ctDNA targeted to ILC with imaging that is more sensitive to ILC compared with FDG PET. If there is an 
association, this would be a significant area for future research since ctDNA expression could then be 
utilized to select patients who should undergo this advanced imaging or serve to improve specificity in 
lieu of confirmatory biopsy if the association with verified detection rate of metastasis was particularly 

 
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robust. Finally, understanding if this correlation is associated with upregulated amino acid metabolism 
and/or tumor neoangiogenesis as characterized by molecular imaging could yield valuable insights into 
the biologic behavior of ILC. 
2.2 Background
Invasive lobular carcinoma (ILC) comprises 10-15% of breast cancer. While mammography, ultrasound, 
breast MRI, and whole body FDG PET form the backbone of breast imaging, there are limitations in the 
imaging of lobular cancer histology, especially for distant disease (figure 1) [22, 23]. ILC is diagnosed at 
a later and more advanced stage compared with IDC and is typically hormone receptor positive [1]. Up 
to 50% of patients with the CDH1 genetic mutation will 
develop ILC [1]. The development of metastatic 
disease is likely facilitated by the loss of E-cadherin in 
most 
ILC 
cells 
[24]. 
Similarly, 
while 
(18F) 
fluorodeoxyglucose positron emission tomography 
(FDG-PET) has also assumed an important role in 
whole body staging, detection of recurrent disease 
and monitoring therapy response, limitations for 
lobular cancer are well described in the literature [2, 
25]. These limitations are secondary to indolent 
growth and diffuse growth patterns, making lobular 
cancer metastasis difficult to distinguish from 
background activity. In a study by Ulaner in 146 
patients with ILC, FDG PET had only a modest effect 
on upstaging and did not reveal additional nodal 
disease. Compared with IDC patients who were 
upstaged in 22% of cases to stage 4 in a separate cohort, only 6% of ILC patients had unsuspected 
distant metastasis that were FDG PET positive [26]. 
Fluciclovine
Due to the limitations of FDG PET for the staging of breast cancer in earlier studies, we have investigated 
molecular imaging targeting other aspects of the metabolome [27, 28]. Amino acids are involved in many 
aspects of human nutrition including the synthesis of proteins. Amino acid metabolism has been 
demonstrated to be upregulated in many tumors [13]. Since amino acid metabolism is also upregulated 
in breast cancer, molecular imaging utilizing natural or synthetic amino acid radiotracers is an attractive 
approach [28, 29]. Emory University is a center of innovation for amino acid transport PET imaging, 
having developed anti-1-amino-3-[18F]fluorocyclobutane-1-carboxylic acid (anti-3-[18F]FACBC or 
fluciclovine), a synthetic amino acid analog PET radiotracer, which was subsequently FDA approved for 
restaging of prostate cancer [30]. Liang reported fluciclovine uptake in breast carcinoma cell lines which 
correlated with malignant potential as well as in orthotopic MDA-MB-231 breast carcinoma xenografts 
[16]. With funds from a Winship Glenn Family Breast Center Pilot Grant in 2014-2015, we studied uptake 
characteristics of fluciclovine in breast carcinoma, confining the study to evaluation of the primary breast 
lesions and locoregional disease. 
A total of 12 patients with 13 primary lesions were studied at Emory [5]. At diagnosis, 9/12 patients 
had primary breast cancer while 3/12 had local recurrence. Of the malignant lesions, there were 7/13 
invasive ductal carcinoma (IDC), 5/13 invasive lobular carcinoma (ILC) and 1/13 invasive metaplastic 
carcinoma with sarcomatous differentiation (IMC). Histologic sampling occurred in 9 locoregional lymph 
nodes (4 benign and 5 malignant). Of the malignant nodes, 3 were ILC and 2 were IDC. Although 
fluciclovine uptake in IDC and ILC were both higher than in benign breast lesions, IDC had higher uptake 
than ILC in the primary tumor. However, this was reversed with nodal disease in which metastatic 
ILC nodes had higher uptake than IDC nodes (ILC mean SUVmax (±SD) of 6.2±0.8; IDC mean 
SUVmax of 3.3±2.6). Interestingly, uptake by fluciclovine in ILC locoregional metastatic disease 
was also greater than uptake in the primary lesion. Figure 2 is an example of a patient with ILC who 
had fluciclovine uptake in a known primary breast lesion and a biopsy proven metastatic axillary node 
reported negative on outside MR and ultrasound. 
Figure 1
Subtle mesenteric infiltration (arrow) 
missed on PET/CT, biopsy proven 

 
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Four patients also underwent clinical FDG PET-CT. Fluciclovine and FDG activity were compared in 8 
biopsy proven lesions (5 malignant and 2 benign primary, 1 node). The malignant lesions included 3 IDC, 
2 ILC and 1 IMC. For ILC, mean SUVmax was 4.6 (±3.5) with fluciclovine compared to 2.6 (±1.0) for 
FDG. This finding was surprising and intriguing since uptake was greater in ILC with fluciclovine 
PET than with FDG. Figure 3 is an example of greater uptake in an ILC intramammary nodal metastasis 
with fluciclovine than with FDG PET-CT. 
In our pilot study, we unexpectedly discovered greater uptake in ILC with fluciclovine than 
with FDG PET, and greater uptake with fluciclovine in metastasis versus the primary lesion, and 
this observation was not limited to our center. Simultaneously, fluciclovine PET was also being 
studied in breast cancer by the Ulaner Group at Memorial Sloan Kettering. Results were published in the 
same issue of the Journal of Nuclear Medicine as our paper along with an accompanying editorial [6, 31]. 
In that series 27 women with locally advanced IDC (n = 19) or ILC (n = 8) underwent fluciclovine PET/CT. 
All primary tumors were radiotracer avid. Fluciclovine PET detected 20/21 patients with axillary nodal 
metastases and also detected pathologically proven extraaxillary nodal disease in 3 patients, including 2 
previously unsuspected internal mammary nodes. In the 4 patients with ILC who underwent FDG and 
fluciclovine PET, primary ILCs demonstrated fluciclovine avidity (median SUVmax, 6.1; range, 4.5–10.9) 
greater than FDG avidity (median SUVmax, 3.7; range, 1.8–6.0). Thus, in total from both cohorts, 13 
patients with ILC were studied. There was greater uptake in ILC lesions on fluciclovine versus FDG 
PET. And while IDC had greater fluciclovine uptake than ILC in the primary breast lesion; in nodal 
metastases, this relationship reversed and ILC in nodal disease had greater fluciclovine uptake 
than IDC. Furthermore, ILC nodal metastasis had greater fluciclovine uptake than in the primary 
ILC breast lesion. ILC also seems to have better correlation with complete metabolic response to 
neoadjuvant chemotherapy as reported in a separate study [4]. Critically, since these were pilot 
studies, only images of the thorax were obtained. Detection of distant disease was not studied. 
Our proposed trial will specifically address the detection of distant disease and locoregional 
staging.   
PSMA
Prostate specific membrane antigen (PSMA) is a Type 2 transmembrane glycoprotein of the M28 
peptidase family and is overexpressed on the cell surface of prostate cancer [32]. The name prostate-
specific is a misnomer, since other cancers including breast cancer have upregulated PSMA receptors 
due to tumor angiogenesis. Most data regarding PSMA PET with breast cancer pertains to IDC. Wernicke 
studied 92 samples in 106 patients with breast cancer [7]. Immunohistochemistry (IHC) for tumor 
associated vascular endothelial cell PSMA receptors revealed staining in 74% of primary and 100% of 
brain metastasis. There was absence of PSMA staining in 98% of normal breast tissue. Wernicke noted 
there was no difference between PSMA staining for IDC and ILC, yet only 10 ILC samples were included. 
In another study of 72 patients with breast cancer, moderate to strong tumor associated neovasculature 
PSMA staining was present in 67% of distant metastasis versus 36% in primary tumor cells [9]. Yet, only 
12 ILC patients were included and it was not clear if these patients had metastases. In a study of 315 
A
B
Figure 3. Coregistered fluciclovine [A] and 
FDG [B] PET-CT in a 54 yr old woman with a 
2.1cm x 1.1cm right ILC. Fluciclovine [A] had 
higher uptake (SUVmax = 7.1) compared to 
FDG [B] (SUVmax = 3.3) in metastasis to an 
intramammary node (arrows).
A
B
Figure 2. CT [A] and coregistered fluciclovine 
PET-CT [B] in a 49 yr old woman with left ILC. 
SUVmax = 3.5 in the primary mass (short 
arrow) and 5.6 in biopsy proven metastasis to 
a 6 x 11mm left axillary lymph node (long 
arrow).

 
Version 1.0;  Page 10 of 28; 29 July 2020
cases by Tolkach of which 64 tumors were ILC, 64.6% of patients with IDC had PSMA staining, versus 
42.2% of those with ILC [11]. Finally, in one of the few imaging studies, Sathekge reported in 81 lesions 
in 19 breast carcinoma patients that PSMA PET visualized 84% of lesions; yet again, only 2 ILC patients 
were included in this cohort. In a major review in the Journal of Nuclear Medicine, Salas-Fragomeni 
argues “this potential application of PSMA-targeted imaging in breast cancer should perhaps be most 
urgently explored, as the need for reliable agents for systemic staging in lobular breast cancer has been 
long-standing” [8]. Our study will address this urgent question, that of the utility of PSMA PET explicitly 
for ILC metastatic disease, as well as that of fluciclovine PET for ILC. 
ctDNA
The absolute number of circulating tumor cells has been associated with metastatic breast cancer [33, 
34]. A more refined technique, that of circulating tumor DNA (ctDNA), holds promise as a “liquid biopsy,” 
since tumor DNA circulating in the blood is more specific for carcinoma. High levels of ctDNA are reported 
to correlate with tumor size, lymph node involvement, histopathological grade, and clinical staging [35-
40]. Though there have been anecdotal reports of ctDNA and ILC or inclusion of a small number of ILC 
related ctDNA in a larger study, ILC has not been systematically studied [41]. Probing the association of 
ctDNA to tumor burden in ILC with two novel imaging biomarkers (amino acid transport/fluciclovine and 
angiogenesis/PSMA) with more sensitivity than FDG PET for ILC will provide biologic insights in the 
context of this exploratory aim.  
2.3 Potential Risks and Benefits
Benefits:
The primary benefit of participation in this study is more accurate identification of metastatic disease 
which may impact appropriate staging and therapy decisions. If occult metastasis are present but are not 
identified on conventional imaging this would lead to understaging and possible futile curative therapy. 
On the other hand, if conventional imaging identifies metastasis which are in fact false positive for 
neoplasia, and which could have been more definitively characterized by molecular imaging, this may 
lead to overstaging and inappropriate withholding of curative therapy. 
Study Specific Procedure Risks:
1) Radiation Risks: The principal risk associated with a radiation dose is the possibility of developing a 
radiation-induced cancer later in life. However, the additional risk of radiation-induced cancer from these 
diagnostic procedures is low compared to the risks from the disease itself as well as systemic therapy or 
radiation therapy. 
Human dosimetry data for fluciclovine from FDA package insert: The (radiation absorbed) effective dose 
resulting from the administration of the recommended activity of 370 MBq (10 mCi) of fluciclovine is 8 
mSv (22 microSv/MBq). For an administered activity of 370 MBq (10 mCi), the highest-magnitude 
radiation doses are delivered to the pancreas, cardiac wall, and uterine wall: 38 mGy, 19 mGy, and 17 
mGy, respectively. 
Human dosimetry data for Ga-PSMA from Joint EANM and SNMMI Procedure Guideline for Prostate 
Cancer Imaging: Version 1.0 [42]. Based on the available studies, the coefficient for effective dose from 
68Ga-PSMA averages is 2.0 × 10−2 mSv/MBq, resulting in an average effective radiation dose of 3 mSv 
for an administered activity of 150 MBq. For a typical dose of 5 mCi (185 MBq) that we will be 
administering, the effective dose equivalent would be 3.7 mSv. The organs with the highest dose are 
urinary bladder wall (0.13-0.173 mSv/MBq) and kidney (0.122-0.262 mSv/MBq).
2) Allergic or other reactions to radiotracer: Although the risk is extremely small, it is possible to develop 
an allergic reaction to the fluciclovine or Ga-PSMA.  This can result in hives, rash, itching and difficulty 
breathing which may require emergency medical treatment. There have been no previous instances of 
allergic reaction. In prior studies with both radiotracers, the risk of adverse events which can be attributed 
to the radiotracer is extremely low, and of minimal medical impact such as burning at IV site or dysgeusia. 

 
Version 1.0;  Page 11 of 28; 29 July 2020
3) Risk related to IV for PET scan: A small amount of radioactive material will be injected by either a 
hand-held needle or a machine. Such injections are generally quite safe, but any injection involves some 
risks. The injection could harm a nerve, artery or vein, or cause infection. The radioactive material could 
also leak from veins, causing swelling and discomfort. 
4) False positive on PET: It is possible that a lesion identified on the experimental PET procedures may 
be false positive, thus potentially leading to an inappropriate change in stage. Yet, no lesion will be acted 
upon which could change therapeutic intent unless confirmed via histology or advanced imaging such as 
MR for certain bone lesions. See below for risks associated with biopsy or MR. 
Non-Study Specific Procedure Risks:
1) Biopsy associated risk: If a suspect lesion is identified on either or both PET scans, definitive histologic 
proof in the safest manner possible will be sought if clinically indicated. Risks of biopsy, though small in 
expert hands, include bleeding, infection and even rarely death. Note that before biopsy is considered, 
careful scrutiny of the already obtained CT or MR will be undertaken to determine if an unsuspected 
anatomic correlate can be identified. This will then prompt a discussion between the patient and primary 
clinician on the benefits versus risks to definitively determine if a metastasis is present which could affect 
therapy. Biopsy will be undertaken in the least invasive manner possible as a standard of care procedure, 
reimbursed by third party payers.  
2) MR: If a lesion is not deemed safe or possible to biopsy, MR may then be performed (e.g. for bone or 
liver). MR, even with the administration of Gadolinium is considered a safe and effective noninvasive 
imaging procedure. In addition to the small possible reactions to contrast material if used, claustrophobia 
while in the magnet is a potential risk, as well as reaction from metal within the body. Yet at Emory, 
special screening is performed before MR to determine if the patient has tattoos or any metal items in 
the body such as implants, pacemakers, clips or shrapnel, to make sure the MRI scan is done safely.

 
Version 1.0;  Page 12 of 28; 29 July 2020
3. Objectives and Endpoints
3.1 Study Objectives:
Our major goal in this proposed investigation is to use advanced molecular imaging to better 
detect metastases in patients with invasive lobular cancer. 
Specific Aim 1: Improve detection of metastasis with fluciclovine and Ga-PSMA PET versus best 
standard of care conventional imaging, as confirmed with histology. We will image 20 patients with 
ILC who have either: a) clinical or imaging suspicion of metastatic disease; or b) proven metastatic 
disease but in whom there is suspicion of an even greater tumor burden that could change therapy 
approach. Abnormal foci which correlate to anatomic lesions previously unsuspected on conventional 
imaging will undergo optional biopsy as clinically indicated in the safest manner possible (if not already 
done) to determine the verified detection rate of metastatic disease.
Specific Aim 2: Determine concordance and discordance of ILC detection with Ga-PSMA versus 
fluciclovine PET, as confirmed with histology. We will compare verified detection rates for 
metastasis between fluciclovine and PSMA PET modalities. This may necessitate a second biopsy of a 
distant lesion discordant between the 2 PET techniques. We will also analyze images for biodistribution 
in the primary lesion(s) for both radiotracers.
Exploratory Aim 3: Establish the role of molecular detection by ctDNA mutations including 
PIK3CA, ESR1, HER2, and AKT1 in characterizing the degree of tumor burden as identified by 
metabolic amino acid transport and/or tumor neovasculature receptor imaging [12]. We will collect 
peripheral blood for ctDNA in all patients and determine if ctDNA correlates with presence or absence of 
metastases and tumor burden as identified by fluciclovine (metabolic) and/or PSMA (angiogenesis) PET. 
3.2 Study Endpoints:
Primary endpoint: 
Determine verified detection rate for metastasis of invasive lobular cancer using fluciclovine PET 
and PSMA PET for imaging (Specific Aims 1 and 2)
Secondary endpoints: 
Correlation of ctDNA results with presence or absence of metastasis (Specific Aim 3)
Correlation of ctDNA results with results of imaging (Specific Aim 3)

 
Version 1.0;  Page 13 of 28; 29 July 2020
4. Study Design
4.1 Overall Design
We will undertake a study with 20 patients with treatment naïve biopsy proven ILC who have either: a) 
clinical or imaging suspicion of metastatic disease; b) biopsy proven metastatic disease but in whom 
there is suspicion of an even greater tumor burden that could change therapy approach. All patients will 
be recruited from the Winship Glenn Family Breast Cancer Program at any Emory facility. All patients will 
undergo Ga PSMA PET followed at minimum of 10 hours (to allow for radiotracer decay) by fluciclovine 
PET. (This may also happen in the opposite order, in which case 20 hours after fluciclovine will be needed 
to allow for decay.) There is no need for randomization since each patient will undergo both imaging 
techniques, and the results will be interpreted blindly. Thus, radiopharmacy scheduling will dictate the 
order of the radiotracers to ensure the patient will undergo both imaging modalities in the shortest time 
frame. Two nuclear medicine physician co-investigators, Drs. Schuster and Muzahir, will independently 
interpret the 20 cases, each reading half (the other reading the other half) of the fluciclovine and PSMA 
images (to avoid bias in always reading the same type of imaging). The readers will be blinded to all 
imaging including the other PET. All suspicious lesions with nonphysiologic activity above background 
for locoregional spread and distant disease will be identified and rated for suspicion of malignancy on a 
6 point Likert scale (where 1 = definitely benign and 6 = definitely malignant). Both the primary lesion and 
all suspected metastases will be measured on an advanced workstation and metabolic uptake 
parameters (SUV, MTV, TLA) recorded. After both studies have been interpreted blindly, the third nuclear 
medicine physician co-investigator, Dr. Ulaner, will blindly interpret deidentified PET studies on every 
patient (alternating for a total of 10 each fluciclovine and Ga-PSMA PET). Afterward, an unblinded 
reconciliation interpretation will take place with all 3 readers. To ensure balance, 10 of the studies will be 
reviewed for consensus first showing Ga-PSMA, then fluciclovine; 10 of the studies will be reviewed in 
the opposite manner. In this manner, a preliminary sense of kappa can be developed between the third 
and the other 2 readers, yet will guarantee the highest degree of patient care as there are currently no 
validated criteria for interpreting fluciclovine or PSMA PET for breast cancer. This approach will help us 
adapt existing prostate specific criteria to that of breast cancer. Concordant and discordant lesions 
between each PET study and conventional imaging will be reported and potential biopsy sites, if any, 
recommended. The most distant and accessible lesion may undergo biopsy in the safest manner possible 
(if not already done) as clinically indicated after discussion between the primary oncologist and patient. 
Preference for biopsy will be given to discordant lesions. Uncommonly, in some lesions not deemed safe 
to biopsy, characteristic appearance on correlative imaging will be accepted as truth (e.g., MRI for certain 
skeletal or liver lesions). In select cases, a second biopsy of a discordant distant lesion may be required. 
For example, if there are 2 potentially impactful lesions which are fluciclovine positive/PSMA negative 
and fluciclovine negative/PSMA positive, respectively. Note that before biopsy is considered, careful 
scrutiny of the already obtained CT or MR will be undertaken to determine if an unsuspected anatomic 
correlate can be identified. This will then prompt a discussion between the patient and the referring 
clinician co-investigator on the benefits versus risks to definitively determine if a metastasis is present 
which could affect therapy. Biopsy may be undertaken in the least invasive manner as standard of care 
as clinically indicated, reimbursed by third party payers. Uptake parameters will be correlated with all 
proven primary, locoregional, and distant lesions. Verified detection rates for metastasis will be calculated 
for each modality. 
In addition, we will collect 20 ml peripheral blood for ctDNA analysis in all patients at visit 1 (or possibly 
2) to be initially processed and stored by Dr. Torres’ lab for off-site specialty analysis. Plasma levels of 
ctDNA will be measured using a targeted 28 gene panel to detect point mutations including mutations in 
PIK3CA, ESR1, HER2, and AKT1, genes associated with hormone therapy resistance and ILC [38, 39]. 
We will examine ctDNA results for association with presence or absence of locoregional metastasis on 
imaging as confirmed with histology, presence or absence of distant metastasis on imaging as confirmed 
with histology, correlation of presence or absence and absolute ctDNA with PET uptake.

 
Version 1.0;  Page 14 of 28; 29 July 2020
4.2 Scientific Rationale for Study Design
Our preliminary data support a key scientific premise that metabolic imaging with amino acid PET has 
shown promise in the detection of ILC. There is also emerging data that tumor neovasculature imaging 
with PSMA PET may improve detection of ILC. We hypothesize that metabolic imaging with amino 
acid transporter PET will improve staging of ILC, particularly for distant metastases, compared to 
best standard of care conventional imaging (usually CT or MR and possible bone scan), and which 
typically does not include FDG PET due to low sensitivity. We also hypothesize that receptor 
directed PSMA imaging of tumor associated neovasculature in ILC will reveal unique information 
to complement metabolic interrogation with fluciclovine PET. Improved staging will facilitate 
more appropriate management decisions.
To test these hypotheses with the highest scientific rigor, we propose a feasibility trial with 
fluciclovine and Ga-PSMA PET strategies centered on detection of metastasis in patients with advanced 
ILC using histology as the gold standard. As an exploratory aim, we will also correlate the occurrence of 
circulating tumor DNA (ctDNA) mutations including PIK3CA, ESR1, HER2, and AKT1 with presence of 
metastasis and tumor burden. We will explore if a multiparametric strategy with ctDNA and imaging will 
help define which patients should undergo molecular imaging and also identify those who may forego 
biopsy if distant lesions are detected on imaging. We expect this pilot trial will generate sufficient 
preliminary data to determine feasibility for a definitive NIH sponsored trial developing more accurate 
staging techniques to help modify current practice for imaging of ILC.
4.3 Justification for Dose
The doses administered for each radiotracer: 10 mCi fluciclovine (18F) and 5 mCi for Ga PSMA are based 
upon well established guidelines referenced below. 
4.4 End of Study Definition
The study will be closed to accrual once all 20 patients have been recruited and scanned with PET. End 
of analysis will occur once sufficient followup data has been collected for each patient to determine study 
endpoints. Study will be formally closed once all manuscripts have been published. Though it is likely 
most relevant standard of care followup will be acquired within 1 year, patients will be consented for 5 
year followup through the medical record in the case of future required analysis. There will be no other 
study specific visits after the second PET scan (visit 2), though the patient will be called by the study 
nurse at 5-10 business days to ensure no delayed AEs occurred. 

 
Version 1.0;  Page 15 of 28; 29 July 2020
5. Study Population
Approximately 1400 breast cancer patients are seen yearly at Winship, with approximately 20% of lobular 
histology (280). Of these, approximately 100-150 are advanced. Thus at a conservative recruitment rate 
of 10% (10-15 patients/year), successful accrual is highly feasible. Lobular cancer tends to present at a 
higher stage. Based on historical averages, we expect that 31% of patients will present with Stage 3 and 
37% of patients with Stage 4 disease. We expect approximately 10% dropout either before completion 
of both PET scans or after PET scanning and before biopsy. 
5.1 Inclusion Criteria
___(Y) 1. Treatment naïve biopsy proven ILC Patients with ILC 
___(Y) 2. Either: a) clinical or imaging suspicion of metastatic disease; or b) proven metastatic disease 
but in whom there is suspicion of an even greater tumor burden that could change therapy approach.
___(Y) 3. Ability and willingness to undergo biopsy if needed per standard of care for possible metastasis 
which could change therapy approach.  
___(Y) 4. Age over 18
Non-English-speaking patients may be considered for enrollment in this trial. Subjects with 
Limited English Proficiency LEP may be enrolled and study team members will use Emory IRB 
approved short forms to conduct the consent process.
5.2 Exclusion Criteria
___(N) 1. Pregnancy. Qualitative or quantitative serum or urine pregnancy test will be done in women of 
childbearing potential within 24 hours before PET1.  
1 A female of childbearing potential (FCBP) is a sexually mature woman who: 1) has not undergone a hysterectomy 
or bilateral oophorectomy; or 2) has not been naturally postmenopausal for at least 12 consecutive months (if age 
> 55 years);  if the female subject is < 55 years and she has been naturally postmenopausal for  > 1 year her 
reproductive status has to be verified by additional  lab tests ( < 20 estradiol OR estradiol < 40 with FSH > 40 in 
women not on estrogen replacement therapy).

 
Version 1.0;  Page 16 of 28; 29 July 2020
6.  Registration Procedures
Informed consent is required prior to participation in the study. Patients will sign the written informed 
consent, if possible, at the time of enrollment. In instances where this is not possible, verbal informed 
consent will be first obtained in order to instruct the patient to fast in preparation for the fluciclovine PET 
scan (not required for PSMA). However final written informed consent will be obtained before the PET 
scan. Participants will also be assigned an identification number for screening purposes; data collected 
during the screening process will also be recorded using that number. Patients will then be registered in 
OnCore through usual Winship and department of Radiology and Imaging Sciences guidelines. No 
randomization will be performed. Blinding will only take place for initial study interpretation before 
consensus interpretation by Nuclear Medicine imagers. 
7.  Study Intervention
Radiotracer production: Blue Earth Diagnostics, Ltd manufacturers of fluciclovine has pledged support 
with gratis radiotracer unit doses supplied from PETNET Solutions. 
Telix Pharmaceuticals (US) Inc, manufacturer of Ga-PSMA, has pledged support with gratis radiotracer 
kits which will be compounded by the Emory CSI radiopharmacy. 
Fluciclovine
PETNET Solutions, located on the Emory campus, produces commercial doses of fluciclovine which is 
FDA-approved for the detection of recurrent prostate cancer.  Unit doses of this material will be provided 
for the study. The IRB has decided that a formal IND exemption is not required. 
The following is provided as justification for IND exemption: This is an early phase study in which 20 
female patients will undergo fluciclovine and PSMA PET on separate days. We have already studied 12 
female patients with fluciclovine in the past for breast cancer under an Emory IRB approved protocol 
without safety concerns. At the time, the fluciclovine radiotracer was manufactured at the Emory CSI 
radiopharmacy under Dr. Schuster’s existing IND 072437. Yet, we will now be administering FDA 
approved commercial doses supplied by the commercial radiopharmacy PETNET to which our IND would 
not be applicable. Thus, this is off-label research of an FDA approved product for recurrent prostate 
cancer. The patient will undergo pregnancy testing within 24 hours before PET as appropriate per 
Winship and Radiology regulations. Note also that 27 women had also been studied for this indication at 
Sloan Kettering in the past without complication. Three female normal volunteers and a number of other 
female patients with other cancers have been studied with fluciclovine under our IND without complication 
and this data had been used to obtain FDA approval in terms of safety. Thus, we believe an IND 
exemption is in order since: 1) The investigation is not intended to be reported to FDA as a well-controlled 
study in support of a new indication for use nor intended to be used to support any other significant 
change in the labeling for the drug; 2) The drug that is undergoing investigation is lawfully marketed as a 
prescription drug product, and the investigation is not intended to support a significant change in the 
advertising for the product; 3) The investigation is conducted in compliance with the requirements 
concerning the promotion and sale of drugs; 4) The investigation is conducted in compliance with the 
requirements for IRB review and informed consent; 5) The investigation does not intend to invoke 21 
CFR 50.24, a waiver of informed consent in an emergency room setting.
PSMA
68Ga-PSMA will be produced under Dr. Schuster’s IND #143137 via a lyophilized kit produced by Telix 
Pharmaceuticals. Briefly, 68Ga will be eluted from our Eckerd and Ziegler 50 mCi Pharmaceutical Grade 
68Ge/68Ga Generator (or equivalent) located at our in-house radiopharmacy and combined in a vial with 
the premixed lyophilized product including peptide. Our existing PSMA IND #143137 has been amended 
with the study protocol. 

 
Version 1.0;  Page 17 of 28; 29 July 2020
7.1 Study Specific Experimental Procedures
A. Imaging (PET acquisition)
Note that this order may be reversed in certain patients depending on patient and radiopharmacy 
schedule.
PSMA
Patients will undergo PSMA PET-CT after 5 mci (3-7 mCi) IV dose of 68Ga-PSMA. Then after an uptake 
period of approximately 60 minutes, the patient will be encouraged to void. A PET-CT will then be 
completed from pelvis to skull base at approximately 5 minutes per bed position. Fasting is not required 
for this study. 
Fluciclovine
On a separate day to allow for radiotracer decay, and within 15 days of PET Visit 1, a fluciclovine PET-
CT at approximately 4 min post IV injection of 10 mCi fluciclovine will be completed from pelvis to skull 
base at approximately 3 minutes per table position. Fasting except for water (and medications) will be 
required for at least 4 hours before the PET scan. 
B. Circulating Tumor DNA
Step #1 - Blood Draw will typically occur on day of first PET scan. 
1.
20ml of peripheral blood (or two 10 ml Cell-Free DNA BCT Streck tubes) is collected by any 
standard phlebotomy technique from a peripheral access point or from a central line by trained 
personnel.
2.
Tubes are inverted about 10 times immediately after collection.
3.
Samples are then prepared for transportation to the laboratory for processing (within 24 hours).
Step #2 - Plasma Processing (in Laboratory)
1.
Perform this step once for each patient: transfer 1 mL whole blood with a pipette to a pre-labeled 
2 mL cryogenic vial, round bottom, self-standing. This 1 ml will be stored frozen per below as a 
backup sample. Then the remainder gets processed as following. 
2.
Streck tubes are centrifuged at room temperature for 10 min at 1600 (±150) g.
3.
After centrifugation, remove tubes from centrifuge and transfer supernatant of the Streck tubes to 
one fresh 10 ml polypropylene centrifuge tube without disturbing the cellular layer using a 
disposable serological pipette or disposable bulb pipette. 
4.
Centrifuge the plasma in the 10 ml centrifuge tube at room temperature for 10 min at 3000 (±150) 
g.  
5.
After centrifugation, remove tubes from centrifuge and transfer supernatant to a fresh 10 ml 
centrifuge tube without disturbing the cellular layer using a disposable serological pipette or 
disposable bulb pipette. After transferring the plasma to a new 10 ml centrifuge tube as described, 
gently mix plasma and record total plasma volume (~8-10 ml plasma per 20 ml blood).
6.
Transfer 1 ml plasma aliquots with a pipette to 2 ml pre-labeled cryogenic vials.
7.
Place plasma tubes into storage box and freeze plasma in freezer upright in storage box at -80°C 
or colder. Six-hour time storage at -20°C is possible.

 
Version 1.0;  Page 18 of 28; 29 July 2020
Step #3 - Specimen Storage
1.
Sample are maintained continuously at -80°C or colder.
2.
When outside the freezer, such as when transferring to a different freezer in another location or 
preparing for shipment, boxes containing tubes should be covered with dry ice.
3.
Freezer or dry ice specimen storage container temperature must be checked and monitored. 
Document any deviation from protocol.
4.
The freezer or dry ice storage box containing the specimens will either be locked or in a secure 
area accessible only to authorized study staff.
5.
A backup storage plan will be in place in the event of freezer failure.
8. Study assessments and Procedures.
8.1 Schedule of Procedures
See above schedule of activities in Section 1.3 for study specific experimental procedures.
Standard of Care Procedures
1) Conventional imaging is broadly defined as mammography, ultrasound, bone scan, FDG PET, CT 
and/or MR: Any or all of these imaging studies may have been performed as standard of care at 
enrollment. These are not under control of study design. Conventional imaging and reports within 6 
months of enrollment (ideally 2 months) will be reviewed to correlate to PET imaging. No additional 
interpretation will take place. Imaging reports for these studies will be utilized for comparative analysis. 
2) Biopsy: After the PET studies, patients with abnormal findings on the PET scans will have the most 
distant and accessible lesion identified so that the patient may undergo biopsy in the safest manner 
possible (if not already done) as clinically indicated. Preference for biopsy will be given to discordant 
lesions. In some lesions not deemed safe to biopsy, characteristic appearance on correlative imaging will 
be accepted as truth (e.g., MRI for certain skeletal or liver lesions). In select cases, a second biopsy of a 
discordant distant lesion may be required. Note that before biopsy is considered, careful scrutiny of the 
already obtained CT or MR will be undertaken to determine if an unsuspected anatomic correlate can be 
identified. This will then prompt a discussion between the patient and the referring clinician co-investigator 
on the benefits versus risks to definitively determine if a metastasis is present which could affect therapy. 
Biopsy will be undertaken in the least invasive manner as standard of care, reimbursed by third party 
payers.
3) Therapy: All subsequent therapy for breast cancer will be standard of care and beyond the scope of 
this investigation. 

 
Version 1.0;  Page 19 of 28; 29 July 2020
9. Measurement of Effect
Image Analysis: Two nuclear medicine physician co-investigators, Drs. Schuster and Muzahir, will 
independently interpret the 20 cases, each reading half (the other reading the other half) of the fluciclovine 
and PSMA images (to avoid bias in always reading the same type of imaging). The readers will be blinded 
to all imaging including the other PET. All suspicious lesions with nonphysiologic activity above 
background for locoregional spread and distant disease will be identified and rated for suspicion of 
malignancy on a 6 point Likert scale (where 1 = definitely benign and 6 = definitely malignant). Both the 
primary lesion and all suspected metastases will be measured on an advanced workstation and metabolic 
uptake parameters (SUV, MTV, TLA) recorded. After both studies have been interpreted blindly, the third 
nuclear medicine physician co-investigator, Dr. Ulaner, will blindly interpret deidentified PET studies on 
every patient (alternating for a total of 10 each fluciclovine and PSMA PET). Afterward, an unblinded 
reconciliation interpretation will take place with all 3 readers. To ensure balance, 10 of the studies will be 
reviewed for consensus first showing Ga-PSMA, then fluciclovine; 10 of the studies will be reviewed in 
the opposite manner. In this manner, a preliminary sense of kappa can be developed between the third 
and the other 2 readers, yet will guarantee the highest degree of patient care as there are currently no 
validated criteria for interpreting fluciclovine or PSMA PET for breast cancer. This approach will help us 
adapt existing prostate specific criteria to that of breast cancer. Concordant and discordant lesions 
between each PET study and conventional imaging will be reported and potential biopsy sites, if any, 
recommended. 
Correlation of histology with images: Uptake parameters as above will be correlated with all proven 
primary, locoregional, and distant lesions.
ctDNA analysis: Plasma levels of ctDNA will be measured using targeted assays to detect point 
mutations including mutations in PIK3CA, ESR1, HER2, and AKT1, genes associated with hormone 
therapy resistance and ILC [12, 38, 39, 43]. Because ILC are predominantly hormone receptor positive, 
hormone resistance is particularly relevant because it predicts for higher rate of distant metastasis. Dr. 
Mylin Torres, will provide expertise in ctDNA analyses. Deidentified specimens will be shipped to the 
Circulating Tumor Cell Center at Massachusetts General Hospital for fee for service ctDNA processing.    
Correlation of ctDNA results with results of imaging: We will examine ctDNA results for association 
with presence or absence of locoregional metastasis on imaging as confirmed with histology, presence 
or absence of distant metastasis on imaging as confirmed with histology, correlation of presence or 
absence and absolute ctDNA with PET uptake as determined by the following indices: SUVmax, 
SUVmean, SUVpeak, Total Lesion Activity for the index primary, locoregional and distant metastatic 
lesion if any, and correlation to summed indices as a reflection of total metabolic tumor burden. ctDNA 
will also be correlated with CI. 

 
Version 1.0;  Page 20 of 28; 29 July 2020
10. Statistical considerations 
Specific Aims 1 and 2
Analysis plan: The objectives of Aims 1 and 2 are to compare verified detection rates between 
fluciclovine and PSMA PET versus conventional imaging, as confirmed with histology (Aim 1), and 
determine concordance and discordance of ILC detection with PSMA versus fluciclovine PET, as 
confirmed with histology (Aim 2). Paired detection rates will be assessed using McNemar’s test.  
Detection rates will be reported for each method, and 95% confidence intervals will be estimated using 
the Clopper-Pearson approach. Rates of concordant/discordant observations will be reported. The 
Likert rating-scale data also allows for determination of sensitivity, specificity, PPV, NPV and Receiver 
Operating Characteristic (ROC) Area Under the Curve (AUC) values to compare. 
Sample size and power: We will image 20 patients with ILC for this study. This is a small, exploratory 
study, with a goal of obtaining preliminary data to estimate effect size. Thus, there is no power analysis 
for the study aims. If we find a 20% difference in detection rate with a 40% discordant rate, we would 
require 67 patients in a larger study to achieve 80% power with a Type I error rate of 0.05 using 
McNemar’s test. Discordant rates of 50% and 60% with a difference in detection rate of 20% would 
require 83 and 99 patients, respectively, to achieve 80% power. If the difference in detection rate is 
15%, the required sample sizes would range from 118 to 175 for discordant rates of 40% to 60% to 
achieve 80% power.  If the difference in detection rate is 25%, the required sample sizes would range 
from 42 to 63 for discordant rates of 40% to 60% to achieve 80% power.
Exploratory Aim 3
Analysis plan: In Aim 3, ctDNA parameters will be summarized descriptively using mean, median, 
min/max, IQR, and standard deviation. ctDNA will be correlated with PET uptake parameters using 
Pearson’s or Spearman’s correlation coefficient (where appropriate), as well as chi-squared tests, 
Fisher’s exact tests, or ANOVA if any variables are further categorized. In addition, generalized linear 
models will be fit to evaluate the relationship between ctDNA parameters and PET uptake parameters, 
controlling for relevant characteristics.  
Sample size and power: We will image 20 patients with ILC for this study.  This is a small, exploratory 
study, with a goal of obtaining preliminary data to estimate effect size. Thus, there is no power analysis 
for the study aims.  If we find a correlation coefficient of 0.3 between a specific ctDNA parameter and 
PET update parameter, we would need 84 patients to detect that correlation coefficient vs. a null value 
of 0 with 80% power and a Type I error of 0.05.  We would need 46 patients to detect a correlation 
coefficient of 0.4 vs. a null value of 0 to achieve 80% power.
11. Adverse Events:  List and Reporting Requirements 
Adverse event data collection and reporting, which are required as part of every clinical trial, are done to 
ensure the safety of patients enrolled in the studies as well as those who will enroll in future studies using 
similar agents. Adverse events are reported in a routine manner at scheduled times during a trial (please 
follow directions for routine reporting provided in the full Data Safety and Monitoring Plan (DSMP)). 
Additionally, certain adverse events must be reported in an expedited manner for timely monitoring of 
patient safety and care. The following sections provide information about expedited reporting.
Special note for radiotracers given during this trial: The risk of AEs or SAEs from PET 
radiotracers is exceedingly low. A significant shift from baseline which can be attributable 
to the radiotracer injection and not the patient’s medical condition will be considered an 
unexpected AE. An event greater than 20 hours post scan for fluciclovine and 10 hours for 
Ga-PSMA will not be considered a related to study procedure AE since the radiotracer has 
effectively decayed by 20 hours and 10 hours respectively. Reporting will follow relevant FDA 
and manufacturer guidelines as outlined below.
A serious adverse event is any medical occurrence which is fatal, is immediately life threatening, requires 
hospitalization (or prolongs an existing hospitalization), results in persistent significant disability or 
incapacity, is a congenital abnormality or a birth defect, or is considered medically significant by a 
physician.

 
Version 1.0;  Page 21 of 28; 29 July 2020
The definition of serious adverse event (experience) also includes important medical event. Medical and 
scientific judgment will be exercised in deciding whether expedited reporting is appropriate in other 
situations, such as important medical events that may not be immediately life-threatening or result in 
death or hospitalization but may jeopardize the patient or may require intervention to prevent one of the 
other outcomes listed in the definition above. These should also usually be considered serious. Examples 
of such events are intensive treatment in an emergency room or at home for allergic bronchospasm; 
blood dyscrasias or convulsions that do not result in hospitalization; or development of drug dependency 
or drug abuse.
Attribution:  The determination of whether an adverse event is related to a study imaging agent.  The 
categories are:
Definite:  The adverse event is clearly related to the study imaging agent.
Probable:  The adverse event is likely related to the study imaging agent.
Possible:  The adverse event may be related to the study imaging agent.
Unlikely:  The adverse event is doubtfully related to the study imaging agent.
Unrelated:  The adverse event is clearly not related to the study imaging agent.
Unexpected Adverse Event:  An adverse event, the nature or severity of which is not consistent with the 
applicable product information.  
Investigational Agent:  A protocol drug administered under an Investigational New Drug Application (IND).  
Commercial Agent:  An agent not provided under an IND but obtained from a commercial source.
Determination of Reporting Requirements
Reporting requirements may include the following considerations: 1) whether the patient has received an 
investigational or commercial agent; 2) the characteristics of the adverse event including the grade 
(severity), the relationship to the study imaging agent (attribution), and the prior experience 
(expectedness) of the adverse event; 3) the Phase (1, 2, or 3) of the trial; and 4) whether or not 
hospitalization or prolongation of hospitalization was associated with the event.
Steps to determine if an adverse event is to be reported in an expedited manner:
Step 1: Identify the type of event using the NCI Common Toxicity Criteria for Adverse Event Reporting 
Version 5.0 (CTCAE v5.0). The CTCAE v5.0 provides descriptive terminology and a grading scale for 
each adverse event listed. A copy of the CTCAE v5.0 can be downloaded from the CTEP home page 
(http://ctep.cancer.gov). Additionally, if assistance is needed, the NCl has an Index to the CTCAE v5.0 
that provides help for classifying and locating terms. All appropriate treatment locations should have 
access to a copy of the CTCAE v5.0.
Step 2: Grade the event using the NCI CTCAE  v5.0.
Step 3: Determine whether the adverse event is related to the protocol imaging agent (investigational or 
commercial). 
Step 4: Determine the prior experience of the adverse event. Expected events are those that have been 
previously identified as resulting from administration of the agent. An adverse event is considered 
unexpected, for expedited reporting purposes only, when either the type of event or the severity of the 
event is not listed in the investigator brochure for an investigational agent or the drug package insert for 
a commercial agent.
Step 5: Review Expedited reporting requirements below to determine if there are any protocol-specific 
requirements for expedited reporting of specific adverse events that require special monitoring.
Step 6: Determine if the protocol treatment given prior to the adverse event included investigational 
agent(s), a commercial agent(s), or a combination of investigational and commercial agents.
Reporting Methods:
The Principal Investigator is responsible for evaluating all reports of adverse events which occur in 
patients enrolled on this study and reporting those events which are serious to Telix Pharmaceuticals or 

 
Version 1.0;  Page 22 of 28; 29 July 2020
to Blue Earth  as outlined below and the Emory University IRB according to the guidelines below. SAEs 
must be reported as required by the WCI DSMP.
Expedited reporting requirements:  All grade 4-5 adverse events (unexpected and expected) and grade 
2-3 adverse events that are unexpected must be reported as above within 24 hours of occurrence.
Reporting to Institutional Review Board (IRB):  All SAEs must be reported by the Principal Investigator to 
the Emory University IRB as required by their regulations and the conditions of approval for the protocol.
Reporting to FDA: The Sponsor- Investigator must notify the FDA in a written IND safety report of any 
adverse event which is both serious and unexpected.  Each written notification must be made as soon 
as possible but no later than 15 calendar days of first becoming aware of the event.  Reports should be 
submitted using the FDA Form 3500A (available on the FDA website at www.fda.gov/medwatch.  
Reporting may be done online or via mail or fax.     
Fluciclovine: The Investigator will report all Serious Adverse Events occurring in a subject on the day of 
or within XX days following the Agent administration to Bracco, the parent company for Blue Earth 
Diagnostics (“Bracco”) by telephone (+44 1483 212151), FAX (+44 1483 212178) or e mail 
(drugsafetyus@blueearthdx.com). 
Events will be reportedBracco within 24 hours of the investigator becoming aware of the events 
occurrence. Bracco will prepare an individual single case report (ISCR) in compliance with applicable 
regulations. A copy of the ISCR will be sent by Bracco to the Investigator. The Investigator is responsible 
for informing the ethics committee of serious events occurring during the study in compliance with local 
regulations. Bracco will report the event, if appropriate, to the regulatory authority in compliance with local 
regulations. The sponsor will also report SAEs to the FDA per regulations as noted in Section 10 above.
Ga PSMA: Emory has provided a copy of the detailed Clinical Trial protocol to Telix Pharmaceuticals, as 
amended from time-to-time, as well as a copy of the annual safety reporting (Safety Reporting) involving 
the use of the Kit. Emory shall provide an annual report to Telix, summarizing the Adverse Event (AE) 
profile in relation to the use of the Kit, specifically noting whether there was attribution of the AE (however 
serious) to the Kit.
In the event of a Serious Adverse Event (SAE) or death occurring any time within 7 days after 
administration of 68Ga-PSMA-11 prepared using the Kit, and not related to disease progression, Emory 
shall 
report 
to 
Telix 
Designated 
Contact 
(Bernard 
Lambert; 
(571) 
294 
4646; 
bernard.lambert@telixpharma.com)
(a)
Within seven (7) calendar days of the date of awareness of a life-threatening adverse 
event or death (Date of Awareness), regardless of apparent causality to the use of the 
Kit.
(b)
Within fifteen (15) calendar days of the Date of Awareness, any other SAEs, regardless of 
apparent causality to the use of the Kit.
(c)
Within seven (7) calendar days upon communication of any other safety-related report, 
issues or queries related to the Kit that are either raised by, or communicated to, regulatory 
authorities or ethics committees.
(d)
Within seven (7) calendar days after submission, the Development Safety Update Report 
(DSUR) submitted to the FDA.
Any unexpected fatal (Grade 5) or unexpected, life-threatening (Grade 4) adverse event must be reported 
to the FDA as soon as possible but no later than 7 calendar days of first becoming aware of the event. 
Reports should be submitted using the FDA Form 3500A via fax at 1-800-332-0178

 
Version 1.0;  Page 23 of 28; 29 July 2020
12. Data Reporting / Regulatory Requirements
Patients will be monitored by the technologists and/or study nurse before and after the studies for any 
adverse events/reactions.  They will be given contact phone numbers to call if they experience any 
problems (i.e. problems with the IV site, any allergic reaction symptoms).  Basic monitoring of adverse 
events during the 68Ga-PSMA-11 PET visit will be performed as pursuant to FDA agreement. This will 
consist of direct observation by our study staff during the visit with documentation of any adverse events 
or lack thereof in the electronic medical record. 
The Data and Safety Monitoring Committee (DSMC) of the Winship Cancer Institute of Emory University 
will oversee the conduct of this study. This committee will review all pertinent aspects of study conduct 
including patient safety, compliance with protocol, data collection and efficacy. 
The DSMC will review the charts of 10% of patients enrolled to the study and two of the first 5 patients 
entered to the study. Reviews will occur annually for studies that are moderate risk. High risk studies will 
be reviewed every 6 months. The committee reserves the right to conduct additional audits if necessary, 
at any time-point. The Principal Investigator is responsible for notifying the DSMC about the accrual of 
patients when the first 5 have been entered to the study. 
The charter for the Winship DSMC is available upon request to the investigator or other study-related 
personnel.  
As with our ongoing clinical trial, for the current proposal we will adhere strictly to Winship’s Data Safety 
Monitoring Plan which is provided in detail at the following link: 
https://winshipcancer.emory.edu/research/clinical-trials-office/data-and-safety-monitoring-
committee.html
Data Safety Monitoring Board: 
Note that our proposed trial is an early phase clinical trial that will be conducted completely within the 
Emory system, so a formal and independent Data Safety Monitoring Board (DSMB) [which is required for 
multi-site studies or Phase III trials] is not required for our proposal. As described above, the DSMC of 
Winship Cancer Institute of Emory University will oversee the conduct of our study.  
Fidelity to Protocol and Data Integrity:
Clinical trial performance and fidelity to the protocol, will be monitored by the DSMC of Winship Cancer 
Institute of Emory University.  Integrity of the data will be maintained by rigorous peer-review internally 
by all the clinical co-investigators in Radiation Oncology, Radiology/Nuclear Medicine, Surgical Oncology, 
and Medical Oncology. 
13. Ethics and Protection Of Human Subjects
The study will be approved by the Emory IRB per standard institutional requirements and informed written 
consent obtained. 
To maintain patient confidentiality, medical records will be accessed only by IRB approved study 
personnel (e.g. CRC). Partial HIPAA waiver will be obtained to allow screening of provider schedules for 
identification of potentially eligible research subjects. Upon medical record review and identification of a 
potentially eligible research subject, patient's full name, MRN, EMPI, and full dates (e.g. DOB, procedure 
dates, admission dates, etc.) will be stored on a screening log and used by study coordinators for the 
subsequent study activities. The screening log will be kept on the HIPAA compliant shared folder and will 
be accessible only to limited IRB approved study personnel (e.g CRC). Sensitive data will always be 
stored on the HIPAA compliant shared folder and will never be stored on local or portable drives that are 
not encrypted per Emory IS standards. 

 
Version 1.0;  Page 24 of 28; 29 July 2020
Each study participant will be assigned a unique study identification (ID) number at the time that informed 
consent is given. Personal identifying information, study data, including the unique study ID, will be 
entered in the Microsoft Database that will be kept on the HIPAA compliant shared folder accessible to 
approved study personnel only. Only de-identified data will be shared with those outside the study team 
to ensure adequate protection of sensitive data.
Study identifiers will be kept indefinitely. 
To maintain participant confidentiality, no identifying information about any of the study participants will 
be published. Any data published (including demographic information about the study sample as a whole) 
will be in aggregate/summary form only. 
Note also that Dr. Gary Ulaner of MSKCC and Dr. Aditya Bardia of the Circulating Tumor Cell 
Center at Massachusetts General Hospital, will only be exposed to deidentified data, samples, 
and images. As such they would not be considered “engaged” in human subjects research. As a 
result, they do not require inclusion on the IRB per our consultation with the Emory IRB.
Incidental Findings
a.   All incidental findings noted by the imager will be discussed with the attending physician who 
is a co-investigator and documented in the electronic medical record. 
b.   The oncologist will educate the patient about the nature of the incidental finding, how to seek 
care from a clinician or specialist, obtaining health insurance to secure treatment, and/or 
referral to a clinical specialist, if one is required. 
c.   Language to this effect is present in the consent form.
PET findings will be used for research purposes within the context of the clinical trial as noted above. All 
incidental findings will be recorded in the clinical research form and emergent incidental findings will be 
communicated to the patient’s physician who is also an investigator on this study.
Compensation for time and effort: Each patient will be compensated $50 as per diem for travel expenses 
per scan (total of $100 for both scans).

 
Version 1.0;  Page 25 of 28; 29 July 2020
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Version 1.0;  Page 27 of 28; 29 July 2020
APPENDIX A   Abbreviations and definition of terms
The following abbreviations and special terms are used in this study Clinical Study Protocol.
Abbreviation or 
special term
Explanation
AE
Adverse event
Akt
Protein kinase B (or PKB)
CRFs
Case Report Forms
CT
Computed tomography
CTCAE
Common Terminology Criteria for Adverse Event
ctDNA
Circulating tumor DNA
DSMB
Data Safety Monitoring Board 
DSMC
Data and Safety Monitoring Committee 
DSMP
Data Safety and Monitoring Plan
ECOG
Eastern Cooperative Oncology Group
eCRF
Electronic case report form
ESR1
Estrogen Receptor alpha
FCBP
Female of childbearing potential
FDG
fluorodeoxyglucose
FDA
Food and Drug Administration
FSH
Follicle-stimulating hormone
GCP
Good Clinical Practice
hCG
Human chorionic gonadotropin
HIPAA
Health Insurance Portability Accountability Act
ICF
Informed consent form
IDC
invasive ductal carcinoma
IHC
Immunohistochemistry
IL
Interleukin
ILC
Invasive lobular carcinoma

 
Version 1.0;  Page 28 of 28; 29 July 2020
Abbreviation or 
special term
Explanation
IMC
invasive metaplastic carcinoma
IND
Investigational New Drug
IRB
Institutional Review Board
ISCR
individual single case report
IV
Intravenous
MBq
Megabecquerel
mCi
Millicurie
MRI
Magnetic resonance imaging
NCCN
National Comprehensive Cancer Network
NCI
National Cancer Institute
NIH
National Institutes of Health
PET
Positron Emission Tomography
PI3Ks
Phosphoinositide 3-kinases 
PSMA
Prostate specific membrane antigen
RNA
Ribonucleic acid
SAE
Serious adverse event
SUV
Standardized uptake value
TLA
Total lesion activity
VEGF
Vascular endothelial growth factor