Conditions: Brain Tumor
Allocation: NON_RANDOMIZED | Intervention model: SINGLE_GROUP
Enrollment: 10
Healthy volunteers: no

Brief summary:
The hypothesis of this exploratory clinical trial in patients with high-grade a primary brain tumor who receive chemoradiation is that the PET imaging agents \[18F\]Fluciclovine and/or \[18F\]FLT will be a better predictor of tumor response than standard MRI based brain tumor response criteria. When used in conjunction, the two PET agents may be better able to predict tumor aggressiveness and thus overall survival than the use of individual-tracer PET biomarkers. This may eventually lead to improved assessment of response (including time to progression and overall survival) and differentiation of tumor recurrence/progression from treatment effect (pseudoprogression).

Detailed description:
The standard treatment approach for patients with high-grade primary brain tumors includes maximum feasible surgical resection, followed by 6 weeks of concurrent cranial irradiation, and daily low-dose temozolomide chemotherapy; followed by 12 cycles of high-dose temozolomide administered for 5 consecutive days every 4 weeks (Stupp et al., 2005). Contrast-enhanced MRI is the current standard for evaluating the success of therapy and monitoring for tumor recurrence. MRI is typically obtained prior to initial surgery, within 24 hours after surgery, at the conclusions of cranial irradiation, and then every 8 weeks during temozolomide chemotherapy until evidence of recurrence. Despite this careful clinical and radiographic surveillance, and despite decades of research into the histologic and molecular classification of primary brain tumors, our ability to predict tumor behavior remains very limited. Some gliomas will result in overall survival times of only months, whereas other histologically-identical gliomas may yield survivals of years to decades (Curran et al., 1993, Carson et al., 2007). Current assessment of tumor response to therapy is also poor. Patients with complete radiographic response after cranial irradiation often progress rapidly post-irradiation. In contrast, some patients with enhancing masses at the end of chemoradiotherapy may respond dramatically to further chemotherapy alone; or the masses may even disappear in the absence of further therapy, so called "tumor pseudoprogression" (Chamberlain et al., 2007). This confounding situation demonstrates a need for better assessment of tumor response.

Improvements in the ability to predict tumor behavior prior to the start of therapy would allow more efficient and effective tumor surveillance; better prognostication; and more appropriate assignment of patients to conventional, aggressive, or investigational therapies early in their clinical courses. This would provide huge economic and social benefits, and could afford decisive insights into brain tumor physiology and biology.

Similarly, the ability to identify, earlier and more accurately, whether individual patients were responding to therapy would allow prompt discontinuation of ineffectual treatments and institution of potentially more effective therapies.

Previous efforts using imaging for such tasks have generally been limited to a single modality (e.g. MRI) and/or single-tracer (e.g. FDG-PET). However, there is a significant and growing body of evidence that complementary imaging of multiple aspects of tumor physiology (i.e. using multiple PET tracers) can provide greatly enhanced information over imaging with a single modality or tracer alone. In solid tumors, complex interactions exist between blood flow, metabolic activity, and oxygen status which affect metastatic and proliferative activity. Heterogeneous tumors may contain both slow-growing and fast-growing regions that present different profiles of proliferation rates and amino acid uptake.

Arms:
arm 1: Participants enrolled in Group A will undergo a maximum of 6 scans throughout this study. \[18F\]FLT- and \[18F\]Fluciclovine-PET/CT scans at up to three imaging time points: (1) prior to any tumor-directed therapy, (2) within 1-4 weeks after the conclusion of the intitial (\~6-8 weeks) chemoradiotherapy, and (3) following suspected recurrence/progression versus treatment effect (pseudoprogression) shown with MRI, within 6 months of the completion of chemoradiation arm 2: Participants enrolled in Group B will undergo 2 scans as part of this study. \[18F\]FLT- and \[18F\]Fluciclovine-PET/CT scans will be acquired at a single timepoint, at the time of suspected pseudoprogression.

Interventions: intervention 1: [18F]FLT intervention 2: [18F]Fluciclovine
Intervention descriptions: intervention 1: PET exams of \[18F\]FLT will be acquired in each patient at up to three time points: (1) prior to any tumor-directed therapy, either prior to surgery or immediately after surgery providing a complete surgical resection was not performed and confirmed by a post-operative contrast MRI scan where residual tumor \> 1.0 cm in diameter was present and prior to any tumor-directed therapy; (2) at the conclusions of the initial (\~6-8 weeks) chemoradiotherapy; and (3) patients with MRI-documented possible recurrence/progression versus treatment effect (pseudoprogression) within 6 months from the time of completion of chemoradiation. intervention 2: PET exams of \[18F\]Fluciclovine will be acquired in each patient at up to three time points: (1) prior to any tumor-directed therapy, either prior to surgery or immediately after surgery providing a complete surgical resection was not performed and confirmed by a post-operative contrast MRI scan where residual tumor \> 1.0 cm in diameter was present and prior to any tumor-directed therapy; (2) at the conclusions of the initial (\~6-8 weeks) chemoradiotherapy; and (3) patients with MRI-documented possible recurrence/progression versus treatment effect (pseudoprogression) within 6 months from the time of completion of chemoradiation.

Protocol document:
Prot_SAP_002.pdf: 
[18F]Fluciclovine and [18F]FLT Assessment of Primary High-Grade Brain Tumors 
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Page 1 of 56 
 
 
 
 
 
Title: 
 
[18F]Fluciclovine and [18F]FLT PET/CT Assessment of Primary High-Grade Brain 
Tumors 
 
 
 
 
Jeffrey Yap, PhD Principal Investigator 
 
 
 
 
 
 
Imaging Agents to be used: 
 
            [18F] anti-FACBC ([18F]Fluciclovine) 
                  IND # - 136875 (Yap) 
 
 
 
3’-deoxy-3’-[18F]fluorothymidine ([18F]FLT) 
 
 
 
IND # - 76,843 (Yap) 
 
 
University of Utah IRB # 104704 
 
 
Historical Protocol Versions 
Version 1: 12/10/2017 
Version 2: 04/18/2018 
Version 3: 12/13/2018 
Version 4: 10/16/2019 
Version 5: 04/15/2024 
 


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1. OBJECTIVES 
5 
1.1. PRIMARY OBJECTIVES 
5 
1.2. THE HYPOTHESES TO BE TESTED AND OBJECTIVES 
6 
2. BACKGROUND 
7 
2.1. MALIGNANT BRAIN TUMORS 
7 
2.2. BRAIN TUMOR IMAGING WITH MAGNETIC RESONANCE IMAGING (MRI) 
8 
2.3 BRAIN TUMOR PSEUDOPROGRESSION 
8 
2.4. OVERVIEW OF THE PET TRACERS [18F]FLT AND [18F]FLUCICLOVINE 
8 
2.5. COMPLEMENTARY VALUE OF MULTIPLE PET TRACERS 
12 
3.  PHARMACOLOGY AND SAFETY OF [18F]FLT AND [18F]FLUCICLOVINE AND RADIATION DOSIMETRY OF [18F]FLUCICLOVINE 
AND [18F]FLT 
13 
3.1. PHARMACOLOGY AND SAFETY 
13 
3.2. HUMAN RADIATION DOSIMETRY 
17 
4. RATIONALE, GOALS AND OBJECTIVES 
20 
4.1. CLINICAL RATIONALE 
20 
4.2. HYPOTHESES 
20 
4.3. EXPLORATORY OBJECTIVES 
21 
5. TRIAL DESIGN 
22 
5.1. PATIENT ELIGIBILITY 
22 
5.2. INCLUSION CRITERIA 
23 
5.3. EXCLUSION CRITERIA 
24 
5.4. PATIENT REGISTRATION 
24 
5.5. STUDY PROCEDURES, SCHEDULE OF EVENTS, TRACER ADMINISTRATION:  ROUTE AND DOSING 
25 
5.6. DATA COLLECTION 
26 
6. METHODS FOR EVALUATION OF IMAGING STUDIES 
27 
6.1. [18F]FLT-PET IMAGING 
27 
6.2. [18F]FLUCICLOVINE PET IMAGING 
29 
6.3. MULTI-TRACER ASSESSMENTS IN CONJUNCTION 
30 
7. DATA ANALYSIS AND STATISTICS 
30 
7.1. VALIDATION OF MULTI-TRACER PET IMAGING TECHNOLOGY 
30 
7.2 DATA ANALYSIS 
30 
7.3. JUSTIFICATION OF SAMPLE SIZE 
31 
8. REGULATORY AND REPORTING REQUIREMENTS 
32 
8.1. INFORMED CONSENT 
32 
8.2 INSTITUTIONAL REVIEW 
33 
8.3 DATA AND SAFETY MONITORING PLAN 
33 
8.4 ADVERSE EVENTS / SERIOUS ADVERSE EVENTS 
33 
8.5 SAE REPORTING REQUIREMENTS 
35 
8.6 EXPEDITED ADVERSE EVENT REPORTING 
36 
8.7 PROTOCOL AMENDMENTS 
36 
8.8 PROTOCOL DEVIATIONS 
37 
8.9 FDA ANNUAL REPORTING 
37 
8.10 CLINICAL TRIALS DATA BANK 
37 
8.11 RECORD KEEPING 
37 


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9. PET TRACER PRODUCTION 
37 
9.1. [18F]FLT 
37 
9.2. [18F]FLUCICLOVINE 
38 
10. REFERENCES 
41 
11. APPENDICES 
49 
APPENDIX A: ELIGIBILITY CHECK LIST 
50 
APPENDIX B: SCHEDULE OF EVENTS 
51 
APPENDIX C: [18F]FLT INFUSION FLOW CHART FOR STAFF 
52 
APPENDIX D: [18F]FLUCICLOVINE INFUSION FLOW CHART FOR STAFF 
53 
APPENDIX E: [18F]FLT PATIENT ADVERSE EVENT QUESTIONNAIRE 
54 
APPENDIX F: [18F]FLUCICLOVINE PATIENT ADVERSE EVENT QUESTIONNAIRE 
55 
APPENDIX G: NCI COMMON TOXICITY CRITERIA 
56 
 
 
 


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PROTOCOL SIGNATURE 
 
I confirm that I have read this protocol, and I will conduct the study as outlined herein and according to the 
ethical principles stated in the latest version of the Declaration of Helsinki, the applicable ICH guidelines 
for good clinical practice, and the applicable laws and regulations of the federal government. I will promptly 
submit the protocol to the IRB for review and approval. Once the protocol has been approved by the IRB, I 
understand that any modifications made during the course of the study must first be approved by the IRB 
prior to implementation except when such notification is made to remove an immediate hazard to the 
subject.   
 
I will provide copies of the protocol and all pertinent information to all individuals responsible to me who 
assist in the conduct of this study.  I will discuss this material with them to ensure that they are fully 
informed regarding the study treatment, the conduct of the study, and the obligations of confidentiality. 
 
Note: This document is signed electronically through submission and approval by the Principal Investigator 
in the University of Utah IRB Electronic Research Integrity and Compliance Administration (ERICA) 
system. 


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1. OBJECTIVES 
1.1. Primary Objectives 
Positron emission tomography (PET) is a molecular imaging modality that can probe various aspects of 
tumor function and biology using a variety of radiolabeled imaging agents (“tracers”). Oncologic PET 
imaging has seen a dramatic rise in clinical utilization over the past decade for cancer detection, staging, and 
evaluating residual or recurrent disease following therapy. These clinical scans use the FDA approved PET 
radiopharmaceutical [18F]fluoro-2-deoxy-D-glucose (FDG), which accumulates in cells in proportion to 
GLUT transporter and hexokinase activity. FDG thus provides a measure of tissue glucose metabolism. On 
May 27, 2016 [18F] anti-FACBC ([18F]Fluciclovine) with the brand name of Axumin was approved for 
detection of recurrent prostate cancer (Axumin 2016). ([18F]Fluciclovine is a synthetic L-leucine analogue 
that is transported into cells but is not incorporated into proteins. Primary brain tumors have upregulated 
amino acid utilization and thus increased [18F]Fluciclovine uptake.  Another PET tracer that has received 
significant attention in research is 3’-deoxy-3’-[18F]fluorothymidine ([18F]FLT).  The uptake, 
retention/washout, and ultimate biodistribution of these tracers ([18F]Fluciclovine and 18F]FLT) are each 
related to different functional or molecular processes. As such, each can be used to probe a different aspect 
of tumor biology: [18F]FLT directly assesses tumor proliferation (Shields et al., 1998, Rasey et al., 2002, 
Schwartz et al., 2003, Chen et al., 2005, Choi et al., 2005, Jacobs et al., 2005) and [18F]Fluciclovine 
provides a measure of tumor growth related to amino acid transport and utilization (Shoup and Goodman, 
1999, Akhurst et al., 2006, Meirelles et al., 2006, Oka et al., 2007, Yu  2009, Yu 2010, Oka et al., 2012). 
 
Assessing response, particularly early response, to therapy in malignant glioma is a challenging problem. 
Typically the extent of MRI contrast enhancement in malignant gliomas has been used as an indicator of 
therapeutic response (Grant et al., 1997). Several response criteria have been developed for the assessment 
of malignant gliomas including the Macdonald criteria (Macdonald et al., 1990) and more recently the 
Response Assessment in Neuro-Oncology Working Group (RANO) criteria (Wen et al., 2010). The major 
modification proposed by the RANO guidelines was the assessment of increasing fluid-attenuated inversion 
recovery (FLAIR) signal intensity change as evidence of tumor progression. This was particularly helpful 
when antiangiogenic therapy became a treatment option for malignant brain tumors. The RANO criteria still 
have many issues making their implementation difficult particularly in clinical trials (Pope and Hessel, 
2011). Another known problem with the current response criteria is the fact that there is often viable tumor 
extending beyond the enhancing zone.  Biopsy studies have shown that viable tumor can extend beyond the 
margin of T2 signal change in glioblastomas (Lunsford et al., 1986, Watanabe et al., 1992) and can extend 
up to 2.5 cm beyond the area of T2 signal change. There are reports of tumor being identified in regions with 
a normal T1 signal in 16% of biopsies and a normal T2 signal in 4% of biopsies (Kelly et al., 1987).        
 
To date there have been no published human studies assessing the use of [18F]Fluciclovine or [18F]FLT to 
assess response to chemoradiation in primary high-grade brain tumors.  This study is therefore exploratory 
as it will (1) define the semi-quantitative change in [18F]FLT and [18F]Fluciclovine uptake which is 
consistent with response based on standard brain tumor response criteria. (2) define the semi-quantitative 
change in [18F]FLT and/or [18F]Fluciclovine uptake which predicts time to progression and overall survival. 
The standard response assessment will utilize standard MRI techniques in patients with high-grade brain 
tumors receiving standard chemoradiation and (3) assess the ability of [18F]FLT or [18F]Fluciclovine alone 
or together to predict true progression from pseudoprogression in patients with this clinical dilemma. The 
objective is more clearly described below. By assessing a biologic process such as proliferation with 
[18F]FLT and amino acid uptake with [18F]Fluciclovine, we may be able to better define viable tumor and 
better assess response and overall survival to chemoradiation.  
 


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This exploratory study will be in 30 patients with suspected or pathologically confirmed high-grade primary 
brain tumors.  Two different groups of patients will be able to enroll in the trial.  The first group, Group A, 
will be for patients who have not yet received any therapy for their suspected or newly found brain tumors.  
Group B will enroll patients with pathologically proven malignant brain tumor (WHO Grade III or IV glial-
based tumors) who have undergone chemoradiation and have a contrast enhancing mass ≥ 1.0 cm in greatest 
diameter concerning for pseudoprogression.  MRI-documented possible recurrence/progression versus 
treatment effect (pseudoprogression) within 6 months from the time of completion of chemoradiation.  
 
Group A will have three different patient cohorts.  The first, for patients where there is compelling evidence, 
based on the MRI and/or CT imaging, that a high-grade primary brain tumor is present. Pathologic 
confirmation will occur with subsequent biopsy or surgery.  The second cohort of patients are individuals 
where the tumor is felt to be inoperable and a biopsy is performed but no surgery. The third cohort of 
patients are post-surgery where a complete surgical resection was not possible. This will be confirmed by a 
post-operative contrast MRI/CT scan where residual tumor > 1.0 cm in diameter is present.  
 
The study will include PET imaging procedures at up to three possible time points: (1) at “baseline” prior to 
any tumor-directed chemoradiotherapy (2) At the conclusion of the initial chemoradiotherapy (1-4 weeks 
post last treatment); and (3) at any time of MRI-documented possible recurrence/progression (> 1.0 cm in 
greatest diameter) versus treatment effect (pseudoprogression) within 6 months from the time of completion 
of chemoradiation. Also, patients who were not originally enrolled in the study for baseline and conclusion 
of therapy imaging will be eligible to enroll at the time of MRI-documented possible recurrence/progression 
versus treatment effect (pseudoprogression) if within 6 months from the time of completion of 
chemoradiation.  
 
A number of quantitative and semi-quantitative imaging biomarkers for each tracer will be computed at 
each imaging time point, and the change in each biomarker between time-points will also be computed. 
These data will be compared with clinical endpoints (standard RANO criteria, survival, time to 
progression), and with tumor biologic information (histology, WHO grade, vascularity, MIB-1, VEGF, 
EGFR, p53, IDH status) in cases when tumor tissue becomes available from standard care. These 
preliminary and exploratory data will provide information regarding semi-quantitative changes in uptake 
consistent with standard response, the potential value of concurrent multiple PET biomarkers for predicting 
tumor behavior prior to the start of therapy, for improved prognostication, for more efficient and effective 
tumor surveillance, and/or for more appropriate assignment of patients to conventional, aggressive, or 
investigational therapies early in their clinical courses. This initial data will be important for design of 
subsequent clinical trials using [18F]Fluciclovine and/or [18F]FLT to assess response to chemoradiation in 
high-grade primary brain tumor.  
 
1.2. The Hypotheses to be Tested and Objectives 
The hypothesis of this exploratory clinical trial in patients with high-grade a primary brain tumor who 
receive chemoradiation is that the PET imaging agents [18F]Fluciclovine and/or [18F]FLT will be a better 
predictor of tumor response than standard MRI based brain tumor response criteria. When used in 
conjunction, the two PET agents may be better able to predict tumor aggressiveness and thus overall 
survival than the use of individual-tracer PET biomarkers. This may eventually lead to improved assessment 
of response (including time to progression and overall survival) and differentiation of tumor 
recurrence/progression from treatment effect (pseudoprogression). 
 


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The primary endpoint of the study is that a 25% reduction in SUVmax or SUVmean for each of the PET 
imaging tracers will be predictive of increased overall survival.  
 
Secondary Exploratory Objectives 
 
Assess the association between overall survival and change in each of the following quantitative markers for 
[18F]Fluciclovine and [18F]FLT:  K1-k4, Knet and Ktrans, from baseline to post therapy. 
Assess the association between progression free survival and change in each of the following quantitative 
markers for [18F]Fluciclovine and [18F]FLT:  K1-k4, Knet and Ktrans, from baseline to post therapy. 
Assess the ability of [18F]FLT and/or [18F]Fluciclovine alone or together to predict true progression from 
pseudoprogression in patients with this clinical dilemma. 
A 25% reduction in SUVmax or SUVmean for each of the PET imaging tracers will be more predictive of 
overall survival than the current MRI based response criteria.  
A 25% reduction in SUVmax or SUVmean for each of the PET imaging tracers will be more predictive of 
time to progression than the current MRI based response criteria.  
 
2. BACKGROUND 
2.1. Malignant Brain Tumors 
Despite significant advances in the understanding of brain tumor biology and genetics as well as 
improvements in surgical techniques, radiotherapy administration, and chemotherapy methods; most 
primary brain tumors remain incurable. Most primary brain tumors are highly infiltrative neoplasms, and are 
therefore unlikely to be cured by local treatments such as surgery, focal radiotherapy, radiosurgery, or 
brachytherapy. 
 
The clinical presentation of a brain tumor is highly dependent on its location within the nervous system; but 
includes signs/symptoms that relate directly to compression and/or infiltration of specific functional areas of 
brain parenchyma, increased intracranial pressure, hydrocephalus, cerebral edema, and/or vascular 
complications from the tumor. The most common symptoms include headaches, cognitive deficits, 
nausea/vomiting, seizures, balance disturbances, and focal neurologic deficits. Once clinically suspected of 
having such a lesion, a patient usually undergoes an imaging procedure, typically a computed tomography 
(CT) scan or a magnetic resonance (MR) scan. Neurosurgical intervention, in the form of either a brain 
biopsy or craniotomy with resection, is the next step to obtain a tissue diagnosis and, if warranted, to 
decrease the tumor mass. 
 
There are approximately 16,900 new cases of primary CNS tumors diagnosed in the United States each 
year. Numerous studies have documented an increase in the incidence of these tumors in the last decades 
(Sanson‐Fisher et al., 2000). About 50% of these lesions are supratentorial high-grade gliomas. Anaplastic 
astrocytoma (AA) (WHO Grade III) and glioblastoma (GBM) (WHO Grade IV) are the most common glial 
primary brain tumors with at least 80% of malignant gliomas being GBMs (Brock et al., 1998, Ichimura et 
al., 1998). Patients with newly diagnosed malignant gliomas are usually treated with biopsy or surgical 
resection followed by radiation therapy and/or chemotherapy; depending on their functional status. Despite 
these interventions, the median survivals for newly diagnosed GBM and AA are 50 and 150 weeks, 
respectively. Chemotherapy has been shown to increase the proportion of long-term survivors from less than 
5% to between 15-20 % (Treat et al., 2012). Once a patient has failed initial therapy, salvage therapy is 
difficult and most patients die of tumor progression within 4-12 months (Group, 2002). 
 


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2.2. Brain Tumor Imaging with Magnetic Resonance Imaging (MRI) 
When considering a broader approach to the diagnosis and treatment of these tumors and other CNS lesions, 
the blood-brain barrier (BBB) is an important obstacle to the delivery of both therapeutic and diagnostic 
imaging agents. The BBB is a unique feature of CNS capillaries that results from a continuous layer of 
endothelial cells bound together with tight junctions that allow very little transcellular or pericellular 
transport of blood-borne molecules (Neuwelt, 1989, Banks et al., 1999). The blood-tumor barrier in both 
primary cerebral tumors and in cerebral metastatic disease is often highly permeable compared to the 
normal BBB (Long, 1979, Neuwelt, 1989, Groothuis et al., 1991). This disruption of the integrity of the 
BBB allows the contrast agents typically used in MRI and CT imaging to leak from the vascular space and 
accumulate in the perivascular and intercellular spaces in tumors, causing altered signal known as 
enhancement in both CT and MRI imaging. The most commonly performed study today to assess the 
integrity of the blood brain barrier is a T1 MRI study obtained post-injection of gadolinium DTPA. There 
are currently several commercially-available and FDA-approved gadolinium based MRI contrast agents 
marketed and licensed in the United States for brain imaging. These imaging agents are non-specific; and, 
since there is also loss of integrity of the BBB in radiation necrosis and treatment effect, MRI imaging 
cannot be used to unequivocally differentiate tumor recurrence from radiation necrosis/treatment effect 
(Chaskis et al., 2009, da Cruz et al., 2011, Caroline and Rosenthal, 2012). Newer MRI techniques to assess 
pseudoprogression have been tried (see below), however, these have not found widespread clinical use (Chu 
et al., 2013, Gahramanov et al., 2013, Park et al., 2015, Yun et al., 2015). Magnetic resonance spectroscopy 
(MRS) has also been used in an attempt to differentiate radiation necrosis/treatment effect from recurrent 
tumor; unfortunately, the results were less than impressive (da Cruz et al., 2011, Sawlani et al., 2012). 
Consequently, both national coverage requests to the Center for Medicare and Medicaid Services (CMS) to 
reimburse MRI spectroscopy in the past decade have been denied. 
 
2.3 Brain Tumor Pseudoprogression 
Treatment of high-grade brain tumors (GBM and AA) with chemoradiation (temozolomide/radiation) can 
cause changes in tumor vascular permeability which alter the pattern of gadolinium enhancement, 
mimicking tumor progression or recurrence. These temporary vascular permeability changes usually 
improve or stabilize on follow-up MRI and have been termed “pseudoprogression” (Hoffman et al., 1979, 
De Wit et al., 2004, Chamberlain et al., 2007, Brandes et al., 2008, Brandsma et al., 2008, Fine et al., 2008, 
Brandsma and van den Bent, 2009, Chaskis et al., 2009, Wen et al., 2010) to distinguish them from true 
tumor progression. The lack of a definitive imaging modality to distinguish these post-treatment 
radiographic imaging changes (PTRIC), including pseudoprogression and radiation necrosis/treatment 
effect, from true tumor progression presents a major unmet clinical need in the care of GBM patients (Wen 
et al., 2010). To date, no single imaging modality (including MRI, MRS, and PET) has been able to 
consistently and accurately distinguish between PTRIC from true tumor progression. At present, only direct 
examination of tumor histopathology can accurately and consistently discriminate true tumor progression 
from PTRIC. Furthermore, there is no clear understanding of the molecular, biochemical, and cellular 
mechanisms mediating PTRIC. This clinical trial will hopefully provide a better understanding of the 
mechanisms responsible for this clinical dilemma and for the development of a novel PET imaging 
approach to distinguish PTRIC from true tumor progression. 
 
2.4. Overview of the PET Tracers [18F]FLT and [18F]Fluciclovine 
The use of more specifically targeted imaging agents, such as PET radiopharmaceuticals, has great potential 
for overcoming the limitations of MRI imaging in brain tumors 
 


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2.4.1. [18F]FLT as an Imaging Agent 
3’-deoxy-3’-[18F]fluorothymidine: [18F]FLT (mol. wt. 244.2 for [F-19]FLT) is a structural analog of the 
DNA constituent, thymidine. [18F]FLT is a radiolabeled imaging agent that has been utilized for 
investigating cellular proliferation with PET (Shields et al., 1998, Rasey et al., 2002). Although [18F]FLT is 
not incorporated into DNA, it is trapped in the cell due to phosphorylation by thymidine kinase, a part of the 
proliferation pathway. As such it has the potential to image proliferating tumor in proportion to the DNA 
synthesis rate. In the case of the brain, [18F]FLT is not taken up or retained in normal brain tissue due to the 
intact blood-brain barrier as well as the paucity of proliferative activity . This gives [18F]FLT a distinct 
advantage compared to FDG as it should only be concentrated in viable proliferating tumor, allowing for 
improved differentiation of radiation necrosis from recurrence. 
 
[18F]FLT has not been marketed in the United States and to the best of our knowledge, there has been no 
marketing experience with this drug in other countries. The radiopharmaceutical product, [18F]FLT is the 
only active ingredient and it is dissolved in a solution of  < 10 mL of 92% 0.01 M phosphate buffered saline 
(PBS): 8% ethanol (v:v). The drug solution is stored at room temperature in a sealed, sterile, pyrogen-free 
syringe with an expiration time of 8 hours. The injectable dose of [18F]FLT for this study will typically be < 
0.1 mCi/kg of fluorine-18 not to exceed 10.0 mCi, with a specific activity greater than 200 Ci/mmol at the 
time of injection. The three required IND qualification runs performed in the cyclotron facility at the 
Huntsman Cancer Institute (HCI) had specific activities of 5600 Ci/mmol, 5223 Ci/mmol, and 4659 
Ci/mmol (end of synthesis). In the dose of [18F]FLT only a small fraction of the FLT molecules are 
radioactive. The amount of injected drug is < 12.2 µg (<50 mmol) of FLT. [18F]FLT is administered to 
subjects by intravenous injection of < 10 mL. There is no evidence that non-radioactive and radioactive FLT 
molecules display different biochemical behavior. 
 
 
 
FLT undergoes relatively little degradation after injection aside from production of the glucuronide in the 
liver [Shields 2005]. Thymidine phosphorylase in the blood does not degrade FLT. The mathematical model 
which can be used to describe the uptake of [18F]FLT is therefore relatively simple since the only metabolite 
in the plasma that needs to be measured is the glucuronide conjugate. 
 
CH3
HN
O
N
18F
HO
O
O
CH3
HN
O
N
HO
O
O
CH3
HN
O
N
HO
O
O
OH
OH
11C
2-[C-11]-Thymidine
Thymidine
FLT


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Retention of [18F]FLT is reflected in the flux constant, FluxFLT or KFLT, and transport is measured by 
K1FLT: 
 
 
 
Flux constant:  KFLT = K1FLT × k3FLT / (k2FLT + k3FLT). 
 
Preliminary modeling work from investigators in Seattle is illustrated in the model diagrams above (Muzi et 
al., 2005a, Muzi et al., 2005b). The kinetic modeling requires dynamic imaging and arterial plasma 
sampling or sampling via the heated hand method for arterialization of venous blood (Phelps et al., 1979). 
 
To date there have been no human studies assessing the use of [18F]FLT to assess response to 
chemoradiation in primary high-grade brain tumors. Several studies have been performed assessing brain 
tumor treatment response in patients receiving chemotherapy and/or bevacizumab (Chen et al., 2007, 
Schwarzenberg et. al., 2012, Wardak et. al., 2014).  A single study by Chen (Chen et al., 2007) used 
[18F]FLT to predict response in malignant glioma in patients receiving Bevacizumab and Irinotecan as 
second line therapy.  The patients were treated with biweekly cycles of bevacizumab and irinotecan and 
were prospectively studied with [18F]FLT-PET at baseline, after 1 to 2 weeks, and after 6 weeks from start 
of treatment. A more than 25% reduction in tumor [18F]FLT uptake as measured by standardized uptake 
value was defined as a metabolic response. In this study there were nine responders (47%) and 10 non-
responders (53%). Responders survived three times as long as non-responders (10.8 v 3.4 months; P = .003), 
and tended to have a prolonged progression-free survival (P =.061). Both early and later [18F]FLT -PET 
responses were more significant predictors of overall survival (1 to 2 weeks, P = .006; 6 weeks, P =.002), 
compared with the MRI responses (P =.060 for both 6-week and best responses). A second study was 
published as an extension of the Chen study assessing response using [18F]FLT and [18F]FDOPA. This study 
showed the superiority of using kinetic modelling and multiple linear regression analysis to improve the 
predictive ability of the radiopharmaceuticals. Kinetic parameters from FLT were more predictive of overall 
survival than those from FDOPA. Using information from both radiopharmaceuticals resulted in a better 
three predictor model (adjusted R2 = 0.83) than using information from FDOPA alone (adjusted R2 = 0.41), 
and only marginally different from using information from FLT alone (adjusted R2 = 0.82). Another study 
by Schwarzenberg (Schwarzenberg et al, 2012) assessed early survival prediction in patients receiving 
bevacizumab chemotherapy. In this study early and late changes in tumor [18F]F-FLT uptake were more 
predictive of overall survival than MRI criteria (P < 0.001 and P = 0.01, respectively). [18F]F-FLT uptake 
changes were also predictive of progression-free survival (P < 0.001). The median overall survival for 
responders was 3.3 times longer than for non-responders based on [18F]F-FLT PET criteria (12.5 vs. 3.8 
months, P < 0.001). A potential limitation of these studies was the use of standardized uptake value rather 
than full kinetic analysis in brain tumors (Peck et al., 2015). This is due to the fact that [18F]FLT is restricted 
at the blood-brain-barrier. This results in a transport-dominated SUV image because transport of [18F]FLT is 
the rate-limiting path to uptake. Studies utilizing full kinetic analysis would be helpful to test whether there 
A 
[FLT] 
Free  
FLT 
FLT 
(FLT-M P) 
Plasma 
Tissue 
k3FLT 
k4FLT 
K1F LT 
k2FLT 
B 
P 
K1FLT ·  k3FLT  
 
k2FLT + k3FLT  
 
FluxFLT = 


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is any uptake of [18F]FLT by passive diffusion, allowing phosphorylation to be the rate determining step in 
the metabolism of [18F]FLT (Peck et al., 2015).  
 
2.4.2. [18F]Fluciclovine as an Imaging Agent 
Anti-FACBC is a synthetic L-leucine analogue that is transported into cells but is not incorporated into 
proteins. It can be labelled with 18F for PET imaging of amino acid transport and is thus known as 
[18F]Fluciclovine. It exhibits excellent in vitro uptake into the DU145 prostate carcinoma cell line and into 
orthotopic implanted prostate tumors in nude mice (Oka et al., 2007). The uptake of [18F]Fluciclovine is 
mediated via the amino acid transporter (AAT) responsible system. [18F]Fluciclovine uptake has been 
investigated in cell culture (Oka et al., 2012, Oka et al., 2014) and gene knock down studies (Okudaira et al., 
2011). The sodium-dependent transporter ASCT2 and the sodium-independent system LAT1 have been 
identified as the dominant transporters involved (Ono et al., 2013). These findings are further corroborated 
using heterologous expression systems (Okudaira et al., 2013). Since amino acid uptake is enhanced in 
malignancy, it accumulates faster and to a higher degree in cancer cells than normal cells, making it a 
promising biomarker for cancer diagnosis by PET imaging. Only a small fraction is excreted through the 
urinary pathway in the first hours after injection and it is not taken up to a high degree by normal brain 
tissue; therefore, unlike [18F] fluorodeoxyglucose (FDG), PET imaging with [18F]Fluciclovine may be useful 
for the detection and diagnosis of prostate cancer and malignant gliomas. The ability of [18F]Fluciclovine to 
detect malignant lesions in humans has been assessed in preliminary studies by the Emory group and others. 
[18F]Fluciclovine is reported to have utility in the evaluation of primary and metastatic cancers in the brain, 
due to its low brain background uptake and high uptake by brain tumors (Shoup et al., 1999, Akhurst et al., 
2006, Meirelles et al., 2006). More recently, it has been reported that PET imaging with [18F]Fluciclovine is 
useful in assessing metastatic or recurrent prostate cancer in the prostate bed, lymph nodes and bone 
(Schuster et al., 2007, Nanni et al., 2013, Ono et al., 2015a). The data suggest that the LAT system is highly 
active in these types of malignancies and that [18F]Fluciclovine is a promising radiotracer for PET imaging 
of brain and prostate tumors. 
 
To date there have been no published human studies assessing the use of [18F]Fluciclovine to assess 
response to chemoradiation in glioma. In an autoradiography animal study by Ono  (Ono et al., 2015b) 
utilizing anti-14C-FACBC (rather than [18F]Fluciclovine), there was a significant reduction in anti-14C-
FACBC uptake (represented by the differential absorption ratio (DAR)) in animals treated with 
chemotherapy. In these experiments a human xenograft rat glioma model utilizing U87 and U87R (TMZ-
resistant subculture) cells were inoculated into the right and left basal ganglia. Single agent and combination 
therapy was performed with temozolomide, interferon-β, and bevacizumab. Tumor to non-tumor ratios 
(T/NT) were also determined. The DAR of anti-14C-FACBC was significantly higher in tumors than in non-
involved cortices. 14C-FACBC accumulation was lower after single-agent treatment and lower still after 
combination therapy. Treatment with temezolomide alone significantly decreased the T/NT for the DAR of 
U87 tumors, whereas the T/NT ratios for those of U87R tumors remained unchanged. Temozolomide and 
interferon-β combination treatment significantly decreased anti-14C-FACBC uptake in both U87R and U87 
tumors. Addition of bevacizumab to the temozolomide and interferon-β combination further decreased anti-
14C-FACBC uptake in both tumor types. In another study, Doi (Doi et al., 2015) showed that 
[18F]Fluciclovine-PET visualization of gliomas was possible and occurred irrespective of blood-brain 
integrity. This is important since radiopharmaceuticals that cross the BBB, such as Flucilovine, will be taken 
up by low-grade tumors which in many instances will have equivocal findings with MRI due to an intact 
blood-brain barrier.  In the study, the tumor-to normal brain uptake ratio of [18F]Fluciclovine for the most 
part correlated with tumor cell density. The authors felt that [18F]Fluciclovine PET combined with MRI will 
be valuable for preoperative glioma delineation. The authors reiterated the fact that surgery will continue to 
be the first-line therapy for both low-grade and high-grade gliomas. With its unique ability to show uptake 


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even with an intact blood-brain barrier, [18F]Fluciclovine has the ability to delineate the extent of tumor in 
low-grade tumors.  Improved delineation of the tumor, particularly low-grade tumors, will have potential 
clinical utility.   
 
2.5. Complementary Value of Multiple PET Tracers 
There is a significant and growing body of evidence that complementary imaging of more than one tracer 
(“multi-tracer PET”) would provide enhanced information over FDG imaging alone for many cancer 
imaging applications (Wong et al., 2002). We highlight certain examples: 
 
• Kubota et al.(Kubota et al., 1993) studied 18F-FDG or 18F-fluorouridine, 14C-methionine, 3H-
thymidine, and 67Ga-citrate in rat tumors, finding differing and complementary information for each 
tracer. Similar work by d’Argy et al.(d'Argy et al., 1988) with FDG, thymidine, methionine, and 
toremifene (an estrogen receptor-avid agent), also demonstrated complementary value of these tracers. 
 
• Mankoff et al.(Mankoff et al., 2002, Mankoff et al., 2003) found that the ratio of the metabolic rate of 
FDG to blood flow (from 15O-water) was the best predictor of macroscopic complete response of 
advanced breast cancer to neoadjuvant chemotherapy, and that this ratio is a more robust indicator of 
tumor status than either tracer alone. 
 
• Kubota et al.(Kubota et al., 1999) evaluated the imaging potential of agents for hypoxia (18F-MISO), 
glucose metabolism (14C-2-deoxyglucose) and proliferation (11C-Met) in a rat tumor model: 
“Combined use of these tracers might provide better information on tumor characteristics. The ratio 
of FMISO uptake to 2DG (FDG) uptake may reflect the proportion of hypoxic tissue in the tumor, the 
hypoxic fraction. This fraction has been used in the field of radiation biology as an important index to 
predict radiosensitivity. On the other hand, the ratio of Met uptake to 2DG (FDG) uptake may 
correspond to the percentage of proliferative tissue, the growth fraction.  This is also an important 
parameter with which to describe the growth kinetics of tumor tissue.” 
 
Figure 1.  Distribution model proposed by Kubota et al. 
(Kubota et al., 1999) highlighting the complementary value 
of multi-tracer PET imaging using tracers for hypoxia, 
proliferation, and glucose metabolism. The combinations 
of these three tracers provides the hypoxic fraction and 
growth fractions of the tumor, which will enhance grading 
prognosis, and especially treatment planning and 
monitoring with PET. 
 
 
• Dimitrakopoulou-Strauss et al. (Dimitrakopoulou-Strauss et al., 2001) studied imaging with FDOPA, 
FDG, and 15O-water in melanoma patients, finding that detectability of metastases was enhanced when 
both FDOPA and FDG were used. The accompanying editorial (Graham et al., 2003) highlights the 
potential of multi-tracer PET. 
 
• In non-Hodgkin’s lymphoma, Leskinen-Kallio et al. (Leskinen-Kallio et al., 1991) found that 11C-
methionine (MET) was preferable for detection, but FDG was superior for distinguishing tumor grade. 
Aronen et al. (Aronen et al., 2000) likewise found that MET gave better detection and FDG better 
grading in brain tumors. 
 
Proliferating
Anoxic
Hypoxic
Normoxic
Viable
FDG
FMISO
MET


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• Narayanan et al. (Narayanan et al., 2002) found that combined FDG + MET was useful for grading 
astrocytomas, and MET was better than FDG for delineating low-grade tumor boundaries. The same effect 
was observed by Heiss et al. (Heiss et al., 1996), with amino acid tracers (11C-MET, 18F-tyrosine) offering 
better detection and FDG better grading. 
 
• In prostate cancer, Oyama et al.(Oyama et al., 2002) found 11C-acetate (ACE) more sensitive for primary 
disease and local metastases, and FDG preferable for distant metastases and tumor grade. 
 
• In hepatocellular tumors, Ho et al. (Ho et al., 2003) found that ACE had a sensitivity of 87.3%, compared 
to only 47.3% for FDG. Well differentiated lesions had high lesion-to-liver uptake ratios by ACE and low 
by FDG, whereas poorly differentiated lesions had high FDG uptake and low ACE uptake. 
 
• PET can detect physiologic changes in tumors that occur immediately in response to therapy, providing 
early (subclinical) assessment of the effectiveness of therapy (reviewed in (Young et al., 1999, Hoekstra 
et al., 2000, Lammertsma, 2001, Stokkel et al., 2001, Van der Hiel et al., 2001, Eary et al., 2002)). 
Much of this work has focused on FDG-PET (Ichiya et al., 1991, Chaiken et al., 1993, Blasberg, 1994, 
Letocha et al., 1994, Price et al., 1995, Findlay et al., 1996, Ichiya et al., 1996, Jones et al., 1996, 
Mortimer et al., 1996, Mankoff et al., 1997, Mitsuhashi et al., 1997, Schulte et al., 1999, Franzius et al., 
2000, Nair et al., 2000, Vlassenko et al., 2000, Krohn et al., 2001, Mortimer et al., 2001, Mankoff et al., 
2002, Mankoff et al., 2003), but Eary et al.(Eary et al., 2002) and Lammertsma (Lammertsma, 2001) 
highlight the importance of imaging cellular proliferation, perfusion and hypoxia in addition to glucose 
metabolism for this task. 
 
3.  PHARMACOLOGY AND SAFETY OF [18F]FLT and [18F]Fluciclovine and 
RADIATION DOSIMETRY OF [18F]Fluciclovine and [18F]FLT 
3.1. Pharmacology and Safety 
3.1.1. Pharmacology of [18F]FLT in Human 
The pharmacology of FLT is based on its action as an inhibitor of DNA synthesis (Langen et al., 1968, 
Langen et al., 1971, Langen and Graetz, 1972, Matthes et al., 1987, Matthes et al., 1988). Intracellular 
metabolism of FLT produces FLT phosphates, but these nucleotides inhibit endogenous DNA polymerases 
because they lack a 3’-hydroxyl substituent. This results in premature chain termination for DNA (Matthes 
et al., 1987, Sundseth et al., 1996). These biochemical properties can account for FLT’s prominent 
hematological and liver toxicity (Faraj et al., 1994, Flexner et al., 1994, Sundseth et al., 1996). The 
pharmacology of FLT is similar to that of the widely used prescription HIV-antiviral drug azidothymidine 
(AZT) (Lundgren et al., 1991, Kong et al., 1992). Both FLT and AZT are 3’-deoxythymidine analogs that 
act as inhibitors of DNA synthesis and are cleared from the body in the same way. However, FLT was 
significantly more cytotoxic than AZT in test cell lines (Faraj et al., 1994). Cellular uptake of FLT and 
thymidine was greater than that of AZT. Transport of FLT and thymidine across cell membranes occurs 
both by active transport and by simple diffusion (Kong et al., 1992). 
 
3.1.2. Toxicity of FLT in Humans 
FLT was investigated as an anti-AIDS drug in humans (Flexner et al., 1994). Toxic effects and death were 
reported for some subjects receiving FLT during randomized concentration controlled trials during a 16-
week treatment of oral multi-dosing. Doses of 0.125 mg/Kg every 12h, produced a mean cumulated drug 
exposure (AUC12: area under curve) of 417 ng-h/mL. At this level, serious (grade 3) hematologic toxicity 


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occurred in 6 of 10 subjects. At 300 ng-h/mL, grade 2 or greater (fall in hemoglobin to < 9.4 g/dL) 
developed within 4 weeks in 9 of 12 subjects. At 200 ng-h/mL almost no clinically significant anemia 
developed, but dose-limiting granulocytopenia (< 750 granulocytes/mm3) occurred in 5 of 15 subjects. Mild 
peripheral neuropathy occurred in 2 of 10 subjects at 50 ng-h/mL, but was not dose-limiting. FLT drug trials 
were terminated after the unexpected death of 2 subjects of hepatic failure. One patient assigned to 200 ng-
h/mL developed progressive liver failure and died after 12 weeks of FLT therapy. A second subject, who 
received a fixed dose of 10 mg/day, developed progressive liver failure and died at about the same time. All 
surviving subjects were followed closely for 4 weeks after stopping FLT and none had evidence of clinically 
significant liver disease or other adverse effects. Overall, 25 of the 44 subjects receiving at least two doses 
of FLT, completed the 16 week study without clinically significant adverse effects. 
 
In the human studies of [18F]FLT, no toxicities were reported at the doses and specific activities provided in 
numerous publications. However, it must be emphasized that of the 26 papers reviewed as part of the NCI 
initial IND submission for [18F]FLT only half (n=13) mentioned specific activity of the [18F]FLT. None of 
these published manuscripts specifically mentioned whether any assessments of safety or toxicity were 
performed. 
 
In a recent study performed at the University of Washington, Turcotte and colleagues (Turcotte et al., 2007) 
preliminarily assessed the toxicity of [18F]FLT in twenty patients with proven or suspected diagnosis of non-
small cell lung cancer. All patients gave written informed consent to the [18F]FLT injection, subsequent PET 
imaging, and blood draws. Blood samples were collected for each patient at multiple times before and after 
[18F]FLT-PET. These samples were assayed for comprehensive metabolic panel, total bilirubin, and 
complete blood and platelet counts. In addition, a standard neurological examination was performed for 
each patient by a qualified physician before and immediately after [18F]FLT-PET. All [18F]FLT doses were 
calculated based on patient weight (2.59MBq/kg = 0.07 mCi/kg) with a maximal dose of 185 MBq (5mCi). 
Starting with the [18F]FLT injection, dynamic PET images were acquired for 90 or 120 minutes. By placing 
a region-of-interest in the center of the left ventricular chamber, blood time-activity curves were generated 
for each patient from the dynamic PET data and then extrapolated to 720 minutes. This provided a measure 
of the area under the [18F]FLT concentration curve for 12 hours (AUC12). A separate estimation of the 
AUC12 was also obtained from sequential blood samples collected during PET data acquisition. The 
AUC12 values estimated from imaging data were not significantly different from those found from serial 
measures of [18F]FLT blood concentrations (p= 0.66). No side effects were reported by patients or 
witnessed. No change was observed in the neurological status of patients. Only albumin, red blood cell 
count, hemoglobin, and hematocrit showed a statistically significant decrease over time. These changes 
were attributed to IV hydration during PET imaging and to subsequent blood loss at surgery. 
 
The single dose AUC12 values derived from blood samples ranged from 0.22 to 1.34 ng-h/mL with a mean 
of 0.80 ng-h/mL. This range corresponds to 0.46% to 2.7% of the Flexner clinical trial AUC12 of 50 ng-
h/mL. The AUC12 of a single 10 mCi radiotracer dose of [18F]FLT is small compared to the AUC12 of the 
least toxic therapeutic trial dose. This is because the Flexner trial administered [18F]FLT every 12 hours for 
112 days with the only toxicity, peripheral neuropathy, developing at a mean of 40 days. At the lowest 
acceptable [18F]FLT specific activity (200 Ci/mmol), the [18F]FLT dose from 10 mCi is < 24.4 µg (< 50 
nmol) for one [18F]FLT PET study. The maximum dose from three 10.0 mCi injections of [18F]FLT in a 70 
kg subject, the maximum number doses that a single subject could receive under this protocol, is <20% of 
the 12 hour dose and less than 0.2% of the cumulative 40 day dose associated with any reported toxic effect 
in humans.  The only reported toxic effect was a mild, non-dose limiting peripheral neuropathy in 2/10 
subjects at 50 ng-h/mL from oral administration of FLT every 12 hour for 16 weeks. Based on these data, 
we conclude that FLT imaging will not lead to clinically detectable toxic effects. In addition, a recent small 
study assessing possible toxicities of [18F]FLT in primary brain tumor patients was also published from 


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investigators at the University of Washington (Spence et al., 2008). No significant toxicities to [18F]FLT 
were noted in this twelve patient study.  
 
There is some literature on the mutagenic properties of FLT. Ehrlich ascites tumor cells incubated with 10 
µM FLT for extended periods (12, 24, 36 h: AUC 120, 240, 360 nmol-h/mL) showed chromosome damage 
(Bäumlein and Wobus, 1976). The most prominent effects were breaks and gaps, however, much less 
damage was seen if a recovery time was included (12, 24 h) and the damage could also be largely reversed 
by post-treatment with thymidine (10 µM). FLT anabolism, FLT incorporation into DNA, and the effects of 
FLT on cellular genome integrity have been studied in cultured CEM (CD4+ human lymphoblastoid) cells. 
FLT concentrations of 10 and 100 µM produced chromosome fragmentation characteristic of cells 
undergoing apoptosis. In contrast, at 1 µM FLT, the level of fragmentation was similar to the controls 
without FLT exposure. Despite prominent levels of intracellular FLT anabolites, the fraction of FLT in 
DNA was low (10-6 total). At the lowest acceptable [18F]FLT specific activity (200 Ci/mmol), the dose to 
patients (< 10 mCi) will correspond to an initial, maximal plasma concentration of about 10 nM. This is 100 
times lower than the level of FLT where no chromosomal damage was seen in CEM cells (1 µM). Based on 
these data, the administration of 10 mCi of [18F]FLT to humans does not pose a probable threat of 
mutagenesis. 
 
3.1.3. Pharmacology of [18F]Fluciclovine in Humans 
The information provided here in is abstracted from the [18F]Fluciclovine Investigator Drug Brochure 
Version 5 (2017) and Axumin prescribing information (Axumin 2016). Three studies have evaluated 
biodistribution and radiation dosimetry in healthy volunteers (Nye et al., 2007, Asano et al., 2011, 
McParland et al., 2013). In general, the results of these studies are consistent. The primary evidence on the 
biodistribution and radiation dosimetry from the phase I study conducted by GE Healthcare (McParland et 
al., 2013, Sörensen et al., 2013) is reviewed in more detail below. This trial (GE-148-001) in 6 healthy 
volunteers and 6 subjects with biopsy-proven prostate cancer started in June 2009 and the clinical phase was 
completed in November 2009. The first part of the trial investigated the safety, biodistribution, and internal 
radiation dosimetry of [18F]Fluciclovine injection in 6 healthy volunteers (3 males and 3 females). Overall, 
there was no evidence indicating that [18F]Fluciclovine is metabolized in vivo. 
 
Qualitative assessment of the distribution of [18F]Fluciclovine activity in all 6 subjects showed that the 
distribution was largely uniform throughout the body with the exception of the liver, pancreas, lung, red 
bone marrow, and heart wall. The initial uptake of [18F]Fluciclovine in each subject was assessed at the first 
imaging time point, on average 7.9 mins (6.5 to 10.2 mins) post injection. Initial uptake of [18F]Fluciclovine 
activity in the liver, pancreas, and the red bone marrow of the thoracic and lumbar vertebrae and skull was 
immediately evident in all subjects. There was very little brain uptake, 1.6% (0.7% to 2.2%). With 
increasing time post injection, distributed uptake was apparent and, on the basis of the anatomical 
distribution, was mostly associated with skeletal muscle. This uptake could not be isolated for quantification 
as it was not possible to segment the muscle in the whole-body image. Hence, muscle was included in the 
“remaining tissues” category. Uptake in the spleen was apparent in some subjects. Uptake in the uterus was 
apparent in some female subjects and initial activity uptake in the uterus was limited, 1.2% (0.3% to1.7%). 
It was assumed, based on the general skeletal muscle uptake, to be associated with the muscle of the uterine 
wall. The four organs with the highest initial uptake of [18F]Fluciclovine were the liver at 13.8% (8.3% to 
17.1%), red bone marrow at 11.1% (4.8% to 20.4%), lung at 7.1% (5.2% to 8.6%), and pancreas at 4.2% 
(3.4% to 5.2%). Based on comparison with washout of activity from the whole-blood samples, washout of 
activity from the lung was consistent with washout of activity from the pulmonary blood content. 
 


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Excretion of [18F]Fluciclovine activity was limited and was only found for the renal pathway, reaching 3.2% 
(1.1% to 7.4%) at the last imaging time point an average of 4.2 hours post injection (3.8 to 4.7 hours). The 
small amount of activity entering the gastrointestinal (GI) tract could not be clearly identified due to the 
surrounding activity in muscle and mesentery. On the basis of activity washout from the liver and the 
pancreas, and assuming that all of this activity was to enter the duodenum through hepatobiliary and 
pancreatic transport, the activity excreted via the GI tract was estimated to be of the order of 10 % or less. 
Although this could not be accurately quantified, any activity present in the contents of the GI tract was 
grouped within the “remaining tissues” category for evaluation of internal radiation dosimetry. No 
differences considered to be of likely clinical significance were noted between the 3 males and the 3 
females. 
 
3.1.4. Toxicity of [18F]Fluciclovine in Humans 
The information provided here in is abstracted from the [18F]Fluciclovine Investigator Drug Brochure, 
Version 5 (2017) and Axumin prescribing information (Axumin 2016). Fourteen-day repeat-dose toxicity 
studies in two mammalian species (rat and dog) showed no important treatment-related adverse effects. 
Genotoxicity tests showed that no mutagenic or clastogenic effects were elicited by [18F]Fluciclovine or its 
major impurity, AH114760. No adverse effects were observed during local tolerance tests using the 
intravenous and paravenous administration route in rabbits. [18F]Fluciclovine also showed no potential for 
hemolysis of human blood. 
 
Pregnant or nursing women: Reproductive toxicology studies have not been performed with 
[18F]Fluciclovine. However, since [18F]Fluciclovine is a radiopharmaceutical, it is assumed that fetal 
toxicity or embryo fetal toxicity may result if it is administered to a pregnant woman. Therefore, 
[18F]Fluciclovine injection is not suitable for administration to pregnant or nursing women. 
 
Genotoxic and mutagenic potential: The results of mutagenicity and clastogenicity studies performed with 
[18F]Fluciclovine, including the Ames, MLA and rat in vivo bone marrow micronucleus assays, did not 
reveal either mutagenic or clastogenic potential. No carcinogenicity studies have been conducted, because 
[18F]Fluciclovine injection is intended as a diagnostic imaging agent for infrequent administration. 
 
Findings in safety pharmacology and toxicology studies: Nonclinical toxicology and safety 
pharmacology studies with [18F]Fluciclovine have not identified any specific target organs or adverse effects 
on nervous, respiratory, or cardiovascular systems. As such, no potential [18F]Fluciclovine injection-related 
safety risks for humans have been identified on the basis of the nonclinical data. 
 
Expected risks to subjects participating in clinical studies: No serious adverse reactions have been 
reported from any investigator sponsored studies or from the compassionate use program in Norway. The 
risks to subjects mainly relate to the intravenous injection and intravenous blood sampling procedures, and 
the radiation emitted by [18F]Fluciclovine. Intravenous injection and the use of an intravenous cannula are 
known to carry a small risk of infection and hematoma. The exposure to radiation will not exceed that which 
is considered acceptable in accordance with appropriate guidelines. 
 
AEs arising from the industry-sponsored studies described above which are considered to be potential 
adverse reactions to [18F]Fluciclovine are listed in Table 1. Of these events, dysgeusia, headache, redness at 
the injection site, and sensation of burning during injection are considered to be adverse drug reactions 
potentially related to the investigational product and are included in the summary Table 1 below (Reference 
Safety Information). [18F]Fluciclovine remains under clinical investigation. Investigators should be alert to 
the potential for additional adverse reactions to be observed as clinical experience continues 


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([18F]Fluciclovine Investigator’s Brochure, Version 05; May 30, 2017 and Axumin prescribing information 
2016). Based on the pharmacokinetics of [18F]Fluciclovine and of AH114760, the safety reporting window 
for monitoring for potential adverse reactions is set at 28 days post injection. 
 
Table 1.  Reference Safety Information 
 
Frequency 
Intensity 
 
 
 
 
 
System organ 
class/Preferred 
term 
Number of 
Subjects (%) 
Total N = 1153 
Mild 
Moderate 
Severe 
Any Event 
12 (1%) 
12 
0 
0 
General disorders and 
administration site 
conditions 
7(0.6%) 
7 
0 
0 
Injection-site reactions 
7 (0.6%) 
7 
0 
0 
Nervous system disorders 
4 (0.3%) 
4 
0 
0 
Dysgeusia 
4 (0.3%) 
4 
0 
0 
Respiratory Organ, 
Thoracic and Mediastinal 
Disorders  
 
2 (0.2%) 
2 
0 
0 
Parosmia  
 
2 (0.2%) 
2 
0 
0 
*1 patient reported both dysgeusia and parosmia 
 
3.2. Human Radiation Dosimetry 
The information on dosimetry calculations for each individual tracer is presented in sections 3.2.1 and 3.2.2. 
The CT dosimetry calculations used for attenuation correction used in conjunction with the PET imaging is 
provided in section 3.2.3. The cumulative dosimetry (Table 2) for a maximum of 6 possible PET/CT 
examinations (baseline imaging of each tracer, imaging of each tracer after completion of therapy, and 
imaging of each tracer at the time of pseudoprogression) is then presented in section 3.2.4. The dosimetry 
for both [18F]FLT and [18F]Fluciclovine is based on published studies and the biodistribution and radiation 
dosimetry provided in the [18F]Fluciclovine Investigator Drug Brochure, Version 5, 5/30/2017 and Axumin 
prescribing information 2016.  A CT is required for attenuation correction and the dosimetry tables include 
the available data that has been revised based on ICRP 106.  
 
3.2.1. Human Radiation Dosimetry of [18F]FLT 
Eighteen patients (11 men, 7 women) with known or suspected lung cancer were prospectively studied with 
[18F]FLT-PET imaging at the University of Washington from March 2000 to April 2002 (Vesselle et al., 
2003).  Biodistribution data from these 18 patient studies were used for dosimetry calculations. The amount 
of injected [18F]Fluorothymidine activity used in this protocol will be approximately 370 MBq (10 mCi). 
The dosimetry for this compound was previously published in ICRP Publication 128 (Mattsson, 2015). The 
greatest organ absorbed doses for a 370 MBq (10 mCi) injection of [18F]Fluorothymidine are the liver (18 
mGy) and the kidneys (16 mGy), while the effective dose is 5.6 mSv (Table 2). 
 


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3.2.2. Human Radiation Dosimetry of [18F]Fluciclovine 
Three studies have evaluated biodistribution and radiation dosimetry in healthy volunteers (Nye et al., 2007, 
Asano et al., 2011, McParland et al., 2013). In general, the results of these studies are consistent. The 
primary evidence on the biodistribution and radiation dosimetry from the phase I study conducted by GE 
Healthcare (McParland et al., 2013, Sörensen et al., 2013) was used to determine the human radiation 
dosimetry. The amount of injected [18F]Fluciclovine activity used in this protocol will be approximately 370 
MBq (10. mCi). A summary of dosimetry studies for this compound was previously published and is used to 
estimate radiation dose and risks in this protocol (McParland et al., 2013). The Blue Earth Diagnostics 
Investigator’s Drug Brochure for [18F]Fluciclovine also uses the dosimetry from this paper. The greatest 
organ absorbed doses for a 370 MBq (10 mCi) injection of [18F]Fluciclovine are the pancreas (38 mGy) and 
the heart (19 mGy), while the effective dose is 8.2 mSv (Table 2). 
 
3.2.3 Human Radiation Dosimetry for CT-based Attenuation Correction for PET 
The study will be performed on the research GE Discovery 710 PET/CT scanner in the Molecular Imaging 
Suite at HCI. Each PET imaging study will require CT imaging for attenuation correction. After positioning 
the patient in the gantry of the PET/CT system, a topogram of the head will be obtained to confirm the 
correct positioning and identify the anatomic range for a single PET bed position covering the entire brain. 
A helical CT scan is then performed over the same anatomic range corresponding to the PET scan in order 
to perform for attenuation correction. The PET emission scan will commence immediately after the CT 
scan. The CT acquisition parameters will be 120 kVp, 0.5s rotation speed, 35 mA tube current, 64 x 0.625 
mm collimation, and a pitch of 0.984. This results in a CT Dose Index (CTDIvol) of 2.81 mGy based on a 
16 cm phantom. A standard length of 1 PET bed position results in a Dose Length Product (DLP) of 55.3 
mGy x cm. The estimated effective dose using the DLP scaling method is 0.12 mSv (AAPM Report No. 96, 
2008). In order to estimate the absorbed doses of individual organs and the resulting effective dose, the 
ImPACT Scan CT Patient Dosimetry Calculator (Version 1.0.4) was used with the specific acquisition 
parameters used in this protocol and the NRPB Monte Carlo dose data sets for the GE Lightspeed VCT 
scanner produced in report SR250 dosimetry tables (Jones et al., 1991). A correction factor has been applied 
to account for the difference in the CTDIvol derived from ImPACT Calculator and that reported on the GE 
scanner. Note that the topogram contributes a negligible radiation exposure to the helical CT exam and was 
not included in the dose estimates. The greatest organ absorbed doses for a single head CT scan using the 
aforementioned acquisition parameters are the eye lenses (2.4 mGy) and the brain (2.4 mGy), while the 
effective dose is 0.10 mSv (Table 2). 
 
3.2.4. Cumulative Radiation Dosimetry for [18F]FLT/CT + [18F]Fluciclovine/CT 
The dosimetry (mGy/10.0 mCi) for each radiotracer is summarized in Table 2. The proximate maximum 
administered activity for each tracer will be as follows: 10.0 mCi [18F]FLT and 10 mCi [18F]Fluciclovine. 
There is a maximum total of 6 PET/CT scans possible on this study. This includes baseline [18F]FLT/CT 
and [18F]Fluciclovine/CT, immediate post therapy [18F]FLT/CT and [18F]Fluciclovine/CT, and [18F]FLT/CT 
and [18F]Fluciclovine/CT at the time of pseudo-progression. A group of patients may only receive the 
[18F]FLT/CT and [18F]Fluciclovine/CT at the time of pseudo-progression. A low-dose CT is used for 
attenuation correction and will be obtained for each PET study.   
 
 


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 Table 2. Cumulative Radiation Dosimetry 
IRB #104740 
[18F]Fluciclovine
-PET 
[18F]FLT-PET Low Dose Head 
CT 
PET/CT Total 
Activity (mCi) 
10 
10 
- 
- 
Number of Scans: 
3 
3 
6 
 
 
 
Organ 
Single Scan 
Absorbed dose 
(mGy) 
Single Scan 
Absorbed 
dosemGy) 
Single Scan 
Absorbed 
dose(mGy) 
Protocol 
Absorbed dose 
(mGy) 
Adrenals 
5.99 
5.92 
0.000367 
35.74 
Bladder 
9.32 
8.51 
0.000009 
53.50 
Bone surfaces 
8.58 
7.03 
0.626846 
50.60 
Brain 
3.22 
3.03 
2.377692 
33.03 
Breasts 
5.07 
3.03 
0.001513 
24.32 
Gallbladder 
6.18 
6.66 
0.000069 
38.52 
Stomach 
5.18 
4.44 
0.000223 
28.86 
Small intestine 
4.88 
4.81 
0.000013 
29.08 
Colon 
4.70 
4.44 
0.000011 
27.42 
Heart 
19.13 
4.44 
0.001441 
70.72 
Kidneys 
5.07 
15.91 
0.000159 
62.94 
Liver 
12.40 
17.76 
0.000360 
90.47 
Lungs 
12.77 
4.07 
0.005260 
50.54 
Muscles 
3.92 
3.63 
0.051156 
22.95 
Oesophagus 
4.63 
3.63 
0.003603 
24.77 
Ovaries 
4.92 
4.44 
0.000000 
28.08 
Pancreas 
37.81 
5.55 
0.000245 
130.09 
Red marrow 
9.14 
9.62 
0.151308 
57.18 
Skin 
2.96 
2.78 
0.158513 
18.16 
Spleen 
8.81 
8.14 
0.000346 
50.84 
Testes 
6.44 
3.26 
0.000000 
29.08 
Thymus 
4.63 
3.63 
0.003603 
24.77 
Thyroid 
3.81 
3.48 
0.100872 
22.47 
Uterus 
16.50 
4.44 
0.000009 
62.83 
Effective dose (mSv) 
8.18 
5.55 
0.10 
41.79 
 


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4. RATIONALE, GOALS AND OBJECTIVES 
4.1. Clinical Rationale 
The standard treatment approach for patients with high-grade primary brain tumors includes maximum 
feasible surgical resection, followed by 6 weeks of concurrent cranial irradiation, and daily low-dose 
temozolomide chemotherapy; followed by 12 cycles of high-dose temozolomide administered for 5 
consecutive days every 4 weeks (Stupp et al., 2005). Contrast-enhanced MRI is the current standard for 
evaluating the success of therapy and monitoring for tumor recurrence. MRI is typically obtained prior to 
initial surgery, within 24 hours after surgery, at the conclusions of cranial irradiation, and then every 8 
weeks during temozolomide chemotherapy until evidence of recurrence. Despite this careful clinical and 
radiographic surveillance, and despite decades of research into the histologic and molecular classification of 
primary brain tumors, our ability to predict tumor behavior remains very limited. Some gliomas will result 
in overall survival times of only months, whereas other histologically-identical gliomas may yield survivals 
of years to decades (Curran et al., 1993, Carson et al., 2007). Current assessment of tumor response to 
therapy is also poor. Patients with complete radiographic response after cranial irradiation often progress 
rapidly post-irradiation. In contrast, some patients with enhancing masses at the end of chemoradiotherapy 
may respond dramatically to further chemotherapy alone; or the masses may even disappear in the absence 
of further therapy, so called “tumor pseudoprogression” (Chamberlain et al., 2007). This confounding 
situation demonstrates a need for better assessment of tumor response. 
 
Improvements in the ability to predict tumor behavior prior to the start of therapy would allow more 
efficient and effective tumor surveillance; better prognostication; and more appropriate assignment of 
patients to conventional, aggressive, or investigational therapies early in their clinical courses. This would 
provide huge economic and social benefits, and could afford decisive insights into brain tumor physiology 
and biology. Similarly, the ability to identify, earlier and more accurately, whether individual patients were 
responding to therapy would allow prompt discontinuation of ineffectual treatments and institution of 
potentially more effective therapies. 
 
Previous efforts using imaging for such tasks have generally been limited to a single modality (e.g. MRI) 
and/or single-tracer (e.g. FDG-PET). However, there is a significant and growing body of evidence that 
complementary imaging of multiple aspects of tumor physiology (i.e. using multiple PET tracers) can 
provide greatly enhanced information over imaging with a single modality or tracer alone.  In solid tumors, 
complex interactions exist between blood flow, metabolic activity, and oxygen status which affect 
metastatic and proliferative activity. Heterogeneous tumors may contain both slow-growing and fast-
growing regions that present different profiles of proliferation rates and amino acid uptake.  
 
4.2. Hypotheses 
The hypothesis of this exploratory clinical trial in patients with high-grade a primary brain tumor who 
receive chemoradiation is that the PET imaging agents [18F]Fluciclovine and/or [18F]FLT will be a better 
predictor of tumor response than standard MRI based brain tumor response criteria. When used in 
conjunction, the two PET agents may be better able to predict tumor aggressiveness and thus overall 
survival than the use of individual-tracer PET biomarkers. This may eventually lead to improved assessment 
of response (including time to progression and overall survival) and differentiation of tumor 
recurrence/progression from treatment effect (pseudoprogression). 
 


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4.3. Exploratory Objectives 
4.3.1. Exploratory Objectives 
The primary objective of the study is to show that a 25% reduction in SUVmax or SUVmean for each of the 
PET imaging tracers will be predictive of overall survival  
 
Secondary Exploratory Objectives 
 
• Assess the association between overall survival and change in each of the following quantitative 
markers for [18F]Fluciclovine and [18F]FLT:  K1-k4, Knet and Ktrans, from baseline to post therapy. 
• Assess the association between progression free survival and change in each of the following 
quantitative markers for [18F]Fluciclovine and [18F]FLT:  K1-k4, Knet and Ktrans, from baseline to post 
therapy. 
• Assess the ability of [18F]FLT and/or [18F]Fluciclovine alone or together to predict true progression 
from pseudoprogression in patients with this clinical dilemma. 
• A 25% reduction in SUVmax or SUVmean for each of the PET imaging tracers will be more 
predictive of longer overall survival than the current MRI based response criteria.  
• A 25% reduction in SUVmax or SUVmean for each of the PET imaging tracers will be more 
predictive of time to progression than the current MRI based response criteria.  
 
Two different groups of patients will be able to enroll in the trial.  The first group, Group A, will be for 
patients who have not yet received any therapy for their suspected or newly found brain tumors.  Group B 
will enroll patients with pathologically proven malignant brain tumor (WHO Grade III or IV glial-based 
tumors) who have undergone chemoradiation and have a contrast enhancing mass ≥ 1.0 cm in greatest 
diameter concerning for pseudoprogression.  MRI-documented possible recurrence/progression versus 
treatment effect (pseudoprogression) within 6 months from the time of completion of chemoradiation.  
 
Group A will have three different patient cohorts.  The first, for patients where there is compelling evidence, 
based on the MRI and/or CT imaging, that a high-grade primary brain tumor is present. Pathologic 
confirmation will occur with subsequent biopsy or surgery.  The second cohort of patients are individuals 
where the tumor is felt to be inoperable and a biopsy is performed but no surgery. The third cohort of 
patients are post-surgery where a complete surgical resection was not possible. This will be confirmed by a 
post-operative contrast MRI/CT scan where residual tumor > 1.0 cm in diameter is present.  
 
A number of quantitative and semi-quantitative imaging biomarkers for each tracer will be computed at 
each imaging time point, and the change in each biomarker between time-points will also be computed. 
These data will be compared with changes in tracer uptake before and after therapy and used to determine 
the semi-quantitative change in [18F]FLT and [18F]Fluciclovine uptake which is consistent with response 
based on standard brain tumor response. In addition the imaging biomarkers will be used to determine if 
they are predictive endpoints (survival, time to progression), and with tumor biologic information 
(histology, WHO grade, vascularity, MIB-1, VEGF, EGFR, p53, IDH status) in cases when tumor tissue 
becomes available from standard care. These data will provide pilot information regarding the potential 
value of concurrent multiple PET biomarkers for predicting tumor behavior prior to the start of therapy, for 
improved prognostication, for more efficient and effective tumor surveillance, and/or for more appropriate 
assignment of patients to conventional, aggressive, or investigational therapies early in their clinical courses. 
 
All patients will receive standard tumor-directed therapy and will be monitored with conventional imaging 
according to standard of care, which may include MRI and FDG-PET as described earlier. The [18F]FLT or 


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[18F]Fluciclovine PET research scans will not affect clinical decisions. A database of demographic, clinical, 
and molecular biologic information for each patient will be created. Tissue samples, when available from 
routine care, will undergo detailed histopathologic evaluations, with results prospectively and systematically 
recorded. All patients will be followed until the end of the trial (at least 12 months post last imaging 
assessment) for important clinical endpoints as listed in Table 3. 
 
Table 3. PET Imaging Biomarkers, Clinical and Biologic Information Database 
PET Imaging Biomarkersa 
Clinical and Biologic Information 
 
Age at diagnosis, performance status, tumor size and location, radiographic 
characteristics, extent of surgery, dates and types of therapy. 
 
Tumor histology and WHO grade, 
vascularity, MIB-1, VEGF, EGFR, p53, IDH status 
Clinical and radiographic response, concomitant medications; time to 
progression, overall survival 
[18F]FLT (proliferation):  SUVmax, 
SUVmean,SUVFLT , K net
FLT  
 
[18F]Fluciclovine amino acid uptake:  
         SUVmax, SUVmean, SUVFLUC, 
KFLUCnet  
 
                                    
 
a Quantitative and semi-quantitative imaging biomarkers  
 
The data from multiple PET biomarkers combined with histologic and clinical data will enable identification 
and development of PET biomarker profiles which correlate with (and might be predictive of) tumor 
aggressiveness and clinical outcomes. The three imaging time point assessments serve multiple uses in this 
initial exploratory study. If baseline imaging can predict tumor aggressiveness and clinical outcomes, such 
imaging may potentially be used to guide selection of conventional, aggressive, or investigational therapies. 
The second time point, within 1-4 weeks after 6wks of chemoradiotherapy, will provide a measurement of 
tumor response that may potentially differentiate tumors that will progress rapidly from those that will not 
(changes in imaging biomarkers between exams 1 and 2 will likely be important for this determination). 
Finally, the multi-tracer array at the time of treatment effect (pseudoprogression) versus true 
recurrence/progression may yield new insights into the physiology and biology of these tumors, and multi-
tracer imaging may help to differentiate recurrence from radiation necrosis/treatment effect 
(pseudoprogression). We believe the complementary value of the two tracers in conjunction has much 
greater potential for meeting these goals than any one tracer alone. For example, tumors with high 
proliferation ([18F]FLT) and/or growth ([18F]Fluciclovine) may respond best to anti-EGFR-directed agents 
or conventional cytotoxic chemotherapies. Of specific interest, is there a multi-tracer PET signature that 
distinguishes between treatment effect (pseudoprogression) and true progression/recurrence that will have 
important clinical utility? These questions will be studied in detail, developing formal hypotheses that will 
be tested in future phase II/III clinical trials. 
 
5. TRIAL DESIGN 
5.1. Patient Eligibility 
Two different groups of patients will be able to enroll in the trial.  The first group, Group A, will be for 
patients who have not yet received any therapy for their suspected or newly found brain tumors.  Group B 
will enroll patients with pathologically proven malignant brain tumor (WHO Grade III or IV glial-based 
tumors) who have undergone chemoradiation and have a contrast enhancing mass ≥ 1.0 cm in greatest 
diameter concerning for pseudoprogression.  MRI-documented possible recurrence/progression versus 
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Group A will have three different patient cohorts.  The first, for patients where there is compelling evidence, 
based on the MRI and/or CT imaging, that a high-grade primary brain tumor is present. Pathologic 
confirmation will occur with subsequent biopsy or surgery.  The second cohort of patients are individuals 
where the tumor is felt to be inoperable and a biopsy is performed but no surgery. The third cohort of 
patients are post-surgery where a complete surgical resection was not possible. This will be confirmed by a 
post-operative contrast MRI/CT scan where residual tumor > 1.0 cm in diameter is present.  
 
Eight to ten patients per year will be enrolled, for a total of 30 patients. Providers within the Radiation 
Oncology and Neuro-Oncology departments will identify and recruit patients who may be eligible 
participants. 
 
5.2. Inclusion Criteria 
• Two adult patient groups will be eligible for inclusion in this study:  
o Group A: Patients where there is compelling evidence, based on the MRI and/or CT imaging, 
that a high-grade primary brain tumor is present. Pathologic confirmation will occur with 
biopsy or surgery. 
Patients whose tumor is felt to be inoperable and a biopsy is performed but no surgery. 
Patients with a newly diagnosed primary malignant brain tumor (WHO Grade III or IV) who 
will be receiving chemoradiation and who either did not undergo surgical resection or 
underwent incomplete resection with residual tumor > 1.0 cm in greatest diameter by contrast 
MRI postoperatively.  
o Group B: Patients with pathologically proven malignant brain tumor (WHO Grade III or IV 
glial-based tumors) who have undergone chemoradiation and have MRI-documented 
possible recurrence/progression versus treatment effect (pseudoprogression) within 6 months 
from the time of completion of chemoradiation.  
• Patients must be 18 years or older for inclusion in this study. There is little experience with the 
safety of [18F]FLT or [18F]Fluciclovine in children, and the risks associated with radiation exposure 
may be increased for children under 18 years old as well. 
• Patients must document their willingness to be followed for up to 24 months after recruitment by 
signing informed consent documenting their agreement to have clinical endpoints and the results of 
histopathologic tissue analysis (when tissue becomes available from routine care) entered into a 
research database. 
• All patients, or their legal guardians, must sign a written informed consent and HIPAA authorization 
in accordance with institutional guidelines. 
• Determination of pregnancy status: Female patients who are not postmenopausal or surgically sterile 
will undergo a serum pregnancy test prior to each set of [18F]FLT and [18F]Fluciclovine PET scans. 
A negative test will be necessary for such patients to undergo research PET imaging. 
• Pre-imaging laboratory tests must be performed within 28 days prior to the [18F]FLT imaging 
procedure. These must be less than 4.0 times below or above the upper or lower limit range for the 
respective laboratory test for entry into the study (unless clinically not relevant). In those instances 
where a laboratory value is outside of this range, then such a patient will be ineligible for enrollment 
unless at the discretion of the PI the abnormal lab value is of no clinical significance to the safety or 
integrity of the study results. For follow-up scanning sessions after therapy has been instituted, 
laboratory testing will also be required due to the use of [18F]FLT. The patients have brain tumors 
and will have received chemoradiation; therefore, many routine laboratory tests may not be within 
the typical normal range. As such, a factor of 4.0 times above or below the upper or lower value for 
the normal range for any laboratory test will also be used to determine the acceptable range for the 


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2nd and possibly 3rd imaging time points (unless clinically not relevant as explained above). The 
baseline laboratory testing will include liver enzymes (ALT, AST), bilirubin (total), serum 
electrolytes, CBC with platelets, prothrombin time, partial thromboplastin time, BUN, and 
creatinine. For those patients receiving coumadin or another anticoagulant the upper limit for 
prothrombin time or partial thromboplastin time must not exceed 6 times the upper limit of the 
normal range. (Appendix E, Laboratory Study Results).  
 
5.3. Exclusion Criteria 
• Patients with known allergic or hypersensitivity reactions to previously administered 
radiopharmaceuticals. Patients with significant drug or other allergies or autoimmune diseases may 
be enrolled at the Investigator’s discretion. 
• Patients who are pregnant or lactating or who suspect they might be pregnant. Serum pregnancy tests 
will be obtained prior to each set of multi-tracer PET scans in female patients that are not 
postmenopausal or surgically sterile. 
• Adult patients who require monitored anesthesia for PET scanning. 
• Patients who are too claustrophobic to undergo PET scanning. 
• Known HIV positive patients due to the previous toxicity noted with [18F]FLT in this patient group. 
 
5.4. Patient Registration 
Following appropriate pre-registration evaluation, a signed consent, HIPAA authorization, and completed 
eligibility checklist will be obtained (Appendix A). New patient registrations will be submitted to the 
clinical trials office by the study coordinator, who will record study data on all patients entered into the 
study and complete subsequent forms. 
 


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5.5. Study Procedures, Schedule of Events, Tracer Administration:  Route and Dosing 
 
 
5.5.1. Initial Visits Prior to PET Imaging 
The patient will be under the care of their treating neuro-oncologist. The baseline measures of liver function, 
renal function, and electrolytes will be collected if not previously obtained within 28 days of the initial 
[18F]FLT imaging visit. The following additional patient data will be obtained: histological diagnosis (when 
available following surgery), age at radiological diagnosis, gender, and other treatment modalities used. 
Clinical (non-research related) imaging studies, such as contrast MRI and/or standard clinical FDG PET, 
will be collected according to standard of care. 
 
5.5.2. Multi-Tracer PET Exams 
Multi-tracer PET exams of  [18F]FLT and [18F]Fluciclovine will be acquired in each patient at up to three 
time points: (1) prior to any tumor-directed therapy, either prior to surgery or after surgery providing a 
complete surgical resection was not performed and confirmed by a post-operative contrast MRI scan where 
residual tumor > 1.0 cm in diameter was present and prior to any tumor-directed therapy; (2) within 1-4 
weeks after the conclusions of the initial (~6-8 weeks) chemoradiotherapy; and (3) patients with MRI-
documented possible recurrence/progression versus treatment effect (pseudoprogression) within 6 months 
from the time of completion of chemoradiation. The abnormality must be > 1.0 cm in diameter by contrast 
MRI or CT or show changes on non-enhancing MRI sequences (T2 or FLAIR).  
 
Patients who were originally not enrolled to study baseline and conclusion of therapy imaging will be 
eligible to participate at the time of MRI-documented possible recurrence/progression versus treatment 
effect (pseudoprogression) within 6 months from the time of completion of chemoradiation. The 
abnormality must be > 1.0 cm in diameter by contrast MRI or CT or show changes on non-enhancing MRI 
sequences (T2 or FLAIR).  


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5.5.3. Day of Research PET Scan(s) 
The patient will have the appropriate IV access placed for radiotracer administration. The patient will then 
be positioned comfortably on the PET imaging table with the PET head-holder attachment. A CT scan for 
PET attenuation correction will be acquired using x-ray CT (PET/CT scanner). Dynamic PET scans of each 
tracer will then be acquired, which requires administration of the PET tracer(s) while the scanner is 
acquiring in dynamic mode. The details of tracer administration are summarized in Table 4. 
 
Table 4. PET Tracer Administration:  Route, Dosing, and Blood Sampling 
Tracer 
Route 
Manner 
Injected Dose 
Scan Mode 
[18F]FLT 
[18F]Fluciclovine 
IV 
IV 
~60s push 
~60s push 
~10 mCi 
~10 mCi 
Dynamic 
Dynamic 
 
Each radiotracer will be prepared by the investigational drug radiochemists in the PET cyclotron facility at 
HCI on the day of the scanning session. The tracer will be administered to the patient by a physician, 
nuclear medicine technologist, or trained research personnel in the PET imaging suite. Each tracer 
administration will be pushed by hand as a manually timed infusion (~60s) as indicated by Table 4, where 
each infusion is designed to optimize dynamic PET efficacy for that tracer. 
 
Adverse events potentially related to the administration of [18F]FLT or [18F]Fluciclovine will be recorded at 
the conclusion of the [18F]FLT or [18F]Fluciclovine study. (Appendix B: Schedule of Events; Appendix D: 
[18F]FLT Infusion Flow Chart, Appendix G: [18F]Fluciclovine Infusion Flow Chart). 
 
5.6. Data Collection 
5.6.1. Safety Data Recorded During [18F]FLT-PET and [18F]Fluciclovine-PET Scans 
Data collected on visit(s) for [18F]FLT-PET and [18F]Fluciclovine-PET scanning will be recorded on the 
[18F]FLT or [18F]Fluciclovine Protocol flow chart and will include: vital signs (including heart rate, blood 
pressure, and temperature) recorded just prior to the [18F]FLT or [18F]Fluciclovine infusion and then after 
the completion of the [18F]FLT and [18F]Fluciclovine imaging study. In addition to monitoring the above-
mentioned safety parameters, an emergency code cart is located in the PET suite and can be utilized by the 
PET imaging team, as well as accessed by the code team at the institution should an emergent situation 
arise. 
 
Additional information to be collected will include: 
• Questioning of patient regarding adverse events immediately after each [18F]FLT and 
[18F]Fluciclovine administration. 
• Patients may be discharged after the completion of the [18F]FLT or [18F]Fluciclovine imaging 
procedure if stable and if no adverse events are noted on questioning. Patients will be evaluated 
for adverse events after the completion of the [18F]FLT and [18F]Fluciclovine scans.  
 
5.6.2. Tissue Collection and Analysis 
All patients enrolled on this study will have undergone a neurosurgical procedure (either biopsy or tumor 
resection) in order to obtain a histological diagnosis of their tumors as part of standard care. Tissue 
specimens will be analyzed by the attending neuropathologist as part of routine care, who will assign a 


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histologic diagnosis and tumor grade according to World Health Organization (WHO) criteria. If sufficient 
tissue is available, specimens are routinely evaluated immunohistochemically for MIB-1, EGFR, p53, and 
IDH status; and by conventional light microscopy for vascularity. These results are routinely recorded on 
the surgical pathology report.  
 
6. METHODS FOR EVALUATION OF IMAGING STUDIES 
Each PET imaging study will be evaluated using qualitative, semi-quantitative, or fully-quantitative analysis 
techniques that have previously been used by us and other groups, and represent the standard and accepted 
means of evaluating static- and dynamic-PET images for these tracers. These assessments are described 
below for each tracer. 
 
6.1. [18F]FLT-PET Imaging 
6.1.1. [18F]FLT Qualitative Visual Assessment 
Qualitative (visual) assessment of level of uptake in brain tumor and normal brain will be performed.  
Regarding the initial question of tumor presence the following scale will be used: 1= definite tumor, 2 = 
probably tumor, 3 equivocal for tumor, 4 = probably not tumor, 5 = definitely not tumor. For the immediate 
post therapy imaging [18F]FLT scan the same scales will be used. In the second group of patients where 
recurrence/progression (pseudoprogression) visual assessment on the [18F]FLT-PET study will be 
interpreted as to confidence of tumor recurrence: 5 = definitely recurrence/progression, 4= probably 
recurrence/progression, 3= unable to differentiate recurrence/progression, 2= probably treatment 
effect/necrosis, 1= definitely treatment effect/necrosis. 
 
6.1.2. [18F]FLT Semi-Quantitative Assessment 
Semi-quantitative assessment of [18F]FLT uptake will be performed by calculating SUVmax and SUVmean. A 
region-of-interest (ROI) will be drawn over any tumor mass, necrotic region, or other abnormal region(s) on 
the summed static [18F]FLT PET image. For each ROI, standardized uptake values (SUVs) with body 
surface-area correction will be obtained for both the maximum and mean voxel value in ROI: 
 
 
SUVmax =
Amax
InjectedDose BSA   
 
SUVmean =
Amean
InjectedDose BSA  
 
 
BSA = W 0.425 × H 0.725 × 0.00718  
 
 
 
Amax  = maximum voxel value in ROI (µCi/mL; mCi/L; or MBq/L) 
 
 
Am ean  = mean voxel value in ROI (µCi/mL; mCi/L; or MBq/L) 
 
 
BSA  = body surface area, computed from height H (cm) and weight W (kg) 
 
Ratios of the SUV of tumor to that of white matter (T/WM) and cortex (T/C) will also be computed. The 
change in SUVs for pre-treatment (baseline) and post-treatment images will be computed and correlated 
with available histologic and clinical data. 
 


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6.1.3. [18F]FLT Fully-Quantitative Assessment 
Quantitative assessment of [18F]FLT uptake will be performed by mathematical modeling of time activity 
curves for [18F]FLT and its metabolites in blood and tissues. Several methods of analysis are being 
considered for evaluating [18F]FLT images and results. A plausible scheme is outlined in the figure below. 
 
 
 
The compartments describe the kinetic behavior of [18F]FLT. Its transport across cell membranes is 
governed by two rate constants, K1 and k2. The phosphorylation of [18F]FLT is governed by the rate 
constant k3. Evidence from FLT experiments in dogs suggests that [18F]FLT is retained in cells without 
significant dephosphorylation (k4). We will initially assume that k4 is zero to simplify the modeling 
equations; the Akaike Information Criterion (AIC) will be used to determine if the time-activity curves 
support the more complex model (k4≠0), which will then be used as indicated. 
 
We will investigate the contribution of the labeled metabolite. If the labeled metabolite is restricted to the 
plasma space and urine and if the rate constant k4 is negligible, then the [18F]FLT flux constant, KFLT 
(mL/min/g), will be computed using graphical analysis (Patlak et al., 1983, Patlak and Blasberg, 1985). In 
the event that the [18F]FLT metabolite contribution in tissue is significant, we will investigate the use of a 
metabolite-corrected flux constant as a measure of [18F]FLT uptake (Mankoff et al., 1997, Muzi et al., 
2005a, Muzi et al., 2005b, Shields et al., 2005, Muzi et al., 2006).  
 
The parameters of interest for this model are those directly linked to DNA synthesis– specifically, k3 and 
Kflux, as they represent the phosphorylation of [18F]FLT to reflect cellular proliferation. We will correlate 
these rates with proliferative (MIB-1) indices on histological tissue and S phase fraction determined by flow 
cytometry when these are available. 
 
Procedure for [18F]FLT plasma metabolite analyses: Dynamic arterial or heated hand blood sampling will be 
performed (Phelps et al., 1979). It is anticipated that the majority of patients will be assessed with the heated 
hand methodology thus eliminating the need for radial artery catheterization. Approximately 23 (1-2 mL) 
samples will be drawn over a period of 70 minutes with approximately 12 samples drawn in the first 2-3 
minutes post injection and additional samples obtained at 4, 5, 7, 10, 15, 20, 30, 45, 60, and 70 minutes after 
injection. This sampling routine allows for determination of the whole blood and serum time activity curves. 
The whole blood samples will be counted and then centrifuged to separate plasma from cells. Aliquots of 
plasma (0.5 mL) will be counted for radioactivity. Another 0.5 mL of each plasma sample will be diluted 
with 3.5 mL of water and the resulting diluted plasma sample passed sequentially through a QMA cartridge 
followed by a C18 SepPak brand solid phase extraction cartridge. Then 4 mL of water will be passed 
sequentially through the cartridges to rinse them. The 8 mL of combined eluents will be counted to measure 
any [18F] breakthrough. The QMA and C18 SepPaks will then be separated. [18F]FLT glucuronide conjugate 
will be eluted from the QMA cartridge by passing 4 mL of 0.25M aqueous sodium citrate through the 
cartridge. [18F]FLT will be eluted by passing 4 mL of ethanol through the C18 SepPak. All of the eluates 
will be counted for radioactivity. It should be noted that in certain individuals it is not possible to obtain 
adequate venous access to perform the dynamic blood sampling. In the event that adequate venous access is 
not possible, this analysis will not be required. 
 
k4
FLT
Plasma
FLT
Cellular
Exchangeable
FLT
K1
FLT
k2
FLT
Cellular
~Trapped
FLT Nucleotides
k3
FLT


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6.2. [18F]Fluciclovine PET Imaging 
6.2.1. [18F]Fluciclovine Qualitative Visual Assessment 
The uptake of [18F]Fluciclovine is mediated by the sodium-independent “L” large-neutral amino acid 
transport (LAT) system. Since amino acid uptake is enhanced in malignancy, it accumulates faster and to a 
higher degree in cancer cells than normal cells, making it a promising biomarker for cancer diagnosis by 
PET imaging. Only a small fraction is excreted through the urinary pathway in the first hours after injection 
and it is not taken up to a high degree by normal brain tissue; therefore, unlike [18F] fluorodeoxyglucose 
FDG, PET imaging with [18F]Fluciclovine may be useful for the detection and diagnosis of prostate cancer 
and malignant gliomas. The ability of [18F]Fluciclovine to detect malignant lesions in humans has been 
assessed in preliminary studies by the Emory group and others. [18F]Fluciclovine is reported to have utility 
in the evaluation of primary and metastatic cancers in the brain, due to its low brain background uptake and 
high uptake by brain tumors (Shoup et al., 1999, Akhurst et al., 2006, Meirelles et al., 2006). 
 
The following scale will be used to qualitatively assess tracer uptake in areas of abnormality, including 
tumor mass and necrotic regions, as related to normal brain regions: 1= definite tumor, 2 = probably tumor, 
3 equivocal for tumor, 4 = probably not tumor, 5 = definitely not tumor. In the second group of patients 
where recurrence/progression (pseudoprogression) visual assessment on the [18F]Fluciclovine-PET study 
will be interpreted as to confidence of tumor recurrence: 5 = definitely recurrence/progression, 4 = probably 
recurrence/progression, 3 = unable to differentiate recurrence/progression, 2 = probably treatment 
effect/necrosis, 1 = definitely treatment effect/necrosis. 
 
6.2.2. [18F]Fluciclovine Semi-Quantitative Assessment 
Semi-quantitative assessment of [18F]Fluciclovine uptake will be performed by calculating SUVmax and 
SUVmean in the same manner as for [18F]FLT described earlier. SUVs will be calculated for summed static 
images from 70 minutes post injection. The change in SUVs for pre-treatment (baseline) and post-treatment 
images will be computed and correlated with available histologic and clinical data. 
 
6.2.3. [18F]Fluciclovine Fully-Quantitative Assessment 
Fully-quantitative compartment modeling analysis of the dynamic [18F]Fluciclovine-PET images will be 
performed according to the two-compartment model shown below, which has been previously described 
(Sasajima et al., 2013, Sörensen et al., 2013). As with the other tracers, parameters of interest include 
individual rate parameters (K1, k2-k4) and the net uptake parameters. The primary imaging endpoints for 
[18F]Fluciclovine are not yet fully understood, and will in part be elucidated by this study. Of interest, the 
second-order retention (k3) and reversible (k4) parameters for this tracer have high potential for 
differentiating inflammatory processes occurring after radiotherapy from residual tumor activity. 
 
Compartment modeling techniques will be applied to the dynamic PET data to obtain estimates of kinetic 
rate parameters (K1, k2-k4) and FDG net uptake (Knet). Individual rate parameters will be computed using 
full compartment-modeling techniques according to the two-tissue compartment model shown below. 
Commercially available PET analysis software (e.g. PMOD Technologies LTD., Zurich, Switzerland) will 
be used. The net uptake parameter will be computed from both full compartment modeling and Patlak 
graphical analysis (Patlak et al., 1983, Patlak and Blasberg, 1985), both of which are standard and accepted 
methods. 
 


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Procedure for [18F]Fluciclovine plasma analyses: Dynamic arterial or heated hand blood sampling will be 
performed (Phelps et al., 1979). It is anticipated that the majority of patients will be assessed with the heated 
hand methodology thus eliminating the need for radial artery catheterization. Approximately 23 (1-2 mL) 
samples will be drawn over a period of 70 minutes with approximately 12 samples drawn in the first 2-3 
minutes post injection and additional samples obtained at 4, 5, 7, 10, 15, 20, 30, 45, 60, and 70 minutes after 
injection. This sampling routine allows for determination of the whole blood and serum time activity curves. 
The whole blood samples will be counted and then centrifuged to separate plasma from cells. Aliquots of 
plasma (0.5 mL) will be counted for radioactivity. It should be noted that in certain individuals it is not 
possible to obtain adequate venous access to perform the dynamic blood sampling.  In the event that 
adequate venous access is not possible, this analysis will not be required.  
 
6.3. Multi-Tracer Assessments in Conjunction 
The exploratory aims of this study will assess the value of multi-tracer imaging endpoints, used in 
conjunction, over individual PET tracers for tumor characterization, predicting tumor aggressiveness, 
determination of functional changes that occur in response to therapy, and differentiating between 
recurrent/progressive tumor and treatment effect/necrosis (pseudoprogression). All three phases of image 
assessment will be studied: qualitative visual assessment (e.g. image scoring), semi-quantitative measures of 
tracer uptake (SUVs), and fully-quantitative measures (K1-k4; Knet). In each case, combinations of imaging 
endpoints will be considered in conjunction, such as SUV ratios of tracer pairs, and compared with 
histologic measurements and clinical outcomes.  
 
7. DATA ANALYSIS AND STATISTICS 
7.1. Validation of Multi-Tracer PET Imaging Technology 
This study will explore several hypotheses and provide preliminary exploratory data in the value of the two 
PET tracers [18F]FLT and [18F]Fluciclovine alone and in conjunction to assess response and differentiate 
tumor recurrence/progression from treatment effect/necrosis (pseudoprogression). The broader question of 
validating precisely what physiological quantity or process that each tracer/imaging endpoint measures and 
how these correlate with clinical endpoints and histologic data, when available, is the purpose of the study. 
 
7.2 Data Analysis 
Qualitative and Visual Assessment. The static images for each tracer will be visually assessed for any 
artifacts. The images will then be scored as described earlier for each tracer based on the level of uptake and 
confidence of tumor presence. Bland-Altman plots will be prepared. 
 
Semi- and Fully-Quantitative Assessments.  
 
Primary Endpoint 
 
The primary endpoint of the study is that a 25% reduction in SUVmax or SUVmean for each of the PET 
imaging tracers will be predictive of increased overall survival. These endpoints will be analyzed by 


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Kaplan-Meier analysis and logrank tests. Cox proportional hazards regression will also be used to estimate 
hazard ratios. Each analysis will be performed at the two sided 0.0125 significance level to adjust for four 
comparisons.  
 
Secondary Exploratory Objectives 
 
No multiple comparison adjustment will be performed for analysis of secondary endpoints. 
 
• Assess the association between overall survival and change in each of the following quantitative 
markers for [18F]Fluciclovine and [18F]FLT:  K1-k4, Knet and Ktrans, from baseline to post therapy. 
This association will be addressed by Kaplan-Meier curves and associated logrank tests. Cox 
proportion hazards regression will be used to estimate hazard ratios.  
• Assess the association between progression free survival and change in each of the following 
quantitative markers for [18F]Fluciclovine and [18F]FLT:  K1-k4, Knet and Ktrans, from baseline to 
post therapy. This association will be addressed by Kaplan-Meier curves and associated logrank 
tests. Cox proportion hazards regression will be used to estimate hazard ratios. 
• Assess the ability of [18F]FLT and/or [18F]Fluciclovine alone or together to predict true progression 
from pseudoprogression in patients with this clinical dilemma. True progression will be separated 
from pseudoprogression by subsequent MRI imaging. Receiver operating characteristic curve (ROC) 
analysis with true progression as outcome and each imaging marker or best combination as predictor 
will be used for this assessment. Bootstrap methods will be used to assess the standard error.  
• A 25% reduction in SUVmax or SUVmean for each of the PET imaging tracers will be more predictive 
of longer overall survival, than the current MRI based response criteria. This endpoint will be 
addressed by informal comparison of Kaplan-Meier curves, log rank p-values, and hazard ratios 
from separate analyses of overall survival for each imaging parameter.  
• A 25% reduction in SUVmax or SUVmean for each of the PET imaging tracers will be more predictive 
of time to progression than the current MRI based response criteria. This endpoint will be addressed 
by informal comparison of Kaplan-Meier curves, log rank p-values, and hazard ratios from separate 
analyses of time to progression for each imaging parameter.  
 
7.3. Justification of Sample Size 
(Sample size calculations were prepared by Kenneth Boucher, Ph.D. of the HCI Biostatistics Resource) 
 
Our justification of sample size is based on the data from Chen et al and Schwarzenberg et al. (Chen et al., 
2007, Schwarzenberg et al., 2012) in which a more than 25% reduction in tumor [18F]FLT uptake as 
measured by standardized uptake value was defined as a metabolic response. In this study there were nine 
responders (47%) and 10 non-responders (53%). Responders survived three times as long as non-responders 
(10.8 v 3.4 months; P = .003), and tended to have a prolonged progression-free survival (P =.061). Both 
early and later [18F]FLT -PET responses were more significant predictors of overall survival (1 to 2 weeks, 
P = .006; 6 weeks, P =.002), compared with the MRI responses (P =.060 for both 6-week and best 
responses). A potential limitation of this study was the use of standardized uptake value rather than full 
kinetic analysis in brain tumors (Peck et al., 2015). We will use full kinetic modelling and hopefully provide 
even more robust response data for [18F]FLT. Since there are no human studies utilizing [18F]Fluciclovine to 
measure response we have extrapolated from the available human xenograft rat glioma study of Ono et al. 
(2015b) utilizing anti-14C-FACBC with U87 and U87R (TMZ-resistant subculture) cells which were 
inoculated into the right and left basal ganglia. In autoradiographic experiments the DAR of anti-14C-
FACBC was significantly higher in tumors than in non-involved cortices. 14C-FACBC accumulation was 
lower after single-agent treatment and lower still after combination therapy. Treatment with temezolomide 


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alone significantly decreased the T/NT for the DAR of U87 tumors, whereas the T/NT ratios for those of 
U87R tumors remained unchanged. Temozolomide and interferon-β combination treatment significantly 
decreased anti-14C-FACBC uptake in both U87R and U87 tumors. Addition of bevacizumab to the 
temozolomide and interferon-β combination further decreased anti-14C-FACBC uptake in both tumor types.  
 
With an initial sample size of 30, we estimate that approximately 75% of the subjects (N = 22) will have 
both a [18F]Fluciclovine-PET and [18F]FLT-PET after completion of initial chemoradiotherapy. Our 
justification of sample size is based on data on the prediction of OS using PET reported in Chen et al. (Chen 
et al., 2007). In this study more than 25% reduction in tumor [18F]FLT uptake as measured by standardized 
uptake value was defined as a metabolic response. In this study there were nine responders (47%) and 10 
non-responders (53%). Responders survived three times as long as non-responders (10.8 v 3.4 months; P = 
.003), and tended to have a prolonged progression-free survival (P =.061). Both early and later [18F]FLT -
PET responses were more significant predictors of overall survival (1 to 2 weeks, P = .006; 6 weeks, P 
=.002), compared with the MRI responses (P =.060 for both 6-week and best responses). In univariate and 
multivariate Cox regression analysis, lack of FLT reduction at 6 weeks was the best predictor of reduced 
OS, with adjusted hazard ratio = 4.955 (p =0.02).  Assuming 50% of subjects are metabolic responders, 
median survival of 3.4 months in non-responders and two years of follow up, a sample size of N = 22 
subjects with follow up PET scans will provide 85% power to detect a hazard ratio of 4.9 using a two-sided 
logrank test at the 0.0125 significance level.  
 
8. REGULATORY AND REPORTING REQUIREMENTS 
The AE reporting time frame for this study with two investigational radiopharmaceuticals is 24 hours post 
injection of each tracer. This is defined in FDA regulation (see section 8.4.1). Per the approval of IND 76, 
843 for [18F]FLT on 2/1/07 the following reporting of unexpected fatal or life threatening events, serious 
adverse events, and serious and unexpected adverse events will occur: (1) Reporting any unexpected fatal or 
life threatening adverse experience associated with the use of [18F]FLT by telephone or fax no later than 7 
calendar days after initial receipt of the information. (2) Reporting any adverse experience associated with 
the use of [18F]FLT, that is both serious (SAE) and unexpected in writing no later than 15 calendar days 
after initial receipt of the information. (3) Submitting annual reports. The reportable events will also be 
submitted to the IRB using the University of Utah ERICA online system:  
https://erica.research.utah.edu/erica/Rooms/DisplayPages/LayoutInitial?Container=com.webridge.entity.Ent
ity%5BOID%5B5FD2DA60262617429607E459C0E09D92%5D%5D 
 
The same reporting process and requirements will be used for [18F]Fluciclovine.  
 
The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse 
Events (CTCAE) version 5.0 with subsequent modifications will be utilized for adverse event reporting 
(http://ctep.cancer.gov/reporting/index.html). 
 
All appropriate treatment areas will have access to a copy of the CTCAE version 5.0 with modifications. A 
list of adverse events that have occurred or might occur (Reported Adverse Events and Potential Risks) can 
be found in Section 6 (Methods for Evaluation of Imaging Studies). 
 
8.1. Informed Consent 
Informed consent will be obtained from all research participants prior to performing any study procedures 
using the most recent IRB approved version. 
 


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8.2 Institutional Review 
Study will be approved by the Institutional Review Board of University of Utah. 
 
8.3 Data and Safety Monitoring Plan 
A Data and Safety Monitoring Committee (DSMC) is established at Huntsman Cancer Institute (HCI) and 
approved by the NCI to assure the well-being of patients enrolled on Investigator Initiated Trials that do not 
have an outside monitoring review.  Roles and responsibilities of the DSMC are set forth in the NCI 
approved plan.  The activities of this committee include a quarterly review of adverse events including 
SAEs, important medical events, significant revisions or amendments to the protocol, and approval of 
cohort/dose escalations.  If the DSMC and/or the PI have concerns about unexpected safety issues, the study 
will be stopped and will not be resumed until the issues are resolved.  The DSMC also reviews and approves 
audit reports generated by the Research Compliance Office. 
 
8.3.1 Data Reporting 
This study will be monitored by the Principal Investigator using the ERICA system. In addition, the study 
will be monitored by the HCI Data and Safety Monitoring Committee. Cumulative data will be submitted 
electronically to ERICA, the HCI Data and Safety Monitoring Committee, and the IRB as required. 
 
8.4 Adverse Events / Serious Adverse Events 
This study will utilize the CTCAE (NCI Common Terminology Criteria for Adverse Events) version 5.0 for 
AE and SAE reporting. An electronic copy of the CTCAE version 5.0 can be downloaded from: 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_50 
 
8.4.1 Adverse Events (AE) 
An adverse event is the appearance or worsening of any undesirable sign, symptom, or medical condition 
occurring after receiving the radioactive tracer(s).  For the purposes of this study, the terms toxicity and 
adverse event are used interchangeably.  Medical conditions/diseases present before starting the study are 
only considered adverse events if they worsen after being injected with the radioactive tracer(s).  Abnormal 
laboratory values or test results constitute adverse events only if they induce clinical signs or symptoms, are 
considered clinically significant, or require therapy. 
 
Adverse event reporting for studies assessing diagnostic radiopharmaceuticals have different criteria than 
for therapeutic drugs. The NCI CTEP requires that AE’s be reported for up to ten half-lives after the 
administration of the tracer rounded UP to the nearest whole day.  In this instance, the half-life of the 18F 
radioisotope is 110 minutes, with ten half-lives equivalent to 18.33 hours.  This study will therefore collect 
adverse events from the time the radiopharmaceutical  is administered and 24 hours afterword, even if the 
event is not considered to be related to the tracer. 
 
The collection of any adverse events will begin when a patient receives their first dose of [18F]FLT or 
[18F]Fluciclovine and will end 24 hours after receiving that dose.  As there is the potential for each patient to 
receive up to six scanning visits, each constituting a radioactive tracer to be injected into the patient, the 
adverse events will be monitored for 24 hours after each injection and subsequent scan.  Those adverse 
events that are not associated with [18F]FLT and [18F]Fluciclovine that do not require expedited reporting 
will be reported in the routine manner to the IRB. 
 


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The occurrence of adverse events should be sought by non-directive questioning of the patient at each visit 
or phone contact during the study.  Adverse events also may be detected when they are volunteered by the 
patient during or between visits within the AE reporting period or through physical examination, laboratory 
tests, or other assessments.  As far as possible, each adverse event should be evaluated to determine: 
 
1. The severity grade based on CTCAE v.5 (Grade 1-5). 
2. Its relationship to the study radioactive tracer(s) (definite, probably, possible, unlikely, not related) 
3. Its duration (start and end dates or if continuing at final exam). 
4. Action taken (no action taken; study tracer dosage adjusted/temporarily interrupted; study tracer 
permanently discontinued due to this adverse event; concomitant medication taken; non-drug 
therapy given; hospitalization/prolonged hospitalization). 
5. Whether it constitutes an SAE. 
 
All adverse events will be treated appropriately. Once an adverse event is detected, it should be followed 
until its resolution, and assessment should be made at each visit (or more frequently, if necessary) of any 
changes in severity, the suspected relationship to the study tracer, the interventions required to treat it, and 
the outcome. 
 
Information about common side effects already known about the tracer is described in the Pharmacology 
and Safety of [18F]FLT and [18F]Fluciclovine and Radiation Dosimetry of [18F]FLT and [18F]Fluciclovine 
(Section 3).  This information will be included in the patient informed consent and will be discussed with 
the patient during the study as needed. 
 
All adverse events will be immediately recorded in the patient research chart. 
 
8.4.2 Serious Adverse Event (SAE) 
Information about all serious adverse events within the reporting period will be collected and recorded.  A 
serious adverse event is an undesirable sign, symptom, or medical condition which: 
• Is fatal or life-threatening. 
• Results in persistent or significant disability/incapacity. 
• Is medically significant, i.e., defined as an event that jeopardizes the patient or may require medical 
or surgical intervention to prevent one of the outcomes listed above. 
• Causes congenital anomaly or birth defect. 
• Requires inpatient hospitalization or prolongation of existing hospitalization, unless hospitalization 
is for: 
o Routine treatment or monitoring of the studies indication, not associated with any 
deterioration in condition (procedures such as central line placements, paracentesis, pain 
control). 
o Elective or pre-planned treatment for a pre-existing condition that is unrelated to the 
indication under study and has not worsened since the start of study drug. 
o Treatment on an emergency outpatient basis for an event not fulfilling any of the definitions 
of a SAE given above and not resulting in hospital admission. 
o Social reasons and respite care in the absence of any deterioration in the patient’s general 
condition. 
 
 
Note: All deaths on study during the AE reporting window will be reported using expedited reporting 
regardless of causality.  Attribution to treatment or other cause will be provided. 


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Fatal and life-threatening events will be reported to the IRB within 24 hours of notification of the event, 
indicating that a full report will follow. Any unexpected fatal or life threatening adverse event associated 
with the use of  [18F]FLT of [18F]Fluciclovine will be reported to the FDA by telephone or fax no later than 
7 calendar days after initial receipt of the information. All reportable adverse events will be submitted to the 
FDA & IRB within the required timeframe by as mandated by the FDA and IRB.  
 
Toxicities which fall within the definitions listed above must be reported as an SAE regardless if they are 
felt to be related to the radioactive tracer(s) or not.  Toxicities unrelated to the radioactive tracer(s) that do 
NOT fall within the definitions above, must simply be documented as AEs in the patient research chart. 
 
8.5 SAE Reporting Requirements 
SAEs must be reported to the DSMC, the FDA, the IRB and Blue Earth Diagnostics, according to the 
requirements described below: 
 
A MedWatch 3500 A form must be completed and submitted to compliance@hci.utah.edu as soon as 
possible, but no later than 10 days of first knowledge or notification of event (5 days for fatal or life 
threatening event). 
 
*MedWatch 3500A form can be found at: 
http://www.fda.gov/dowloads/Safety/MedWatch/HowToReport/DowloadForms/ucm082728.pdf 
 
DSMC Notifications: 
• An HCI Research Compliance Officer (RCO) will process and submit the MedWatch form to the 
proper DSMC member as necessary for each individual study. 
• The RCO will summarize and present all reported SAEs according to the Data and Safety 
Monitoring Plan at the quarterly DSMC meeting. 
 
FDA Notifications: 
• Adverse events occurring during the AE reporting period of a clinical study that meet the following 
criteria will be promptly reported to the FDA:  
o Serious 
o Unexpected 
o Definitely, Probably, or Possibly Related to the investigational drug 
o Fatal or life-threatening events that meet the criteria above will be reported within 7 calendar 
days after first knowledge of the event by the investigator; followed by as complete a report 
as possible within 8 additional calendar days. 
o All other events that meet the criteria above will be reported within 15 calendar days after 
first knowledge of the event by the investigator. 
o The RCO will review the MedWatch report for completeness, accuracy and applicability to 
the regulatory reporting requirements. 
o The RCO will ensure the complete, accurate and timely reporting of the event to the FDA. 
o The Regulatory Coordinator will submit the report as an amendment to the IND application. 
o All other adverse events and safety information not requiring expedited reporting that occur 
or are collected during the course of the study will be summarized and reported to the FDA 
through the IND Annual Report. 
 
IRB Notification: 


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• Events meeting the University of Utah IRB reporting requirements 
(http://www.research.utah.edu/irb/) will be submitted through the IRB’s electronic reporting system 
within 10 working days. 
 
Sponsor/Drug Manufacturer Notifications: 
• All SAEs within the 24 hour reporting period following [18F]Fluciclovine PET/CT must be reported 
within 24 hours of the Site Study Team becoming aware of the event.  Contact information for the 
study team is as follows: 
o Blue Earth Diagnostics 
SAE Email: Drugsafety@pharsafer.com 
Tel: 1-855 AXUMIN1 (1 855 298 6461)  
Fax: +44 (0) 1483 212178 
 
 
8.6 Expedited Adverse Event Reporting 
8.6.1 Expedited Reporting Guidelines – Phase 1/2 studies with Investigational Agents 
The guidelines given in Table 5 below will be followed. Hospitalization will be defined as any medical 
event equivalent to CTCAE (Version 5) grade 3, 4, or 5 that precipitated hospitalization (or prolongation of 
existing hospitalization).  
 
 
 
Table 5. 
 
UNEXPECTED EVENT 
 
EXPECTED EVENT 
GRADES 2 – 3 
Attribution of Possible, 
Probable or Definite 
GRADES 4 and 5 
Regardless of 
Attribution 
GRADES 1 - 3 
GRADES 4 and 5 
Regardless of 
Attribution 
 
Expedited report within 7 
working days. 
 
(Grade 1 Adverse Event 
Expedited Reporting NOT 
required.) 
 
Report by phone to 
IRB within 24 hrs.  
Expedited report to 
follow within 10 
working days. 
 
 
 
 
Adverse Event 
Expedited 
Reporting NOT 
required. 
 
Expedited report, 
including Grade 5  
 
 
. 
 
For Hospitalization only – Any medical event equivalent to CTCAE grade 3, 4, or 5 that precipitated 
hospitalization (or prolongation of existing hospitalization) must be reported regardless of designation as 
expected or unexpected and attribution. 
 
8.7 Protocol Amendments 
Any amendments or administrative changes in the research protocol during the period, for which the IRB 
approval has already been given, will not be initiated without submission of an amendment for IRB review 
and approval. 


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These requirements for approval will in no way prevent any immediate action from being taken by the 
investigator in the interests of preserving the safety of all patients included in the trial. 
 
Any amendments to the protocol that significantly affect the safety of subjects, scope of the investigation, or 
the scientific quality of study are required to submit the amendment for FDA review. 
 
8.8 Protocol Deviations 
A protocol deviation (or violation) is any departure from the defined procedures and treatment plans as 
outlined in the protocol version submitted and previously approved by the IRB.  Protocol deviations have 
the potential to place participants at risk and can also undermine the scientific integrity of the study thus 
jeopardizing the justification for the research.  Protocol deviations are unplanned and unintentional events. 
The inability to obtain the dynamic blood sampling for [18F]FLT and/or [18F]Fluciclovine will not be 
considered a protocol deviation.   
 
Because some protocol deviations pose no conceivable threat to participant safety or scientific integrity, 
reporting is left to the discretion of the PI within the context of the guidelines below.  The IRB requires the 
prompt reporting of protocol deviations which are: 
• Exceptions to eligibility criteria. 
• Intended to eliminate apparent immediate hazard to a research participant or 
• Harmful (caused harm to participants or others, or place them at increased risk of harm – including 
physical, psychological, economic, or social harm), or 
• Possible serious or continued noncompliance. 
 
8.9 FDA Annual Reporting 
An annual progress report will be submitted to the FDA within 60 days of the anniversary of the date that 
the IND went into effect. (21 CFR 312.22). 
 
8.10 Clinical Trials Data Bank 
The study will be registered on http://clinicaltrials.gov and the NCI CTRG (Clinical Trials Reporting 
Program) by the Clinical Trials Office. 
 
8.11 Record Keeping 
Per 21 CFR 312.57, Investigator records shall be maintained for a period of 2 years following the date a 
marketing application is approved; or, if no application is filed or the application is not approved, until 2 
years after the investigation is discontinued and the FDA is notified. 
 
9. PET TRACER PRODUCTION 
9.1. [18F]FLT 
9.1.1. Study Agent 
This protocol utilizes 3’-deoxy-3’-[18F]fluorothymidine, [18F]FLT, as an imaging agent for measuring 
cellular proliferation in primary high-grade brain tumors. The agent is administered by intravenous injection 


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of <10 mL solution of 0.1 mCi/kg, up to approximately 10 mCi maximum, of [18F]FLT. The non-
radioactive [F-19]FLT in the injectate will be < 6.1 µg (25 nmol). The [18F]FLT is formulated in a solution 
of 92% 0.01 M phosphate buffered saline (PBS), USP with 8% ethanol, USP (v:v). The injectate may be 
diluted with isotonic saline for injection, USP. 
 
9.1.2. Reported Adverse Events and Potential Risks 
No adverse events have been reported for [18F]FLT at the dosage described for this study. As described in 
part 3.2 of this document, non-radioactive FLT has been investigated as an anti-AIDS drug and some 
adverse effects, namely a reversible peripheral neuropathy, were observed in subjects exposed to 50 ng-
h/mL plasma over a course of 16 weeks. The dose anticipated for this study will be nominally 1 ng-h/mL for 
a single injection. The radiation exposure associated with this study is described in part 3.5 of this document 
and is comparable to the dose for other widely used clinical nuclear medicine procedures.  
 
9.1.3. Production of the Radiopharmaceutical 
The 3’-deoxy-3’-[18F]fluorothymidine [18F]FLT used in this study will be prepared locally by the PET 
Radiochemistry Group at the University of Utah. The precursors for the radiosynthesis include F-18 
prepared at the Huntsman Cancer Institute cyclotron from proton irradiation of [O-18]water and an organic 
precursor, 5’-O-Benzoyl-2, 3’-anhydrothymidine, supplied by ABX. The only other reagents used in the 
synthesis are potassium carbonate, water, dimethyl sulfoxide, acetonitrile, kryptofix [2.2.2] (a phase transfer 
agent), USP absolute ethanol, USP PBS, USP sterile water for injection, and USP saline for injection. The 
radiopharmaceutical product is a clear and colorless liquid that is stored at room temperature in a sterile 
serum vial. The [18F]FLT has an expiration time of eight hours after sterile filtration or when the Ci/mmol or 
mCi/mL fall below the specified limits. 
 
9.1.4. Agent Accountability 
[18F]FLT is a radiopharmaceutical produced in the cyclotron facility at the Huntsman Cancer Institute. The 
agent is investigational and approved by the FDA under IND # 76,843. The organic precursor for [18F]FLT 
is provided by ABX in Germany. The precursor is stored in a controlled temperature refrigerator in a locked 
and secure room and is inventoried with a chain of custody maintained from the time of receipt. Each 
radiosynthesis is done by University of Utah cyclotron and radiochemistry staff and the product [18F]FLT in 
a dose calibrated syringe will be released after passing all required quality control assays to the physician 
who will be responsible for administering the appropriate amount (Jeffrey Yap, PhD or his designee). The 
quality control tests that must be passed prior to release of the product [18F]FLT for injection include the 
radioactive purity, the radiochemical purity, sterilizing filter integrity, tests for kryptofix, DMSO, 
acetonitrile, pyrogens and particulates. The [18F]FLT dose is drawn up into a syringe, assayed for mCi at the 
time of injection, and administered to the research subject. 
 
9.2. [18F]Fluciclovine 
9.2.1. Study Agent 
[18F]Fluciclovine is a fluorine-18 labelled PET diagnostic agent supplied as a ready-to-inject solution in 
either vials or syringes. The maximum dose volume is 5 ml. The drug substance is [18F]Fluciclovine. 
Presence of the syn-isomer is minimized in the production process. The formulation of [18F]Fluciclovine 
drug product contains trisodium citrate buffer in water. The pH of the drug product is 3.5-6.0. The 
radiochemical purity (RCP) is greater than 95% throughout the shelf-life (up to 8 hours). The pH is kept low 
to avoid the risk of degradation of [18F]Fluciclovine. [18F]Fluciclovine injection is manufactured by 


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automated radiosynthesis followed by formulation with buffer and aseptic dispensing in a remotely 
controlled system. Fluorine-18 decays by positron emission (β+ decay, 96.7%) and orbital electron capture 
(3.3%) with a half-life of approximately 110 minutes (mins). The positron undergoes annihilation with an 
electron to produce two gamma photons each of energy 511 keV (193.4% emission).  
 
[18F]Fluciclovine injection is a sterile, aqueous solution of [18F]Fluciclovine and excipients for intravenous 
administration. The product is supplied with a radioactive content of 200 MBq/ml (5.4 mCi/ml) at the 
reference date and time in a syringe or a glass vial sealed with a synthetic rubber closure and aluminum 
overseal and then withdrawn into syringes at the clinical site. Each vial or syringe is transported in a lead or 
tungsten shield. The quality control (QC) analysis of a sample of the drug product may be performed in 
parallel with transportation of the drug product to the study site. The investigator (or nominated deputy) will 
receive release/reject information for the drug product. Only product for which confirmation of release has 
been received shall be used. Where the product is transported as a single patient dose, the dose will be 
measured in a dose calibrator before administration. Where the product is transported in its original 
container the volume of injection for each patient is calculated and withdrawn into a shielded syringe 
immediately before injection. The calculation is based on the radioactive content, the half-life of fluorine-18 
(109.8 mins), the reference date and time, the prescribed dose and the time of injection. Each patient dose 
will contain up to approximately 370 MBq (10.0 mCi) at the time of administration. The doses will contain 
no more than 170 μg/ml [18F]Fluciclovine and related substances. The maximum administered dose volume 
is 5 ml. 
 
9.2.2. Reported Adverse Events and Potential Risks 
No serious adverse reactions have been reported from any investigator sponsored studies or from the 
compassionate use program in Norway as described in part 3.1.4 of this document. The risks to subjects 
mainly relate to the intravenous injection and intravenous blood sampling procedures, and the radiation 
emitted by [18F]Fluciclovine. Intravenous injection and the use of an intravenous cannula are known to carry 
a small risk of infection and hematoma. The exposure to radiation will not exceed that which is considered 
acceptable in accordance with appropriate guidelines. 
 
9.2.3. Production of the Radiopharmaceutical 
The [18F]Fluciclovine used in this study will be prepared locally by the PET Radiochemistry Group at the 
University of Utah. The precursors for the radiosynthesis include F-18 prepared at the Huntsman Cancer 
Institute cyclotron from proton irradiation of [O-18]water and an organic precursor supplied in the cassette 
to be used on the GE FastLab synthesis module. [18F]Fluciclovine is manufactured by automated 
radiosynthesis on the GE FastLab followed by formulation with buffer and aseptic dispensing in a remotely 
controlled system. The formulation of [18F]Fluciclovine drug product contains trisodium citrate buffer in 
water. The pH of the drug product is 3.5-6.0. The radiochemical purity (RCP) is greater than 95% 
throughout the shelf-life (up to 8 hours). The pH is kept low to avoid the risk of degradation of 
[18F]Fluciclovine. The radiopharmaceutical product is a clear and colorless liquid that is stored at room 
temperature in a sterile serum vial. The [18F]Fluciclovine has an expiration time of eight hours after sterile 
filtration.   
 
9.2.4. Agent Accountability 
[18F]Fluciclovine is a radiopharmaceutical produced in the cyclotron facility at the Huntsman Cancer 
Institute. The agent is investigational and approved by the FDA under a pending IND.   
 


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The shelf-life of [18F]Fluciclovine is up to 10 hours from the end of production and the product must not be 
used beyond this limit. [18F]Fluciclovine should be stored at 15-25°C in a shielded container. All non-
radioactive containers (shielding, transport cans) must be returned to the manufacturing site. Containers that 
are radioactive or that contained radioactive products must be destroyed at either the study site or another 
designated facility, after the study and after overall drug accountability has been completed by the sponsor 
or its representative. Waste must be disposed of according to national regulations for radioactive material. 
Precautions for the safe handling of radioactive materials should be observed. 
 
Each radiosynthesis is done by University of Utah cyclotron and radiochemistry staff and the product 
[18F]Fluciclovine in a dose calibrated syringe will be released after passing all required quality control 
assays to the physician who will be responsible for administering the appropriate amount (Jeffrey Yap, PhD 
or his designee). The quality control tests that must be passed prior to release of the product 
[18F]Fluciclovine for injection include the radioactive purity, the radiochemical purity, sterilizing filter 
integrity, tests impurities, pyrogens and particulates. The [18F]Fluciclovine dose is drawn up into a syringe, 
assayed for mCi at the time of injection, and administered to the research subject. 
 


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11. APPENDICES 
 
 
APPENDIX A 
 
 
 
Eligibility Check List 
 
 
 
APPENDIX B 
 
 
 
Schedule of Events 
 
 
 
APPENDIX C 
 
 
 
[18F]FLT Infusion Flow Chart for Staff  
 
 
 
APPENDIX D 
 
 
 
[18F]Fluciclovine Infusion Flow Chart for Staff  
 
APPENDIX E 
 
 
 
[18F]FLT Patient Adverse Event Questionnaire 
 
 
 
APPENDIX F 
                  [18F]Fluciclovine Patient Adverse Event Questionnaire  
 
APPENDIX G 
                  NCI Common Toxicity Criteria  
 
 
 
 


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APPENDIX A: Eligibility Check List 
Patient Name: 
_________________________________     MRN: _________________________ 
 
Person Confirming Eligibility: _______________________________     Date:_________________ 
 
_____ (Yes/No)    Patient is > 18 years old. 
 
_____ (Yes/No)    Group A. Either patients where there is compelling evidence, based on the MRI and/or CT 
imaging, that a high-grade primary brain tumor is present. Pathologic confirmation will occur 
with biopsy or  surgery.  OR patients whose tumor is felt to be inoperable and a biopsy is  
performed but no surgery.  OR patients with a newly diagnosed primary malignant brain tumor  
WHO Grade III or IV) who will be receiving chemoradiation and who either did not undergo  
surgical resection or underwent incomplete resection with residual tumor > 1.0 cm in greatest 
diameter by contrast MRI postoperatively.  
 
_____ (Yes/No) 
Group B. Patients with pathologically proven malignant brain tumor (WHO Grade III or IV glial 
based tumors) who have undergone chemoradiation and have MRI-documented (contrast 
enhancing mass  ≥ 1.0 cm in greatest diameter) possible recurrence/progression versus treatment 
effect (pseudoprogression) within 6 months from the  time of completion of chemoradiation.    
 
_____ (Yes/No)    Patient is willing to have their clinical records reviewed for at least 24 months after enrollment. 
 
_____ (Yes/No)    Patient, or their legal guardians, have signed a written informed consent and HIPAA 
authorization in accordance with institutional guidelines. 
 
_____ (Yes/No)    If patient is female, she must be postmenopausal for a minimum of one year, surgically sterile, or  
                                 is willing to undergo a serum pregnancy test prior to each imaging session. 
 
_____ (Yes/No)    Patient is not lactating. 
 
_____ (Yes/No)    Pre- [18F]Fluciclovine and [18F]FLT PET laboratory tests not greater than 4.0× the upper or lower 
values in the normal range: liver enzymes (AST, ALT, ALK), bilirubin (total), serum electrolytes, 
CBC with platelets, prothrombin time, partial thromboplastin time, BUN, and creatinine.    For 
those patients receiving coumadin or another anticoagulant the upper limit for prothrombin time 
or partial thromboplastin time must not exceed 6 times the upper limit of the normal range. 
 
  _____ (Yes/No)    Patient does not have allergic or hypersensitivity reactions to previously administered 
radiopharmaceuticals. 
 
_____ (Yes/No)    Patient does not require monitored anesthesia for PET/CT imaging. 
 
_____ (Yes/No)    Patient is not too claustrophobic to undergo PET/CT imaging  
 
_____ (Yes/No)    Patient is NOT known to be HIV positive. 
 
Completed by______________________________________________    Date: _______________________ 
 
 
Physician Verification  ______________________________________    Date: _______________________ 
 
 
3rd Party Verification: ________________________________________    Date: _______________________ 
 


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APPENDIX B: Schedule of Events 
 
Screen 
Imaging Session 1 
Imaging Session 2 
Imaging Session 3 
 
 
[18F]FLT [18F]Fluciclovine 
[18F]FLT 
[18F]Fluciclovine 
[18F]FLT [18F]Fluciclovine 
Assessment/ 
Procedures 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
Inclusion/ 
Exclusion Criteria 
X 
 
 
 
 
 
 
Laboratory 
Monitoring 
X1, 2 
 
 
X1,3 
 
X1,4, 7 
 
Infusion of 
[18F]FLT 
 
X 
 
X 
 
X 
 
[18F]FLT PET/CT 
Imaging 
 
X 
 
X 
 
X 
 
Infusion of 
[18F]Fluciclovine 
 
 
X 
 
X 
 
X 
[18F]Fluciclovine 
PET/CT Imaging 
 
 
X 
 
X 
 
X 
Vital Signs5 
 
X5 
X5 
X5 
X5 
X5 
X5 
AE Assessment 
 
X 
X 
X 
X 
X 
X 
Concomitant 
Medication 
 
X6 
X6 
X6 
 
1. 
Laboratory tests will include: (AST, ALT), bilirubin (total), serum electrolytes, CBC with platelets, prothrombin time, partial 
thromboplastin time, BUN, and creatinine. Female patients who are not postmenopausal or surgically sterile will undergo a serum 
pregnancy test prior to each imaging session ([18F]FLT and [18F]Fluciclovine PET scans). The values for the imaging sessions 
must be within 4.0 X the upper or lower normal limits (unless clinically not relevant). For those patients receiving coumadin or 
another anticoagulant the upper limit for prothrombin time or partial thromboplastin time must not exceed 6 times the upper limit 
of the normal range. 
2. 
Laboratory tests from the screening visit will be used if the first [18F]FLT imaging session are performed within 28 days of the 
screening laboratory tests.  Labs will be redrawn prior to [18F]FLT imaging session 1 if they are outside of the 28 day window 
prior to the scan. 
3. 
Within 96 hours prior to the second [18F]FLT imaging session the laboratory assessments will be performed. The values for this 
assessment must be within 4.0 X the upper or lower normal limits (unless clinically not relevant).  For those patients receiving 
coumadin or another anticoagulant the upper limit for prothrombin time or partial thromboplastin time must not exceed 6 times 
the upper limit of the normal range.   
4. 
Within 96 hours prior to the possible third [18F]FLT imaging session the laboratory assessments will be performed. The values for 
this assessment must be within 4.0 X the upper or lower normal limits (unless clinically not relevant). For those patients receiving 
coumadin or another anticoagulant the upper limit for prothrombin time or partial thromboplastin time must not exceed 6 times 
the upper limit of the normal range.   
 
5. 
Includes: Heart Rate, Blood Pressure, and Temperature.  Vitals signs will be collected prior to infusion and again at the 
completion of the imaging study. Height and weight will be recorded at the beginning of an imaging session.  
6. 
To be collected prior to the first imaging time point of each session, regardless of whether [18F]FLT or [18F]Fluciclovine is the 
first imaging to take place. 
7. 
For patients enrolling under Group B, laboratory tests from the screening visit will be used if they are performed within 28 days 
of the imaging session. 
 
Multi-Tracer PET Imaging Visit:  
 
Imaging of the two PET tracers will be performed on separate days. The two days of imaging will be 
scheduled according to scanner and cyclotron availability, and all days of imaging will be obtained within 
one to two weeks of each other. 


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APPENDIX C: [18F]FLT Infusion Flow Chart for Staff 
 
Subject Name and MRN: _____________________________________           Subject Study ID (if enrolled): ______________  
                    
 
 
Projected Study Start Date: ______________________         Referring MD: _________________________________________ 
 
                                                                                                
[18F]Fluciclovine and [18F]FLT PET/CT Assessment of Primary High-Grade Brain Tumors 
 
IMAGING VISIT 1 (Baseline):   
 
 
 
 
 
IMAGING VISIT 2 (Follow-up): 
 
 
IMAGING VISIT 3 (Pseudoprogression):   
 
 
 
 
 
 
 
 
Administered dose of [18F]FLT   ___________ mCi 
 
 
Specific Activity of [18F]FLT   _____________Ci/mmol 
 
                                                                                              
Initials of Person 
Making Assessment 
Time 
Study 
Temp 
BP 
HR 
Weight 
Height 
 
 
Baseline 
 
 
 
 
 
 
 
End of 
Study 
 
 
 
NA 
NA 
 
 
 
  Completed by: ______________________________________________                      Date: ___________________________  
 
 
 
 
 
PI Signature:  ________________________________________________                      Date:  __________________________ 
The infusion and imaging procedure will be terminated in any patient who exhibits anaphylaxis, significant hypotension 
(systolic blood pressure less than 80 mmHg, dyspnea, chest pain, grand mal seizure or an O2 saturation lower than 80%. 


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APPENDIX D: [18F]Fluciclovine Infusion Flow Chart for Staff 
 
Subject Name and MRN: _____________________________________           Subject Study ID (if enrolled): ______________  
                    
 
 
Projected Study Start Date: ______________________         Referring MD: _________________________________________ 
  
                                                                                               
[18F]Fluciclovine and [18F]FLT PET/CT Assessment of Primary High-Grade Brain Tumors 
 
IMAGING VISIT 1 (baseline):   
 
 
          IMAGING VISIT 2 (follow-up): 
 
IMAGING VISIT 3 (Pseudoprogression):   
 
 
 
 
 
 
 
 
 
Administered dose of  [18F]Fluciclovine   ___________ mCi 
 
 
Specific Activity of [18F]Fluciclovine   _____________Ci/mmol 
 
                                                                                              
Initials of Person 
Making Assessment 
Time 
Study 
Temp 
BP 
HR 
Weight 
Height 
 
 
Baseline 
 
 
 
 
 
 
 
End of 
Study 
 
 
 
NA 
NA 
 
 
  Completed by: ______________________________________________                      Date: ___________________________  
 
 
 
 
PI Signature:  ________________________________________________                      Date:  __________________________
The infusion and imaging procedure will be terminated in any patient who exhibits anaphylaxis, significant hypotension 
(systolic blood pressure less than 80 mmHg, dyspnea, chest pain, grand mal seizure or an O2 saturation lower than 80%. 


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APPENDIX E: [18F]FLT Patient Adverse Event Questionnaire  
Subject Name and MRN: _____________________________________           Subject Study ID (if enrolled): ______________  
                    
 
Projected Study Start Date: ______________________         Referring MD: _________________________________________ 
 
IMAGING VISIT 1 (baseline): 
 
IMAGING VISIT 2 (follow-up):  
IMAGING VISIT 3 (Pseudoprogression): 
 
POSSIBLE ADVERSE EVENT 
Y =Yes      Comment on possible AE 
N = No 
Body as a Whole: 
Pain (Abdominal) 
 
Pain (Chest/Breast) 
 
 
PAIN (OTHER SITE) 
 
Fever 
 
Injection site reaction 
 
 
Cardiovascular System: 
Vasodilation (flushing) 
 
 
Digestive System: 
Nausea 
 
Diarrhea 
 
Vomiting 
 
 
Respiratory System: 
Dyspnea (Shortness of breath) 
 
 
Skin and Appendages: 
Rash 
 
Pruritus (Itching) 
 
Urticaria (Hives) 
 
Sweating  
 
Cyanosis (discoloration of 
fingers/toes) 
 
 
Central Nervous System 
 
Visual disturbances 
 
Numbness of feet 
 
Numbness of fingers/hands 
 
Weakness of feet 
 
Weakness of fingers 
 
Burning sensation in feet 
 
Burning sensation of fingers 
 
 
Performed by: ___________________________________            Date: ________________ 
 
PI Signature: _______________________________________            Date:  __________________ 


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APPENDIX F: [18F]Fluciclovine Patient Adverse Event Questionnaire  
 
Subject Name and MRN: _____________________________________           Subject Study ID (if enrolled): ______________  
                    
 
Projected Study Start Date: ______________________         Referring MD: _________________________________________ 
 
 
IMAGING VISIT 1 (baseline): 
 
IMAGING VISIT 2 (follow-up):  
IMAGING VISIT 3 (Pseudoprogression): 
POSSIBLE ADVERSE EVENT 
Y =Yes      Comment on possible AE 
N = No 
Body as a Whole: 
Pain (Abdominal) 
 
Pain (Chest/Breast) 
 
 
PAIN (OTHER SITE) 
 
Fever 
 
Injection site reaction 
 
 
Cardiovascular System: 
Vasodilation (flushing) 
 
 
Digestive System: 
Nausea 
 
Diarrhea 
 
Vomiting 
 
 
Respiratory System: 
Dyspnea (Shortness of breath) 
 
 
Skin and Appendages: 
Rash 
 
Pruritus (Itching) 
 
Urticaria (Hives) 
 
Sweating  
 
Cyanosis (discoloration of 
fingers/toes) 
 
 
Central Nervous System 
 
Visual disturbances 
 
Numbness of feet 
 
Numbness of fingers/hands 
 
Weakness of feet 
 
Weakness of fingers 
 
Burning sensation in feet 
 
Burning sensation of fingers 
 
 
Performed by: ___________________________________            Date: ________________ 
 
PI Signature: _______________________________________            Date:  __________________ 


[18F]Fluciclovine and [18F]FLT Assessment of Primary High-Grade Brain Tumors 
Version 04/15/2024 
 
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APPENDIX G: NCI Common Toxicity Criteria 
 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/CTCAE_v5_
Quick_Reference_5x7.pdf