A Pilot Study of 18Fluorine-Fluciclovine Positron Emission Tomography/Computed Tomography for Staging Muscle Invasive Bladder Cancer Preceding Radical Cystectomy
Phase: EARLY_PHASE1
Conditions: Bladder Cancer, Urothelial Carcinoma
Sponsor: Brigham and Women's Hospital

Official Title: A Pilot Study of 18Fluorine-Fluciclovine Positron Emission 
Tomography/Computed Tomography for Staging Muscle Invasive Bladder Cancer Preceding 
Radical Cystectomy. 
 
NCT Number: 04018053 
Document Date: 06/10/2022 

NCI Protocol #: N/A
DF/HCC Protocol #: 19-208
Protocol Version Date: June 10, 2022
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Responsible Data Manager: pending
Agent(s):  Fluciclovine F18 (Axumin®, Blue Earth Diagnostics Inc; commercial) 
Study Exempt from IND Requirements per 21 CFR 312.2(b).  
Protocol Type / Version # / Version Date:
Amendment / Version #6 / June 10, 2022

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Protocol Version Date: June 10, 2022
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SCHEMA

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TABLE OF CONTENTS
SCHEMA.........................................................................................................................................3
1.        OBJECTIVES.......................................................................................................................6
1.1
Study Design............................................................................................................6
1.2
Primary Objectives...................................................................................................6
1.3
Secondary Objectives...............................................................................................6
2.
BACKGROUND .................................................................................................................6
2.1
Urothelial carcinoma of the bladder ........................................................................6
2.2
Current imaging methods to stage bladder cancer...................................................7
2.3
18Fluorine-Fluciclovine............................................................................................7
2.4
Rationale for staging bladder cancer with 18F-fluciclovine.....................................8
2.5
Correlative Studies Background ..............................................................................8
3.
PARTICIPANT SELECTION.............................................................................................9
3.1
Eligibility Criteria....................................................................................................9
3.2
Exclusion Criteria ....................................................................................................9
3.3
Inclusion of Women and Minorities ......................................................................10
4.
REGISTRATION PROCEDURES ...................................................................................10
4.1
General Guidelines for DF/HCC Institutions ........................................................10
4.2
Registration Process for DF/HCC Institutions.......................................................11
4.3
General Guidelines for Other Investigative Sites ..................................................11
4.4
Registration Process for Other Investigative Sites.................................................11
5.
IMAGING PLAN ..............................................................................................................11
5.1
Summary................................................................................................................11
5.2
18F-fluciclovine PET/CT imaging..........................................................................11
5.3
Surgery...................................................................................................................12
5.4
Investigational Imaging Agent Administration .....................................................12
5.5
Duration of Study Participation .............................................................................13
5.6
Duration of Follow Up...........................................................................................13
5.7
Criteria for Taking a Participant Off Study ...........................................................13
6.
DOSING DELAYS/DOSE MODIFICATIONS ...............................................................14
6.1
Anticipated Toxicities............................................................................................14
6.2
Toxicity Management............................................................................................15
6.3
Dose Modifications................................................................................................16
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................16
7.1
Expected Toxicities................................................................................................16
7.2
Adverse Event Characteristics...............................................................................16
7.3
Adverse Event Reporting.......................................................................................17
7.4
Reporting to Hospital Risk Management...............................................................17

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7.5
Routine Adverse Event Reporting .........................................................................17
7.6
Additional Adverse Event Reporting.....................................................................17
8.
IMAGING AGENT INFORMATION ..............................................................................18
8.1
18F-fluciclovine ......................................................................................................18
9.
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ........................................20
10.
STUDY CALENDAR .......................................................................................................20
11.
MEASUREMENT OF EFFECT .......................................................................................21
11.1
Pathological staging of bladder cancer ..................................................................21
12.
DATA REPORTING / REGULATORY REQUIREMENTS...........................................21
12.1
Data Reporting.......................................................................................................21
12.2
Data Safety Monitoring..........................................................................................22
13.
STATISTICAL CONSIDERATIONS...............................................................................22
13.1
Study Design, Objectives, and Endpoints..............................................................22
13.2
Data Analysis Plan.................................................................................................23
13.3
Sample Size, Accrual Rate and Study Duration ....................................................23
13.4
Stratification Factors..............................................................................................25
13.5
Analysis of Primary Endpoints..............................................................................25
13.6
Analysis of Secondary Endpoints..........................................................................25
13.7
Reporting and Exclusions ......................................................................................26
14.
PUBLICATION PLAN .....................................................................................................26
15.
RFERENCES.....................................................................................................................26
APPENDIX A ........................................................................PERFORMANCE STATUS CRITERIA
............................................................................................................................................30
APPENDIX B .......................................................................................AXUMIN PACKAGE INSERT
..............................................................................................................................................1

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1.        OBJECTIVES
1.1
Study Design
This is a prospective, pilot study.
1.2
Primary Objectives
To estimate the agreement rate of detecting metastatic disease between 18F-fluciclovine-PET/CT 
and histopathology from radical cystectomy for detecting locoregional metastatic disease in 
patients with cT2-T4N0M0 muscle invasive bladder cancer by conventional CT/MRI imaging.
1.3
Secondary Objectives
1.3.1 To estimate the rate of detection of suspected distant metastatic disease by 18F-fluciclovine
-PET/CT in patients with cT2-T4N0M0 muscle invasive bladder cancer by conventional 
CT/MRI imaging.
1.3.2 To investigate the relationship between 18F-fluciclovine uptake on PET/CT and pathologic 
tumor stage and size of primary bladder tumor after radical cystectomy.
1.3.3 To investigate the relationship between 18F-fluciclovine uptake on PET/CT and the 
presence/absence of a ASCT2 and LAT1 amino acid transporters in the resected primary bladder 
tumors. 
2.
BACKGROUND
2.1
Urothelial carcinoma of the bladder
Urothelial carcinoma of the bladder is the fifth most common malignancy in the United States, 
with an estimated 75,000 new cases and 15,000 deaths in a year [1]. Approximately 25% of new 
cases are muscle-invasive bladder cancer (MIBC), while the rest are non-muscle-invasive 
bladder cancer (NMIBC). Accurate staging of bladder cancer is paramount in deciding the 
appropriate course of management. While NMIBC is generally managed by cystoscopic 
resection with intravesical BCG or chemotherapy, the standard treatment for MIBC is radical 
cystectomy (RC) preceded by neoadjuvant chemotherapy (NAC) in cisplatin-eligible patients [2-
4].  It is estimated that ~30% of patients undergoing RC may have nodal metastases [5, 6].    
Metastatic disease has exhibited improved outcomes when treated with first-line systemic 
cisplatin-based chemotherapy and salvage post-platinum therapy using one of multiple 
programmed death (PD)-1 and PD-ligand (L)-1 inhibitors (pembrolizumab, atezolizumab, 
durvalumab, nivolumab, avelumab) [7-13]. Additionally, pembrolizumab and atezolizumab are 
approved for the first-line therapy of cisplatin-ineligible patients based on durable responses [14, 
15]. Therefore, pre-operative identification of those with node-positive metastatic disease will 
enhance prognostic risk stratification and tailoring of neoadjuvant therapy, for example with the 
consideration of clinical trials of more aggressive and promising neoadjuvant combination 

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regimens and tailoring the extent of lymph node dissection based on the extent of node-positive 
disease.   
2.2
Current imaging methods to stage bladder cancer
Current imaging methods used to stage bladder cancer predominantly include computed 
tomography (CT) and magnetic resonance imaging (MRI). While both are useful in locoregional 
staging, both exhibit limitations. CT and MRI rely on nodal enlargement as an indicator of 
disease involvement. Non-enlarged lymph nodes harboring disease can be falsely negative by 
anatomic imaging. Conversely, false positive lymphadenopathy from benign etiologies can 
preclude eligible patients from RC. CT and MRI sensitivity for detection of nodal metastasis 
ranges from 31-50% and specificity ranges from 68-100% [6].  Some reports suggest that 40% of 
those with bladder confined disease by imaging have extravesical and/or pathologically node-
positive disease at RC [16-20].  
Fluorodeoxyglucose (18FDG)-positron emission tomography (PET) is a functional imaging 
modality that has excellent sensitivity for detecting metastatic disease when combined with 
anatomic CT or MRI [21-25]. However, effective use of FDG-PET/CT in bladder cancer is 
limited by the fact that FDG is excreted in urine. Saturation of the imaging signal from within the 
bladder and urinary tract can affect accurate assessment of locoregional FDG uptake. 
Furthermore, elevated FDG uptake can also be seen in benign inflammatory conditions and 
within the gastrointestinal tract, which can also confound staging assessment.  Correspondingly, 
FDG-PET is not approved by regulatory agencies to stage bladder cancer [4]. 
Given the limitations of both conventional structural CT/MRI and FDG-PET to stage urinary 
tract cancers, an alternative functional imaging marker without these limitations is highly 
desirable and warrants investigation.
2.3
18Fluorine-Fluciclovine
Anti-1-amino-3-F-18-fluorocyclobutane-1-carboxylic acid (FACBC or 18F-fluciclovine) is a 
novel radiotracer for imaging amino acid transporters, which can be over-expressed in cancer 
[26]. 18F-fluciclovine was recently approved by the Food and Drug Administration for the 
detection of biochemically recurrent prostate cancer [27, 28] and it may have a role in 
complementing MRI for staging localized prostate cancer [29] and radiation planning [30]. 18F-
fluciclovine is predominantly transported in cells via the L-system amino acid transporters, 
ASCT (alanine-serine-cysteine transporters)-2 and LAT (large neutral amino acid transporters)-
1, with peak uptake in tumors 5 to 20 minutes after injection.
In addition to prostate cancer, 18F-fluciclovine has been shown to accumulate in other tumor 
types, including breast, lung and colon cancer [31].  A unique feature of 18F-fluciclovine 
biodistribution is low bladder uptake at early post injection timepoints when elevated uptake in 
tumors is already seen [32, 33].  Additional advantages of 18F-fluciclovine over other naturally 
occurring amino acid PET imaging agents, such as 11C methionine, include a longer radiolabel 
half-life (110 minutes for 18F agents vs 20 minutes for 11C) such that an on-site cyclotron is not 
required [34]. Other 11C agents have also been tested, including 11C choline and 11C acetate 

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PET/CT have been tested for staging bladder cancer with equivocal results [35].  
No adverse effects to patients that is directly attributable to 18F-fluciclovine have been reported 
to date [36].
2.4
Rationale for staging bladder cancer with 18F-fluciclovine
Amino acid transporters are overexpressed in many cancers [26]. Diets rich in the amino acids L-
isoleucine and L-leucine have been shown to promote bladder cancer in rats [37]. The LAT-1 
transporter has been implicated as responsible for leucine uptake in T24 bladder cancer cells 
[38]. Inhibition of LAT-1 can prevent cell growth in bladder cancer cell lines [38], and 
overexpression of LAT-1 can predict progression of urinary tract cancer [39, 40]. These findings 
suggest that the amino acid transporters are a viable imaging target in bladder cancer.
18F-fluciclovine has been shown to be a promising imaging agent in multiple cancer types. The 
biodistribution profile of 18F-fluciclovine shown in prior studies suggests that it may be useful 
for imaging bladder cancer. Experiments with a rat orthotopic prostate cancer model compared 
the uptake of 18F-fluciclovine with that of FDG and found that target-to-background ratio was 
higher for 18F-fluciclovine with only minimal bladder accumulation. Compared to FDG, 18F-
fluciclovine is only minimally eliminated by the kidneys during the typical imaging time course 
suggesting that it may be more optimal than FDG for imaging renal and urinary tract 
malignancies. 18F- fluciclovine is physiologically found in the pancreas, liver, bone marrow, and 
muscle, with negligible uptake in kidneys, bowel, and shows delayed urinary excretion even 
when compared with 11C choline, leading to a more favorable distribution profile in the abdomen 
and pelvis [32]. 
Taken together, these studies suggested that 18F-fluciclovine-PET/CT may be a viable imaging 
strategy to accurately stage muscle invasive bladder cancer. 
2.5
Correlative Studies Background
LAT1 and ASCT2 expression are determined by immunohistochemical (IHC) staining with anti-
LAT1 and anti‐ASCT2 monoclonal antibodies, respectively. Immunostaining with these 
antibodies are based on standard protocols and have been described in detail previously [41]. 
Briefly, 5 unstained slides per patient consisting of 5 µM thick sections of formalin fixed 
paraffin embedded (FFPE) tissue are obtained from deparaffinized and rehydrated sections. 
These sections are treated with 0.3% hydrogen peroxide (H2O2) in methanol for 30 min to block 
endogenous peroxidase activity. To expose the antigens, sections are autoclaved in 
ethylenediaminetetraacetic acid (pH 8.0) for 5 min and cooled for 30 min. After rinsing in 
phosphate‐buffered saline, the sections are incubated with anti‐LAT1 and anti‐ASCT2 antibodies 
overnight followed by secondary staining with horseradish peroxidase-conjugated antibodies. 
Visualization of staining is carried out using 0.02% 3,3′‐diaminobenzidine tetrahydrochloride 
and 0.01% H2O2 in 0.05 M Tris‐HCl (pH 7.4). Negative control tissue sections were stained as 
described above, except that the primary antibodies are omitted. The LAT1 and ASCT2 
expression scores are assessed by the extent of staining as follows: 0, less than 5% of tumor area 
stained; 1, 5–10% stained; 2, 11–25% stained; 3, 26–50% stained; and 4, 51% or more stained. 

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3.
PARTICIPANT SELECTION
3.1
Eligibility Criteria 
Participants must meet the following criteria on screening examination to be eligible to 
participate in the study:
3.1.1
Participants must have histologically or cytologically confirmed urothelial carcinoma 
of the bladder.
3.1.2
Participants must have cT2-T4N0 disease at the time of the study, as defined by 
conventional CT or MRI imaging. Patients must have no definite evidence of 
locoregional or distant metastatic disease at the time of study eligibility, as defined by 
conventional imaging. 
3.1.3
Radical cystectomy must be planned for the patient after the planned 18F-fluciclovine-
PET/CT.
3.1.4
Patients may or may not have had prior neoadjuvant therapy prior to this study.
3.1.5
Age ≥18 years.  Since no dosing or adverse event data are currently available on the 
use of 18F-fluciclovine in participants <18 years of age, and the majority of bladder 
cancer occur in the adult population [42], children are excluded from this study but 
will be eligible for future pediatric trials.  
3.1.6
ECOG performance status ≤2 (Karnofsky ≥60%, see Appendix A)
3.1.7
Ability and willingness to comply with the study procedures.
3.1.8
The effects of 18F-fluciclovine on the developing human fetus are unknown. For this 
reason and because radiopharmaceuticals may be teratogenic, women of childbearing 
potential and men must agree to use adequate contraception (barrier method of birth 
control; abstinence) prior to study entry and for 24 hours after the PET/CT scan is 
completed.  Should a woman become pregnant or suspect she is pregnant while 
participating in this study, she should inform her treating physician immediately.  
3.1.9          Ability to understand and the willingness to sign a written informed consent 
document
3.2
Exclusion Criteria
Participants who exhibit any of the following conditions at screening will not be eligible for 
admission into the study:

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3.2.1
Participants with other known malignancy that has required treatment in the past 3 
years.
3.2.2
Pregnant women are excluded from this study because 18F-fluciclovine is a 
radiopharmaceutical with the potential for teratogenic effects.  Because of the 
radiation exposure to a nursing infant from 18F-fluciclovine, women who are 
breastfeeding are also excluded from this study.
3.2.3
History of allergic reactions attributed to compounds of similar chemical or biologic 
composition to 18F-fluciclovine.
3.2.4
Uncontrolled intercurrent illness including, but not limited to, ongoing or active 
infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac 
arrhythmia, or psychiatric illness/social situations that would limit compliance with 
study requirements.
3.2.5
Contraindications for PET/CT including:

Severe claustrophobia
3.2.6
Any past or current condition that in the opinion of the study investigators would 
confound the results of the study or pose additional risk to the patient by their 
participation in the study.
3.3
Inclusion of Women and Minorities
Both men and women of all races and ethnic groups are eligible for this trial.
4.
REGISTRATION PROCEDURES 
4.1
General Guidelines for DF/HCC Institutions
Institutions will register eligible participants in the Clinical Trials Management System (CTMS) 
OnCore. Registrations must occur prior to the initiation of any protocol-specific therapy or 
intervention. Any participant not registered to the protocol before protocol-specific therapy or 
intervention begins will be considered ineligible and registration will be denied.
An investigator will confirm eligibility criteria and a member of the study team will complete the 
protocol-specific eligibility checklist.
Following registration, participants may begin protocol-specific therapy and/or intervention. 
Issues that would cause treatment delays should be discussed with the Overall Principal 
Investigator (PI). If the subject does not receive protocol therapy following registration, the 
subject must be taken off-study in the CTMS (OnCore) with an appropriate date and reason 
entered.  

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4.2
Registration Process for DF/HCC Institutions
Applicable DF/HCC policy (REGIST-101) must be followed.  
4.3
General Guidelines for Other Investigative Sites
N/A
4.4
Registration Process for Other Investigative Sites
N/A
5.
IMAGING PLAN
5.1
Summary
All procedures will be performed on an outpatient basis. Expected toxicities and potential risks 
are described in Section 6 (Expected Toxicities and Dosing Delays/Dose Modification). 
This is a pilot study investigating 18F-fluciclovine PET/CT for imaging patients with muscle 
invasive bladder cancer. The investigational imaging procedure in this study is 18F-fluciclovine-
PET/CT.  
After the participant meets eligibility criteria for the study and informed written consent is 
obtained, the participant will undergo the study procedure (18F-fluciclovine-PET/CT imaging). 
18F-fluciclovine-PET/CT will be obtained within 6 weeks of the standard-of-care 
diagnostic staging procedures (MRI and/or diagnostic CT).
Participants will then undergo radical cystectomy per standard-of-care as determined by the 
medical oncologist.  At the time of radical cystectomy, which usually includes pelvic lymph 
node dissection, standard evaluation of the surgical specimen will determine pathologic staging. 
We will assess the results of the standard evaluation of the surgical specimen for metastatic 
disease. These results will be compared with staging as determined by 18F-fluciclovine PET/CT 
and standard-of-care diagnostic imaging.
The participant will have completed all study requirements once tissue is obtained at radical 
cystectomy.  
5.2
18F-fluciclovine PET/CT imaging
5.2.1 The 18F-fluciclovine-PET/CT imaging procedure is described in Section 5.4.
Participants will be instructed to refrain from strenuous exercise for the 24 hours 
preceding the PET/CT exam, since amino acid transporters expression have been 

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noted to increase in skeletal muscle for that period after exercise [43]. Participants 
will be NPO for 4 hours prior to administration of 18F-fluciclovine.
5.2.2
Participants will be provided with instructions regarding what precautions to take
at home once they receive the study agent. As with all radiopharmaceuticals 
administered, participants will receive a card stating that they have received a 
radiopharmaceutical for medical purposes which includes, but is not limited to, 
radiopharmaceutical, dosage, suggested time of detection, suggested 
contamination item storage duration, and institutional and departmental contact 
information.
Given the short half-life and diagnostic dose of 18F-fluciclovine (110 minutes), no 
special precautions regarding excreta or contact with other humans are necessary.
5.3
Surgery
After completion of the 18F-fluciclovine-PET/CT scan, participants will undergo standard-of-care 
radical cystectomy surgery as determined by medical oncology and urology. If surgical plans (for 
example, the presence of unsuspected extensive disease precluding cystectomy) are altered based 
on clinical findings during surgery, the patient may remain evaluable if a lymph node dissection 
is performed as determined by the study team. Results from the standard-of-care evaluation of 
the pathologic specimens will be collected and reviewed. This includes evaluation of 
pathological samples for metastatic cancer burden.
5.4
Investigational Imaging Agent Administration
10 millicurie (mCi) of 18F-fluciclovine will be administered via slow push over 10 seconds 
through a peripheral intravenous line. [33]. This is performed immediately prior to imaging. No 
specific pre-administration lab parameters or procedures for the patient is required prior to agent 
administration.
5.4.1
Image Acquisition Details:
Immediately after the injection of the radiopharmaceutical, dynamic PET/CT images of 
the pelvis will be obtained for 15 minutes. Subsequently, PET/CT images will be 
obtained from the pelvis to the base of skull in the caudal to cranial direction.  CT will be 
obtained for attenuation correction per standard institutional PET/CT policies. PET 
images will be acquired in 3D mode at 3-5 minutes per bed position.  
As noted in Section 8.1, this image acquisition protocol is based on the pharmacokinetics 
of the agent in male patients with prostate cancer. While we do not anticipate significant 
differences in the kinetics of the agent in patients with bladder cancer, appropriate 
adjustments to the imaging acquisition parameters will be made if significant differences 
are observed on initial imaging studies.

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5.4.2
Image Analysis Details:
18F-fluciclovine-PET/CT images will be reviewed for lesions, defined as focal areas of 
18F-fluciclovine uptake not correlating with normal anatomy or physiology. Lesions will 
be scored on a 5-point scale for diagnostic certainty (0, definitely benign; 1, probably 
benign; 2, equivocal; 3, probably malignant; and 4, definitely malignant).  
Three-dimensional regions of interest will be placed in areas of abnormal tracer uptake 
scored as 3-5 and the level of 18F-fluciclovine uptake recorded. Measures of 18F-
fluciclovine will include: maximal standardized uptake value (SUVmax, body weight), 
metabolic tumor volume (MTV; volume of tumor [cm3] with SUV >42% of SUVmax), 
SUVmean (within the MTV), and total lesion avidity (TLA; calculated as MTV × 
SUVmean). Tumor to background ratios will also be calculated using blood pool, muscle 
and bone marrow as reference normal organs.
5.5
Duration of Study Participation 
Duration of study participation will be until the time that surgical specimens are obtained at the 
time of radical cystectomy. 
Participants may be removed from study participation prior to the scheduled completion if one of 
the following events occurs:  
•
Unacceptable adverse event(s),
•
Participant demonstrates an inability or unwillingness to comply with the study
                  procedures,
•
Participant decides to withdraw from the study, 
•
Condition that would result in confounding information for study procedure
                  interpretation as determined by the study investigators,
•
General or specific changes in the participant's condition render the participant 
unacceptable for continuing study procedures in the opinion of the treating 
investigator.
5.6
Duration of Follow Up
After completing the study procedures, patients will be followed for at least 30 days. Continued 
standard of care will be provided by the treating physician. Participants removed from the study 
for unacceptable adverse events determined to be related to the study drug will be followed until 
resolution or stabilization of the adverse event. 
5.7
Criteria for Taking a Participant Off Study
Participants will be removed from study when any of the following criteria apply:

Lost to follow-up

Withdrawal of consent for data submission

Death

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The reason for taking a participant off study, and the date the participant was removed, must be 
documented in the case report form (CRF). In addition, the study team will ensure Off 
Treatment/Off Study information is updated in OnCore in accordance with DF/HCC policy 
REGIST-101. If a participant is taken off study prior to undergoing the research 18F-fluciclovine-
PET/CT scan, the participant may be replaced.
6.
DOSING DELAYS/DOSE MODIFICATIONS
Dose delays and modifications will be made as indicated in the following table(s). The 
descriptions and grading scales found in the revised NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 4.0 will be utilized for dose delays and dose modifications. A 
copy of the CTCAE version 4.0 can be downloaded from the CTEP website
 http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
If possible, symptoms should be managed symptomatically. In the case of toxicity, appropriate 
medical treatment should be used (including anti-emetics, anti-diarrheals, etc.).
The study procedure is 18F-fluciclovine PET/CT imaging.  All adverse events experienced by 
participants will be collected from the time of the administration of the study agent (18F-
fluciclovine) until 24 hours after 18F-fluciclovine PET/CT images are obtained, regardless of 
their attribution/relatedness).  Adverse events related to standard-of-care of FDA-approved 
procedures for comparison with 18F-fluciclovine PET/CT imaging (i.e., Diagnostic CT scans, 
MR imaging) will not be reported as part of this trial.  Beyond 24 hours after 18F-fluciclovine 
PET/CT imaging, adverse events that are determined to be related to the participant’s systemic 
therapy or underlying condition and are determined not to be related to the agents and procedures 
in this protocol, will not be reported, as the investigational procedure is a diagnostic imaging 
procedure.  
Adverse events related to radical cystectomy surgery will not be reported as part of this trial.  
Participants continuing to experience toxicity related to 18F-fluciclovine at the off-study visit 
may be contacted for additional assessments until the toxicity has resolved or is deemed 
irreversible. 
6.1
Anticipated Toxicities
6.1.1
Expected Adverse Events from 18F-fluciclovine
18F-fluciclovine will be administered in tracer quantities. As such, the tracer dose of 18F-
fluciclovine is expected to have no significant effect on normal human physiology and we 
do not anticipate any adverse events [36].
18F-fluciclovine is an FDA-approved imaging agent for evaluation of metastatic prostate 
cancer. Adverse reactions were reported in ≤1% of subjects during clinical studies with Axumin 
(Appendix B). The most common adverse reactions were injection site pain, injection site 

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erythema and dysgeusia. To date, no adverse events have been reported that have been 
directly attributable to 18F-fluciclovine [36], 
6.1.2
Expected Adverse Events from Intravenous Line Placement
•
Minor discomfort
•
Bleeding at the injection site
•
Infection at the injection site
•
Bruising at the injection site
•
Claustrophobia
•
Discomfort
6.1.3
Expected Adverse Events from Radiation from PET/CT scans
The estimated effective dose from 18F-fluciclovine is approximately effective dose per 
unit administered activity is 22.1 μSv/MBq [44]. 
Additional exposure from the CT component of the combined 18F-fluciclovine PET/CT 
scan is approximately 10 mSv.
Table 1. Dosimetry for 18F-fluciclovine [44]
Organ
(μGy/MBq)
Liver
33.5±4.2 
Kidneys
13.7±0.8
Spleen
23.8±11.7
Bladder wall
25.2±17
Lungs
34.5±5.3
Whole body
12.6±0.2
Effective dose
22.1±2.1
6.2
Toxicity Management
Allergic reactions and anaphylaxis to 18F-fluciclovine will be managed following standard 
institutional guidelines for contrast reaction management (DFCI Department of Imaging Policy 
5.31).  
If a study participant has a severe allergic reaction or anaphylactic reaction to 18F-fluciclovine 
and PET/CT images are unable to be obtained, the participant will be removed from the study.

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All other toxicities and adverse events will be managed as medically necessary.  Supportive care 
will be administered as medically necessary.
6.3
Dose Modifications
The dosage of 18F-fluciclovine is never modified.
7.
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial.  The 
following list of reported and/or potential AEs (Section 7.1) and the characteristics of an 
observed AE (Section 7.2) will determine whether the event requires expedited reporting in 
addition to routine reporting.
7.1
Expected Toxicities
Local injection site pain 
Local injection site erythema
Dysgeusia 
7.2
Adverse Event Characteristics

CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 will be utilized for AE reporting.  All appropriate treatment areas should have access 
to a copy of the CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be 
downloaded from the CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.

For expedited reporting purposes only:  
-
AEs for the agent(s) that are listed above should be reported only if the adverse event 
varies in nature, intensity or frequency from the expected toxicity information which 
is provided.
-
Other AEs for the protocol that do not require expedited reporting are outlined in the 
next section (Expedited Adverse Event Reporting) under the sub-heading of Protocol-
Specific Expedited Adverse Event Reporting Exclusions.

Attribution of the AE:
-
Definite – The AE is clearly related to the study treatment.
-
Probable – The AE is likely related to the study treatment.
-
Possible – The AE may be related to the study treatment.
-
Unlikely – The AE is doubtfully related to the study treatment.
-
Unrelated – The AE is clearly NOT related to the study treatment.

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7.3
Adverse Event Reporting 
7.3.1
In the event of an unanticipated problem or life-threatening complications 
treating investigators must immediately notify the Overall PI.
7.3.2
Investigators must report to the Overall PI any adverse event (AE) that 
occurs after the initial dose of study treatment, during treatment, or within 30 days of the 
last dose of treatment on the local institutional SAE form. 
7.3.3
DF/HCC Adverse Event Reporting Guidelines
Investigative sites within DF/HCC will report AEs directly to the DFCI Office for Human 
Research Studies (OHRS) per the DFCI IRB reporting policy. 
. 
7.3.4
Protocol-Specific Adverse Event Reporting Exclusions
For this protocol only, the AEs/grades occurring from the time of 18F-fluciclovine 
administration until 24 hours after 18F-fluciclovine administration will be reported.  
However, they still must be reported through the routine reporting mechanism (i.e. case 
report form).
7.4
Reporting to Hospital Risk Management
Participating investigators will report to their local Risk Management office any participant 
safety reports, sentinel events or unanticipated problems that require reporting per institutional 
policy. 
7.5
Routine Adverse Event Reporting
All Adverse Events must be reported in routine study data submissions to the Overall PI on the 
toxicity case report forms.  AEs reported through expedited processes (e.g., reported to the 
IRB, FDA, etc.) must also be reported in routine study data submissions.
7.6
Additional Adverse Event Reporting
In addition to the reporting of Adverse Events as described in 7.5, any observed Serious Adverse 
Events that occur following the receipt of 18F-fluciclovine, whether related to the product or not, 
PI or designee shall notify BED within twenty four (24) hours of knowledge of any SAEs. SAE 
reports to BED must be recorded and faxed or scanned and emailed to::
Bracco Diagnostics Inc. Drug Safety Unit
E mail:  Drugsafetyus@BlueEarthDx.com
Fax: +1-609-514-2522

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8. IMAGING AGENT INFORMATION
A list of the adverse events and potential risks associated with 18F-fluciclovine can be found in 
Section 7.1 and Appendix B
8.1
18F-fluciclovine
8.1.1
Description
18Fluorine-Fluciclovine is (1r, 3r)-1-amino-3[18F]fluorocyclobutane-1-carboxylic acid. 
The molecular weight is 132.1. 
The following pharmacokinetic information for 18F-fluciclovine is based on experience in 
male patients with prostate cancer:
Following intravenous administration, 18F-fluciclovine distributes to the liver (14% of 
administered activity), pancreas (3%), lung (7%), red bone marrow (12%) and 
myocardium (4%). With increasing time, 18F-fluciclovine distributes to skeletal 
muscle. 
Following intravenous administration, the tumor-to-normal tissue contrast is highest 
between 4 and 10 minutes after injection, with a 61% reduction in mean tumor uptake 
at 90 minutes after injection. 
Across the first four hours post-injection, 3% of administered radioactivity is excreted 
in the urine. Across the first 24 hours post-injection, 5% of administered radioactivity 
is excreted in the urine.
           
It should be noted that the pharmacokinetics of 18F-fluciclovine is unknown in patients 
with bladder cancer at this time, and may not follow the distribution pattern described 
above. While we do not anticipate significant differences for bladder cancer patients, 
appropriate adjustments to the imaging parameters will be made as necessary if this 
pattern is dramatically different on initial studies.
8.1.2
Form
18F-fluciclovine is supplied as a clear, colorless injection in a ready to use syringe that 
contains a single patient-specific dose of  18F-fluciclovine at calibration time and date. 
18F-fluciclovine is supplied by PETNET for Blue Earth Diagnostics Ltd. Prior to study 
opening, Blue Earth Diagnostics will work with Dana Farber Cancer Institute and 
PETNET to set up a study-specific account for this trial.  This account should be used for 
ordering doses for the study. The BED study number (BED-IIT-355) should referenced 
when ordering doses.
8.1.3
Storage and Stability

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18F-fluciclovine is stored at controlled room temperature (USP) 20°C to 25°C (68°F to 
77°F). 18F-fluciclovine does not contain a preservative. 18F-fluciclovine is stored within 
the original container in radiation shielding. 
8.1.4
Compatibility
18F-fluciclovine is being administered in this study in tracer quantities for diagnostic 
imaging purposes. No drug interactions are expected between 18F-fluciclovine and other 
drug regimens that each patient may be receiving.
8.1.5
Handling
18F-fluciclovine should be handled and administered only by nuclear medicine personnel 
trained to handle radioactive material.
 
8.1.6
Availability
18F-fluciclovine will be provided by Blue Earth Diagnostics Ltd as part of a research 
agreement for this study.
8.1.7
Administration
10 millicurie (mCi, ± 20%) of 18F-fluciclovine will be administered via slow push over 10 
seconds intravenously. A peripheral IV line is preferred. A butterfly line or established 
IV line may be used as long as the patency is established. The use of a power port 
needs to be approved by the study team.
8.1.8
Ordering
The study investigators will order the 18F-fluciclovine from PETNET per institutional 
policies for the ordering of radiopharmaceuticals. Prior to study opening, Blue Earth 
Diagnostics will work with Dana Farber Cancer Institute and PETNET to set up a study-
specific account for this trial.  This account should be used for ordering doses for the 
study. The BED study number (BED-IIT-355) should referenced when ordering doses.
8.1.9
Accountability
A computer-based system for logging receipt and administration of 
radiopharmaceuticals per DFCI Imaging/Nuclear Medicine will be used for the 
accountability of the unit doses of the study radiopharmaceutical, 18F-fluciclovine, 
received from Blue Earth Diagnostics, Ltd.  
8.1.10
Destruction and Return
Unused 18F-fluciclovine will be disposed of following institutional policies for the 
disposal of radioactive waste.

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BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES
Correlation of imaging data with pathological specimens obtained at the time of radical 
cystectomy will be performed as described in Sections 2.5, 5.3 and 13.2.  
10.
STUDY CALENDAR
Phase
Pre-study
Study day
After Study
Within 6 weeks of 
Diagnostic 
CT/MRI
Obtain informed consent
x
Review inclusion/exclusion 
criteria
x
Physical exam1
x
Review of prior/concomitant 
medications1
x
x
Qualitative β-hCG
x5
Height/weight2
x
18F-Fluciclovine PET/CT
x
Diagnostic CT/ MRI
x1
Collection of surgical 
specimens 
x6
Review adverse events4
x
x
1Physical exams, review of prior/concomitant medications, serum creatinine, 
serum or urine β-hCG at baseline, vital signs as part of the physical exam, and 
diagnostic CT/MRI obtained pre-study are part of the standard-of-care for 
patients with bladder cancer. We will be collecting and reviewing the data from 
these standard procedures to compare to 18F-fluciclovine PET/CT imaging 

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results.  Those with ECOG performance status ≤2 (Karnofsky ≥60%, see 
Appendix A) will be eligible. A urine pregnancy test will be performed where 
indicated. 
2Height and weight will be obtained as part of the PET/CT scan procedures. 
4Adverse events will be collected from the time of administration of 18F-
fluciclovine until 24 hours after 18F-fluciclovine-PET/CT imaging.
5Female participants of childbearing potential must have a negative qualitative 
pregnancy test (β-hCG) at the Study Day. Results of a standard-of-care β-hCG 
may be used for the injection of 18F-fluciclovine if performed within 24 hours 
prior. Females who are two years post tubal ligation or postmenopausal for at 
least one year are not considered to be of childbearing potential and do not need 
to undergo pregnancy testing. 
6 This is part of the standard-of-care radical cystectomy.  
MEASUREMENT OF EFFECT
11.1
Pathological staging of bladder cancer
Pathological staging of bladder cancer is not the primary objective of this study. It will be used 
for comparison and correlation with imaged-based staging on 18F-fluciclovine PET/CT and 
standard-of-care MRI and diagnostic CT.
DATA REPORTING / REGULATORY REQUIREMENTS
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements).
12.1
Data Reporting
12.1.1
Method
The study team in the Department of Imaging will collect, manage, and perform quality 
checks on the data for this study.
The following databases will be utilized:
· Partners’ Research Patient Data Registry (RPDR) – Clinical database that indexes results from 
the Partners’ hospitals, including BWH. This will be used to augment the pathology and 
laboratory data from the CRIS database.
· Epic – Epic will be used to collect patient medical history as detailed in the “Methods of Data 
Collection” section below.

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· Centricity Patient Archiving and Communication Systems (PACS) – Radiology system for 
SOC image storage as well as only anatomical imaging analysis on fluciclovine PET/CT studies. 
This system will be used by the study radiologists to collect and analyze subjects’ imaging data 
and gather tumor measurements.
· Hermes PACS – DFCI Imaging PACS for all of Nuclear Medicine, PET/CT, and non-SOC data 
of all imaging modalities that can’t be stored on Centricity. All fluciclovine project data, 
including data received from outside hospitals, will be placed into a dedicated archive on 
Hermes. This data will be read from Hermes storage using Hermes viewing/analysis software.
· DFCI Imaging PET/CT database – A database of patient information and scanning parameters. 
The database contains data for all PET and PET/CT acquisitions performed at DFCI Imaging. It 
will be used to retrieve this information when it is needed.
12.1.2
Responsibility for Data Submission
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or 
data forms to the Office of Data Quality (ODQ) in accordance with DF/HCC policies
  
12.2
Data Safety Monitoring
The DF/HCC Data and Safety Monitoring Committee (DSMC) will review and monitor toxicity 
and accrual data from this study. The committee is composed of medical oncologists, research 
nurses, pharmacists and biostatisticians with direct experience in cancer clinical research.  
Information that raises any questions about participant safety will be addressed with the Overall 
PI and study team.
The DSMC will review each protocol up to four times a year with the frequency determined by 
the outcome of previous reviews. Information to be provided to the committee may include: up-
to-date participant accrual; current dose level information; DLT information; all grade 2 or 
higher unexpected adverse events that have been reported; summary of all deaths occurring 
within 30 days of intervention for Phase I or II protocols; for gene therapy protocols, summary of 
all deaths while being treated and during active follow-up; any response information; audit 
results, and a summary provided by the study team. Other information (e.g. scans, laboratory 
values) will be provided upon request.  
13.
STATISTICAL CONSIDERATIONS
13.1
Study Design, Objectives, and Endpoints
This is a prospective pilot study evaluating 18F-fluciclovine-PET/CT for detecting metastatic 
disease in patients with cT2-T4N0M0 muscle invasive bladder cancer.
The primary objective of this study is to estimate the agreement rate of metastatic disease status 
between 18F-fluciclovine-PET/CT and histopathology from radical cystectomy for detecting 

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locoregional metastatic disease in patients with cT2-T4N0M0 muscle invasive bladder cancer by 
conventional CT/MRI imaging.  Secondary objectives include estimating of the rate of detection 
of suspected distant metastatic disease by 18F-fluciclovine-PET/CT, analysis of the association of 
18F-fluciclovine uptake on PE/CT in primary bladder tumor with pathologic tumor grade/size after 
radical cystectomy, and analysis of 18F-fluciclovine uptake on PET/CT to the presence/absence of 
a ASCT2 and LAT1 amino acid transporters in the resected primary bladder tumors. 
The primary endpoints are metastatic disease status by 18F-fluciclovine-PET/CT and pathologic 
stage at radical cystectomy.  The agreement rate is defined as the number of scans that are 
considered positive metastatic disease by both 18F-fluciclovine-PET/CT and histopathology and 
those considered negative metastatic disease by both 18F-fluciclovine-PET/CT and 
histopathology, out of all scans performed. A positive 18F-fluciclovine-PET/CT is defined as 
evidence of metastatic disease either regionally in the pelvic lymph nodes or distantly to bone, 
lungs, viscera, or lymph nodes outside of the pelvis.  The ability of 18F-fluciclovine-PET/CT for 
detecting locoregional metastatic disease will be defined as the presence of disease detected 
locoregionally on 18F-fluciclovine-PET/CT.  Secondary endpoints include visualization 
(present/absent) and number of distant metastases on 18F-fluciclovine-PET/CT. The amount of 
18F-fluciclovine accumulation in the primary bladder tumor on PET will be quantified using the 
maximum standardized uptake value (SUVmax). Tumor grade/size and the presence of amino 
acid transporters will be assessed histopathologically with specimens from cystectomy as part of 
standard-of-care.
13.2
Data Analysis Plan
The primary and first secondary analyses will include all 16 eligible and treated patients with 
completed 18F-fluciclovine-PET/CT scans and pathology dissection. Among these 16 patients, 
those who undergo cystectomy will be included for secondary analysis 2 and 3. 
We will tabulate the metastatic disease status by 18F-fluciclovine-PET/CT (limited to bladder, 
locoregional nodes positive, or distant) and pathologic stage at radical cystectomy and compute 
the agreement rate, along with the corresponding 90% exact binomial confidence interval (CI). 
The proportion of patients who show positive 18F-fluciclovine-PET/CT will be computed, along 
with the corresponding 90% exact binomial confidence interval.  This similar methodology will 
be used to evaluate the proportion of agreement compared to histopathology and the rates of 
locoregional disease and suspected distant metastatic disease detected by 18F-fluciclovine-
PET/CT.  In addition, treating pathologic and/or biopsy findings as the reference standard, we will 
estimate the sensitivity and specificity of 18F-fluciclovine-PET/CT for detection of metastatic 
disease.  
The analysis of the relationship of 18F-fluciclovine uptake on PET/CT (SUVmax) in primary 
bladder tumor to pathologic tumor grade/size after radical cystectomy will be analyzed using the 
Kruskal-Wallis test. The Wilcoxon-Rank-Sum test will be used to assess the association between 
SUVmax and the presence/absence of ASCT2 and LAT1 amino acid transporters in the resected 
primary bladder tumors.

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13.3
Sample Size, Accrual Rate and Study Duration
The planned sample size is 16 participants with completed 18F-fluciclovine-PET/CT scans and 
pathology dissection. Patients who did not complete the scans or without pathology dissection are 
unevaluable and will be replaced. Considering 4 patients who may be unevaluable and replaced, 
up to 20 patients can be enrolled. The estimated accrual rate is 1 participant/month, and accrual is 
expected to complete in another 16 months. Participants who undergo 18F-fluciclovine-PET/CT 
but have altered surgical plans due to clinical findings during surgery may remain evaluable and 
not replaced if a lymph node dissection was performed as determined by the study investigators. 
This will not affect the primary study objective given the primary objective is the agreement rate 
between PET/CT and pathology for metastases. Participants who do not undergo cystectomy will 
not be included for secondary objectives (2 and 3).
We will employ a Simon’s 2-stage design to test the hypothesis that the agreement rate of 
metastatic disease status between 18F-fluciclovine-PET/CT and histopathology will be 75%, 
compared to a null agreement rate of 50%. In the first stage we will enroll 10 patients, and if 6 or 
more scans among those 10 are in agreement regarding metastatic disease status, we will open the 
second stage of accrual to enroll an additional 6 patients, making a total sample size of 16 
patients. If fewer than 6 scans are in agreement in the first stage then the study may be stopped or 
the study design may be amended to enroll additional patients. In order to consider the study a 
success, a total of 11 or more scans need to be in agreement among all 16 scans. This design has a 
0.62 probability of stopping early under the null, and has 79.9% power while maintaining an 
overall one-sided type I error rate of 0.101. The agreement rate between 18F-fluciclovine-PET/CT 
and histopathology is expected to be 75% (or higher) and the corresponding 90% CI will be (0.52, 
0.91).
 
Accrual Targets
Sex/Gender
Ethnic Category
Females
Males
Total
Hispanic or Latino
+
=
Not Hispanic or Latino
+
=
Ethnic Category: Total of all 
subjects
(A1) +
(B1)=
(C1)
Racial Category
American Indian or Alaskan 
Native
+
=
Asian
+
=
Black or African American
+
=
Native Hawaiian or other Pacific 
Islander
+
=
White
+
=

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Racial Category: Total of all 
subjects
(A2) +
(B2)=
(C2)
(A1 = A2)
(B1 = B2)
(C1 = C2)
13.4
Stratification Factors
N/A 
13.5
Analysis of Primary Endpoints
See section 13.1
13.6
Analysis of Secondary Endpoints
Secondary objectives are to:
1. To determine the rate of detection of suspected distant metastatic disease by 18F-fluciclovine-
PET/CT in patients with cT2-T4N0M0 muscle invasive bladder cancer by conventional CT/MRI 
imaging.
Endpoints: 
1) Visualization and number of distant metastases on 18F-fluciclovine-PET/CT
Visualization of distant metastases on 18F-fluciclovine-PET/CT will be binary-categorized as 
present/absent.  In the categorization, a qualitative scale (0-4) may be accounted and an optimal 
cut-off may be examined.  We will compute sensitivity to compare 18F-fluciclovine-PET/CT 
with standard imaging modalities for distant metastases. The number of distant metastases will 
be descriptively shown by imaging modalities.
2.  To correlate 18F-fluciclovine uptake on PET/CT in primary bladder tumor with pathologic 
primary tumor stage and size after radical cystectomy.
Endpoints: 
•
18F-fluciclovine SUVmax in primary bladder tumor
•
Primary tumor stage and size at radical cystectomy
The amount of 18F-fluciclovine accumulation in the primary bladder tumor on PET will be 
quantified using the maximum standardized uptake value (SUVmax). Tumor grade will be 
assessed histopathologically with specimens from cystectomy as part of standard-of-care. The 
correlations between 18F-fluciclovine uptake and tumor grade will be evaluated using Spearman 
correlation coefficients. 
3. To correlate 18F-fluciclovine uptake on PET/CT to the presence/absence of ASCT2 and LAT1 
amino acid transporters in the resected primary bladder tumors.

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Endpoints:  
•
18F-fluciclovine SUVmax in primary bladder tumor
•
Presence/absence of ASCT2 and LAT1 amino acid transporter in resected primary bladder 
tumors
The Wilcoxon-Rank-Sum test will be used to assess the association between SUVmax and the 
presence/absence of a ASCT2 and LAT1 amino acid transporters in the resected primary bladder 
tumors.
13.7
Reporting and Exclusions
13.7.1 Evaluation of Toxicity
All participants will be evaluable for toxicity from the time of administration of 18F-fluciclovine.
13.7.2 Evaluation of the Primary Efficacy Endpoint
All participants in who undergo 18F-fluciclovine-PET/CT and radical cystectomy will be 
evaluable for the primary study endpoint.
14.
PUBLICATION PLAN
This is a pilot study and the results will be prepared for publication after all study data answering 
a specific endpoint has been collected. The plan for publication may first be in abstract form 
followed by full manuscript publication. The Principal Investigator is primarily responsible for 
the publication of the study results.
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APPENDIX A
PERFORMANCE STATUS CRITERIA
ECOG Performance Status Scale
Karnofsky Performance Scale
Grade
Descriptions
Percent
Description
100
Normal, no complaints, no evidence 
of disease.
0
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction.
90
Able to carry on normal activity; 
minor signs or symptoms of disease.
80
Normal activity with effort; some 
signs or symptoms of disease.
1
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work).
70
Cares for self, unable to carry on 
normal activity or to do active work.
60
Requires occasional assistance, but 
is able to care for most of his/her 
needs.
2
In bed <50% of the time.  
Ambulatory and capable of all 
self-care, but unable to carry out 
any work activities.  Up and about 
more than 50% of waking hours.
50
Requires considerable assistance and 
frequent medical care.
40
Disabled, requires special care and 
assistance.
3
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours.
30
Severely disabled, hospitalization 
indicated.  Death not imminent.
20
Very sick, hospitalization indicated. 
Death not imminent.
4
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair.
10
Moribund, fatal processes 
progressing rapidly.
5
Dead.
0
Dead.

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