Conditions: Brain Metastases
Keywords: Recurrent Brain Metastases Diagnostic Positron Emission Tomography (PET) Scan
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 23
Healthy volunteers: no

Brief summary:
An Open-label, Single-arm, Single-dose, Prospective, Multicenter Phase 2b Study to Establish Image Interpretation Criteria for 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 MegaBecquerel (MBq) (5 Millicurie (mCi)) ± 20%, delivered as an intravenous bolus

Protocol document:
Prot_002.pdf: 
BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 1 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
 
 
 
CLINICAL STUDY PROTOCOL 
 
An Open-label, Single-arm, Single-dose, Prospective, Multicenter Phase 2b Study to 
Establish Image Interpretation Criteria for 18F-fluciclovine Positron Emission 
Tomography (PET) in Detecting Recurrent Brain Metastases after Radiation Therapy 
(PURSUE) 
 
BED-FLC-219 
 
Phase: 2b 
 
IND Number: 145265 
 
Protocol Version and Date: Protocol Version 3.0, 10 June 2021 
 
 
 
Sponsor: Blue Earth Diagnostics 
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. 
 
 
 




BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 3 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
CONTENTS 
LIST OF ABBREVIATIONS ....................................................................................... 7 
PROTOCOL SYNOPSIS ........................................................................................... 10 
STUDY SCHEMA...................................................................................................... 17 
1. 
BACKGROUND ..................................................................................... 18 
1.1. 
Overview of Clinical Studies ................................................................... 19 
2. 
STUDY RATIONALE ............................................................................ 20 
2.1. 
Risk-Benefit Assessment ......................................................................... 22 
2.1.1. 
Benefit ...................................................................................................... 22 
2.1.2. 
Risk .......................................................................................................... 22 
3. 
STUDY OBJECTIVES ........................................................................... 23 
3.1. 
Primary Objective .................................................................................... 23 
3.2. 
Secondary Objectives .............................................................................. 23 
4. 
STUDY DESIGN .................................................................................... 24 
4.1. 
Study Overview ....................................................................................... 24 
4.2. 
Concurrent Enrollment with BED-FLC-312 ........................................... 25 
5. 
CRITERIA FOR EVALUATION ........................................................... 26 
5.1. 
Primary Efficacy Endpoint ...................................................................... 26 
5.2. 
Secondary Efficacy Endpoints ................................................................. 26 
5.3. 
Safety Evaluations ................................................................................... 26 
6. 
SUBJECT SELECTION .......................................................................... 27 
6.1. 
Study Population ...................................................................................... 27 
6.2. 
Inclusion Criteria ..................................................................................... 27 
6.3. 
Exclusion Criteria .................................................................................... 28 
7. 
CONCURRENT MEDICATIONS .......................................................... 29 
7.1. 
Allowed Medications and Treatments ..................................................... 29 
7.2. 
Prohibited Medications and Treatments .................................................. 29 
8. 
STUDY TREATMENTS......................................................................... 30 
8.1. 
Method of Assigning Subjects to Treatment Groups .............................. 30 
8.2. 
Blinding ................................................................................................... 30 
8.3. 
Formulation of Test Product .................................................................... 30 
8.4. 
Packaging and Labelling .......................................................................... 30 
8.5. 
Supply of Study Drug at the Site ............................................................. 30 
8.6. 
Dosage/Dosage Regimen ......................................................................... 30 
8.7. 
Dispensing ............................................................................................... 31 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 4 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
8.8. 
Administration Instructions ..................................................................... 31 
8.9. 
Storage ..................................................................................................... 31 
8.10. 
Study Drug Accountability ...................................................................... 31 
8.11. 
Measures of Treatment Compliance ........................................................ 32 
8.12. 
End Of Trial ............................................................................................. 32 
9. 
STUDY PROCEDURES AND GUIDELINES....................................... 33 
9.1. 
Clinical Assessments ............................................................................... 36 
9.1.1. 
Concomitant Medications ........................................................................ 36 
9.1.2. 
Demographic and Baseline Characteristics ............................................. 36 
9.1.3. 
ECOG Performance Status ...................................................................... 36 
9.1.4. 
Medical History ....................................................................................... 36 
9.1.5. 
Imaging Studies ....................................................................................... 36 
9.1.6. 
Vital Signs ............................................................................................... 38 
9.1.7. 
SoC Craniotomy / Surgery....................................................................... 38 
9.2. 
Clinical Laboratory Measurements.......................................................... 38 
9.2.1. 
Pregnancy Test ......................................................................................... 38 
10. 
EVALUATIONS BY VISIT ................................................................... 39 
10.1. 
Visit 1 Screening .................................................................................... 39 
10.2. 
Visit 2- Day 1: IMP Administration and 18F-fluciclovine PET Scan (1 to 
28 days after Screening) .......................................................................... 40 
10.3. 
Visit 2  Day 1 or up to 3 days after 18F-fluciclovine Administration: On-
Study MRI Brain Scan ............................................................................. 40 
10.4. 
Visit 3  Safety Follow-up (+1 to 3 days after IMP Injection/PET) ....... 41 
10.5. 
Visit 4  SoC Craniotomy (Pre-planned, 1 to 21 days after IMP 
Injection/PET) .......................................................................................... 41 
10.6. 
Early Withdrawal ..................................................................................... 42 
11. 
IMAGING PROTOCOL ......................................................................... 43 
11.1. 
18F-fluciclovine PET Scan Patient Preparation ........................................ 43 
11.2. 
PET/CT Scanner ...................................................................................... 43 
11.3. 
18F-fluciclovine Injection Administration................................................ 43 
11.4. 
18F-fluciclovine PET Acquisition ............................................................ 43 
11.5. 
On-Study MRI Acquisition...................................................................... 43 
11.6. 
Image Transfer ......................................................................................... 43 
11.7. 
Image Evaluability ................................................................................... 43 
11.8. 
Central Image Analysis ............................................................................ 43 
12. 
ADVERSE EVENT REPORTING AND DOCUMENTATION ............ 46 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 5 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
12.1. 
Adverse Events ........................................................................................ 46 
12.2. 
AE Severity .............................................................................................. 46 
12.3. 
Adverse Event Relationship to IMP ........................................................ 47 
12.4. 
Serious Adverse Event (SAE) ................................................................. 47 
12.4.1. 
Serious Adverse Event Reporting ............................................................ 47 
12.4.2. 
Suspected Unexpected Serious Adverse Reactions (SUSAR) ................ 47 
12.4.3. 
Pregnancy ................................................................................................ 48 
13. 
DISCONTINUATION AND REPLACEMENT OF SUBJECTS .......... 49 
13.1. 
Discontinuation/Withdrawal from the Study ........................................... 49 
13.2. 
Replacement of Subjects .......................................................................... 49 
13.3. 
Lost to Follow-up .................................................................................... 49 
14. 
PROTOCOL VIOLATIONS ................................................................... 51 
15. 
STATISTICAL METHODS AND CONSIDERATIONS ...................... 52 
15.1. 
Estimation of Sample Size ....................................................................... 52 
15.2. 
Analysis Sets ............................................................................................ 52 
15.3. 
Subject Disposition .................................................................................. 52 
15.4. 
Demographic and Baseline Characteristics ............................................. 52 
15.5. 
Analysis of Primary Endpoint ................................................................. 52 
15.6. 
Analysis of Secondary Endpoint(s) ......................................................... 53 
15.7. 
Planned Interim Analysis ......................................................................... 54 
16. 
DATA COLLECTION, RETENTION AND MONITORING ............... 55 
16.1. 
Data Collection Instruments .................................................................... 55 
16.2. 
Data Management Procedures ................................................................. 55 
16.3. 
Data Quality Control and Reporting ........................................................ 55 
16.4. 
Archiving of Data .................................................................................... 56 
16.5. 
Availability and Retention of Investigational Records ............................ 56 
16.6. 
Monitoring and Auditing ......................................................................... 56 
16.7. 
Subject Confidentiality ............................................................................ 56 
17. 
REGULATORY, ETHICAL AND STUDY OVERSIGHT 
CONSIDERATIONS ............................................................................... 58 
17.1. 
Institutional Review Boards and Independent Ethics Committees.......... 58 
17.2. 
Amendments ............................................................................................ 58 
17.3. 
Subject Information and Consent/Assent ................................................ 58 
17.4. 
Post-trial Care .......................................................................................... 59 
17.5. 
Publications .............................................................................................. 59 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 6 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
17.6. 
Investigator Responsibilities .................................................................... 60 
18. 
REFERENCES ........................................................................................ 62 
APPENDIX 1. ECOG PERFORMANCE STATUS................................................ 66 
APPENDIX 2. PROTOCOL AGREEMENT .......................................................... 67 
 
LIST OF TABLES 
Table 1. Schedule of Study Procedures ...................................................................... 34 
Table 2. CTCAE (V5) AE Severity Grading .............................................................. 46 
 
LIST OF FIGURES 
Figure 1. Schema of Study Design ............................................................................. 17 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 7 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
LIST OF ABBREVIATIONS 
ADR 
Adverse drug reaction 
AE 
Adverse event 
ALARA 
As low as reasonably achievable 
ASCT2 
Alanine-serine-cysteine-transporter 2 
BED 
Blue Earth Diagnostics 
CBP 
Child-bearing potential 
CE 
Contrast enhancement 
CLR 
Complete lesion response 
CT 
Computerized tomography 
CTCAE 
Common Terminology Criteria for Adverse Events  
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic Case Report form 
EDC 
Electronic data capture 
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 
HCG 
Human chorionic gonadotropin 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 8 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
ICF 
Informed consent form 
ICH 
International Council for Harmonisation 
IEC 
Independent Ethics Committee 
IIT 
Investigator-initiated trial 
IEAS 
Imaging Evaluable Analysis Set 
IMP 
Investigational medicinal product 
IRB 
Institutional Review Board 
IV 
Intravenous 
LAR 
Legally authorized representative 
LAT1 
L-type Amino Acid Transporter 1 
MBq 
MegaBecquerel 
mCi 
Millicurie 
MedDRA 
Medical Dictionary for Regulatory Activities 
MR 
Magnetic resonance 
MRI 
Magnetic resonance imaging 
NCCN 
National Comprehensive Cancer Network 
NPV 
Negative predictive value 
PET 
Positron emission tomography 
PPV 
Positive predictive value 
RANO 
Response Assessment in Neuro-Oncology 
ROC 
Receiver operating characteristic 
SAE 
Serious adverse event 
SAP 
Statistical Analysis Plan 
SAF 
Safety Analysis Set 
SoC 
Standard of care 
SoT 
Standard of truth 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 9 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
SRS 
Stereotactic radiosurgery 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
SUV 
Standardized uptake value 
SUVmax 
Maximum standardized uptake value 
SUVpeak 
Peak standardized uptake value 
SUVmean 
Mean standardized uptake value 
TAC 
Time activity curve 
TBR 
Tumor background ratio 
TBRmax 
Maximum tumor to background ratio 
TBRpeak 
Peak tumor to background ratio 
USA 
United States of America 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 10 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
PROTOCOL SYNOPSIS 
Study Title 
An open-label, single-arm, single-dose, prospective, multicenter 
Phase 2b study to establish image interpretation criteria for 
18F-fluciclovine positron emission tomography (PET) in detecting 
recurrent brain metastases after radiation therapy (PURSUE) 
Phase 
2b 
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 
patients with solid tumor brain metastases previously treated with 
radiation therapy, designed to define image interpretation criteria 
for 18F-fluciclovine PET in detecting recurrent brain metastases. 
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 magnetic resonance imaging (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, including radiation 
necrosis (Galldiks, 2019). These treatment-related changes have 
similar appearance 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 




BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 12 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
recurrence, and low 18F-fluciclovine uptake in lesions with 
treatment-related change (Patel, 2018).   
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. 
Primary Objectives 
and Outcome 
Measures 
Objective: 
To establish visual image 
interpretation criteria for 
18F-fluciclovine PET in 
detecting recurrent brain 
metastases. 
Outcome Measure: 
Diagnostic performance 
(sensitivity, specificity, positive 
predictive value [PPV] and 
negative predictive value [NPV]) of 
different thresholds of lesion 
18F-fluciclovine uptake on visual 
reads (as defined by the different 
combinations of degree of uptake) 
versus standard of truth (SoT) 
derived from central 
histopathological analysis of all 
lesions, at subject-level and lesion-
level.
Secondary Objectives 
and Outcome 
Measures 
Objectives: 
To establish other image 
interpretation criteria for 
18F-fluciclovine PET in 
detecting recurrent brain 
metastases.  
Outcome Measures: 
Diagnostic performance 
(sensitivity, specificity, PPV and 
NPV) of different thresholds of 
quantitative and dynamic measures 
of lesion 18F-fluciclovine uptake 
versus SoT derived from central 
histopathological analysis of all 
lesions, at subject-level and 
lesion-level.
To assess subject-level 
diagnostic performance of 
18F-fluciclovine PET in 
detecting recurrent brain 
metastases. 
Subject-level sensitivity, 
specificity, PPV and NPV of 
18F-fluciclovine PET for detecting 
recurrent brain metastases, based 
on combined application of the 
established image interpretation 
criteria.


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 13 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
To assess lesion-level 
diagnostic performance of 
18F-fluciclovine PET in 
detecting recurrent brain 
metastases. 
Lesion-level sensitivity, specificity, 
PPV and NPV of 18F-fluciclovine 
PET for detecting recurrent brain 
metastases, based on combined 
application of the established image 
interpretation criteria. 
 
To assess the safety of 
18F-fluciclovine injection in 
the patient population. 
Safety of 18F-fluciclovine injection 
in the patient population. 
Study Sites 
Approximately 10 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: Up to 9 months 
Subject Duration: 2 months per subject (1 day for adverse event 
[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. 
 Screening (Visit 1). 
2. Eastern Cooperative Oncology Group (ECOG) 
performance status 0-2. 
3. 
(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), or in the salvage setting (repeat radiation 
therapy), with or without previous or concurrent systemic 
treatments. 
7. A reference lesion considered by the site investigator to be 
equivocal for recurrent brain metastasis, as determined by 




BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 15 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 a history of 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. 
6. Applicable to subjects actively on the BED-FLC-312 
study: the subject at time of Screening, is within the BED-
FLC-312 Visit 4 window (i.e. up to and including Day 22).
Planned Interim 
Analyses 
Not applicable. 
STATISTICS 
Primary Analysis 
Plan 
Diagnostic performance (sensitivity, specificity, PPV and NPV) 
of different thresholds of lesion 18F-fluciclovine uptake on visual 
reads (as defined by the different combinations of degree of 
uptake) versus SoT derived from central histopathological 
analysis of all lesions, at subject-level and lesion-level, will be 
calculated. Subject-level diagnostic performance will be 
calculated based on reference lesion data, while lesion-level 
diagnostic performance will be calculated based on data of all 
sampled lesions. Receiver operating characteristic (ROC) analysis 
of SoT against the quantitative measures of lesion 18F-fluciclovine 
uptake will be performed. For dynamic measures of lesion 
18F-fluciclovine uptake represented by time activity curve (TAC) 
type, diagnostic performance for each TAC category will be 
calculated. The Image Interpretation Committee will review these 
analyses to establish appropriate image interpretation criteria. 
Subsequently, for the diagnostic performance secondary 
endpoints, sensitivity, specificity, PPV and NPV for a 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 16 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
combination of the established image interpretation criteria will 
be calculated based on the recommended thresholds, using 
standard definitions. 
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, 
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, through 1 day 
following IMP administration. Adverse event verbatim terms will 
be mapped to preferred terms and system organ classes using the 
current Medical Dictionary for Regulatory Activities (MedDRA). 
Severity of AEs will be graded using the NCI- Common 
Terminology Criteria for Adverse Events (CTCAE) v5.0. The 
frequency of TEAEs will be summarized overall, by system organ 
class and preferred term, and by severity grade and relationship to 
IMP. 
Rationale for Number 
of Subjects 
Up to 40 subjects will be enrolled. Sufficient numbers of subjects 
will be included in the study in order to gain sufficient data to 
establish image interpretation criteria. No formal statistical 
hypotheses are stated. 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 17 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
STUDY SCHEMA 
Figure 1. Schema of Study Design 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 craniotomy / surgery.  
Administer Investigational Product and Perform PET 
Scan. Refer to Table 1, Schedule of Study Procedures 
Visit 2: 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:  
Follow-up 
Day 2 to 4 
Day 1 + 1 to 3 days. Follow-up safety contact.  
Refer to Table 1, Schedule of Study Procedures 
Visit 4: SoC 
Craniotomy / 
Surgery 
Day 2 to Day 22 
Pre-planned SoC craniotomy / Neurosurgical Intervention 
1 to 21 days after Day 1 
Final Assessments 
Refer to Table 1, Schedule of Study Procedures 




BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 19 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 patients with 9 lesions and another in 12 patients with 
17 lesions. In the larger study, all patients 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 patients with 
indeterminate MRI, was suggested to be potentially useful in this setting. A retrospective 
analysis of 21 patients 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 patients. 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 
patients 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 patients exposed to 18F-fluciclovine in clinical trials. The 
doses administered range from 51.8 to 485 MBq, with the majority of patients 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%) patients 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 is conducting the current clinical study 
(BED-FLC-219) as the Phase 2 study in a clinical program to support the use of 
18F-fluciclovine PET imaging to detect brain metastases in adults with suspected recurrence 
after radiation therapy.  


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 20 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 US (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 
residual and 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 increase 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 
can be similar to that in normal grey matter. The sensitivity of detection of lesions is further 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 21 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 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; 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 
PET tracer uptake, and in the case of 18F-FET, incorporate the interpretation of dynamic scan 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 22 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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. 
The purpose of the current study is to take initial steps to address this area of diagnostic 
unmet need by defining image interpretation criteria for 18F-fluciclovine PET in detecting 
recurrent brain metastases in adults with suspected recurrence after radiation therapy. 
2.1. 
Risk-Benefit Assessment 
2.1.1. 
Benefit 
The 18F-fluciclovine PET scans may provide further clinical information regarding the 
If such information arises, this may be considered by the responsible clinician to help direct 
 management, including guiding the planned surgery. This may provide a direct 
benefit to the subject. 
2.1.2. 
Risk 
The risks from the imaging studies to patients mainly relate to the intravenous (IV) injection 
and the radiation emitted by the radiopharmaceutical and the 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 
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-
management, there is a potential risk of additional resections/biopsies including those that are 
ultimately unnecessary. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 23 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
3. 
STUDY OBJECTIVES 
3.1. 
Primary Objective 
The primary objective is 
 To establish visual image interpretation criteria for 18F-fluciclovine PET in 
detecting recurrent brain metastases. 
3.2. 
Secondary Objectives 
The secondary objectives are: 
 To establish other image interpretation criteria for 18F-fluciclovine PET in 
detecting recurrent brain metastases. 
 To assess subject-level diagnostic performance of 18F-fluciclovine PET in 
detecting recurrent brain metastases. 
 To assess lesion-level diagnostic performance of 18F-fluciclovine PET in detecting 
recurrent brain metastases. 
 To assess the safety of 18F-fluciclovine injection in the patient population. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 24 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 define image 
interpretation criteria for 18F-fluciclovine PET in detecting recurrent brain metastases (when 
read with standard MRI for anatomical reference). 
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. Sites are encouraged to enroll subjects with a variety 
of primary tumor types. In order to ensure variation in tumor types on the study population, 
the number of subjects with lung cancer will be capped at approximately 60% of enrolled 
subjects. 
On-site investigators will prospectively annotate and measure 
on a 
post-radiation treatment MRI scan and on the SoC MRI brain scan to confirm eligibility. The 
lesion which is: 
 equivocal for recurrent metastasis on SoC MRI (according to inclusion 
criterion #7, see Section 6.2); 
 intended for pre-planned SoC craniotomy; and 
 if >1 equivocal lesion is intended for resection, the largest of these lesions. 
If >1 equivocal lesion is intended for resection, the on-site investigator will also annotate and 
measure 
SoC MRI brain scan, 
defined by the same radiological criteria for a reference lesion per inclusion criterion #7 (see 
Section 6.2). 
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 as anatomical reference for the 18F-fluciclovine PET scan. The 
Visit 2 On-Study MRI should be done 
 days after Visit 
2 PET and completed before the pre-planned SoC craniotomy. A safety follow-up (Visit 3) 
1 to 3 days after Visit 2 PET will be made for AE evaluation by telephone call or in person (if 
same day as scheduled SoC craniotomy). 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 craniotomy. The Visit 2 PET must 
be organized to take place a minimum of 1 day and maximum of 21 days before pre-planned 
SoC craniotomy. 
The on-site investigator will review the 18F-fluciclovine PET scan to identify, annotate and 
measure potential additional lesions, not previously reported on SoC MRI 
. Additional PET lesions should be annotated only where judged suggestive of 
brain metastasis, warranting confirmation according to SoC practice, be neurosurgically 
feasible and accordingly, planned for biopsy or resection. 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 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 25 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 
image registration. 
For each subject, the final visit will usually be the preplanned SoC craniotomy/surgery 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 and will not be 
recorded, including all medications and SoC procedure-related events occurring at and 
beyond Visit 4, and any SoC follow-up.   
Subject-level SoT will be defined by central histopathological diagnosis of the reference 
lesion. Lesion-level SoT will be defined by central histopathological diagnosis of all sampled 
lesions (reference lesions, MRI surgical lesions, additional PET lesions). Separately, central 
image analysis will define qualitative and quantitative lesion uptake of 18F-fluciclovine, for 
each resected lesion. These results will be reviewed by an Imaging Interpretation Committee 
to form image interpretation criteria. 
4.2. 
Concurrent Enrollment with BED-FLC-312 
Subjects who are enrolled in BED-FLC-312 and during its Follow-Up period are deemed to 
require a SoC craniotomy for diagnostic evaluation of an equivocal lesion, may be enrolled 
into BED-FLC-219 while remaining enrolled in BED-FLC-312. Subjects may be enrolled 
any time during the BED-FLC-312 Follow-Up period after Day 22 (i.e., after the full 21-day 
maximum timeframe for completion of the BED-FLC-312 Visit 4 neurosurgical procedure). 
For absolute clarity, this will be irrespective of the applicability of BED-FLC-312 Visit 4 to 
the subject. Such subjects will be required to sign an informed consent form and meet all 
eligibility requirements for BED-FLC-219, including availability of the SoC MRI within the 
specified timeframe and reference lesion definitions. All BED-FLC-219 procedures remain 
applicable to any such concurrently enrolled subjects. Interim enrollment in BED-FLC-219 
nt ongoing participation in BED-FLC-312. 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 26 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
5. 
CRITERIA FOR EVALUATION 
5.1. 
Primary Efficacy Endpoint 
  Diagnostic performance (sensitivity, specificity, positive predictive value [PPV] 
and negative predictive value [NPV]) of different thresholds of lesion 
18F-fluciclovine uptake on visual reads (as defined by the different combinations 
of degree of uptake) versus SoT derived from central histopathological analysis of 
all lesions, at subject-level and lesion-level. 
5.2. 
Secondary Efficacy Endpoints 
 Diagnostic performance (sensitivity, specificity, PPV and NPV) of different 
thresholds of quantitative and dynamic measures of lesion 18F-fluciclovine uptake 
versus SoT derived from central histopathological analysis of all lesions, at 
subject-level and lesion-level. 
 Subject-level sensitivity, specificity, PPV and NPV of 18F-fluciclovine PET scan 
for detecting recurrent brain metastases, based on combined application of the 
established image interpretation criteria. 
 Lesion-level sensitivity, specificity, PPV and NPV of 18F-fluciclovine PET for 
detecting recurrent brain metastases, based on combined application of the 
established image interpretation criteria.  
 Safety of 18F-fluciclovine injection in the patient 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 adverse events 
(SAEs and AEs, respectively). 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 27 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
6. 
SUBJECT SELECTION 
6.1. 
Study Population 
Subjects with solid tumor brain metastases previously treated with radiation therapy who are 
scheduled for SoC craniotomy and 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 0-2. 
3. Subject or 
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), or in the salvage setting (repeat radiation 
therapy), with or without 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**, 
on contrast-enhanced MRI, and meeting one of 
the following radiological criteria: 
a. 
increase in the longest diameter of the reference lesion on contrast-
enhanced MRI relative to nadir***, where the longest diameter at nadir is 
 
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. 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 28 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
***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 craniotomy 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 child-bearing potential 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 child-bearing 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. 
6. Applicable to subjects actively on the BED-FLC-312 study: the subject at time of 
Screening, is within the BED-FLC-312 Visit 4 window (i.e., up to and including 
Day 22). 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 29 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
7. 
CONCURRENT MEDICATIONS 
All subjects should be maintained on the same medications throughout the entire study 
period, as medically feasible, with no introduction of new chronic therapies.  
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.  




BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 31 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 Image 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. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 32 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
8.11. 
Measures of Treatment Compliance 
Not Applicable. This is a single dose study, administered by site staff. 
8.12. 
End Of Trial 
For each subject, the last visit will usually be Visit 4/Day 2-22 SoC craniotomy/surgery. All 
ongoing follow-up and any further treatment will be in accordance with SoC. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                           Page 33 of 67 
 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 the Health Insurance Portability and Accountability Act (HIPAA) 
authorization must be signed and dated by the subject or LAR (Section 17.3). 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                                                                                                               Page 34 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
 Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 Craniotomy 
 
Before Visit 1 
Screening 
Within 28 to 1 
days before Visit 
2 Day 1 
1 to 21 days 
before SoC 
craniotomy/surger
y Visit 4 Day 3 
Day of  
18F-fluciclovine 
injection or up to  
 3 days after 
1 to 3 days after  
18F-fluciclovine injection, 
before SoC Craniotomy 
At time of  
pre-planned SoC 
craniotomy/surgery and after 
Visit 2 MRI and Visit 3 
Follow-Up 
SoC MRI 
X 
 
 
 
 
 
Informed Consent 
 
X 
 
 
 
 
Confirm inclusion/exclusion 
criteria 
 
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 
 
 
 
Order 18F-fluciclovine dose 
 
X 
 
 
 
 
Eligibility Review g 
 
X 
X 
 
 
 
18F-fluciclovine Injection 
 
 
X 
 
 
 
18F-fluciclovine PET Brain 
Scan 
 
 
X 
 
 
 
Adverse Events 
 
 
X 
 
X 
 
On-Study MRI Brain Scan 
 
 
 
X 
 
 
Annotate Reference Lesion on 
SoC MRI Brain Scan 
 
X 
 
 
 
 
Annotate MRI surgical lesions 
(i.e. other lesions intended for 
resection per SoC [if 
applicable]) 
 
X 
 
 
 
 
Identify and annotate additional 
PET lesions (i.e. potential 
additional lesions not 
previously reported on SoC 
MRI [if applicable]) h 
 
 
 
X 
 
 
Confirm sites of reference 
lesion and MRI surgical lesions 
on Visit 2 scans 
 
 
 
X 
 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                                                                                                               Page 35 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
 Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
 
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 Craniotomy 
 
Before Visit 1 
Screening 
Within 28 to 1 
days before Visit 
2 Day 1 
1 to 21 days 
before SoC 
craniotomy/surger
y Visit 4 Day 3 
Day of  
18F-fluciclovine 
injection or up to  
 3 days after 
1 to 3 days after  
18F-fluciclovine injection, 
before SoC Craniotomy 
At time of  
pre-planned SoC 
craniotomy/surgery and after 
Visit 2 MRI and Visit 3 
Follow-Up 
Safety Follow-up Phone Call 
 
 
 
 
X 
 
SoC Craniotomy/Surgery 
 
 
 
 
 
X 
Record sites of all sampled 
lesions  
 
 
 
 
 
X 
Document Neurosurgical 
Procedure 
 
 
 
 
 
X 
Record local histopathology 
results and send specimens to 
central histopathology 
laboratory 
 
 
 
 
 
X 
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. 
c Treatment history for previous cancer to include previous treatments for brain metastases and previous cancer treatments for primary tumor 
d A standard of care ECOG assessment may be recorded as the Screening ECOG if the assessment was performed within 28 days of Screening. 
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 child-bearing potential. 
g Eligibility review to include collection and review of prior pre- and post-treatment MRI brain scans as needed to confirm eligibility. Prior pre- and post-treatment MRI brain 
scans and current SoC MRI images to be uploaded to central imaging core lab. 
h 
otential additional lesions identified by the on-site investigator on 18F-fluciclovine PET scan (with Visit 2 MRI for anatomical reference), not 
previously reported on SoC MRI, should be judged suggestive of brain metastasis, warranting confirmation according to SoC practice, and be neurosurgically feasible and 
accordingly, planned for biopsy / resection. 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 36 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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. 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 
treatments of 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
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 reference lesion nadir or showing 
complete lesion 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 MRI surgical 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 pre-planned SoC craniotomy; and 
c. if >1 equivocal lesion is intended for resection, the largest of these lesions. 
If >1 equivocal lesion is pre-planned for resection as SoC, the on-site investigator will also 
annotate and measure the other lesions (termed 
) on the SoC MRI brain 
scan, defined by the same radiological criteria for a reference lesion per inclusion criterion #7 
(see Section 6.2). 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 37 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
Site will perform and upload a dynamic 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 
PET lesions should be annotated only where judged suggestive of brain metastasis, 
warranting confirmation according to SoC practice, be neurosurgically feasible and 
accordingly, planned for biopsy/resection. 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 
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 MRI surgical 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 specify the anatomical location. 
9.1.5.1. 
18F-fluciclovine PET Scan 
Dynamic PET/CT or PET/MRI scan of the brain is 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 central 
imaging core lab by 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 Image 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 
On-study 
MRI will consist of MRI sequences per the minimum standard consensus recommendations 
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, and  
 FLAIR and/or T2-weighted MRI, and 
 Diffusion-weighted imaging. 
On-study MRI brain scan will be uploaded to central imaging core lab by site investigator/site 
staff. In addition to reviewing the 18F-fluciclovine PET scan to identify potential additional 
PET lesions, the site investigator will confirm the sites of the reference lesion and if 
applicable, MRI surgical lesions (i.e., other lesions already planned for resection per SoC). 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 38 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
Full details on the imaging protocol are provided in the Image Acquisition Standards. If 
available, scans acquired on a PET/MRI are allowed. 
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 Craniotomy / Surgery 
Craniotomy procedure to take place as planned for SoC, for surgical resection of the 
reference lesion and MRI surgical lesions, and biopsy / surgical resection of any additional 
PET lesions if clinically applicable. 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 per SoC and will send a 
representative set of specimens with their matching lesion identifiers to the central laboratory 
for analysis. Refer to Sample Handling Laboratory Manual.  
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 child-bearing 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). 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 39 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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. 
LAR) and obtain written informed 
consent and HIPAA authorization and assent, if appropriate. 
2. Assign the subject a unique subject number. 
3. Record demographics data. 
4. Record medical 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- and post-treatment MRI brain scans as needed for 
investigator to confirm eligibility and current SoC MRI. Upload MRI images to 
central imaging core lab and annotate and measure reference lesion and MRI surgical 
lesions. 
10. Record or assess ECOG performance status. 
11. Perform urine HCG pregnancy test (female subjects of child-bearing potential only). 
12. Confirm inclusion / exclusion criteria. 
13. Schedule 18F-fluciclovine PET scan and order IMP for delivery on day of PET scan. 
14. Schedule subject for Visit 2 (IMP + PET) to occur within 42 days of 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 pre-planned SoC craniotomy; and 
c. if >1 equivocal lesion is intended for resection, the largest of these lesions. 
If >1 equivocal lesion is pre-planned for resection as SoC, the on-site investigator will also 
annotate the other lesions (termed 
) on the current SoC MRI brain 
scan, defined by the same radiological criteria for a reference lesion per inclusion criterion #7 
(see Section 6.2). 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 40 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
10.2. 
Visit 2- Day 1: IMP Administration and 18F-fluciclovine PET Scan 
(1 to 28 days after Screening) 
Subjects who have provided informed consent and who meet all eligibility criteria will be 
enrolled into the study. At the time the subject is deemed to be eligible, 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. Perform assessment of vital signs with subject resting at least 5 minutes prior to IMP 
administration. The vital signs should be collected between 5 to 60 minutes before 
PET scan. 
5. IMP + PET: Administer 18F-fluciclovine injection, followed by 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. 
6. Monitor and record any adverse events. 
7. 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 PET scan. 
8. Upload 18F-fluciclovine PET brain scan to central imaging core lab. 
9. Review of 18F-fluciclovine PET brain scan by site investigator to identify potential 
additional PET lesions will be performed with the Visit 2 MRI scan (for anatomical 
reference), see Section 10.3. 
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. It should take place before the Visit 4  SoC 
craniotomy. 
1. Perform Visit 2 on-study MRI brain scan as outlined in Section 11.5. 
2. Upload Visit 2 on-study MRI brain scan to central imaging core lab 
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 MRI surgical lesions on Visit 2 scans. 
The on-site investigator will review the 18F-fluciclovine PET scan to identify and annotate 
suggestive of metastasis, warranting confirmation according to SoC practice, be 
neurosurgically feasible and accordingly, planned for biopsy/resection. Undertaking 
biopsy / resection of additional PET lesion(s) identified on the 18F-fluciclovine PET 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 41 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 
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 
craniotomy 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 Craniotomy (Pre-planned, 1 to 21 days after IMP 
Injection/PET) 
Visit 4  SoC Craniotomy 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 resection of reference lesion, and if applicable, MRI surgical 
lesions pre-planned for resection.      
If SoC resection of the reference lesion and/or MRI surgical lesions could not be 
performed during the procedure, record the contributing clinical factors, and 
whether a SoC biopsy was performed in lieu 
b. 
biopsy/resection.* 
c. If applicable, record neuronavigation system used for neurosurgical procedure. 
d. 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). Where biopsy was performed, 
upload image/screenshot of neuronavigation system depicting stereotactic 
coordinates of biopsy. 
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 lab study imaging 
platform on the previously annotated scans. 
3. Process specimens per SoC and local laboratory standard practice.  
4. Ship a representative set of specimens to the central laboratory per the Sample 
Processing Manual.  


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 42 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
5. Record results of local histopathology analysis. 
*Undertaking biopsy/resection of additional PET lesion(s) identified on the 
18F-fluciclovine PET scan is at the discretion of on-site investigators. The lesions should 
be judged suggestive of brain metastasis, warranting confirmation according to SoC 
practice, be neurosurgically feasible and accordingly, planned for biopsy/resection. The 
subject will receive post-procedural management per institutional SoC. 
10.6. 
Early Withdrawal  
Early withdrawal is defined as discontinuing from study without having SoC 
craniotomy/surgery on or before Day 22. 
 Record any AEs or changes to concomitant medication occurring during safety 
reporting period. 
 Record reason for early withdrawal. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 43 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
11. 
IMAGING PROTOCOL 
Full details on the imaging protocol are in the Image Acquisition Standards (PET scan 
imaging and processing only). 
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 Pharmacy Manual for administration instructions. 
11.4. 
18F-fluciclovine PET Acquisition 
For the dynamic PET acquisition, patients 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 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.  
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 parameters detailed in 
the Image Acquisition Standards.   
11.8. 
Central Image Analysis 
Central image analysis of 18F-fluciclovine PET, with Visit 2 on-study MRI used for 
anatomical reference, will be performed by 3 independent readers blinded to all clinical 
information. 
1. Central image analysis of Visit 2 PET, with Visit 2 on-study MRI used for anatomical 
reference (i.e. using the on-study MRI to show the anatomical location of the 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 44 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
 
PET 
 
Qualitative analysis: 3 independent reader evaluation, with Visit 2 on-study MRI 
used for anatomical reference. 
 
18F-fluciclovine uptake of all sampled lesions, including the reference lesion, will 
be determined from static imaging data (e.g., from 10-20 minutes post-injection), 
using the following definitions: 
 Absent  18F-fluciclovine uptake indistinguishable from surrounding 
structurally normal brain parenchyma 
 Mild  18F-fluciclovine uptake above surrounding structurally normal brain 
parenchyma, up to or similar to blood pool in the venous sinuses 
 Moderate  18F-fluciclovine uptake higher than blood pool, up to or similar to 
parotid glands (if parotid gland uptake is asymmetrical, uptake in the gland 
with higher uptake is used as reference). 
 Marked  18F-fluciclovine uptake higher than parotid glands. 
 Time activity curve (TAC) based on 18F-fluciclovine uptake of all sampled 
lesions, including the reference lesion, on dynamic imaging data will be derived 
from the following image acquisition frame sequences: first 10 min after 
injection - 12 frames of 5 s, 6 frames of 10 s, 6 frames of 30 s and 5 frames of 
60 s; from 10 min to 30 min after injection  5 minute acquisition frames (Law, 
2018). 
 Type 0: absent uptake 
 Type I: continuous increase in uptake throughout the study 
 Type II: initial uptake followed by plateau later in the study 
 Type III: relatively rapid uptake with reduction in uptake later in the study 
Quantitative analysis 
 Lesion(s) and background structures will be outlined visually on static imaging 
data (e.g., from 10-20 minutes post-injection). This will be performed by the same 
3 independent readers performing the qualitative analysis. 
 Perform quantitative measure of background activity: mean standardized uptake 
value (SUVmean). 
 Perform quantitative measures of lesion 18F-fluciclovine uptake, including: 
maximum standardized uptake value (SUVmax), peak standardized uptake value 
(SUVpeak), maximum tumor: background ratio (TBRmax; tumor SUVmax: SUVmean 
background), peak tumor: background ratio (TBRpeak; tumor SUVpeak, background 
SUVmean). 
2. Central histopathology 
 Specimens will be centrally reviewed. 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. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 45 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
3. Image interpretation criteria 
 Central image analysis and central histopathologic correlate results will be 
reviewed by a separate Image Interpretation Committee (including representatives 
from the Sponsor and expert readers) to determine a set of image interpretation 
criteria based on the described visual reads, quantitative and dynamic measures. 
Additional details on the central image analysis and Image Interpretation Committee will be 
provided in the Image Interpretation Standards and Image Interpretation Committee Plan, 
respectively. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 46 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 
is of greater severity or frequency than expected based on the 
 
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 
i
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 
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; 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.   


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 47 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 [ADR]) 
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 at 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.  
12.4.2. 
Suspected Unexpected Serious Adverse Reactions (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 
  


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 48 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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, 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 
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. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 49 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 eCRF. 
Refer to Section 10.6 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, or are withdrawn or 
discontinued from the study, prior to completed reference lesion resection at 
craniotomy/surgery, may be replaced.  
13.3. 
Lost to Follow-up 
A subject will be considered lost to follow-up if he or she fails to complete the Visit 3 post-
IMP safety follow-up phone call or fails to continue through to Visit 4/Day 2-22 for SoC 
craniotomy/surgery and is unable to be contacted by the study site staff.  
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 prior to 
pre-scheduled Visit 4/Day 2-22 SoC craniotomy/surgery and ascertain if the 
subject wishes to and/or should continue in the study 
 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


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 50 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
or local equivalent methods). These contact attempts should be documented in the 
subject
 
 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 20 days post-IMP administration. 
 Has not received IMP and is beyond 28 days of pre-study SoC MRI. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 51 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 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 uti
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 52 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 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, minimum and maximum. Categorical data will be 
summarized using frequency counts and percentages. 
15.1. 
Estimation of Sample Size 
Up to 40 subjects are planned for enrollment. The sample size is not calculated based on 
formal statistical hypothesis testing criteria but is estimated to provide sufficient numbers of 
subjects and data to establish image interpretation criteria.  
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 Safety Analysis Set (SAF) will be used for all safety analyses, and include all subjects 
who have been dosed with IMP (with or without the associated PET scan).   
The Imaging Evaluable Analysis Set (IEAS) includes all subjects who have been dosed with 
IMP, have evaluable Visit 2 on-study PET scan (see Section 11.7) , and an evaluable central 
histopathology report (defined in the Histopathology Charter) from the sample obtained from 
the Visit 4 SoC craniotomy. 
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 
history, prior treatment history) will be summarized using appropriate summary statistics for 
the FAS, SAF and IEAS.  
15.5. 
Analysis of Primary Endpoint 
The primary endpoint is the diagnostic performance (sensitivity, specificity, PPV and NPV) 
of different thresholds of lesion 18F-fluciclovine uptake on visual reads (as defined by the 
different combinations of degree of uptake) versus SoT derived from central 
histopathological analysis of all lesions, at subject-level and lesion-level.  


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 53 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
Three thresholds of lesion 18F-fluciclovine uptake, derived from visual reads in the 
3 independent reader evaluation, will be considered: 
Threshold 
Combination of degree of uptake 
Mild or higher uptake 
Absent uptake vs (Mild + Moderate + Marked uptake) 
Moderate or higher uptake 
(Absent + Mild uptake) vs (Moderate + Marked uptake) 
Marked uptake 
(Absent + Mild + Moderate uptake) vs Marked uptake 
Estimates and the 95% exact confidence intervals for sensitivity, specificity, PPV and NPV 
for each threshold will be calculated using standard definitions: 
 
Tumor Present (SoT) 
Tumor Absent (SoT) 
Threshold Met  
True positive 
False positive 
Threshold Not Met  
False negative 
True negative 
 Sensitivity (% of true positives) = true positive / (true positive + false negative) 
 Specificity (% of true negatives) = true negative / (true negative + false positive) 
 PPV = true positive / (true positive + false positive) 
 NPV = true negative / (true negative + false negative) 
Subject-level diagnostic performance will be calculated based on reference lesion data, while 
lesion-level diagnostic performance will be calculated based on data of all sampled lesions. 
Lesions annotated on the scans will be matched to the histopathology results based on the 
lesion identifiers assigned by the study imaging platform at time of annotation. The results of 
these analyses will be reviewed by the separate Image Interpretation Committee (consisting 
of representatives from the Sponsor, contracted imaging core lab, and expert readers) to 
determine image interpretation criteria based on visual reads. 
15.6. 
Analysis of Secondary Endpoint(s) 
In order to establish other image interpretation criteria for 18F-fluciclovine PET in detecting 
recurrent brain metastases, receiver operating characteristic (ROC) analysis of SoT against 
the quantitative measures of lesion 18F-fluciclovine uptake will be performed. For dynamic 
measures of lesion 18F-fluciclovine uptake represented by TAC type, applying each TAC 
category as a diagnostic criterion, estimates and the 95% exact confidence intervals for 
sensitivity, specificity, PPV and NPV for each of the 4 TAC types will be calculated using 
the standard definitions stated above.  
These analyses will be reviewed by the separate Image Interpretation Committee to determine 
a set of image interpretation criteria based on the quantitative and dynamic measures. 
Subsequently, for the diagnostic performance secondary endpoints, estimates and the 95% 
exact confidence intervals for sensitivity, specificity, PPV and NPV will be calculated based 
on the recommended thresholds from the following combination of interpretation criteria (if 
featured in the interpretation criteria): 
1. Visual assessment of level of lesion 18F-fluciclovine uptake 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 54 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
2. Strongest performing quantitative measure (based on ROC analysis)  
Subject-level diagnostic performance will be calculated based on reference lesion data, while 
lesion-level diagnostic performance will be calculated based on data of all sampled lesions.  
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. Adverse event verbatim terms will be coded to preferred 
terms and system organ classes using the latest version of Medical Dictionary for Regulatory 
Activities (MedDRA). Severity of AEs will be graded using the NCI-CTCAE v5.0. The 
frequency of TEAEs will be summarized overall, by system organ class and preferred term, 
and by severity. IMP-related AEs and SAEs will also be summarized. 
15.7. 
Planned Interim Analysis 
Not applicable. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 55 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 
and upload the MRI 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 (and other previous cancers, if 
applicable) 
 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 
-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, but 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 (EDC) system directly. 
The study database will be updated in accordance with the resolved queries. All changes to 
the study database will be documented. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 56 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 
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  (or designee ) 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. 
Additional subject confidentiality issues (if applicable) are covered in the Clinical Study 
Agreement. 
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 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 57 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
all applicable privacy regulations (e.g., Health Insurance Portability and Accountability Act 
of 1996, etc.). 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 58 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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, 
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. 
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., 
, 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 application 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. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 59 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
The investigator (or an appropriately qualified designee) will explain the study to the subject 
LAR, and answer any 
questions that arise. 
, or 
, comprehension, of the purposes, procedures, and potential risks of the study 
and the rights of research subjects. Subjects 
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 (
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. 
LAR, may 
LAR is not capable of 
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, 
LAR, and that informed consent was freely given 
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 origina
.  
LAR, will be informed in a timely manner if new information 
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 rele
information and consent form should receive IRB/IEC approval / favorable opinion prior to 
use. 
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 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 60 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 
ochure, 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; 
subject to review through-out the trial. 
5. Submit a written progress report at least annually and in accordance with the 
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 
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. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 61 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
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 or until notified by the Sponsor. 
12. Provide monitors, auditors, IRB/IEC and regulatory authorities direct access to all 
requested study-related records. 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 62 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
18. 
REFERENCES 
1. Arvold ND, Lee EQ, Mehta MP, et al. Updates in the management of brain 
metastases. Neuro Oncol. 2016;18(8):1043-65.   (Arvold, 2016) 
2. Bach-Gansmo T, Nanni C, Nieh PT, et al. Multisite experience of the safety, detection 
rate and diagnostic performance of Fluciclovine (18F) positron emission 
tomography/computerized tomography imaging in the staging of biochemically 
recurrent prostate cancer. J Urol. 2017;197(3):676-83.   (Bach-Gansmo, 2017) 
3. Bogsrud TV, Londalen A, Brandal P, Leske H, Panagopoulos I, Borghammer P, et al. 
18F-Fluciclovine PET/CT in suspected residual or recurrent high-grade glioma. Clin 
Nucl Med. 2019.   (Bogsrud, 2019) 
4. Brown C, Dempsey MF, Gillen G, et al. Investigation of 18F-FDG 3D mode PET 
image quality versus acquisition time. Nuc Med Commun. 2010;31 (3 ):254-9.   
(Brown, 2010) 
5. Ceccon G, Lohmann P, Stoffels G, Judov N, Filss CP, Rapp M, et al. Dynamic O-(2-
18F-fluoroethyl)-L-tyrosine positron emission tomography differentiates brain 
metastasis recurrence from radiation injury after radiotherapy. Neuro Oncol. 
2017;19(2):281-8.   (Ceccon, 2017) 
6. Cicone F, Minniti G, Romano A, Papa A, Scaringi C, Tavanti F, et al. Accuracy of 
F-DOPA PET and perfusion-MRI for differentiating radionecrotic from progressive 
brain metastases after radiosurgery. Eur J Nucl Med Mol Imaging. 
2015;42(1):103-11.   (Cicone, 2015) 
7. Dequesada IM, Quisling RG, Yachnis A, Friedman WA. Can standard magnetic 
resonance imaging reliably distinguish recurrent tumor from radiation necrosis after 
radiosurgery for brain metastases? A radiographic-pathological study. Neurosurgery. 
2008;63(5):898-903; discussion 4.   (Dequesada, 2008) 
8. FDA/NBTS. FDA-NBTS Public Workshop: product development for central nervous 
system (CNS) metastases. Available at: https://www.fda.gov/drugs/news-events-
human-drugs/fda-nbts-public-workshop-product-development-central-nervous-
system-cns-metastases [Accessed: 31 July 2019]. 2019. (FDA/NBTS, 2019) 
9. Fuchs BC, Bode BP. Amino acid transporters ASCT2 and LATl in cancer: partners in 
crime? Semin Cancer Biol. 2005;15(4):254-66.   (Fuchs, 2005) 
10. Galldiks N, Stoffels G, Filss CP, Piroth MD, Sabel M, Ruge MI, et al. Role of O-(2-
(18)F-fluoroethyl)-L-tyrosine PET for differentiation of local recurrent brain 
metastasis from radiation necrosis. J Nucl Med. 2012;53(9):1367-74.    
(Galldiks, 2012) 
11. Galldiks N, Langen KJ, Albert NL, et al. PET imaging in patients with brain 
metastasis-report of the RANO/PET group. Neuro Oncol. 2019;21(5):585-95. 
(Galldiks, 2019) 
12. Heiss WD. Clinical impact of amino acid PET in gliomas. J Nucl Med. 
2014;55(8):1219-20.   (Heiss, 2014) 
13. Henderson F, Brem S, O'Rourke DM, Nasrallah M, Buch VP, Young AJ, et al. 18F-
Fluciclovine PET to distinguish treatment-related effects from disease progression in 
recurrent glioblastoma: PET fusion with MRI guides neurosurgical sampling. Neuro-
Oncology Practice. 2019:1-6.   (Henderson, 2019) 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 63 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
14. Kann BH, Park HS, Johnson SB, et al. Radiosurgery for brain metastases: changing 
practice patterns and disparities in the United States. J Natl Compr Cane Netw. 2017; 
15(12): 1494-502.   (Kann, 2017) 
15. Karlberg A, Berntsen EM, Johansen H, Skjulsvik AJ, Reinertsen I, Dai HY, et al. 
18F-FACBC PET/MRI in diagnostic assessment and neurosurgery of gliomas. Clin 
Nucl Med. 2019;44(7):550-9.   (Karlberg, 2019) 
16. Kaufmann TJ, Smits M, Boxerman J, Huang R, Barboriak DP, Weller M, et al. 
Consensus recommendations for a standardized brain tumor imaging protocol for 
clinical trials in brain metastases. Neuro Oncol. 2020; 
noaa030, https://doi.org/10.1093/neuonc/noaa030. (Kaufmann, 2020) 
17. Kocher M, Soffietti R, Abacioglu U, Villa S, Fauchon F, Baumert BG, et al. Adjuvant 
whole-brain radiotherapy versus observation after radiosurgery or surgical resection 
of one to three cerebral metastases: results of the EORTC 22952-26001 study. J Clin 
Oncol. 2011;29(2):134-41.   (Kocher, 2011) 
18. Kondo A, Ishii H, Aoki S, et al. Phase Ila clinical study of [18F]fluciclovine: efficacy 
and safety of a new PET tracer for brain tumors. Ann Nucl Med. 2016;30(9):608-18.   
(Kondo, 2016) 
19. Langen KJ, Galldiks N. Update on amino acid PET of brain tumours. Curr Opin 
Neurol. 2018;31(4):354-61.   (Langen, 2018) 
20. Langen KJ, Galldiks N, Hattingen E, Shah NJ. Advances in neuro-oncology imaging. 
Nat Rev Neurol. 2017;13(5):279-89.   (Langen, 2017) 
21. Law I, Albert NL, Arbizu J, Boellaard R, Drzezga A, Galldiks N, et al. Joint 
EANM/EANO/RANO practice guidelines/SNMMI procedure standards for imaging 
of gliomas using PET with radiolabelled amino acids and [18F]FDG: version 1.0. Eur 
J Nucl Med Mol Imaging. 2019;46(3):540-57.   (Law, 2018) 
22. Lin NU, Lee EQ, Aoyama H, Barani IJ, Barboriak DP, Baumert BG, et al. Response 
assessment criteria for brain metastases: proposal from the RANO group. Lancet 
Oncol. 2015;16(6):e270-8.   (Lin, 2015)  
23. Lizarraga KJ, Allen-Auerbach M, Czernin J, DeSalles AA, Yong WH, Phelps ME, et 
al. (18)F-FDOPA PET for differentiating recurrent or progressive brain metastatic 
tumors from late or delayed radiation injury after radiation treatment. J Nucl Med. 
2014;55(1):30-6.   (Lizarraga, 2014) 
24. McParland BJ, Wall A, Johansson S, et al. The clinical safety, biodistribution and 
internal radiation dosimetry of [18F]fluciclovine in healthy adult volunteers. Eur J 
Nucl Med Mol Imaging. 2013;40(8): 1256-64.   (McParland, 2013) 
25. Michaud L, Beattie BJ, Akhurst T, Dunphy M, Zanzonico P, Finn R, et al. (18)F-
Fluciclovine ((18)F-FACBC) PET imaging of recurrent brain tumors. Eur J Nucl Med 
Mol Imaging. 2020;47: 1353-67.    (Michaud, 2020) 
26. Minamimoto R, Saginoya T, Kondo C, Tomura N, Ito K, Matsuo Y, et al. 
Differentiation of brain tumor recurrence from post-radiotherapy necrosis with 11C-
methionine PET: visual assessment versus quantitative assessment. PLoS One. 
2015;10(7):e0132515.   (Minamimoto, 2015) 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 64 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
27. NCCN 2019. NCCN Guidelines® Central Nervous System Cancers (5 March 2019). 
Available at: https://www.nccn.org/professionals/physician gls/default.aspx 
[Accessed: 31 July 2019].   (NCCN, 2019) 
28. Oka S, Okudaira H, Yoshida Y, Schuster DM, Goodman MM, Shirakami Y. 
Transport mechanisms of trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid 
in prostate cancer cells. Nucl Med Biol. 2012;39(1):109-19.   (Oka, 2012) 
29. Oka S, Okudaira H, Ono M, Schuster DM, Goodman MM, Kawai K, et al. 
Differences in transport mechanisms of trans-1-amino-3-
[18F]fluorocyclobutanecarboxylic acid in inflammation, prostate cancer, and glioma 
cells: comparison with L-[methyl-11C]methionine and 2-deoxy-2-[18F]fluoro-D-
glucose. Mol Imaging Biol. 2014;16(3):322-9.   (Oka, 2014) 
30. Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, Carbone PP. 
Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin 
Oncol. 1982;5:649-55.   (Oken, 1982) 
31. Okudaira H, Shikano N, Nishii R, Miyagi T, Yoshimoto M, Kobayashi M, et al. 
Putative transport mechanism and intracellular fate of trans-1-amino-3-18F-
fluorocyclobutanecarboxylic acid in human prostate cancer. J Nucl Med. 
2011;52(5):822-9.   (Okudaira, 2011) 
32. Ono M, Oka S, Okudaira H, Schuster DM, Goodman MM, Kawai K, et al. 
Comparative evaluation of transport mechanisms of trans-1-amino-3-
[18F]fluorocyclobutanecarboxylic acid and L-[methyl-11C]methionine in human 
glioma cell lines. Brain Res. 2013;1535:24-37.   (Ono, 2013) 
33. Parent EE, Benayoun M, Ibeanu I, Olson JJ, Hadjipanayis CG, Brat DJ, et al. 
18F-Fluciclovine PET discrimination between high- and low-grade gliomas. EJNMMI 
Res. 2018;8(1):67.   (Parent, 2018) 
34. Patel D, Nye J, Schuster D, et al. Fluciclovine PET imaging of intracranial metastases. 
J Nucl Med 2018;59(suppl 1): Abstract 1097 and Conference Poster.   (Patel, 2018) 
35. Peng L, Parekh V, Huang P, Lin DD, Sheikh K, Baker B, et al. Distinguishing true 
progression from radionecrosis after stereotactic radiation therapy for brain 
metastases with machine learning and radiomics. Int J Radiat Oncol Biol Phys. 
2018;102(4):1236-43.   (Peng, 2018) 
36. Pope WB. Brain metastases: neuroimaging. Handb Clin Neurol. 2018;149:89-112.   
(Pope, 2018) 
37. Romagna A, Unterrainer M, Schmid-Tannwald C, Brendel M, Tonn JC, Nachbichler 
SB, et al. Suspected recurrence of brain metastases after focused high dose 
radiotherapy: can 18F-FET- PET overcome diagnostic uncertainties? Radiat Oncol. 
2016;11(1):139.   (Romagna, 2016) 
38. Schuster DM, Votaw JR, Halkar RK, et al. Uptake of the synthetic PET amino acid 
radiotracer 1-amino-3-l 8F-fluorocyclobutane-1-carboxylic acid (18F-FACBC) within 
primary and metastatic brain cancer compared with 18F-fluorodeoxyglucose 
(18F-FDG). J Nucl Med. 2003;44(suppl): Abstract 167P.   (Schuster, 2003) 
39. Schuster DM, Nanni C, Fanti S, et al. Anti-1-amino-3-18F-fluorocyclobutane-1-
carboxylic acid: physiologic uptake patterns, incidental findings, and variants that 
may simulate disease. J Nuc Med 2014;55:1986-92.   (Schuster, 2014) 


BED-FLC-219 Protocol Version 3.0, 10 June 2021                                             Page 65 of 67 
CONFIDENTIAL Blue Earth Diagnostics  
Based on Protocol Template, SOP-MED-002-AD-2, Version 3 
40. Sinha R, Sage W, Watts C. The evolving clinical management of cerebral metastases. 
Eur J Surg Oncol. 2017;43(7):1173-85.   (Sinha, 2017) 
41. Stockham AL, Tievsky AL, Koyfman SA, Reddy CA, Suh JH, Vogelbaum MA, et al. 
Conventional MRI does not reliably distinguish radiation necrosis from tumor 
recurrence after stereotactic radiosurgery. J Neurooncol. 2012;109(1):149-58.   
(Stockham, 2012) 
42. Suh JH. Stereotactic radiosurgery for the management of brain metastases. N Engl J 
Med. 2010;362(12):1119-27.   (Suh, 2010) 
43. Teoh E, Morotti M, Bridges E, Jones D, Miar A, Wigfield S, et al. Fluciclovine (18F) 
is a marker for high-affinity glutamine transporter ASCT2-mediated amino acid 
transport in breast cancer. J Nucl Med. 2017;58(suppl 1):1028.   (Teoh, 2017) 
44. Terakawa Y, Tsuyuguchi N, Iwai Y, Yamanaka K, Higashiyama S, Takami T, et al. 
Diagnostic accuracy of 11C-methionine PET for differentiation of recurrent brain 
tumors from radiation necrosis after radiotherapy. J Nucl Med. 2008;49(5):694-9.   
(Terakawa, 2008) 
45. Tsuyuguchi N, Terakawa Y, Uda T, Nakajo K, Kanemura Y. Diagnosis of brain 
tumors using amino acid transport PET imaging with (18)F-fluciclovine: A 
comparative study with L-methyl-(11)C-methionine PET imaging. Asia Ocean J Nucl 
Med Biol. 2017;5(2):85-94.   (Tsuyuguchi, 2017) 
46. Vellayappan B, Tan CL, Yong C, Khor LK, Koh WY, Yeo TT, et al. Diagnosis and 
management of radiation necrosis in patients with brain metastases. Front Oncol. 
2018;8:395.   (Vellayappan, 2018) 
47. Verma N, Cowperthwaite MC, Burnett MG, et al. Differentiating tumor recurrence 
from treatment necrosis: a review of neuro-oncologic imaging strategies. Neuro 
Oncol. 2013;15(5):515-34.   (Verma, 2013) 
48. Wakabayashi T, Iuchi T, Tsuyuguchi N, et al. Diagnostic performance and safety of 
positron emission tomography Using 18F-Fluciclovine in patients with clinically 
suspected high- or low-grade gliomas: A multicenter phase IIb trial. Asia Ocean J 
Nucl Med Biol. 2017;5(1 ): 10-21.   (Wakabayashi, 2017) 
49. Walker AJ, Ruzevick J, Malayeri AA, Rigamonti D, Lim M, Redmond KJ, et al. 
Postradiation imaging changes in the CNS: how can we differentiate between 
treatment effect and disease progression? Future Oncol. 2014;10(7):1277-97.   
(Walker, 2014) 
50. Wen PY, Macdonald DR, Reardon DA, Cloughesy TF, Sorensen AG, Galanis E, et al. 
Updated response assessment criteria for high-grade gliomas: response assessment in 
neuro-oncology working group. J Clin Oncol. 2010;28(11):1963-72.   (Wen, 2010) 
51. Zhang J, Liu H, Tong H, et al. Clinical applications of contrast-enhanced perfusion 
MRI techniques in gliomas: recent advances and current challenges. Contrast Media 
Mol Imaging. 2017;2017:7064120.   (Zhang, 2017) 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021
Page 66 of 67
 
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. 
 


BED-FLC-219 Protocol Version 3.0, 10 June 2021
Page 67 of 67
 
APPENDIX 2. PROTOCOL AGREEMENT 
I have read the protocol specified below. In my formal capacity as Investigator, my duties include 
providing Blue Earth Diagnostics 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-219 
 
Protocol Title:  An open-label, single-arm, single-dose, prospective, multicenter Phase 2b study to 
establish image interpretation criteria for 18F-fluciclovine positron emission tomography (PET) in 
detecting recurrent brain metastases after radiation therapy (PURSUE) 
 
Protocol Version:  3.0 
Protocol Date:  09 June 2021  
 
 
 
 
Investigator Signature  
 
Date 
 
Print Name and Title 
Site #
 
Site Name 
 
Address
 
 
 
 
Phone Number
 
Email 
 
 
 
 
 

SAP_003.pdf: 




Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 3 of 22
 
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 .................................................................................. 5 
6.1 
Overall Study Design ........................................................................... 5 
6.2 
Schedule of Assessments ...................................................................... 8 
6.3 
Treatment(s) ......................................................................................... 8 
6.3.1 
Treatment(s) Administered ........................................................... 8 
6.3.2 
Method of Assigning Subjects to Treatment Groups ................. 8 
6.4 
Efficacy and Safety Variables ............................................................. 9 
6.4.1 
Efficacy Variable(s) ....................................................................... 9 
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 ......................................................... 17 
7.5 
Safety Evaluation ............................................................................... 18 
7.5.1 
Extent of Exposure ....................................................................... 18 
7.5.2 
Adverse Events ............................................................................. 19 
7.5.3 
Deaths, Other Serious Adverse Events, and Other Significant 
Adverse Events ............................................................................. 19 
7.5.4 
Clinical Laboratory Evaluation .................................................. 20 
7.5.5 
Vital Signs ..................................................................................... 20 
7.5.6 
Prior and Concomitant Medications .......................................... 20 
7.6 
Determination of Sample Size ........................................................... 21 
7.7 
Changes in the Conduct of the Study or Planned Analyses ........... 21 
8 
Reference List .......................................................................................... 21 
 
 
 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 4 of 22
 
3 LIST OF ABBREVIATIONS  
 
Abbreviation 
Definition  
AE 
Adverse Event 
ATC 
Anatomical Therapeutic Chemical 
BMI 
Body Mass Index 
COVID-19 
Coronavirus Disease of 2019 
CSR 
Clinical Study Report 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
electronic Case Report Form 
FAS 
Full Analysis Set 
HCG 
Human Chorionic Gonadotropin 
ICH 
International Council for Harmonisation 
IEAS 
Imaging Evaluable Analysis Set 
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 
NPV 
Negative Predictive Value 
PET 
Positron Emission Tomography 
PPV 
Positive Predictive Value 
PT 
Preferred Term 
ROC 
Receiver Operating Characteristic 
SAE 
Serious Adverse Event 
SAF 
Safety Analysis Set 
SAP 
Statistical Analysis Plan 
SAS  
Statistical Analysis Software  
SoC 
Standard of Care 
SOC 
System Organ Class 
SoT 
Standard of Truth 
SUV 
Standardized Uptake Value 
SUVmax 
Maximum Standardized Uptake Value 
SUVpeak 
Peak Standardized Uptake Value 
SUVmean 
Mean Standardized Uptake Value 
TAC 
Time Activity Curve 
TBR 
Tumor Background Ratio 
TBRmax 
Maximum Tumor to Background Ratio 
TBRpeak 
Peak Tumor to Background Ratio 
TEAE 
Treatment-Emergent Adverse Event 
TESAE 
Treatment-Emergent Serious Adverse Event 
WHODrug 
World Health Organization Drug Dictionary 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 5 of 22
 
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-219 (An Open-label, Single-arm, Single-
dose, Prospective, Multicenter Phase 2b Study to Establish Image Interpretation 
Criteria for 18F-fluciclovine Positron Emission Tomography (PET) in Detecting 
Recurrent Brain Metastases After Radiation Therapy (PURSUE)). 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, listings, and figures, 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, figures, 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 establish visual image interpretation criteria for 18F-fluciclovine PET in 
detecting recurrent brain metastases. 
5.2 
Secondary Study Objectives 
The secondary objectives of this study are: 
 To establish other image interpretation criteria for 18F-fluciclovine PET in 
detecting recurrent brain metastases.  
 To assess subject-level diagnostic performance of 18F-fluciclovine PET in 
detecting recurrent brain metastases.  
 To assess lesion-level diagnostic performance of 18F-fluciclovine PET in 
detecting recurrent brain metastases.  
 To assess the safety of 18F-fluciclovine injection in the subject population.
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 define 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 6 of 22
 
image interpretation criteria for 18F-fluciclovine PET in detecting recurrent brain
metastases (when read with standard magnetic resonance imaging (MRI) for 
anatomical reference).  
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. Sites are 
encouraged to enroll subjects with a variety of primary tumor types. In order to ensure 
variation in tumor types on the study population, the number of subjects with lung 
cancer will be capped at approximately 60% of enrolled subjects.   
On-
on a post-radiation treatment MRI scan and on the SoC MRI brain scan to confirm 
 
 equivocal for recurrent metastasis on SoC MRI (according to inclusion 
criterion #7, protocol section 6.2);  
 intended for pre-planned SoC craniotomy; and  
 if >1 equivocal lesion is intended for resection, the largest of these lesions.  
If >1 equivocal lesion is intended for resection, the on-site investigator will also 
annotate and measure the other lesions (termed 
MRI brain scan, defined by the same radiological criteria for a reference lesion per 
inclusion criterion #7 (see protocol section 6.2). 
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 as anatomical reference for the 18F-fluciclovine 
PET scan. The Visit 2 On-Study MRI should be done ideally on the same day, 
 days after Visit 2 PET and completed before the pre-planned SoC 
craniotomy. A safety follow-up (Visit 3) 1 to 3 days after Visit 2 PET will be made 
for adverse event (AE) evaluation by telephone call or in person (if same day as 
scheduled SoC craniotomy). 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 craniotomy. The 
Visit 2 PET must be organized to take place a minimum of 1 day and maximum of 21 
days before pre-planned SoC craniotomy.  
The on-site investigator will review the 18F-fluciclovine PET scan to identify, 
annotate and measure potential additional lesions, not previously reported on SoC 
only where judged suggestive of brain metastasis, warranting confirmation according 
to SoC practice, be neurosurgically feasible and accordingly, planned for biopsy or
resection. 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 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 7 of 22
 
stable or partially responding), 18F-fluciclovine activity contiguous with a known 
lesion (previously annotated or otherwise) and apparently incongruent with the  
explained by image registration.    
For each subject, the final visit will usually be the preplanned SoC 
craniotomy/surgery 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 and will not be recorded, including all medications and SoC 
procedure-related events occurring at and beyond Visit 4, and any SoC follow-up.   
Subject-level standard of truth (SoT) will be defined by central histopathological 
diagnosis of the reference lesion. Lesion-level SoT will be defined by central 
histopathological diagnosis of all sampled lesions (reference lesions, MRI surgical 
lesions, additional PET lesions). Separately, central image analysis will define 
qualitative and quantitative lesion uptake of 18F-fluciclovine, for each resected lesion. 
These results will be reviewed by an Imaging Interpretation Committee to form image 
interpretation criteria. 
Concurrent Enrollment with BED-FLC-312  
Subjects who are enrolled in BED-FLC-312 and during its Follow-Up period are 
deemed to require a SoC craniotomy for diagnostic evaluation of an equivocal lesion, 
may be enrolled into BED-FLC-219 while remaining enrolled in BED-FLC-312. 
Subjects may be enrolled any time during the BED-FLC-312 Follow-Up period after 
Day 22 (i.e., after the full 21-day maximum timeframe for completion of the BED-
FLC-312 Visit 4 neurosurgical procedure). For absolute clarity, this will be 
irrespective of the applicability of BED-FLC-312 Visit 4 to the subject. Such subjects 
will be required to sign an informed consent form and meet all eligibility 
requirements for BED-FLC-219, including availability of the SoC MRI within the 
specified timeframe and reference lesion definitions. All BED-FLC-219 procedures 
remain applicable to any such concurrently enrolled subjects. Interim enrollment in 
BED-FLC-219 does not impact the subj
BED-FLC-312. 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 8 of 22
 
6.2 
Schedule of Assessments 
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. 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 9 of 22
 
6.4 
Efficacy and Safety Variables 
6.4.1 
Efficacy Variable(s) 
6.4.1.1 
Primary Efficacy Variable 
The primary efficacy endpoint is:  
 Diagnostic performance (sensitivity, specificity, positive predictive value 
[PPV] and negative predictive value [NPV]) of different thresholds of lesion 
18F-fluciclovine uptake on visual reads (as defined by the different 
combinations of degree of uptake) versus SoT derived from central 
histopathological analysis of all lesions, at subject-level and lesion-level. 
18F-fluciclovine uptake of all sampled lesions, including the reference lesion, will be 
determined from static imaging data (e.g., from 10-20 minutes post-injection), using 
the following definitions:  
- 
Absent  18F-fluciclovine uptake indistinguishable from surrounding 
structurally normal brain parenchyma  
- 
Mild  18F-fluciclovine uptake above surrounding structurally normal brain
parenchyma, up to or similar to blood pool in the venous sinuses  
- 
Moderate  18F-fluciclovine uptake higher than blood pool, up to or similar to
parotid glands (if parotid gland uptake is asymmetrical, uptake in the gland 
with higher uptake is used as reference).  
- 
Marked  18F-fluciclovine uptake higher than parotid glands. 
Three thresholds of lesion 18F-fluciclovine uptake, derived from visual reads in the 3 
independent reader evaluation, will be considered: 
Threshold 
Combination of degree of uptake 
Mild or higher uptake 
Absent uptake vs (Mild + Moderate + Marked uptake)
Moderate or higher uptake 
(Absent + Mild uptake) vs (Moderate + Marked uptake)
Marked uptake 
(Absent + Mild + Moderate uptake) vs Marked uptake
For each threshold, a 2x2 table will be created for each reader at subject level and also 
at lesion level.  
 
Tumor Present (SoT) 
Tumor Absent (SoT)
Threshold Met 
True positive 
False positive 
Threshold Not Met 
False negative 
True negative 
Sensitivity, specificity, PPV and NPV for each threshold will be calculated using 
standard definitions: 
 Sensitivity (% of true positives) = true positive / (true positive + false 
negative)  


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 10 of 22
 
Specificity (% of true negatives) = true negative / (true negative + false 
positive)  
 PPV = true positive / (true positive + false positive)  
 NPV = true negative / (true negative + false negative)  
Subject-level diagnostic performance will be calculated based on reference lesion 
data, while lesion-level diagnostic performance will be calculated based on data of all 
sampled lesions.  
6.4.1.2 
Secondary Efficacy Variables 
Secondary efficacy endpoints include the following: 
 Diagnostic performance (sensitivity, specificity, PPV and NPV) of different
thresholds of quantitative and dynamic measures of lesion 18F-fluciclovine 
uptake versus SoT derived from central histopathological analysis of all 
lesions, at subject-level and lesion-level.  
 Subject-level sensitivity, specificity, PPV and NPV of 18F-fluciclovine PET 
scan for detecting recurrent brain metastases, based on combined application 
of the established image interpretation criteria.  
 Lesion-level sensitivity, specificity, PPV and NPV of 18F-fluciclovine PET for 
detecting recurrent brain metastases, based on combined application of the 
established image interpretation criteria.   
Dynamic measures of lesion 18F-fluciclovine uptake 
Time activity curve (TAC) based on 18F-fluciclovine uptake of all sampled lesions, 
including the reference lesion, on dynamic imaging data will be derived from the 
following image acquisition frame sequences: first 10 min after injection - 12 frames 
of 5s, 6 frames of 10s, 6 frames of 30s and 5 frames of 60s; from 10 min to 30 min 
after injection  5 minute acquisition frames.   
- 
Type 0: absent uptake  
- 
Type I: continuous increase in uptake throughout the study  
- 
Type II: initial uptake followed by plateau later in the study  
- 
Type III: relatively rapid uptake with reduction in uptake later in the study  
For each TAC type, a 2x2 table will be created for subject level and also lesion level, 
for each reader. Each TAC type will be considered separately (eg. Type 0 vs Type 
I/II/III). In addition a combination will be evaluated: Type 0/I vs Type II/III. 
 
Tumor Present (SoT) 
Tumor Absent (SoT)
TAC Type X 
True positive 
False positive 
TAC Not Type X 
False negative 
True negative 
Sensitivity, specificity, PPV and NPV for each TAC type will be calculated in the 
same way as the primary endpoint. 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 11 of 22
 
Quantitative analysis  
Lesion(s) and background structures will be outlined visually on static imaging data 
(e.g., from 10-20 minutes post-injection), and the measures recorded will include: 
 lesion maximum standardized uptake value (SUVmax),  
 lesion peak standardized uptake value (SUVpeak),  
 background mean standardized uptake value (SUVmean),  
 maximum tumor: background ratio (TBRmax; tumor SUVmax: SUVmean 
background),  
 peak tumor: background ratio (TBRpeak; tumor SUVpeak : background 
SUVmean).   
 
 
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 (SAEs and AEs, respectively). 
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 Invest
of greater severity or frequency than expected based on the information in the 
 
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 (ie. Day 2). Any unresolved AE at Visit 3 Safety Follow-
up beyond Day 2 will be followed until resolution or stabilization. The investigator 
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.


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 12 of 22
 
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 2-day post-treatment Safety 
Follow-up phone call (Visit 3), and at early termination when applicable. Dose, route, 
frequency of 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, figures, and listings, presented in rich text 
format, and using recommended ICH numbering. Output specifications for all tables, 
figures, 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 and figures will be summarized for all subjects in the specified analysis set. In 
general, all data collected 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.  


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 13 of 22
 
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 Safety Analysis Set (SAF) will be used for all safety analyses, and will include all 
subjects who have been dosed with IMP (with or without the associated PET scan).   
The Imaging Evaluable Analysis Set (IEAS) includes all subjects who have been 
dosed with IMP, have an evaluable Visit 2 on-study PET scan, and an evaluable 
central histopathology report (defined in the Histopathology Charter) from the sample 
obtained from the Visit 4 SoC craniotomy.    


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 14 of 22
 
7.3 
Study Subjects 
7.3.1 
Disposition of Subjects 
Subject disposition will be summarized for all subjects who entered the study, by 
study site and overall. Summaries will include the number of subjects enrolled, 
failed/completed screening, and 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, by study site and overall. 
7.3.3 
Demographic and Other Baseline Characteristics 
Demographic variables including age, sex, ethnicity and race will be summarized for 
the FAS, SAF and IEAS. 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, 
. 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, SAF, and IEAS; 
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).  
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, osteosarcoma, thyroid, 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 15th of the month for the purpose of the 
calculation. If day and month are both not available then the 1st July 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 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 15 of 22
 
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 
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 MRI surgical 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 
 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 




Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 17 of 22
 
The results of these analyses will be reviewed by the separate Image Interpretation 
Committee as described in the study protocol to determine image interpretation 
criteria based on visual reads. 
7.4.4 
Secondary Endpoint Analysis Methods  
In order to establish other image interpretation criteria for 18F-fluciclovine PET in 
detecting recurrent brain metastases, receiver operating characteristic (ROC) analysis 
of SoT against the quantitative measures of lesion 18F-fluciclovine uptake will be 
performed for each reader. A ROC curve will be produced for lesion SUVmax, 
SUVpeak, TBRmax and TBRpeak, with each point corresponding to a specific threshold, 
max value vs < SUVmax value. The AUC of each curve will be calculated 
using the trapezoidal method and will be annotated on the plot. Sensitivity, 
specificity, PPV, and NPV will be listed for each value of lesion SUVmax, SUVpeak, 
TBRmax and TBRpeak. 
For TBRmax and TBRpeak, pairwise inter-rater agreement will be assessed using 
Pearson correlation coefficients. 
For dynamic measures of lesion 18F-fluciclovine uptake represented by TAC type, 
applying each TAC category as a diagnostic criterion, estimates and the 95% exact 
confidence intervals for sensitivity, specificity, PPV and NPV for each of the 4 TAC 
types will be calculated and presented, by reader, on both subject and lesion levels.
Lesion-level confidence intervals will be based on the method by Zhou et al..  
In addition, pairwise inter-reader agreements at the lesion level for the 3 readers will 
Kappa and the associated 95% confidence interval will be calculated. 
These analyses will be reviewed by the separate Image Interpretation Committee to 
determine a set of image interpretation criteria based on the quantitative and dynamic 
measures.  
Subsequently, for the diagnostic performance secondary endpoints, estimates and the 
95% exact confidence intervals for sensitivity, specificity, PPV and NPV will be 
calculated based on the recommended thresholds from the following combination of 
interpretation criteria (if featured in the interpretation criteria):  
1. Visual assessment of level of lesion 18F-fluciclovine uptake  
2. Strongest performing quantitative measure (based on ROC analysis)  
7.4.5 
Statistical/Analytical Issues 
7.4.5.1 
Handling of Dropouts or Missing Data 
No imputations will be performed on missing data; all analyses will be based on 
observed data only. 
7.4.5.2 
Interim Analyses and Data Monitoring 
Not applicable. 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 18 of 22
 
7.4.5.3 
Multicenter Studies 
This is a multicenter study, with approximately 10 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 
The summaries of different thresholds of lesion 18F-fluciclovine uptake, and time 
activity curve types, on visual reads vs standard or truth, at the lesion-level, will be 
repeated on the following subgroups:  
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 
Calculations will only be performed when there are at least 10 observations in the 
subgroup. 
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. Total administered 
activity, in mCi and MBq, will be summarised, along with whether there were any 
injection site reactions during or immediately after dose, and if a dynamic PET/CT or 
PET/MRI scan was performed. Other details will be listed. 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 19 of 22
 
7.5.2 
Adverse Events 
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. 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 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: 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 20 of 22
 
A by-subject listing of all deaths that occurred during the study, including 
cause 
 A by-subject listing of all serious adverse events 
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 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 21 of 22
 
available then level 3 will be used; if level 3 and 4 are not available then level 2 will 
be used. 
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 
 
7.6 
Determination of Sample Size 
Up to 40 subjects are planned for enrollment. The sample size is not calculated based 
on formal statistical hypothesis testing criteria, but is estimated to provide sufficient 
numbers of subjects and data to establish image interpretation criteria.   
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 
 


Blue Earth Diagnostics
Statistical Analysis Plan
BED-FLC-219 
 
Version 2.0; 01Jul2021
 
Confidential 
 
Page 22 of 22
 
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;