Imaging CCR2 Receptors With 64Cu-DOTA-ECL1i in Head and Neck Cancer
Phase: PHASE1
Conditions: Head and Neck Cancer
Sponsor: Washington University School of Medicine

VI. PROTOCOL  
 
 
Imaging CCR2 Receptors with 64Cu-DOTA-ECL1i in Head and Neck Cancer  
 
Principal Investigator 
Farrokh Dehdashti, M.D. 
Washington University School of Medicine 
660 S. Euclid, Campus Box 8223 
St. Louis, MO  63110 
Telephone: 314-362-1474 
E-mail: dehdashtif@wustl.edu 
Protocol Version # 5 
HRPO # 201912003 Version Date: 08/28/2023 
 
Sub-Investigators 
Ryan Jackson, M.D. 
Department of Surgery (Head & Neck) 
Telephone: (314) 362-7509 
Email: jackson.ryan@wustl.edu 
 
Yongjian Liu Ph.D. 
Department of Radiology 
Tel: (314) 362-8431 
Email: yongjianliu@wustl.edu 
 
Ningying Wu, MD, PhD (statistician) 
Division of Biostatistics 
Telephone: (314) 747-8162 
Email: wun@wustl.edu 
 
Rebecca Chernock, M.D. 
Department of Pathology & Immunology 
Telephone: (314) 362-0101 
Email: rchernock@wustl.edu 
 
 
Kooresh I. Shoghi, Ph.D. 
Department of Radiology 
Telephone: (314) 362-8990 
Email: shoghik@wustl.edu 
 
Sidharth Puram, MD, PhD 
Department of Otolaryngology 
Telephone: (314) 362-7395 
Email: sidpuram@wustl.edu 
Chieh-Yu Lin, MD, PhD 
Department of Pathology & Immunology 
Telephone: (314) 273-6993 
Email: chieh-yu@wustl.edu  
Richard Laforest, PhD.  
Department of Radiology 
Telephone: 314-362-8423 
Email: rlaforest@wustl.edu  
Study Drug: 64Cu-DOTA-ECL1i 
Applicable IND(s): 137620 – Dr. Robert Gropler 
Clinical Trials.gov #: NCT04217057 
 
 

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Imaging CCR2 Receptors with 64Cu-DOTA-ECL1i in Head and Neck Cancer  
Protocol Revision History 
 
Initial submission version:  10/14/2019 
 
Date 
Protocol Section  
Summary of Changes 
Submission 
Information  
11/13/2019 6.10.0 Regulatory and Reporting 
Requirements 
Updated to match 
SOP for Siteman 
Cancer Center 
Received 11/2019 
Pending RDRC 
review 
Pre HRPO Review 
Pre FDA submission 
FDA submitted Jan 
2020 
  
04/14/2020 
Title Page 
Update HRPO 
number, CT.Gov 
number, protocol 
version number and 
date 
PRMC approved 
07/22/2020 
 FDA 
Dec 2020 
04/14/2020 
Footer all pagers 
Update date and add 
page numbers 
PRMC approved 
07/22/2020 FDA 
Dec 2020  
4/14/2020 
6.1.0 Background 
Additional 
information on 
immunostaining for 
CCR2  
PRMC approved 
07/22/2020 
FDA Dec 2020  
4/14/2020 
6.2.0 Objectives 
Addition of an 
objective related to 
assessing the 
relationship between 
CCR2 expression 
and HPV status  
PRMC approved 
07/22/2020 
FDA Dec 2020 
04/14/2020 
6.5.2 Patient Population 
Add information on 
analysis of HPV+ 
and HPV- tissue 
samples 
PRMC approved 
07/22/2020 
 FDA  Dec 2020 
4/14/2020 
6.5.9 Immunohistochemical 
Evaluation of Chemokinetic 
Receptors 
Adding a 
retrospective section 
to assess the 
expression of CCR2 
in HPV+ and HPV- 
HNSCC metastasis 
PRMC approved 
07/22/2020 
FDA  Dec 2020 

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Renewal Version 2a Dated 11/02/2020 
11/09/2020 
Title Page 
Update protocol 
version number and 
date for renewal 
submission  
PRMC approved 
11/25/2020 
 FDA Feb 2022 
Protocol Amendment Version 3 03/31/21 
03/31/21 
Title Page 
Update protocol 
version number and 
date for renewal 
submission  
 
03/31/21 
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Update version 
number and date 
 
03/31/21 
Throughout protocol 
Correct 
syntax/punctuation 
 
03/31/21 
Study Coordinators 
Remove Helen 
Kaemmerer, add 
Austin Turner 
 
03/31/21 
Schema 
Revise to include 
patients who will 
receive other 
definitive therapy  
 
03/31/21 
6.4.0 Inclusion Criteria/ 
Exclusion Criteria 
Revise to include 
patients who will 
receive other 
definitive therapy 
 
03/31/21 
6.5.2 Patient Population 
Revise to include 
patients who will 
receive other 
definitive therapy 
 
03/31/21 
6.5.8 Safety & Clinical 
Follow-Up 
Revise to include 
patients who will 
receive other 
definitive therapy 
 
03/31/21 
6.12.0 Data Submission 
Schedule 
Add Tissue 
Collection Form and 
submission schedule 
 
03/31/21 
6.13 
Study Calendar 
Add tissue collection 
 
 
Renewal Version 3a Dated 10/12/2021 

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4 
10/12/2021 
Title Page 
Update protocol version 
number and date for 
renewal submission  
PRMC approved 
11/02/2021 
FDA Feb 2022  
Amendment V4 02/15/2022 
02/15/2022 
Title Page 
Update protocol version 
number and date for 
submission  
Oncore Updated Feb 
15, 2022 
FDA Feb 2022 
Title Page 
Update Study team 
members (statistician, 
pathology, radiology 
team) 
Footer all pagers 
Update version number 
and date 
Renewal Version 4a 09/12/2022 
09/12/2022 
Title Page 
Update protocol version 
number and date for 
renewal submission  
PRMC 10/11/2022 
FDA 02/06/2023  
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Update version number 
and date 
Renewal / Amendment Version 5 08/28/2023 
08/28/2023 
Title Page 
Update protocol version 
number and date for 
renewal submission 
Remove study coordinators 
Pending PRMC 
Pending FDA  
Footer all pagers 
Update version number and 
date Fix pagination error(s) 
Formatting all pages  
Document consistency fix 
spacing etc 
Contents 
Update table of Contents 
6.5.3 PET 
Imaging 
6.13 Study 
Calendar 
Clarify metabolites are 
collected whenever 
possible 
6.8.0 Risks and Benefits 
Fix typographical error 
referencing PDAC 
&Clarify metabolites are 
collected whenever 
possible  
 
 

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Contents 
SCHEMA ...................................................................................................................................6 
6.1.0 Background .....................................................................................................................7 
6.2.0 Objectives ..................................................................................................................... 10 
6.3.0 Statement of Qualifications ............................................................................................ 10 
6.4.0 Inclusion Criteria/ Exclusion Criteria ............................................................................. 10 
6.4.1 Inclusion of Women and Minorities ........................................................................... 11 
6.4.2 Registration Procedures & Screen Failures................................................................. 11 
6.5.0 METHODS ................................................................................................................... 11 
6.5.1 Study Design ............................................................................................................. 11 
6.5.2 Patient Population ...................................................................................................... 11 
6.5.3 PET Imaging ............................................................................................................. 12 
6.5.4 PET Image Processing and Analysis .......................................................................... 13 
6.5.5 Vital Signs ................................................................................................................. 14 
6.5.6 Clinical Laboratory Testing ....................................................................................... 14 
6.5.7 Electrocardiograms (ECGs) ....................................................................................... 15 
6.5.8 Safety & Clinical Follow-Up ..................................................................................... 16 
6.5.9 Immunohistochemical Evaluation of Chemokinetic Receptors ................................... 16 
6.6.0 Drug Preparation ........................................................................................................... 16 
6.7.0 Statistics ........................................................................................................................ 17 
6.8.0 Risks and Benefits ......................................................................................................... 17 
6.9.0 Record Keeping ............................................................................................................. 18 
6.10.0 Regulatory and Reporting Requirements ........................................................................ 18 
6.10.1 
Sponsor – Investigator / WU PI Reporting Requirements ....................................... 19 
6.10.1.1 
Reporting to the Human Research Protection Office (HRPO) at Washington 
University) 19 
6.10.1.2 
Reporting to the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University ................................................................................. 19 
6.10.1.3 
Reporting to the FDA ..................................................................................... 19 
6.10.2 
Exceptions to Expedited Reporting ........................................................................ 20 
6.11.0 Data and Safety Monitoring ........................................................................................... 20 
6.12.0 Data Submission Schedule ............................................................................................. 21 
6.13 
Study Calendar .............................................................................................................. 22 
6.14 
References ..................................................................................................................... 23 
APPENDIX A: Definitions for Adverse Event Reporting .......................................................... 25 
APPENDIX B: Reporting Timelines ...........................................................................................3 
 
 
 

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SCHEMA 
 
Imaging CCR2 Receptors with 64Cu-DOTA-ECL1i in Head and Neck Cancer  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Research 64Cu-DOTA-ECL1i-
PET/CT  
Patients with New Diagnosis of Head & Neck Cancer who are 
Candidates for Surgical Resection Or non-surgical Candidates 
with Sufficient Biopsy for Analysis of CCR2 (n=20) 
Baseline Standard of Care Clinical and 
Radiological Studies Including CT and/or MRI 
Tumor Tissue Analysis  
For Assessment of CCR2 Expression 
Per available tissue from either SOC Surgical Resection OR 
SOC Diagnostic Biopsy   

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6.1.0 Background  
Chemokines, also known as chemoattractant cytokines, are a super family of small secreted 
proteins initially characterized by their ability to induce leukocyte migration. They are small 
proteins known as cytokines that have chemoattractant properties and, thus, are called chemokines. 
One of their most critical functions is governed by the specific amino acid sequence (or motif) of 
Glu-Leu-Arg (or ELR) immediately preceding the first cysteine of their CXC motif. ELR+ CXC 
chemokines are considered angiogenic, whereas ELRCXC chemokines are considered 
angiostatic1. To date, more than 50 chemokines and 20 chemokine receptors have been identified. 
Chemokines and their receptors have a highly conserved sequence throughout the genetic tree 2. 
They are grouped into 4 distinct groups (C, CC, CXC, and CX3C), which are based on the number 
and position of conserved cysteine residues near the NH2 terminus of the molecule 3. The specific 
effects of the chemokines on their target cells are mediated by members of a family of 7-
transmembrane-spanning, G-protein–coupled chemokine receptors. Most chemokines are able to 
bind with high affinity to multiple receptors while their receptors usually are restricted to a single 
subclass and induce an intensive signaling cascade of activated second messengers that lead to cell 
motility and multiple other functional effects in the target cells. Chemokines insert their cellular 
trafficking and processes are influenced significantly by chemokines via their receptors.  
Chemokine Receptors: Most chemokine receptors bind specifically, but indiscriminately 
to their chemokine ligands. Chemokine receptors exist in many epithelial and hematopoietic cells. 
These receptors are members of the 7-TM GPCR super family. Like chemokines, chemokine 
receptors also share structurally conserved/homologous regions, which allow their subgrouping 
classification. In general, upon binding to their ligands, chemokine receptors undergo 
conformational changes that allow the binding of G proteins to intracellular loop epitopes and the 
carboxy terminal tail of the receptors 3. The second intracellular loop of the receptors contains an 
Asp-Arg-Tyr-Leu-Ala-Ile-Val (DRYLAIV) motif, which is missing in the nonsignaling receptors 
and does not permit G-protein coupling 2 .The chemokine receptors that do not bind to G proteins 
may act in synchrony with other proteins to support their effects. Examples of these alternative 
cohorts include b-arrestin, integrins, and growth factor receptors 4-9.  
Chemokines are vital for leukocyte migration and activation, and physiologically they have 
been implicated in various immune, inflammatory and activation responses such as allergic 
disease, atherosclerosis, and wound healing. Furthermore, chemokines play an important role in 
the neoplastic processes of many organs and in determining the location of the metastatic spread 
of cancer cells. For example in squamous cell carcinoma of head and neck, chemokine receptors 
orchestrate critical functions in oral tumorigenesis by favoring angiogenesis, tumor growth and 
metastasis 10,11. In prostate cancer, multiple chemokines and their receptors (including CCR5, 
CCR7, CCR9, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR7) are involved in the 
multistep process of metastasis 5,8,12-15. In cervical cancer, CXCR4 receptor has been shown to play 
an important role during cervical cancer invasion 16. In addition, CXCR7 expression has been 
shown to predict poor disease-free and disease-specific survival in cervical cancer patients, and 
might be a promising new therapeutic marker. In a large majority of cases, CXCR7 is co-expressed 
with CXCR4 and/or EGFR, supporting the hypothesis that these receptors assist in CXCR7 signal 
transduction 17. CXCR4 has been found to be upregulated in several other cancers such as breast, 
prostate, lung, bladder, ovarian, renal, oesophageal, colorectal, and pancreatic cancer, and 
lymphoma, melanoma, osteosarcoma, neuroblastoma, etc. 18. In addition, high level of CXCR4 
expression correlated with tumor progression and metastasis 19,20, as well as poor prognosis and 
resistance to chemotherapy 21,22. Thus, these receptors provide an important target for imaging and 
treatment of various types of cancers.  
 
CCR2 in Head & Neck Cancer: The importance of chemokines and their cognate 
receptors in head and neck cancers is being revealed by increasing amount of studies. Recently, 

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the expression of CXCR2 was reported substantially higher than that in paraneoplastic tissue in 
laryngeal squamous cell carcinoma 23. Elevated expression was significantly related with lymph 
node metastasis, histological grade, and 5-year survival 23. Thus, CXCR2 expression could be 
considered as a potent prognostic marker for laryngeal squamous cell carcinoma patients 23. 
Several studies have also documented the significance of CXCR4 in HNSCC tumor progression 
and organ-specific metastasis24,25. Wang et al investigated the expression of CXCR4 in 
nasopharyngeal carcinoma tissues, and they found that CXCR4 expression was elevated in tumor 
tissues and the increased expression was correlated with metastatic rates in patients as well as poor 
overall survival 26. This finding was consistent with another study which reported that CXCR4 
mRNA was significantly higher in HNSCC tissues than in paraneoplastic tissues and its expression 
were associated with lymph node metastasis and distant metastasis 27. These findings clearly 
demonstrate that CXCR4 could also be used to predict prognosis and metastasis in HNSCC 
patients.  
Chen et al. reported that a 64I mutation of the CC chemokine receptor-2 (CCR2) is 
significantly associated with oral cancer susceptibility 28. Also, another group demonstrated that 
mutation of MCP-1 gene, which encodes CCR2 ligands, increased the expression of MCP-1 
resulting in elevated CCR2 activity which could increase the risk for developing oral squamous 
carcinoma29. The authors predicted that MCP-1 and CCR2 could be used as genetic markers for 
diagnosis of oral carcinoma squamous29. In addition, Fujita et al. found that The CCL2-CCR2 axis 
is associated with lymphatic metastasis in oral squamous cell carcinoma30. Yang et al. have shown 
that CCL2-CCR2 axis could promote nasopharyngeal carcinoma (metastasis by activating 
ERK1/2-MMP2/9 pathway. This study helps to develop novel therapeutic targets for distant 
metastasis in nasopharyngeal carcinoma31. Another CC chemokine receptor, i.e., CCR7, which 
plays a critical role in the migration of activated dendritic cells to regional lymph nodes, was also 
found elevated in HNSCC tumor tissues compared with paraneoplastic tissues and the elevated 
expression of CCR7 was correlated with lymph metastasis and tumor tissue differentiation status27. 
Another study evaluated the expression of CCR7 in primary and metastatic tumor cell lines and 
biopsies from both primary and metastatic lesions, and it was found that CCR7 expression was 
elevated in metastatic cells and tissues27. These findings reveal an important role of CCR7 in 
mediating the metastasis and tumor malignancy in head and neck squamous cell cancer patients. 
Taken together, accumulating evidence indicates that the altered expressions of various chemokine 
receptors (and their cognate ligands as well) in head and neck squamous cell cancer are associated 
with different cellular events including cell survival, tumor progression, and metastasis. The 
expression of chemokine receptors is associated with tumor cell differentiation and prognosis of 
head and neck squamous cell cancer patients. Moreover, their expression is also, to some degree, 
histologically specific, i.e., the expression of these chemokine receptors indicates it is the 
squamous cell carcinoma rather than other types (such as adenoid cystic carcinoma)32. These 
features, along with other tumor biomarkers, may help oncologists to assess their patients more 
thoroughly 
In the last few decades, human papillomavirus (HPV) has emerged as the key etiologic 
factor driving the rising incidence of oropharyngeal squamous cell cancer 33, 34 HPV-related 
oropharyngeal squamous cell cancer has emerged as a distinct clinical entity of head and neck 
cancer with expected high survival. This recognition has led to the investigation of whether a 
population of patients can be identified who can safely undergo treatment de-escalation, in an 
effort to minimize long-term treatment toxicity while maintaining excellent survival. The 
relationship of HPV and CCR2 has not been established in head and neck cancer. As the number 
of patients in this study is limited and we often do not know the HPV status of the patients, it will 
be extremely important to assess the CCR2 expression in the cancer tissues of HPV+ and HPV- 
patients to understand better the relationship of CCR2 expression and HPV. In future studies, we 
will be able to assess whether presence of CCR2 in each group is predictive of worse prognosis 

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and more aggressive therapy is needed. This will be very important as treatment deintensification 
for HPV+ squamous cell cancer of head and neck cancer is being evaluated.  
 
 
Positron Emission Tomography (PET) Using CCR-Based Tracers: PET imaging of 
tumor metabolism has been extensively used for staging, treatment planning and monitoring of 
response in various types of malignancies. Due to the importance of chemokine receptors, attention 
has been made to develop radiolabeled compounds that targets these receptors.  
 
 
 
64Cu-DOTA-ECL1i-PET/CT: 
Our group has developed a CCR2 specific PET radiotracer based on the peptide, ECL1i 
(d(LGTFLKC)) and radiolabeled with 64Cu (64Cu-DOTA-ECL1i) 35. The CCR2 detection 
sensitivity and specificity has been demonstrated in multiple preclinical studies and ex vivo human 
tissues 36-38. We have tested this tracer in preclinical inflammation models, human pancreatic 
cancer and human head and neck cancer and demonstrated a positive correlation between the PET 
signals and CCR2 expression.  
Ex vivo characterization of 64Cu-DOTA-
ECL1i binding to CCR2 expressed in 
human head and neck cancer tissue: 
CCR2 
immunohistochemistry 
was 
performed on the de-identified human 
cancerous tonsil tissue collected from the 
SCCTPC (Figure 1A). As shown in 
Figure 1B, strong CCR2 (brown color) 
signals were detected in the area of 
tumor cells. Low CCR2 signals were 
also measured in the muscle and soft 
tissue, likely due to the expression of the 
receptors on the infiltrated macrophages 
or inflammatory cells. The control IgG 
staining showed negative signal of 
CCR2, 
confirming 
the 
specific 
expression of CCR2 on these tumor 
cells. The ex vivo binding assay of 64Cu-
DOTA-ECL1i using autoradiography 
showed strong binding to the cancerous 
area with signals in similar profile to the 
CCR2 IHC, indicating the specific 
binding of the tracer to the CCR2 
receptors on tumor cells (Figure 1C). The competitive receptor blocking using non-radiolabeled 
DOTA-ECL1i conjugate in excess amount (DOTA-ECL1i: 64Cu-DOTA-ECL1i molar ratio = 
100:1) significantly blocked the binding to the tissue. Quantitative comparison of counts in binding 
and blocking tissues demonstrated 50.8 ± 0.6% (n=4, p<0.005) blocking efficiency (Binding was 
set as 1 for each specimen), confirming the binding specificity (Figure 1D).  
 
Figure 1. Ex vivo characterization of 64Cu-DOTA-ECL1i
binding to CCR2 in human head and neck cancer tissue. 
(A) H&E. (B) CCR2 DAB staining showed high expression of 
the receptor in the area full of tumor cells. Control IgG staining 
confirmed 
the 
receptor 
staining 
specificity. 
(C)
Autoradiography showed tracer binding to the cancerous area 
of the tissue and the specificity was verified by the blocking. 
(D) Quantitative comparison of counts in binding and blocking 
tissues. 

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Summary: CCR2 is a significant prognostic biomarker in head and neck cancer. Currently there 
is no clinical biomarker to study CCR2, its prognostic significance or to select patients for CCR2-
targeted therapy and to monitor response to such therapy.  Our group has developed a CCR2 
specific PET radiotracer based on the peptide, ECL1i (d(LGTFLKC)) and radiolabeled with 64Cu 
(64Cu-DOTA-ECL1i).  We also have found that 64Cu-DOTA-ECL1i specific binding has been 
demonstrated in human head and neck cancer tissue.  Currently 64Cu-DOTA-ECL1i is being 
studied in several clinical settings under IND (#137620) at Washington University.  Our group is 
studying 64Cu-DOTA-ECL1i in patients with head and neck cancer.  The use of 64Cu-DOTA-
ECL1i has proven to be safe in our ongoing clinical trials with favorable radiation dosimetry (0.064 
rem/ mCi) and is compatible with other imaging tracers such as 18F-Fludeoxyglucose (FDG) 
 
6.2.0 Objectives 
We are planning to perform a trial with goals to evaluate tumor uptake of 64Cu-DOTA-
ECL1i in patients with squamous cell head and neck cancer with at least one measurable lesion of 
≥ 1.5 cm. We also will correlate the tumor uptake of 
64Cu-DOTA-ECL1i with 
immunohistochemical staining for CCR2 receptors in the tumor specimens.  
Study objectives:  
6.2.1 To determine the diagnostic quality and CCR2 expression detection rate of 64Cu-
DOTA-ECL1i-PET/CT images.  
6.2.2 To evaluate the relationship 
between 
64Cu-DOTA-ECL1i uptake and 
immunohistochemical staining for various types of chemokine receptors such as 
CCR2.  
6.2.3  To assess the association of CCR2 expression and HPV status. CCR2 
immunohistochemical staining in HPV+ and HPV- squamous cell cancer of the 
head and neck.  To better assess the analysis of tissue prospectively we will 
retrospectively review a limited number of identified samples obtained from the 
Department of Pathology at Washington University (20 total samples).   
 
6.3.0 Statement of Qualifications  
The study will be done under eIND (#137620).  Robert J. Gropler, M.D., Washington 
University School of Medicine, St. Louis, Missouri is the physician-investigator of the IND.  
 
6.4.0 Inclusion Criteria/ Exclusion Criteria 
Adult patients (men or women) with confirmed new diagnosis of squamous cell head and 
neck cancer who are being evaluated for surgical resection or definitive therapy  are eligible 
to participate in this study.   
 
Inclusion Criteria:  
• Adult patient 18 years of age or older 
• Cytology or biopsy-proven squamous cell head and neck cancer scheduled to be treated 
with standard of care surgery. Patients who are not surgical candidates should have 
adequate tissue from tumor biopsy for analysis of CCR2.  
•  Lesion size of at least 1.5 cm (treatment naïve)  
• Able to give informed consent 
• Not currently pregnant or nursing:  Subject must be surgically sterile (has had a 
documented bilateral oophorectomy and/or documented hysterectomy), post-
menopausal (cessation of menses for more than 1 year), non-lactating, or of 

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childbearing potential for whom a urine pregnancy test (with the test performed within 
the 24 hour period immediately prior to administration of 64Cu-DOTA-ECL1i is 
negative  
 
Exclusion Criteria: 
• Patients with other invasive malignancies, with the exception of non-melanoma skin 
cancer, who had (or have) any evidence of the other cancer present within the last 2 
years 
• Unable to tolerate approximately 90 min (total time) of PET/CT imaging. 
 
6.4.1 Inclusion of Women and Minorities 
Both men and women are eligible. Members of all races and ethnic groups are 
eligible for this trial. 
 
6.4.2 Registration Procedures & Screen Failures 
Initial eligibility will be confirmed by the study coordinator.  The PI, Co-PI or other 
engaged study investigator will review and sign an eligibility checklist to confirm 
the patient is eligible to participate on study.  All enrolled subjects will be given a 
unique study specific patient identification and registered through the Siteman 
Cancer Center OnCore database at Washington University.  Screen failures are 
those subjects who consent to and undergo any study related procedures.  A subject 
who signs consent in clinic but declines to participate after further discussion of the 
imaging protocol and study requirements with the study coordinator is not 
considered a screen failure and will not be registered to the protocol since no study 
procedures took place.   
 
 
6.5.0 METHODS  
6.5.1 Study Design  
A single center, open-label, baseline-controlled diagnostic imaging study designed to 
assess the diagnostic quality and CCR2 expression detectability of 64Cu-DOTA-ECL1i 
imaging with PET/CT. In addition, data will be collected to build the ongoing safety 
profile of 64Cu-DOTA-ECL1i and uptake will be correlated to in vitro assays of 
chemokines receptor from clinically obtained biopsy/surgical specimen. 
     
6.5.2 Patient Population  
Twenty adult individuals with new diagnosis of squamous cell of the head and neck 
cancer who are scheduled for definitive therapy (surgery for fresh tissue collection or 
other standard of care therapy who have  adequate tissue from tumor biopsy for analysis 
of CCR2) and have not received treatment for their cancer will be studied.  Written 
informed consent will be obtained prior to the PET imaging session.  64Cu-DOTA-
ECL1i-PET/CT imaging consisting of a dynamic scan centered at the level of the 
known tumor followed by a limited body scan of the head/neck and upper chest will be 
performed in each patient.  64Cu-DOTA-ECL1i -PET/CT images will also be correlated 
with all available imaging studies to assess lesion localization.   
 

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To aid prospective tissue analysis a total of 20 samples (10 each: HPV+ and HPV-) 
will be obtained from the Department of Pathology at Washington University and 
immunohistochemical staining of CCR2 will be performed.  
 
6.5.3 PET Imaging  
All subjects will have a minimum of 24 hours to review the consent form before 
agreeing to participate in this research project. All PET imaging will be performed with 
a PET/CT scanner with preference given to scanners located in the Center for Clinical 
Imaging Research Center (CCIR) located on the 10th floor West Pavilion of Barnes-
Jewish Hospital.   
 
All patients will undergo routine clinical staging as dictated by the treating physician. 
The results of 64Cu-DOTA-ECL1i-PET/CT will not be provided to the patient or the 
treating oncologist/surgeon unless, in the judgment of the principal investigator, the 
images demonstrate an unsuspected abnormality that may warrant further evaluation.  
 
Serum Stability and Metabolite Analysis (whenever possible): 64Cu-DOTA-ECL1i has 
been shown to be stable up to 60 min in preclinical studies.   Our preclinical studies 
showed 95% of the tracer was intact after 1 hour incubation with mouse serum. In order 
to assess serum stability and for metabolite analysis of 64Cu-DOTA-ECL1i in this 
patient population, 2 venous samples (approximately 1 mL each) will be obtained (1 
within the first 5 min of imaging and 1 at the completion of the first hour of imaging). 
Additional blood samples may be obtained based on results of the 2 samples to increase 
the time points, if necessary. 
 
For each PET/CT study, when collected, high performance liquid chromatography 
(HPLC) analysis of the serum stability and metabolite profile will be performed by Dr. 
Liu Lab 37,38. Whole blood will be centrifuged; plasma and packed red cells will be 
separated and the radioactivity in each portion will be recorded.  A 200 µL aliquot of 
plasma will be diluted with 200 µL MilliQ water and passed through a 0.22 micron 
filter prior to HPLC injection.  Eluent fractions will be collected every 1 minute and 
analyzed by gamma counting.     
 
6.5.3.1 PET Imaging Procedure 
Following administration of 8-10 mCi of the radiotracer 64Cu-DOTA-
ECL1i-PET/CT, all twenty subjects who enter on study will undergo a 60 minute 
dynamic scan centered over the site of the known tumor.  An additional limited 
scan (top of head to upper/mid-chest) will be performed approximately 1-3 hours 
after administration. 
 
For dynamic imaging, the subject will be placed supine on the imaging table with 
arms secured comfortably by the side of the body.  A low dose spiral CT scan for 
attenuation correction will be obtained centered on the primary tumor site.  The CT 
will consist of a 5-20 second topogram for determining correct anatomical 
positioning followed by a spiral CT at a maximum of 50 mAs. Care dose will be 
calculated for each scan and the care dose imaging parameters will be used if less 
than the maximum 50 mAs is calculated. CT imaging will be acquired with a 
standard 120 kVp. Average spiral CT scan time is 5-30 seconds. Scans are acquired 

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using a 3-5 mm slice thickness.  Immediately after the attenuation CT scan, subjects 
will be injected with 8 – 10 mCi 64Cu-DOTA-ECL1i .  The dose will be followed 
with a normal saline flush of 10-30 ml. A 60 min list mode dynamic imaging 
acquisition will follow study drug injection.  At the end of the imaging session, 
subjects will be encouraged to void.     
 
Subjects will be allowed to rest outside of the scanner until such time subject 
indicates they are willing to start the 2nd scan (1 – 3 hours post injection).  Scan #2 
will consist of a limited body scan from the vertex to upper/mid chest region.  
Following topogram and low dose CT scan for attenuation correction (maximum 
50 mAs with care dose imaging parameters) a 2 bed limited scan from vertex to 
upper / mid chest will be obtained at 3-5 minutes per bed position.     
To add to the existing safety profile of 64Cu-DOTA-ECL1i,  all subjects will 
undergo vital sign measurement, clinical laboratory testing, and ECG testing as 
specified in sections 6.5.5, 6.5.6 and 6.5.7 of the protocol.   
 
6.5.4 PET Image Processing and Analysis  
List mode dynamic PET images will be reconstructed using current scanner 
manufacturer recommendations as follows (frames x seconds) 12x10, 6x30, and 11x300.  
PET images will be evaluated qualitatively (graded on a five-point scale regarding the 
presence or absence of abnormal uptake: 1 = definitely negative; 2 = probably negative; 3 
= equivocal; 4 = probably positive; and 5 = definitely positive) and then, semi-
quantitatively (standardized uptake value (SUV), tumor-to-normal tissue ratio (T/N) or 
tumor-muscle-ratio (T/M)) with the knowledge of the location (correlating with all 
available imaging studies). SUV is a decay-corrected measurement of activity per volume 
of tissue (µCi/mL) divided by the average activity per unit mass in the entire body. The 
overall PET image quality also will be graded (using 4-point scale with 1 being the worse 
and poor quality, not acceptable for diagnostic interpretation and 4 being good image 
quality, similar to routine clinical studies. PET analysis will not be performed in real time, 
but performed in batches or after all subjects have been enrolled, however, the images will 
be evaluated qualitatively for incidental findings.   
The pharmacokinetic of the lesion time-activity curve using graphical analysis and 
tracer kinetic compartmental modeling will be determined.  Classical receptor-ligand 
models will be employed to describe the kinetics of the tracers and different models to 
optimize goodness-of-fit will be evaluated.  Goodness-of-fit will be judged by a nested F-
test for the number of parameters, Akaike Information Criterion (AIC), and the Schwarz 
Criteria.1-tissue with two kinetic rate constants and 2-tissue with 4 rate constants 
compartmental reversible models will be used to fit the data using the blood input function 
as input function.  The blood time activity curve will be determined noninvasively from a 
large 3D ROI traced over the aorta (OR carotid artery) and corrected for metabolites per 
above analyses, if available.  The measured lesion time-activity curves will be fitted to the 
1- or 2-compartment model prediction and added to the plasma component weighted by 
the tissue blood fraction Vb.  Best fit will be used to determine the distribution volume 
(DV) from the 1-tissue model (with DV = K1/k2), and we will determine the DV and the 
non-displaceable binding potential (BP) from the 2-tissue model (with DV = (K1/k2)(1+BP) 
and BP = k3/k4).  The graphical analysis using Logan and Patlak analysis to determine the 
volume of distribution (DV) also will be performed. We will further use both the plasma 
time activity curve and a reference region to determined DV and DVR, respectively with 
non-displaceable BP = DVR -1.  A possible region of reference will be a suitably located 

Version #5 08/28/2023 
 
14 
muscle in the same field of view as head and neck cancer. All quantitative and semi-
quantitative metrics of 64Cu-DOTA-ECL1i-PET/CT will be correlated with CCR2 receptor 
expression and binding affinity of each biopsy-proven tumor specimen.   
 
6.5.5 Vital Signs 
All vital signs will be recorded on the case report form. Vital signs may be obtained 
with the subject in the supine or upright position. Care will be taken to obtain subsequent 
recordings with the subject in the same position (supine or upright). Although allergic or 
other immediate adverse reactions are not anticipated, subjects will be monitored for at 
least 60 min post injection. Vital signs will be obtained at 3 time points pre-injection 
(anticipated to be within 30 mins prior to injection of 64Cu-DOTA-ECL1i although it might 
be longer if delays in quality control testing result in delay of study drug injection), at the 
completion of dynamic imaging and at study discharge.    Vital signs will include the 
following: heart rate, systolic blood pressure, diastolic blood pressure, respiratory rate, and 
body temperature. The following changes from baseline will be considered noteworthy:   
 
Heart rate: 
 
 
> 30 beats per min 
 
Systolic blood pressure 
> 40 mm Hg 
 
Diastolic blood pressure 
> 30 mm Hg 
Noteworthy changes will be documented on the Principal Investigator Data Safety 
Review Form. The Principal Investigator will indicate on the form whether or not the 
changes in vital signs are clinically significant. If clinically significant, the principal 
investigator will assess the causality of the change to the injection of 64Cu-DOTA-ECL1i.. 
If a clinically significant change of a vital sign is noted and assessed as being attributable 
to 64Cu-DOTA-ECL1i injection by the Principal Investigator on the Data Safety and 
Review CRF, it will be reported on the adverse event log.  
 
6.5.6 Clinical Laboratory Testing 
Laboratory tests will consist of the following: standard CBC, and comprehensive 
metabolic panel obtained at the following time points:  
• Baseline: 
Baseline labs may be obtained anytime within 21 days or 
immediately prior to 64Cu-DOTA-ECL1i injection and can be done as 
standard of care or for research purposes.   
• Discharge: At least 60 minutes post injection or prior to study discharge.   
Approximately 15 mL of blood will be collected for clinical laboratory testing, serum stability 
/ metabolite analysis.     
 
Labs obtained at baseline prior to injection of 64Cu-DOTA-ECL1i will be compared to labs 
obtained at study discharge.  The following changes from baseline clinical laboratory values 
are considered to be noteworthy and require assessment as to clinical significance when they 
fall outside of normal limits. Clinically significant changes in laboratory values which are 
attributed to 64Cu-DOTA-ECL1i injection should be followed up until they return to baseline 
or normal levels, or until follow-up is no longer warranted. Laboratory values that are abnormal 
at baseline but move into normal range will not be considered clinically significant.  If a 
clinically significant change of a laboratory value is noted, it will be reported on the PI Data 
Safety and Review Form so that the PI can assess the change as being attributable to 64Cu-
DOTA-ECL1i injection.  All clinically significant laboratory changes as identified below 

Version #5 08/28/2023 
 
15 
which are identified by the PI as being attributable to 64Cu-DOTA-ECL1i will be recorded on 
the adverse event log.   
Clinically Significant Laboratory Values 
Analyte 
Change from baseline 
Hemoglobin 
> 2g/dL 
WBCs 
> 1 K/mm3 
Neutrophils  
> 10 % 
Lymphocytes  
> 10% 
Platelets 
> 50 K/mm3 
Creatinine 
> 0.75 mg/dL 
BUN 
> 20 mg/dL 
Calcium 
> 1mg/dL 
Sodium 
> 5 mmol//L 
Potassium 
> 0.5 mmol/L 
CO2 
> 4 mmol/L 
ALT (SGPT)  
> 150 IU/L 
AST (SGOT) 
> 100 IU/L 
Alkaline Phosphatase 
> 150 IU/L 
Total Bilirubin 
> 0.5 mg/dL 
Albumin  
> 1g/dL 
 
6.5.7 Electrocardiograms (ECGs)  
A standard 12-lead ECG will be obtained on all subjects at baseline (within 
approximately 30 mins prior to injection of 64Cu-DOTA-ECL1i although it might be 
longer if delays in quality control testing result in delay of study drug injection), and at 
least 60 minutes post injection or prior to study discharge.    
The following table lists criteria for normal limits and clinically notable limits 
for ECGs in adults.  
 
Criteria for Normal Limits and Clinically Notable Limits for ECGs in Adults 
 
Normal Limits (msec) 
Notable Limits (msec) 
ECG Variables 
Low 
High 
Low  
High 
PR interval 
120 
200 
<120 
>200 
QRS interval 
50 
100 
< 50 
>100 
RR interval 
600 
1000 
<600 
>1000 
QT interval 
No lower limit 
≥ 460 
No lower limit 
≥ 460 

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16 
ECG’s which have limits noted to be outside of the table above will be recorded on the PI Data 
Safety and Review Form where the PI will review and assess as being attributable to 64Cu-DOTA-
ECL1i injection.  Any ECG recordings identified by the PI as being attributable to 64Cu-DOTA-
ECL1i  will be recorded on the adverse event log. 
 
6.5.8 Safety & Clinical Follow-Up 
 
To continue collecting data for the safety profile of 64Cu-DOTA-ECL1i a follow up phone 
call or in person visit to assess for adverse events will occur at least 24 hours following 
administration of 64Cu-DOTA-EL1i.  Taking into consideration both the physical half-life (13 
hours) and the much quicker biological half-life optimal follow up time is 48-120 hours post 
injection but follow up as soon as 24 hours and as late as 14 days post administration are allowed.  
As this is a verbal secondary assessment follow up phone call or visit beyond 14 days will be 
accepted with proper documentation of delay(s) such as subject underwent surgical resection and 
unavailable due to surgery and subsequent recovery.  It is anticipated that some subjects may be 
scheduled for surgery as soon as 12 hours following 64Cu-DOTA-ECL1i administration and 
imaging so care will be taken to separate surgical related signs and symptoms from those which 
might be attributed to 64Cu-DOTA-ECL1i administration and PET/CT imaging.   
 
Clinically, subject’s medical record will be followed to document surgical resection or the start of 
definitive therapy.   
 
 
6.5.9 Immunohistochemical Evaluation of Chemokinetic Receptors 
To evaluate the relationship between 64Cu-DOTA-ECL1i uptake and expression of CCR2 
receptors in head and neck tumor samples, IHC staining in all patients who have available tissue 
will be performed using techniques routinely performed in Dr. Liu’s Laboratory 37,38. Intensity of 
CCR2 signal in IHC will be assessed by study pathologist, Dr. Chernock and categorized into 4 
categories (0=absent/faint, 1=weak, 2=moderate, 3=strong). Any cells that stained positively with 
CCR2 will be assessed and the final score will be based on an overall assessment of CCR2 IHC 
on all these cells.  It is anticipated that most subjects will have tumor available from planned 
surgical resection or prior biopsy however standard of care clinical analysis of resected tumor will 
take priority over research tissue testing.  Tumor testing will occur in batches or at the end of study 
recruitment depending on availability of testing supplies.   
To better understand how CCR2 expression correlates with HPV status and to correlate 
with samples obtained prospectively a small retrospective tissue analysis study will also be 
performed.  We will obtain tissue from 10 HPV+ and 10 HPV- squamous cell head and neck cancer 
subjects from the Department of Pathology at Washington University.  These samples will be 
analyzed, as described above, to establish baseline expectations on the correlation of CCR2 
expression to HPV status.   
  
 
6.6.0 Drug Preparation  
64CuCl2 will be produced by the Cyclotron Facility at Washington University School of 
Medicine, Mallinckrodt Institute of Radiology. 64Cu-DOTA-ECL1i will be produced in the 
Biologic Therapy Core Facility (ISO 7, class 10,000) at Washington University School of 
Medicine following SOPs and batch production records. To produce 64Cu-DOTA-ECL1i, 
64CuCl2 (20-70 mCi) will be buffered with 20 mM sodium acetate stabilized with gentisic acid 
and then added to DOTA-ECL1i (30 μg) to bring the total volume to 500 μL (we do not expect 
any interference with CCR2/5 inhibitor therapeutic effect, because we use a trace amount of 

Version #5 08/28/2023 
 
17 
DOTA-ECL1i in comparison to 600 mg therapeutic dose, which is 20,000 higher than the 
imaging dose). This reaction is incubated in a 45ºC heated shaker (ThermoMixer, Eppendorf) 
for 45 minutes at 950 rpm during which time the DOTA chelator binds with 64Cu. The product 
will be evaluated by pre-release specifications and post-release specifications to control the 
quality of the radiotracer for clinical study. This study will be done under an eIND (#137620) 
for 64Cu-DOTA-ECL1i 
 
6.7.0 Statistics  
Demographic and clinical characteristics of the enrolled patients will be summarized using 
descriptive statistics with frequency and percentages used for categorical variables, median (25th, 
75th) for ordinal variables and mean (standard deviation) for continuous variables. The diagnostic 
quality of images and CCR2 expression detection will be assessed qualitatively and semi-
quantitatively for all participants and these measures (described above) will be summarized for a 
lesion using descriptive statistics. The association between 64Cu-DOTA-ECL1i uptake and tissue 
signal intensity for CCR2 and other chemokine receptors will be evaluated using a Spearman 
correlation coefficient. Collection of additional data for the safety profile will not undergo formal 
statistical review.    
The primary goal of this single center, open-label, baseline-controlled diagnostic imaging 
study is to assess the diagnostic quality and CCR2 expression detectability of 64Cu-DOTA-ECL1i 
in head and neck cancer patients. The study is primarily descriptive and the sample size is based 
on feasibility of recruitment. However, a sample size of 20 achieves 80% power to detect a 
correlation of 0.61 using a two-sided hypothesis test with a significance level of 0.05.  
 
6.8.0 Risks and Benefits 
Targeting CCR2 is emerging as a promising therapeutic strategy in many types of cancer, 
but accurate patient selection for this strategy is critical. The potential risks from this imaging 
protocol are expected to be very low. Expected risks include discomfort from the placement of 
intravenous catheter(s) for injection of 64Cu-DOTA-ECL1i, radiation exposure from the injection 
of 64Cu-DOTA-ECL1i and CT for attenuation correction scans, and discomfort from lying still on 
the imaging table. A temporary altered taste sensation may be associated with the IV injection of  
64Cu-DOTA-ECL1i.   Blood samples will be obtained for safety profile, whenever possible 
additional samples are collected for serum stability and metabolites testing.  There is a slight risk 
of bruising and a remote risk of infection from the placement of the intravenous catheter(s).  
Although unlikely, there is a rare chance of having patients suffer an allergic or other immediate 
adverse reaction to 64Cu-DOTA-ECL1i.  To minimize the risks to subjects, intravenous catheters 
will only be placed by trained personnel. All studies will take place under the supervision of the 
Principal Investigator or a collaborating physician. Additionally, the PET scanner is located within 
the hospital where advanced life support personnel and equipment are available for immediate use.  
Radiation Exposure: This research project involves radiation exposure to participants from 
the i.v. injection of 64Cu-DOTA-ECL1i. The total amount of radiation exposure received from one 
10 mCi injection is 0.64 rem.  Additional radiation exposure from 2 CT scans for attenuation 
correction will result in an additional 0.27 rem of exposure (including dose from positioning 
topogram – 0.09 rem from 1 bed dynamic scan + 0.18 rem from 2 bed limited scan)  Total radiation 
exposure from one 10 mCi injection of 64Cu-DOTA-ECL1i and 2 limited low dose CT scans for 
attenuation correction is 0.91 rems or approximately 18% of the annual dose for a radiation worker.   
The patients entered on this study will not directly benefit from 64Cu-DOTA-ECL1i-
PET/CT imaging, the overall and long-term benefits to future patients with head and neck cancer 
far outweigh the minimal risks anticipated.  The minimal risks should be far outweighed by the 

Version #5 08/28/2023 
 
18 
potential benefit to society.  It is anticipated that this study will contribute to the understanding of 
the performance of the 64Cu-DOTA-ECL1i-PET/CT in evaluating CCR2 expression in head and 
neck cancers that will have important impact on the selection of the patients for appropriate mode 
of therapy.  Ultimately, 
64Cu-DOTA-ECL1i-PET/CT may provide clinically important 
information to predict/assess the effectiveness of CCR2-targeted therapy.   
The results of the PET imaging studies to be performed as part of the proposed research 
are not intended to be used in the diagnostic evaluation or treatment planning of individual patients.  
The results of the PET studies will not be provided to the patient or the treating oncologist or 
surgeon unless the CT images demonstrate an unsuspected, potentially life-threatening 
abnormality that warrants further investigation and/or urgent therapy.  Dr. Dehdashti will assess 
all imaging results in light of other clinical and radiologic data and will inform the responsible 
attending physician as soon as possible if the information derived from the experimental imaging 
studies is judged to be of critical importance to the care of individual patients. 
 
6.9.0 Record Keeping 
It is anticipated that 20 subjects will be entered into this study. Initial review of images for 
quality and uptake will occur in real time with additional analysis performed in batches or after 
enrollment has been completed.  The pathological analysis of the CCR2 will be performed after 
study enrollment has been completed on the standard of care specimens obtained at surgical 
resection or from diagnostic biopsy. Data collected to ensure the safety profile of 64Cu-DOTA-
ECL1i is accurate will be assessed from the time prior to study drug injection through imaging 
discharge.   
 A patient file will be kept in the Research Coordinators’ suite located on the 10th floor of 
the Mallinckrodt Institute of Radiology in Barnes-Jewish Hospital South Campus. All digital data 
associated with 64Cu-DOTA-ECL1i-PET/CT is maintained on the PET acquisition computer. Raw 
data files are backed up on password protected encrypted external hard drive(s). All imaging data 
may also backed up on DVD after it has been transferred to a desktop PC. Processed data will be 
stored on the Principal Investigator's password protected desktop.  CRF's and patient files will be 
stored in the study coordinators' office. Entrance to the PI's and coordinators' offices requires 
admission through two separate locked doors.  Standard laboratory assessment (CBC + CMP) will 
be processed locally with results added to the subjects electronic medical record.  ECG’s are 
obtained for study purposes only and are not included in the electronic medical record.   
 
 
6.10.0 Regulatory and Reporting Requirements   
 
The entities providing oversight of safety and compliance with the protocol require reporting as 
outline below.  Please refer to Appendix A for definitions and Appendix B for grid of reporting 
timelines.  Adverse events will be tracked from start of treatment through study discharge.  All 
adverse events must be recorded on the toxicity tracking case report form (CRF) with the 
exception of: 
• 
Baseline adverse events, which shall be recorded on the appropriate study CRF 
• 
Events related to surgery or any testing or procedures performed in preparation for 
surgery 
 
Reporting requirements for Washington University study team may be found in Section 6.10.1   
 

Version #5 08/28/2023 
 
19 
6.10.1 Sponsor – Investigator / WU PI Reporting Requirements  
6.10.1.1 Reporting to the Human Research Protection Office (HRPO) at Washington 
University) 
Reporting will be conducted in accordance with Washington University IRB Policies. 
 
Pre-approval of all protocol exceptions unless they meet the definition of unforeseen 
imaging exception as described in Appendix A must be obtained prior to implementing 
the change.   
 
6.10.1.2 Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at 
Washington University 
The Washington University Sponsor Investigator / PI (or designee) is required to 
notify the QASMC of any unanticipated problems involving risks to participants or 
others occurring at WU or any BJH or SLCH institution that have been reported to 
and acknowledged by HRPO.  (Unanticipated problems reported to HRPO and 
withdrawn during the review process need not be reported to QASMC.) 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via 
email to qasmc@wustl.edu.  Submission to QASMC must include the myIRB form 
and any supporting documentation sent with the form. 
 
 
6.10.1.3 Reporting to the FDA 
The conduct of the study will comply with all FDA safety reporting requirements.  PLEASE 
NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER FROM REPORTING 
REQUIREMENTS FOR HRPO/QASMC.  It is the responsibility of the Washington University 
principal investigator to report to the FDA as follows: 
 
• 
Report any unexpected fatal or life-threatening suspected adverse reaction (refer to Appendix 
A for definitions) no later than 7 calendar days after initial receipt of the information.   
• 
Report a suspected adverse reaction that is both serious and unexpected (SUSAR, refer to 
Appendix A) no later than 15 calendar days after it is determined that the information 
qualifies for reporting.  Report an adverse event (refer to Appendix A) as a suspected adverse 
reaction only if there is evidence to suggest a causal relationship between the drug and the 
adverse event, such as: 
o A single occurrence of an event that is uncommon and known to be strongly associated 
with drug exposure 
o One or more occurrences of an event that is not commonly associated with drug exposure 
but is otherwise uncommon in the population exposed to the drug 
o An aggregate analysis of specific events observed in a clinical trial that indicates those 
events occur more frequently in the drug treatment group than in a concurrent or 
historical control group 
• 
Report any findings from epidemiological studies, pooled analysis of multiple studies, or 
clinical studies that suggest a significant risk in humans exposed to the drug no later than 15 
calendar days after it is determined that the information qualifies for reporting. 
• 
Report any findings from animal or in vitro testing that suggest significant risk in humans 
exposed to the drug no later than 15 calendar days after it is determined that the information 
qualifies for reporting.  

Version #5 08/28/2023 
 
20 
• 
Report any clinically important increase in the rate of a serious suspected adverse reaction of 
that listed in the protocol or IB within 15 calendar days after it is determined that the 
information qualifies for reporting. 
 
Submit each report as an IND safety report in a narrative format or on FDA Form 3500A or in an 
electronic format that FDA can process, review, and archive.  Study teams must notify the 
Siteman Cancer Center Protocol Development team of each potentially reportable event within 1 
business day after initial receipt of the information, and must bring the signed 1571 and FDA 
Form 3500A to the Siteman Cancer Center Protocol Development team no later than 1 business 
day prior to the due date for reporting to the FDA. 
 
Each notification to FDA must bear prominent identification of its contents (“IND Safety 
Report”) and must be transmitted to the review division in the Center for Drug Evaluation and 
Research (CDER) or in the Center for Biologics Evaluation and Research (CBER) that has 
responsibility for review of the IND.  Relevant follow-up information to an IND safety report 
must be submitted as soon as the information is available and must be identified as such 
(“Follow-up IND Safety Report”). 
 
 
6.10.2 Exceptions to Expedited Reporting 
Events that do not require expedited reporting as described in Section 1.1 include: 
• 
planned hospitalizations 
• 
hospitalizations < 24 hours 
• 
respite care 
• 
events related to disease progression 
• Unforeseen imaging exceptions as described in Appendix A 
Events that do not require expedited reporting must still be captured in the study files/ 
case report forms.   
 
6.11.0 Data and Safety Monitoring 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, 
the Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the 
Washington University Quality Assurance and Safety Monitoring Committee (QASMC) semi-
annually beginning six months after accrual has opened (if at least one patient has been enrolled) 
or one year after accrual has opened (if no patients have been enrolled at the six-month mark). 
 
For imaging studies, the Principal Investigator will review all patient data at least every six 
months, and provide a semi-annual report to the QASMC. This report will include: 
• 
HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
• 
Date of initial HRPO approval, date of most recent consent HRPO approval/revision, date 
of HRPO expiration, date of most recent QA audit, study status, and phase of study 
• 
History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
• 
Study-wide target accrual and study-wide actual accrual 
• 
Protocol activation date 

Version #5 08/28/2023 
 
21 
• 
Average rate of accrual observed in year 1, year 2, and subsequent years 
• 
Expected accrual end date and accrual by cohort 
• 
Objectives of protocol with supporting data and list the number of participants who have 
met each objective 
• 
Measures of efficacy (phase I studies only if efficacy is objective of the protocol) 
• 
Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
• 
Power analysis and/or interim analysis (if described in the protocol) 
• 
Summary of toxicities separated by cohorts with the number of dose-limiting toxicities 
indicated 
• 
Abstract submissions/publications 
• 
Summary of any recent literature that may affect the safety or ethics of the study  
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC 
according to institutional guidelines. 
 
6.12.0 Data Submission Schedule  
 
Case report forms with appropriate source documentation will be completed according to the 
schedule listed in this section. 
 
Case Report Form 
Submission Schedule 
Eligibility check List 
Prior to registration  
Original Consent Form 
Prior to registration 
PET Imaging Form  
Real Time completion of events as they occur 
PET Imaging Follow up 
Form 
Real time to comply with protocol requirements 
Adverse Event Log  
After follow up has been completed  
PI Data Safety Review 
After follow up and AE forms have been completed  
Tissue Collection Form  
At time of surgery or requested after follow up from 
standard of care biopsy sample  
Image Analysis Form  
Per PI Schedule  
 
 
 

Version #5 08/28/2023 
 
22 
6.13 
Study Calendar  
 
Procedure 
Baseline(a) 
Imaging(b) 
Follow up 
Phone 
Call/Visit (d) 
Tissue 
Collection  
Informed Consent 
X 
 
 
 
Medical History 
X 
 
 
 
Radiology Reports 
X 
 
 
 
Pathology Report(s) 
X 
 
 
 
Blood for Laboratory/ 
Serum Stability & 
Metabolite Analysis  
X(c) 
X 
 
 
Vital Signs(d) 
X 
X 
 
 
ECG(e) 
X 
X 
 
 
PET/CT Imaging  
 
X 
 
 
AE Assessment  
X 
X 
X 
 
SOC Tissue Collection (f) 
 
 
 
X 
(a) Baseline is defined as period from initial subject consent until injection of 64Cu-
DOTA-ECL1i Baseline for vital signs and ECG is within approximately 30 mins prior 
to injection of 64Cu-DOTA-ECL1i.  Baseline blood for laboratory samples is within 21 
days up to immediately prior to 64Cu-DOTA-EL1i administration.  
(b) Imaging day is defined as period of time from injection of 64Cu-DOTA-ECLi1 
through completion of imaging on that day.   
(c) Baseline lab is CBC/CMP prior to study drug injection.  Serum Stability /Metabolite 
samples are drawn post injection and post scan #1 whenever possible.  Discharge labs 
are repeat CBC/CMP drawn at least 60 minutes post injection.   
(d) Follow up will occur at least 24 hours following administration of 64Cu-DOTA-
ECL1i optimal follow up time is 48-120 hours however follow up as soon as 24 hours 
and as late as 14 days is acceptable.   
(e) To be performed prior to and at least 60 min after administration of 64Cu-DOTA-
ECL1i.   
(f) Fresh tissue is collect at the time of surgical resection.  If other therapy is planned 
tissue or slides will be requested from standard of care biopsy samples. 
 
 

Version #5 08/28/2023 
 
23 
6.14 
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25 
APPENDIX A: Definitions for Adverse Event Reporting 
 
A. 
Adverse Events (AEs) 
 
As defined in 21 CFR 312.32: 
 
Definition: any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered drug-related. 
 
Grading: the descriptions and grading scales found in the revised NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for all toxicity reporting.  A copy 
of the CTCAE version 5.0 can be downloaded from the CTEP website. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms listed that 
should be used are those provided by the Department of Health and Human Services’ Office for 
Human Research Protections (OHRP).  A copy of this guidance can be found on OHRP’s website: 
http://www.hhs.gov/ohrp/policy/advevntguid.html 
 
B. 
Suspected Adverse Reaction (SAR) 
 
As defined in 21 CFR 312.32: 
 
Definition: any adverse event for which there is a reasonable possibility that the drug caused the 
adverse event.  “Reasonable possibility” means there is evidence to suggest a causal relationship 
between the drug and the adverse event.  “Suspected adverse reaction” implies a lesser degree of 
certainty about causality than adverse reaction, which means any adverse event caused by a drug. 
 
C. 
Life-Threatening Adverse Event / Life Threatening Suspected Adverse Reaction  
 
As defined in 21 CFR 312.32: 
 
Definition: any adverse drug event or suspected adverse reaction is considered “life-threatening” 
if, in the view of the investigator, its occurrence places the patient at immediate risk of death. It 
does not include an adverse event or suspected adverse reaction that, had it occurred in a more 
severe form, might have caused death. 
 
D. 
 Serious Adverse Event (SAE) or Serious Suspected Adverse Reaction 
 
As defined in 21 CFR 312.32: 
 
Definition:  an adverse event or suspected adverse reaction is considered “serious” if, in the view 
of the investigator, it results in any of the following outcomes: 
o Death 
o A life-threatening adverse event 

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26 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
o A congenital anomaly/birth defect 
o Any other important medical event that does not fit the criteria above but, based upon 
appropriate medical judgment, may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed above 
 
E. 
Protocol Exceptions 
Definition: A planned change in the conduct of the research for one participant. 
Unforeseen Imaging Exception:  Research imaging protocols which involve the injection 
of radioactive tracers can produce unique situations not common to standard treatment 
protocols.  In the event a situation occurs which requires deviation from this protocol – for 
example less than expected tracer production, problems with the scanner, patient unable to 
tolerate the imaging protocol as described, the Principal Investigator will have final 
authority over whether or not a study is completed.  Any protocol deviations will be 
documented on the PET imaging data form.  Deviation such as less than expected tracer 
production can be accounted for during data analysis and will not necessarily result in 
cancellation of the scan.  Similarly, adjusting imaging procedures to accommodate patient 
comfort can also be accounted for at the time images are reviewed.   
 
Except as described above, pre-approval of all protocol exceptions must be obtained prior to 
the event 
 
F. 
Deviation 
Definition: Any alteration or modification to the IRB-approved research without prospective IRB 
approval.  The term “research” encompasses all IRB-approved materials and documents including 
the detailed protocol, IRB application, consent form, recruitment materials, questionnaires/data 
collection forms, and any other information relating to the research study. 
 
A minor or administrative deviation is one that does not have the potential to negatively impact 
the rights, safety, or welfare of participants or others or the scientific validity of the study. 
 
A major deviation is one that does have the potential to negatively impact the rights, safety, or 
welfare of participants or others or the scientific validity of the study. 
 
 

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3 
APPENDIX B: Reporting Timelines 
 
Expedited Reporting Deadlines  
Event 
HRPO 
QASMC 
FDA 
Serious AND 
unexpected 
suspected adverse 
reaction 
 
 
Report no later than 15 
calendar days after it is 
determined that the 
information qualifies for 
reporting 
Unexpected fatal or 
life-threatening 
suspected adverse 
reaction 
 
 
Report no later than 7 
calendar days after initial 
receipt of the information 
Unanticipated 
problem involving 
risk to participants 
or others 
Report within 10 working 
days.  If the event results in 
the death of a participant 
enrolled at WU/BJH/SLCH, 
report within 1 working day. 
Report via email 
after IRB 
acknowledgment 
 
Major deviation 
Report within 10 working 
days.  If the event results in 
the death of a participant 
enrolled at WU/BJH/SLCH, 
report within 1 working day. 
 
 
A series of minor 
deviations that are 
being reported as a 
continuing 
noncompliance 
Report within 10 working 
days.   
 
 
Protocol exception 
Approval must be obtained 
prior to implementing the 
change 
 
 
Clinically important 
increase in the rate 
of a serious 
suspected adverse 
reaction of that list 
in the protocol or IB 
 
 
Report no later than 15 
calendar days after it is 
determined that the 
information qualifies for 
reporting 
 
Complaints 
If the complaint reveals an 
unanticipated problem 
involving risks to 
participants or others OR 
noncompliance, report 
within 10 working days.  If 
the event results in the death 
of a participant enrolled at 
WU/BJH/SLCH, report 
within 1 working day.  
Otherwise, report at the time 
of continuing review. 
 
 

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4 
Expedited Reporting Deadlines  
Event 
HRPO 
QASMC 
FDA 
Breach of 
confidentiality 
Within 10 working days. 
 
 
Incarceration 
If withdrawing the 
participant poses a safety 
issue, report within 10 
working days.   
 
If withdrawing the 
participant does not 
represent a safety issue and 
the patient will be 
withdrawn, report at 
continuing review. 
 
 
 
 
 

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5 
 
 
Routine Reporting Requirements 
Event 
HRPO 
QASMC 
FDA 
Adverse event or 
SAE that does not 
require expedited 
reporting 
If they do not meet the 
definition of an unanticipated 
problem involving risks to 
participants or others, report 
summary information at the 
time of continuing review 
Adverse events will 
be reported in the 
toxicity table in the 
DSM report which 
is typically due 
every 6 months. 
The most current toxicity 
table from the DSM 
report is provided to the 
FDA with the IND’s 
annual report. 
Minor deviation 
Report summary information 
at the time of continuing 
review. 
 
 
Complaints 
If the complaint reveals an 
unanticipated problem 
involving risks to 
participants or others OR 
noncompliance, report within 
10 working days.  If the 
event results in the death of a 
participant enrolled at 
WU/BJH/SLCH, report 
within 1 working day.  
Otherwise, report at the time 
of continuing review. 
 
 
Incarceration 
If withdrawing the 
participant poses a safety 
issue, report within 10 
working days.   
 
If withdrawing the 
participant does not represent 
a safety issue and the patient 
will be withdrawn, report at 
continuing review.