Clinical trial NCT04410133
Conditions: Brain Metastases
Keywords: Recurrent Brain Metastases Diagnostic Positron Emission Tomography (PET) Scan
Sponsor: Blue Earth Diagnostics
Start date: 2020-10-26; Completion date: 2023-06-05
Enrollment: 151 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
An open-label, single dose, single arm, prospective, multicenter Phase 3 study to establish the diagnostic performance of 18F fluciclovine positron emission tomography (PET) in detecting recurrent brain metastases after radiation therapy

Detailed description:

Arms: arm 1: Single intravenous administration of 18F fluciclovine for PET Scan
Interventions: intervention 1: 18F fluciclovine
Intervention descriptions: intervention 1: 18F fluciclovine injection, 185 MBq (5 mCi) ± 20%, delivered as an intravenous bolus

Protocol documents:
Prot_002.pdf: 
BED-FLC-312 Protocol Version 3.0, 17 March 2023 
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CONFIDENTIAL Blue Earth Diagnostics, Ltd  Based on Protocol Template, SOP-MED-002-AD-2, Version 3
CLINICAL STUDY PROTOCOL
An Open-label, Single-arm, Single-dose, Prospective, Multicenter Phase 3 Study to 
Establish the Diagnostic Performance of 18F-fluciclovine Positron Emission 
Tomography (PET) in Detecting Recurrent Brain Metastases after Radiation Therapy
 
REVELATE 
 
BED-FLC-312 
 
Phase: 3
IND Number: 145265 
Protocol Version and Date: Protocol Version 3.0, 17 March 2023 
 
 
Sponsor: Blue Earth Diagnostics, Ltd 
Confidentiality Statement
This confidential information about an investigational product is provided for the 
exclusive use of investigators of this product and is subject to recall at any time. The 
information in this document may not be disclosed unless applicable laws or regulations 
require such disclosure. Subject to the foregoing, this information may be disclosed only 
to those persons involved in the study who have a need to know, with the obligation not 
to further disseminate this information.
 




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CONTENTS 
LIST OF ABBREVIATIONS.......................................................................................7
PROTOCOL SYNOPSIS ........................................................................................... 10
STUDY SCHEMA...................................................................................................... 20
1. 
BACKGROUND..................................................................................... 21
1.1.
Overview of Clinical Studies...................................................................22
2.
STUDY RATIONALE............................................................................23
2.1. 
Risk-Benefit Assessment......................................................................... 25
2.1.1. 
Benefit...................................................................................................... 25
2.1.2. 
Risk.......................................................................................................... 25
3.
STUDY OBJECTIVES ...........................................................................27
3.1. 
Primary Objective.................................................................................... 27
3.2. 
Secondary Objectives .............................................................................. 27
4.
STUDY DESIGN ....................................................................................28
4.1. 
Study Overview ....................................................................................... 28
5. 
CRITERIA FOR EVALUATION ........................................................... 31
5.1. 
Primary Efficacy Endpoint ......................................................................31
5.2. 
Secondary Efficacy Endpoints.................................................................31
5.3. 
Safety Evaluations ................................................................................... 31
6. 
SUBJECT SELECTION.......................................................................... 32
6.1. 
Study Population...................................................................................... 32
6.2.
Inclusion Criteria .....................................................................................32
6.3. 
Exclusion Criteria.................................................................................... 33
7.
CONCURRENT MEDICATIONS..........................................................34
7.1. 
Allowed Medications and Treatments ..................................................... 34
7.2. 
Prohibited Medications and Treatments .................................................. 34
8. 
STUDY TREATMENTS......................................................................... 35
8.1. 
Method of Assigning Subjects to Treatment Groups ..............................35
8.2. 
Blinding ................................................................................................... 35
8.3. 
Formulation of Test Product .................................................................... 35
8.4. 
Packaging and Labelling .......................................................................... 35
8.5. 
Supply of Study Drug at the Site............................................................. 35
8.6. 
Dosage/Dosage Regimen .........................................................................35
8.7. 
Dispensing ............................................................................................... 36
8.8. 
Administration Instructions .....................................................................36


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8.9. 
Storage..................................................................................................... 36
8.10. 
Study Drug Accountability ...................................................................... 36
8.11.
Measures of Treatment Compliance........................................................37
8.12.
End Of Trial.............................................................................................37
9. 
STUDY PROCEDURES AND GUIDELINES....................................... 38
9.1. 
Clinical Assessments ............................................................................... 42
9.1.1. 
Concomitant Medications ........................................................................ 42
9.1.2.
Demographic and Baseline Characteristics .............................................42
9.1.3.
ECOG Performance Status ......................................................................42
9.1.4. 
Medical History ....................................................................................... 42
9.1.5. 
Imaging Studies ....................................................................................... 42
9.1.6. 
Vital Signs ............................................................................................... 44
9.1.7.
SoC Biopsy/Neurosurgical Intervention..................................................44
9.1.8.
Clinical Follow-Up / Data Abstraction....................................................44
9.2. 
Clinical Laboratory Measurements.......................................................... 45
9.2.1. 
Pregnancy Test......................................................................................... 45
10. 
EVALUATIONS BY VISIT ................................................................... 46
10.1. 
Visit 1 –Screening.................................................................................... 46
10.2. 
Visit 2 - Day 1: IMP Administration and Dynamic PET Scan (1 to 
28 days after Screening) .......................................................................... 47
10.3. 
Visit 2 – Day 1 or up to 3 days after 18F-fluciclovine Administration: On-
Study MRI Brain Scan .............................................................................47
10.4. 
Visit 3 – Safety Follow-up (+1 to 3 days after IMP Injection/PET) ....... 48
10.5.
Visit 4 – SoC Biopsy/Neurosurgical Intervention (if applicable, pre-
planned 1 to 21 days after IMP Injection/PET) ....................................... 48
10.6. 
Visit 5 – Month 6 Follow-up (183 days ± 14 days) ................................. 49
10.7.
Early Withdrawal.....................................................................................49
11. 
IMAGING PROTOCOL ......................................................................... 51
11.1. 
18F-fluciclovine PET Scan Patient Preparation........................................ 51
11.2. 
PET/CT Scanner...................................................................................... 51
11.3. 
18F-fluciclovine Injection Administration................................................ 51
11.4. 
18F-fluciclovine PET Acquisition ............................................................51
11.5. 
On-Study MRI Acquisition...................................................................... 51
11.6. 
Image Transfer ......................................................................................... 51
11.7. 
Image Evaluability ................................................................................... 51
11.8. 
Central Evaluations.................................................................................. 51


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11.8.1.
Central Histopathology ............................................................................ 51
11.8.2.
Central Truth Panel .................................................................................. 52
11.8.3.
Blinded Image Evaluation .......................................................................52
11.8.4.
Central Neuroradiologist Reader .............................................................52
12. 
ADVERSE EVENT REPORTING AND DOCUMENTATION............ 53
12.1. 
Adverse Events ........................................................................................ 53
12.2. 
AE Severity.............................................................................................. 53
12.3.
Adverse Event Relationship to IMP ........................................................54
12.4.
Serious Adverse Event (SAE) .................................................................54
12.4.1.
Serious Adverse Event Reporting ............................................................ 54
12.4.2.
Suspected Unexpected Serious Adverse Reaction (SUSAR).................. 55
12.4.3.
Pregnancy ................................................................................................ 55
13.
DISCONTINUATION AND REPLACEMENT OF SUBJECTS ..........56
13.1.
Discontinuation/Withdrawal from the Study...........................................56
13.2. 
Replacement of Subjects.......................................................................... 56
13.3. 
Lost to Follow-up .................................................................................... 56
14. 
PROTOCOL VIOLATIONS................................................................... 58
15. 
STATISTICAL METHODS AND CONSIDERATIONS ...................... 59
15.1. 
Estimation of Sample Size....................................................................... 59
15.2. 
Analysis Sets............................................................................................ 59
15.3. 
Subject Disposition.................................................................................. 59
15.4.
Demographic and Baseline Characteristics .............................................59
15.5. 
Analysis of Primary Endpoint ................................................................. 60
15.6.
Analysis of Secondary Endpoint(s) .........................................................60
15.6.1.
Subject-Level Positive Predictive Value / Negative Predictive Value .... 60
15.6.2.
Lesion-Level Diagnostic Performance ....................................................60
15.6.3.
Subgroup Analysis of Diagnostic Performance Data.............................. 60
15.6.4.
Clinical Utility ......................................................................................... 60
15.6.5.
Inter-Reader / Intra-Reader Agreement ................................................... 61
15.6.6.
Safety ....................................................................................................... 61
15.7. 
Planned Interim Analysis......................................................................... 61
16. 
DATA COLLECTION, RETENTION AND MONITORING ............... 62
16.1. 
Data Collection Instruments ....................................................................62
16.2. 
Data Management Procedures ................................................................. 62
16.3. 
Data Quality Control and Reporting ........................................................ 62


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16.4. 
Archiving of Data .................................................................................... 63
16.5. 
Availability and Retention of Investigational Records ............................ 63
16.6.
Monitoring and Auditing.........................................................................63
16.7.
Subject Confidentiality............................................................................63
17. 
REGULATORY, ETHICAL AND STUDY OVERSIGHT 
CONSIDERATIONS............................................................................... 64
17.1. 
Institutional Review Boards and Independent Ethics Committees..........64
17.2.
Amendments............................................................................................64
17.3.
Subject Information and Consent/Assent ................................................64
17.4. 
Post-trial care ........................................................................................... 65
17.5. 
Publications.............................................................................................. 65
17.6. 
Investigator Responsibilities.................................................................... 66
18.
REFERENCES ........................................................................................68
APPENDIX 1.
ECOG PERFORMANCE STATUS................................................72
APPENDIX 2. PROTOCOL AGREEMENT .......................................................... 73
LIST OF TABLES 
Table 1. Schedule of Study Procedures ...................................................................... 39
Table 2. CTCAE (V5) AE Severity Grading .............................................................. 53
LIST OF FIGURES 
Figure 1. Schema of Study Design ............................................................................. 20


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LIST OF ABBREVIATIONS 
AE
Adverse event 
ALARA 
As low as reasonably achievable 
ASCT2 
Alanine-serine-cysteine-transporter 2 
BED 
Blue Earth Diagnostics, Ltd
BIE 
Blinded image evaluation 
BTIP-BM 
Brain tumor imaging protocol for clinical trials in brain 
metastases  
CBP 
Child-bearing potential 
CE 
Contrast enhancement 
CI 
Confidence interval 
CLR 
Complete lesion response 
CT 
Computerized tomography 
CTCAE 
Common Terminology Criteria for Adverse Events  
CTP 
Central Truth Panel 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic Case Report form 
EU 
European Union 
18F
Isotope of fluorine
FACBC 
18F-labeled 1-amino-3-fluorocyclobutane-1-carboxylic acid 
FAS
Full Analysis Set
FDA 
Food and Drug Administration 
FDG 
Fluorodeoxyglucose 
18F-FDOPA 
18F-fluoro-dihydroxyphenylalanine 
18F-FET
18F-fluoro-ethyltyrosine
FLAIR 
Fluid-attenuated inversion recovery 
GCP 
Good Clinical Practice 


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HCG 
Human chorionic gonadotropin 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
ICF 
Informed consent form
ICH 
International Council for Harmonisation 
IEC 
Independent Ethics Committee 
IIT 
Investigator-initiated trial 
IMP 
Investigational medicinal product 
IRB 
Institutional Review Board 
IV 
Intravenous 
LAR 
Legally Authorized Representative 
LAT1 
L-type amino acid transporter 1 
LITT 
Laser interstitial thermal therapy  
MBq 
MegaBecquerel 
mCi 
Millicurie 
MedDRA 
Medical Dictionary for Regulatory Activities 
MR 
Magnetic resonance 
MRI 
Magnetic resonance imaging 
NBTS 
National Brain Tumor Society 
NCCN 
National Comprehensive Cancer Network 
NCI 
National Cancer Institute 
NMP 
Nihon Medi-Physics Co., Ltd 
NPA 
Negative percent agreement 
NPV 
Negative predictive value 
PET 
Positron emission tomography 
PPA 
Positive percent agreement 
PPV 
Positive predictive value 


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PT 
Preferred term
RANO 
Response Assessment in Neuro-Oncology 
SAE 
Serious adverse event
SAP 
Statistical Analysis Plan
SAF 
Safety Analysis Set 
SoC 
Standard of Care 
SoR 
Standard of Reference 
SoT 
Standard of Truth 
SRS 
Stereotactic radiosurgery 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TEAE 
Treatment-emergent adverse event 
USA 
United States of America 


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PROTOCOL SYNOPSIS 
Study Title
An open-label, single-arm, single-dose, prospective, multicenter 
Phase 3 study to establish the diagnostic performance of 
18F-fluciclovine positron emission tomography (PET) in 
detecting recurrent brain metastases after radiation therapy 
(REVELATE)
Phase 
3
Sponsor 
Blue Earth Diagnostics, Ltd 
Funding Organization
Blue Earth Diagnostics, Ltd 
Study Design 
This is a prospective, open-label, single-arm, single-dose study 
in subjects with solid tumor brain metastases previously treated 
with radiation therapy, designed to establish the diagnostic 
performance of 18F-fluciclovine PET (read with standard 
magnetic resonance imaging [MRI] for anatomical reference) in 
detecting recurrent brain metastases where MRI is equivocal.
Number of Subjects 
Approximately 150 subjects will be enrolled, to obtain 
130 evaluable subjects. 
Study Rationale 
Brain metastases are the most common intracranial tumor in 
adults, occurring in up to 40% of patients with cancer 
(Suh, 2010; Sinha, 2017), with approximately 200,000 patients 
affected each year in the United States of America (USA) 
(Arvold, 2016). Following localized treatment of brain 
metastases (increasingly, stereotactic radiosurgery [SRS] 
alongside neurosurgical resection [Kann, 2017; NCCN, 2019]), 
close follow-up with serial MRI of the brain is performed to 
evaluate for recurrent disease. Conventional MRI is currently 
recommended as the main imaging test (NCCN, 2019) following 
localized treatment, as it is widely available and offers high 
spatial resolution, with presence of recurrent disease suggested 
by increased contrast enhancement (CE) depicting 
anatomical/structural information. However, conventional MRI 
(CE-T1 and fluid-attenuated inversion recovery [FLAIR]/T2-
weighted sequences) has limited specificity due to the incidence 
of treatment-related changes, primarily radiation necrosis 
(Galldiks, 2019). These treatment-related changes have similar 
appearances to true recurrence of disease on conventional MRI, 
including CE, origin near the primary tumor site, vasogenic 
edema, growth over time, and mass effect (Langen, 2017; 
Langen, 2018; Pope, 2018). 
No specific feature or combination of features on conventional 
MRI has been established to differentiate between disease 
recurrence and treatment-related changes including radiation 
necrosis and pseudoprogression (Verma, 2013). Specificity of 
conventional MRI to diagnose recurrent tumor after SRS using 




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confirmed and it is approved for clinical use in this condition. 
18F-fluciclovine is also reported to have utility in the evaluation 
of primary and metastatic cancers in the brain due to its low 
normal brain background uptake and increased uptake by brain 
tumors (Parent, 2018; Patel, 2018; Tsuyuguchi, 2017). In 
particular, one study performed specifically in subjects with 
previously treated brain metastases and an MRI indeterminate 
for recurrence reported increased 18F-fluciclovine uptake in all 
lesions with recurrence, and low 18F-fluciclovine uptake in 
lesions with treatment-related change (Patel, 2018). However, 
the datasets of this study were too small to perform robust 
characterization of diagnostic performance nor inform image 
interpretation criteria. A separate Phase 2b study 
(BED-FLC-219) is being conducted by Blue Earth Diagnostics, 
Ltd to establish image interpretation criteria.  
18F-fluciclovine has been demonstrated to be transported by 
amino acid transporters alanine-serine-cysteine-transporter 2 
(ASCT2) and L-type amino acid transporter 1 (LAT1), which 
are high-affinity glutamine and leucine transporters, respectively 
(Schuster, 2014). Given the known increased expression of 
ASCT2 and LAT1 in many tumor types (including the 3 most
common sources of brain metastases: lung, breast and skin 
cancer) (Fuchs, 2005), and aforementioned early clinical study 
data, it is anticipated that PET imaging with 18F-fluciclovine will 
be useful in detecting recurrent brain metastases. 
Throughout the present Phase 3 study, 18F-fluciclovine PET will 
be read with standard MRI for anatomical reference. Standard 
MRI sequences performed on study will include contrast 
enhanced T1-weighted, T2-weighted/FLAIR and diffusion-
weighted imaging (Kaufmann, 2020).
Primary Objective 
and Outcome 
Measure
Objective: 
1. To assess the negative 
percent agreement 
(NPA) and positive 
percent agreement 
(PPA) of 
18F-fluciclovine PET in 
detecting recurrent 
brain metastases on a 
subject-level. 
Outcome Measure: 
1. Subject-level NPA and 
PPA (equivalent to 
specificity and 
sensitivity, 
respectively) of 
18F-fluciclovine PET in 
detecting recurrent 
brain metastases.  
Secondary Objectives 
and Outcome 
Measures 
Objectives:
1. To assess other 
diagnostic performance 
parameters of 
18F-fluciclovine PET in 
detecting recurrent 
Outcome Measures: 
1. Subject-level positive 
predictive value (PPV) 
and negative predictive 
value (NPV) of 
18F-fluciclovine PET 


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brain metastases on a 
subject-level.  
for detecting recurrent 
brain metastases.  
2. To assess lesion-level 
diagnostic performance 
of 18F-fluciclovine PET 
in detecting recurrent 
brain metastases.
2. Lesion-level PPA, 
NPA, PPV and NPV of 
18F-fluciclovine PET 
for detecting recurrent 
brain metastases. 
3. To assess subject-level 
diagnostic performance 
of 18F-fluciclovine PET 
in detecting recurrent 
brain metastases in 
different clinical 
settings.  
3. Sub-group analyses of 
subject-level PPA, 
NPA, PPV, and NPV 
of 18F-fluciclovine 
PET, according to 
primary tumor type and 
concurrent 
immunotherapy. 
4. To evaluate the added 
clinical usefulness of 
18F-fluciclovine PET in 
evaluation of subjects 
with suspected 
recurrent brain 
metastases.
4. Clinical usefulness:
(a) Number of days taken 
by the site to establish 
presence/absence of 
metastasis by clinical 
follow-up.  
(b) Proportion of subjects 
with additional 
metastases identified 
on 18F-fluciclovine 
PET in addition to 
standard of care brain 
MRI. 
(c) Proportion of subjects 
whose prospective 
diagnostic management 
plan changes following 
18F-fluciclovine PET. 
5. To establish inter- and 
intra-reader 
reproducibility of 
18F-fluciclovine PET 
image interpretation for 
detecting recurrent 
brain metastases.
5. Inter-reader and intra-
reader agreement 
statistics (kappa 
coefficient).  
 
6. To assess the safety of 
18F-fluciclovine 
injection in the subject 
population.
6. Treatment-emergent 
adverse events 
(TEAEs) following 
18F-fluciclovine 
injection in the subject 
population.


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Safety Evaluations 
Safety will be assessed from the time of 18F-fluciclovine 
administration until 1 day post 18F-fluciclovine administration 
based on reported serious adverse events (SAEs) and non-
serious adverse events (AEs).
Study Sites
Approximately 18 sites / US Only
Investigational 
Medicinal Product(s), 
including Control 
Products 
Investigational Medicinal Product:  
18F-fluciclovine injection, 185 MBq (5 mCi) ± 20%, delivered as 
an intravenous bolus
Control Product: Not applicable.
Study and Subject 
Duration 
Study Duration: Approximately 20 months
Subject Duration: 7 months per subject (1 day for AE 
monitoring following 18F-fluciclovine PET scan) 
Subject Population 
Subjects with solid tumor brain metastases previously treated 
with radiation therapy who meet the inclusion and exclusion 
criteria will be eligible for participation in this study.   
Subject Selection 
Inclusion Criteria: 
1. 
 
2. Eastern Cooperative Oncology Group (ECOG) 
performance status of 0-2. 
3. Subject or subject’s Legally Authorized Representative 
(LAR) is willing and able to provide written informed 
consent. 
4. Previous history of solid tumor brain metastasis of any 
origin. 
5. Histopathological confirmation of the primary solid 
tumor or a metastatic site within 4 years of Screening 
(Visit 1). Cancer of unknown primary is excluded. 
6. Previous radiation therapy of brain metastatic lesion(s) 
completed at least 8 weeks before Screening. Form of 
radiotherapy may include stereotactic radiosurgery or 
whole brain radiotherapy, previously delivered as 
primary (initial) treatment, in the adjuvant setting (e.g., 
post-surgery), salvage setting (repeat radiation therapy), 
in or out of the context of previous or concurrent 
systemic treatments. 
7. A reference lesion considered by the site investigator to 
be equivocal for recurrent brain metastasis, as 
determined by a recent* standard of care (SoC) MRI 
brain scan**, measuring 
 mm in longest diameter on 
contrast-enhanced MRI, and meeting one of the 
following radiological criteria: 
a.
reference lesion on contrast-enhanced MRI relative to 




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chemotherapy, anti-estrogens, or ovarian 
suppression. 
10. All subjects, male and female, who are not surgically 
sterilized or postmenopausal as defined above, must 
agree to abstain from sexual conduct for 24 hours 
post-18F-fluciclovine injection.
Exclusion Criteria: 
1. Pregnant or breastfeeding during participation in the 
study, or, where applicable, unwilling to abstain from 
sexual conduct for 24 hours post-18F-fluciclovine 
injection.
2. Subjects with any medical condition or circumstance that 
the investigator believes may confound the data 
collected. 
3. Subjects with active hematological malignancy or cancer 
of unknown primary. 
4. Subject has received, or is scheduled to receive, another 
investigational medicinal product (IMP) from 1 month or 
within 5 half-lives of the other IMP (whichever is 
shorter) before 18F-fluciclovine injection to the 
completion of Visit 3.* 
*
Subjects receiving concurrent systemic therapies, 
including immunotherapies, are permitted for study 
entry as long as the concurrent therapy is not an 
IMP. 
5. Known contraindications to a contrast-enhanced MRI 
procedure. 
Planned Interim 
Analyses
Not applicable.
Study Definitions of 
Term(s) 
Reference Lesion
The reference lesion is defined as the lesion which is:  
a.
Equivocal for recurrent metastasis on SoC MRI 
(according to inclusion criterion #7).  
b. Intended for SoC biopsy/neurosurgical intervention 
procedure (if planned). If >1 lesion is intended for 
biopsy/intervention, the largest of these lesions will 
be the reference lesion. 
c.
Where clinical follow-up is planned, the largest of all 
equivocal lesions (if >1 is present) will be the 
reference lesion.  


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If >1 equivocal lesion is under evaluation as SoC, the on-site 
investigator will also annotate ‘other equivocal lesions’ on the 
pre-study SoC MRI brain scan, defined as follows: 
a.
Equivocal for recurrent metastasis on SoC MRI 
(according to the same radiological criteria for a 
reference lesion per inclusion criterion #7). 
b. Where SoC biopsy/neurosurgical intervention 
procedure is planned, these are the other equivocal 
lesions pre-planned for biopsy/intervention. 
c.
Where SoC clinical follow-up is planned, these are 
the other equivocal lesions pre-planned for SoC 
clinical follow-up 
The on-site investigator will also review the 18F-fluciclovine 
PET scan to identify potential additional lesions, not previously 
reported on SoC MRI (termed ‘additional PET lesions’). 
Additional PET lesions should be annotated only where judged 
suggestive of brain metastasis, warranting confirmation by 
clinical follow-up, biopsy or resection, according to SoC 
practice. Undertaking biopsy/resection of additional PET 
lesion(s) identified on the 18F-fluciclovine PET scan is at the 
discretion of on-site investigators.  
STATISTICS 
Primary Analysis 
Plan 
Analysis of Primary Endpoint 
The primary endpoint for the study is subject-level NPA and 
PPA (equivalent to specificity and sensitivity, respectively) of 
18F-fluciclovine PET in detecting recurrent brain metastases. 
Subject-level NPA and PPA will be calculated based on blinded 
image evaluation (BIE) of 18F-fluciclovine PET compared to 
standard of reference (SoR) on the reference lesion. 
Where NPA0 and PPA0 are performance goals for NPA and 
PPA, respectively, performance goals of 50% (NPA0) for NPA 
and 50% (PPA0) for PPA were selected as acceptable thresholds 
given the setting where SoC imaging is already equivocal. The 
analyses for NPA and PPA will be performed using the one-
sample binomial exact test. In addition to the rates, exact two-
sided 95% confidence intervals (CIs) according to the 
Clopper-Pearson method will also be provided. If the pre-
defined NPA and PPA goals are met by the same two of three 
readers (both tests reach statistical significance for the same two 
readers), the study will be considered to have successfully 
demonstrated the effectiveness of 18F-fluciclovine PET in 
detecting recurrent brain metastases. 
Analysis of Secondary Endpoints 
Subject-level PPV and NPV of 18F-fluciclovine PET for 
detecting recurrent brain metastases will be calculated based on 
BIE of 18F-fluciclovine PET compared to SoR on the reference 




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will be presented for the initial read vs re-read of a subset of 
PET scans for each reader. 
Analysis of Safety
Safety will be assessed from the time of 18F-fluciclovine 
administration until 1 day post-18F-fluciclovine administration 
based on reported serious and non-serious adverse events (SAEs 
and AEs, respectively).  
For the evaluation of safety, TEAEs are defined as those AEs 
with onset after the dose of IMP, or existing events that 
worsened after the dose of IMP during the study, up to 1 day 
following IMP administration. Verbatim terms of the AEs will 
be coded to preferred terms (PTs) and system organ classes 
(SOCs) using the latest version of Medical Dictionary for 
Regulatory Activities (MedDRA), for data summary purposes. 
Severity of AEs will be graded using the NCI-CTCAE v5.0. The 
frequency of TEAEs will be summarized overall, by SOC and 
PT, and by severity. IMP-related AEs and SAEs will also be 
summarized.
Rationale for Number 
of Subjects 
A one-sample binomial exact test will be used to test the 
following hypotheses in NPA and PPA of 18F-fluciclovine PET: 
NPA
 H0(NPA
0
 H1(NPA): NPA of PET >NPA0
PPA
H0(PPA
0
 H1(PPA): PPA of PET >PPA0
Where NPA0 and PPA0 are performance goals for NPA and 
PPA, respectively.  
Performance goals of 50% (NPA0) for NPA and 50% (PPA0) for 
PPA were selected as acceptable thresholds given the setting 
where SoC imaging is already equivocal. The lower limits for 
the exact 95% CIs (Clopper-Pearson method) for both NPA and 
PPA should be greater than 50%. Assuming an NPA of at least 
70% and a PPA of at least 70% for 18F-fluciclovine PET would 
need to be observed, a sample size of 130 would achieve 90% 
power at a 1-sided significance level of 0.025 to reject the 
performance goals for both NPA and PPA, assuming a brain 
metastasis prevalence rate of 0.5. 


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STUDY SCHEMA 
Figure 1. Schema of Study Design 
*As applicable; Visit 4 may not apply to all subjects.  
IMP, investigational medicinal product; MRI, magnetic resonance imaging; PET, positron emission 
tomography; SoC, standard of care. 
Prior to 
Enrollment 
SoC MRI
Visit 1: Screening
Day -28 to -1 
0 to 41 days after SoC MRI. 1 to 28 days before Visit 2. 
Obtain informed consent. Conduct Screening assessments, 
including inclusion and exclusion criteria; disease history. 
Refer to Table 1, Schedule of Study Procedures 
Visit 2: IMP 
Administration / 
PET
Day 1
1 to 42 days after SoC MRI. 1 to 21 days before planned 
SoC biopsy or neurosurgical intervention  
Administer Investigational Product and Perform PET 
Scan. Refer to Table 1, Schedule of Study Procedures 
Visit 2: 
On-Study MRI 
Day 1 + 0 to 
3 days 
MRI brain (same day as PET or up to 3 days after PET) 
Refer to Table 1, Schedule of Study Procedures 
Visit 3: 
Safety Follow-up 
Day 2 to 4 
Day 1 + 1 to 3 days. Safety Follow-up contact.
Refer to Table 1, Schedule of Study Procedures 
Visit 4: SoC 
Biopsy / 
Neurosurgical 
Intervention* 
Day 2 to 22 
Pre-planned SoC Neurosurgical Intervention 1 to 21 days 
after Day 1  
Refer to Table 1, Schedule of Study Procedures 
Visit 5: Month 6  
Follow-up 
6 months after Visit 2 18F-fluciclovine PET.  
Follow-up clinical data collection 
Refer to Table 1, Schedule of Study Procedures 




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1.1. 
Overview of Clinical Studies 
Two small investigator-initiated trials (IITs) conducted at Emory University have evaluated 
18F-fluciclovine for PET imaging of brain metastases in adults (Schuster, 2003; Patel, 2018), 
with results suggesting efficacy. Both studies reported increased 18F-fluciclovine uptake in 
brain metastases: one study in 5 subjects with 9 lesions and another in 12 subjects with 
17 lesions. In the larger study, all subjects had prior treatment for brain metastases and had a 
magnetic resonance imaging (MRI) indeterminate for recurrent disease. Using either biopsy 
or clinical follow-up as the standard of truth (SoT), 15/15 18F-fluciclovine positive lesions 
were determined to be recurrent disease. On the other hand, the remaining 2 lesions were 
determined to be radiation necrosis, both of which had low 18F-fluciclovine uptake.
Apart from brain metastases, other studies have also reported the use of 18F-fluciclovine in 
other neuro-oncology settings (Parent, 2018; Karlberg, 2019; Michaud, 2020; 
Bogsrud, 2019). This includes one IIT in suspected recurrent primary brain tumor (N=27) 
(Michaud, 2020), where tumor uptake of 18F-fluciclovine provided significantly higher image 
contrast to 11C-methionine. 18F-fluciclovine, based on a subset of 3 subjects with 
indeterminate MRI, was suggested to be potentially useful in this setting. A retrospective 
analysis of 21 subjects with suspected residual or recurrent high grade glioma 
(Bogsrud, 2019) reported high 18F-fluciclovine uptake in histopathologically / clinically 
confirmed disease, including the detection of small satellite tumors not previously visualized 
with MRI in 4 subjects. A recent report of 2 cases has also described the successful use of 
18F-fluciclovine PET-MRI–guided biopsy in post-treatment glioblastoma to distinguish areas 
of tumor recurrence from areas of predominant treatment effect (Henderson, 2019). Industry 
sponsored studies include 4 studies conducted by Nihon Medi-Physics Co., Ltd (NMP) in 
subjects with treatment-naïve glioma: NMK36-BT-P201 (N=5) (Kondo, 2016), 
MK36-BT-P202 (N=40) (Wakabayashi, 2017), NMK36-BT-P301 and NMK36-BT-P302. 
These studies collectively suggest the potential for 18F-fluciclovine to delineate extent of 
treatment-naïve primary brain tumor beyond contrast-enhancing regions on MRI. 
As a marketed product, 18F-fluciclovine has been administered to more than 75,000 patients. 
Safety data are available from 1203 subjects exposed to 18F-fluciclovine in clinical trials. The 
doses administered range from 51.8 to 485 MBq, with the majority of subjects receiving 
approximately 370 MBq for the imaging of prostate cancer, and approximately 180 MBq for 
glioma in Studies NMK36-BT-P201, NMK36-BT-P202, NMK36-BT-P301 and 
NMK36-BT-P302. No deaths and no serious adverse events (SAEs) attributable to 
18F-fluciclovine injection have been reported. Adverse events (AEs) have generally been mild 
in intensity. Based on the accumulated information, injection site reactions, dysgeusia and 
parosmia are considered to be adverse reactions associated with receipt of 18F-fluciclovine 
injection in humans; these events occurred in 17/1203 (1.4%) subjects overall. 
Based upon preliminary data from the 2 IITs conducted at Emory University (Schuster, 2003; 
Patel, 2018), and supported by experience reported from the collective clinical studies of 
18F-fluciclovine in other brain tumor settings (Parent, 2018; Karlberg, 2019; Michaud, 2020; 
Bogsrud, 2019; Henderson, 2019), BED has a clinical program in development to support the 
use of 18F-fluciclovine PET imaging to detect brain metastases in adults with suspected 
recurrence after radiation therapy. Within this clinical program, BED is conducting a Phase 2 
study (BED-FLC-219) designed to define image interpretation criteria, and the present 
Phase 3 study (BED-FLC-312) designed to establish the diagnostic performance of 
18F-fluciclovine PET (read with standard MRI for anatomical reference) in detecting 
recurrent brain metastases where MRI is equivocal. 


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2. 
STUDY RATIONALE 
Brain metastases are the most common intracranial tumor in adults, occurring in up to 40% of 
patients with cancer (Suh, 2010; Sinha, 2017), with approximately 200,000 patients affected 
each year in the USA (Arvold, 2016). Following localized treatment of brain metastases 
(increasingly, stereotactic radiosurgery [SRS] alongside neurosurgical resection [Kann, 2017; 
NCCN, 2019]), close follow-up with serial MRI of the brain is performed to evaluate for 
recurrent disease. Conventional MRI is currently recommended as the main imaging test 
(NCCN, 2019) following localized treatment, as it is widely available and offers high spatial 
resolution, with presence of recurrent disease suggested by increased contrast enhancement 
(CE) depicting anatomical/structural information. However, conventional MRI (CE-T1 and 
fluid-attenuated inversion recovery [FLAIR]/T2-weighted sequences) has limited specificity 
due to the incidence of treatment-related changes, primarily radiation necrosis 
(Galldiks, 2019). These treatment-related changes have similar appearances to true recurrence 
of disease on conventional MRI, including CE, origin near the primary tumor site, vasogenic 
edema, growth over time, and mass effect (Langen, 2017; Langen, 2018; Pope, 2018). 
No specific feature or combination of features on conventional MRI has been established to 
differentiate between disease recurrence and treatment-related changes, including radiation 
necrosis and pseudoprogression (Verma, 2013). Specificity of conventional MRI to diagnose 
recurrent tumor after SRS using visual reads has been reported to be as low as 19% 
(Peng, 2018), with attempts at validating permutations of neuroradiologist-defined 
measurements reporting specificities of 32% to 41% (Dequesada, 2008; Stockham, 2012). 
Alongside the estimated 25% incidence rate of radiation necrosis (Vellayappan, 2018), rates 
of true local recurrence of disease are similar, ranging from 27% to 31% (Kocher, 2011; 
Brown, 2010). Therefore, the true prevalence of recurrent disease post-radiotherapy where 
conventional MRI indicates the possibility of recurrence, can be estimated to be 
approximately 50%.  
Guidelines and recommendations established by the Response Assessment in 
Neuro-Oncology (RANO) group recognize the limitations of conventional MRI, and in view 
of the lack of substantial evidence, do not yet advocate any advanced imaging technique 
when MRI features are suggestive, but equivocal, for recurrence of brain metastases 
(Wen, 2010; Lin, 2015). The management of patients with MRI findings suggestive, but 
equivocal, for recurrent brain metastases includes clinical monitoring or biopsy sampling 
(Lin, 2015). Close clinical monitoring with repeat imaging may be performed in the hope that 
scan appearance resolves over time, but this is at the expense of delayed therapeutic decision 
making. Further, neurosurgical biopsy sampling is an invasive procedure that carries the 
associated risk of further deteriorating quality-of-life for patients already undergoing 
aggressive treatment (of their primary cancer) (Verma, 2013).
National Comprehensive Cancer Network (NCCN) guidelines list advanced MRI scans 
(magnetic resonance [MR] spectroscopy, MR perfusion) and PET imaging as potentially 
useful techniques in differentiating tumor from treatment effect (NCCN, 2019); however, the 
availability, acquisition, processing, interpretation and, therefore, diagnostic performance of 
advanced MRI scans vary considerably (Zhang, 2017). In addition, the only PET imaging 
agent approved by the US Food and Drug Administration (FDA) for use in brain tumor 
imaging is 18F-fluorodeoxyglucose (FDG), which has notable limitations. Use of 18F-FDG in 
brain tumor imaging is limited in sensitivity and specificity, as differentiation of tumor from 
non-tumorous tissue is often difficult due to the high metabolic rate of normal brain 
parenchyma and inflammatory tissue (Galldiks, 2019). 18F-FDG uptake in low-grade tumors 


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can be similar to that in normal grey matter. The sensitivity of detection of lesions is further 
decreased by the high variance of 18F-FDG uptake and its heterogeneity within a single tumor 
(Heiss, 2014). 
Given this area of great diagnostic unmet need, accurate imaging to differentiate disease 
recurrence from treatment-related changes is valuable for several reasons: 
 Identifying treatment-related changes is important to avoid unneeded treatment 
(e.g., surgery) and erroneously premature termination of potentially effective 
treatment (Walker, 2014). 
 Accurate scans can inform the management decision of cessation of non-effective 
treatments, to minimize morbidity from treatment side effects (Galldiks, 2019) 
and reduce the economic burden. 
 Timely diagnosis of true recurrence will allow prompt stratification of patients to 
further therapies (Galldiks, 2019), which may maximize therapeutic benefit and 
clinical outcome. 
 Given the high morbidity and mortality of patients with brain metastases and, 
therefore, the need for high quality clinical research, such imaging will be pivotal 
in determining suitability for clinical trial entry, and accurate characterization of 
investigational therapeutic efficacy (Lin, 2015). This particular need for certainty 
on optimal clinical trial endpoints has been recognized by the FDA and National 
Brain Tumor Society (NBTS), leading to recent joint efforts to stimulate much 
needed research and development in this area (FDA/NBTS, 2019). 
 Aiding the physician to risk-stratify continuation of a therapeutic regimen (where 
treatment-related changes can be confidently diagnosed). This is of particular 
value in the context of a treatment with a significant side effect profile. 
Limited clinical research has been conducted with other amino acid PET tracers in brain 
metastases, possibly in part due to their highly limited availability in the US. Studies of brain 
metastases with amino acid PET tracers 18F-fluoro-ethyltyrosine (18F-FET), 
18F-fluoro-dihydroxyphenylalanine (18F-FDOPA) and 11C-methionine have all been 
described, with demonstration of increased tracer uptake and sensitivities of 79% to 95%, 
specificities of 84% to 93% for the detection of brain metastases where MRI is suggestive of 
recurrent disease (Terakawa, 2008; Galldiks, 2012; Lizarraga, 2014; Cicone, 2015; 
Minamimoto, 2015; Romagna, 2016; Ceccon, 2017). In recent recommendations by the 
RANO working group, amino acid PET was recommended as useful in distinguishing 
post-therapeutic reactive changes following radiotherapy from recurrent brain metastases 
(Galldiks, 2019). This was based on review of Level 2 evidence on 18F-FET, 18F-FDOPA and 
11C-methionine. Transport mechanisms of these tracers have overlap with 18F-fluciclovine, in 
that these are predominantly LAT1-mediated. Furthermore, known cross-affinity for various 
amino acids between the 2 transporters support the expectation for 18F-fluciclovine to 
demonstrate comparable performance in brain metastases, akin to other amino acid PET 
tracers and known 18F-fluciclovine imaging characteristics in glioma. Thus, the investigation 
of 18F-fluciclovine in the imaging of brain metastases is of considerable clinical relevance, 
particularly when considering promising results from the aforementioned IITs of 
18F-fluciclovine in brain metastases.
Amongst the previously cited studies using 18F-FET, 18F-FDOPA and 11C-methionine, 
different interpretation criteria for optimum detection of disease recurrence have been 
reported. Furthermore, these criteria have been based on different methods of quantifying 


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PET tracer uptake, and in the case of 18F-FET, incorporate the interpretation of dynamic scan 
data (time-activity curves). Therefore, existing heterogeneous data on optimum image 
interpretation criteria in other amino acid PET tracers cannot be directly applied to 
18F-fluciclovine. Within this clinical program, BED is conducting a Phase 2 study 
(BED-FLC-219) designed to define image interpretation criteria.
18F-fluciclovine has a well-established safety profile and a wide distribution network in the 
US where it is routinely used as standard of care (SoC) for imaging in biochemical recurrence
of prostate cancer.
Throughout this study, 18F-fluciclovine PET will be read with standard MRI for anatomical 
reference. Standard MRI sequences performed on study will include contrast enhanced 
T1-weighted, T2-weighted/ FLAIR and diffusion-weighted imaging (Kaufmann, 2020).
The purpose of the current study is to establish the diagnostic performance of 18F-fluciclovine 
PET (read with standard MRI for anatomical reference) in detecting recurrent brain 
metastases where MRI is equivocal.
2.1. 
Risk-Benefit Assessment 
2.1.1. 
Benefit
The 18F-fluciclovine PET scans may provide further clinical information regarding the 
subject’s disease status that may not have been appreciated using other standard of care tests. 
If such information arises, this may be considered by the responsible clinician to help direct 
the subject’s management, including guiding any planned neurosurgical intervention. This 
may provide a direct benefit to the subject.
2.1.2.
Risk
The risks from the imaging studies to subjects mainly relate to the intravenous (IV) injection 
and the radiation emitted by the radiopharmaceutical and the computerized tomography (CT) 
transmission scan (when the PET scan is acquired on a PET/CT scanner). There is a potential 
risk of a hypersensitivity reaction requiring the availability of resuscitative equipment and 
personnel. Intravenous injection carries a small risk of infection and hematoma.
The mean effective dose per unit administered activity of 18F-fluciclovine is 22.1
(McParland, 2013). An administered activity of 5 mCi (185 MBq) will result in an effective 
dose of 4.1 mSv. If PET/CT is used, the effective dose due to CT acquisition should be in 
accordance with ALARA (as low as reasonably achievable) principles. The maximum 
effective dose due to a CT transmission scan will vary from site to site, but as a guide, a dose 
ranging from 0.5 mSv to 2.0 mSv would be expected. Combined with the effective dose from 
18F-fluciclovine (4.1 mSv), the total effective dose of the 18F-fluciclovine PET-CT scan of 
6.1 mSv is in line with other common brain nuclear medicine procedures.  
Due to the risk of radiation exposure, women who are either pregnant or breast feeding are 
excluded from participation. All subjects, male and female, who are not surgically sterilized 
or postmenopausal, must agree to abstain from sexual conduct for 24 hours 
post-18F-fluciclovine injection.   
Image interpretation errors can occur with 18F-fluciclovine PET brain imaging. Physiological
distribution of 18F-fluciclovine does not rule out the presence of recurrent metastasis and 
18F-fluciclovine uptake within a lesion does not confirm the presence of recurrent metastasis. 
18F-fluciclovine uptake is not specific for brain metastases and may occur with other types of 


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malignancy in or around the brain. There may be uptake in other conditions such as active 
multiple sclerosis, brain abscess, and meningioma. Accordingly, as the responsible clinician 
may consider information from the 18F-fluciclovine PET scan to direct the patient’s 
management, there is a potential risk of additional resections/biopsies including those that are 
ultimately unnecessary. 


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3. 
STUDY OBJECTIVES 
3.1. 
Primary Objective 
The primary objective is:
1. To assess the negative percent agreement (NPA) and positive percent agreement 
(PPA) of 18F-fluciclovine PET in detecting recurrent brain metastases on a 
subject-level.
3.2. 
Secondary Objectives 
The secondary objectives are:
1. To assess other diagnostic performance parameters of 18F-fluciclovine PET in 
detecting recurrent brain metastases on a subject-level. 
2. To assess lesion level diagnostic performance of 18F-fluciclovine PET in detecting 
recurrent brain metastases.
3. To assess subject-level diagnostic performance of 18F-fluciclovine PET in detecting 
recurrent brain metastases in different clinical settings. 
4. To evaluate the added clinical usefulness of 18F-fluciclovine PET in evaluation of 
subjects with suspected recurrent brain metastases. 
5. To establish inter- and intra-reader reproducibility of 18F-fluciclovine PET image 
interpretation for detecting recurrent brain metastases. 
6. To assess the safety of 18F-fluciclovine injection in the subject population. 


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4. 
STUDY DESIGN 
4.1. 
Study Overview 
This is a prospective, open label, single arm, single dose study in subjects with solid tumor 
brain metastases previously treated with radiation therapy, designed to establish the 
diagnostic performance of 18F-fluciclovine PET (read with standard MRI for anatomical 
reference) in detecting recurrent brain metastases where MRI is equivocal.
Subjects with a history of brain metastases previously treated with primary, adjuvant or 
repeat (salvage) radiation therapy, with a recent SoC brain MRI found to be equivocal for 
recurrent brain metastasis, and who meet all inclusion criteria and none of the exclusion 
criteria, will be consented and enrolled. The study population will be balanced to represent 
different primary tumor types of any origin, with a minimum of approximately 15% of the 
cohort each with lung cancer, breast cancer and melanoma. The number of subjects with lung 
cancer will be capped at approximately 50% of enrolled subjects.  
Investigator will prepare a diagnostic management plan prior to the 18F-fluciclovine PET scan 
and will update the plan following the scan. All eligible subjects will receive an 
18F-fluciclovine PET scan (Visit 2 PET) within 42 days of SoC MRI. Subjects will then 
undergo a repeat study-specific brain MRI scan (Visit 2 On-Study MRI) to be used for 
anatomical reference, ideally on the same day, otherwise 
 days after Visit 2 PET and 
completed before any pre-planned neurosurgical intervention when applicable. A Safety 
Follow-up (Visit 3) 1 to 3 days after IMP injection/PET (Visit 2 PET) will be made for AE 
evaluation by telephone call or in person (if same day as other scheduled SoC appointments). 
AEs occurring from the time of 18F-fluciclovine administration until 1 day post-
18F-fluciclovine administration will be recorded. The Safety Follow-up must be completed 
before the pre-planned SoC biopsy/neurosurgical intervention, if applicable. Subjects who 
experience an SAE or an AE that persists at Visit 3, will be followed until resolution or 
stabilization of these events. All ongoing follow-up and any further treatment will be in 
accordance with SoC.
As MRI is not specific for tumor recurrence in this setting, current clinical practice is mainly 
focused on close clinical follow-up to determine the presence of recurrence. Alternatively, 
biopsy or direct neurosurgical intervention of the abnormality may be performed. This study 
will utilize all available diagnostic and follow-up clinical data to establish the underlying 
diagnosis.  The planned diagnostic method will be recorded at Screening and assessed for any 
changes following 18F-fluciclovine PET scan.
If biopsy/neurosurgical intervention has been pre-planned per SoC, the Visit 2 PET will be 
organized to take place a minimum of 1 day and maximum of 21 days before pre-planned 
SoC biopsy or neurosurgical intervention. 
On-site investigators will prospectively annotate and measure the ‘reference lesion’ on a 
post-radiation treatment MRI scan and on the pre-study SoC MRI scan to confirm eligibility.  
The ‘reference lesion’ is defined as the lesion which is: 
a. Equivocal for recurrent metastasis on SoC MRI (according to inclusion criterion 
#7, see Section 6.2).  
b. Intended for SoC biopsy/neurosurgical intervention procedure (if planned). If 
>1 lesion is intended for biopsy/intervention, the largest of these lesions will be 
the reference lesion.  


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c.
Where clinical follow-up is planned, the largest of all equivocal lesions (if >1 is 
present) will be the reference lesion.  
If >1 equivocal lesion is under evaluation as SoC, the on-site investigator will also annotate 
‘other equivocal lesions’ on the pre-study SoC MRI scan, defined as follows:
a. Equivocal for recurrent metastasis on SoC MRI (according to the same 
radiological criteria for a reference lesion per inclusion criterion #7, see 
Section 6.2). 
b. Where SoC biopsy/neurosurgical intervention procedure is planned, these are the 
other equivocal lesions pre-planned for biopsy/intervention.
c. Where SoC clinical follow-up is planned, these are the other equivocal lesions 
pre-planned for SoC clinical follow-up. 
The on-site investigator will also review the 18F-fluciclovine PET scan to identify potential 
additional lesions, not previously reported on SoC MRI (termed ‘additional PET lesions’). 
Additional PET lesions should be annotated only where judged suggestive of brain 
metastasis, warranting confirmation by clinical follow-up, biopsy or resection, according to 
SoC practice. Undertaking biopsy/resection of additional PET lesion(s) identified on the 
18F-fluciclovine PET scan is at the discretion of on-site investigators. Moreover, an additional 
PET lesion should be discrete and spatially separate from known, pre-existing lesions. In 
particular, the following should not be annotated as Additional PET lesions: known pre-
existing non-progressive lesions (considered at site level as stable or partially responding), 
18F-fluciclovine activity contiguous with a known lesion (previously annotated or otherwise) 
and apparently incongruent with the lesion’s outline on correlating MRI, particularly where 
this may be reasonably explained by image registration.
For subjects due for pre-planned SoC biopsy/neurosurgical intervention, the procedure will 
be at Visit 4. The subject will receive post-procedural management per institutional SoC. All 
ongoing follow-up and any further treatment will be in accordance with SoC. Samples 
obtained during this Visit 4 will be subject to central histopathological evaluation.    
As with subjects who undergo pre-planned SoC biopsy/neurosurgical intervention, subjects 
pre-planned for clinical follow-up to evaluate the equivocal lesions will undergo follow-up 
performed as SoC per local practice. This is anticipated to vary between subjects.  
The final visit for each subject, including subjects who had undergone pre-planned SoC 
biopsy/neurosurgical intervention, will usually be the Month-6 Follow-up at Visit 5. For 
study purposes, clinical data accrued during SoC clinical follow-up, pertaining to the ongoing 
assessment, eventual confirmation of diagnosis, and treatment of the lesions, will be 
collected. The data will comprise brain imaging scans, treatment regimens, histopathological 
reports and mortality status. The period of data accrual will be up to 6 months following 
18F-fluciclovine PET, or death, whichever comes first. Subjects still considered equivocal at 
site level at Month 6 (Visit 5) will undergo a study MRI. 
With the exception of PET scans, the clinical follow-up data and, where applicable, central 
histopathology results, will be reviewed by a Central Truth Panel (CTP) to assign a final
diagnosis to each lesion, and form the Standard of Reference (SoR). Separately, 
18F-fluciclovine PET scans will be centrally read in a blinded image evaluation (BIE), using 
image interpretation criteria established from the separate Phase 2 study (BED-FLC-219). 
Diagnostic performance primary and secondary endpoints will then be derived from BIE scan 
reads matched against SoR. Subject-level SoR will be defined by CTP diagnosis of the 


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reference lesion. Lesion-level SoT will be defined by CTP diagnosis of all lesions (reference 
lesions, other equivocal lesions, additional PET lesions). 


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5. 
CRITERIA FOR EVALUATION 
5.1. 
Primary Efficacy Endpoint 
1. Subject-level NPA and PPA (equivalent to specificity and sensitivity, respectively) of 
18F-fluciclovine PET in detecting recurrent brain metastases.
5.2.
Secondary Efficacy Endpoints
1. Subject-level positive predictive value (PPV) and negative predictive value (NPV) of 
18F-fluciclovine PET for detecting recurrent brain metastases.
2. Lesion-level PPA, NPA, PPV and NPV of 18F-fluciclovine PET for detecting 
recurrent brain metastases.
3. Sub-group analyses of subject-level PPA, NPA, PPV and NPV of 18F-fluciclovine 
PET, according to primary tumor type and concurrent immunotherapy. 
4. Clinical usefulness: 
a. Number of days taken by the site to establish presence/absence of metastasis by 
clinical follow-up. 
b. Proportion of subjects with additional metastases identified on 18F-fluciclovine 
PET in addition to standard of care brain MRI.
c. Proportion of subjects whose prospective diagnostic management plan changes 
following 18F-fluciclovine PET. 
5. Inter-reader and intra-reader agreement statistics (kappa coefficient). 
6. Treatment-emergent adverse events following 18F-fluciclovine injection in the subject 
population.
5.3. 
Safety Evaluations 
Safety will be assessed from the time of 18F-fluciclovine administration until 1 day 
post-18F-fluciclovine administration based on reported serious and non-serious AEs (SAEs 
and AEs, respectively). 


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6. 
SUBJECT SELECTION 
6.1. 
Study Population 
Subjects with solid tumor brain metastases previously treated with radiation therapy who 
meet the inclusion and exclusion criteria will be eligible for participation in this study.  
6.2.
Inclusion Criteria
1. 
 
2. Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. 
3. Subject or subject’s Legally Authorized Representative (LAR) is willing and able to 
provide written informed consent. 
4. Previous history of solid tumor brain metastasis of any origin. 
5. Histopathological confirmation of the primary solid tumor or a metastatic site within 
4 years of Screening (Visit 1). Cancer of unknown primary is excluded. 
6. Previous radiation therapy of brain metastatic lesion(s) completed at least 8 weeks 
before Screening. Form of radiotherapy may include stereotactic radiosurgery or 
whole brain radiotherapy, previously delivered as primary (initial) treatment, in the 
adjuvant setting (e.g., post-surgery), salvage setting (repeat radiation therapy), in or 
out of the context of previous or concurrent systemic treatments.
7. A reference lesion considered by the site investigator to be equivocal for recurrent 
brain metastasis, as determined by a recent* SoC MRI brain scan**, measuring 
in longest diameter on contrast-enhanced MRI, and meeting one of the following 
radiological criteria: 
a. 
in the longest diameter of the reference lesion on 
contrast-enhanced MRI relative to nadir***, where the longest diameter at nadir is 
10mm
b. 
contrast-enhanced MRI relative to nadir, where the longest diameter at nadir is 
<10mm 
c. a contrast enhancing lesion at site of previous radiotherapy following complete 
lesion response (CLR, defined as disappearance of the irradiated contrast 
enhancing lesion on contrast-enhanced MRI).
*SoC MRI brain scan must be completed within no more than 42 days before study 
PET scan.
**The SoC MRI brain scan must consist of conventional sequences, defined as:
 T1-weighted without and with contrast enhancement, and 
FLAIR and/or T2-weighted.  
If SoC also includes advanced/investigational MRI sequences, including but 
not limited to perfusion, spectroscopy, diffusion weighted imaging, and 
susceptibility weighted imaging, trial entry is permitted as long as the other 
inclusion criteria are met.


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***Nadir is defined as the smallest size after radiotherapy, measured by single longest 
diameter on an available contrast-enhanced MRI.
8. Subject requires further confirmatory diagnostic procedures to confirm brain MRI 
findings and is planned for:
a. Biopsy or neurosurgical intervention (i.e., craniotomy, laser interstitial thermal 
therapy where peri-procedural biopsy is planned) as SoC, or
b. Clinical follow-up as SoC.  
9. Females of child-bearing potential (CBP) to have negative pregnancy test (urine) 
before on-study 18F-fluciclovine PET scan.
Note: Women of CBP are defined as sexually mature women without prior 
hysterectomy or who have had any evidence of menses in the past 12 months. 
However, women who have been amenorrheic for 12 or more months are still 
considered to be of childbearing potential if the amenorrhea is possibly due to 
prior chemotherapy, anti-estrogens, or ovarian suppression. 
10. All subjects, male and female, who are not surgically sterilized or postmenopausal as 
defined above, must agree to abstain from sexual conduct for 24 hours 
post-18F-fluciclovine injection.
6.3. 
Exclusion Criteria 
1. Pregnant or breastfeeding during participation in the study, or, where applicable, 
unwilling to abstain from sexual conduct for 24 hours post-18F-fluciclovine injection.
2. Subjects with any medical condition or circumstance that the investigator believes 
may confound the data collected.
3. Subjects with active hematological malignancy or cancer of unknown primary.
4. Subject has received, or is scheduled to receive, another IMP from 1 month or within 
5 half-lives of the other IMP (whichever is shorter) before 18F-fluciclovine injection to 
the completion of Visit 3.*
*Subjects receiving concurrent systemic therapies, including immunotherapies, are 
permitted for study entry as long as the concurrent therapy is not an IMP. 
5. Known contraindications to a contrast-enhanced MRI procedure.


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7. 
CONCURRENT MEDICATIONS 
All concurrent medications are allowed except for treatments noted in the exclusion criteria 
(Section 6.3) and as noted in the prohibited medications section below. 
7.1.
Allowed Medications and Treatments
Standard therapy for solid tumor brain metastases (e.g., systemic therapies, including 
immunotherapies) is allowed except for treatments noted in the exclusion criteria described 
above and as noted in the prohibited medications section below.
7.2.
Prohibited Medications and Treatments
The following medications are prohibited during the study and administration will be 
considered a protocol violation: 
 any concurrent therapy that is an IMP, prohibited from 1 month or within 5 
half-lives of the other IMP (whichever is shorter) before 18F-fluciclovine injection 
to the completion of Visit 3. 




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8.7. 
Dispensing 
When the study site receives the dose, both prior to and after administration, the activity in 
the syringe will be measured using an appropriate dose calibrator. Should the activity be less 
than 148 MBq (4 mCi) or the volume required exceed 5 mL of undiluted material or the 
administration be after the labeled expiration time, the scan should not be performed.
Additional details for handling the IMP are provided in the Pharmacy Manual. 
8.8.
Administration Instructions 
Position the subject supine, with arms alongside the body and the head stabilized 
appropriately. The entire brain, including the cerebellum, should be in the field of view. A 
venous cannula will be inserted, and the subject will receive an administered activity of 
185 MBq (5 mCi) ± 20% of 18F-fluciclovine. The 18F-fluciclovine will be administered as an 
IV bolus injection followed by a 10 mL flush of normal saline solution.
Additional details are provided in the Imaging Acquisition Standards.  
8.9. 
Storage 
The shelf-life of 18F-fluciclovine injection is up to 10 hours from the end of production and 
the product must not be administered beyond this limit. 18F-fluciclovine should be stored at 
 
All non-radioactive containers (shielding, transport cans) must be returned to the 
manufacturing site. Shipping containers that are radioactive must be decontaminated or 
allowed to decay prior to return to the manufacturing site. Used product syringes and other 
radioactive materials must be destroyed at either the study site or another designated facility. 
Waste must be disposed of according to national and local regulations for radioactive 
material. 
Precautions for the safe handling of radioactive materials should be observed. 
Additional details are provided in the Pharmacy Manual. 
8.10.
Study Drug Accountability
An accurate and current accounting of the dispensing and disposal of study drug for each 
subject will be recorded on the Investigational Drug Accountability Record. These records 
will be maintained at the radiopharmacy, hot lab or nuclear medicine department responsible 
for receipt and dispensation of study drug. Records should include at a minimum:
Dates of receipt, lot number and quantities received from Sponsor or designee;
Dates, subject numbers, and amount of 18F-fluciclovine dispensed for 
administration to specific subjects, including administered dose activity; 
 If applicable, for any unused IMP, dates, lot numbers, and reason dose was not 
administered.
The investigator is responsible for ensuring that study drug is administered only to subjects 
included in this study in accordance with the study protocol. 
Throughout the study, drug accountability will be performed by appropriate Sponsor 
representative(s) and when appropriate, reconciliation will be performed. Additional details 
are provided in the Pharmacy Manual. 


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8.11. 
Measures of Treatment Compliance 
Not Applicable. This is a single dose study, administered by site staff. 
8.12.
End Of Trial
Subjects will be followed to 6 months per SoC. Month-6 Follow-up data abstraction 
(6 months following 18F-fluciclovine PET) will be conducted in order to collect diagnostic, 
treatment, and response information which will be provided to a CTP for review. Subjects 
whose reference or other equivocal lesion(s) continue to be considered equivocal at site level 
at Visit 5 / Month 6 will undergo a study MRI.


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9. 
STUDY PROCEDURES AND GUIDELINES 
A schedule of events representing the required study procedures to be performed for the 
duration of the study is provided in Table 1.  
Prior to conducting any study-related activities, written informed consent (and assent, if 
applicable) including Health Insurance Portability and Accountability Act (HIPAA) 
authorization must be signed and dated by the subject or LAR (Section 17.3).


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Table 1. Schedule of Study Procedures 
 
Day -42 to -1 
SoC MRI 
Visit 1 
Day -28 to -1 
Screening 
Visit 2 
Day 1 
IMP + PET 
Visit 2  
Day 1 to 4 
On-Study MRI 
Visit 3
Day 2 to 4 
Safety  
Follow-Up 
Phone Call
Visit 4 
Day 2 to 22 SoC 
Biopsy / 
Neurosurgical 
Intervention  
(if applicable)
Visit 5
Month 6 
(183 days ± 
14 days) 
Follow-up 
 
Before Visit 1 
Screening
Within 28 to 1 
day before Visit 2 
Day 1
1 to 21 days before 
SoC optional 
Biopsy / 
Neurosurgical 
Intervention Visit 4 
Day 3
Day of  
18F-fluciclovine 
injection or up to  
3 days after
1 to 3 days after  
18F-fluciclovine 
injection, before 
optional SoC 
Biopsy 
/Neurosurgical 
Intervention 
(if applicable)
If applicable, at 
time of  
pre-planned SoC 
Biopsy / 
Neurosurgical 
Intervention and 
after Visit 2 MRI 
and Visit 3 
Follow-Up
Through 6 months 
after 
18F-fluciclovine 
PET
SoC MRI
X
Informed Consent
X
Confirm inclusion/exclusion 
criteria
X
X
Demographics a
X
Baseline Characteristics b
X
Medical / Disease History c
X
X
ECOG Performance Status d
X
Concomitant Medications
X
X
X
Vital Signs e
X
Pregnancy Test (Urine) f
X
X
Diagnostic Management Plan
X
X
Order 18F-fluciclovine dose
X
Eligibility Review g
X
18F-fluciclovine Injection
X
Dynamic PET Brain Scan
X
Adverse Events
X
X
On-Study MRI Brain Scan
X
X h
Annotate Reference Lesion 
on SoC MRI Brain Scan 
X
Annotate other equivocal 
lesions (if applicable) i
X


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Table 1. Schedule of Study Procedures 
ECOG, Eastern Cooperative Oncology Group; IMP, investigational medicinal product; MRI, magnetic resonance imaging; PET, positron emission tomography; SoC, 
standard of care. 
a Demographic information recorded at Screening will include age, sex, race and ethnicity. 
b Baseline assessments will include pre-scan body weight and height. Pre-scan body weight and height will be collected at Visit 2.
c Treatment history for previous cancer to include previous treatments for brain metastases and previous cancer treatments for primary tumor. Any changes to the subject’s 
medical condition between Screening (Visit 1) and the start of 18F-fluciclovine administration (Visit 2) should be recorded as updated medical history.
d A standard of care ECOG assessment may be recorded as the Screening ECOG if the assessment was performed within 28 days of Screening.
 
Day -42 to -1 
SoC MRI 
Visit 1 
Day -28 to -1 
Screening 
Visit 2 
Day 1 
IMP + PET 
Visit 2  
Day 1 to 4 
On-Study MRI 
Visit 3
Day 2 to 4 
Safety  
Follow-Up 
Phone Call
Visit 4 
Day 2 to 22 SoC 
Biopsy / 
Neurosurgical 
Intervention  
(if applicable)
Visit 5
Month 6 
(183 days ± 
14 days) 
Follow-up 
 
Before Visit 1 
Screening
Within 28 to 1 
day before Visit 2 
Day 1
1 to 21 days before 
SoC optional 
Biopsy / 
Neurosurgical 
Intervention Visit 4 
Day 3
Day of  
18F-fluciclovine 
injection or up to  
3 days after
1 to 3 days after  
18F-fluciclovine 
injection, before 
optional SoC 
Biopsy 
/Neurosurgical 
Intervention 
(if applicable)
If applicable, at 
time of  
pre-planned SoC 
Biopsy / 
Neurosurgical 
Intervention and 
after Visit 2 MRI 
and Visit 3 
Follow-Up
Through 6 months 
after 
18F-fluciclovine 
PET
Identify and annotate 
additional PET lesions (i.e. 
potential additional lesions 
not previously reported on 
SoC MRI [if applicable]) j 
X 
 
 
Safety Follow-up Phone Call 
X 
SoC Biopsy/Neurosurgical 
Intervention 
 
 
 
 
X
 
Document Neurosurgical 
Procedure
X
Record local histopathology 
results and send specimens to 
central histopathology 
laboratory
X
Neurological Deterioration 
Assessment
X
Clinical Follow-Up / Data 
abstraction k
X


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e Vital signs (body temperature, blood pressure, pulse and respirations) will be collected after resting for at least 5 minutes before IMP injection, and after resting for at least 
5 minutes following PET scan. 
f Females of childbearing potential.
g Eligibility review to include collection and review of prior post-treatment MRI brain scans as needed to confirm eligibility. Prior post-treatment MRI brain scans and 
current SoC MRI images to be uploaded to central imaging core lab. 
h  A study MRI will be performed for any subject whose reference or other equivocal lesion(s) is/are still considered equivocal at site level at Visit 5 / Month 6 unless a SoC 
MRI was performed within the preceding 3 weeks. 
i  If >1 equivocal lesion is under evaluation as SoC, the on-site investigator will also annotate ‘other equivocal lesions’ on the Visit 2 On-Study MRI brain scan, defined as 
follows: 
a) Equivocal for recurrent metastasis on SoC MRI (according to the same radiological criteria for a reference lesion per inclusion criterion #7). 
b) Where SoC biopsy/neurosurgical intervention procedure is planned, these are the other equivocal lesions pre-planned for biopsy/intervention. 
c) Where SoC clinical follow-up is planned, these are the other equivocal lesions pre-planned for SoC clinical follow-up. 
j  Additional PET lesions are potential additional lesions identified by the on-site investigator on 18F-fluciclovine PET scan, not previously reported on SoC MRI, and judged 
to be suggestive of brain metastasis, warranting confirmation by clinical follow-up, biopsy or resection according to SoC.  
k Relevant clinical and imaging data will be uploaded for up to 6 months following 18F-fluciclovine PET scan.


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9.1. 
Clinical Assessments 
9.1.1.
Concomitant Medications
All concomitant medication and concurrent therapies will be documented at Screening 
(Visit 1), at IMP administration (Visit 2) and at the post-treatment Safety Follow-up phone 
call (Visit 3), and at early termination when applicable.  In addition to recording all 
concomitant medications as described, treatments and therapies for the brain lesion(s) and 
primary tumor will be recorded through Month 6 (Visit 5) as described in Section 9.1.8. 
Dose, route, frequency of administration, and indication for administration and dates of 
medication will be captured. 
9.1.2. 
Demographic and Baseline Characteristics  
Demographic information recorded at Screening will include age, sex, race and ethnicity. 
Baseline assessments of pre-scan body weight and height will be collected at Visit 2.  
9.1.3. 
ECOG Performance Status 
Conduct ECOG Performance Status assessment at Screening (Visit 1) or record a standard of 
care ECOG Performance Status if the assessment was performed within 28 days prior to 
Screening (Visit 1) (see Appendix 1). 
9.1.4. 
Medical History  
Record medical and disease history at Screening (Visit 1). Include history for previous brain 
metastases and brain metastases treatments, diagnosis of primary tumor and previous cancer 
treatments for primary tumor, and any other previous cancers, as applicable. Brain metastases 
and primary tumor histories should include dates of initial diagnosis, initial tumor stage, dates 
and type of definitive therapy and adjuvant treatment (if given). Any changes to the subject’s 
medical condition between Screening (Visit 1) and the start of 18F-fluciclovine administration 
(Visit 2) should be recorded as updated medical history. 
9.1.5. 
Imaging Studies
Sites will be instructed to submit historic SoC imaging data required to confirm eligibility. 
Submitted MRI scans should confirm the reference lesion criteria outlined in Section 6.2. 
Therefore, these should be the MRI scan confirming nadir or showing complete response, the 
current SoC MRI brain scan performed before Screening (Visit 1), and may include the 
pre-treatment MRI if available. Sites will annotate and measure the reference lesion and other 
equivocal lesions on the current SoC MRI brain scan using the imaging core lab study 
imaging platform.
The reference lesion is defined as the lesion which is:
a.
Equivocal for recurrent metastasis on SoC MRI (according to inclusion criterion 
#7, see Section 6.2). 
b. Intended for SoC biopsy/neurosurgical intervention procedure (if planned). If >1 
lesion is intended for biopsy/intervention, the largest of these lesions will be the 
reference lesion. 
c.
Where clinical follow-up is planned, the largest of all equivocal lesions (if >1 is 
present) will be the reference lesion. 


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If >1 equivocal lesion is under evaluation as SoC, the on-site investigator will also annotate 
‘other equivocal lesions’ on the pre-study SoC MRI brain scan, defined as follows: 
a. Equivocal for recurrent metastasis on SoC MRI (according to the same 
radiological criteria for a reference lesion per inclusion criterion #7, see 
Section 6.2). 
b. Where SoC biopsy/neurosurgical intervention procedure is planned, these are the 
other equivocal lesions pre-planned for biopsy/intervention. 
c. Where SoC clinical follow-up is planned, these are the other equivocal lesions 
pre-planned for SoC clinical follow-up. 
Sites will perform and upload a 18F-fluciclovine PET brain scan on Day 1 (Visit 2), and an 
on-study MRI brain scan performed 0 to 3 days after the 18F-fluciclovine PET scan (Visit 2). 
The site investigator will review the 18F-fluciclovine PET scan to identify potential additional 
lesions, not previously reported on SoC MRI (termed ‘additional PET lesions’). Additional 
PET lesions should be annotated only where judged suggestive of brain metastasis, 
warranting confirmation by clinical follow-up, biopsy or resection, according to SoC practice. 
Moreover, an additional PET lesion should be discrete and spatially separate from known, 
pre-existing lesions. In particular, the following should not be annotated as Additional PET 
lesions: known pre-existing non-progressive lesions (considered at site level as stable or 
partially responding), 18F-fluciclovine activity contiguous with a known lesion (previously 
annotated or otherwise) and apparently incongruent with the lesion’s outline on correlating 
MRI, particularly where this may be reasonably explained by image registration.
The investigator will then annotate the additional PET lesions on the 18F-fluciclovine PET 
scan using the imaging core lab study imaging platform. The site investigator will also 
confirm the sites of the reference and other equivocal lesions on the Visit 2 scans. 
For each annotated lesion, a lesion identifier will be automatically created and assigned by 
the imaging core lab study imaging platform. For each annotated lesion, the site will also be 
required to define the anatomical location.
Subjects whose reference or other equivocal lesion(s) continue to be considered at the 
site-level to be equivocal for brain metastasis at Month 6 (Visit 5) will have a final research 
MRI unless an SoC MRI was performed within the prior 3 weeks.
9.1.5.1.
18F-fluciclovine PET Scan
Dynamic PET/CT or PET/MRI scan of the brain to be performed at Visit 2 following 
18F-fluciclovine injection. If the PET scan is performed on a PET/CT scanner, a CT scan of 
the brain will also be performed for attenuation correction. Site investigator will review the 
18F-fluciclovine PET scan to identify potential additional PET lesions (this will be done with 
the Visit 2 MRI scan for anatomical reference). The PET scan will be uploaded to the central 
imaging core laboratory by the site investigator/site staff. The selected PET scanner must be 
qualified by the study management team prior to any imaging studies. Full details on the 
imaging protocol are provided in the Imaging Acquisition Standards. 
9.1.5.2.
On-Study MRI
Throughout this study, 18F-fluciclovine PET will be read with standard MRI for anatomical 
reference. Therefore, all subjects will receive an on-study, study-specific brain MRI scan at 
Visit 2, ideally on the same d
-study 
MRI will consist of MRI sequences per the minimum standard consensus recommendations 


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for a standardized brain tumor imaging protocol for clinical trials in brain metastases 
(BTIP-BM) (Kaufmann, 2020).
These will include:
 T1-weighted MRI without and with contrast enhancement, 
FLAIR and/or T2-weighted MRI, and
Diffusion-weighted imaging.
On-study MRI brain scans will be uploaded to the central imaging core laboratory by the site 
investigator/site staff. In addition to reviewing the 18F-fluciclovine PET scan to identify 
potential additional PET lesions, on the Visit 2 MRI, the site investigator will confirm the 
sites of the reference and other equivocal lesions initially defined on the SoC MRI. Full 
details on the imaging protocol are provided in the Imaging Acquisition Standards. If 
available, scans acquired on a PET/MRI are allowed. 
A final on-study MRI will be performed at Visit 5 (Month 6) only for any surviving subject 
whose reference or other equivocal lesion(s) remain equivocal at site level, unless an SoC 
MRI was performed within the preceding 3 weeks. 
9.1.6. 
Vital Signs 
Vital signs, including body temperature, blood pressure, pulse and respirations, will be 
collected at Visit 2 (IMP injection + PET) after resting for at least 5 minutes before IMP 
injection and after resting for at least 5 minutes following PET scan. The vital signs should be 
collected between 5 to 60 minutes before and after the PET scan. 
9.1.7. 
SoC Biopsy/Neurosurgical Intervention 
Biopsy/resection of the reference / other equivocal lesions is to take place as planned for SoC. 
Neurosurgical intervention procedures include craniotomy and laser interstitial thermal 
therapy (LITT) where peri-procedural biopsy is performed. Biopsy/surgical resection of any 
additional PET lesions may also be performed if clinically applicable. Where a craniotomy is 
planned, en bloc resection is to be performed where possible. Where en bloc resection is not 
possible, the surgeon should try and send as much of the sample as possible to enable optimal 
histopathology assessment. Care should be taken to follow the Sample Handling Laboratory 
Manual to label all samples sent to the site pathology laboratory, and to reflect the locations 
sampled and lesion identifiers assigned by the imaging core lab study imaging platform. 
The site pathology laboratory will perform histopathology analysis on lesion samples per SoC 
and will send a representative set of specimens to the central laboratory for analysis. Refer to 
the Sample Handling Laboratory Manual.   
9.1.8. 
Clinical Follow-Up / Data Abstraction 
Clinical follow-up will be performed as SoC per local practice and is anticipated to vary 
between subjects. For study purposes, clinical data accrued during the period of clinical 
follow-up, pertaining to the ongoing assessment, eventual confirmation of diagnosis, and 
treatment of the lesions, will be collected. The data will comprise of brain imaging scans, 
treatment regimens, histopathological reports and mortality status, see Table 1. Treatment 
regimens will pertain to treatments and therapies for the brain lesion(s) and primary tumor. 
These will include and are not limited to systemic treatments such as chemotherapy, 
immunotherapy and corticosteroids; and localized therapies such as radiotherapy, LITT and 


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craniotomy, see Section 9.1.1. The period of data accrual will be up to 6 months following 
18F-fluciclovine PET, or death, whichever comes first. 
At this visit, an assessment will be performed to evaluate if the patient had experienced 
neurological deterioration potentially related to progression of brain metastases over the 
study period. This assessment will be performed by a site investigator who is a 
board-certified neuro-oncologist, radiation oncologist, medical oncologist or neurosurgeon. 
As with the other items at this Visit, this assessment will be based on an investigator review 
of the subject medical record and will take place in the physical absence of the subject.
Based on a review of the subject medical record at the Month 6 Follow-Up visit, the 
investigator will ascertain if the patient had demonstrated neurological deterioration over the 
6-month period. This must be evidenced by one of the following as assessed by the 
investigator:   
1. Notable clinical deterioration corresponding to a significant decline in Karnofsky 
Performance Status: 
a. 
 
b. 
or 
c. 
2. New or persistent/worsening focal neurological deficit with neuro-anatomical 
correlation to the site of the reference lesion 
If one of the criteria are met, the investigator must also confirm that this impairment: 
 Lasted for at least 7 days, and 
 Was not attributable to co-morbid events, treatment-related toxicity, changes in 
corticosteroid dose or progression of systemic disease, and
 Was not altered by therapies for radiation necrosis 
If none of the criteria are met (i.e., assessed as no neurological deterioration potentially 
related to brain metastases), the date of the most recent clinical record to the time of 
assessment (e.g., a clinical record at 4 weeks prior to Visit 5), which supports the 
investigator’s assessment, should be recorded.
Results of this assessment of neurological deterioration will be used to support diagnostic 
evaluation at Month 6. 
9.2. 
Clinical Laboratory Measurements 
9.2.1. 
Pregnancy Test 
A human chorionic gonadotropin (HCG) urine pregnancy test will be obtained from female 
subjects who are of childbearing age prior to their participation in the study at Screening, and 
again on Visit 2 before IMP administration (or on the day prior to IMP administration). 


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10. 
EVALUATIONS BY VISIT 
10.1. 
Visit 1 –Screening  
Screening to take place following SoC MRI brain scan and up to 28 days before Visit 2. 
Visit 1 – Screening may be performed at Day -1 (1 day prior to Visit 2 PET scan) only if 
next-day delivery of the 18F-fluciclovine dose is confirmed by the site. 
1. Review the study with the subject (subject’s LAR) and obtain written informed 
consent and HIPAA authorization and assent, if appropriate. 
2. Assign the subject a unique subject number. 
3. Record demographic data. 
4. Record medical / disease history.
5. Record concomitant medications. 
6. Record histopathological confirmation of the primary tumor or a metastatic site within 
4 years of Screening. 
7. Record previous cancer treatments for primary tumor (and other previous cancers, if 
applicable). 
8. Record previous diagnoses and treatments for brain metastases. 
9. Collect and review prior pre-treatment (if available), post-treatment and current SoC 
MRI brain scans as needed for the investigator to confirm eligibility. Upload MRI 
images to the central imaging core laboratory and annotate reference lesion, measure 
reference lesion, and annotate other equivocal lesions. 
10. Record or assess ECOG performance status. A standard of care ECOG assessment 
may be recorded as the Screening ECOG if the assessment was performed within 
28 days of Screening.
11. Perform urine HCG pregnancy test (female subjects of child-bearing potential only). 
12. Confirm eligibility against the inclusion / exclusion criteria. 
13. Investigator records the prospective Diagnostic Management Plan as noted following 
SoC MRI.
14. Schedule 18F-fluciclovine PET scan and order IMP for delivery on day of PET scan.
15. Schedule subject for Visit 2 (IMP + PET) to occur within 42 days of the SoC MRI, 
and provide the subject with 18F-fluciclovine PET scan preparation instructions (see 
Section 11.1). 
The reference lesion is defined as the lesion which is:
a.
Equivocal for recurrent metastasis on SoC MRI (according to inclusion criterion 
#7, see Section 6.2).  
b. Intended for SoC biopsy/neurosurgical intervention procedure (if planned). If >1 
lesion is intended for biopsy/intervention, the largest of these lesions will be the 
reference lesion. 
c. Where clinical follow-up is planned, the largest of all equivocal lesions (if >1 is 
present) will be the reference lesion.  


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If >1 equivocal lesion is under evaluation as SoC, the on-site investigator will also annotate 
‘other equivocal lesions’ on the SoC MRI brain scan, defined as follows: 
a.
Equivocal for recurrent metastasis on SoC MRI (according to the same 
radiological criteria for a reference lesion per inclusion criterion #7, see 
Section 6.2). 
b. Where SoC biopsy/neurosurgical intervention procedure is planned, these are the 
other equivocal lesions pre-planned for biopsy/intervention. 
c.
Where SoC clinical follow-up is planned, these are the other equivocal lesions 
pre-planned for SoC clinical follow-up. 
10.2. 
Visit 2 - Day 1: IMP Administration and Dynamic PET Scan (1 to 
28 days after Screening) 
At Visit 2-Day 1, the following activities will be performed:
1. The subject will prepare for the 18F-fluciclovine PET scan according to preparation 
instructions (see Section 11.1).
2. Record any changes in concomitant medications and medical history since Screening 
(Visit 1).
3. Perform urine HCG pregnancy test (female subjects of child-bearing potential only) 
unless performed 1 day prior to IMP administration. 
4. Confirm eligibility against the inclusion / exclusion criteria. 
5. Perform assessment of vital signs with the subject resting at least 5 minutes prior to 
IMP administration. The vital signs should be collected between 5 to 60 minutes 
before the PET scan. 
6. IMP + PET: Administer 18F-fluciclovine injection, followed by a dynamic PET scan 
of the brain. If the PET scan is performed on a PET/CT scanner, a CT scan of the 
brain will also be performed for attenuation correction. 
7. Monitor and record any adverse events.
8. Perform assessment of vital signs with subject resting at least 5 minutes after PET 
scan. The vital signs should be collected between 5 to 60 minutes after the PET scan.
9. Upload 18F-fluciclovine PET brain scan to central imaging core laboratory. 
10. Review of 18F-fluciclovine PET brain scan by the site investigator to identify potential 
additional PET lesions will be performed with the Visit 2 MRI scan (for anatomical 
reference).  
11. Investigator updates Diagnostic Management Plan following review of 
18F-fluciclovine PET brain scan.
10.3. 
Visit 2 – Day 1 or up to 3 days after 18F-fluciclovine 
Administration: On-Study MRI Brain Scan 
Visit 2 on-study MRI brain scan should take place on the same day as the 18F-fluciclovine 
PET scan (Day 1) or up to 3 days after. Where applicable, it should take place before the 
Visit 4 – SoC biopsy/neurosurgical intervention. 
1. Perform Visit 2 on-study MRI brain scan as outlined in Section 11.5.


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2. Upload Visit 2 on-study MRI brain scan to the central imaging core laboratory. 
3. Review of 18F-fluciclovine PET brain scan (with Visit 2 MRI for anatomical 
reference) by site investigator to identify potential additional PET lesions, not 
previously reported on SoC MRI. 
4. Confirm sites of reference lesion and other equivocal lesions on Visit 2 scans. 
The on-site investigator will review the 18F-fluciclovine PET scan to identify and annotate 
potential additional lesions, not previously reported on SoC MRI (termed ‘additional PET 
lesions’). Additional PET lesions should be annotated only where judged suggestive of brain 
metastasis, warranting confirmation by clinical follow-up, biopsy or resection, according to 
SoC practice. Undertaking biopsy/resection of additional PET lesion(s) identified on the 
18F-fluciclovine PET scan is at the discretion of on-site investigators. Moreover, an additional 
PET lesion should be discrete and spatially separate from known, pre-existing lesions. In 
particular, the following should not be annotated as Additional PET lesions: known 
pre-existing non-progressive lesions (considered at site level as stable or partially 
responding), 18F-fluciclovine activity contiguous with a known lesion (previously annotated 
or otherwise) and apparently incongruent with the lesion’s outline on correlating MRI, 
particularly where this may be reasonably explained by image registration. 
10.4. 
Visit 3 – Safety Follow-up (+1 to 3 days after IMP Injection/PET) 
1. Clinical safety review by telephone call (or in person if same day as scheduled SoC 
biopsy, Visit 2 MRI, or other planned visit) to assess concomitant medications and 
AEs.
2. Record any changes in concomitant medications since Screening (Visit 1). 
3. Record any AEs which have occurred during the safety reporting period (from the 
time of 18F-fluciclovine administration until 1 day post 18F-fluciclovine 
administration). For scheduling purposes, the subject may be contacted up to 3 days 
after IMP injection, but the assessment is to be limited to the first day after IMP 
injection only. 
10.5.
Visit 4 – SoC Biopsy/Neurosurgical Intervention (if applicable, 
pre-planned 1 to 21 days after IMP Injection/PET) 
Where this visit is applicable, SoC biopsy/neurosurgical intervention should also take place 
after both the Visit 2 on-study MRI brain scan and Visit 3 Safety Follow-up. 
1. Document neurosurgical procedure: 
a. Record sites of reference lesion and other equivocal lesions which underwent SoC 
biopsy, and where applicable, ‘additional PET lesion(s)’, if performed. 
b. Record sites of reference lesion and other equivocal lesions which underwent 
resection, and where applicable, ‘additional PET lesion(s)’, if performed.*
* Undertaking biopsy/resection of additional PET lesion(s) identified on the 
18F-fluciclovine PET scan is at the discretion of the on-site investigators. The 
lesions should be judged suggestive of brain metastasis, warranting confirmation 
according to SoC practice. The subject will receive post-procedural management 
per institutional SoC. 
c. If applicable, record neuronavigation system used for neurosurgical procedure. 


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d. Where biopsy was performed, upload image/screenshot of neuronavigation system 
depicting stereotactic coordinates of biopsy. Where resection was performed, 
upload image/screenshot of neuronavigation system depicting the surgical 
resection plan/approach (if neuronavigation was not used for resection, a 
representative screenshot of the post-procedure MRI demonstrating the resection 
cavity is suitable). 
e. Upload neurosurgical procedural report. 
f. Record any clinical factors associated with the neurosurgical intervention which 
impacted the type of procedure performed.
2. Label specimens with lesion ID as referenced by the imaging core laboratory study 
imaging platform on the previously annotated scans. 
3. Process specimens per SoC and local laboratory standard practice. Histopathological 
report from SoC biopsy/neurosurgical intervention will be recorded.
4. Ship a representative set of specimens to the central laboratory per the Sample 
Processing Manual. 
10.6. 
Visit 5 – Month 6 Follow-up (183 days ± 14 days) 
All subjects will be followed for 6 months (183 days ± 14 days) following 18F-fluciclovine 
PET in order to collect diagnostic information (brain imaging scans, histopathological 
reports) and treatment regimens which will be provided to the Central Truth Panel 
(Section 11.8). Treatment regimens will pertain to treatments and therapies for the brain 
lesion(s) and primary tumor. These will include and are not limited to systemic treatments 
such as chemotherapy, immunotherapy and corticosteroids; and localized therapies such as 
radiotherapy, LITT and craniotomy.  
The date and nature of the diagnosis (presence or absence of metastasis) of the reference, 
other equivocal and additional PET (if applicable) lesions reached by site will be collected. 
An assessment of neurological deterioration will be recorded. The subject mortality status 
will also be recorded, with collection of date and cause of death (if applicable).  
Determination of a subject’s mortality status must be verifiable from the medical record, 
including confirmation of death where applicable or, where alive, a documented clinical 
interaction (e.g. imaging scan, blood test, consult) taking place on or beyond Day 169.  If the 
site is unable to verify mortality status from the medical record, additional steps will be 
undertaken, including a review of vital records or a documented phone call to the patient or 
the patient’s family.
Therefore, this Visit comprises predominantly of investigator review of the subject medical 
record and will take place in the physical absence of the subject. Subjects whose reference or 
other equivocal lesion(s) continue to be considered at the site level to be equivocal for brain 
metastasis at Month 6 (Visit 5) will have a final research MRI unless an SoC MRI was 
performed within the prior 3 weeks.
10.7. 
Early Withdrawal  
Early withdrawal is defined as discontinuing from study within 168 days of Visit 2. Death is 
not considered as early withdrawal. 


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Record any AEs or changes to concomitant medication occurring during safety 
reporting period. 
Record reason for early withdrawal.
Record clinical follow-up data to date of early withdrawal as detailed in Visit 5 
evaluations (see Section 10.6 ). 


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11. 
IMAGING PROTOCOL 
Full details on the imaging protocol are in the Image Acquisition Standards. 
11.1.
18F-fluciclovine PET Scan Patient Preparation
Patients should be advised not to eat for at least 4 hours prior to administration of 
18F-fluciclovine injection.  Patients may have water or other clear fluids within this time 
window.
11.2. 
PET/CT Scanner 
A dedicated hybrid PET scanner (e.g., PET/CT, PET/MRI) is mandatory. The selected PET 
scanner must be qualified by the study management team. 
11.3.
18F-fluciclovine Injection Administration
See Section 8.8 and the Pharmacy Manual for administration instructions.
11.4.
18F-fluciclovine PET Acquisition
For the dynamic PET acquisition, subjects will be imaged for approximately 30 minutes. For 
the CT acquisition (if acquired on a PET/CT scanner), an unenhanced (no IV contrast) CT 
will be employed.
11.5. 
On-Study MRI Acquisition 
All subjects will receive an on-study, study-specific brain MRI scan at Visit 2 consisting of 
standard MRI sequences including (1) T1-weighted MRI without and with contrast 
enhancement, (2) FLAIR and/or T2-weighted MRI, and (3) diffusion-weighted imaging, 
ideally on the same day as the PET scan or up to a maximum of 3 days after the PET scan. 
A final on-study MRI will be performed at Visit 5 (Month 6) only for any surviving subject 
whose reference or other equivocal lesion(s) remain equivocal at site level, unless an SoC 
MRI was performed within the preceding 3 weeks.
11.6. 
Image Transfer 
Following the completion of PET and on-study MRI imaging at the study site, the scan data 
will be sent to the Imaging Core Lab using either the Imaging Management Solution software 
or on physical media by courier.
11.7. 
Image Evaluability 
Both PET and on-study MRI scans will be assessed by the Imaging Core Lab for evaluability 
based on the Image Quality Standards. All data will undergo quality control including 
technical analysis, indication and protocol specific criteria, based on the parameters detailed 
in the Image Acquisition Standards.  
11.8. 
Central Evaluations 
11.8.1.
Central Histopathology 
As described in Section 10.5, a representative set of specimens obtained in subjects 
undergoing Visit 4 (SoC biopsy/neurosurgical intervention) will be shipped to a central 


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laboratory. This is to allow for central histopathology review. Detailed specification of this 
workflow, and assignment of recurrent metastasis (positive) vs no recurrent metastasis 
(negative) based on the central review, will be laid out prospectively in a histopathology 
charter.
11.8.2.
Central Truth Panel
An independent panel consisting of an experienced neuro-oncologist, neuroradiologist, 
neuropathologist, neurosurgeon, and radiation oncologist with expertise in treating brain 
tumors will serve as a Central Truth Panel. 
 The panel will establish a charter to prospectively define objective data review 
parameters including pre-specified MRI evaluation criteria.
 The panel will review the central histopathology results (where applicable) and 
clinical data (including site histopathology reports, follow-up MRI scans, and 
cancer-related therapies) to confer a diagnosis of recurrent metastasis (positive) vs 
no recurrent metastasis (negative) for each reference lesion, other equivocal 
lesion(s), and additional PET lesions. 
 The clinical data will encompass a 6-month period following 18F-fluciclovine 
PET, or up to the time when the subject dies, whichever comes first.
The Central Truth Panel will have no access to the 18F-fluciclovine PET scans, nor 
the study record of lesion diagnosis reached by the site. 
11.8.3.
Blinded Image Evaluation 
A 3-reader central, independent BIE of the 18F-fluciclovine PET scans will be performed, 
diagnosing recurrent metastasis (positive) vs no recurrent metastasis (negative) for each 
reference lesion, other equivocal lesion(s) and additional PET lesion(s), using the Visit 2 
MRI for anatomical reference. 
Image criteria used in the BIE will be informed by a Phase 2b study 
(BED-FLC-219).
Inter- and intra-reader concordance will be measured.
The readers will not have access to clinical history, prior clinical imaging, any 
site-generated diagnostic evidence, nor the final patient outcome.
11.8.4.
Central Neuroradiologist Reader
A neuroradiologist reader will independently review the reference lesion on the historical and 
Screening SoC MRI scans to assess each subject’s baseline equivocal status. This 
independent determination will be applied to a sensitivity analysis of the primary endpoint.


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12. 
ADVERSE EVENT REPORTING AND DOCUMENTATION 
12.1. 
Adverse Events 
An AE is any untoward medical occurrence in a clinical investigation subject administered a 
pharmaceutical product and that does not necessarily have a causal relationship with the 
treatment. An AE is therefore any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom or disease temporally associated with the administration of an 
investigational product, whether or not related to that investigational product. An unexpected 
AE is one of a type not identified in nature, severity, or frequency in the current 
Investigator’s Brochure or is of greater severity or frequency than expected based on the 
information in the Investigator’s Brochure.
The investigator will probe, via discussion with the subject, for the occurrence of AEs during 
each subject visit within the AE collection period. The collection period for all AEs will 
begin after initiation of 18F-fluciclovine injection and end 1 day post 18F-fluciclovine 
administration (i.e., Day 2). Any unresolved AE at Visit 3 Safety Follow-up beyond Day 2 
will be followed until resolution or stabilization. The investigator will record the information 
in the site’s source documents. Adverse events will be recorded in the subject electronic Case 
Report form (eCRF). Adverse events will be described by duration (start and stop dates and 
times), severity, outcome, treatment and relation to study drug. 
12.2. 
AE Severity 
The National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) 
Version 5 (November 27, 2017) will be used to assess and grade event severity, including 
laboratory abnormalities judged to be clinically significant. For events not specifically 
mentioned in CTCAE, the general severity grading is provided below (Table 2).
Table 2. CTCAE (V5) AE Severity Grading
Severity (Toxicity Grade)
Description
Grade 1
Mild; asymptomatic or mild symptoms; clinical or 
diagnostic observations only; intervention not indicated.
Grade 2
Moderate; minimal, local or non-invasive intervention 
indicated; limiting age-appropriate instrumental ADL.*
Grade 3
Severe or medically significant but not immediately 
life-threatening; hospitalization or prolongation of 
hospitalization indicated; disabling; limiting self-care 
ADL**.
Grade 4
Life-threatening consequences; urgent intervention 
indicated. 
Grade 5
Death related to AE. 
ADL, activities of daily living; AE, adverse event; CTCAE, Common Terminology Criteria for Adverse Events. 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, managing 
money, etc. 
** Self-care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking medications, 
and not bedridden.   


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12.3. 
Adverse Event Relationship to IMP 
The relationship of an AE to IMP will be categorized as follows:
Related: when there is a reasonable possibility of a causal relationship between IMP 
administration and an AE (i.e., adverse drug reaction). 
Not related: when an AE does not follow a reasonable temporal sequence from IMP 
administration or when an AE can be reasonably explained by other factors including 
underlying disease, concomitant drugs or concurrent treatment. 
12.4. 
Serious Adverse Event (SAE) 
An SAE is defined as any adverse event occurring at any dose that results in any of the 
following outcomes:
 death
a life-threatening adverse experience
inpatient hospitalization or prolongation of existing hospitalization
a persistent or significant disability/incapacity
 a congenital anomaly/birth defect  
Other important medical events may also be considered an SAE when, based on appropriate 
medical judgment, they jeopardize the subject or require intervention to prevent one of the 
outcomes listed above.  
12.4.1. 
Serious Adverse Event Reporting
Study sites will document all SAEs that occur (whether or not related to study drug) on an 
SAE Report Form. The collection period for all SAEs will begin after initiation of 
18F-fluciclovine injection and end 1 day post 18F-fluciclovine administration (i.e., Day 2). 
After this point, the investigator is not required to actively solicit SAE information from 
subjects. However, if they should become aware of an SAE that they suspect is related to the 
IMP, then they should report it. Any unresolved SAE beyond Day 2 at Visit 3 Safety Follow-
up phone call, will be followed until resolution or stabilization. 
All SAEs must be reported within 24 hours of the site study team becoming aware of the 
event by sending the completed SAE Report Form by fax, or scanned and emailed, to:
Bracco Diagnostics Inc. Drug Safety Unit: 
drugsafetyus@blueearthdx.com
Fax: +1 609-514-2522
Additional and further requested information (follow-up or corrections to the original event) 
will be detailed on a new SAE Report Form and faxed/emailed to the same address.
For the US, in accordance with the standard operating procedures and policies of the local 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) the site investigator 
will report SAEs to the IRB/IEC. 


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12.4.2.
Suspected Unexpected Serious Adverse Reaction (SUSAR)
A SUSAR is a serious adverse reaction, the nature and severity of which is not consistent 
with the reference safety information set out in the Investigator’s Brochure.  
The Sponsor will be responsible for reporting all SUSARs to the relevant authorities, and 
other parties, as applicable. 
Investigators will be informed of all SUSARs for the relevant IMP for all studies sponsored 
by Blue Earth Diagnostics, Ltd, whether or not the event occurred in the current study. 
12.4.3.
Pregnancy
In the unlikely event of a pregnancy arising from sexual conduct performed within 24 hours 
following IMP administration, it will require expedited reporting to the sponsor’s 
pharmacovigilance department within the same timelines as an SAE. Women who become
pregnant between the Screening visit and Visit 2 should be withdrawn from the study. All 
reported pregnancies in study participants should be followed and the outcome reported using 
the same form. If the outcome of the pregnancy meets any of the criteria for seriousness, it 
must also be reported as an SAE.  Examples of pregnancy outcomes that are SAEs:  
 Reports of congenital anomalies or developmental delay, in the fetus or the child.  
 Reports of fetal death and spontaneous abortion.
 Reports of suspected adverse reactions in the neonate that are classified as serious. 


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13. 
DISCONTINUATION AND REPLACEMENT OF SUBJECTS 
13.1. 
Discontinuation/Withdrawal from the Study  
Subjects are free to withdraw from participation in the study at any time, for any reason, 
specified or unspecified, and without prejudice. 
An investigator may discontinue or withdraw a subject for the following reasons:   
 Significant protocol violation or non-compliance. 
 If any clinical AE, laboratory abnormality, or other medical condition or situation 
occurs such that continued participation in the study would not be in the best 
interests of the subject (investigator decision). 
 Protocol violation requiring discontinuation of study treatment. 
 If the subject meets the exclusion criteria (either newly developed or not 
previously recognized) that precludes further study participation. 
 Lost to follow-up.
 Sponsor request for early termination of study. 
 Subject requests to be withdrawn from the study.
 Positive pregnancy test (females).
The reason for subject discontinuation or withdrawal from the study, if known, will be 
recorded in the subject’s source documents and in the eCRF.
Refer to Section 10.7 for early withdrawal procedures.
13.2. 
Replacement of Subjects 
Subjects who sign the informed consent form (ICF) and do not receive the study scan may be 
replaced. Subjects who sign the ICF and receive the study scan, but the scan is not considered 
evaluable (refer to Section 11 of the Imaging Protocol), may be replaced. Subjects who sign 
the ICF, and receive the study scan, and subsequently withdraw early (as defined in
Section 10.7), or are withdrawn or discontinued from the study, may be replaced. 
13.3. 
Lost to Follow-up 
A subject will be considered lost-to-follow-up if there are no follow-up data at Visit 5 
(183 days ± 14 days) and the subject is unable to be contacted by the study site staff. Death 
will not be considered lost to follow-up. 
The following actions must be taken if a subject fails to complete a required study visit: 
 The site will attempt to contact the subject and reschedule the missed visit (if 
within acceptable visit windows), and ascertain if the subject wishes to and/or 
should continue in the study.
 Where a pre-scheduled Visit 4/Day 3-22 SoC biopsy/neurosurgical intervention is 
planned, the site will attempt to contact the subject and reschedule the missed visit 
prior to the SoC biopsy/neurosurgical intervention, and ascertain if the subject 
wishes to and/or should continue in the study. 


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Before a subject is deemed lost to follow-up, the investigator or designee will 
make every effort to regain contact with the subject (where possible, 3 telephone 
calls and, if necessary, a certified letter to the subject’s last known mailing address 
or local equivalent methods). These contact attempts should be documented in the 
subject’s medical record or study file.  
 Should the subject continue to be unreachable, he or she will be considered to 
have withdrawn from the study with a primary reason of lost to follow-up. A 
subject will be considered unreachable when: 
 Has received IMP and is beyond 183 days post-IMP administration. 
 Has not received IMP and is beyond 42 days of pre-study SoC MRI. 


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14. 
PROTOCOL VIOLATIONS  
A protocol violation occurs when the subject, investigator, or Sponsor fails to adhere to 
significant protocol requirements affecting the inclusion, exclusion, subject safety and 
primary endpoint criteria. Protocol violations for this study include, but are not limited to, the 
following:
 Failure to meet inclusion/exclusion criteria. 
 Use of a prohibited concomitant medication. 
 Use of a non-validated PET scanner or imaging technique. 
Significant non-compliance from Good Clinical Practice (GCP) guidelines will also result in 
a protocol violation. The Sponsor will determine if a protocol violation will result in 
withdrawal of a subject. 
When a protocol violation occurs, it will be discussed with the investigator and reported to 
the Sponsor for further assessment of the violation and any potential impact on ongoing 
protocol requirements and/or on the utility of the subject’s collected study data. 


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15. 
STATISTICAL METHODS AND CONSIDERATIONS 
Prior to database lock and analysis of the final study data, a detailed Statistical Analysis Plan 
(SAP) will be prepared and approved, describing all the analyses to be performed. The SAP 
will document any changes to the analyses described below. Continuous data will be 
summarized using mean, standard deviation, median, and minimum and maximum values. 
Categorical data will be summarized using frequency counts and percentages. 
15.1.
Estimation of Sample Size
Approximately 150 subjects will be enrolled, to obtain 130 evaluable subjects. 
A one-sample binomial exact test will be used to test the following hypotheses in NPA and 
PPA of 18F-fluciclovine PET:
NPA 
H0(NPA
0
H1(NPA): NPA of PET >NPA0
PPA
 H0(PPA
0
 H1(PPA): PPA of PET >PPA0 
Where NPA0 and PPA0 are performance goals for NPA and PPA, respectively.  
Performance goals of 50% (NPA0) for NPA and 50% (PPA0) for PPA were selected as 
acceptable thresholds given the setting where SoC imaging is already equivocal. The lower 
limits for the exact 95% confidence intervals (CI) (Clopper-Pearson method) for both NPA 
and PPA should be greater than 50%. Assuming an NPA of at least 70% and a PPA of at least 
70% for 18F-fluciclovine PET would need to be observed, a sample size of 130 would achieve 
90% power at a 1-sided significance level of 0.025 to reject the performance goals for both 
NPA and PPA; assuming a brain metastasis prevalence rate of 0.5. 
15.2.
Analysis Sets
The Full Analysis Set (FAS) will include all enrolled subjects dosed with IMP with an 
evaluable Visit 2 on-study PET scan. The FAS will be used for primary and secondary non-
safety endpoints (including diagnostic performance and inter-reader and intra-reader 
assessment for the PET scans). 
The Safety Analysis Set (SAF) will include all subjects dosed with IMP (with or without the 
associated PET scan). The SAF will be used for all safety analyses.  
15.3. 
Subject Disposition 
Subject disposition will be summarized for all subjects who entered the study. Summaries 
will include the number and percentage of subjects in each analysis set, completing the study, 
and discontinuing the study early by the primary reason for discontinuation. 
15.4. 
Demographic and Baseline Characteristics 
Demographic variables (including age, sex, ethnicity and race) and baseline characteristics 
(e.g., height, weight, body mass index [to be calculated], ECOG performance status, medical 


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history, prior treatment history) will be summarized using appropriate summary statistics for 
the FAS and SAF. 
15.5. 
Analysis of Primary Endpoint 
The primary endpoint for the study is subject-level NPA and PPA (equivalent to specificity 
and sensitivity, respectively) of 18F-fluciclovine PET in detecting recurrent brain metastases. 
Subject-level NPA and PPA will be calculated based on BIE of 18F-fluciclovine PET 
compared to SoR on the reference lesion.
Where NPA0 and PPA0 are performance goals for NPA and PPA, respectively. Performance 
goals of 50% (NPA0) for NPA and 50% (PPA0) for PPA were selected as acceptable 
thresholds given the setting where SoC imaging is already equivocal. The analyses for NPA 
and PPA will be performed using the one-sample binomial exact test. In addition to the rates, 
exact two-sided 95% CIs according to the Clopper-Pearson method will also be provided. If 
the pre-defined NPA and PPA goals are met by the same two of three readers (both tests 
reach statistical significance for the same two readers), the study will be considered to have 
successfully demonstrated the effectiveness of 18f-fluciclovine PET in detecting recurrent 
brain metastases.  
15.6. 
Analysis of Secondary Endpoint(s) 
15.6.1.
Subject-Level Positive Predictive Value / Negative Predictive Value 
Subject-level PPV and NPV of 18F-fluciclovine PET for detecting recurrent brain metastases 
will be calculated based on BIE of 18F-fluciclovine PET compared to SoR on the reference 
lesion. The point estimates for PPV and NPV, together with the 95% 2-sided exact CI 
(Clopper-Pearson method) will be presented.
15.6.2. 
Lesion-Level Diagnostic Performance 
Lesion-level diagnostic performance (lesion level PPA, NPA, PPV and NPV) will be 
calculated based on BIE of 18F-fluciclovine PET compared to SoR on all lesions (reference, 
other equivocal and additional PET lesions).
15.6.3. 
Subgroup Analysis of Diagnostic Performance Data 
Subgroup analyses of subject-level PPA, NPA, PPV, and NPV of 18F-fluciclovine PET, 
according to primary tumor type and concurrent immunotherapy, will also be presented.
15.6.4.
Clinical Utility
Clinical utility endpoints include the following, and will be analyzed as described: 
 Number of days taken by the site to establish presence/absence of metastasis by 
clinical follow-up will be defined as the time from administration of 
18F-fluciclovine to the date the presence or absence of metastasis is determined at 
site level. If the presence or absence of metastasis is not ascertained by the time 
the subject reaches the end of the follow-up or when the subject prematurely 
withdraws from the study, the subject will be censored on the date he/she 
withdraws from the study. Kaplan-Meier methodology will be used to handle the 


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censoring of the data, and to estimate the median time to establish 
presence/absence of metastasis. 
 Proportion of subjects with additional metastases identified on 18F-fluciclovine 
PET but not on SoC MRI. The estimate of the percentage and the associated 95% 
2-sided exact CI will be presented.
 Proportion of subjects whose prospective diagnostic management plan changes 
following 18F-fluciclovine PET, by comparing the diagnostic management plans 
before and after 18F-fluciclovine PET. The estimate of the percentage and the 
associated 95% 2-sided exact CI will be presented.
15.6.5. 
Inter-Reader / Intra-Reader Agreement 
Cohen’s kappa statistic will be calculated to assess inter-reader and intra-reader agreement on 
18F-fluciclovine PET scan central reads. The statistic will be presented for each pairwise 
comparison of the 3 readers for the inter-reader agreements, at the subject-level 
(i.e., reference lesion only) and also at lesion-level (i.e., all lesions defined by site). Similar 
statistics will be presented for the initial read vs re-read of a subset of PET scans for each 
reader. 
15.6.6. 
Safety 
Treatment-emergent adverse events (TEAEs) are defined as those AEs with onset after the 
dose of IMP, or existing events that worsened after the dose of IMP during the study, up to 1 
day following IMP administration. Verbatim terms of the AEs will be coded to preferred 
terms (PTs) and system organ classes using the latest version of Medical Dictionary for 
Regulatory Activities (MedDRA), for data summary purposes. Severity of AEs will be graded 
using the NCI-CTCAE v5.0. The frequency of TEAEs will be summarized overall, by system 
organ classes and PT, and by severity. IMP-related AEs and SAEs will also be summarized. 
15.7. 
Planned Interim Analysis 
Not applicable.


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16. 
DATA COLLECTION, RETENTION AND MONITORING 
16.1. 
Data Collection Instruments 
The investigator will prepare and maintain adequate and accurate source documents designed 
to record all observations and other pertinent data for each subject administered the study 
drug. Particular care should be taken to ensure all data points are recorded in source 
documentation, especially those which are not part of standard practice.
The investigator will collect relevant pre- and post-treatment MRI brain scans at Screening, 
on-study PET and MRI scans and SoC imaging during study and upload the images to the 
central imaging core lab. 
Documentation of subject cancer history must include: 
 Type of primary tumor that gave rise to the brain metastasis. 
 Previous treatments for brain metastases. 
 Previous cancer treatments for primary tumor. 
 Histopathological confirmation of the primary tumor or a metastatic site within 
4 years of Screening. 
Study personnel at each site will enter data from source documents corresponding to a 
subject’s visit into the protocol-specific eCRF when the information corresponding to that 
visit is available. 
Subjects will not be identified by name in the study database or on any study documents to be 
collected by the Sponsor and will be identified by a site number and subject number.
If a correction is required for an eCRF, the time and date stamps track the person entering or 
updating eCRF data and creates an electronic audit trail. 
The investigator is responsible for all information collected on subjects enrolled in this study. 
All data collected during the course of this study must be reviewed and verified for 
completeness and accuracy by the investigator.
16.2.
Data Management Procedures
The data will be entered into a validated database. The Data Management group will be 
responsible for data processing, in accordance with procedural documentation. Database lock 
will occur once quality assurance procedures have been completed.
All procedures for the handling and analysis of data will be conducted using good computing 
practices meeting FDA guidelines for the handling and analysis of data for clinical trials.
16.3. 
Data Quality Control and Reporting 
After data have been entered into the study database, a system of computerized data 
validation checks will be implemented and applied to the database on a regular basis. Queries 
are entered, tracked, and resolved through the electronic data capture system directly. The 
study database will be updated in accordance with the resolved queries. All changes to the 
study database will be documented.


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16.4. 
Archiving of Data 
The database is safeguarded against unauthorized access by established security procedures; 
appropriate backup copies of the database and related software files will be maintained. 
Databases are backed up by the database administrator in conjunction with any updates or 
changes to the database. 
At critical junctures of the protocol (e.g., production of interim reports and final reports), data 
for analysis is locked and cleaned per established procedures. 
16.5. 
Availability and Retention of Investigational Records 
The investigator should maintain adequate and accurate source documents and study records 
that include all pertinent observations on each of the site’s study subjects. Source data should 
be attributable, legible, contemporaneous, original, accurate and complete. Changes to source 
data should be traceable, should not obscure the original entry, and should be explained if 
necessary. 
The investigator / institution should maintain the study documents as specified in Section 8 of 
International Council for Harmonisation (ICH) GCP E6 (R2) and as required by the 
applicable regulatory requirement(s). The investigator should take measures to prevent 
accidental or premature destruction of these documents.
Upon request of the monitor, auditor, IRB/IEC, or regulatory authority, the investigator must 
make available for direct access all requested study-related records.
Study records should be retained for a minimum of 2 years after the last approval of a 
marketing application in an ICH region and until there are no pending or contemplated 
marketing applications in an ICH region or until at least 2 years have elapsed since the formal 
discontinuation of clinical development of the study intervention / at least 25 years after the 
end of the clinical trial. These documents should be retained for a longer period, however, if 
required by applicable local regulations. No records will be destroyed without the written 
consent of the Sponsor, if applicable. It is the responsibility of the Sponsor to inform the 
investigator when these documents no longer need to be retained.
The medical files of subjects shall be archived in accordance with applicable local 
regulations.
16.6.
Monitoring and Auditing
Monitoring visits will be conducted by representatives of the Sponsor according to ICH GCP 
and relevant regulations. By signing this protocol, the investigator grants permission to the 
Sponsor’s (or designee’s) monitors and auditors, as well as the IRB/IEC and regulatory 
authorities to conduct on-site monitoring and/or auditing and provide direct access to all 
requested study-related records. 
16.7. 
Subject Confidentiality 
In order to maintain subject confidentiality, only a site number and subject number will 
identify all study subjects on eCRFs and other documentation submitted to the Sponsor.  
Clinical information will not be released without written permission of the subject, except as 
necessary for monitoring by Regulatory Authorities. The investigator must also comply with 
all applicable privacy regulations (e.g., HIPAA of 1996, etc.). 


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17. 
REGULATORY, ETHICAL AND STUDY OVERSIGHT 
CONSIDERATIONS 
The study will be conducted in accordance with ICH GCP and all applicable regulations. The 
investigator will assure that no deviation from, or changes to the protocol will take place 
without prior agreement from the Sponsor and documented approval from the relevant 
regulatory authority (if applicable) and IRB/IEC, except where necessary to eliminate an 
immediate hazard(s) to the trial subjects. All personnel involved in the conduct of this study 
have completed ICH GCP training, relevant to their role. 
17.1.
Institutional Review Boards and Independent Ethics Committees
Any documents that the IRB/IEC may need to fulfill its responsibilities (such as protocol, 
protocol amendments, Investigator’s Brochure, consent forms, information concerning 
subject recruitment, payment or compensation procedures, or other pertinent information) 
will be submitted to the IRB/IEC.
The IRB/IECs written unconditional approval / favorable opinion of the study, and any 
additional local approvals (e.g., hospital management, Radiation Safety Committee, etc.), 
must be obtained prior to shipment of study drug to the site and prior to any subjects 
undergoing study-specific procedures. The investigator will obtain assurance of IRB/IEC 
compliance with regulations. Note: Regulatory authority approvals may also be required.
The IRB/IEC’s standard operating procedures and policies will be followed for the 
submission of SAEs and progress reports during the conduct of the study. 
An end of study notification will be submitted per regulatory requirements.  
17.2. 
Amendments 
Any decision to amend the clinical trial application and / or associated documents 
(e.g., protocol, informed consent form, CRF, Investigator’s Brochure, etc.) will be made by 
the Sponsor. 
The relevant regulations will be followed to determine what approvals from regulatory, 
IRB/IEC or local bodies are required. All required approvals will be obtained prior to 
implementation of the amendment, except as necessary to eliminate immediate safety hazards 
to subjects in accordance with ICH E6 (R2), 4.5 and applicable regulatory requirements. The 
Sponsor will notify each participating investigator site when the amendment can be 
implemented.
All changes to the consent form will be IRB/IEC approved; a determination will be made 
regarding whether a new consent needs to be obtained from subjects who provided consent, 
using a previously approved consent form.
17.3. 
Subject Information and Consent/Assent  
In obtaining and documenting subject informed consent, the investigator must comply with 
the applicable regulatory requirement(s), ICH GCP and the ethical principles that have their 
origin in the Declaration of Helsinki. 
Subject information and consent forms, and any other written material provided to the 
subject, must be approved by the relevant IRB/IEC (and by any other body as required by 
national regulations) prior to the start of the study at each study site. 


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The investigator (or an appropriately qualified designee) will explain the study to the subject 
or, if the subject is unable to provide informed consent, the subject’s LAR, and answer any 
questions that arise. A verbal explanation will be provided in terms suited to the subject’s, or 
subject’s LAR’s, comprehension, of the purposes, procedures, and potential risks of the study 
and the rights of research subjects. Subjects (and the subject’s LAR) will have the 
opportunity to carefully review the written information and consent form, to discuss the study 
with their family or surrogates, and be given ample time to think about the study and ask 
questions before agreeing to participate. 
Subjects (and the subject’s LAR) must be informed that participation is voluntary and that 
they may withdraw from the study at any time, without prejudice. The rights and welfare of 
the subjects will be protected by emphasizing to them that the quality of their medical care 
will not be adversely affected if they decline to participate in this study. 
Prior to the subject undergoing any study-specific procedures, the written informed consent 
form must be signed and personally dated by the subject, or their LAR, and by the person 
who conducted the informed consent discussion. The informed consent process will also be 
documented in the source document (including the date/time consent was obtained). 
If a subject is unable to read or if a LAR is unable to read, an impartial witness should be 
present during the entire informed consent discussion. The subject, or subject’s LAR, may 
orally consent to the subject’s participation, if the subject, or subject’s LAR is not capable of 
providing of signing and personally dating the consent form. Once the subject, or subject’s 
LAR, has provided consent, the witness should also sign and personally date the consent 
form. By signing the consent form the witness attests that the information sheet / consent 
form was accurately explained to, and apparently understood by, the subject, or subject’s 
LAR, and that informed consent was freely given by the subject, or subject’s LAR. 
The distribution of the signed information sheet /consent form will be as required by any 
applicable local regulations. Otherwise a copy of the signed informed consent document will 
be given to the subject and the original maintained with the subject’s records.  
The subject or subject’s LAR, will be informed in a timely manner if new information 
becomes available that may be relevant to the subject’s willingness to continue participation 
in the study. The communication of this information will be documented in the source 
documentation. The written subject information/consent form and any other written 
information provided to the subjects should be revised whenever important new information 
becomes available that may be relevant to the subject’s consent. Any revised written subject 
information and consent form should receive IRB/IEC approval / favorable opinion prior to 
use. The subject, or subject’s LAR, should sign and personally date any revised consent form 
and receive a copy (or original, if required by applicable regulations). 
17.4. 
Post-trial care   
18F-fluciclovine is a single-use diagnostic agent. No additional care for trial subjects is 
therefore planned once their participation through Visit 3 Safety Follow-Up has ended. All 
subjects will receive SoC treatment in-line with their medical condition as determined by 
their physician.
17.5. 
Publications  
The preparation and submittal for publication of manuscripts containing the study results 
shall be in accordance with a process determined by mutual written agreement among the 


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study Sponsor and participating institutions. The publication or presentation of any study 
results shall comply with all applicable privacy laws, including, but not limited to, the Health 
Insurance Portability and Accountability Act of 1996 and the General Data Protection 
Regulation (EU) 2016/679.  
17.6.
Investigator Responsibilities
By signing the Protocol Agreement form, the investigator agrees to: 
1. Comply with ICH GCP principles and all applicable regulatory requirements; be 
familiar with the appropriate use of the investigational product(s) as described in the 
protocol, Investigator’s Brochure, and any other information sources provided. 
2. Personally conduct or supervise the study; maintain a list of appropriately qualified 
persons to whom significant trial-related duties are delegated. Ensure all persons 
assisting with the study are adequately informed about the protocol, the 
investigational product(s), and their study-related duties. 
3. Ensure that all study-related medical decisions are made by a qualified physician who 
is an investigator or sub-investigator for the study; ensure that adequate medical care 
is provided to a subject for any adverse events.
4. Obtain the written approval / favorable opinion of the IRB/IEC before the study starts; 
provide the IRB/IEC with the current Investigator’s Brochure and all documents 
subject to review through-out the trial.
5. Submit a written progress report at least annually and in accordance with the 
IRB/IEC’s request; submit an end of trial notification / final report to the IRB/IEC at 
the end of the study.
6. Conduct the study in compliance with the approved protocol and not implement any 
deviation from, or changes to the protocol without the agreement of the Sponsor, and 
prior approval / favorable opinion of the IRB/IEC of an amendment, except where 
necessary to eliminate an immediate hazard to a study subject, or when the change(s) 
involve only logistical or administrative aspects of the trial. 
a.
Promptly (immediately) notify the Sponsor and IRB/IEC of any actions taken to 
eliminate an immediate hazard to a study subject in accordance with ICH E6 (R2), 
4.5 and applicable regulatory requirements. 
b. Comply with ICH GCP, applicable regulatory requirements and the ethical 
principles that have their origin in the Declaration of Helsinki, when obtaining and 
documenting informed consent. 
7. Ensure that investigational product(s) are: stored as specified by the Sponsor and in 
accordance with applicable regulatory requirement(s); used in accordance with the 
protocol and that adequate records are maintained. 
8. Immediately report all SAEs to the Sponsor unless otherwise specified in the protocol 
or other document (e.g., Investigator’s Brochure). Comply with applicable regulatory 
requirement(s) related to the reporting of unexpected serious adverse drug reactions to 
regulatory authorities and the IRB/IEC. 
9. If the study is prematurely terminated or suspended for any reason, promptly inform 
study subjects and assure appropriate therapy and follow-up, as required; follow ICH 
GCP and required regulatory requirements to notify the Sponsor and IRB/IEC. 


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10. Maintain adequate and accurate source documents and study records that include all 
pertinent observations on each of the study subjects; source data should be 
attributable, legible, contemporaneous, original, accurate and complete. Changes to 
source data must be traceable, should not obscure the original entry and should be 
explained if necessary.
11. Retain essential documents for at least 2 years of a marketing application /at least 
25 years from the end of the clinical trial or until notified by the Sponsor.
12. Provide monitors, auditors, IRB/IEC and regulatory authorities direct access to all 
requested study-related records.


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18. 
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APPENDIX 1.
ECOG PERFORMANCE STATUS
ECOG PERFORMANCE STATUS a
Grade 
ECOG 
0 
Fully active, able to carry on all pre-disease performance without restriction. 
1
Restricted in physically strenuous activity but ambulatory and able to carry out 
work of a light or sedentary nature, e.g., light house work, office work. 
2
Ambulatory and capable of all self-care but unable to carry out any work 
activities. Up and about more than 50% of waking hours. 
3
Capable of only limited self-care, confined to bed or chair more than 50% of 
waking hours. 
4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or 
chair. 
5 
Dead 
ECOG = Eastern Cooperative Oncology Group. 
a As published in Oken, 1982. 
 


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CONFIDENTIAL Blue Earth Diagnostics, Ltd 
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
APPENDIX 2.
PROTOCOL AGREEMENT
I have read the protocol specified below. In my formal capacity as Investigator, my duties include 
providing Blue Earth Diagnostics, Ltd with the information, as outlined in the protocol. It is 
understood that all information pertaining to the study will be held strictly confidential and that this 
confidentiality requirement applies to all study staff at this site. Furthermore, on behalf of the study 
staff and myself, I agree to maintain the procedures required to carry out the study in accordance 
with accepted GCP principles, applicable regulatory requirements, and to abide by the terms of this 
protocol. 
Protocol Number:  BED-FLC-312 
 
Protocol Title:  An Open-label, Single-arm, Single-dose, Prospective, Multicenter Phase 3 Study to 
Establish the Diagnostic Performance of 18F-fluciclovine Positron Emission Tomography (PET) in 
Detecting Recurrent Brain Metastases After Radiation Therapy (REVELATE) 
 
Protocol Version:  3.0 
Protocol Date:  17 March 2023  
 
 
 
 
Investigator Signature  
 
Date 
 
Print Name and Title 
Site # 
 
 
Site Name 
 
Address
Phone Number 
 
Email 
 
 
 
 
 
 

SAP_003.pdf: 
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STATISTICAL ANALYSIS PLAN 
Blue Earth Diagnostics 
BED-FLC-312 
 
 
 
Protocol Title: 
An Open-label, Single-arm, Single-dose, Prospective, 
Multicenter Phase 3 Study to Establish the Diagnostic 
Performance of 18F-fluciclovine Positron Emission 
Tomography (PET) in Detecting Recurrent Brain Metastases 
after Radiation Therapy (REVELATE) 
Protocol Version 
and Date: 
Version 2.0; 06 July 2021 
Sponsor: 
 
Blue Earth Diagnostics, 
 
 
 
Prepared By: 
 
Precision for Medicine, Oncology and Rare Disease 
 
 
Document Version 
and Date: 
Version 3.0; 13 July 2021 
 




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2 TABLE OF CONTENTS 
 
1 
Statistical Analysis Plan Approval ....................................................................................... 2 
2 
Table of Contents ................................................................................................................... 3 
3 
List of Abbreviations ............................................................................................................. 4 
4 
Introduction ............................................................................................................................ 5 
5 
Study Objectives ..................................................................................................................... 5 
5.1 
Primary Study Objective .................................................................................................. 5 
5.2 
Secondary Study Objectives............................................................................................. 5 
6 
Investigational Plan ................................................................................................................ 6 
6.1 
Overall Study Design ........................................................................................................ 6 
6.2 
Schedule of Assessments ................................................................................................... 8 
6.3 
Treatment(s) ...................................................................................................................... 9 
6.3.1 
Treatment(s) Administered ....................................................................................... 9 
6.3.2 
Method of Assigning Subjects to Treatment Groups .............................................. 9 
6.4 
Efficacy and Safety Variables ........................................................................................ 10 
6.4.1 
Efficacy Variable(s) .................................................................................................. 10 
6.4.2 
Description of Safety Variables ............................................................................... 11 
6.5 
Data Quality Assurance .................................................................................................. 12 
7 
Statistical Methods ............................................................................................................... 12 
7.1 
General Methodology ..................................................................................................... 12 
7.1.1 
Reporting Conventions ............................................................................................ 12 
7.1.2 
Standard Calculations .............................................................................................. 13 
7.2 
Analysis Sets .................................................................................................................... 13 
7.3 
Study Subjects ................................................................................................................. 14 
7.3.1 
Disposition of Subjects ............................................................................................. 14 
7.3.2 
Protocol Deviations................................................................................................... 14 
7.3.3 
Demographic and Other Baseline Characteristics ................................................ 14 
7.4 
Efficacy Evaluation ......................................................................................................... 16 
7.4.1 
Datasets Analyzed .................................................................................................... 16 
7.4.2 
Measurements of Treatment Compliance .............................................................. 16 
7.4.3 
Primary Efficacy Endpoint Analysis Methods ...................................................... 16 
7.4.4 
Secondary Endpoint Analysis Methods .................................................................. 17 
7.4.5 
Statistical/Analytical Issues ..................................................................................... 21 
7.5 
Safety Evaluation ............................................................................................................ 22 
7.5.1 
Extent of Exposure ................................................................................................... 22 
7.5.2 
Adverse Events ......................................................................................................... 22 
7.5.3 
Deaths, Other Serious Adverse Events, and Other Significant Adverse 
Events ........................................................................................................................ 23 
7.5.4 
Clinical Laboratory Evaluation .............................................................................. 24 
7.5.5 
Vital Signs ................................................................................................................. 24 
7.5.6 
Concomitant Medications ........................................................................................ 24 
7.6 
Determination of Sample Size ........................................................................................ 25 
7.7 
Changes in the Conduct of the Study or Planned Analyses ........................................ 25 
8 
Reference List ....................................................................................................................... 25 
 
 
 


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3 LIST OF ABBREVIATIONS  
 
Abbreviation 
Definition  
AE 
Adverse Event 
ATC 
Anatomical Therapeutic Chemical 
BIE 
Blinded Image Evaluation 
BMI 
Body Mass Index 
COVID-19 
Coronavirus Disease of 2019 
CSR 
Clinical Study Report 
CTP 
Central Truth Panel 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
electronic Case Report Form 
FAS 
Full Analysis Set 
HCG 
Human Chorionic Gonadotropin 
ICH 
International Council for Harmonisation 
IMP 
Investigational Medicinal Product 
LITT 
Laser Interstitial Thermal Therapy 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRI 
Magnetic Resonance Imaging 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
NPA 
Negative Predictive Agreement 
NPV 
Negative Predictive Value 
PET 
Positron Emission Tomography 
PPA 
Positive Predictive Agreement 
PPV 
Positive Predictive Value 
PT 
Preferred Term 
SAE 
Serious Adverse Event 
SAF 
Safety Analysis Set 
SAP 
Statistical Analysis Plan 
SoC 
Standard of Care 
SOC 
System Organ Class 
SoR 
Standard of Reference 
SoT 
Standard of Truth 
TEAE 
Treatment-Emergent Adverse Event 
TESAE 
Treatment-Emergent Serious Adverse Event 
WHODrug 
World Health Organization Drug Dictionary 
 
 
 


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4 INTRODUCTION 
The purpose of this statistical analysis plan (SAP) is to provide comprehensive and 
detailed descriptions of the methods and presentation of data analyses proposed for Blue 
Earth Diagnostics Protocol BED-FLC-312 (An Open-label, Single-arm, Single-dose, 
Prospective, Multicenter Phase 3 Study to Establish the Diagnostic Performance of 18F-
fluciclovine Positron Emission Tomography (PET) in Detecting Recurrent Brain 
Metastases after Radiation Therapy (REVELATE)). Descriptions of planned analyses are 
provided in order to avoid post hoc decisions that may affect the interpretation of the 
statistical analysis. The statistical methods applied in the design and planned analyses of 
this study are consistent with the International Council for Harmonisation (ICH) 
guideline Statistical Principles for Clinical Trials (E9) (1998).  
This SAP will be finalized prior to data analysis to provide full details, including 
templates for tables and listings, to be presented in the clinical study report (CSR). Any 
changes between the statistical methods provided in the clinical study protocol and this 
SAP will be explained herein; any changes or deviations from this SAP relative to the 
final analysis will be fully documented in the CSR. Minor changes or deviations from the 
templates for tables and listings need not be documented in the CSR. 
5 STUDY OBJECTIVES 
5.1 
Primary Study Objective 
The primary objective of this study is:  
• To assess the negative percent agreement (NPA) and positive percent 
agreement (PPA) of 18F-fluciclovine PET in detecting recurrent brain 
metastases on a subject-level. 
5.2 
Secondary Study Objectives 
The secondary objectives of this study are: 
• To assess other diagnostic performance parameters of 18F-fluciclovine PET in 
detecting recurrent brain metastases on a subject-level.  
• To assess lesion-level diagnostic performance of 18F-fluciclovine PET in 
detecting recurrent brain metastases.  
• To assess subject-level diagnostic performance of 18F-fluciclovine PET in 
detecting recurrent brain metastases in different clinical settings.  
• To evaluate the added clinical usefulness of 18F-fluciclovine PET in 
evaluation of subjects with suspected recurrent brain metastases. 
• To establish inter- and intra-reader reproducibility of 18F-fluciclovine PET 
image interpretation for detecting recurrent brain metastases. 
• To assess the safety of 18F-fluciclovine injection in the subject population. 


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6 INVESTIGATIONAL PLAN 
6.1 
Overall Study Design 
This is a prospective, open-label, single-arm, single-dose study in subjects with solid 
tumor brain metastases previously treated with radiation therapy, designed to establish 
the diagnostic performance of 18F-fluciclovine PET (read with standard magnetic 
resonance imaging [MRI] for anatomical reference) in detecting recurrent brain 
metastases where MRI is equivocal. 
Subjects with a history of brain metastases previously treated with primary, adjuvant or 
repeat (salvage) radiation therapy, with a recent standard of care (SoC) brain MRI found 
to be equivocal for recurrent brain metastasis, and who meet all inclusion criteria and 
none of the exclusion criteria, will be consented and enrolled. The study population will 
be balanced to represent different primary tumor types of any origin, with a minimum of 
approximately 15% of the cohort each with lung cancer, breast cancer and melanoma. 
The number of subjects with lung cancer will be capped at approximately 50% of 
enrolled subjects.  
Investigator will prepare a diagnostic management plan prior to the 18F-fluciclovine PET 
scan and will update the plan following the scan. All eligible subjects will receive an 18F-
fluciclovine PET scan (Visit 2 PET) within 42 days of SoC MRI. Subjects will then 
undergo a repeat study-specific brain MRI scan (Visit 2 On Study MRI) to be used for 
anatomical reference, ideally on the same day, and potentially at the same time, as a 
PET/MRI scan, otherwise ≤3 days after Visit 2 PET and completed before any pre-
planned neurosurgical intervention when applicable. A Safety Follow-up (Visit 3) 1 to 3 
days after investigational medicinal product (IMP) injection/PET (Visit 2 PET) will be 
made for adverse event (AE) evaluation by telephone call or in person (if same day as 
other scheduled SoC appointments). AEs occurring from the time of 18F-fluciclovine 
administration until 1 day post-18F-fluciclovine administration will be recorded. The 
Safety Follow-up must be completed before the pre-planned SoC biopsy/neurosurgical 
intervention, if applicable. Subjects who experience a serious adverse event (SAE) or an 
AE that persists at Visit 3, will be followed until resolution or stabilization of these 
events. All ongoing follow-up and any further treatment will be in accordance with SoC. 
As MRI is not specific for tumor recurrence in this setting, current clinical practice is 
mainly focused on close clinical follow up to determine the presence of recurrence. 
Alternatively, biopsy or direct neurosurgical intervention of the abnormality may be 
performed. This study will utilize all available diagnostic and follow-up clinical data for 
up to six months post PET scan, to establish the underlying diagnosis.  The planned 
diagnostic management plan will be recorded at Screening and assessed for any changes 
following 18F-fluciclovine PET scan. 
If biopsy/neurosurgical intervention has been pre-planned per SoC, the Visit 2 PET will 
be organized to take place a minimum of 1 day and maximum of 21 days before pre-
planned SoC biopsy or neurosurgical intervention. 


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On-site investigators will prospectively annotate and measure the ‘reference lesion’ on a 
post-radiation treatment MRI scan and on the pre-study SoC MRI scan to confirm 
eligibility.   
The ‘reference lesion” is defined as the lesion which is: 
a. Equivocal for recurrent metastasis on SoC MRI (according to inclusion 
criterion #7, see protocol section 6.2).  
b. Intended for SoC biopsy/neurosurgical intervention procedure (if planned). If 
>1 lesion is intended for biopsy/intervention, the largest of these lesions will 
be the reference lesion.  
c. Where clinical follow-up is planned, the largest of all equivocal lesions (if >1 
is present) will be the reference lesion.  
If >1 equivocal lesion is under evaluation as SoC, the on-site investigator will also 
annotate ‘other equivocal lesions’ on the pre-study SoC MRI scan, defined as follows: 
a. Equivocal for recurrent metastasis on SoC MRI (according to the same 
radiological criteria for a reference lesion per inclusion criterion #7, see 
protocol section 6.2). 
b. Where SoC biopsy/neurosurgical intervention procedure is planned, these are 
the other equivocal lesions pre-planned for biopsy/intervention. 
c. Where SoC clinical follow-up is planned, these are the other equivocal lesions 
pre-planned for SoC clinical follow-up. 
The on-site investigator will also review the 18F-fluciclovine PET scan to identify 
potential additional lesions, not previously reported on SoC MRI (termed ‘additional PET 
lesions’). Additional PET lesions should be annotated only where judged suggestive of 
brain metastasis, warranting confirmation by clinical follow-up, biopsy or resection, 
according to SoC practice. Undertaking biopsy/resection of additional PET lesion(s) 
identified on the 18F-fluciclovine PET scan is at the discretion of on-site investigators. 
Moreover, an additional PET lesion should be discrete and spatially separate from 
known, pre-existing lesions. In particular, the following should not be annotated as 
Additional PET lesions: known pre-existing non-progressive lesions (considered at site 
level as stable or partially responding), 18F-fluciclovine activity contiguous with a known 
lesion (previously annotated or otherwise) and apparently incongruent with the lesion’s 
outline on correlating MRI, particularly where this may be reasonably explained by 
image registration. 
For subjects due for pre-planned SoC biopsy/neurosurgical intervention, the procedure 
will be at Visit 4. The subject will receive post-procedural management per institutional 
SoC. All ongoing follow-up and any further treatment will be in accordance with SoC. 
Samples obtained during this Visit 4 will be subject to central histopathological 
evaluation.    
As with subjects who undergo pre-planned SoC biopsy/neurosurgical intervention, 
subjects pre-planned for clinical follow up to evaluate the equivocal lesions will undergo 
follow-up performed as SoC per local practice. This is anticipated to vary between 
subjects.  


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The final visit for each subject, including subjects who had undergone pre-planned SoC 
biopsy/neurosurgical intervention, will usually be the Month-6 Follow-up at Visit 5. For 
study purposes, clinical data accrued during SoC clinical follow-up, pertaining to the 
ongoing assessment, eventual confirmation of diagnosis, and treatment of the lesions, will 
be collected. The data will comprise brain imaging scans, treatment regimens, 
histopathological reports and mortality status. The period of data accrual will be up to 6 
months following 18F-fluciclovine PET, or death, whichever comes first. Subjects still 
considered equivocal at site level at Month 6 (Visit 5) will undergo a study MRI. 
With the exception of PET scans, a set of clinical follow-up data (specified in the Central 
Truth Panel charter) and, where applicable, central histopathology results, will be 
reviewed by a Central Truth Panel (CTP) to assign a final diagnosis to each lesion, and 
form the Standard of Reference (SoR). Separately, 18F-fluciclovine PET scans will be 
centrally read in a blinded image evaluation (BIE), using image interpretation criteria 
established from the separate Phase 2 study (BED-FLC-219). Diagnostic performance 
primary and secondary endpoints will then be derived from BIE scan reads matched 
against SoR. Subject-level SoR will be defined by CTP diagnosis of the reference lesion. 
Lesion-level SoR will be defined by CTP diagnosis of all lesions (reference lesions, other 
equivocal lesions, additional PET lesions). 
6.2 
Schedule of Assessments 
 
 


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6.3 
Treatment(s) 
6.3.1 
Treatment(s) Administered 
Subjects will receive a single dose of 18F-fluciclovine by injection, 185 MBq (5 mCi) ± 
20%, delivered as an intravenous bolus by site staff. 
6.3.2 
Method of Assigning Subjects to Treatment Groups 
Not applicable. All subjects will receive a single dose of 18F-fluciclovine. 


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6.4 
Efficacy and Safety Variables 
6.4.1 
Efficacy Variable(s) 
6.4.1.1 
Primary Efficacy Variable 
The primary efficacy endpoint is:  
• Subject-level NPA and PPA (equivalent to specificity and sensitivity 
respectively) of 18F-fluciclovine PET in detecting recurrent brain metastases.  
Subject level NPA and PPA will be calculated based on BIE of 18F-fluciclovine PET 
compared to SoR on the reference lesion, for each of the 3 readers, as follows:  
 
 
SoR - Positive 
SoR - Negative 
Reader X – Positive 
True positive 
False positive 
Reader X – Negative 
False negative 
True negative 
• PPA (% of true positives) = true positive / (true positive + false negative)  
• NPA (% of true negatives) = true negative / (true negative + false positive)  
6.4.1.2 
Secondary Efficacy Variables 
Secondary efficacy endpoints include the following: 
• Subject level positive predictive value (PPV) and negative predictive value 
(NPV) of 18F-fluciclovine PET for detecting recurrent brain metastases. 
o PPV = true positive / (true positive + false positive)  
o NPV = true negative / (true negative + false negative)  
• Lesion-level PPA, NPA, PPV and NPV of 18F-fluciclovine PET for detecting 
recurrent brain metastases 
• Sub-group analyses of subject-level PPA, NPA, PPV and NPV of 18F-
fluciclovine PET, according to primary tumor type and concurrent 
immunotherapy. 
• Clinical usefulness: 
o Number of days taken by the site to establish presence/absence of 
metastasis by clinical follow-up. 
o Proportion of subjects with additional metastases identified on 18F-
fluciclovine PET in addition to standard of care brain MRI. 
o Proportion of subjects whose prospective diagnostic management plan 
changes following 18F-fluciclovine PET. 


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• Inter-reader and intra-reader agreement statistics (kappa coefficient). 
• Treatment-emergent adverse events (TEAEs) following 18F-fluciclovine 
injection in the subject population 
6.4.2 
Description of Safety Variables 
Safety will be assessed from the time of 18F-fluciclovine administration until 1 day post-
18F-fluciclovine administration based on reported serious and non-serious adverse events. 
Vital signs, concomitant medications, and pregnancy test results will also be recorded.  
6.4.2.1 
Adverse Events 
An AE is any untoward medical occurrence in a clinical investigation subject 
administered a pharmaceutical product and that does not necessarily have a causal 
relationship with the treatment. An AE is therefore any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom or disease temporally associated 
with the administration of an investigational product, whether or not related to that 
investigational product. An unexpected AE is one of a type not identified in nature, 
severity, or frequency in the current Investigator’s Brochure or is of greater severity or 
frequency than expected based on the information in the Investigator’s Brochure.  
The investigator will probe, via discussion with the subject, for the occurrence of AEs 
during each subject visit within the AE collection period. The collection period for all 
AEs will begin after initiation of 18F-fluciclovine injection and end 1 day post 18F-
fluciclovine administration (i.e., Day 2). Any unresolved AE at Visit 3 Safety Follow-up 
beyond Day 2 will be followed until resolution or stabilization. The investigator will 
record the information in the site’s source documents. Adverse events will be recorded in 
the subject electronic Case Report form (eCRF). Adverse events will be described by 
duration (start and stop dates and times), severity, outcome, treatment, and relation to 
study drug. 
6.4.2.2 
Vital Signs 
Vital signs including body temperature, blood pressure, pulse and respirations will be 
collected at Visit 2 (IMP injection + PET) after resting for at least 5 minutes before IMP 
injection, and after resting for at least 5 minutes following PET scan. The vital signs 
should be collected between 5 to 60 minutes before and after the PET scan. 
6.4.2.3 
Concomitant Medication 
All concomitant medication and concurrent therapies will be documented at Screening 
(Visit 1), at IMP administration (Visit 2) and at the post-treatment Safety Follow-up 
phone call (Visit 3), and at early termination when applicable. In addition to recording all 
concomitant medications as described, treatments and therapies for the brain lesion(s) and 
primary tumor will be recorded through Month 6 (Visit 5). Dose, route, frequency of 


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administration, and indication for administration and dates of medication will be 
captured. 
6.4.2.4 
Laboratory Parameters 
A human chorionic gonadotropin (HCG) urine pregnancy test will be obtained from 
female subjects who are of childbearing age prior to their participation in the study at 
Screening, and again on Visit 2 before IMP administration (or on day prior to IMP 
administration). 
6.5 
Data Quality Assurance 
Report summaries will be generated using validated Base SAS software, version 9.4 or 
higher, on a PC or server-based platform. Additional validated software may be used to 
generate analyses, as needed.  
All SAS programs that create outputs or supporting analysis datasets will be validated by 
a second statistical programmer or biostatistician. At a minimum, validation of programs 
will consist of a review of the program log, review of output or dataset format and 
structure, and independent confirmatory programming to verify output results or dataset 
content. Additionally, all outputs will undergo a review by a senior level team member 
before finalization. 
The content of the source data will be reviewed on an ongoing basis by project statistical 
programmers and statisticians. Data will be checked for missing values, invalid records, 
and extreme outliers through defensive programming applications, analysis-based edit 
checks, and other programmatic testing procedures. All findings will be forwarded to the 
project data manager for appropriate action and resolution.  
7 STATISTICAL METHODS 
7.1 
General Methodology 
Data will be analyzed by Precision for Medicine biostatistics personnel. Statistical 
analyses will be reported with tables and listings, presented in rich text format, and using 
recommended ICH numbering. Output specifications for all tables and listings will be in 
conformance with guidelines specified by the ICH in Appendix 7 of the Electronic 
Common Technical Document Specification (Apr 2003).  
7.1.1 
Reporting Conventions 
Tables will be summarized for all subjects in the specified analysis set. In general, all 
data collected on the eCRF plus data received by Precision for Medicine from the image 
management system, and any derived data will be presented in subject data listings, for 
all enrolled subjects. Listings will be ordered by subject number, and assessment or event 
date.  


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In general, continuous variables will be summarized to indicate the study population 
sample size (N), number of subjects with available data (n), mean, SD, median, 
minimum, and maximum values. Categorical variables will be summarized by the 
population size (N), number of subjects with available data (n), number of subjects in 
each category, and the percentage of subjects in each category. Unless otherwise noted, 
the denominator to determine the percentage of subjects in each category will be based on 
the number of subjects in the specified analysis set.  
Non-zero percentages will be rounded to one decimal place. Rounding conventions for 
presentation of summary statistics will be based on the precision of the variable of 
summarization, as it is collected in its rawest form (i.e., on the eCRF or as provided 
within an external file) and are outlined as follows: 
• The mean and median will be rounded to one more decimal place than the 
precision of the variable of summarization; 
• Standard deviation will be rounded to two more decimal places than the 
precision of the variable of summarization; and 
• Minimum and maximum values will be presented using the same precision as 
the variable of summarization. 
Other statistics (e.g., CIs) will be presented using the same general rules outlined above, 
or assessed for the most appropriate presentation based on the underlying data. 
7.1.2 
Standard Calculations 
Where appropriate, the calculated study day of each assessment or event will be 
presented with the assessment or event date on subject data listings, where study day will 
be determined as: 
• The assessment/event date minus the date of IMP, if the assessment/event date 
is prior to the date of IMP; and 
• The assessment/event date minus the date of IMP, plus one, if the 
assessment/event date is on or after the date of IMP. 
7.2 
Analysis Sets 
The Full Analysis Set (FAS) will include all enrolled subjects dosed with IMP with an 
evaluable Visit 2 on-study PET scan. The FAS will be used for primary and secondary 
non-safety endpoints (including diagnostic performance and inter-reader and intra-reader 
assessment for the PET scans).   
The Safety Analysis Set (SAF) will include all subjects dosed with IMP (with or without 
the associated PET scan). The SAF will be used for all safety analyses.    


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7.3 
Study Subjects 
7.3.1 
Disposition of Subjects 
Subject disposition will be summarized for all subjects who entered the study. Summaries 
will include the number and percentage of subjects in each analysis set, completing the 
study, and discontinuing the study early by the primary reason for discontinuation. 
Withdrawals relating to coronavirus disease of 2019 (COVID-19) will be included. 
7.3.2 
Protocol Deviations 
Deviations from the protocol, including those specifically relating to COVID-19, and 
relevant details will be tracked throughout the study and listed. Critical and major 
deviations will be summarized for the full analysis set. 
7.3.3 
Demographic and Other Baseline Characteristics 
Demographic variables including age, sex, ethnicity and race will be summarized for the 
FAS and SAF. Age will be as recorded on the eCRF and will not be re-calculated. 
Age will be summarized using descriptive statistics, and age group (<65 years old, ≥65 
years old). Sex, ethnicity, and race will be summarized with the number and percentage 
of subjects in each parameter category.   
Baseline characteristics include height, weight, body mass index (BMI), Eastern 
cooperative oncology group (ECOG) performance status, medical history, and prior 
treatment history. BMI will be calculated as: weight (kg) / [height (cm) / 100]2. Height, 
weight, BMI and ECOG will be summarized for the FAS and SAF; prior treatment 
history and medical history will be summarized for the FAS only. Height, weight, and 
BMI at baseline will be summarized using descriptive statistics. ECOG performance 
status will be summarized with the number and percentage of subjects with each score (0 
to 4).  
The number of subjects with confirmed equivocal reference lesions by central 
neuroradiologist will be summarized. 
The prevalence data (% positive reference lesions according to SoR) will be summarized. 
Primary tumor origin and the corresponding details such as disease staging and molecular 
profiles, where applicable, will be summarized, along with the extra-cranial (systemic) 
disease status and time since first known date of diagnosis of brain metastasis(es). 
Primary tumor origin will be categorized as Lung Cancer (non-small cell lung cancer 
[NSCLC] + small cell lung cancer [SCLC]), NSCLC, SCLC, Breast Cancer, Melanoma, 
and Other (colorectal, pancreatic, kidney, testes, cervix, ovary, head & neck, sarcoma, 
other). Time (months) since first known date of diagnosis will be calculated as (screening 
date – first known date of diagnosis of brain metastasis(es))/30.4375. If the date of 
diagnosis is a partial date and day is not available, then day will be imputed as the 1st of 


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the month for the purpose of the calculation. If day and month are both not available then 
the 1st January will be imputed. 
Prior cancer treatments for primary tumor will be summarized with the number and 
percentage of subjects in each of the following categories:  
• Surgery 
• Lymph nodes dissection 
• Radiotherapy 
• Other ablative techniques 
• Drug therapy  
For the drug therapy category, each drug will be coded using the World Health 
Organization drug dictionary, WHODrug Global B3 Mar. 1, 2020, grouped into one of 
the following categories and summarized along with preferred term. The grouped 
categories will be provided for analysis by a medical expert: 
• Chemotherapy 
• Immunotherapy 
• Targeted Therapy 
• Other Therapy 
Similarly, concomitant cancer treatments for primary tumor will be summarized by type 
of treatment (Chemotherapy, Immunotherapy, Targeted Therapy, Other Therapy) and 
preferred term. The data will come from the Concomitant Medication and Cancer Drug 
Therapies page, and will include data with the indication for “Treatment for Primary 
Cancer”. 
For prior therapies for brain lesions considered as equivocal (i.e. study lesions), the 
number and percentage of subjects with the following radiotherapies will be summarized: 
• Stereotactic radiosurgery 
• Whole brain radiotherapy 
• Prophylactic cranial irradiation 
• Other 
The number of lesions (reference lesions and other equivocal lesions) that have 
undergone stereotactic radiosurgery will also be counted. The number of subjects and 
lesions that have undergone laser interstitial thermal therapy (LITT) will be counted. 
The number and percentage of subjects with other prior therapies for any non-study 
lesions, will be summarized using the following categories: 
• Craniotomy 


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• Laser interstitial thermal therapy 
• Stereotactic radiosurgery 
• Whole brain radiotherapy 
• Prophylactic cranial irradiation 
• Other 
Medical history terms on case report forms will be mapped to preferred terms and system 
organ classes using the Medical Dictionary for Regulatory Activities (MedDRA, version 
23.0) and will be summarized by system organ class and preferred term. Medical history 
data will be listed and include start date, end date, ongoing at study start (including 
grade), preferred term and system organ class. 
7.4 
Efficacy Evaluation 
7.4.1 
Datasets Analyzed 
All efficacy summaries will be based on the FAS. A data listing of subjects excluded 
from the FAS, to include the reason for exclusion, will be presented. 
7.4.2 
Measurements of Treatment Compliance 
Not applicable. This is a single dose study, administered by site staff. 
7.4.3 
Primary Efficacy Endpoint Analysis Methods 
The primary endpoint for the study is subject-level NPA and PPA (equivalent to 
specificity and sensitivity, respectively) of 18F-fluciclovine PET in detecting recurrent 
brain metastases. 
Subject-level NPA and PPA will be calculated based on BIE of 18F-fluciclovine PET 
compared to SoR on the reference lesion. Imputations for SoR indeterminate findings 
will be performed according to section 7.4.5.1.  
The analyses for NPA and PPA will be performed using the one-sample binomial exact 
test to test the following hypotheses: 
NPA 
• H0(NPA): NPA of PET ≤NPA0 
• H1(NPA): NPA of PET >NPA0 
PPA 
• H0(PPA): PPA of PET ≤PPA0 
• H1(PPA): PPA of PET >PPA0 


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where NPA0 and PPA0 are performance goals for NPA and PPA, respectively. 
Performance goals of 50% (NPA0) for NPA and 50% (PPA0) for PPA have been selected 
as acceptable thresholds given the setting where SoC imaging is already equivocal.  
Exact two-sided 95% CIs according to the Clopper-Pearson method will be provided. If 
the pre-defined NPA and PPA goals are met by the same two of three readers (both tests 
have lower limits for the exact 95% confidence intervals greater than 50% for the same 
two readers), the study will be considered to have successfully demonstrated the 
effectiveness of 18F-fluciclovine PET in detecting recurrent brain metastases. 
7.4.4 
Secondary Endpoint Analysis Methods  
7.4.4.1 
Subject-Level Positive Predictive Value / Negative Predictive Value 
Subject-level PPV and NPV of 18F-fluciclovine PET for detecting recurrent brain 
metastases will be calculated based on BIE of 18F-fluciclovine PET compared to SoR on 
the reference lesion. The point estimates for PPV and NPV, together with the 95% 2-
sided exact confidence interval (Clopper-Pearson method) will be presented. 
7.4.4.2 
Lesion-Level Diagnostic Performance 
Lesion-level diagnostic performance (lesion-level PPA, NPA, PPV and NPV) will be 
calculated based on BIE of 18F-fluciclovine PET compared to SoR on all lesions 
(reference, other equivocal and additional PET lesions) as determined by CTP (i.e. if CTP 
decides that the lesion SoR is indeterminate, then the lesion will not be counted in this 
lesion-level diagnostic performance). Point estimates and 95% 2-sided confidence 
intervals will be presented. 
The confidence interval will be calculated based on the methodology described by Zhou et 
al, for lesion-level PPA, NPA, PPV and NPV. The formula for PPV is: 
 
𝑃𝑃𝑉
̂ = ∑𝑇𝑃𝑝
𝑃
∑𝑃𝑜𝑠𝑝
𝑃
⁄
 
𝑉𝑎𝑟
̂ (𝑃𝑃𝑉
̂) =
1
𝑃(𝑃−1) ∑(𝑃𝑜𝑠𝑝
𝑃𝑜𝑠
̅̅̅̅̅
⁄
)
2
(𝑃𝑃𝑉𝑝
̂ −𝑃𝑃𝑉
̂)
2
𝑃
 
 
With:  
 
P 
 
 
: (number of) Patients 
 
p 
 
 
: individual patient 
 
Posp 
 
 
: Number of positive regions for patient p 
 
TPp 
 
 
: Number of true positive regions for patient p 
 
𝑃𝑜𝑠
̅̅̅̅̅ = ∑𝑃𝑜𝑠𝑝𝑃
⁄
𝑃
 
: Mean cluster size 
 
𝑃𝑃𝑉𝑝
̂ = 𝑇𝑃𝑝𝑃𝑜𝑠𝑝
⁄
 
: Patient PPV 
The example in Zhou et al. is coded in SAS in Appendix 1. 


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7.4.4.3 
Subgroup Analysis of Diagnostic Performance Data 
Subgroup analyses of subject-level PPA, NPA, PPV, and NPV of 18F-fluciclovine PET, 
will also be presented. Subgroups will be as follows, and the calculations will only be 
performed when there are at least 10 observations in the subgroup: 
Age Group: 
• <65 years old 
• ≥ 65 years old 
Sex: 
• Male 
• Female 
Race: 
• American Indian or Alaska Native 
• Asian 
• Black or African American 
• Native Hawaiian or Other Pacific Islander 
• White 
Primary Tumor Type: 
• Melanoma 
• Breast 
• Lung Cancer (NSCLC and SCLC) 
• Gastrointestinal (Colorectal and Pancreatic) 
• Genitourinary (Bladder, Kidney, Testes) 
• Non-Lung/Breast/Melanoma 
Concurrent Immunotherapy: 
• Received Concurrent Immunotherapy 
• Did Not Receive Concurrent Immunotherapy 


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7.4.4.4 
Clinical Usefulness 
7.4.4.4.1 
Number of Days taken by the site to establish presence/absence of 
metastasis by clinical follow-up 
The number of days taken by the site to establish presence/absence of metastasis for each 
lesion, by clinical follow up, will be defined as the time from administration of 18F-
fluciclovine to the date the presence or absence of metastasis is determined in each lesion 
by the site. Analysis will be performed at the lesion-level (including all lesions - 
reference, other equivocal, additional PET) recorded on the lesion annotation page of the 
eCRF, and at the subject-level (based on the reference lesion only). 
If the presence or absence of metastasis is not ascertained by the time the subject reaches 
the end of the follow-up or when the subject prematurely withdraws from the study, the 
lesion/subject will be censored on the date the subject withdraws from the study. The 
number of diagnoses ascertained and the number censored will be summarized. Kaplan-
Meier methodology will be used to handle the censoring of the data, and to estimate the 
median time to establish presence/absence of metastasis. The range of days taken to 
establish presence/absence will be presented for all lesions/subjects and also for 
uncensored lesions/subjects. 
7.4.4.4.2 
Proportion of subjects with additional metastases identified on 18F-
fluciclovine PET in addition to standard of care brain MRI.  
Subjects with additional metastases identified on 18F-fluciclovine PET but not on SoC 
MRI are those with at least one lesion with type = ‘Additional PET Lesion’ on the lesion 
annotation eCRF page, which has SoR = ‘positive’ according to the CTP. Lesions that are 
deemed to have SoR = ‘indeterminate” by CTP will not be considered as additional 
metastases. 
The estimate of the proportion of subjects and the associated 95% 2-sided exact 
confidence interval (Clopper-Pearson) will be presented. 
7.4.4.4.3 
Proportion of subjects whose prospective diagnostic management plan 
changes following 18F-fluciclovine PET. 
The proportion of subjects whose prospective diagnostic management plan changes 
following 18F-fluciclovine PET will be established by comparing the diagnostic 
management plan of the reference lesion before and after 18F-fluciclovine PET.  
The intended diagnostic management plan on the reference lesion will be recorded at visit 
1 as craniotomy, biopsy, biopsy in context of LITT, or clinical follow-up. At visit 2, post 
18F-flucicolvine PET, the diagnostic plan will be recorded again. If a different option has 
been chosen, or if there are any additional/new subject management activities planned 
then this will be counted as a change.   
The estimate of the proportion of subjects and the associated 95% 2-sided exact 
confidence interval (Clopper-Pearson) will be presented. 


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7.4.4.5 
Inter-Reader / Intra-Reader Agreement (Blinded Image Evaluation) 
For inter-reader agreement, pairwise comparisons of the 18F-fluciclovine PET scan 
central reads for the 3 readers (i.e. Reader 1 vs Reader 2, Reader 1 vs Reader 3, and 
Reader 2 vs Reader 3) will be performed at the subject-level (i.e., reference lesion only) 
and also at the lesion-level (i.e., all lesions defined by site). The number and percentage 
of each combination of results (Positive [1st reader] / Positive [2nd reader], 
Positive/Negative, Negative/Positive, Negative/Negative) will be presented, along with 
the number and percentage of results in agreement and disagreement. Cohen’s kappa 
statistic will be calculated and presented for each pairwise comparison of the 3 readers, 
with the corresponding 95% CI and p-value. 
Similar statistics will be presented for intra-reader agreement (10% of subjects) – 
comparisons will be between the initial read vs re-read of a subset of PET scans for each 
reader. 
7.4.4.6 
Sensitivity Analyses 
7.4.4.6.1 
Analysis 1: Subject-Level NPA and PPA on subjects with 
histopathology or follow-up MRI prior to CNS-directed therapy 
Subject-level NPA and PPA (equivalent to specificity and sensitivity, respectively) of 
18F-fluciclovine PET in detecting recurrent brain metastases will be calculated for 
subjects who either have histopathology available, or have follow-up MRI acquired 
before new or altered CNS-directed therapy and have lesions with at least 20% increase 
or 30% decrease in the longest diameter relative to the SoC baseline MRI.  
The SoR will be based on CTP determination on the two corresponding domains: 
histopathology and on MRI findings before new or altered CNS-directed therapy. 
• SoR will be positive if one domain is positive and the other domain is positive or 
non-contributory. 
• SoR will be negative if one domain is negative and the other domain is negative 
or non-contributory. 
• SoR will be indeterminate if both domains are non-contributory, or if one domain 
is positive and the other domain is negative. 
7.4.4.6.2 
Analysis 2: Subject-Level NPA and PPA on subjects without 
contributory MRI findings after new or altered CNS-directed therapy 
Subject-level NPA and PPA (equivalent to specificity and sensitivity, respectively) of 
18F-fluciclovine PET in detecting recurrent brain metastases will be calculated for 
subjects who: 
• have no CNS-directed therapy during follow-up, or 
• have non-contributory findings in MRIs acquired after new or altered CNS-
directed therapy. 




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To check the validity of the results based on the site’s determination of the reference 
equivocal lesions, these lesions will be measured and confirmed by the central 
neuroradiologist. A sensitivity analysis on the main endpoint of subject level PPA and 
NPA, using only subjects with central neuroradiologist confirmation of the reference 
equivocal lesions, will be performed if there is discrepancy between the site confirmation 
of reference equivocal lesions vs the central neuroradiologist confirmation. 
For lesion level analysis, none of the lesions will be imputed if the SoR designation from 
the CTP is missing or indeterminate. Lesion-level analysis will only include lesions with 
SoR designation of positive or negative by the CTP. 
7.4.5.2 
Interim Analyses and Data Monitoring 
Not applicable. 
7.4.5.3 
Multicenter Studies 
This is a multicenter study, with approximately 18 sites expected to participate. Data 
collected from all study sites will be pooled for data analysis.  
7.4.5.4 
Multiple Comparisons/Multiplicity 
There will be no adjustments for multiple comparisons in the efficacy analysis for this 
study.  
7.4.5.5 
Examination of Subgroups 
Subgroup analysis will be performed as described in section 7.4.4.3. 
7.5 
Safety Evaluation 
Safety analysis will be carried out for the Safety analysis set, to include all subjects who 
have been dosed with IMP. Subjects who do not complete the study, for whatever reason, 
will have all available data up until the time of termination included in the analysis.  
7.5.1 
Extent of Exposure 
Each subject will receive a single dose of 18F-fluciclovine by injection, 185 MBq (5 mCi) 
± 20%, delivered as an intravenous bolus by site staff. IMP administration data will be 
summarized, and details of administration will also be listed. 
7.5.2 
Adverse Events 
Treatment-emergent adverse events are defined as those AEs with onset after the dose of 
IMP, or existing events that worsened after the dose of IMP during the study, up to 1 day 
following IMP administration. Events reported with a partial onset date (e.g., month and 
year are reported but the day is missing) will be considered to be treatment-emergent if it 


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cannot be confirmed that the event onset was prior to the dose of IMP based on the 
available date entries. 
Adverse event verbatim terms will be coded to preferred terms and system organ classes 
using the latest version of MedDRA, version 23.0. Severity of AEs will be graded using 
the National Cancer Institute common terminology criteria for adverse events (NCI-
CTCAE) v5.0. 
Summaries that are displayed by system organ class (SOC) and preferred term (PT) will 
be ordered by descending incidence of SOC and PT within each SOC, and then 
alphabetically for SOC, and PT within SOC. Summaries of the following types will be 
presented: 
• Overall summary of subject incidence of TEAEs by severity, relationship, and 
action taken, subject incidence of treatment-emergent serious adverse events 
(TESAEs) by relationship; 
• Subject incidence of TEAEs by MedDRA system organ class and preferred 
term; 
• Subject incidence of TEAEs by NCI-CTCAE grade, MedDRA system organ 
class, and preferred term; 
• Subject incidence of TEAEs related to IMP by MedDRA system organ class, 
and preferred term;  
• Subject incidence of TESAEs by MedDRA system organ class and preferred 
term; 
• Subject incidence of TESAEs related to IMP by MedDRA system organ class 
and preferred term; 
At each level of summarization (e.g., any AE, SOC, and PT), subjects experiencing more 
than one TEAE will be counted only once. For each subject and each adverse event, the 
worst severity recorded will be attributed and used in the by-severity summaries.   
A by-subject listing of all adverse events (including non-treatment-emergent events) will 
be provided.  This listing will include: subject identifier, age, sex, race, adverse event 
(SOC, PT, and verbatim term), date of onset and study day of onset, date of resolution 
and study day of resolution, duration, severity, seriousness, action taken, outcome and 
causality. 
7.5.3 
Deaths, Other Serious Adverse Events, and Other Significant Adverse 
Events 
The following listings will be created: 
• A by-subject listing of all deaths and cause of death that occurred during the 
study, including the 6-month follow-up 
• A by-subject listing of all serious adverse events 


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Listings will follow the format described for adverse events in Section 7.5.2. 
7.5.4 
Clinical Laboratory Evaluation 
Urine pregnancy test results will be listed. 
7.5.5 
Vital Signs 
Vital signs recorded values and their respective changes from the pre-dose values will be 
summarized using descriptive statistics. All results will be listed. 
7.5.6 
Concomitant Medications 
Medications will be coded using WHODrug Global B3 Mar. 1, 2020. 
 
Medications will be considered as prior medications and concomitant medications based 
on the following rules: 
Medication Start date 
Medication Stop date 
Prior 
Medication 
Concomitant 
Medication 
Prior to date of IMP 
Prior to date of IMP 
Yes 
No 
Prior to date of IMP 
On or after date of IMP 
Yes 
Yes 
On or after date of IMP 
Any 
No 
Yes 
The following imputation rules will be used for determining prior/concomitant 
medications if the medication start or stop dates are incomplete: 
• If medication start and/or stop dates are missing or partial, the dates will be 
compared as far as possible with the date of IMP.  Medications will be assumed to 
be Concomitant only, unless there is clear evidence (through comparison of 
partial dates) to suggest that the medication started prior to the dose of IMP.   
• If there is clear evidence to suggest that the medication started prior to the dose of 
IMP, the medication will be assumed to be both Prior and Concomitant, unless 
there is clear evidence to suggest that the medication stopped prior to the dose of 
IMP.   
• If there is clear evidence to suggest that the medication stopped prior to the dose 
of IMP, the medication will be assumed to be Prior only. 
All prior and concomitant medications will be listed by subject.  Concomitant 
medications will be summarized by anatomical therapeutic chemical (ATC) Class (level 
4) and generic drug name, using the safety analysis set. If level 4 is not available then 
level 3 will be used; if level 3 and 4 are not available then level 2 will be used. 


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Concomitant drug therapies relating to the treatment or corticosteroids for brain 
metastasis / lesions will be summarized separately, in the same way described above. The 
data will come from the Concomitant Medication and Cancer Drug Therapies eCRF 
page, and will include data with the indication for “Treatment for Brain Metastasis / 
Lesion” or “Corticosteroids for Brain Metastasis / Lesion”. 
7.6 
Determination of Sample Size 
Approximately 150 subjects will be enrolled, to obtain 130 evaluable subjects. 
A one-sample binomial exact test will be used to test the following hypotheses in NPA 
and PPA of 18F-fluciclovine PET: 
 
NPA 
• H0(NPA): NPA of PET ≤NPA0 
• H1(NPA): NPA of PET >NPA0 
PPA 
• H0(PPA): PPA of PET ≤PPA0 
• H1(PPA): PPA of PET >PPA0 
Where NPA0 and PPA0 are performance goals for NPA and PPA, respectively.  
Performance goals of 50% (NPA0) for NPA and 50% (PPA0) for PPA were selected as 
acceptable thresholds given the setting where SoC imaging is already equivocal. The 
lower limits for the exact 95% confidence intervals (Clopper-Pearson method) for both 
NPA and PPA should be greater than 50%. Assuming a NPA of at least 70% and PPA of 
at least 70% for 18F-fluciclovine PET would need to be observed, a sample size of 130 
would achieve 90% power at a 1-sided significance level of 0.025 to reject the 
performance goals for both NPA and PPA; assuming a brain metastasis prevalence rate of 
0.5.  
7.7 
Changes in the Conduct of the Study or Planned Analyses 
There were no changes to the study conduct or planned analyses identified within the 
development of this SAP, relative to the descriptions provided within the clinical study 
protocol. 
8 REFERENCE LIST 
Zhou XH, Obuchowski NA and McClish, DK. Statistical Methods in Diagnostic 
Medicine. Wiley, New York, 2002 pp 104-6 
 


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Appendix 1: SAS program to reproduce data in Zhou et al. 
data one; 
input TN 
No_Polyps; 
Cards; 
1 
1 
2 
2 
2 
2 
1 
1 
2 
2 
2 
2 
1 
1 
1 
1 
1 
1 
1 
1 
2 
2 
0 
1 
2 
3 
2 
2 
1 
1 
1 
1 
1 
1 
2 
2 
1 
2 
0 
2 
1 
1 
2 
2 
2 
2 
2 
2 
0 
1 
;;;; 
run; 
 
proc sql; 
   create table two as 
      select  
         TN , 
         No_Polyps, 
         TN/No_Polyps as Sei_hat, 
         Se_hat, 
         No_Polyps/mNo as Ni_N, 
         (calculated Ni_N)*(calculated Ni_N)  
             * (calculated Sei_hat-Se_hat)* (calculated Sei_hat-Se_hat) as fc, 
         sum(calculated fc) as sfc, 
         calculated sfc/(n*(n-1)) as varSe_hat 
      from  
         (select  
            sum(TN) as sTN,  
            sum(No_Polyps) as sNo, 
            mean(No_Polyps) as mNo, 
            count(*) as n, 
            calculated sTN/calculated SNo as Se_hat 
            from one ) as o,  
         one; 
quit;  
 
data final; 
   set two; 
   if _n_=1; 
   keep Se_hat varSe_hat; 
run;