Clinical trial NCT03387514
Conditions: Renal Cell Carcinoma
Keywords: Clear Cell Kidney Cancer RCC
Sponsor: University of Wisconsin, Madison
Start date: 2018-12-08; Completion date: 2021-06-29
Enrollment: 5 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The primary objective of this research is to evaluate response to systemic therapy, including anti-angiogenesis therapy and/or immune-based therapies via 18F-DCFPyL prostate-specific membrane antigen (PSMA)-based positron emission tomography/computed tomography (PET/CT) in patients with metastatic renal cell carcinoma (RCC) and to compare qualitatively with conventional imaging response criteria - Response Evaluation Criteria In Solid Tumors (RECIST 1.1) and histopathological endpoints including isolation, enumeration and staining of Circulating Tumor Cells (CTC).

Detailed description:
Response of systemic therapy, including anti-angiogenesis therapy and/or immune-based therapies will be quantified using PSMA-based PET imaging using a novel agent,18F-DCFPyL, as a non-invasive imaging biomarker of tumor neovasculature to functionally monitor renal cell cancer neovasculature in patients undergoing systemic anti-angiogenesis therapy. PSMA PET will be compared with response to anti-angiogenesis therapy using conventional imaging computed tomography(CT)-based RECIST1.1 criteria as well as histopathological endpoints (tumor vascular density, immunohistochemical staining for PSMA and neovascularization (cluster of differentiation(CD)105, CD31). Whole body PSMA PET/CT scans will be obtained at baseline, following adjuvant anti- angiogenic therapy and when the patient becomes refractory to treatment.

The rationale and time points for obtaining PET scans is planned with respect to the typical natural history of metastatic RCC. This project will obtain information from tumors that are responding to anti-angiogenesis therapy and those resistant to treatment.

Arms: arm 1: 18F-DCFPyL whole body PET/CT scan at three time-points
Interventions: intervention 1: PSMA-based 18F-DCFPyL PET tracer for PET/CT exams
Intervention descriptions: intervention 1: 18F-DCFPyL whole body PET/CT scan administered at the following timepoints:

PET1 - Prior to scheduled nephrectomy

PET2 - to establish a new baseline PET before systemic therapy

* PET2A - Post-surgery and prior to start of standard of care systemic therapy
* PET2B - 12-16 weeks from start of first line systemic therapy (immune-based or anti-angiogenic)

PET3 - If first line systemic therapy did not include anti-angiogenesis therapy and new systemic therapy does include anti-angiogenesis therapy

* PET3A - Prior to start of additional anti-angiogenesis therapy
* PET3B - 12-16 weeks from the start of additional anti-angiogenesis therapy

PET4 - obtained at clinical progression or 2 years following initial systemic therapy

Protocol documents:
Prot_SAP_000.pdf: 
1 
 
Functional Microscale Organotypic Assays to Predict 
Patient Response to Anti-Angiogenesis Therapies 
 
UW IRB Tracking # 2017-1343 
UWCCC Tracking # UW17104 
NCT03387514 
Protocol Version Date: 13 October 2020 
 
 
Principal Investigator: 
 
Steve Cho, MD – Associate Professor (CHS) 
University of Wisconsin, School of Medicine & Public Health 
Department of Radiology 
 
Co-Principal Investigators: 
 
David Beebe, PhD – Professor 
University of Wisconsin, School of Medicine & Public Health 
Department of Radiology 
 
E. Jason Abel, MD, FACS – Associate Professor 
University of Wisconsin, School of Medicine & Public Health 
Department of Urology 
 
Christos Kyriakopoulos, MD – Assistant Professor 
University of Wisconsin, School of Medicine & Public Health 
Department of Hematology/Oncology 
 
KyungMann Kim, PhD – Professor 
University of Wisconsin, School of Medicine & Public Health 
Department of Biostatistics and Medical Informatics 
 
Wei Huang, MD – Professor (CHS) 
University of Wisconsin, School of Medicine & Public Health 
Department of Pathology and Laboratory Medicine 
 
 
 
 
Funding Sponsor: NIH 
 
 
 
 
 
 


2 
 
Protocol Revision History 
Version Date 
Summary of Revisions Made 
20 Sep 2017 
Original Version 
25 Oct 2017 
UWCCC PRMC requested modifications 
03 May 2018 
Blood samples will be collected in addition to the tissue samples by 
UWCCC TSB  
22 April 2019 
Added background information for additional PET/CT imaging for 
immune-based systemic therapies and included additional FDG PET/CT 
imaging time-points for immune-based systemic therapies 
13 Oct 2020 
Remote consent procedures; clarification of inclusion criteria 
 
 


3 
 
Table of Contents 
 
LIST OF ABBREVIATIONS 
STATEMENT OF COMPLIANCE 
PROTOCOL SUMMARY 
1.0 
Introduction 
2.0 
Rationale 
3.0 
Objectives 
4.0 
Research Design and Methods 
5.0 
Study Procedures 
6.0 
Risks and Benefits 
7.0 
Adverse Events and Safety Issues 
8.0 
Privacy and Confidentiality Protections 
9.0 
Data and Safety Monitoring 
10.0 Statistical Considerations 
11.0 Drug Formulation and Procurement 
12.0 References 
 
 


4 
 
LIST OF ABBREVIATIONS 
AE 
Adverse event 
CAEPR 
Comprehensive Adverse Events and Potential Risks 
CDx 
Companion diagnostic 
CSRAs 
Chemotherapy Sensitivity and Resistance Assays 
CT 
Computed tomography 
CTCAE 
Common Toxicity Criteria for Adverse Events 
DSMB/DSMC 
Data Safety Monitoring Board/Committee 
FDA 
Food and Drug Administration 
GE 
General Electric 
GFR 
Glomerular filtration rate 
HIPAA 
Health Insurance Portability and Accountability Act 
IEC 
International Electrotechnical Commission 
IRB 
Institutional Review Board 
IV 
Intravenous 
IND 
Investigational New Drug 
kg 
kilogram 
MBq 
megabecquerel  
mCi 
millicurie 
mGy 
milligray 
MicroC3 
microfluidic-cis-co-culture 
mL 
milliliter 
mmol 
millimole 
mRCC 
Metastatic Renal Cell Carcinoma 
PACS 
Picture Archiving and Communication System 
PET 
Positron Emission Tomography 
PI 
Principal Investigator 
PSMA 
Prostate-specific membrane antigen 
RCC 
Renal Cell Carcinoma 
REM 
Roentgen Equivalent Mammal 
RF 
Radio frequency 
RPF 
Radiopharmaceutical Production Facility 
SAE 
Serious Adverse Event 
UP 
Unanticipated Problem 
UW 
University of Wisconsin 
UWHC 
University of Wisconsin Hospital and Clinics 
 
 
 
 
 


5 
 
STATEMENT OF COMPLIANCE 
 
The signature below constitutes the approval of this protocol and the attachments, and provides the 
necessary assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements and applicable US federal regulations and ICH guidelines. 
 
I agree to ensure that all staff members involved in this study are informed about their obligations in 
meeting the above commitments. 
 
 
Principal Investigator:  Steve Y. Cho, MD 
 
 
Signed: 
 
 
 
 
 
 
Date: 
 
 
__________________________________   
 
__________________ 
 
 
Steve Y. Cho, M.D. 
Department of Radiology, Nuclear Medicine Section 
University of Wisconsin School of Medicine and Public Health 
University of Wisconsin Carbone Comprehensive Cancer Center 
 
 


6 
 
PROTOCOL SUMMARY 
Title 
Functional Microscale Organotypic Assays to Predict Patient Response to Anti-
Angiogenesis Therapies. 
IND Sponsor-
Investigator 
Steve Y. Cho, MD 
Co-Investigators 
David Beebe, PhD  
E. Jason Abel, MD  
Christos Kyriakopoulos, MD  
KyungMann Kim, PhD 
Wei Huang, MD 
Participating Site 
University of Wisconsin-Madison  
Patient Population 
Patients diagnosed with metastatic clear cell RCC that are surgical candidates 
Accrual Objective 
This research will enroll 15 subjects 
Study Design 
Phase 2, single-center, open-label study  
Study Duration 
The estimated accrual period is over 3 years 
Primary Objective 
The primary objective of this research is to evaluate response to systemic therapy, 
including anti-angiogenesis and/or immune-based therapies, via 18F-DCFPyL 
PSMA-based PET/CT in patients with metastatic renal cell carcinoma and to 
compare qualitatively with conventional imaging response criteria (RECIST 1.1) and 
histopathological endpoints including isolation, enumeration and staining of 
Circulating Tumor Cells (CTC). 
Secondary 
Objectives 
1. Measure therapy response of patient specific uVESSEL model 
2. Evaluate the predictive power and validate the uVESSEL model 
Inclusion Criteria 
• Patients diagnosed with locally advanced (≥cT3) or metastatic clear cell RCC, as 
proven by biopsy. 
• Adults, 18 years of age or older. 
• Surgical candidates who have clinical indication for nephrectomy and standard 
of care biopsy of metastatic disease followed by possible standard of care 
systemic anti-angiogenesis based treatment regimen 
• Have consented to participate in the UWCCC Biobank. 
Exclusion Criteria 
• Patients who have received prior RCC systemic therapies 
• Prior history of prostate cancer 
• Prior history of any other malignancy within the last 2 years, other than skin 
basal cell or cutaneous superficial squamous cell carcinoma that has not 
metastasized and superficial bladder cancer 
• Unable to lie flat during or tolerate PET/CT 
• Serum creatinine > 2 time the upper limit of normal 
Treatment 
Description 
18F-DCFPyL whole body PET/CT scan administered at the following timepoints: 
1) Prior to scheduled nephrectomy 
2) After completion of systemic antiangiogenesis and/or immune-based  
therapy 
3) When patient is refractory to treatment 
Study Procedures 
• 
18F-DCFPyL whole body PET/CT scan 
• Review of relevant imaging and medical record information 
Statistical 
Considerations 
A total of 15 patients will be recruited into the clinical study. This is based on the 
feasibility and availability of suitable patients over the funding period with 


7 
 
approximately five patients per year.  With 15 patients, the mean of quantitative 
endpoints such as tumor size and SUV parameters will have standard error that is 
0.258 times the sample standard deviation, and the width of a 95% confidence 
interval for the probability of binary qualitative endpoints such as objective 
response will be less than 0.253. 
 


8 
 
1.0 
INTRODUCTION  
 
Renal cell carcinoma (RCC) is the third most common genitourinary malignancy in the United States with 
~64,000 new cases and ~14,000 deaths per year (Siegel 2017).  Despite the increase in the number of 
treatment options, median overall survival for patients with metastatic RCC (mRCC) remains at less than 
2 years (Heng 2013).   
 
The purpose of this research is to build on previous success with using patient’s own cells to predict their 
response to first line therapies for the treatment of multiple myeloma (MM).  This research represents a 
transdisciplinary approach that leverages the recent development of a practical, functional, and adaptable 
in vitro/ex vivo model of RCC vascularization, coupled with novel prostate-specific-membrane antigen 
(PSMA)-based PET imaging of tumor neovasculature, to evaluate the model’s ability to predict an 
individual patient’s response to anti-angiogenic therapeutics. 
 
2.0 
RATIONALE 
Recently, David Beebe’s Laboratory has shown in a retrospective study that harnessing the micro scale 
to retain critical multi-cellular signaling, e.g. stromal-cancer, 
allows the use of a patient’s own cells to predict that patient’s 
response to first line therapy, i.e. bortezomib, in multiple 
myeloma (MM) (Pak 2015). There are multiple cell types in the 
bone marrow microenvironment, including bone marrow stromal 
cells (BMSCs), macrophages, osteoclasts, and other immune 
cells, which secrete factors, e.g. cytokines, known to regulate 
signaling pathways that may contribute to chemotherapy 
resistance in MM (Asimakopoulos 2013, Hideshima 2007, Hope 
2014, Kim 2012, Markovina 2010). Therefore, in the Pak (2015) 
study, Beebe’s lab used a simple, yet novel, micro scale co-
culture system to create a companion diagnostic (CDx) to 
measure the drug response of cancer cells taken from the 
patient (Fig. 1, top).  We will work together with David Beebe 
and his lab to adapt and apply an analogous approach (Fig. 1, 
bottom) using a micro scale organotypic co-culture model 
(subsequently referred to as uVESSEL) to predict response to 
anti-angiogenic therapies in RCC.  
 
RCC is the third most common genitourinary malignancy in the United States with ~64,000 new cases 
and ~14,000 deaths per year (Siegel 2017). Clear cell RCC accounts for up to 85% of all RCCs and is 
different from MM in important ways. Clear cell RCC tumors are highly vascularized with blood vessels 
comprising approximately 25% of the tumor volume, increasing the supply of nutrients and driving tumor 
growth and metastasis. These tumor-associated blood vessels are enlarged, lack hierarchy, have a leaky 
endothelial layer, have abnormal disconnected sprouts, and have up-regulated soluble factor secretion 
when compared to their normal counterparts (Dudley 2012). Thus, endothelial cells and neovascularity 
are key components of the RCC tumor biology and, therefore, anti-angiogenic drugs have been the 
Fig. 1 - Top - Patient specific co-culture system 
used to predict response to first-line therapy in 
MM (Pak 2015). Bottom - Analogous approach 
for RCC with endothelial cells as target (and 
readout) for response to anti-angiogenic drugs.  


9 
 
preferred approach to treat RCC. Anti-angiogenic drugs are designed to target existing blood vessels 
and prevent the generation of new blood vessels in addition to normalizing the vessels within the tumor 
to improve the delivery of chemotherapeutics in a combination therapy (Hida 2016). Thus, while cancer 
cells were the therapeutic target in MM, endothelial cells are the therapeutic target in RCC (Fig. 1). Yet 
as decades of testing with Chemotherapy Sensitivity and Resistance Assays (CSRAs) attest (Editorial 
2013) and recent American Society of Clinical Oncology (ASCO) guidelines confirm (Burstein 2011), 
examining the target cell in isolation is insufficient to predict patient response reliably. However, David 
Beebe’s recent MM work (Pak 2015), as well as work by Ramamoorthy (2015), has shown that inclusion 
of patient specific cancer-stroma interactions can enable accurate prediction of drug response. In this 
study, we will include the tumor milieu via incorporation of tissue explants in our micro scale RCC CDx 
(Fig. 1, bottom). 
Despite increased clinical options, overall outcomes remain poor in RCC highlighting the need for 
predictive assays - Over the last decade, treatment options for mRCC have increased with the 
development of targeted agents that inhibit tumor angiogenesis. Unfortunately, median overall survival 
for mRCC patients remains less than 2 years (Heng 2013) and there remains an urgent need to improve 
treatment of mRCC patients. The first line agents pazopanib and sunitinib are multi-targeted small 
molecule, tyrosine kinase inhibitors (TKI) that inhibit a broad range of receptors for pro-angiogenic factors 
including VEFGR, PDGFR, and c-kit among others. These therapies are thought to act primarily or 
exclusively on tumor endothelial cells, decreasing tumor blood supply (Huang 2010). Axitinib and 
cabozantinib have similar (and also distinct) mechanisms of action, but clinically these agents are used 
as a second-line therapy only when primary agents fail. Importantly, wide variation in treatment responses 
to different agents has been demonstrated in clinical trials with anti-angiogenic therapies (Heng 2013). 
Currently, no biomarkers are available to predict which therapy will provide the best response to individual 
tumors and a “trial and error” approach is used to select initial treatments for mRCC patients. This 
standard paradigm is based on randomized clinical trial data demonstrating better clinical outcomes. The 
disadvantages of this empiric (and population-based) approach to selecting initial treatments in mRCC is 
also magnified by the rapid development of ten FDA approved therapies in ten years. This rapid 
expansion of new agents quickly outpaced the ability to evaluate these treatments in clinical trials. In 
2017, clinicians and patients now have many choices for systemic treatment of mRCC, but desperately 
need tools for selecting the best treatments for individual patients. A better approach would be to test 
each patient with a CDx to determine which of the many available drugs is most appropriate for that 
patient, resulting in precision medicine for treatment of patients with mRCC. 
As discussed above, the molecular pathogenesis of RCC is intrinsically linked to angiogenesis and the 
use of anti-angiogenic drugs. Our micro scale model will recapitulate patient vasculature and we need a 
method to measure vascularization in vivo in patients in order to evaluate the predictive value of our 
model. Current conventional imaging response criteria originally developed to assess response to 
chemotherapy using computed tomography (CT), such as RECIST1.1, are limited for determining 
treatment response to anti-angiogenesis agents (Ammari 2014). In order to monitor response in patients 
we can exploit the highly vascularized tumors in RCC, using a positron emission tomography (PET) 
radiotracer targeting the tumor neovasculature to reliably image RCC metastatic lesions. A new PET 
radiotracer for imaging angiogenesis targets the cell surface protein, prostate-specific membrane antigen 
(PSMA), which, despite the specificity implied by its name, is highly expressed on the tumor 
neovasculature of a number of solid tumors including a variety of RCC subtypes but most pronounced in 
clear cell RCC (Baccala 2007, Al-Ahmadie 2008).  Recent studies by ourselves and others have reported 


10 
 
PSMA-based PET detection of metastatic RCC (Siva 2017, Gorin 2017, Rhee 2016, Rowe 2015 & 2016). 
The development of PET imaging compounds targeted to the vascular endothelium allows direct imaging 
of tumor neovasculature, avoiding the limitations of indirect methods such as traditional contrast 
enhanced imaging. Thus, PSMA PET imaging addresses a major unmet clinical need in metastatic RCC 
for a reliable non-invasive functional imaging method for metastatic tumor detection and response to 
therapy, especially for anti-angiogenesis treatments. 
 
In addition to anti-angiogenesis agents, immunotherapy has also been shown to play an important role 
in the treatment of patients with metastatic RCC. Nivolumab is an anti-programmed cell death 1 (PD-1) 
monoclonal antibody that was approved by FDA in 2015 based on a phase III clinical trial that showed 
improvement in survival compared to the mTOR inhibitor everolimus in patients whose disease had 
developed resistance to anti-angiogenesis therapies (Motzer 2015). More recently, combination 
immunotherapy with nivolumab and ipilimumab, which is an anti-cytotoxic T-lymphocyte-associated 
protein 4 (CTLA-4) monoclonal antibody, demonstrated improvement in survival compared to sunitinib 
(Motzer 2018) and eventually became the preferred standard-of-care first-line treatment for patients with 
metastatic RCC. Finally, two additional trials that were published in March of 2019 showed that the 
combination of axitinib with anti-PD-1 immunotherapy (pembrolizumab and avelumab respectively) [Rini 
2019; Motzer, 2019] showed superiority compared to sunitinib and are currently pending approval by 
FDA as first-line therapies for metastatic RCC. In this rapidly evolving field, it is unclear how the most 
recent studies will impact the care of these patients, however systemic therapy with anti-angiogenesis 
agents will continue to play an important role. 
 
3.0 
OBJECTIVES  
3.1 
Primary Objective: To evaluate response to systemic therapy, including anti-
angiogenesis and/or immune-based therapies, via 18F-DCFPyL PSMA-based PET/CT in patients 
with metastatic renal cell carcinoma and to compare qualitatively with conventional imaging 
response criteria (RECIST 1.1) and other histopathological endpoints including isolation, 
enumeration and staining of Circulating Tumor Cells (CTC). 
 
Response of systemic immune-based and/or anti-angiogenesis therapies will be quantified using PSMA-
based PET imaging using a novel agent,18F-DCFPyL, as a non-invasive imaging biomarker of tumor 
neovasculature to functionally monitor  renal  cell cancer neovasculature in patients undergoing systemic 
anti-angiogensis therapy.  PSMA PET will be compared with response to anti-angiogenesis therapy using 
conventional imaging computed tomography(CT)-based RECIST1.1 criteria (Eisenhauer 2009) as well 
as histopathological endpoints (tumor vascular density, immunohistochemical staining for PSMA and 
neovascularization (CD105, CD31). Whole body PSMA PET/CT scans will be obtained at baseline, 
following adjuvant anti- angiogenic therapy and when the patient becomes refractory to treatment. 
 
The rationale and time points for obtaining PET scans is planned with respect to the typical natural history 
of metastatic RCC.  This project will obtain information from tumors that are responding to anti-
angiogenesis therapy and those resistant to treatment. 
PET1 is obtained pre-surgically to establish the baseline tumor PET and CT characteristics prior to 


11 
 
surgical removal of the primary renal tumor and metastatic sites (e.g. LNs). Surgery technique is not 
altered for this study. 
PET2A is obtained post-surgery and prior to start of first line standard-of-care (SOC) systemic therapy, 
either immune-based and/or anti-angiogenesis therapy. If the first line SOC systemic therapy is an 
immune-based therapy, a PSMA-based 18F-DCFPyL PET/CT will be obtained as PET2A and PET2B 
imaging time points, with potential PET3A and PET3B imaging as possible intended anti-angiogenesis 
therapy. The purpose of PET2 imaging is to establish a new baseline PET before first line SOC systemic 
therapy commences, given that post-surgical healing is a complex process that also includes 
angiogenesis and post-surgical inflammatory changes with potential changes in PET imaging. PET2 is 
necessary to characterize the existing metastatic tumors and any metastatic tumors which develop 
between PET1 and beginning systemic therapy. Clinical imaging with standard imaging is also obtained 
at this time as per standard care.  
PET2B is obtained at 12-16 weeks from start of first-line SOC systemic therapy, either immune-based 
and/or anti-angiogenesis therapy (with associated SOC conventional diagnostic chest/abdomen/pelvis 
CT) to evaluate mRCC tumors response to treatment. This time point is chosen given that less than 10% 
of patients will have progressive disease on SOC conventional imaging at their initial evaluation. 
PET3A and PET3B are obtained as follows, if the first line SOC systemic therapy did not include an anti-
angiogenesis therapy and if this new systemic therapy includes an anti-angiogenesis therapy: 
PET3A is obtained prior to start of additional systemic therapy including anti-angiogenesis 
therapy. The purpose of PET3A imaging is to establish a new baseline PET before this systemic 
therapy commences. PET3A is necessary to characterize the existing metastatic tumors and any 
metastatic tumors which develop between PET2B and start of this systemic therapy. Clinical 
imaging with standard imaging is also obtained at this time as per standard care. 
PET3B is obtained at 12-16 weeks from start of this new systemic therapy including anti-
angiogenesis therapy (with associated SOC conventional diagnostic chest/abdomen/pelvis CT) 
to evaluate mRCC tumors response to treatment. 
PET4 is obtained at clinical progression per (RECIST 1.1) or 2 years following initial systemic therapy, 
whichever occurs first. In patients with progressive disease, PET imaging will be used to evaluate 
changes in index lesions. In recent review (NEJM 2017) of clinical trials for first line mRCC therapies, the 
median time to progression on therapy is less than one year (range 3.1-11 months), with median OS 
about 2 years (range 7.3- 29 months). (Krajewski 2014 and Choueiri 2017). 
  
3.2 
Secondary Objectives:  
 
3.2.1 Measure therapy response of patient specific uVESSEL model 
Patient-derived uVESSEL models (from primary & metastatic sites) and  inc orporating patient-
derived immune cells  will be tested for response to anti-angiogenic therapies including pazopanib, 
sunitinib, cabozantinib, and axitinib. Response will be quantified via multiple endpoints, e.g. vessel 
sprouting, PSMA expression, cell death. 
 
3.2.2 Evaluate the predictive power and validate the uVESSEL model 
 


12 
 
While a number of novel biomimetic approaches show promise in their ability to recapitulate 
physiologically relevant behavior, clinical validation is needed to determine if such approaches are 
predictive. Using the clinical, pathological and imaging endpoints under 3.1, we will develop predictive 
models using responses to uVESSEL model above. More specifically we will use the objective 
response (dichotomous endpoint of yes or no) measured using PSMA-based PET/CT as a dependent 
variable in a logistic regression model with response to uVESSEL model from above as an independent 
predictor to segregate uVESSEL responses into two groups corresponding to responders and 
non-responders to anti-angiogenic therapies. 
 
3.2.3 Evaluate the predicative power and validate in-vitro immune therapy models 
 
At the moment we are expanding our uVESSEL patient-specific organotypic models (Fig. 2) to contain 
more cell types from the tumor microenvironment (cancer epithelial, endothelial, immune, stromal) in 
similar 
proportions 
as 
found in vivo. 
Patient tissue can be 
disaggregated, 
specific 
cell types isolated and 
placed in the uVESSEL 
model 
(Bischel 
2014) 
(Jimenez-Torres 
et 
al 
2019). Tumor spheroids 
can be created from a 
mixture of tumor epithelial 
cells and immune cells, to 
mimic 
tumor-infiltrating 
leukocytes, embedded in 
a hydrogel matrix (Sung 
2014). Tumor spheroids 
model solid tumors and 
replicate the hypoxia and 
nutrient starvation observed in vivo in RCC (Fig. 2). Hypoxia influences the microenvironment by 
significantly suppressing the anti-tumor response by increasing regulatory and immunosuppressive 
immune cell phenotypes and reducing cytotoxic lymphocyte functions (Noman 2015).  Likewise, the 
uVESSEL model allows for the creation of lumen structures within a hydrogel matrix that can be lined 
with endothelial cells to form microvessel structures, mimicking the highly vascularized nature of RCC 
(Jimenez-Torres 2019). Thus, we can create a carefully controlled model tumor microenvironment made 
entirely of primary cells all from the same patient (Fig. 2) that can be treated with various immune 
checkpoint inhibitors, TKI’s or combinations thereof with readouts examining tumor growth, immune cell 
phenotype and function and angiogenesis. Our functional endpoints include spheroid size 
variation/viability, as well as the angiogenic sprouting endpoint already developed for TKI predictive 
value. Both outputs can be expressed as a dichotomous endpoint of responders/non-responders for the 
validation of uVESSEL performance with patient response as observed in PET/CT. 
 
 
Fig. 2. A) Left: model scheme showing a tumor spheroid flanked by 2 endothelial lumens. 
Right: a 4-microdevice array. Confocal images of B) a tumor spheroid (blue) and immune 
cells (green). C) tumor organoid (green) with hypoxic core (red). D) lateral endothelial 
lumen. 


13 
 
4.0 
RESEARCH DESIGN AND METHODS:  
Study Design: This study is designed as a single cohort study without randomization or stratification. 
Study population:  A total of 15 patients diagnosed with clear cell RCC will be enrolled in this study.  
Achieving the objectives outlined above will require the collection of tissue from the UWCCC Biobank, 
the review of subject medical records, and subject participation in up to four research visits lasting 
approximately 2 – 3 hours. 
Inclusion Criteria: 
• 
Patients diagnosed with locally advanced (≥cT3) or metastatic clear cell RCC, as proven by 
biopsy. 
• 
Adults, 18 years of age or older. 
• 
Surgical candidates who have clinical indication for nephrectomy and standard-of-care biopsy of 
metastatic disease followed by possible standard of care systemic anti-angiogenesis based 
treatment regimen  
• 
Have consented to participate in the UWCCC Biobank. 
 
Exclusion Criteria 
• 
Patients who have received prior RCC systemic therapies 
• 
Prior history of prostate cancer 
• 
Prior history of any other malignancy within the last 2 years, other than skin basal cell or 
cutaneous superficial squamous cell carcinoma that has not metastasized and superficial 
bladder cancer 
• 
Unable to lie flat during or tolerate PET/CT 
• 
Serum creatinine > 2 times the upper limit of normal 
 
Subject identification and recruitment  
GU Oncology staff will identify patients who meet the eligibility criteria and will introduce them to this 
research opportunity.  Those interested will be referred to a study team member who will review the 
purpose of the research as well as details about participation.   
 
Informed Consent and Subject Information 
The consent process will occur prior to the administration of research PET/CT procedures.  Potential 
subjects will meet with a study team member to review the information in the consent form including study 
procedures, risks associated with participation, alternatives to participation and whom to contact for 
additional information.  Any questions will be addressed prior to the start of any study procedures and all 
subjects will be reminded that participation is optional and they can change their mind at any time. 
 
The consent process will occur prior to the administration of research procedures.  During the COVID-19 
pandemic, consent procedures will be conducted by phone, when possible, to minimize face-to-face 
contact subjects have with the research team.  A copy of the consent form will be mailed or emailed to 
subjects prior to the scheduled consent phone call. If emailed, the consent form will be encrypted.  A 
study team member will call potential subjects at the scheduled time to review the information in the 
consent form including study procedures, risks associated with participation, alternatives to participation 
and whom to contact for additional information.  Any questions will be addressed during the course of the 
phone call and subjects will be encouraged to contact the study team with any questions or concerns 


14 
 
they might have at any time.  Upon completion of the consent process, a copy of the signed consent form 
will be provided in one of the following ways depending on each subject preferences and capabilities: 
• 
Electronic signature will be provided using DocuSign 
• 
Subject will be asked to scan a copy of the entire consent document and email it back to 
the study team.  
• 
Subject will be asked to take a photo of the signature page and email a copy to the study 
team.  
• 
If subjects are unable to provide an electronic copy of the signed consent form, they will 
be asked to bring a copy of the form to the research visit. 
 
 
5.0 
STUDY PROCEDURES 
This project was designed to seamlessly integrate with the current pathway and standard of care both 
surgically and with medical oncology treatments. The patient will be evaluated in Dr. Abel’s clinic and 
discussed with Drs. Kyriakopoulos, Cho and Huang in the weekly multi-disciplinary conference from 12-
1 pm every Thursday. Suitable patients will be invited to participate in this research by someone involved 
in their care.  Those interested in participating will be referred to a member of Dr. Cho’s research team.  
Patients enrollment in the UWCCC Biobank program will be conducted in accordance with the Biobank 
protocol. 
Subjects will obtain standard of care (SOC) labs and pre-operative imaging as well as research PSMA-
PET imaging prior to surgery.  Surgery will proceed as per SOC and will not have extra procedures 
specific to this project. Patients will have time to recover following surgery and receive SOC imaging. 
Metastatic RCC patients will havea 2nd PSMA-PET (PET2A) greater than 4 weeks post-surgery and prior 
to initiation of systemic therapy. Patients with locally advanced disease will not have PET2A until 
metastatic RCC identified with SOC imaging. The rationale for the second PSMA PET prior to start of 
systemic therapy (PET2A) is to establish a new baseline PET.  We want to establish any potential post-
surgical PET findings from PET1 to PET2A (ex: post-surgical PET changes, change in PSMA PET uptake 
at sites of remaining measurable disease, new potential lesions).  This will be followed by a third PSMA 
PET (PET2B) obtained at 12-16 weeks from start of first-line SOC systemic therapy, either immune-
based 
and/or 
anti-angiogenesis 
therapy 
(with 
associated 
SOC 
conventional 
diagnostic 
chest/abdomen/pelvis CT) to evaluate mRCC tumors response to treatment. This time point is chosen 
given that less than 10% of patients will have progressive disease on SOC conventional imaging at their 
initial evaluation. 
If the first line SOC systemic therapy did not include an anti-angiogenesis therapy and if this second 
systemic therapy includes anti-angiogenesis therapy, an additional 4th and 5th PET imaging will be 
obtained as PET3A and PET3B. PET3A is obtained prior to start of additional systemic therapy including 
anti-angiogenesis therapy, to establish a new baseline PET before this systemic therapy commences, to 
characterize the existing metastatic tumors and any metastatic tumors which develop between PET2B 
and start of this systemic therapy. PET3B is obtained at 12-16 weeks from start of this second systemic 
therapy including anti-angiogenesis therapy (with associated SOC conventional diagnostic 
chest/abdomen/pelvis CT) to evaluate mRCC tumors response to treatment. 


15 
 
Following recovery from surgery the patients will initiate systemic therapy as per SOC.  The patients will 
receive systemic therapy with an antiangiogenic agent as per first-line or later SOC therapy. There are 
currently 4 oral antiangiogenic agents approved by FDA for the treatment of patients with metastatic RCC 
(pazopanib, sunitinib, cabozantinib, axitinib) and patients on this study will receive one of those 4 agents 
as per physician’s preference. 
 
Study procedures will include a tissue analyses as well as the use of PET/CT imaging to monitor subject’s 
clinical response to treatment.  For tissue, immune cell and CTC analyses, investigators will be working 
with the UWCCC Biobank staff to acquire blood samples and tissue samples donated from the kidney 
with primary tumor, lymph nodes, and any other metastatic site from which tissue was collected. 
 
Subjects will be asked to complete four to six research visits that will include the administration of a PSMA 
PET/CT exam.  There will be an initial baseline 18F-DCFPyL PSMA PET/CT (PET1) prior to nephrectomy 
and SOC biopsy of metastatic sites as clinically indicated, followed by a second 18F-DCFPyL PSMA 
PET/CT (PET2A) prior to start of systemic anti-angiogenesis and/or immune-based therapy.  A third on-
treatment 18F-DCFPyL PSMA PET/CT (PET2B) will be performed at 12-16 weeks from start of systemic 
therapy (with associated SOC diagnostic chest/abdomen/pelvis CT).  If system  The fourth (or sixth if 
there is PET3A and PET3B) 18F-DCFPyL PSMA PET/CT (PET4) will be performed at the time of disease 
progression as determined by conventional imaging such as CT or MRI; or two years after initiation of 
therapy (whichever one comes first). 
 
Blood and Tissue Collection 
We are collaborating with the UWCCC Translational Science BioCore (TSB) for sample collection as this 
research is consistent with their mission “to facilitate cancer-related basic science and translational 
research within the UWCCC through the integration of human biospecimen collection and storage with 
associated histology, molecular, morphometric and consultative services”.  The TSB team is uniquely 
equipped to appropriately identify and consent patients and to collect and properly handle/process the 
samples without disrupting the clinical workflow.  As a result, this research will only target patients that 
have previously consented to allow additional and residual tissue samples to be included in the UWCCC 
Biobank.  Biobank personnel will provide blood and the sample(s) collected from the kidney with primary 
tumor, lymph nodes, and any metastatic sites that are excised.  
 
PET/CT Imaging 
Each PET/CT research visit is estimated to take 2-3 hours to complete.  Women of childbearing potential 
will be administered a pregnancy test prior to the start of imaging procedures.  Subjects will be asked to 
fast for 4 to 6 hours and to stay well hydrated prior to each visit.  Upon their arrival to the WIMR facilities, 
an IV will be placed to allow injection of the PET tracer.  After the IV administration of less than 9 mCi of 
18F-DCFPyL radiotracer by slow IV push, subjects will be required to sit quietly for 1-hour to allow for the 
biodistribution of the tracer.  After approximately 50 minutes, subjects will be given the option of using 
the restroom prior to being positioned in the PET/CT scanner.  Once positioned, a whole body (defined 
in this protocol as vertex-of-skull to mid-thigh) PET scan will be obtained with corresponding low-dose 
non-IV contrast CT imaging with the PET/CT obtained for PET attenuation correct and anatomic co-
registration.   
 


16 
 
A member of the study team will follow-up with all subjects approximately 1 to 3 days after their PET/CT 
exam by phone to inquire about delayed adverse events.  If the subject is experiencing any late occurring 
adverse events, they will be asked to return to the research clinic or urology clinic for a follow-up exam.  
 
Review of Medical Records 
Medical records will be reviewed to obtain medical history including physical exams, medications (all 
concomitant medications before and after each of the PET/CT scans, treatment medications for RCC), 
laboratory tests (metabolic chemistry, hematology, pathology), imaging results (all available CT, MRI, 
ultrasound, PET/CT) and information related to the diagnosing, treatment, or outcomes of the study 
subject’s diagnosis, treatment and clinical follow-up up to two years from the time of initiation of systemic 
anti-angiogenesis therapy. 
 
Remuneration 
Subjects will receive $50 per scan, for a total of up to $300, as compensation for their participation in 
this study. 
 
Withdrawal/Termination from Study 
Reasons for withdrawal or termination  
 
Enrolled patients will be removed from the study in the following circumstances: 
• 
The patient does not complete the imaging examinations per study protocol 
• 
The subject withdraws consent 
• 
Exclusion criteria are discovered after registration but prior to the research examinations 
• 
The PI or co-investigators determine it is within the best interest of the subject to remove 
them from the research. 
 
Study Procedures Table 
Study 
Procedures 
Screening1 
Baseline 
Prior to surgery 
VISIT 2a and 
2b 
Prior to and during 
1st systemic 
therapy 
VISIT 3a and 
3b 
Prior to and during 
2nd systemic 
therapy 
VISIT 42 
Informed Consent5 
X 
 
 
 
 
Clinical History 
X 
 
 
 
 
PET/CT Exam 
 
X 
X4 
X4 
X 
Tissue Collection 
 
 
X3 
 
 
1. 
Screening visit may be completed on the same day as the baseline visit 
2. 
To be done at the time of disease progression or within 2 years, whichever occurs first. 
3. 
Tissue collection will occur post SOC surgery and will be coordinated with the UWCCC TSB. 
4. 
PET/CT scan obtained prior to systemic therapy (immune-based and/or anti-angiogenesis therapy) and at 12-16 
weeks into therapy 
5. 
During the COVID-19 pandemic, consent procedures will be conducted by phone, when possible, to minimize face-to-
face contact subjects have with the research team.  An encrypted copy of the consent form will be mailed or emailed 
to subjects prior to the scheduled consent phone call.   
 
 


17 
 
6.0 
RISKS AND BENEFITS 
PET: 
Detailed organ dosimetry derived from four representative patients is included in the table below.  Of 
note, the effective dose from 18F-DCFPyL was 0.0165 mSv/MBq or 6.1 mGy (0.61 rem) for an injected 
dose of 370 MBq (10 mCi), or 5.49 mGy or 5.49 mSv (0.549 rem) for 333 MBq (9 mCi) which is the 
highest dose administered for each scan in this study.   
Dosimetry:   
Highest radiation dose was estimated for the kidneys (0.0945 mGy/MBq) followed by urinary bladder wall 
(0.0864 mGy/MBq), submandibular glands (0.0387 mGy/MBq) and liver (0.0380 mGy/MBq). The mean 
absorbed dose to the bone marrow was 0.01 mGy/MBq.  In comparison to the published data on our first 
generation agent, 18F-DCFBC, 18F-DCFPyL shows significantly lower (under 50 %) doses in most 
radiosensitive organs such as the thymus, ovaries, red marrow and osteogenic cells. Less radiation dose 
was also measured in lower large intestinal wall, small intestine, stomach wall, lungs, muscle and skin. 
Radiation dose from the two radiotracers was similar (within +/-50 %) in the gallbladder wall, upper large 
intestinal wall, spleen, testicles, thyroid, and pancreas.  Radiation dose from 18F-DCFPyL was higher in 
the adrenals, kidneys, liver, pancreas, spleen, and bladder wall. 
 
Organ 
Absorbed 
Dose 
(mGy/MBq) 
 
Organ 
Absorbed 
Dose 
(mGy/MBq) 
Adrenals 
3.11E-02 
 
Ovaries 
8.89E-03 
Brain 
2.19E-03 
 
Pancreas 
2.44E-02 
Breasts 
4.57E-03 
 
Parotid Glands 
2.68E-02 
Gallbladder Wall 
1.44E-02 
 
Red Marrow 
1.04E-02 
Heart Wall 
1.29E-02 
 
Skin 
4.05E-03 
Kidneys 
9.45E-02 
 
Small Intestine 
9.13E-03 
Lacrimal Glands  
3.50E-02 
 
Spleen 
1.85E-02 
Lens 
1.25E-03 
 
Stomach Wall 
1.16E-02 
Liver 
3.80E-02 
 
Submandibular Glands 
3.87E-02 
LLI Wall 
1.05E-02 
 
Testes 
1.01E-02 
Lungs 
1.08E-02 
 
Thymus 
5.56E-03 
Muscle 
6.32E-03 
 
Thyroid 
8.56E-03 
Osteogenic Cells 
9.58E-03 
 
ULI Wall 
1.67E-02 
Urinary Bladder Wall 
 
8.64E-02 
 
Uterus 
1.15E-02 
 
CT: 
Risks associated with CT include exposure to 10.0 mSv of radiation from the low-dose scan required for 
attenuation correction calculations.  Thus, the total whole body effective dose equivalent for a PSMA 
PET/CT is approximately 15.5 mSv (5.5 mSv from PSMA-PET plus 1.0 mSv from CT).  This corresponds 
to approximately 4 years of natural background radiation.  The total whole body effective dose equivalent 
for 2 and 3 PSMA PET/CT scans performed over a one year time period is 31.0 mSv and 46.5 mSv, 
respectively, and remain below the annual occupational dose limit for radiation workers (50 mSv) in a 
one year time period.  If a patient receives a 4th or 5th PSMA PET/CT scan during a one year time period 
due to inclusion of PET3A and 3B timepoints for PET/CT imaging at time of 2nd systemic therapy, the 


18 
 
total whole body effective dose equivalent will be 62 mSv and 77.5 mSv, respectively.   The PET4 
timepoint PSMA PET/CT will be performed at the time of tumor recurrence or two years after initiation of 
therapy (whichever one comes first). (Median OS for metastatic RCC patients is approximately 24 
months.)  
 
7.0 
ADVERSE EVENTS AND SAFETY ISSUES 
Definition of adverse events and potential risks: The term “adverse event” is defined in the 
International Conference on Harmonization Guideline for Good Clinical Practice as follows: “Any 
untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical 
product and which does not necessarily have a causal relationship with this treatment.  An adverse event 
(AE) can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of a medicinal (investigational) product whether 
or not related to the medicinal (investigational) product”.  
Medical conditions of a subject that exist prior to receiving the investigational imaging tracer will be 
recorded as medical history.  After administration of the investigational imaging tracer, all new medical 
conditions are considered adverse events.  Adverse events occurring in this trial will be recorded in data 
collection forms, regardless of whether or not they are thought to be associated with the investigational 
imaging tracer throughout the study period.  However, the Investigator also will assess the possible 
relationship between the adverse event and the investigational imaging tracer. 
For the purposes of this protocol, an adverse event (AE) is any untoward medical occurrence (e.g., sign, 
symptom, disease, syndrome, intercurrent illness, abnormal laboratory finding) that emerges or worsens 
relative to pre-imaging baseline. We anticipate minimal adverse effects or toxicity with the investigational 
imaging tracer 18F-DCFPyL in this study.  
Definition of Serious Adverse Events (SAE), SAE list: Any adverse drug experience, occurring at any 
dose that results in any of the following: 
• 
Death 
• 
Is life-threatening (an event in which the patient was at immediate risk of death; it does not 
refer to an event which hypothetically might have caused death if it were more severe) 
• 
Requires or prolongs inpatient hospitalization  
• 
Results in a persistent or significant disability/incapacity 
• 
Is a congenital anomaly/birth defect 
• 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, 
they may jeopardize the patient or subject and/or may require intervention to prevent one of 
the outcomes listed. 
 
All other AEs will be treated as “non-serious” and are described in the final study report.  An SAE that is 
definitely, probably or possibly related to the investigational imaging tracer will be reported immediately 
(within 24 hours of its determination) by telephone or fax by the Principal Investigator. 
 


19 
 
AE Characteristics: Toxicity will be graded according to the NCI Common Toxicity Criteria (CTCAE), 
version 4.03 (Appendix A; which can also be accessed and downloaded via the website: 
http://ctep.cancer.gov/reporting/ctc.html. The Investigators will monitor the occurrence of adverse 
events during the course of each patient in the study. The Investigators need to assess whether there is 
a reasonable possibility that the study imaging agent caused or contributed to an AE using the following 
criteria as a guide: 
• 
Definite: The adverse event is clearly related to the investigational agent. 
• 
Probable: There is a clinically plausible time sequence between the onset of the AE and 
administration of imaging tracer.  The AE is unlikely to be caused by the concurrent/underlying 
illness, other drugs, or procedures.  
• 
Possible: There is a clinically plausible time sequence between the onset of the AE and 
administration of imaging tracer, but the AE could also be attributed to the 
concurrent/underlying disease, other drugs, or procedures.  “Possibly” should be used when 
the administration of the study imaging tracer is one of several biologically plausible causes 
of the AE. 
• 
Unlikely: The adverse event is doubtfully related to the investigational imaging tracer. 
• 
Not Related: Another cause of the AE is the most plausible and/or a clinically plausible 
temporal sequence is inconsistent with the onset of the AE and the investigational imaging 
tracer; and/or a causal relationship is considered biologically implausible. 
 
If any adverse event is considered to be definitely, possibly or probably related to the investigational 
imaging tracer, that event will be followed until resolution or until it is deemed chronic or irreversible by 
the Investigator.  If an event is unlikely or not related to the investigational imaging tracer, the event will 
not be reported. 
 
All AE will be documented and recorded in the medical record and will include: 
 
I. 
Specific reaction according to NCI Common Toxicity Criteria, version 4.0. 
II. 
Duration of the reaction 
 
III. 
Severity/grade according to NCI Common Toxicity Criteria, version 4.03. If the adverse event 
is not addressed by the Common Toxicity Criteria, the severity should be rated as mild (grade 
1), moderate (grade 2), severe (grade 3), or life threatening (grade 4). 
IV. 
Relation of reaction to study agent 
V. 
Management of reaction, including any interruption or dose modification of the study drugs. 
 
Event Severity: In addition to classifying an adverse event as serious or non-serious, the Investigator 
and/or designated personnel will grade the adverse event to describe The Maximum intensity of the 
event.  Specific criteria for the intensity of adverse events are described in the Common Toxicity Criteria 
(CTC:  National Cancer Institute).  Investigators should follow these criteria for grading and reporting 
adverse events. 
An event may be severe, but may not be serious (e.g., severe headache).  The severity  of an adverse 
event will be assessed by study personnel using their best clinical judgment and includes but is not limited 
to taking into account the patient’s past history, the patient’s chart, interviews with the patient and 


20 
 
caregivers, and direct observation.  The Investigator will also be asked to assess the possible relationship 
between the adverse event and the administration of the investigational imaging tracer. 
Adverse Event Reporting: Per 21CFR312.32(c), all adverse events deemed both serious and 
unexpected associated with the use of the drug must be reported to the FDA and to all participating 
investigators as soon as possible and in no event later than 15 days. 
The FDA will be notified of any unexpected fatal or life threatening experience associated with the drug 
as soon as possible but in no event later than 7 calendar  days.  The UW HS-IRB will be notified in 
accordance with posted institutional policy. 
Adverse Events:  Serious and Non Serious Adverse events will be recorded, regardless of whether or 
not they are thought to be related to the investigational imaging tracer.  While all adverse events will be 
tabulated and reported in the study final report, serious adverse events will be reported in the course of 
the trial.  Adverse events that meet criteria of a serious adverse event listed above will also be recorded 
and reported. 
The Investigator will inform the IRB of all adverse events attributed to the investigational imaging tracer 
in accordance with posted institutional policy. 
 
8.0 
PRIVACY AND CONFIDENTIALITY PROTECTIONS 
Research procedures will be conducted in private areas and we are only collecting the minimum amount 
of information necessary to conduct the study. No sensitive data (HIV status, illicit drug use) will be 
collected. Identifiable records will only be available to study team members. 
Data are not being shared outside of UW except through publications, in which case the data do not 
identify individual subjects. Study data will be managed by the PI and co-PIs. Data will not be placed on 
unencrypted laptop computers. Coded electronic data will be kept on the secure network drives. A 
separate file linking codes to subject identity will be kept in the same manner, in a folder separate from 
study data. At the end of 7 years study data will be de-identified by destroying the link to identifiable 
information. Until that time data will remain coded and the file linking codes to subject identity kept as 
well.  
All samples and images will be given a unique study number that cannot be linked to the patients’ medical 
records in any way. Imaging research images will be stored on PACS under this study number only. The 
investigators will have access to the patients’ electronic medical records that are relevant to this study. 
Study records will be kept for seven years. Study data will be managed by the PI and co-PI. 
Subject’s identifiable information will be linked to a study code.  The study code will be used to identify 
the subject and research images.  Research images may be used in scientific presentations, but all 
images will be de-identified. The data will be stored on the Principal Investigator’s (PI or co-PI) password 
protected computer in her locked office. The code will be stored separate from the data on the PI’s 
computer in her locked office.   
 


21 
 
9.0 
DATA AND SAFETY MONITORING 
Safety monitoring of research protocols investigating novel PET tracers utilize a combination of tools. 
These are framed to provide adequate clinical trial oversight ensuring that the research team is complying 
with the conditions of IRB approval and FDA requirements. The tools enable frequent evaluations of 
study-related activities, identification of deviations or noncompliance with the conduct of the study or 
protocol, review of adverse events tracking and reporting, and review of drug production and 
accountability. 
 
These tools include: 
• 
Study visit checklists – Research Coordinators use study visit checklists to ensure that all study 
required procedures and processes are met before, during and after the subject has been seen. 
Items listed on these checklists include informed consent obtained, consent questions answered 
(and by whom), vitals recorded, medications reviewed, etc. These checklists are reviewed by the 
study coordinator and are signed off for each study subject by the coordinator monitoring the 
study to ensure that protocols are followed accurately and include limited variance from protocol.  
 
• 
Safety monitoring – The safety monitoring of subjects enrolled in the research includes both 
internal and external safety checks governing the administration of 18F-DCFPyL. Internal 
monitoring is performed by co-investigators and external monitoring by a team of four physicians 
with the appropriate broad clinical and technical. The internal monitoring plan includes a brief 
review of each subject visit after it occurs.  In this regard, the study team will discuss data quality, 
18F-DCFPyL production and administration, and subject compliance and tolerance of research 
procedures.  
 
The external monitoring plan will be coordinated by the Department of Radiology Medical Imaging 
Research Support (MIRS) administrative team.  MIRS is a working group that provides 
investigators with the support needed to conduct clinical research that uses imaging as an 
outcome measure.  Members include a core administrative group (physicians, scientists, imaging 
modality managers, regulatory and administrative support) and ad hoc members recruited when 
their expertise is required for the review/monitoring of a research project. MIRS administrative 
team in addition to a 4-person committee of physicians/scientists will meet annually with 
representatives from the research team, to discuss research progress, AE/SAE reports, as well 
as other data reports compiled by the study team.  If an AE/SAE occurs, an ad hoc committee 
meeting will be organized to discuss whether the event is considered serious, whether it can be 
attributed to research procedures, whether it constitutes non-compliance on the part of the study 
team, and the plan for resolution and a future remediation plan. 
 
As part of this plan, the monitor(s) will verify that:  
• 
The rights and well-being of human subjects are protected  
• 
Reported study data are accurate, complete, and verifiable from source documents  
• 
The conduct of the study is in compliance with the currently approved protocol, GCPs, 
applicable regulatory requirements, and guidelines for clinical research studies at the 
University of Wisconsin-Madison and its affiliates  


22 
 
 
 
10.0 
STATISTICAL CONSDERATIONS 
As noted in section 3.0, the primary objective is to evaluate response to anti-angiogenesis therapy using 
PSMA-based PET.  Other endpoints such as qualitative objective response based on RECIST 1.1 and 
quantitative CT-based tumor size and SUV parameters will also be evaluated.  The secondary objectives 
are 1) to measure therapy response of patient-derived uVESSEL models using multiple endpoints such 
as vessel sprouting, PSMA expression and cell death and 2) to evaluate the predictive power and validate 
the uVESSEL model for clinical outcomes. 
10.1 
Sample size justification 
A total of 15 patients will be recruited into this clinical study. This is based on the feasibility and availability 
of suitable patients over the funding period with approximately five patients per year.  With 15 patients, 
the width of a 95% confidence interval for the probability of qualitative response based on PSMA-PET 
and objective response based on RECIST 1.1 will be less than 0.253 and the mean of quantitative 
endpoints such as CT-based tumor size and SUV parameters will have standard error that is 0.258 times 
the sample standard deviation. 
10.2 
Statistical analysis  
Statistical analysis will by necessity (n=15) and nature (predictive modeling of response using patient-
derived ex-vivo uVESSEL responses as predictors, among others) be exploratory and descriptive 
including qualitative comparison between responses based on PSMA-PET and RECIST 1.1 without a 
formal paired test of statistical significance. 
Measures of patient response to anti-angiogenics via clinical endpoint such as response based on PSMA-
PET (primary endpoint) and objective response based on RECIST 1.1 and histopathological endpoints 
(tumor vascular density, immunohistochemical staining for PSMA and neovascularization (CD30, CD34)) 
will be summarized with descriptive statistics (response rate and confidence interval), and changes in 
these endpoints, PSMA staining from baseline to end of treatment and follow-up) will also be summarized 
with descriptive statistics. Measures of tumor neovascularization via PSMA-based PET/CT imaging will 
be summarized with descriptive statistics over time and analyzed using generalized linear mixed-effects 
models for longitudinal measures. Qualitative 18F-DCFPyL PSMA PET/CT imaging will be recorded in a 
three-point scale, and statistical analysis will be performed with the equivocal score of 1 considered as 
positive or negative.  Histopathological endpoints will be qualitatively graded by our study pathologist on 
a zero to four scale with four being the highest level of immunohistochemical staining or 
neovascularization. 
Partial responses are likely from conventional clinical imaging in mRCC patients. Standard imaging 
techniques define responses to therapy based on size changes of target lesions using RECIST1.1. 
Metastatic tumors may respond to anti-angiogenic treatment but not change substantially in size (unlike 
cytotoxic therapies for which RECIST was designed). To address this limitation in current imaging, we 
combine standard imaging with functional imaging protocols to evaluate whether angiogenesis 
decreases in individual metastatic lesions regardless of changes in tumor size.  PSMA PET signal is 
used as a surrogate for changes in tumor neovasculature. These functional imaging findings will be 


23 
 
confirmed pathologically by evaluating angiogenesis/ PSMA expression in tumor samples and patient 
models. Using this approach, we will evaluate functional anti-angiogenesis responses to therapies.  
For the uVESSEL models (secondary objective 1), generalized linear mixed-effects models for 
longitudinal measures taken four times will be used for various endpoints with treatment (control plus four 
anti-angiogenics), dose (three levels each replicated three times), and plate (primary vs metastatic 
crossed by normal vs tumor) as the main factors. This will allow evaluation of the effects of treatment and 
dose and the differences between primary and metastatic samples and between normal and tumor 
vessels.  We will also include these two models as the main factor in a combined generalized linear 
mixed-effects model, which will allow direct comparison of the biomimetic and the organotypic models.   
Predictive modeling (secondary objective 2) will thus include the actual tumor size change (in a typical 
waterfall plot) and the dichotomy of disease control vs no disease control in the predictive model for tumor 
response in addition to the dichotomy of responders vs. non-responders in functional studies.   
Due to limited sample size (n=15) and because not all patients are treated with the same anti-angiogenic 
therapy, further reducing the sample size for each anti-angiogenic therapy, one cannot expect the 
predictive analysis to give a clear discrimination between responders and non-responders to anti-
angiogenic therapies.  As an alternative, we will also use clustering analysis based on k-means or 
Gaussian mixture models which are less sensitive to small sample sizes as in Pak (2015). 
 
Using the clinical, pathological and imaging endpoints as dependent variables, we will develop predictive 
models using responses to the uVESSEL model as independent predictor variables. More specifically, 
we will use the objective response as a dichotomous endpoint as a dependent variable in a logistic 
regression model with response to the uVESSEL model as an independent predictor. This approach is 
equivalent to linear discriminant analysis. Although we expect about 70% of eligible patients will receive 
pazopanib and the rest sunitinib, we can still use the objective response as the dependent variable in 
logistic regression as both pazopanib and sunitinib are expected to give a similar response rate of around 
30% in this patient population.  In addition each patient sample will be subject to both agents in the 
uVESSEL model and provide response to be used as predictors in logistic regression modeling.  
10.3 
Feasibility 
In 2016, there were 184 new patients with renal cell carcinoma seen at UWHC, with between 33-50% of 
patients having locally advanced or metastatic tumors. There are no competing studies for the current 
project, and we anticipate no issues with planned enrollment. 
 
11.0 
DRUG FORMULATION AND PROCUREMENT 
Investigation Product 
The investigational product will be obtained from the University of Wisconsin Radiopharmaceutical 
Production Facility (RPF).  The RPF is located in the Wisconsin Institute for Medical Research Building.  
The FDA IND for this protocol will be cross-filed with the current Johns Hopkins University IND for 18F-
DCFPyL, IND # 121064 to reference the Pharmacology and Toxicology, dosimetry, and Previous Human 
Experience sections of that IND. 


24 
 
 
18F-DCFPyL is eluted with 1 mL of ethanol followed by 10 mL of 0.9% sodium chloride, via a sterilizing 
0.22μ filter into a sterile vial containing 4 mL 0.9% sodium chloride.  Test radiopharmaceutical will be 
administered intravenously via slow I.V. push.  The maximum mass dose of the ligand corresponding 
to the 9 mCi dose of 18F-DCFPyL will be less than 3.98 µg per administration, although the actual 
dose will be significantly less depending on the specific activity achieved during each radiosynthesis 
run.  The lowest limit for specific-activity (SA) we will use at the time of injection is 1000 mCi/µmole, 
although the validation and phase I study radiosynthesis SA was significantly higher than 1000 
mCi/µmole. 
 
Preclinical Toxicology 
A toxicity report “14-Day Study to Determine Toxicity of DCFPyL from a Single 
IV Dose in Sprague Dawley Rats” was prepared by SoBran Inc. (Study No. SB-MP-001).  The 
following summary is taken from that report. 
 
The purpose of this study was to evaluate the toxicity of DCFPyL three and fifteen days following a 
single intravenous dose in rats.  This study consisted of two test article treatment groups of ten male 
and female Sprague-Dawley rats per group dosed with DCFPyL at 0.1 and 0.5 mg/kg.  An additional 
group of ten males and ten females received the vehicle, 5% Dextrose, and served as the control.  All 
rats received a dose volume of 5 mL/kg.  The rats were dosed intravenously once on Study Day 1.  
Five male and five female rats from each group were bled on Study Day 3 and the remaining rats 


25 
 
were bled on Study Day 15.  All animals were euthanized and necropsied following blood collection.  
Parameters evaluated for test article effect included survival, clinical observations, body weight, body 
weight gain, clinical pathology, gross pathology, organ weights, and microscopic pathology. 
 
All rats survived to the scheduled termination and remained bright, alert and responsive during the 
study.  No abnormal findings were indicated during cageside or hands-on observations.  One female 
rat treated with the vehicle control and one female rat treated with 0.1 mg/kg DCFPyL lost weight 
between Days 8 and 15. All rats treated with 5% Dextrose or DCFPyL gained weight during the course 
of the study.  There were no treatment related changes seen in the hematology, coagulation, or clinical 
chemistry data.  Organ weights showed some variance but microscopic findings in the Day 3 and Day 
15 rats were considered incidental and not directly related to the test article.  Under the conditions of 
this study, there were no treatment related findings in Sprague Dawley rats three or fifteen days after 
a single intravenous dose of DCFPyL at 0.1 mg/kg and 0.5 mg/kg.  
18F-DCFPyL was assessed for its ex-vivo pharmacokinetics in non-obese diabetic severe-combined 
immunodeficient (NOD-SCID) mice bearing both PSMA positive PC3-PIP and PSMA negative PC3-
flu xenografts.  Table below shows the percent injected dose per gram of tissue (%ID/g) of 18F-
DCFPyL activity in selected organs. 
 
18F-DCFPyL PSMA-dependent uptake within PSMA positive PC3 PIP xenografts, reaching a value of 
46.7 ± 5.8%ID/g at 30 minutes post-injection (p.i.), which decreased by only about 10% over the 
ensuing 4 hours.  At 60 minutes p.i. the kidney, liver, and spleen displayed the highest uptake.  By 
that time, the urinary bladder also showed relatively high uptake.  However, that uptake includes 
excretion at all-time points.  Rapid clearance from the kidneys was shown, decreasing from 74.1 ± 
6.6%ID/g at 30 minutes to 7.4 ± 0.9%ID/g at 4 hours.  The relatively high values noted in kidney are 
partially due to high expression of PSMA within proximal renal tubules.  The ratio of uptake within 
PSMA positive PIP to PSMA negative flu tumors ranged from 40:1 to more than 1,000:1 over the 4-
hour time period of the study.  A possible explanation for that increased tumor uptake of radiochemical 
over time could be due to ligand-mediated PSMA internalization within tumor cells.  Less retention in 
 


26 
 
kidney relative to tumor over time could be due to a lower degree of internalization in this (normal) 
tissue and/or different metabolism of 18F-DCFPyL, which does not promote retention of radiochemical 
in kidney.  Relatively low bone uptake (<1% ID/g at all-time points) suggests little metabolic 
defluorination of 18F-DCFPyL.  The total effective dose for 333 MBq (9 mCi) of 18F-DCFPyL was 
calculated to be 5.93 mGy (0.593 rem) based on animal models. 
 
Initial Phase I Study Adverse Events: 
An initial phase I study of the biodistribution and dosimetry of 18F-DCFPyL found that in nine patients 
there were no severe adverse events. One patient reported two adverse events that were classified 
as unlikely to be attributable to the radiotracer (mild headache and mild nose bleed, both of which 
resolved without treatment and were considered Grade I by the National Cancer Institute Common 
Terminology Criteria for Adverse Events (NCI CTCAE) Version 4.0). Another participant experienced 
a decrease in platelet count on routine assessment during the post-imaging follow up which, at the 
time of publication, had not resolved but which was attributed to the participant starting treatment for 
prostate cancer. This was also a NCI CTCAE Grade I adverse event.  There were no radiotracer-
related adverse events with monitored heart rate or blood pressure. 
 
Human dosimetry: 18F-DCFPyL human dosimetry reported in Risks/Benefits section. 
 
12.0 
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