First In-human PET Imaging Studies With NIS Reporter [18F]BF4
Phase: PHASE1
Conditions: Myeloma, Endometrial Cancer
Sponsor: Mayo Clinic

 
 
First in-human PET imaging 
studies with NIS reporter 
[18F]BF4 
 
NCT# 02907073 
 
August 10, 2016 

1 
 
 
 
 
 
 
 
 
IRB Protocol 
 
Protocol Number: 
 
15-002494 
 
Date and Version: 
  
August 10, 2016 - Version 4 
 
Test Agent: 
 
Sodium Tetrafluoroborate F-18 Injection, Na [18F]BF4  
 
Title: 
 
 First in-human PET imaging studies with NIS reporter [18F]BF4 
 
Funding Source: 
 
Department of Radiology, Mayo Clinic Rochester 
 
Principal Investigators: 
Timothy R. DeGrado, PhD 
 
 
Val J Lowe, MD 
 
Co-Investigators: 
 
Angela Dispenzieri, MD 
 
 
Jamie Bakkum-Gamez, MD 
 
 
Stephen J Russell, MD, PhD 
 
 
Kah Whye Pen, PhD 
 
 
Huailei Jiang, PhD 
 
 
John J. Schmitz, MD 
 
 
Steven Broski, MD 
 
 
Larry Binkovitz, MD 
 
 
Douglas Collins, MD 
 
 
Jolanta Durski, MD 
 
 
Christopher Hunt, MD 
 
 
Geoffrey Johnson, MD, PhD 
 
 
Brian Mullan, MD 
 
 
Robert Murphy, MD, PhD 
 
 
Mark Nathan, MD 
 
 
Gregory Wiseman, MD 
 
 
Andrew Homb, MD 
 
 
Ajit Goenka, MD 
 
 
Research Coordinators: 
Terry Brinkman 
 
 
Kara Hoff 
 
Protocol Development: 
Kristina Schmidtknecht 
 
 
 
 
 
 
 
 

2 
 
 
 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
3 
 
 
TABLE OF CONTENTS 
 
 
 
 
Page  
 
 
 
 
 
 
1. 
INTRODUCTION 
4 
 
2. 
STUDY OBJECTIVES 
7 
 
3. 
SPONSOR, INVESTIGATOR(s) AND OTHER PARTICIPANTS 
7 
 
4. 
TEST DRUG AND CONTROL AGENTS 
8 
4.1 
Descriptive Name 
8 
4.2 
Radioactive Labeling 
8 
4.3 
Decay Characteristics 
8 
4.4 
Synthesis of Compound [
18F]BF4 
8 
4.5 
Formulation and Dose 
9 
 
5. 
WARNINGS/PRECAUTIONS 
10 
 
6.  
INVESTIGATIONAL PLAN 
10 
6.1 
Overall Design and Plan of Study 
10 
6.2 
Aim 2.  Investigations with [18F]BF4 in Healthy Subjects   
11 
6.2.1 Imaging Protocol 
11 
6.2.2 Planned Dosage and Administration 
11 
6.2.3   Selection of Subjects: Inclusion and exclusion criteria 
11 
6.2.4 Prior and Concomitant Therapy 
12 
6.2.5    Removal of Subjects from Study  
13 
6.2.6 Procedures and Methods for Healthy Subjects 
13 
 
6.2.6.1 Screening 
13 
 
6.2.6.2 Subject Preparation 
13 
 
6.2.6.3 Observations and Imaging 
13 
 
6.2.6.4 Adverse Event Follow-Up – Day 2 
15 
6.3 
Aim 3.  Investigations with [18F]BF4 in Cancer Patients   
16 
6.3.1 Imaging Protocol 
16 
6.3.2 Planned Dosage and Administration 
17 
6.3.3   Selection of Subjects: Inclusion and exclusion criteria 
17 
6.3.4 Prior and Concomitant Therapy 
19 
6.3.5    Removal of Subjects from Study  
19 
6.3.6 Procedures and Methods 
19 
 
6.3.6.1 Screening 
19 
 
6.3.6.2 Subject Preparation 
20 
 
6.3.6.3 Observations and Imaging 
20 
 
6.3.6.4 Adverse Event Follow-Up – Day 4 and Day 9 
21 
 
7.  Informed Consent and Human Subject Information 
21 
8.  Documentation 
22 
9. Adverse Events 
22 

[18F]BF4  - Phase 1/2 
Protocol 
 
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9.1 
Toxicity 
22 
9.2 
Adverse Event Monitoring 
23 
9.3 
Adverse Event Definitions 
23 
9.4 
Adverse Event Documentation 
24 
9.5 
Reporting of Serious Adverse Events 
25 
9.6 
Reporting of AEs and SAEs to the FDA 
25 
 
10. 
STATISTICAL ANALYSIS 
26 
10.1 
Objectives 
26 
10.2 
Data Sets 
26 
10.3 
General Descriptive Statistics 
27 
10.4 
Statistical Analysis Plan 
27 
10.5 
Image Analysis 
28 
10.6 
Metabolite Data 
28 
10.7  Sample size justification 
28 
 
11. 
INVESTIGATOR’S REGULATORY OBLIGATIONS 
28 
11.1 
Investigational Product Control 
28 
 
12. 
APPENDICES 
29 
12.1 
References 
29 
12.2 
Study Flow Chart – Healthy volunteers 
30 
12.3   Study Flow Chart – Myeloma and 
endometrial cancer patients 
31 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
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1.   INTRODUCTION 
With the explosive growth of gene and cell-based therapies, there remains a large unmet clinical need for 
sensitive and quantitative imaging methods for reporter gene monitoring.  Radionuclide-based techniques offer 
the highest sensitivity for monitoring reporter expression, with PET offering the most promise.  A number of 
reporter systems employing PET probes have been described, all with their own strengths and weaknesses.  
The ideal reporter probe would have negligible uptake in all normal tissues, with rapid excretion through the 
kidneys to allow excellent target:background contrast in transduced tissues.  None of the probes developed to 
date exhibit this ideal behavior; each have uptake in one or more normal tissues that would obscure imaging of 
gene transduction in those tissues.  Endogenous sodium/iodine symporter (NIS) expression is high in 
thyroid,salivary glands, and stomach.  These organs would be problematic for reporter imaging.  However, 
there is low expression of NIS in liver, spleen, kidney, skeletal muscle and other organs which can be 
problematic for the other reporter systems.  Regarding imaging sensitivity, the probes that rely on a transport 
or metabolic trapping mechanism (tk, NIS, NET) tend to show higher tissue:blood ratios than probe systems 
that are based on receptor binding (D2R, somatostatin R).   Based on these properties, we believe monitoring 
of NIS transduction using [18F]BF4-PET may have distinct advantages in applications that involve gene therapy 
in liver, spleen, kidneys, bone marrow and skeletal muscle.  Furthermore, since NIS is a self-protein (unlike 
HSV-tk), it minimizes the possibility of immune-mediated destruction of cells encoding the reporter.  On the 
other hand, NIS will not be desirable as a reporter in thyroid, stomach or bladder. 
 
The NIS reporter system has unique properties for monitoring gene transduction.  A number of molecules 
besides radioiodide can be used as imaging probes for NIS, including the SPECT isotope [99mTc]pertechnetate.   
Recently, [18F]BF4 has been revisited as a promising NIS imaging agent for PET [1, 2].  Jauregui-Osoro et al. 
[1] updated the synthesis with newer materials that have become available commercially, but the method 
remained isotopic exchange.  These researchers expected that a maximum specific activity that can be 
achieved with current 18F fluoride production levels is ~1GBq/μmol.   Since NIS is a saturable transporter and 
ultimately the “shelf-life” of a distributed PET probe could be several hours, it is highly desirable that a high 
specific activity synthesis of [18F]BF4 be developed.  We have developed a practical, high-yield method for 
production of high specific activity [18F]BF4.  
 
We have performed preclinical imaging studies to confirm the high avidity uptake of [18F]BF4 using a C6 glioma 
mouse tumor model.  hNIS-transfected C6 glioma cells were implanted subcutaneously in the flank of athymic 
nude mice and allowed to grow to ~5 mm diameter before imaging.  Dynamic PET imaging was performed in 
hNIS-expressing C6 glioma xenografted mice following retro-orbital or IP injection ~1.1 MBq Na [18F]BF4 
(Figure 1). [18F]BF4 showed highly specific uptake in NIS expressing tissues: thyroid, stomach, salivary glands 
and mammary glands.  This distribution exactly matches the well-known normal distribution of radioiodides.  
Urinary excretion was variable.  Uptake increased in tumor over 1 hr.  From biodistribution data, tracer uptake 
at 60 min p.i. was >300 fold higher in C6 tumor expressing NIS relative to control (NIS negative) tumor.  
Uptake of tracer increased in NIS expressing tumor and thyroid with increasing specific activity (Figure 1E).  
These preliminary data corroborate previously published findings and our recent imaging with [18F]BF4 in mice 
bearing osteosarcoma tumors (3) that show that the tetrafluoroborate anion serves as an excellent iodide 
analog for NIS-mediated transport and that [18F]BF4 may serve as a highly specific and reliable PET reporter 
probe for NIS activity in tissues.  They strongly support the translation of [18F]BF4 for clinical imaging.  The 
improvement in specific activity with our newly developed synthesis method will also compensate for prolonged 
distribution times of the radiotracer in a commercial distribution setting. 

[18F]BF4  - Phase 1/2 
Protocol 
 
6 
 
Increasing Specific Activity 
 
 
 
AA 
 
D 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
99mTc SPECT 
A 
B 
C 
E 
Figure 1. [18F]BF4 PET images in (A) normal athymic nude mouse and (B) athymic nude mouse bearing hNIS expressing 
C6 glioma xenograft. Th-Thyroid, St-Stomach, and Bl-Bladder. Opposite tumor not expressing hNIS showed insignificant 
tracer uptake.  (C) 99mTc SPECT image of athymic nude mouse bearing hNIS expressing C6 glioma xenograft.(D) Time-
activity curves in tumor, stomach, thyroid and bladder for different specific activity (SA) preparations of  [18F]BF4.  
Tumor uptake is decreased with decreasing SA.  (E) Effect of specific activity on [18F]BF4 uptake in tumor, thyroid and 
stomach. 
A 
B 
C

[18F]BF4  - Phase 1/2 
Protocol 
 
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With the advent of the high specific activity synthesis method and confirmation of the beneficial imaging 
properties of [18F]BF4 in our own preclinical studies in a NIS-expressing C6 tumor mouse model, we believe 
that [18F]BF4 is ready to be tested for imaging feasibility in human patients.  We therefore propose the first in-
human studies with [18F]BF4 in normal subjects (for biodistribution, metabolism, and dosimetry data), and 
patients with myelomas undergoing experimental treatment with Measles virus (MV) containing NIS as a 
reporter.  99mTc-SPECT scans will be performed in the same patients to provide a comparison of imaging 
sensitivity for the two modalities.  This study will provide essential data on imaging characteristics of [18F]BF4 in 
human patients and provide an initial assessment of detection sensitivity for NIS activity for monitoring 
transduction efficacy of oncoviral therapy. 
 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
8 
 
2.   STUDY OBJECTIVES 
 
We propose to develop 18F-labeled sodium tetrafluoroborate ([18F]BF4) as a more sensitive and quantitative 
probe of NIS activity in tumors.  We currently produce [18F]BF4 at the Mayo Clinic and have demonstrated 
initial promising results in animal studies. This clinical trial is designed to test if [18F]BF4 can detect NIS activity 
in tumors under conditions that would substantially improve cancer therapy by:  1) assessing evidence of viral 
infection and 2) assessing the durability of viral replication in tumor by  monitoring NIS uptake at baseline 
(before virus treatment) and day +9.   [18F]BF4 –PET will be compared with conventional SPECT imaging to 
determine the relative advantage for NIS activity detection and quantification in tumors.  Since [18F]BF4 has not 
been used in human studies to our knowledge, we will establish fundamental pharmacokinetic, biodistribution 
and radiation dosimetry data for the imaging drug.  This data will be necessary to support future regulatory 
submissions.  The specific aims of the R01 proposal submitted to NIH are: 
 
Aim 1. To develop an automated synthesis of high-specific activity [18F]BF4 and establish standard operating 
procedures and documentation for human applications. (This aim concerns radiochemistry development and 
does not involve human subjects, so is not relevant to this IRB application.  We have not renumbered Aims 2 
and 3 to provide consistency between the IRB and the NIH application.) 
 
Aim 2. Define the fundamental biodistribution, metabolism and radiation dosimetry characteristics for [18F]BF4 
in human subjects.  Dynamic trunk [18F]BF4-PET imaging will be performed in 8 normal adult human subjects 
(4 male, 4 female) over a 4 h period.  IV blood samples will be taken and analyzed to determine radiochemical 
stability.  Dosimetry estimates will be generated from the regional uptake data.  Normal NIS-mediated activity 
in thyroid, stomach and salivary glands will be quantified by parametric (compartmental modeling) and 
nonparametric (SUV) methods. 
 
Aim 3. Perform [18F]BF4-PET imaging in 10 patients with myeloma before MV-NIS treatment and 10 patients 
with endometrial cancer before VSV-hINFβ-NIS treatment, and at 9 d following therapy to monitor NIS activity 
in the tumors.  To show the correlation of positive regional uptake with tissue histopathology for NIS, when 
accessible, biopsies will be taken after the day 9 scan.  
NOTE: Tc-99m SPECT scans will be performed for comparison purposes under separate IRB approvals 
(06-005263 and 15-007000) and are not included in this protocol.  
 
3.   SPONSOR, INVESTIGATOR(S) AND OTHER PARTICIPANTS 
The study is sponsored by: 
Radiology Department 
Mayo Clinic 
200 First Street SW 
Rochester, MN  55905 
The Principal Investigator  is:  
 
Timothy R. DeGrado 
 
200 First Street SW 
 
Rochester, MN  55905 
 
degrado.timothy@mayo.edu 
 
One center in the United States will participate. 

[18F]BF4  - Phase 1/2 
Protocol 
 
9 
 
 
4.   TEST DRUG AND CONTROL AGENTS 
 
4.1  Descriptive Name:  Na [18F]BF4  
 
Sodium [18F]BF4  
 
NaBF4 
 
MW = 109.8 amu 
 
4.2 Radioactive Labeling 
 
 
The compound is labeled with fluorine-18 [18F] that decays by positron (β+) emission and has a half-
life of 109.8 min.  The principal photons useful for diagnostic imaging are the 511 keV gamma 
photons, resulting from the interaction of the emitted positron with an electron. 
 
4.3 Decay Characteristics 
The time course of radioactive decay for Fluorine-18 [18F] is shown below in Table 1. 
 
Table 1:  Physical decay chart for Fluorine-18 [18F]. Half -life = 109.8 min. 
 
Min. 
Fraction Remaining 
0 
1.000 
20 
0.8814 
40 
0.7768 
60 
0.6847 
80 
0.6035 
100 
0.5319 
120 
0.4688 
150 
0.3879 
180 
0.3210 
 
4.4  Synthesis of Compound Na [18F]BF4 .  
Synthesis of [18F]BF4 will be performed at the Mayo Clinic Cyclotron Facility in Charlton North building by Dr. 
Huailei Jiang under the supervision of the PI.  An automated synthesis of [18F]BF4 has been developed whereby 
cyclotron-produced [18F]fluoride is trapped on a quaternary methyl ammonium anion exchange (QMA) 
cartridge, then allowed to react with  BF3 freshly pre-formulated in a petroleum ether and tetrahydrofuran (THF) 
(50 : 1) solution.  The resultant [18F]BF4 product is retained on the QMA cartridge.  The cartridge is washed 
with THF and water to remove unreacted BF3 and residual solvent.  The product [18F]BF4 is eluted from the 
cartridge using isotonic saline solution. The final product is passed through two neutral alumina cartridges 
before final sterile filtration to remove unreacted fluoride.   The final radiochemical purity and identity of the 
drug is determined by radiometric ion chromatography HPLC.  Radiochemical purity must exceed 95% in order 
to inject.  Other quality control (QC) tests will include appearance, radionuclidic identity, radiochemical identity 
(rHPLC),  chemical purity (HPLC-CD), radionuclidic purity (HPGe detector), specific activity, pH, residual 
solvents (GC-FID), sterility and pyrogens.  The criteria for passing the QC tests are shown in Table 3.  All tests 

[18F]BF4  - Phase 1/2 
Protocol 
 
10 
 
must be passed for release of the product to be administered to the research subjects.  Three consecutive 
validation syntheses were performed in Feb-Mar, 2016, all passing the designated QC tests. 
 
Table 2. Quality control tests and acceptance criteria for Na [18F]BF4 
 
Test 
Acceptance Criteria 
 
Test 
Acceptance Criteria 
Appearance 
Colorless and free 
from particulate 
matter 
 
Specific activity  
> 0.5 GBq/µmol 
Radionuclidic 
identity  
Half-life is 109.8 ± 
5.5 min  
 
Filter integrity  
> 50 psi 
Radiochemical 
identity  
HPLC: Retention 
time (Rt) of F-18 BF4  
=  ± 10% retention 
time of TFB std 
 
Endotoxins 
< 175 EU/ V3 
Radiochemical purity 
HPLC: Activity of F-
18 TFB peak ≥ 95% 
total radioactivity 
 
 Sterility  
(2 wks later; not used 
for product release)   
Pass 
Radionuclidic purity4 
≥  99.5 % of the 
observed peaks 
should correspond to 
0.511 MeV, 1.022 
MeV, or Compton 
scatter peaks of [18F]  
 
 
 
pH  
4.5 – 7.5 
 
 
 
Residual solvent : 
Acetone 
Petroleum Ether 
THF 
 
≤  0.50 % (w/v) 
≤  0.03 % (w/v) 
≤  0.072 % (w/v) 
 
 
 
 
Radioactive 
Concentration 
1–100 mCi/mL @ EOS  
 
 
 
Osmolality 
(Isotonicity) 
Calculated4  
250-359 mOsmoles/L  
 
 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
11 
 
4.5 Formulation and Dose 
The drug is formulated in a 0.9% sodium chloride, USP injection. [18F]BF4 will be administered i.v. in a tracer 
amount of 9-11 mCi.  The expected specific activity of [18F]BF4 is 1-10 GBq/µmol.  We have set the acceptance 
criterion for specific activity as greater than 1 GBq/µmol. At a 9-11 mCi (333-407 MBq) human dose, the mass 
of [18F]BF4 will be 0.33-0.41 µmol or 36-44 µg.  Higher doses may be administered to correct for the radioactive 
decay of PET drug prior to injection, but the maximum mass dose allowed will be 50 µg. 
 
       5.   WARNINGS/PRECAUTIONS 
 
[18F]BF4 is an experimental imaging agent that will be used at relatively low (tracer) doses. Because 
[18F]BF4  is in the early stages of clinical investigation, it is recommended that subjects receiving [18F]BF4  
for injection be followed closely by means of adverse event reporting, vital signs and laboratory tests. 
 
The potential for drug-drug interactions is not presently known.  Until more data become available, it is 
recommended that drugs with a narrow therapeutic window not be co-administered with [18F]BF4. 
 
There are no data on the effects of [18F]BF4 in human perinatal development.  For this reason, fertile 
females must have a negative pregnancy test at screening to qualify for the study and a negative 
pregnancy test within 48 hours of administration of [18F]BF4 for injection. [18F]BF4 for injection must not 
be administered to females who are pregnant or lactating. 
 
       6.   INVESTIGATIONAL PLAN 
 
6.1  Overall Design and Plan of Study 
 
This study will assess the biodistribution, as well as the pharmacokinetics of [18F]BF4 in 8 healthy 
volunteers (4 male, 4 female), 10 patients with myeloma, and 10 patients with endometrial cancer.   
 
In healthy volunteers, screening assessments will take place over phone.  Subjects who qualify for the 
study will be scheduled for a study visit within 30 days of screening.  At the study visit, healthy 
subjects will have an initial physical exam, urine pregnancy test (if applicable) and will have catheters 
placed for i.v. drug administration and for blood sampling.  Vital signs, and blood samples will be 
obtained. Subjects will then receive an infusion of 9-11 mCi [18F]BF4 for injection over 1 minute and 
PET imaging will begin.  Three PET/CT scanning procedures will be performed over a period of 
approximately 4 hours with two rest breaks in between. Venous blood samples for clinical laboratory 
tests will be taken before radiotracer administration at 1.5 hours post-administration of radiotracer. In 
addition, three venous blood samples will be taken during the PET/CT scanning procedure to 
determine blood pharmacokinetics and metabolite evaluation.  Urine collection will be performed 
immediately after each PET/CT scan.  
 
For cancer patients undergoing virus treatments, subjects will undergo [18F]BF4-PET/CT imaging at 
baseline before virus administration and at day 9 following virus treatment.  Subjects will be screened 
by Drs. Dispenzieri or Bakkum-Gamez within their clinics.  Subjects who qualify for the study will 
return to the clinic within 30 days of screening, have a urine pregnancy test (if applicable) and will 
have catheters placed for i.v. drug administration.  Vital signs will be obtained. All subjects will then 
receive a single i.v. bolus of 9-11 mCi [18F]BF4 for injection and PET/CT imaging will begin. Biopsies 

[18F]BF4  - Phase 1/2 
Protocol 
 
12 
 
will be performed to confirm NIS expression in tissue of tumor regions showing uptake of [18F]BF4 in 
up to five myeloma patients and five endometrial cancer patients when the site is accessible for biopsy. 
 
6.2. Aim 2: Investigations with [18F]BF4 in Healthy Subjects 
 
Healthy volunteers will be studied before cancer patients in Aim 3.   
 
6.2.1. Imaging protocol 
Subjects will be imaged at the following time points to collect biodistribution data: 
 
Time Point 
Image 
-5 → 0 
CT-Transmission Scan over heart 
0 → 45 
Dynamic PET Emission Scan over heart 
45 → 110 
1st Rest Period 
110→ 120 
Total Body CT-Transmission Scan 
120 → 150 
Total Body PET Emission Scan (including head) 
150 → 200 
2nd Rest Period 
200 → 210 
Total Body CT - Transmission Scan 
210 → 240 
Total Body Emission Scan (including head) 
 
6.2.2 Planned Dosage and Duration of Treatment 
 
6.2.2.1 Dosage and Administration 
 
During this study, subjects will receive a single i.v. administration of 9-11 mCi sodium 
[18F]BF4 immediately prior to the first of three PET/CT imaging scans. 
 
6.2.2.2 Rationale for Dosages 
 
The proposed dose for this study is based on comparison to other known imaging agents and is 
supported by the dosimetry estimates generated from the biodistribution experiments following 
administration of [18F]BF4 to rodents. Preliminary dosimetry estimates suggest that the expected 
human exposure for most organs will be approximately 0.05 rem/mCi.  The dose limiting organ is 
estimated to be the stomach with dose of 0.3 rem/mCi.  The estimated human radiation exposure 
after a 11 mCi dose of [18F]BF4 (10.2 mSv) will be comparable or below the range for other 
approved imaging agents, such as 18F-FDG. 
 
[18F]BF4 will be formulated in 0.9% (w/v) sodium chloride solution and administered by i.v. bolus, 
in tracer amount of 9-11 mCi.  The expected specific activity of [18F]BF4 is 1-10 GBq/µmol.  We 
have set the acceptance criterion for specific activity as greater than 1 GBq/µmol. At a 9-11 mCi 
(333-407 MBq) human dose, the mass of [18F]BF4 will be 0.33-0.41 µmol or 36-44 µg.  Higher 
doses may be administered to correct for the radioactive decay of PET drug prior to injection, but the 
maximum mass dose allowed will be 50 µg. 
 
 
 
6.2.3  Selection of Healthy Subjects 

[18F]BF4  - Phase 1/2 
Protocol 
 
13 
 
 
6.2.3.1  Inclusion Criteria 
 
 
1.   Four male and four female healthy volunteers, greater than 21 years of age.  
2.   Subjects must provide written informed consent. 
3.   Willingness to provide all biological specimens as required by the protocol. 
 
 
6.2.3.2  Exclusion Criteria 
 
Volunteers with any of the following are ineligible to enroll in this study: 
 
1.   Have currently clinically significant cancer, neurologic, hepatic, renal, pulmonary, metabolic, 
or endocrine disturbances, especially thyroid disease; 
2.   Current clinically significant cardiovascular disease.  Clinically significant cardiovascular 
disease usually includes one or more of the following: 
a.   cardiac surgery or myocardial infarction within the last 6 months; 
b.   unstable angina; 
c.   coronary artery disease that required a change in medication within the last 3 months; 
d.   decompensated congestive heart failure; 
e.   significant cardiac arrhythmia or conduction disturbance, particularly those resulting in 
atrial or ventricular fibrillation, or causing syncope, near syncope, or other alterations in 
mental status; 
f. severe mitral or aortic valvular disease; 
g.   uncontrolled high blood pressure; 
h.   congenital heart disease; 
 
3.   History of drug or alcohol abuse within the last year, or prior prolonged history of abuse; 
4. Clinically significant infectious disease, including AIDS or HIV infection or previous positive 
test for hepatitis B, hepatitis C, HIV-1, or HIV-2; 
5. Women of childbearing potential must not be pregnant (negative urine β-HCG at the time of 
screen) or lactating over the course of the study.  A commercial urine dipstick test will be 
performed within 48 hours prior to injection of [18F]BF4 unless the screening urine pregnancy 
test falls within 48 hours of  injection.   
6. Volunteers who, in the opinion of the investigator, are otherwise unsuitable for a study of this 
type; 
7. History of severe drug allergy or hypersensitivity; or 
8. Volunteers who had received an investigational medication within the last 30 days or who have 
participated in a clinical trial with any experimental medication or radiopharmaceutical in the 
last 30 days. Additionally, the time between the last dose of the previous experimental 
medication and enrollment (completion of screening assessments) must be at least equal to 5 
times the terminal half-life of the previous experimental medication. 
9.  Volunteers who are taking drugs with narrow therapeutic windows, such as theophylline, or 
warfarin, heparin and other anticoagulant therapies 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
14 
 
 
6.2.4  Prior and Concomitant Therapy 
 
Except as noted below, all medications (prescription or over-the-counter) that 
have been started prior to screening may be continued during the course of the study. Attempts should 
be made to keep the dosage and administration stable throughout the study (from screening through 
the end of the imaging protocol). 
 
All medications that are continued from the start of the study or that are started during the study 
(other than the study medication) must be documented in the case record form on the Concomitant 
Medication Page of the Case Record Form (CRF). 
 
•  Investigators must not enroll patients who are taking drugs with narrow therapeutic windows, such 
as theophylline, or warfarin, heparin and other anticoagulant therapies, and should not prescribe 
these drugs for the duration of the study. 
 
6.2.5  Removal of Subjects from Study 
 
Subjects must be removed from the study if: 
1. Informed consent is withdrawn; or 
2. The investigator believes it is in the best interest of the subject to be removed from the study. 
Subjects may be withdrawn from the study if a serious adverse event occurs. The date and reason 
for discontinuation should be noted on the CRF. Subjects who discontinue prematurely should be 
seen for a final evaluation. 
 
6.2.6   PROCEDURES AND METHODS FOR HEALTHY SUBJECTS 
 
6.2.6.1. Screening:   Day -30 - 0 
Healthy subjects will be recruited from local population by local advertisements. Prospective 
subjects will contact the study coordinator by telephone (see attached recruitment phone script).  If 
subject are deemed eligible for participation, signed informed consent will occur when they arrive 
for the study visit. 
 
Screening assessments will be performed during this recruitment phone call and/or during a 
subsequent phone call within 30 days of the imaging session.  Screening assessments, performed 
over phone, will include: 
•   Demographics (age, sex, education, alcohol and drug use, smoking); 
•   Medical history, concomitant medications; 
 
•    Disease history. 
 
6.2.6.2 Subject Preparation 
• Subjects will be asked to drink at least 2-8 oz glasses of water the morning of the test and prior to 
injection 
• Subjects are not required to fast. 
 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
15 
 
6.2.6.3 Observations and Imaging  
 
The following assessments will be performed (see schedule of assessments 12.2 in 
Appendix): 
 
• Informed consent 
• Physical Exam. A physical examination will be conducted by a Nuclear Medicine physician at the 
subjects’ arrival to the clinic and at the completion of the imaging study. Clinically significant changes 
that occur after injection of [18F]BF4 will be recorded as adverse events through the 24 hours post 
injection period. 
 
• A  pregnancy  test  (urine beta  hCG,  qualitative): will  be  performed  at screening for females who 
are of childbearing potential. A commercial urine dipstick pregnancy test will be performed within 
48 hours of injection if the serum pregnancy test falls outside the 48 hour window. 
 
• Venous catheters will be established in both arms by a Nuclear Medicine Technologist prior to 
[18F]BF4  for injection. 
 
•  Subjects will be positioned in supine orientation in the GE 690XT PET/CT scanner.  For attenuation 
correction and localization, CT of the same region will incorporate Helical CT with pitch of 1 and x-ray 
tube set to 120 kV and 70 mAs and CT of the body will incorporate Helical CT with a pitch of 1 and x-
ray tube set to 120 kVp and 45 mAs.   
 
• Immediately following administration of [18F]BF4 for injection (9-11 mCi over 1 min infusion 
period), subjects will undergo dynamic PET/CT imaging for 45 minutes over the heart with sequence 
20x3s, 12x 10s, 4x30s, 5x60s, 7x300s.  Following the dynamic PET/CT scan, the subjects will be 
asked to void the bladder and allowed to rest outside of the scanner for ~55 minutes (1st rest period). 
Trunk imaging by PET/CT will resume for 30 minutes (~2 h post-injection), followed by a second rest 
period for ~55 minutes.  Trunk  PET/CT imaging will resume for 30 minutes such that PET/CT 
scanning will be commenced at 210 min and completed at 240 min post-injection.  Subjects will void 
urine during the rest periods and the entire void of urine will be collected in a pre-weighed jug for total 
radioactivity counts.  Subjects will void at the end of study and a urine sample will be collected for 
total radioactivity counts. 
 
If it becomes apparent in analyses of the data that the longer post-injection imaging times are not 
contributing valuable data, they may be dropped from further data collections after agreement between 
the study investigators. The investigators will prepare and distribute a written plan for image 
acquisition procedure and parameters as well as case report forms (CRFs) prior to the start of the study. 
 
 
• Vital signs including blood pressure, oral temperature, pulse and respiration rate will be taken by a 
Nuclear Medicine Technologist immediately prior to administration of [18F]BF4 for injection, after 
the first PET/CT scanning sequence (~45 min post-injection), and at the end of the study (after 
completion of PET/CT scanning, ~240 min post-injection); 
 
• Blood samples (venous) for pharmacokinetic and metabolic evaluation will be taken by a Nuclear 
Medicine Technologist at 40, 145, and 235 min after [18F]BF4 administration. Samples will be 
transported to the Molecular Imaging Research lab, Stabile 3-28, for processing.   

[18F]BF4  - Phase 1/2 
Protocol 
 
16 
 
 
• Urine samples will be collected by a Nuclear Medicine Technologist for total radioactivity counts 
after each PET/CT scan at approximately 60, 160, and 240 min post-injection of radiotracer.  The mass 
(g) of urine collected at each time point will be recorded.   The samples will be transferred together 
with the blood samples to Dr. DeGrado’s lab in Stabile building for further analysis.  The radioactivity 
concentration in urine (1 mL each) will be measured (mCi/mL) by using a well counter. The total 
radioactivity excreted in urine will be determined as the product of radioactivity concentration and 
urine volume (assuming a density of 1 g/ mL) for the three collection intervals. 
   
 
• Blood samples will be collected by a Nuclear Medicine Technologist for clinical labs prior to 
administration of [18F]BF4 and at approximately 90 min post-administration of  [18F]BF4  and be 
processed routinely in Mayo Laboratory Medicine and reported in the subjects medical record.  The 
1-2 needle sticks for these samples will be performed by Venipuncture Services.  Three to four 
needles sticks will be made in total, including the sticks required for radiotracer injection and blood 
samples for radioactivity analysis. If the blood laboratory tests provide clinically meaningful 
information, the subject’s primary physician will be notified. 
 
The laboratory tests will include: 
  
 
 
• Hematology (5 mL EDTA):   hemoglobin, hematocrit, RBC, WBC, MCH, MCHC, 
neutrophils, bands, lymphocytes, monocytes, eosinophils, basophils, platelets, morphology, MCV, 
and RBC morphology. 
 
 
• Chemistry (6 mL blood):  total bilirubin, alkaline phosphatase, ALT (SGPT), AST (SGOT), 
urea nitrogen, creatinine, glucose, uric acid, calcium, phosphorus, total protein, albumin, total 
cholesterol, triglycerides, sodium, potassium, bicarbonate, chloride, magnesium, globulin, GGT, 
tryptase. 
 
The analysis of the blood samples for [18F]BF4 levels and radiolabeled metabolites will be 
performed by collecting 3 mL samples in green top tubes and taken at multiple time points (40, 
145, and 235 minutes).  Samples will be immediately placed on ice to stop metabolism and/or 
transport into red blood cells. One mL of each blood sample will be pipetted to a 1.5 mL Eppendorf 
spin tube, spun down, and 0.4 mL plasma transferred by pipette to a test tube for counting. For 
counting of whole blood, 0.4 mL of each whole blood sample will be pipetted to test tube for 
counting. The radioactivity concentration in blood and plasma (1 mL each) will be measured 
(mCi/mL) by using a well counter.    
 
• Subjects will be observed continuously for signs of adverse events or serious adverse events 
throughout the imaging study. 
 
      
6.2.6.4 Adverse Event Follow-Up at 24 h post-administration of [18F]BF4 
 
A scripted phone call will be made by a study coordinator to each subject at approximately 24 hours 
post-injection of [18F]BF4 to check for any adverse events.  Any adverse events will be reported to the 
PI immediately following the phone call. 
 
 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
17 
 
6.3. Aim 3: Investigations with [18F]BF4 in Cancer Patients  
 
6.3.1. Imaging protocol 
 
Subjects will be imaged before virus administration (baseline = Day -0) and Day 9 after 
administration of virus at the following time point to collect biodistribution data: 
 
Time Point 
Image 
0 
Inject [18F]BF4 (9-11  mCi) 
90 → 95 
Body  CT Transmission Scan  
95 → 135 
Trunk PET Emission Scan  
 
Adverse events and serious adverse events will be monitored continuously throughout the imaging study 
and through the 24 hour follow-up phone call.  A Nuclear Medicine co-investigator physician must 
evaluate the subject at patient’s arrival to the clinic for the imaging study and prior to discharge.  Subjects 
who experience an adverse event will not be discharged until the event has resolved or stabilized. 
 
6.3.2 Planned Dosage and Duration of Treatment 
 
6.3.2.1 Dosage and Administration 
 
During this study, subjects will receive two i.v. administrations of 9-11 mCi sodium 
[18F]BF4 immediately prior to imaging. 
 
6.3.2.2 Rationale for Dosages 
 
The proposed dose for this study is based on comparison to other known imaging agents and is 
supported by the dosimetry estimates generated from the biodistribution experiments following 
administration of [18F]BF4 to rodents. Preliminary dosimetry estimates suggest that the expected 
human exposure for most organs will be approximately 0.05 rem/mCi.  The dose limiting organ is 
estimated to be the stomach with dose of 0.3 rem/mCi.  The estimated human radiation exposure 
after a 11 mCi dose of [18F]BF4 (10.2 mSv) will be comparable or below the range for other 
approved imaging agents, such as 18F-FDG. 
 
[18F]BF4 will be formulated in 0.9% (w/v) sodium chloride solution and administered by i.v. bolus, 
in tracer amount of 9-11 mCi.  The expected specific activity of [18F]BF4 is 1-10 GBq/µmol.  We 
have set the acceptance criterion for specific activity as greater than 1 GBq/µmol. At a 9-11 mCi 
(333-407 MBq) human dose, the mass of [18F]BF4 will be 0.33-0.41 µmol or 36-44 µg.  Higher 
doses may be administered to correct for the radioactive decay of PET drug prior to injection, but 
the maximum mass dose allowed will be 50 µg. 
 
6.3.3  Selection of Subjects 
 
6.3.3.1  Inclusion Criteria 
 
 
 
6.3.3.1.1 Aim 3a. Myeloma Patients 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
18 
 
1.  Patients who are currently participating in Mayo Clinic MV-NIS trial (IRBs: 06-005263) and 
agree to enroll in additional [18F]BF4-PET studies.  
2.  Subjects are greater than 21 years of age.  
3. Subjects must provide written informed consent.  
4. All other inclusion criteria are met for study 06-005263. 
5. Agree to provide biopsy of tumor indicated on PET for research purposes, if accessible; 
 
6.3.3.1.2 Aim 3b. Endometrial Cancer Patients 
 
1.  Patients who are currently participating in Mayo Clinic VSV-hINFβ-NIS trial (IRB 15-007000) 
and agree to enroll in additional [18F]BF4-PET studies. 
2.  Subjects are greater than 21 years of age.  
3. Subjects must provide written informed consent.  
4. All other inclusion criteria are met for study 15-007000. 
5. Agree to provide biopsy of tumor indicated on PET for research purposes, if accessible; 
 
6.3.3.2  Exclusion Criteria 
 
 
6.3.3.2.1 Aim 3a. Myeloma Patients 
 
Patients with any of the following are ineligible to enroll in this study: 
 
1.   All exclusion criteria apply from study 06-005263. 
2.   Current clinically significant cardiovascular disease.  Clinically significant cardiovascular 
disease usually includes one or more of the following: 
a.   cardiac surgery or myocardial infarction within the last 6 months; 
b.   unstable angina; 
c.   coronary artery disease that required a change in medication within the last 3 months; 
d.   decompensated congestive heart failure; 
e.   significant cardiac arrhythmia or conduction disturbance, particularly those resulting in 
atrial or ventricular fibrillation, or causing syncope, near syncope, or other alterations in 
mental status; 
f. severe mitral or aortic valvular disease; 
g.   uncontrolled high blood pressure; 
h.   congenital heart disease; 
 
Before enrolling a patient with any of the above conditions, the co- investigator must contact the 
principal investigator 
3.   History of drug or alcohol abuse within the last year, or prior prolonged history of abuse; 
4. Clinically significant infectious disease, including AIDS or HIV infection or previous positive 
test for hepatitis B, hepatitis C, HIV-1, or HIV-2; 
5. Women of childbearing potential must not be pregnant (negative urine β-HCG at the time of 
screen) or lactating over the course of the study.  A commercial urine dipstick test will be 
performed within 48 hours prior to injection of [18F]BF4 unless the screening urine pregnancy 

[18F]BF4  - Phase 1/2 
Protocol 
 
19 
 
test falls within 48 hours of  injection.   
6. Patients who, in the opinion of the investigator, are otherwise unsuitable for a study of this type; 
7. History of severe drug allergy or hypersensitivity; 
8. Patients who had received an investigational medication within the last 30 days or who have 
participated in a clinical trial involving medications other than the related MV-NIS or VSV-
NIS viruses in the last 30 days. Additionally, the time between the last dose of the previous 
experimental medication and enrollment (completion of screening assessments) must be at 
least equal to 5 times the terminal half-life of the previous experimental medication.  
9.Patients with current clinically significant medical comorbidities, that might pose a potential 
safety risk, interfere with the absorption or metabolism of the study medication or limit 
interpretation of the study results.  These include but are not limited to clinically significant 
hepatic, renal, pulmonary, metabolic or endocrine disease, cancer, HIV infection and AIDS. 
 
10. Patients who are taking drugs with narrow therapeutic windows, such as theophylline, or 
warfarin, heparin and other anticoagulant therapies 
 
6.3.3.2.2 Aim 3b. Endometrial cancer patients 
 
Patients with any of the following are ineligible to enroll in this study: 
 
1.   All exclusion criteria apply from VSV-hINFβ-NIS clinical trial (IRB 15-007000). 
2.   Current clinically significant cardiovascular disease.  Clinically significant cardiovascular 
disease usually includes one or more of the following: 
a.   cardiac surgery or myocardial infarction within the last 6 months; 
b.   unstable angina; 
c.   coronary artery disease that required a change in medication within the last 3 months; 
d.   decompensated congestive heart failure; 
e.   significant cardiac arrhythmia or conduction disturbance, particularly those resulting in 
atrial or ventricular fibrillation, or causing syncope, near syncope, or other alterations in 
mental status; 
f. severe mitral or aortic valvular disease; 
g.   uncontrolled high blood pressure; 
h.   congenital heart disease; 
 
Before enrolling a patient with any of the above conditions, the co- investigator must contact the 
principal investigator 
3.   History of drug or alcohol abuse within the last year, or prior prolonged history of abuse; 
4. Clinically significant infectious disease, including AIDS or HIV infection or previous positive 
test for hepatitis B, hepatitis C, HIV-1, or HIV-2; 
5. Women of childbearing potential must not be pregnant (negative urine β-HCG at the time of 
screen) or lactating over the course of the study.  A commercial urine dipstick test will be 
performed within 48 hours prior to injection of [18F]BF4 unless the screening urine pregnancy 
test falls within 48 hours of  injection.   
6. Patients who, in the opinion of the investigator, are otherwise unsuitable for a study of this type; 
7. History of severe drug allergy or hypersensitivity; 

[18F]BF4  - Phase 1/2 
Protocol 
 
20 
 
8. Patients who had received an investigational medication within the last 30 days or who have 
participated in a clinical trial with any experimental medication in the last 30 days. 
Additionally, the time between the last dose of the previous experimental medication and 
enrollment (completion of screening assessments) must be at least equal to 5 times the terminal 
half-life of the previous experimental medication.  
9.Patients with current clinically significant medical comorbidities, that might pose a potential 
safety risk, interfere with the absorption or metabolism of the study medication or limit 
interpretation of the study results.  These include but are not limited to clinically significant 
hepatic, renal, pulmonary, metabolic or endocrine disease, cancer, HIV infection and AIDS. 
10. Patients who are taking drugs with narrow therapeutic windows, such as theophylline, or 
warfarin, heparin and other anticoagulant therapies 
 
 
     6.3.4  Prior and Concomitant Therapy 
Except as noted below, all medications (prescription or over-the-counter) that 
have been started prior to screening may be continued during the course of the study. Attempts should 
be made to keep the dosage and administration stable throughout the study (from screening through 
the end of the imaging protocol). 
 
All medications that are continued from the start of the study or that are started during the study 
(other than the study medication) must be documented in the case record form on the Concomitant 
Medication Page of the Case Record Form (CRF). 
 
6.3.5  Removal of Subjects from Study 
 
Patients must be removed from the study if: 
1. Informed consent is withdrawn; or 
2. The investigator believes it is in the best interest of the subject to be removed from the study. 
Patients may be withdrawn from the study if a serious adverse event occurs. The date and reason 
for discontinuation should be noted on the CRF. Subjects who discontinue prematurely should be 
seen for a final evaluation. 
 
6.3.6   PROCEDURES AND METHODS 
 
6.3.6.1. Screening:   Day -30 - 0 
Patients will be selected from subjects electing to participate in IRB 06-005263 at Mayo Clinic: 
“Phase I/II Trial of Systemic Administration of Edmonston Strain of Measles Virus, Genetically 
Engineered to Express NIS, with or without Cyclophosphamide, in Patients with Recurrent or 
Refractory Multiple Myeloma”, PI: Angela Dispenzieri, MD; or “Phase I Trial of Systemic 
Administration of Vesicular Stomatitis Virus Genetically Engineered to Express NIS and human 
interferon, in Patients with Metastatic and/or Incurable Endometrial and Epithelial Ovarian Cancer”, 
PI: Jamie Bakkum-Gamez, MD, IRB 15-007000.  Prospective subjects will have been screened for 
medical and physical compliance to the inclusion and exclusion criteria.  If subject are deemed 
eligible for participation, participants will be invited to participate and sign informed consent. All 
screening assessments must be performed within 30 days of the imaging session.  Screening 
assessments will include: 

[18F]BF4  - Phase 1/2 
Protocol 
 
21 
 
•   Informed consent; 
•   Demographics (age, sex, education, alcohol and drug use, smoking); 
•   Medical history, physical exam, concomitant medications; 
• Disease history (date/months since symptom onset, date/months since diagnosis); 
 •   Vital signs 
 
6.3.6.2 Subject Preparation – Baseline and Day +9 after virus administration 
• Subjects will be asked to drink at least 2-8 oz glasses of water the morning of the test and prior to 
injection 
• Subjects are not required to fast. 
 
 
6.3.6.3 Observations and Imaging – Baseline and Day +9 after virus administration 
 
The following assessments will be performed (see schedule of assessments 12.3 in 
Appendix): 
 
•  Physical Exam. A physical examination will be conducted by a physician at the patients’ arrival to 
the clinic. Clinically significant changes that occur after injection of [18F]BF4 will be recorded as 
adverse events through the 24 hours post injection period. 
 
• Subjects will void bladder 
 
• Venous catheter will be established in one arm prior to [18F]BF4  for injection. 
 
• Within a dimly lit uptake room, [18F]BF4 (9-11 mCi) will be administered as an infusion 
over 1 minute.  Subjects will remain seated for a 90-min uptake period.  
 
•  Subjects will be position in supine orientation in the PET/CT scanner.  For attenuation correction and 
localization, CT of the same region will incorporate Helical CT with pitch of 1 and x-ray tube set to 
120 kV and 70 mAs and CT of the body will incorporate Helical CT with a pitch of 1 and x-ray tube set 
to 120 kVp and 45 mAs.   
 
• Subjects will undergo a static trunk-region PET/CT imaging for 40 minutes. 
 
• Biospy for histochemical confirmation of viral infection.  The projected numbers of subjects that will 
undergo biopsy are shown in the table below: 
 
Group 
n 
Myeloma w/o biopsy 
5 
Myeloma w/ biopsy 
5 
Endometrial Cancer w/o biopsy 
5 
Endometrial Cancer w/ biopsy 
5 
 
If there is an area that shows focal uptake in tumor on one of the [18F]BF4-PET scans, suggesting that 
there may be measles virus at that location, and if it is in a position in the  body that is easy to biopsy, 
we will exercise the option, with allowance by the subject (see consent form) to perform a needle 

[18F]BF4  - Phase 1/2 
Protocol 
 
22 
 
biopsy to obtain tissue for pathological confirmation of viral infection.  Dr. John Schmitz will perform 
the needle biopsy under guidance of ultrasound or CT, whichever is deemed more appropriate for the 
target location. Biopsies will be performed using standard sterile technique, local anesthesia, and 
potentially moderate sedation with fentanyl and midazolam when appropriate. As with all procedures, 
appropriate medication substitutions will be made based upon documented allergies or prior adverse 
reactions. Two 1-2 cm 18-gauge core samples will be obtained from the targeted tissue using a spring-
loaded biopsy device. A 17-gauge introducer needle may be used to obtain access for the biopsy 
device. Immediate post-biopsy imaging will be obtained with the modality employed, as is standard for 
image guided procedures. The patient will be observed in the radiology post-procedure area according 
to established practice for site biopsied. The primary risks standard to all biopsies will be discussed 
with the patient including but not limited to: bleeding, non-target needle placement/biopsy, and 
adverse medication reaction.  Biopsy sample will be analyzed for NIS expression by 
immunohistochemistry in the lab or Dr. Peng. 
 
• Vital signs including blood pressure, oral temperature, pulse and respiration rate will be taken 
immediately prior to administration of [18F]BF4 for injection, and at the end of the study (after 
completion of PET scanning, ~135 min post-injection);  Nuclear Medicine physician will perform a 
physical examination of patients before their release.  
 
 
• Subjects will be observed continuously for signs of adverse events or serious adverse events 
throughout the imaging study. 
 
6.3.6.4 Adverse Event Follow-Up – 24 h post-administration of [18F]BF4 
 
A scripted phone call will be made to each subject at approximately 24 hours post-injection of [18F]BF4 
to check for any adverse events. 
 
7. Informed Consent and Human Subject Information 
 
NOTE: Since all myeloma and endometrial cancer patients will be recruited from an existing pool of 
subjects already participating in studies at the Mayo Clinic, some of the screening information will 
already be accessible. A review preparatory to research application will used to review this data in 
advance of consent to determine eligibility. 
 
All informed consent forms must be approved by Mayo Clinic Institutional Review Board (IRB).  No 
study related procedures shall be performed prior to completion of the informed consent process, and 
signing of the consent form. A copy of the signed informed consent should be given to the patient 
and/or their caregiver for their records. 
 
Subject will be exposed to radiation but the amount of radiation has a low risk of harmful effect.  The 
amount of Sodium BF4 used in the dose preparation is anticipated to be < 10 µg and no 
pharmacologic effect is anticipated.  Women of childbearing potential must have a negative 
pregnancy test as part of their screening eligibility and within 48 hours of injection of the 
investigational drug [18F]BF4.  Healthy subject will have 3-4 venous needle sticks to insert cannulas 
for drug injection and blood sample draws.  Cancer patients will have one venous needle stick to 
insert cannula for drug injection.  Subjects may experience pain, bruising or rarely infection at the 

[18F]BF4  - Phase 1/2 
Protocol 
 
23 
 
needle site. A total of 20 mL of blood will be drawn from healthy subjects. Urine samples will be 
collected in healthy subjects three times. Imaging may be tiring but every effort will be made to make 
subjects comfortable throughout the scan process.  A head holder will be utilized to help subjects 
remain motionless.   
 
Healthy subjects will be remunerated $200.00 and given parking passes for their time and 
inconvenience associated with study participation.  Patients will be remunerated $300.00 and given 
parking passes for their time and inconvenience associated with the study protocol.  Participants that 
do not complete the study will be provided partial remuneration according to the fraction of the study 
completed. 
 
8. Documentation 
 
PET scan results will be saved in electronic format (DICOM).  All other data required by the protocol 
will be recorded in the CRF.  
 
9. Adverse Events 
 
9.1.   Toxicity 
 
Potassium [18F]BF4 was first reported by Anbar and Guttmann in 1960 (4).  The original 
synthesis was performed by isotopic exchange, producing radiotracer with low specific activity 
(unreported value). Nevertheless, the radiotracer accumulated avidly into rat thyroid, consistent 
with the hypothesis of transport via NIS.  Up to 1 mg of [18F]KBF4 was administered to rats 
with no noted toxicity.  For NaBF4, the acute subcutaneous LD50 in the rat was found to be 
550 mg/kg (5, 6).  The first human exposure to [18F]BF4 in the form of its potassium salt was 
reported by Entzian et al. in 1964 (7).  Again, this radiotracer was synthesized in low specific 
activity (0.13 mCi/mg) by isotopic exchange.  [18F]KBF4 (1-3 mCi, 7-23 mg) was administered 
to ten patients with brain tumors to determine potential to image brain tumors using an early 
coincidence rectilinear scanner.  No adverse events were reported. A typical blood clearance 
curve was reported, showing a rapid clearance phase (T½ = 15 min) in the first 30 min, 
followed by a slower clearance phase out to 5 h (T½ = 290 min) (7).  [18F]KBF4 was found to 
be primarily excreted in the urine with a excretion half-time of approximately 470 min.  Further 
evidence of low toxicity of the BF4
- anion is that it is contained in the formulations of a number 
of FDA approved drugs, including 99mTc-labeled Sestamibi (tetrafluoroborate is its 
counteranion), a myocardial perfusion imaging agent, which is administered in mg quantities to 
more than 6 million patients per year in the U.S. These data support the proposed studies with 
high-specific activity sodium [18F]BF4 (>50 mCi / mg = 10 GBq/µmol), where less than 10 µg 
of NaBF4 will be administered to each patient. 
 
Based on this information, we do not anticipate any pharmacologic effect and therefore, no 
toxicities are anticipated in this study. 
 
9.2.   Adverse Event Monitoring 
 
Each subject must be carefully monitored for adverse events. Healthy volunteers will be studied 

[18F]BF4  - Phase 1/2 
Protocol 
 
24 
 
before cancer patients.  Blood samples will be analyzed in healthy subjects by a broad panel of 
clinical laboratory tests.  Should any significant pharmacology or toxicity be identified in the 
healthy subjects in response to the test radiotracer, we will also include blood testing in cancer 
patients. An assessment will be made by a physician regarding the seriousness, intensity, and 
relationship to the administration of the study medication. 
 
9.3. 
Adverse Event Definitions 
 
Adverse Events 
 
An adverse event is any undesirable experience occurring to a subject during the Phase 
1/2 study, whether or not considered related to the investigational product. 
 
For reporting purposes, study staff will distinguish among pre-existing conditions, study-
emergent adverse events, and treatment-emergent adverse events. 
 
Pre-existing conditions (i.e., undesirable experiences, signs, or symptoms that begin prior to 
the Screening Visit) will be recorded on the medical history and/or physical exam portion of 
the Case Report Form.  These conditions will not be entered on the adverse event portion of 
the Case Report Form unless they worsen in intensity or frequency after the Screening Visit. 
 
Study-emergent adverse events are undesirable experiences, signs or symptoms that begin, 
or worsen in intensity or frequency, after the Screening Visit, and prior to administration of 
study drug at the imaging visit. These will be recorded on the adverse event pages. 
 
Treatment-emergent adverse events are undesirable experiences, signs, or symptoms that 
begin or worsen in intensity or frequency after the administration of study drug. 
 
Serious Adverse Event 
 
A serious adverse event is an adverse event that is fatal or life-threatening, or results in 
hospitalization, prolongation of hospitalization, persistent or significant disability/incapacity, or 
a congenital anomaly/birth defect. A life-threatening adverse event is an adverse event that, in 
the view of the investigator, places the subject at immediate risk of death from the reaction, as it 
occurred.  Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse events when, based upon appropriate 
medical judgment, they may jeopardize the patient or subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
 
 
Relationship to Investigational Product 
 
The assessment of the relationship of an adverse event to the administration of the study drug 
(remote, possible, and probable) is a clinical decision based on all available information at the 
time of the completion of the CRF. The following definitions of the relationship between the 
study drug and the adverse event (including serious adverse events) should be considered: 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
25 
 
•  Remote (unlikely, doubtful, improbable): 
The time course between the administration of the study medication and the occurrence or 
worsening of the adverse event makes a causal relationship unlikely and another cause 
(concomitant drugs, therapies, complications etc.) is probable. 
•  Possible: 
The time course between the administration of the study medication and the occurrence or 
worsening of the adverse event is consistent with a causal relationship but another cause 
(concomitant drugs, therapies, complications etc.) cannot be ruled out; 
 
<or> 
 
The time course between the administration of the study medication and the occurrence or 
worsening of the adverse event is not consistent with a causal relationship but no alternative 
cause can be identified. 
 
•  Probable: 
 
The time course between the administration of the study medication and the occurrence or 
worsening of the adverse event is consistent with a causal relationship and no other cause 
can be identified (concomitant drugs, therapies, complications etc.) 
 
 
If the Investigator is unable to assess causality, the adverse event should be considered 
“Possible” by definition and not “Remote.” 
 
 
Intensity/Severity of an Adverse Event 
 
In addition to assessing the relationship of the administration of the investigational product to 
adverse events, an assessment is required of the intensity (severity) of the event. 
 
The following classifications should be used: 
 
•  Mild: 
A mild adverse event is an adverse event, usually transient in nature and generally not 
interfering with normal activities. 
•  Moderate: 
A moderate adverse event is an adverse event that is sufficiently discomforting to 
interfere with normal activities. 
 
•  Severe: 
A severe adverse event is an adverse event that incapacitates the subject and prevents 
normal activities.   Note that a severe event is not necessarily a serious event.  Nor must a 
serious event necessarily be severe. 
 
9.4.   Adverse Event Documentation 
 
All adverse events must be fully recorded on the Adverse Event Page of the Case Report Form.  
Laboratory test abnormalities considered by the Investigator to be clinically relevant should be 
reported on the Adverse Event page of the Case Report Form.  Signs and symptoms of each AE 

[18F]BF4  - Phase 1/2 
Protocol 
 
26 
 
should be described in detail (e.g., start and stop dates, severity, intensity, relationship to 
investigational product, action taken, and outcome). 
 
Adverse events and laboratory test abnormalities fulfilling the definition of a serious adverse 
event should, in addition, be reported on the Serious Adverse Event Reporting Form. 
 
Mayo Clinic lab standards and clinically determined normal values will be used by the study 
investigators to determine how and if results meet adverse event standards. 
 
9.5.   Reporting of Serious Adverse Events 
 
Serious adverse events must be reported to Mayo Clinic IRB within 5 days of 
learning of it, using the IRB Reportable Event form.   All reportable events meeting 
the Unanticipated Problem Involving Risk to Subjects or Others (UPIRTSO) criteria 
as submitted to the IRB using the Reportable Event form will be sent to an IRB 
Chairperson for review and determination. If the IRB Chairperson determines the 
event is a UPIRTSO, a convened IRB reviews it and either confirms or does not 
confirm the determination. The investigator is notified and the review, 
determination, and investigator communication is documented in IRB electronic 
system.  A UPIRTSO, as confirmed by the convened IRB, is reported to the Mayo 
Clinic Institutional Official and other relevant Federal agencies, when required, 
within 30 days from the date of submission. The investigator is electronically 
notified of this action. 
  
 
9.6 
Reporting of AEs and SAEs to the FDA 
All adverse reactions probably attributable to the use of the radioactive drug in the research study 
shall be immediately (within 7 days) reported by the Radioactive Drug Research Committee to 
the Food and Drug Administration, Center for Drug Evaluation and Research, HFD-160, 5600 
Fishers Lane, Rockville, MD 20857. 
 
 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
27 
 
10. STATISTICAL ANALYSIS 
 
10.1.  Objectives 
 
The primary objective of this study is to evaluate the uptake and distribution, and metabolism of the 
imaging agent [
18F]BF4. This is a preliminary study and all analyses will be exploratory in nature. 
 
10.2.  Data Sets 
 
All subjects that received [
18F]BF4 for injection will be included in the analysis. T i s s u e  
u p t a k e , biodistribution and metabolite analysis will include all subjects for whom there is 
sufficient data to evaluate the parameter in question.  Where possible, tumor uptake will be 
quantified by use of the Standardized Uptake Value = (tissue concentration) / (injected dose/body 
wt. (g)). 
 
10.3.  General Descriptive Statistics  
 
For each of the three cohorts of participants recruited, general descriptive statistics will be calculated. 
The descriptive statistics will include mean, standard deviation, median, and ranges for continuous 
variables, and frequency and percent frequency for categorical variables. In addition, graphical displays 
of the data (e.g., box plots, density plots, etc) will be prepared to better understand the scaling of data 
within each cohort.  
 
10.4.  Statistical Analysis Plan 
 
10.4.1  Primary Analysis 
 
Healthy Volunteers: 
An exploratory ANOVA model with blocking factor for patient will be used to assess effect of gender 
on [18F]BF4 biodistribution.  Biological half-times in major tissues will be estimated from the 3 
imaging time points. 
 
Cancer Patients: 
Biodistribution of uptake in SUV will be descriptively summarized within each patient cohort. An 
exploratory ANOVA model with blocking factor for patient and within patient measurements 
corresponding to measurement site will be fit to the data. Contrasts will be used to compare uptake in 
regions where there should be increased uptake (e.g., tumor site) and should not (normal tissue).  
 
In addition to the summarization of the general uptake, uptake in known or suspected lesions will be 
conducted in the two disease cohorts. Specifically, both [18F]BF4-PET and 99mTc-SPECT scans 
(acquired from separately approved IRB protocols, see note on page 5) will be analyzed by a Nuclear 
Medicine Physician/Radiologist for the following: 
 
1. Number of lesions visualized (areas of positive uptake within tumor relative to background). 
2. Size range of lesions visualized. 
3. Lesion contrast with surrounding structures using the 4-point scale below: 

[18F]BF4  - Phase 1/2 
Protocol 
 
28 
 
 
0: No uptake in tumor observed 
 
1: Low uptake in tumor observed equivalent to background 
 
2: Moderate uptake in tumor observed higher than background 
 
3: High uptake in tumor observed relative to background 
4. Reader confidence using the 4-point scale below: 
 
0: unsure 
 
1: somewhat confident 
 
2: moderately confident 
 
3: very confident 
5. For tumors that show uptake with PET, the amount of uptake will be quantified.  PET allows 
quantitative estimation of SUV = (tissue concentration)/(injected dose/body wt. (g)).  SPECT does not 
allow absolute quantification. 
 
As with the general uptake analysis described above, descriptive statistics will be used to quantity 
these attributes. For regions of interest (ROIs) with corresponding biopsy data available, a preliminary 
diagnostic accuracy study will be conducted. Given the limited patient data available, ROIs will be 
assumed to be statistically independent (i.e., we will conduct a lesion-level analysis without 
accounting for clustering in patients). There is the potential for verification bias if the PET/SPECT 
data is used to guide the biopsy process. This is a limitation of the study design. Receiver operating 
characteristic (ROC) curves will be generated using the lesion contrast scale.  
 
10.4.2 Safety Analysis 
Adverse Events 
 
Any adverse event which occurs within 24 hours of administration of [
18F]BF4 will be reported.  The 
investigator shall immediately report to the Radioactive Drug Research Committee all adverse effects 
associated with the use of the radioactive drug in the research study.  
 
Adverse events will be assessed on a patient-by-patient basis as well as aggregation over all participants 
studied. For the latter, AEs will be summarized in terms of number and percentage of patients 
experiencing an adverse event. The summary will be further broken down by body system and preferred 
term using Common Toxicity Criteria (CTC) terms. Adverse events will also be presented by severity, 
relationship to treatment, and seriousness. All patients that experience serious adverse events or that 
discontinue due to adverse events will be summarized. 
 
Discontinuation 
 
All subjects who discontinued participation prior to completing the study will be listed and their 
discontinuation reasons will be tabulated. 
 
Laboratory Data 
 
Clinically significant changes in laboratory data from Baseline will be summarized. Subjects whose 
laboratory values are outside threshold values will be identified and tabulated. 
 
Vital Signs 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
29 
 
Clinically significant changes in vital signs from baseline will be summarized. 
 
10.5.  Image Analysis 
 
Healthy Volunteers: 
Descriptive statistics will be used to evaluate changes in time of uptake of [18F]BF4 in the heart lung and blood 
pool over time from time of injection to 45 min in healthy subjects.   Biodistribution in other organs will be 
evaluated from the subsequent trunk PET/CT images out to 4 h post-administration.  Tissue uptake will be 
normalized to injected dose through conversion of image data to Standardized Uptake Value [SUV = 
(tissue radioactivity concentration/cc tissue) / (injected dose / g body mass)]. The regions used for 
SUV determination will be of a standardized volume for a representative region within each tissue 
type, and the SUV averaged over that region.  Thus, all subjects will have similar sized regions for 
each tissue type, minimizing the effect of region size.  
 
Cancer Patients: 
In myeloma and endometrial cancer patients, tumor, muscle, and other tissue SUVs will be assessed at 
baseline and at Day +9.  Primary assessments will be made as described above (10.4.1). Changes in 
this SUV assessment from baseline to Day +9 will be analyzed by ANOVA.  We will also do similar 
statistical analysis on tumor:muscle ratios. Radiation dosimetry estimates will be determined from the 
data in healthy human subjects using OLINDA software, with both male and female groups. 
 
10.6.  Metabolite data 
 
Healthy Volunteers: 
The amount of 18F-radioactivity in the form of metabolites other than [18F]BF4 will be measured by 
TLC in plasma over the 4 hour imaging period.  Appropriate metabolic half-live will be calculated and 
descriptive statistics presented. 
 
10.7 Sample Size Justification 
 
This study is not a “powered” study with statistically tested hypotheses. Descriptive statistics will be 
used for measuring biostribution in the healthy subjects. As such, statistical ‘rules of thumb’ are used 
to justify the sample size. Using the ‘rule of thumb’ that recommends using at least 10 observations 
per parameter in a regression model, a sample size of 10 myeloma patients and 10 endometrial cancer 
patients is proposed in order to estimate ANOVA statistics.  The sample size for health controls is 
reduced to 8 with anticipating of pooling the data across patient cohorts and minimizing unnecessary 
exposure.  
 
 
11. INVESTIGATOR’S REGULATORY OBLIGATIONS 
 
11.1. Investigational Product Control 
 
The receipt of clinical supplies (i.e. starting material for [18F]BF4) must be documented at the site. 
 

[18F]BF4  - Phase 1/2 
Protocol 
 
30 
 
All drug supplies for this study should be retained in a safe and secure place at all times during the 
study. [18F]BF4 for injection should be prepared by a qualified radiopharmacist/chemist under GMP 
conditions and dispensed by the pharmacist, the investigator, or by a qualified individual under the 
investigator’s supervision. An up-to-date dispensing record must be maintained.  
 
The PI will cooperate with Mayo Clinic Radiation Safety office for all necessary documentation 
needed for conducting the studies under the auspices of the Mayo Clinic Radioactive Drug and 
Research Committee (RDRC).  The Mayo Clinic Radiation Safety office manages all 
communications with the FDA concerning documentation required for RDRC studies. 
 
12. APPENDICES 
 
12.1. References 
 
 
1. 
Jauregui-Osoro, M., et al., Synthesis and biological evaluation of [18F]tetrafluoroborate: a PET imaging agent for 
thyroid disease and reporter gene imaging of the sodium/iodide symporter. European journal of nuclear medicine 
and molecular imaging, 2010. 37(11): p. 2108-16. 
2. 
Weeks, A.J., et al., Evaluation of [18F]-tetrafluoroborate as a potential PET imaging agent for the human 
sodium/iodide symporter in a new colon carcinoma cell line, HCT116, expressing hNIS. Nuclear medicine 
communications, 2011. 32(2): p. 98-105. 
3.  
Musibay ED, Allen C, Kurokawa C, Hardcastle JJ, Aderca IA, Msaouel P, Bansal A, Jiang H, DeGrado TR, 
Galanis E. Measles edmonston vaccine strain derivatives have potent oncolytic activity against osteosarcoma.  
Cancer Gene Therapy 2014;  21:483-90. 
4.  
Anbar, M., S. Guttmann, and Z. Lewitus, The accumulation of fluoroborate ions in thyroid glands of rats. 
Endocrinology, 1960. 66: p. 888-90. 
5. 
Anbar, M., S. Guttmann, and Z. Lewitus, Effect of monofluorosulphonate, difluorophosphate and fluoroborate 
ions on the iodine uptake of the thyroid gland. Nature, 1959. 183: 1517-8. 
6. 
Duckrey H., Stekar, J., Hunig, S.  Carcinogene wirkung von aethoxy-diazenium-salzen  (O-aethyl-
dialkylnitrosimmonium salzen) an ratten.  Z. Krebsforsh.1973, 80: 17-26.  
7. 
Entzian W, Aronow S, Soloway AH, Sweet WH. A preliminary evaluation of F-18-labeled tetrafluoroborate as a 
scanning agent for intracranial tumors.  J Nucl Med. 1964; 5:542-50.

 
 
Protocol [18F]BF4 – Phase 1/2 – Healthy Volunteer Arm (Aim 2) 
12.2      STUDY FLOW CHART 
 
Day 1 
Day 2 
                                                                                                      Minutes Post [18F]BF4 Administration 
Time Points 
-
30 
-5 
Dose 
0-45 
45-110 
110-
120 
120-150 
150-200 
200-210 
210-240 
240+ 
 
EVALUATIONS 
 
 
 
 
 
 
 
 
 
 
 
 
Signed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical/AD 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
Concomitant Meds 
X 
 
 
 
 
 
 
 
 
 
 
 
Vital Signs1 
X 
 
 
 
X 
45 min 
 
 
 
 
 
 
X 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
 
 
 
 
 
 
 
 
 
 
 
Clinical Labs2 
X 
 
 
 
X 
90 min 
 
 
 
 
 
 
 
Nuc Med Eval 
X 
 
 
 
 
 
 
 
 
 
X 
 
Temperature 
X 
 
 
 
 
 
 
 
 
 
X 
 
Urine Pregnancy 
Test3 
 
X 
 
 
 
 
 
 
 
 
 
 
 
[18F]BF4 Injection 
 
 
X 
 
 
 
 
 
 
 
 
 
Venous blood for 
Radioactivity 
 
 
 
X 
40 min 
 
 
X 
145 min 
 
 
 
X 
235 min 
 
 
Urine for 
Metabolites 
 
 
 
 
 
X 
60 min 
 
 
 
X 
160 min 
 
 
X 
240 min 
 
PET Imaging 
 
 
Heart  
CT 
Trans 
Heart Emission dynamic scan 0-45 
minutes 
~55 
REST 
PERIOD 
 
 
~55 min 
REST 
PERIOD 
 
 
 
 
Biodistribution 
 
 
 
 
 
Body 
Trans 
Body 
Emission 
 
Body 
Trans  
Body 
Emission  
 
 
Adverse Event 
Monitoring 
 
 
 
X------------------------------------------------------------------------------------------------------------------------------------------------------------------------X 
24 hour AE Phone 
Call 
 
 
 
 
 
 
 
 
 
 
 
X 
1. Includes blood pressure, oral temperature, pulse and respiration rate 
2.  Includes Hematology (hemoglobin, hematocrit, RBC, WBC, MCH, MCHC, neutrophils, bands, lymphocytes, monocytes, eosinophils, basophils, platelets, morphology, 
MCV and RBC morphology) and Chemistry (total bilirubin, alkaline phosphatase, ALT(SGPT) AST (SGOT), blood urea nitrogen, creatinine, glucose, uric acid, calcium, 
phosphorus, total protein, albumin, total cholesterol, triglycerides, sodium, potassium, bicarbonate, chloride, magnesium, globulin, GGt and tryptase) testing.  
3.  Not required if screening urine pregnancy test results are within 48 hours prior to [18F]BF4  injection 
 
 

 
 
Protocol [18F]BF4 – Phase 1/2 – Myeloma and Endometrial Cancer Patients Arm (Aims 3a&3b) 
This protocol is the same for baseline (Day -0) and Day +9 post-virus administration 
12.3      STUDY FLOW CHART 
 
Baseline (Day -0) and Day +9 post-virus administration 
Following day 
                                                                                                      Time (min) 
Time Points 
Scre
en 
-
30 
-5 
0 
Dose 
0-90 
90-95 
95-135 
135 
 
EVALUATIONS 
 
 
 
 
 
 
 
 
 
Signed Consent 
X 
 
 
 
 
 
 
 
 
Medical/AD 
History 
X 
 
 
 
 
 
 
 
 
Concomitant Meds 
X 
 
 
 
 
 
 
 
 
Vital Signs1 
X 
X 
 
 
 
 
 
X 
 
Height 
X 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
 
 
 
 
 
 
Nuc Med Eval 
 
X 
 
 
 
 
 
X 
 
Temperature 
 
X 
 
 
 
 
 
 
 
Urine Pregnancy 
Test 
 
X 
X2 
 
 
 
 
 
 
 
[18F]BF4 Injection 
 
 
 
X 
 
 
 
 
 
PET/CT Imaging 
 
 
 
 
Uptake period 
CT 
Imaging 
PET 
Imaging 
 
 
Adverse Event 
Monitoring 
 
 
 
 
X---------------------------------------------------------------------------------------------------------X 
24 hour AE Phone 
Call 
 
 
 
 
 
 
 
 
X 
1. Includes blood pressure, oral temperature, pulse and respiration rate 
2. Not required if screening urine pregnancy test results are within 48 hours prior to [18F]BF4  injection