Clinical trial NCT05048082
Conditions: Lung Cancer
Sponsor: OncoNano Medicine, Inc.
Start date: 2022-04-13; Completion date: 2022-08-31
Enrollment: 24 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This Phase 2 study is an open-label, single-arm trial where each patient is his/her own "intrapatient" control. All patients will receive a single dose of pegsitacianine prior to standard of care surgery.

Detailed description:
This single-arm trial will enroll patients with biopsy confirmed or a high clinical suspicion of a lung malignancy based on MRI, CT and/or PET imaging. A total of up to 40 patients will be enrolled in this trial. The sponsor reserves the right to perform an interim analysis following early enrollment (i.e., n ≤ 10 subjects) to determine the feasibility and benefit of pegsitacianine as an adjunct to standard of care surgical resection of lung malignancies.

The surgeon will begin by attempting to perform their SOC tumor resection. If the tumor is unable to be located under standard white light conditions, an appropriate FDA-cleared near-infrared imaging device may be used to aid in the location of the primary tumor. Localization of the primary tumor, when unable to be located using white light imaging, in conjunction with pathology confirmed presence of disease will be considered a clinically significant event. Following any standard of care procedures conducted under white light only, the near infrared camera will be used to evaluate areas of residual fluorescence that may be resected if suscpicious for metastatic spread or residual margins. The fluorescence status of each specimen will then be correlated to the final pathology for each, and performance metrics of pegsitacianine will be tabulated.

Arms: arm 1: Pegsitacianine 1 mg/ml infused 24-72 hours prior to surgery.
Interventions: intervention 1: pegsitacianine intervention 2: Tumor resection surgery
Intervention descriptions: intervention 1: An intraoperative nanoparticle-based fluorescence imaging agent comprised of micelles covalently conjugated to indocyanine green (ICG). intervention 2: Standard of care surgical removal of cancerous lung lesion

Protocol documents:
Prot_002.pdf: 
 
 
 
 
OncoNano Medicine Inc. 
181 Grand Avenue, Ste 225 
Southlake, Texas 76092 
 
 
Protocol and Protocol Amendments 
 
 
Title 
A Phase 2, Single-Dose, Open-Label Study to 
Evaluate Diagnostic Performance and Safety of 
Pegsitacianine, An Intraoperative Fluorescence 
Imaging Agent for the Detection of Lung 
Malignancies, In Patients Undergoing Routine 
Surgery 
Study Drug 
Pegsitacianine (ONM-100) 
Protocol Amendment 2.0 
25 January 2022 
Original Protocol 
31 August 2021 
 
 
 
 
 


Confidential 
 STUDY PROTOCOL 
A PHASE 2, SINGLE-DOSE, OPEN-LABEL STUDY TO EVALUATE DIAGNOSTIC 
PERFORMANCE AND SAFETY OF PEGSITACIANINE, AN INTRAOPERATIVE 
FLUORESCENCE IMAGING AGENT FOR THE DETECTION OF LUNG MALIGNANCIES, 
IN PATIENTS UNDERGOING ROUTINE SURGERY  
PROTOCOL NUMBER: ON-1005 
Name of Drug: 
Pegsitacianine (ONM-100) 
Phase of Development: 
Phase 2 
Sponsor: 
OncoNano Medicine 
Sponsor Contact: 
Project Manager: 
Medical Monitor: 
Version: 
2 
Date of Protocol: 
25 Jan 2022 
Proprietary Notice: 
The concepts and information contained in this document 
or generated during the study are considered proprietary 
and may not be disclosed in whole or in part without the 
expressed, written consent of OncoNano. 
Compliance Statement: 
The study will be completed according to the guidelines of 
International Conference on Harmonisation Good Clinical 
Practice. Compliance with this standard provides public 
assurance that the rights, safety, and well-being of trial 
patients are protected, consistent with the principles that 
have their origin in the Declaration of Helsinki. 


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SIGNATURE PAGE 
 
 
 
PROTOCOL TITLE: A Phase 2, single-dose, open-label study to evaluate diagnostic 
performance and safety of pegsitacianine, an intraoperative 
fluorescence imaging agent for the detection of lung malignancies, 
in patients undergoing routine surgery 
 
PROTOCOL NUMBER: ON-1005  
 
 
 
 
 
 
 
 
 
VP, Head of Clinical Operations 
 
Date 
 
 
INVESTIGATOR STATEMENT 
  
I agree to conduct the study as outlined in the protocol in accordance with accepted Good 
Clinical Practice, the guidelines and all applicable government regulations including 21 CFR 54.  
 
I have read and understand all sections of the protocol. 
 
 
 
 
 
 
<Principal Investigator’s Name> 
 
 
Date 
 
 



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REVISION HISTORY 
Version Number 
Date 
Summary of Changes 
1    
31 Aug 2021 
Original 
2 
25 Jan 2022 
 
Updated the Medical Monitor 
 
Removal of lab values from exclusion criteria 
 
Updated Inclusion Criteria for clarification 
 
Updated safety information for completed 1002 study  
 
Removed hypothesis testing from sample size 
calculation 
 
Included description of site personnel training and 
responsibilities 
 
Updated Requirements for Part 2 enrollment 
 
Updated Statistical analysis to account for correlation 
 
Clarification of Pegsitacianine as a novel drug 
 
Updated Intent to treat, safety, efficacy populations 
 
Removal of the any additional investigational drug 
restriction 30 days prior 
 
Updated definitions of parameters of Imaging Analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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TABLE OF CONTENTS 
Signature Page ................................................................................................................................ 2 
Table Of Contents ........................................................................................................................... 4 
Protocol Summary .......................................................................................................................... 7 
Abbreviations ................................................................................................................................ 14 
1. 
Introduction ................................................................................................................... 16 
1.1 
Overview ....................................................................................................................... 16 
1.2 
Pegsitacianine Clinical Trial Background .................................................................... 16 
1.3 
Pharmacodynamics and Dose Selection ....................................................................... 18 
2. 
Study Objectives ........................................................................................................... 20 
2.1 
Primary Objective ......................................................................................................... 20 
2.2 
Secondary Objectives.................................................................................................... 20 
2.3 
Exploratory Objectives ................................................................................................. 20 
3. 
Study Plan ..................................................................................................................... 20 
3.1 
Overall Design .............................................................................................................. 20 
3.2 
Imaging Assessments .................................................................................................... 21 
3.3 
Safety Assessments ....................................................................................................... 22 
4. 
Study Drug Dosage and Administration ....................................................................... 22 
4.1 
Prohibited Medications and Restrictions ...................................................................... 22 
4.2 
Study Drug Description ................................................................................................ 22 
4.3 
Study Drug Safety ......................................................................................................... 23 
4.4 
Study Drug Packaging and Storage .............................................................................. 24 
4.5 
Drug Accountability...................................................................................................... 25 
5. 
Subject Enrollment........................................................................................................ 25 
5.1 
Inclusion Criteria .......................................................................................................... 25 
5.2 
Exclusion Criteria ......................................................................................................... 26 
5.3 
Randomization Procedures ........................................................................................... 26 
5.4 
Blinding Procedures ...................................................................................................... 26 
5.5 
Breaking the Blind ........................................................................................................ 26 
5.6 
Subject Withdrawal ....................................................................................................... 26 


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5.6.1 
Reasons for Withdrawal ................................................................................................ 26 
5.6.2 
Handling of Withdrawals .............................................................................................. 27 
5.6.3 
Replacements ................................................................................................................ 27 
5.6.4 
Termination of Study .................................................................................................... 27 
6. 
Study Visits ................................................................................................................... 28 
6.1 
Screening and Enrollment (Days –30 to –1) ................................................................. 28 
6.2 
Pegsitacianine Administration (Day 0) ......................................................................... 28 
6.3 
Surgery and Follow-up Visits ....................................................................................... 29 
6.4 
Early Withdrawal Procedures ....................................................................................... 31 
6.5 
Training ......................................................................................................................... 31 
7. 
Study assessments ......................................................................................................... 31 
7.1 
Demographic Data/Medical History ............................................................................. 31 
7.2 
Physical Examination.................................................................................................... 31 
7.3 
Weight and Height ........................................................................................................ 31 
7.4 
Vital Sign Measurements .............................................................................................. 31 
7.5 
12-lead Electrocardiogram ............................................................................................ 32 
7.6 
Clinical Laboratory Tests .............................................................................................. 32 
7.6.1 
Pregnancy Tests ............................................................................................................ 32 
7.6.2 
Laboratory Measurements ............................................................................................ 32 
8. 
Reporting Adverse Events ............................................................................................ 33 
8.1 
Definitions..................................................................................................................... 33 
8.2 
Adverse Event Reporting .............................................................................................. 34 
8.3 
Assessment of Causality ............................................................................................... 34 
8.4 
Assessment of Severity ................................................................................................. 35 
8.5 
Serious Adverse Event Reporting ................................................................................. 36 
8.5.1 
SAE Reporting Contact Information ............................................................................ 36 
9. 
Statistical Methods ........................................................................................................ 36 
9.1 
Sample Size ................................................................................................................... 36 
9.2 
Populations .................................................................................................................... 36 
9.3 
Rate of Clinically Significant Events ............................................................................ 37 


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9.4 
Imaging Analysis .......................................................................................................... 38 
9.5 
Quantification of Fluorescence Intensity ...................................................................... 39 
9.6 
Diagnostic Imaging Sensitivity Analyses ..................................................................... 39 
9.7 
Safety Analysis ............................................................................................................. 39 
9.8 
Interim Analyses ........................................................................................................... 40 
10. 
Data handling and quality assurance............................................................................. 40 
10.1 
Data Security ................................................................................................................. 40 
10.2 
Case Report Forms ........................................................................................................ 40 
10.3 
Monitoring of the Study ................................................................................................ 40 
10.4 
Inspection of Records ................................................................................................... 41 
10.5 
Study Record Retention ................................................................................................ 41 
11. 
Administrative Considerations...................................................................................... 41 
11.1 
Confidentiality .............................................................................................................. 41 
11.2 
Institutional Review Board/Ethics Committee Approval ............................................. 42 
11.3 
Modification of the Protocol ......................................................................................... 42 
11.4 
Informed Consent.......................................................................................................... 42 
11.5 
Protocol Violations and Deviations .............................................................................. 43 
11.6 
Study Reporting Requirements ..................................................................................... 43 
11.7 
Investigator Documentation .......................................................................................... 43 
11.8 
Study Conduct ............................................................................................................... 44 
11.9 
Publications ................................................................................................................... 44 
12. 
References ..................................................................................................................... 45 
13. 
Appendix 1: Schedule of Events ................................................................................... 46 
14. 
Appendix 2: FDA-Cleared NIR Imaging Devices ........................................................ 48 
14.1 
NOVADAQ SPY Elite ................................................................................................. 48 
14.2 
NOVADAQ SPY-PHI .................................................................................................. 48 
14.3 
NOVADAQ PINPOINT ............................................................................................... 48 
14.4 
da Vinci Firefly ............................................................................................................. 48 
14.5 
Visionsense Iridium ...................................................................................................... 48 
 


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PROTOCOL SUMMARY 
Protocol 
Number 
ON-1005 
Title of Trial 
A Phase 2, single-dose, open-label study to evaluate diagnostic performance and 
safety of pegsitacianine, an intraoperative fluorescence imaging agent, for the 
detection of lung malignancies in patients undergoing routine surgery 
Phase of Clinical 
Trial 
Phase 2 
Indications 
Pegsitacianine is indicated as an imaging agent for intraoperative detection of lung 
malignancies in the thoracic cavity during routine surgical resection. Pegsitacianine 
is administered 
as a single dose 
 prior to surgery. 
Study Centers 
up to 5 centers  
Trial Objectives 
Primary Objective: 
The primary objective of this study is to determine if administration of 
pegsitacianine (  mg/kg) results in a clinically significant event at the level of the 
patient.  
 
Examples of clinically significant events include: 
1. The detection of disease that cannot be detected using standard procedures  
2. The detection of synchronous lesions 
3. The detection of positive surgical margins following primary tumor 
resection 
4. Tumor-negative standard of care (SOC) biopsy 
 
Secondary Objectives: 
Key secondary objectives are to evaluate the safety and toxicity of pegsitacianine. 
Additionally, the sensitivity, specificity, negative predictive values, and positive 
predictive values of the imaging agent will be calculated at the level of the 
individual specimens, including lymph nodes.  
Background 
Pegsitacianine is a novel intraoperative nanoparticle-based fluorescence imaging 
agent that was evaluated during the first-in-human Phase 1 study (ON-1001). ON-
1001 was completed in the Netherlands in patients with solid cancers undergoing 
surgical excision of their tumors. An additional Phase 1 study (ON-1004) evaluating 
the pharmacokinetics in a healthy volunteer population has also been completed. 
Pegsitacianine was further evaluated in a completed Phase 2 study (ON-1002) in 
solid tumors. Pegsitacianine fluorescence is quenched when exposed to normal 
physiological pH but becomes activated following micellar disassembly in the acidic 
tumor microenvironment. Pegsitacianine was demonstrated to be well tolerated and 
showed no dose-limiting toxicities (DLT).  
Trial Design 
This Phase 2 study will be an interventional, open-label, single arm trial where each 
patient is his/her own “intrapatient” control. All patients will receive a single  
mg/kg dose of pegsitacianine 
 prior to 
standard of care (SOC) surgery.  It is recommended by the sponsor to administer 
prophylactic diphenhydramine before administration of pegsitacianine to decrease 
the possibility of an infusion-related reaction. The study will be conducted in two 
Parts. Part 1 will enroll patients undergoing minimally invasive procedures (i.e., 
video-assisted thorascopic surgery [VATS] or robotic surgery). Part 2 will be 
opened at the discretion of the Sponsor and include patients undergoing open 
thoracotomy for the removal of their disease.  Enrollment will be open first to Part 1 
with the opportunity to open Part 2 for enrollment following the demonstration of 


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satisfactory pegsitacianine sensitivity and specificity values of 70% or greater in 
Part 1. 
 
Patients will have a biopsy confirmed diagnosis, or a high clinical suspicion of a 
lung malignancy based on MRI, CT and/or PET imaging. A total of up to 40 patients 
will be enrolled in this trial across both parts. The sponsor reserves the right to 
perform an interim analysis following early enrollment (n ≤ 10 patients) to 
determine the feasibility and benefit of pegsitacianine as an adjunct to SOC 
surgery. 
 
Parts 1 and 2: 
 
The surgeon will attempt to perform their SOC resection of visible tumors. If the 
tumor is unable to be located using standard surgical approaches, an appropriate 
near-infrared (NIR) imaging device may be used to aid in localizing the primary 
tumor. Localization of the primary tumor using NIR imaging in conjunction with 
pathology confirmed presence of disease will be considered a clinically significant 
event.  
 
If the primary tumor is located using standard operative procedures, the tumor will 
be imaged using the NIR camera and removed as per standard of care (i.e., wedge, 
lobectomy, etc.). The primary resection will then be imaged (front, back and 
margins) on a piece of normal tissue within the surgical field at a distance from the 
specimen equal to the working distance of the camera system. Inspection and 
imaging of the resection margins will be performed using the NIR camera system. If 
an area of suspicion is observed, images will be captured of the area, at the working 
distance of the camera away from the margin and documented for future correlation 
to pathological findings. Similarly, the in situ resection margin on the lung will be 
imaged for signs of residual fluorescence that may correspond to disease left behind. 
If found, images will be captured prior to the excision of the margin (in situ) and 
following its resection on a piece of normal tissue (ex situ). Resection of the 
suspicious or fluorescent margin is at the discretion of the surgeon. This procedure 
is to be repeated if additional SOC nodules are planned for resection. An identical 
approach will be followed for any SOC lymph nodes collected during the procedure. 
Lymph node imaging will occur prior to and following the removal of the suspected 
lymph nodes. 
 
Once SOC surgical resection is complete, the surgeon will use the NIR camera 
system to evaluate the thoracic cavity (i.e., chest wall, contralateral lung, bronchi, 
etc.) for indications of residual fluorescence. If additional fluorescent foci are 
observed, the area may be resected, at the discretion of the surgeon, for pathological 
confirmation of disease. Similar to the imaging of margin resections, the suspected 
area will be imaged prior to removal (in situ) and following resection (ex situ) on a 
piece of normal tissue within the thoracic cavity. Additional fluorescent lymph 
nodes, detected during the evaluation of the thoracic cavity, may also be collected 
and imaged both in situ and ex situ prior to being evaluated in pathology. 
 
Collected surgical specimens will be documented by the clinical research 
coordinator, as dictated by the surgeon in the provided Imaging Workbook. The 
fluorescence status of each specimen will be assessed and called out by the surgeon 
(fluorescent or not fluorescent), and similarly documented in the workbook. All 
samples will be sent to pathology for routine analysis. To determine the performance 




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8. Pregnant or lactating 
9. Taking or plan to take medications with known hepatoxicity  
10. Any other significant medical condition the investigator deems 
inappropriate for the trial including any medical or psychiatric conditions 
that would impair informed consent 
Sample Size 
N=40  
 
Up to 40 patients will be enrolled in this study. No set allocation of patients to Part 1 
and Part 2 will be implemented, rather Part 2 will be opened at the discretion of the 
sponsor following the demonstration of satisfactory pegsitacianine sensitivity and 
specificity values of 70% or greater in Part 1. The sponsor reserves the right to 
perform an interim analysis following early enrollment (i.e., n ≤ 10 patients) to 
determine the feasibility and benefit of pegsitacianine as an adjunct to standard 
of care lung malignancy resection. 
Statistical 
Analysis 
The quotient of patients with a CSE over the total number of patients undergoing 
surgery who received any dose of the study drug, and had sufficient imaging data 
(minimum of 1 collected image). Sensitivity and specificity will be determined at 
the specimen level based on correlated fluorescence status and pathological 
outcome.  
 
Pegsitacianine performance data will be evaluated in toto, as well as separately if 
Part 2 of the study is opened to enrollment. Part 1 and Part 2 data will be evaluated 
independently to assess performance when using different imaging systems within 
their respective procedure (i.e., minimally invasive vs. open).  
Schedule of 
Events 
Screening (Day -30 to -1)   
 
Before the study doctor can administer pegsitacianine, he/she must ensure that it is 
safe for the patient to be in the study vis-à-vis information that is already known 
about pegsitacianine. After the patient has agreed to be in the study and signed the 
informed consent form, the study team will perform the following procedures 
(information collected as part of standard of care within a reasonable timeframe that 
meet the presurgical requirements of the institution are also acceptable): 
 
 
Collect demographic data 
 
Record their complete medical history 
 
Perform a complete physical examination 
 
Measure their weight and height and calculate a body mass index 
(BMI) to estimate body fat tissue 
 
Record vital signs (temperature, heart rate, blood pressure, and 
respirations) 
 
Obtain a blood sample for routine laboratory tests, including a 
pregnancy test for females who can become pregnant 
 
Record medications they have been taking, including prescription, 
over-the-counter, and herbal medications 
 
Determine Karnofsky Performance Status (to assess the ability to 
perform ordinary daily tasks) 
 
Confirm that the patient has met all the study criteria to participate 
 
Day 0 (Pegsitacianine administration) 
 
Predose procedures 




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Once the patient is under anesthesia, some procedures will occur that would not 
normally be part of surgery if the patient were not in this study. The SOC surgical 
procedures will be allowed to continue with the addition of the steps listed below. 
 
If the primary tumor is not visually apparent using standard 
approaches, the NIR camera may be used to help locate and guide 
the resection of the primary tumor 
 
If the primary tumor is apparent under standard conditions, the 
suspected area will be imaged using the intraoperative camera 
prior to excision, and on a section of normal tissue at a distance 
equal to the working distance of the camera following excision 
 
Following the primary tumor resection, margins (both on the 
excised primary and the remaining lung) will be examined using 
NIR in an effort to detect the presence of a possible positive 
margin 
 
Any SOC lymph nodes that are to be collected will be imaged 
prior to, and following excision in a manner identical to that 
performed on the primary tumor specimen 
 
Once the SOC primary tumor and lymph node resection is 
complete, the surgeon will examine the thoracic cavity with the 
intraoperative NIR camera in search of additional fluorescent 
disease that was missed during SOC, or additional fluorescent 
lymph nodes 
 
Additional fluorescent lesions will be imaged prior to excision, 
removed, and then imaged on a section of normal tissue in a 
manner analogous to the SOC specimens.  
Day of Discharge  
 
Record the patient’s vital signs 
 
Perform a physical examination  
 
Discuss new or unexpected changes in they are feeling 
 
Obtain blood samples for routine laboratory tests 
 
Record all medications being taken, including prescription, over-
the-counter, and herbal medications 
 
Discuss any new or unexpected changes  
 
Record any AEs 
 
Day 21 (±10 days) after dosing 
 
Record the patient’s vital signs 
 
Perform 12-lead ECG to monitor heart activity 
 
Perform a physical examination  
 
Discuss new or unexpected changes in how they are feeling 
 
Obtain blood samples for routine laboratory tests, including a 
pregnancy test for females who can become pregnant 
 
Record all medications being taken, including prescription, over-
the-counter, and herbal medications 
 
Discuss any new or unexpected changes  
 
Record any AEs 
Training  
Extensive training will be provided to all study staff. Initial training will occur at the 
Site Initiation Visit, followed by on-site training that will take place during the 
initial surgical procedures with Sponsor Representatives present in the operating 
room during the procedures. Sponsor Representatives will ensure all study personnel 


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are adequately trained and follow identical procedures to preserve the integrity of 
the collected data without providing input on the selection of specimens or their 
fluorescent status, these tasks are are the sole responsibility of the operating 
surgeon. Additional retraining may take place if necessary throughout the course of 
the study. Training will include a review of protocol procedures, NIR camera 
operating instructions, documentation of specimens and their fluorescence status, 
and upload of source documentation. All training will be documented and stored 
within the Trial Master File.  


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ABBREVIATIONS 
ABBREVIATION 
TERM 
AE 
Adverse Event 
ALT 
ANOVA 
Alanine Aminotransferase 
Analysis of Variance 
AST 
BMI 
Aspartate Aminotransferase 
Body Mass Index 
BUN 
Blood Urea Nitrogen 
CFR 
Code of Federal Regulations 
CSE 
CT 
CV 
D5W 
DoH 
ECG 
Clinically Significant Event 
Computed Tomography 
Curriculum Vitae 
5% Dextrose in water 
Declaration of Helsinki   
Electrocardiogram 
eCRF 
Electronic Case Report Form 
FDA 
United States Food and Drug Administration 
HNSCC 
ICG 
Head and Neck Squamous Cell Carcinoma  
Indocyanine Green 
ICH 
International Conference on Harmonisation 
IRB 
IV 
Institutional Review Board 
Intravenous 
MFI 
MRI 
NIR 
NPV 
PEG 
PET 
PK 
Mean Fluorescence Intensity 
Magnetic Resonance Imaging 
Near-infrared 
Negative Predictive Value 
Polyethylene Glycol 
Positron Emission Tomography 
Pharmacokinetics 
PMMA 
PPV 
ROC 
SAE 
Polymethylmethacrylate 
Positive Predictive Value 
Receiver Operating Characteristic 
Serious Adverse Event 
SBR 
Specimen-to-Background Ratio 
SOC 
Standard of Care 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
SWI 
TBR  
Sterile Water for Injection 
Tumor-to-Background Ratio 


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ABBREVIATION 
TERM 
TEAE 
VATS 
WMA 
Treatment-Emergent Adverse Event 
Video-Assisted Thorascopic Surgery 
World Medical Association 
 
 
 
























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discretion and after approval by OncoNano’s medical monitor. Justification for this 
decision should be clearly documented in source documents. 
 The subject becomes noncompliant or becomes uncooperative in returning for the 
scheduled study visits. 
 The subject requests withdrawal for any reason. 
The clinical study report will include reasons for subject withdrawals as well as details relevant 
to the subject withdrawal. 
5.6.2 
Handling of Withdrawals 
If a subject withdraws prematurely from the study after receiving the study drug, study staff 
should make every effort to perform the Day 21 (±10 days) assessments. The reason for subject 
withdrawal must be documented in the eCRF. 
If a subject withdraws from the study because of an AE (clinical or laboratory), the subject will 
be asked to return to the clinic for, at a minimum, the evaluations scheduled. If the AE has still 
not resolved, additional follow-up will be performed as appropriate and documented in the 
subject’s medical records. As a minimum requirement, AEs should be followed for 30 days after 
receipt of the subject’s last dose of study drug, until resolution, or when judged to be stable for 
30 days. 
If a subject is lost to follow-up, 3 attempts (i.e., 2 phone calls, then 1 registered letter) to contact 
the subject will be made and documented in the subject’s medical records. 
5.6.3 
Replacements 
Withdrawn patients may be replaced at the discretion of the sponsor.   
5.6.4 
Termination of Study 
If, in the opinion of the investigator, the clinical observations in the study suggest that it may be 
unwise to continue, the investigator may terminate the study after consultation with OncoNano 
Medicine. A written statement fully documenting the reasons for such a termination will be 
provided to OncoNano Medicine. In addition, OncoNano Medicine reserves the right to 
discontinue the study at any time for any reason. Such a termination must be implemented by the 
investigator, if instructed to do so by OncoNano Medicine, in a time frame that is compatible 
with patients’ well-being. 
If the study is terminated, all patients will undergo a complete follow-up examination. Any 
clinically relevant findings, including clinically significant laboratory values and AEs, will be 
followed until resolution, stabilization for 30 days, or until the end of the study (the last study 
evaluation for the last subject), whichever occurs first. 




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Dosing and postdose procedures 
Procedures during and after dosing of pegsitacianine are summarized below: 
 Dosing of pegsitacianine will be administered 
, length of infusion and overall volume will depend on the assigned dose 
and the patient’s body weight. 
 The following postdose assessments and procedures will occur after infusion of 
the study drug has been completed: 
o Record the patient’s vital signs immediately after the infusion and 
30 minutes afterward 
o Discuss any new or unexpected changes in how the patient is feeling 
o Record any AEs 
o Record all medications they are taking, including prescription, over-the-
counter, and herbal medications 
6.3 Surgery and Follow-up Visits  
Procedures at the time of surgery 
 
 
The following procedures will be performed on the day of surgery (before administration of 
anesthesia and before surgery: 
 
 Record the patient’s vital signs  
 Obtain blood for routine laboratory tests  
 Record all medications being taking, including prescription, over-the-counter, and 
herbal medications 
 Discuss with the patient any new or unexpected changes they are feeling.  
 Record any AEs. 
 
Once the patient is under anesthesia, some procedures will occur that would not normally be part 
of surgery if the patient were not in this study. The SOC surgical procedures will be allowed to 
continue with the addition of the imaging steps listed below. 
 
 If the primary tumor is not visually apparent using standard approaches, the NIR 
camera may be used to help locate and guide the resection of the primary tumor. 
o The primary tumor should be imaged prior to excision and following excision 
on a piece of normal tissue from a distance equal to the operating distance of 
the camera.  
 If the primary tumor is apparent under standard conditions, it will be imaged prior 
to resection. Additionally, the resected tissue will be imaged using the 


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intraoperative camera on a section of normal tissue at a distance equal to the 
operating distance of the camera 
 Following the primary tumor resection, margins (both on the excised primary and 
the affected lung) will be examined using NIR in an effort to detect residual 
disease 
 All SOC lymph nodes to be collected during the operative procedure will be 
imaged prior to and following excision in a manner identical to the procedures 
used for imaging the primary resection 
 Once the SOC primary tumor resection is complete, the surgeon will examine the 
thoracic cavity with the intraoperative NIR camera in search of additional 
fluorescent disease, or lymph nodes that were missed during SOC  
 Additional fluorescent lesions, or lymph nodes will be imaged prior to excision, 
removed and imaged on a section of normal tissue in a manner analogous to the 
SOC specimens.  
 For all resected specimens (including SOC primary tumor, margins, SOC lymph 
nodes and additional missed disease), fluorescence status and specimen name will 
be called out by the surgeon for documentation by the clinical research 
coordinator in the provided Imaging Workbook 
 
Day of Discharge 
 Record the patient’s vital signs 
 Perform a physical examination 
 Obtain blood samples for routine laboratory tests 
 Record all medications being taking, including prescription, over-the-counter, and 
herbal medications 
 Discuss any new or unexpected changes they are feeling.  
 Record any AEs. 
 
Day 21 (±10 days) after dosing 
 Record the patient’s vital signs 
 Perform 12-lead ECG to monitor heart activity 
 Perform a physical examination 
 Obtain blood samples for routine laboratory tests, including a pregnancy test for 
females who can become pregnant 
 Record all medications being taking, including prescription, over-the-counter, and 
herbal medications 
 Discuss any new or unexpected changes they are feeling.  


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 Record any AEs 
6.4 Early Withdrawal Procedures 
If a subject is withdrawn from the study before study completion, all procedures scheduled for 
the final visit (Day 21 ±10 days) should be performed if possible. 
6.5 Training 
Extensive training will be provided to all study staff. Initial training will occur at the Site 
Initiation Visit, followed by on-site training that will take place during the initial surgical 
procedures with Sponsor Representatives present in the operating room during the procedures. 
Sponsor Representatives will ensure all study personnel are adequately trained and follow 
identical procedures to preserve the integrity of the collected data without providing input on the 
selection of specimens or their fluorescent status, these tasks are are the sole responsibility of the 
operating surgeon. Additional retraining may take place if necessary throughout the course of the 
study. Training will include a review of protocol procedures, NIR camera operating instructions, 
documentation of specimens and their fluorescence status, and upload of source documentation. 
All training will be documented and stored within the Trial Master File.  
 
7.STUDY ASSESSMENTS  
7.1 Demographic Data/Medical History 
Demographic data and a complete medical history (including drug, alcohol, and tobacco use, as 
well as current use of herbal supplements and multivitamins) will be obtained at Screening. 
7.2 Physical Examination 
A complete physical examination will be performed at Screening, Day of Discharge and on 
Day 21. The complete physical examination may include the following organ or body system 
assessments: skin; head; eyes; ears; nose; throat; thyroid; neurological; chest and lungs; 
cardiovascular; abdomen (liver and spleen); lymph nodes; musculoskeletal; and extremities.   
7.3 Weight and Height 
Body weight (kg) and height (m) will be measured at Screening and weight will again be 
collected on Day 0 prior to dosing. The subject’s body mass index (BMI) will be calculated at 
Screening using the following formula: 
𝐵𝑀𝐼= 𝑊𝑒𝑖𝑔ℎ𝑡 (𝑘𝑔)
(𝐻𝑒𝑖𝑔ℎ𝑡 [𝑚])2 
7.4 Vital Sign Measurements 
Vital sign measurements (systolic and diastolic blood pressure, pulse, respiratory rate, and 
temperature) will be obtained at Screening, Day 0 (Pre/Post dose), Day of Surgery, Day of 
Discharge and Day 21. 
Temperature will be documented in degrees Celsius. 


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Blood pressure and pulse will be measured after a resting period of at least 5 minutes in the 
supine position with a standard mercury sphygmomanometer or an automated oscillometric 
blood pressure monitor.  
7.5 12-lead Electrocardiogram 
A standard safety 12-lead ECG will be conducted on Day 0 (Pre-Dose) and on Day 21. 
Additional ECGs may be performed at the discretion of the investigator. The investigator or 
designee will be responsible for reviewing the ECG to assess whether the ECG is within the 
reference limits and to determine the clinical significance of the results. These assessments will 
be recorded in the eCRF. 
7.6 Clinical Laboratory Tests 
Clinical laboratory tests (hematology and serum chemistry) will be performed by a Local and/or 
Central Laboratory. Blood samples will be collected at Screening, on Day 0, Day of Surgery, 
Day of Discharge and Day 21.  
7.6.1 
Pregnancy Tests 
For female patients, a serum sample or urine dipstick will be collected for the pregnancy test (-
human chorionic gonadotropin) at Screening and blood samples will be collected for pregnancy 
tests on Day 0, and on Day 21. Negative pregnancy test results will be required for patients to 
enroll and continue study participation. 
7.6.2 
Laboratory Measurements 
Blood hematology and serum chemistry tests may be performed using the standard panel of each 
institution (it is not necessary to add analytes if not part of standard panel unless 
ordered/recommended by treating physician). The following clinical laboratory parameters are 
an example of what is to be collected: 


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Clinical Chemistry 
Hematology 
Total protein 
Hemoglobin 
Albumin 
Hematocrit 
Blood urea nitrogen (BUN)  
Erythrocyte count (red blood cells) 
Calcium (total and ionized) 
Differential leukocytes  
Creatinine 
Platelet count 
Creatine kinase 
Total leukocytes (white blood cells) 
Chloride 
 
Total bilirubin 
 
Alkaline phosphatase 
 
Glucose 
 
Sodium 
 
Potassium 
 
Inorganic phosphate 
 
Lactate dehydrogenase 
 
Total Cholesterol 
 
Gamma-glutamyl transferase 
 
Alanine aminotransferase (ALT) 
 
Aspartate aminotransferase (AST) 
 
 
8. REPORTING ADVERSE EVENTS 
All adverse events (AEs) will be recorded in the eCRF, whether they are observed by the 
investigator, reported by the subject, observed from laboratory findings, or collected by other 
means. Adverse events will be monitored beginning on Day 0, following pegsitacianine 
administration, through the end of the study (Day 28). Adverse events will be graded by the 
Investigator using a numerical score according to the defined National Cancer Institute Common 
Terminology Criteria for Adverse Events (NCI CTCAE) Version 5.0 (2017). 
8.1 Definitions 
The investigator is responsible for reporting all AEs that are observed or reported during the 
study, regardless of their relationship to study drug or their clinical significance. 
An AE is defined as any untoward medical occurrence in a subject enrolled into this study 
regardless of its causal relationship to study treatment. Patients will be instructed to contact the 
principal investigator if any symptoms develop. 
A treatment-emergent AE (TEAE) is defined as any event not present before the first exposure to 
the study drug or any event already present that worsens in either intensity or frequency 
following the first exposure to the study drug. 
All AEs that occur beginning on Day 0 must be reported in detail in the eCRF and followed to 
satisfactory resolution or until the principal investigator deems the event to be chronic or the 


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subject to be stable. The description of the AE will include the onset date, duration, date of 
resolution, severity, seriousness, etiology, and the likelihood of relationship of the AE to study 
drug. 
A serious adverse event (SAE) is defined as any event that results in any of the following 
outcomes: 
 Results in death 
 Is life threatening, i.e., the subject was, in the opinion of the investigator, at risk 
of death at the time of the event; it does not refer to an event that, hypothetically, 
might have caused death if it had occurred in a more severe form 
 Requires inpatient hospitalization or prolongs existing hospitalization 
 Results in persistent or significant disability and/or incapacity 
 Is a congenital anomaly/birth defect 
 Is an important medical event 
 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical judgment, they 
may jeopardize the subject and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias, 
convulsions that do not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse. 
8.2 Adverse Event Reporting  
All AEs reported or observed during the study will be recorded in the eCRF. Information to be 
collected includes drug treatment, dosage, type of event, time of onset, investigator-specified 
assessment of severity and relationship to study drug, time of resolution of the event, 
seriousness, any required treatment or evaluations, and outcome. Adverse events resulting from 
concurrent illnesses, reactions to concurrent illnesses, reactions to concurrent medications, or 
progression of disease states must also be reported. All AEs will be followed to adequate 
resolution.  
Any medical condition that is present at the time that the subject is screened but does not 
deteriorate should not be reported as an AE. However, if the condition deteriorates at any time 
during the study after the first exposure to study drug, it should be recorded as an AE. 
8.3 Assessment of Causality 
The investigator’s assessment of an AE's relationship to study drug is part of the documentation 
process, but it is not a factor in determining what is or is not reported in the study. If there is any 
doubt as to whether a clinical observation is an AE, the event should be reported. 


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The relationship or association of the study drug in causing or contributing to the AE will be 
characterized using the following classification and criteria: 
 Unrelated: This relationship suggests that there is no association between the study drug 
and the reported event. 
 Unlikely: This relationship suggests the temporal association between the AE and the study 
drug is such that the study drug is not likely to have any reasonable association with the 
AE. 
 Possible: This relationship suggests that treatment with the study drug caused or 
contributed to the AE, i.e., the event follows a reasonable temporal sequence from the 
time of study drug administration and/or follows a known response pattern to the study 
drug, but could also have been produced by other factors. 
 Probable: This relationship suggests that a reasonable temporal sequence of the event 
with study drug administration exists and, based on the known pharmacological action of 
the drug, known or previously reported adverse reactions to the drug or class of drugs, or 
judgment based on the investigator’s clinical experience, the association of the event with 
the study drug seems likely. 
 Definite: This relationship suggests that a definite causal relationship exists between the 
study drug administration and the AE, and other conditions (concurrent illness, 
progression/expression of disease state, or concurrent medication reaction) do not appear 
to explain the event. 
8.4 Assessment of Severity 
The intensity of the AE will be rated according to CTCAE criteria (Version 5.0) ranging from 
Grade 1 to Grade 5. A brief explanation of the grading is provided below: 
 Grade 1 Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated 
 Grade 2 Moderate; minimal, local or noninvasive intervention indicated; limiting age- 
appropriate instrumental activities of daily living (ADL) 
o Instrumental ADL refers to preparing meals, shopping for groceries or clothes, 
using the telephone, managing money, etc.  
 Grade 3 Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care 
ADL 
o Self-care ADL refers to bathing, dressing and undressing, feeding self, using the 
toilet, taking medications, and not bedridden 
 Grade 4 Life-threatening consequences; urgent intervention indicated.  
 Grade 5 Death related to AE. 
 




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9.3 Rate of Clinically Significant Events 
Determining the rate of clinically significant events (CSE) detected by pegsitacianine at the 
subject-level is the primary objective of this study. CSEs will include pegsitacianine detection of 
primary tumors otherwise undetectable using SOC procedures, detection of occult disease not 
otherwise known by the surgeon to exist, the detection of a positive surgical margin, and 
accurate identification of tumor negative SOC biopsies. A list of CSEs and their definitions is 
provided below. 
 
 Detection of Synchronous Disease – identification of tumor-containing tissue due to 
pegsitacianine fluorescence, confirmed via histopathological analysis, that otherwise 
went undetected during pre-operative imaging (PET, CT, MRI) or by the surgeon during 
SOC surgery. 
o This occurs when a specimen that comes from the thoracic cavity or lymph nodes, 
is (1) collected via fluorescence and (2) is pathologically-confirmed as a tumor. 
Note that any specimen collected from the thoracic cavity following SOC will be 
considered to be collected by fluorescence.  
 
 Detection of Positive Surgical Margin – following the completion of SOC surgery, the 
surgical fields will be examined for residual fluorescence. Detection of a positive surgical 
margin would be those specimens collected as a result of residual pegsitacianine 
fluorescence following the completion of SOC surgery that return a histologically 
positive for tumor outcome.  
o when a margin specimen (either from the primary tumor or a lung resection 
margin) (1) is collected via fluorescence and (2) is pathologically-confirmed as a 
tumor. Note that any lung resection margin collected will be considered to be 
collected by fluorescence.  
 
 Identification/Localization of the Primary Tumor – during SOC surgery, the surgeon will 
examine the affected lung to locate the primary tumor. If the primary tumor is not 
visually apparent using standard imaging methods the surgeon may use the NIR imaging 
device to aid in localization. If the NIR imaging identifies and/or locates the primary 
tumor, which was undetectable using SOC approaches, this will be deemed a CSE. 
o This occurs when the primary tumor is not located using SOC approaches, and is 
instead identified using NIR imaging.  
 
 Tumor Negative SOC Biopsies – during SOC surgery, the surgeon will collect tissue 
specimens that are suspicious for disease. If those samples do not demonstrate 
pegsitacianine fluorescence and are found to be devoid of tumor pathologically, those 
specimens would be included as a tumor negative SOC biopsy. 
o This occurs when any SOC sample (primary tumor, additional SOC specimen, or 
lymph node) that is collected is found to be negative for tumor, and did not fluoresce 
during ex situ imaging.  


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The number and proportion (with corresponding two-sided exact 95% confidence intervals) of 
subjects with any CSE will be reported. 
 
9.4 Imaging Analysis 
Diagnostic performance of pegsitacianine will be assessed at the level of the specimen by 
calculating the ex situ sensitivity, specificity, PPV, and NPV of pegsitacianine in detecting tumor 
containing tissue. The ex situ fluorescence status of collected SOC and pegsitacianine guided 
specimens will be compared to the histological analyses of the collected specimens (which is used 
to determine each sample’s status as true/false positive/negative). For determining these operating 
characteristics, consider the following definitions: 
 False positive is defined as “fluorescence was observed on the specimen but the specimen 
was not found to have tumor via histological analysis”. 
 False negative is defined as “no fluorescence was observed on the specimen but the 
specimen was found to have tumor via histological analysis”. 
 True positive is defined as “fluorescence was observed on the specimen and the specimen 
was found to have tumor via histological analysis”. 
 True negative is defined as “no fluorescence was observed on the specimen and the 
specimen was found not to have tumor via histological analysis”. 
The number of specimens in each category (each combination of true/false positive/negative), 
along with the true/false positive/negative rates (and corresponding exact two-sided 95% 
confidence intervals) will be reported. These rates will be used to determine pegsitacianine’s tumor 
detection sensitivity, specificity, PPV, and NPV  using the following equations: 
        
𝑺𝒆𝒏𝒔𝒊𝒕𝒊𝒗𝒊𝒕𝒚=
# 𝑜𝑓 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠
# 𝑜𝑓 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠+ # 𝑜𝑓𝐹𝑎𝑙𝑠𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠 
 
𝑺𝒑𝒆𝒄𝒊𝒇𝒊𝒄𝒊𝒕𝒚=
#𝑜𝑓 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠
# 𝑜𝑓 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠+ # 𝑜𝑓𝐹𝑎𝑙𝑠𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠  
 
𝑷𝑷𝑽=
# 𝑜𝑓 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠
# 𝑜𝑓 𝑇𝑟𝑢𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠+ # 𝑜𝑓 𝐹𝑎𝑙𝑠𝑒 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠  
 
𝑵𝑷𝑽=
# 𝑜𝑓 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠
# 𝑜𝑓 𝑇𝑟𝑢𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠+ # 𝑜𝑓 𝐹𝑎𝑙𝑠𝑒 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛𝑠 
 
These rates will be reported along with exact two-sided 95% confidence intervals.  
 


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9.5 Quantification of Fluorescence Intensity 
Specimen-to-background (SBR) ratios and tumor-to-background (TBR) ratios will also be 
calculated using white light and NIR images of both SOC and pegsitacianine guided specimens. 
SBRs will be calculated using ex situ images of collected specimens. The equation for calculating 
SBR is listed below.   
𝑺𝑩𝑹=
𝑀𝑒𝑎𝑛 𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 (𝑆𝑝𝑒𝑐𝑖𝑚𝑒𝑛)
𝑀𝑒𝑎𝑛 𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 (𝑁𝑜𝑟𝑚𝑎𝑙 𝑇𝑖𝑠𝑠𝑢𝑒)
TBR is calculated in the same way, but refers specifically to samples that are histologically 
confirmed as tumor.  
The mean fluorescence intensity of the specimen, of the background, and the SBR (or TBR) of 
each specimen will be summarized by the histologically-confirmed tumor status (either “tumor 
present” or “tumor not present/normal tissue”). 
9.6 Diagnostic Imaging Sensitivity Analyses 
In this pilot study we are assuming that any fluorescence is indicative of tumor presence. To assess 
how the amount of observed fluorescence (e.g., the SBR or TBR) may affect the diagnostic 
accuracy of pegsitacianine, a receiver operating characteristic (ROC) analysis will be performed 
on all specimens using the observed thresholds of SBR and TBR. The corresponding ROC curve 
will be provided along with the area under the curve (AUC).  
There is potential for within-subject correlation to bias our estimates of pegsitacianine’s 
diagnostic accuracy. To assess this issue, an ROC curve will be constructed from a mixed model 
that clusters specimens by the subject they originated from. We will compare the AUC of the 
mixed effects ROC curve to the standard ROC curve to assess if within-subject correlation is 
causing significant bias in our analyses. If it is determined that significant bias has been 
introduced, additional analyses on secondary endpoints may be performed to assess the 
diagnostic accuracy of pegsitacianine. 
9.7 Safety Analysis 
Safety data will be summarized by treatment group with descriptive statistics and frequency 
tables. In general, continuous data will be summarized by presenting the number of patients, 
mean, standard deviation, median, minimum, and maximum values. Categorical data will be 
summarized by presenting the number (frequency) and percentage of patients at each level of 
response. Subject disposition will be presented and summarized. 


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Adverse events will be coded using MedDRA, Version 24.0 or higher, by system organ class and 
preferred term, and a listing of all AEs will be generated. Treatment-emergent AEs (TEAEs), 
treatment-related TEAEs, TEAEs by intensity, TEAEs that lead to discontinuation of study drug, 
and SAEs will be summarized by treatment, system organ class, and preferred term. 
The clinical laboratory data will be summarized by time point, treatment group, and change from 
baseline. The values that are below the lower limit or above the upper limit of the reference 
range will be flagged. Those values or changes in values that are considered clinically significant 
by the investigator will also be flagged. Repeated or unscheduled results will not be included in 
the summary statistics but will be included in the individual data listings. 
9.8 Interim Analyses 
A single, internal interim analysis may occur after approximately 10 patients have been enrolled 
in an effort to better understand the feasibility and benefit of pegsitacianine imaging during lung 
malignancy resections. All analyses described herein will be conducted and evaluated by the 
sponsor. After an overall review of the data, a decision will be made by the sponsor to continue 
or terminate enrollment based on the interim findings. The Interim analyses will assess the utility 
of pegsitacianine based on mean fluorescence intensity analysis, calculation of tumor to 
background ratios, calculation of sensitivity and specificity, safety, and investigator feedback. 
The decision to continue or terminate is not based off of any individual endpoint and does not 
correspond to any collection of statistical tests, but instead a synthesis of all available 
information. 
 
10. DATA HANDLING AND QUALITY ASSURANCE 
10.1 
Data Security 
The study data will be collected electronically. This electronic data capture (EDC) system 
complies with ICH GCP, GDPR, and the current 21 CFR Part 11 guidance, Electronic Records 
and Signatures. 
10.2 
Case Report Forms 
As part of the responsibilities assumed by participating in the study, the principal investigator 
agrees to maintain adequate case histories for the patients treated as part of the research under 
this protocol. The principal investigator agrees to maintain accurate eCRFs and source 
documentation as part of the case histories.  
MedTrials Inc. will supply the eCRF. All eCRF information is to be filled in. If an item is not 
available or is not applicable, this fact will be indicated. The eCRF has an electronic audit trail so 
changes can be made until the investigator signs the eCRF. Each completed eCRF must be 
reviewed, signed, and dated by the principal investigator at the completion of the study. The 
completed eCRF will be collected by study monitors as soon as practical after completion. One 
copy will remain at the site in the principal investigator’s files. 
10.3 
Monitoring of the Study 


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The clinical monitor, as a representative of MedTrials Inc. and/or OncoNano Medicine, has the 
obligation to follow the study closely. In doing so, the monitor will visit the principal 
investigator and study facility at periodic intervals, in addition to maintaining necessary contact 
through telephone, email, and letter. The monitor will maintain current personal knowledge of 
the study through observation, review of study records and source documentation, and discussion 
of the conduct of the study with the principal investigator and staff. 
All aspects of the study will be carefully monitored, by MedTrials Inc., OncoNano Medicine or a 
designee, for compliance with applicable government regulation with respect to current 
International Conference on Harmonisation (ICH) harmonised tripartite guideline E6(R1): Good 
Clinical Practice and current standard operating procedures. 
10.4 
Inspection of Records 
The principal investigator and institutions involved in the study will permit trial-related 
monitoring, audits, institutional review board (IRB) review, and regulatory inspection(s) by 
providing direct access to all study records. In the event of an audit, the principal investigator 
agrees to allow MedTrials Inc., OncoNano Medicine, representatives of OncoNano Medicine, 
the United States Food and Drug Administration (FDA), and/or other regulatory agency access to 
all study records. 
The principal investigator should promptly notify MedTrials and OncoNano Medicine of any 
audits scheduled by any regulatory authorities and promptly forward copies of any audit reports 
received to MedTrials Inc. and OncoNano Medicine. 
10.5 
Study Record Retention 
Essential documents should be retained until at least 2 years after the last approval of a 
marketing application in an ICH region and until there are no pending or contemplated marketing 
applications in an ICH region or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the investigational product. These documents should be retained for a 
longer period, however, if required by the applicable regulatory requirements or by an agreement 
with MedTrials Inc. and/or OncoNano Medicine. It is the responsibility of MedTrials Inc. and 
OncoNano Medicine to inform the principal investigator as to when these documents no longer 
need to be retained. 
11. ADMINISTRATIVE CONSIDERATIONS 
The following administrative items are meant to guide the principal investigator in the conduct of 
the trial but may be subject to change based on industry and government standard operating 
procedures or working practice documents or guidelines. Administrative changes will be 
reported to the IRB but will not result in protocol amendments. 
11.1 
Confidentiality 
All laboratory specimens, evaluation forms, reports, and other records will be identified in a 
manner designed to maintain subject confidentiality. All records will be kept in a secure storage 
area with limited access. Clinical information will not be released without the written permission 


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of the subject (or the subject’s guardian), except as necessary for monitoring and auditing by 
OncoNano Medicine, its designee, the FDA, or the IRB. 
The principal investigator and all employees and coworkers involved with this study may not 
disclose or use for any purpose other than performance of the study, any data, record, or other 
unpublished, confidential information disclosed to those individuals for the purpose of the study. 
Prior written agreement from OncoNano Medicine or its designee must be obtained for the 
disclosure of any said confidential information to other parties. 
11.2 
Institutional Review Board/Ethics Committee Approval 
Federal regulations and the ICH guidelines require that approval be obtained from an IRB before 
participation of human patients in research studies. Before the study onset, the protocol, 
informed consent, advertisements to be used for subject recruitment, and any other written 
information regarding this study to be provided to the subject or the subject’s legal guardian 
must be approved by the IRB. Documentation of all IRB approvals and of the IRB compliance 
with ICH E6(R1) will be maintained by the site and will be available for review by OncoNano 
Medicine or its designee. 
All IRB approvals should be signed by the IRB chairman or designee and must identify the IRB 
name and address, the clinical protocol by title and/or protocol number and the date approval 
and/or favorable opinion was granted. 
The principal investigator is responsible for obtaining continued review of the clinical research at 
intervals not exceeding 1 year or otherwise specified by the IRB. The principal investigator must 
supply OncoNano Medicine or its designee with written documentation of continued review of 
the clinical research. 
11.3 
Modification of the Protocol 
Any changes in this research activity, except those necessary to remove an apparent, immediate 
hazard to the subject, must be reviewed and approved by OncoNano Medicine or its designee. 
Amendments to the protocol must be submitted in writing to the principal investigator’s IRB for 
approval before patients are enrolled into an amended protocol. 
11.4 
Informed Consent 
A written informed consent in compliance with Part 50 of Title 21 of the Code of Federal 
Regulations (CFR) shall be obtained from each subject before entering the study or performing 
any unusual or nonroutine procedure that involves risk to the subject. An informed consent 
template may be provided by OncoNano Medicine to investigative sites. If any institution-
specific modifications to study-related procedures are proposed or made by the site, the consent 
should be reviewed by OncoNano Medicine and/or its designee, if appropriate, before IRB 
submission. Once reviewed, the consent will be submitted by the principal investigator to their 
IRB for review and approval before the start of the study. If the informed consent form is revised 
during the course of the study, all active participating patients must sign the IRB-approved 
revised form. 


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Before recruitment and enrollment, each prospective subject will be given a full explanation of 
the study and allowed to read the approved informed consent form. Once the principal 
investigator is assured that the subject understands the implications of participating in the study, 
the subject will be asked to give consent to participate in the study by signing the informed 
consent form. 
The principal investigator shall provide a copy of the original form of the signed informed 
consent to the subject and/or legal guardian.  
11.5 
Protocol Violations and Deviations 
The principal investigator or designee must document and explain in the subject’s source 
documentation any deviation from the approved protocol. The principal investigator may 
implement a deviation from or a change of the protocol to eliminate an immediate hazard to trial 
patients without prior IRB approval. As soon as possible after such an occurrence, the 
implemented deviation or change, the reasons for it, and any proposed protocol amendment(s) 
should be submitted to the IRB for review and approval, to OncoNano Medicine for agreement, 
and to the regulatory authorities, if required. 
A deviation from the protocol is an unintended and/or unanticipated departure from the 
procedures and/or processes approved by OncoNano Medicine and the IRB and agreed to by the 
principal investigator. Deviations usually have an impact on individual patients or a small group 
of patients and do not involve inclusion/exclusion or primary endpoint criteria. A protocol 
violation occurs when there is nonadherence to the protocol that results in a significant, 
additional risk to the subject, when the subject or principal investigator has failed to adhere to 
significant protocol requirements (inclusion/exclusion criteria) and the subject is enrolled 
without prior approval by OncoNano Medicine, or when there is nonadherence to FDA 
regulations and/or ICH E6(R1) guidelines. 
The clinical monitor will document protocol violations and deviations throughout the course of 
monitoring visits. The monitor will notify the principal investigator during a visit and/or in 
writing of all violations and deviations. The IRB should be notified of all protocol violations and 
deviations in a timely manner. 
11.6 
Study Reporting Requirements 
By participating in this study, the principal investigator agrees to submit reports of SAEs 
according to the timeline and method outlined in the protocol. In addition, the principal 
investigator agrees to submit annual reports to their IRB as appropriate. The principal 
investigator also agrees to provide OncoNano Medicine with an adequate report shortly after 
completion of the principal investigator’s participation in the study. 
11.7 
Investigator Documentation 
Before beginning the study, the principal investigator will be asked to comply with ICH 
E6(R1) 8.2 and 21 CFR by providing the following essential documents, including but not 
limited to: 
 An original investigator-signed Investigator Agreement page of the protocol.  


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 An IRB-approved informed consent, samples of site advertisements for 
recruitment for this study, and any other written information regarding this study 
that is to be provided to the subject or legal guardians. 
 IRB approval. 
 Form FDA 1572, fully executed, and all updates on a new fully executed Form 
FDA 1572. 
 Curriculum vitae (CV) for the principal investigator and sub-investigators listed 
on Form FDA 1572. Current licensure must be noted on the CV. They will be 
signed and dated by the principal investigator at study start-up, indicating that 
they are accurate and current. 
 Financial disclosure information to allow OncoNano Medicine to submit 
complete and accurate certification or disclosure statements required under 21 
CFR 54. In addition, the investigators must provide to OncoNano Medicine a 
commitment to promptly update this information if any relevant changes occur 
during the course of the investigation and for 1 year following the completion of 
the study. 
 Laboratory certifications and reference ranges for any local laboratories used by 
the site, in accordance with 42 CFR 493. 
11.8 
Study Conduct 
The principal investigator agrees that the study will be conducted according to the principles of 
the ICH E6(R1) guidelines and the principles of the World Medical Association Declaration of 
Helsinki. The principal investigator will conduct all aspects of this study in accordance with all 
national, state, and local laws or regulations. 
11.9 
Publications 
Following completion of the study, the data may be considered for reporting at a scientific 
meeting or for publication in a scientific journal. In these cases, OncoNano Medicine will be 
responsible for these activities and will work with the investigators to determine how the 
manuscript is written and edited, the number and order of authors, the publication to which it will 
be submitted, and other related issues. OncoNano Medicine has final approval authority over all 
such issues. 
Data are the property of OncoNano Medicine and cannot be published without prior 
authorization, but data and publication thereof will not be unduly withheld. 


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12.  REFERENCES 
 
1 
Society, A. C. Cancer Facts & Figures 2016. (American Cancer Society, Atlanta, 2016). 
2 
Jemal, A. et al. Cancer statistics, 2009. CA Cancer J Clin 59, 225-249, 
doi:10.3322/caac.20006 (2009). 
3 
Aliperti, L. A., Predina, J. D., Vachani, A. & Singhal, S. Local and systemic recurrence is 
the Achilles heel of cancer surgery. Ann Surg Oncol 18, 603-607, doi:10.1245/s10434-
010-1442-0 (2011). 
4 
Ma, X. et al. Ultra-pH-sensitive nanoprobe library with broad pH tunability and 
fluorescence emissions. J Am Chem Soc 136, 11085-11092, doi:10.1021/ja5053158 
(2014). 
5 
Zhao, T. et al. A Transistor-like pH Nanoprobe for Tumour Detection and Image-guided 
Surgery. Nat Biomed Eng 1, doi:10.1038/s41551-016-0006 (2016). 
6 
Li, Y. et al. Molecular basis of cooperativity in pH-triggered supramolecular self-
assembly. Nat Commun 7, 13214, doi:10.1038/ncomms13214 (2016). 
7 
Voskuil, F. J. et al. Exploiting metabolic acidosis in solid cancers using a tumor-agnostic 
pH-activatable nanoprobe for fluorescence-guided surgery. Nat Commun 11, 3257, 
doi:10.1038/s41467-020-16814-4 (2020). 
 
 




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Footnotes 
a For female patients, a serum sample or urine dipstick for a pregnancy test will be collected at screening and on Day 0 and Day 21. 
b Body mass index will be calculated at Screening only. 
c Vital sign measurements will include systolic and diastolic blood pressure, pulse, respiratory rate, and temperature (in degrees Celsius). Blood pressure and 
pulse will be measured after a resting period of at least 5 minutes in the supine position. 
d Patient’s vital signs are to be recorded immediately after the infusion and 30 minutes afterward 
e The Sponsor recommends the use of prophylactic diphenhydramine prior to study drug administration, in an effort to reduce the prevalence of infusion-related 
reactions.   
f Patients will be assessed for AEs occurring from the time of dosing through Day 21 (±10 days)  
g Hematology assessments may include hemoglobin, hematocrit, erythrocyte count (red blood cells), differential leukocytes, platelet count, and total leukocytes 
(white blood cells). 
h 
Pathologic analysis of surgical specimens will take place from the conclusion of surgery and up to 10 days after 
 


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14. APPENDIX 2: FDA-CLEARED NIR IMAGING DEVICES 
NOTE: this list is not comprehensive and does not restrict the use of other FDA-cleared imaging 
device from use in this trial.  
14.1 
NOVADAQ SPY Elite 
The NOVADAQ SPY Elite system is an active device used for intraoperative fluorescence 
imaging. The device is developed for open surgery. The device uses visible (white light) and NIR 
spectra in real-time to visualize circulation and ICG based tumor detection. The device is 
commercially available and 510(k) cleared by the FDA for surgical use. 
14.2 
NOVADAQ SPY-PHI 
The NOVADAQ SPY-PHI (Portable Handheld Imaging) system is an active device used for 
fluorescence imaging. The device uses both visible (white light) and NIR spectra, which can be 
used for visualization of circulation and ICG-based tumor detection during several (surgical or 
non-surgical) procedures. The device is commercially available and 510(k) cleared by the FDA 
for surgical use. 
14.3 
NOVADAQ PINPOINT 
The NOVADAQ PINPOINT is an intraoperative laparoscopic fluorescence imaging system, used 
in conjunction with an injected ICG based fluorescent imaging agent, to assess perfusion and 
visualize structural anatomy using visible (white light) and NIR during cardiothoracic, 
gastrointestinal, breast, and other reconstructive surgical procedures. 
14.4 
da Vinci Firefly  
The da Vinci Firefly system (Intuitive Surgical, California, USA) provides real-time NIR guidance 
through visualization of injectable fluorescence dye. The device uses visible (white light) and NIR 
spectra in real-time to visualize circulation and ICG based tumor detection. The device is 
commercially available and FDA-cleared for visual assessment of vessels, blood flow, and related 
tissue perfusion using NIR imaging.  
14.5 
Visionsense Iridium 
The Visionsense Iridium camera system (Visionsense, Philadelphia, PA, USA) uses 3DHD 
technology intra-operatively for real-time infrared fluorescence and white light imaging 
visualization during live surgery to enhance anatomical feature identification. 
 

SAP_003.pdf: 
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Statistical Analysis Plan v3 
 
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STATISTICAL ANALYSIS PLAN 
 
Protocol: ON-1005 
A Phase 2, single-dose, open-label study to evaluate diagnostic performance and safety of 
pegsitacianine, an intraoperative fluorescence imaging agent for the detection of lung 
malignancies, in patients undergoing routine surgery 
 
SAP Version: 
Version 3 
SAP Date: 
07 NOV 2022 
 
CT Registry Number: NCT04950166 
 
Prepared by 
 
 
 




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SIGNATURES OF APPROVAL 
Study Title: Phase 2, single-dose, open-label study to evaluate diagnostic performance and 
safety of pegsitacianine, an intraoperative fluorescence imaging agent for the detection of lung 
malignancies, in patients undergoing routine surgery 
Protocol Number: ON-1005 
Protocol Version:V3 07 NOV 2022 
 
 
Date: 
Author: 
 
 
 
Date: 
Reviewer: 
 
 
Date: 
Approver: 
 
 
07-Nov-2022
07-Nov-2022
07-Nov-2022


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TABLE OF CONTENTS 
Statistical Analysis Plan .................................................................................................................. 1 
A Phase 2, single-dose, open-label study to evaluate diagnostic performance and safety of 
pegsitacianine, an intraoperative fluorescence imaging agent for the detection of lung 
malignancies, in patients undergoing routine surgery .................................................................... 1 
Document History ........................................................................................................................... 2 
Signatures of Approval ................................................................................................................... 3 
Table of Tables ............................................................................................................................... 7 
List of Abbreviations ...................................................................................................................... 8 
1. Introduction ............................................................................................................................... 10 
2. Study Objectives ....................................................................................................................... 10 
2.1 Objectives and Endpoints ....................................................................................................... 10 
3. Study Description...................................................................................................................... 11 
3.1 Study Design ........................................................................................................................... 11 
3.2 Study Treatment ...................................................................................................................... 13 
4. Sample Size and Power Calculation ......................................................................................... 13 
5. Analysis Endpoints ................................................................................................................... 13 
5.1 Efficacy Endpoints .................................................................................................................. 13 
5.1.1 Clinically Significant Event Endpoints ................................................................................ 13 
5.1.2 Diagnostic Imaging Endpoints ............................................................................................. 14 
5.1.3 Fluorescence Endpoints ....................................................................................................... 15 
5.2 Safety Endpoints ..................................................................................................................... 16 


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6. Analysis Populations ................................................................................................................. 16 
6.1 The Safety Population ............................................................................................................. 16 
6.2 The Intent-to-Treat (ITT) Population...................................................................................... 16 
6.3 The Efficacy Population ......................................................................................................... 16 
7. Analytical Plan and Statistical Methods ................................................................................... 16 
7.1 General Considerations ........................................................................................................... 16 
7.2 Reporting Conventions ........................................................................................................... 17 
7.3 Definition of Baseline ............................................................................................................. 18 
7.4 Handling of Dropouts and Missing, Unused and Spurious Data ............................................ 18 
7.4.1 Dates .................................................................................................................................... 18 
7.5 Adjustment for Multiplicity .................................................................................................... 19 
7.6 Adjustment for Multiple Centers ............................................................................................ 19 
7.7 Patient Disposition .................................................................................................................. 19 
7.8 Protocol Deviations ................................................................................................................. 19 
7.9 Patient Characteristics ............................................................................................................. 20 
7.9.1 Baseline and Demographic Characteristics ......................................................................... 20 
7.9.2 Medical History ................................................................................................................... 21 
7.9.3 Prior and Concomitant Medications and Procedures/Treatments ........................................ 21 
7.9.4 Administration and Exposure of Study Drug....................................................................... 21 
7.9.5 Surgery Details and Specimen Collection ........................................................................... 21 
7.10 Efficacy Endpoint Analyses .................................................................................................. 22 


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7.10.1 Primary Analyses ............................................................................................................... 23 
7.10.2 Secondary Analyses ........................................................................................................... 23 
7.10.3 Exploratory Analyses ......................................................................................................... 23 
7.11 Safety Endpoint Analyses ..................................................................................................... 24 
7.11.1 Adverse Events .................................................................................................................. 24 
7.11.2 Laboratory Data ................................................................................................................. 25 
7.11.3 Vital Signs .......................................................................................................................... 26 
7.11.4 Physical Exam .................................................................................................................... 26 
7.11.5 ECG.................................................................................................................................... 26 
8. Deviations from Analysis as Described in the Protocol ........................................................... 26 
9. Appendix 1: Schedule of Events ............................................................................................... 27 
 
 
 
 


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TABLE OF TABLES 
TABLE 1. OBJECTIVES AND ENDPOINTS ....................................................................................................................... 10 
 
 


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LIST OF ABBREVIATIONS 
ABBREVIATION 
TERM 
AE 
Adverse Event 
ALT 
Alanine Aminotransferase 
ANOVA 
Analysis of Variance 
AST 
Aspartate Aminotransferase 
BMI 
Body Mass Index 
BUN 
Blood Urea Nitrogen 
CFR 
Code of Federal Regulations 
CSE 
Clinically Significant Event 
CT 
Computed Tomography 
CV 
Curriculum Vitae 
D5W 
5% Dextrose in water 
DoH 
Declaration of Helsinki 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
FDA 
United States Food and Drug Administration 
HNSCC 
Head and Neck Squamous Cell Carcinoma 
ICG 
Indocyanine Green 
ICH 
International Conference on Harmonisation 
IRB 
Institutional Review Board 
IRR 
Infusion Related Reaction 
IV 
Intravenous 
MFI 
Mean Fluorescence Intensity 
MRI 
Magnetic Resonance Imaging 
NIR 
Near-infrared 
NPV 
Negative Predictive Value 
PEG 
Polyethylene Glycol 
PET 
Positron Emission Tomography 
PK 
Pharmacokinetics 
PMMA 
Polymethylmethacrylate 
PPV 
Positive Predictive Value 
ROC 
Receiver Operating Characteristic 


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ABBREVIATION 
TERM 
SAE 
Serioust Adverse Event 
SBR 
Specimen-to-Background Ratio 
SOC 
Standard of Care 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
SWI 
Sterile Water for Injection 
TBR  
Tumor-to-Background Ratio 
TEAE 
Treatment-Emergent Adverse Event 
VATS 
Video-Assisted Thorascopic Surgery 
WMA 
World Medical Association 
 
 
 






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Patients will have a biopsy confirmed diagnosis, or a high clinical suspicion of a lung 
malignancy based on CT and/or PET imaging. A total of up to 40 patients will be enrolled in this 
trial. The sponsor reserves the right to perform an interim analysis following early enrollment (n 
surgery. 
The surgeon will attempt to perform their SOC resection of visible tumors. If the tumor is unable 
to be located under standard white light conditions, an appropriate near-infrared (NIR) imaging 
device may be used to aid in the location of the primary tumor. Localization of the primary 
tumor using NIR imaging in conjunction with pathology confirmed presence of disease will be 
considered a clinically significant event.  
If the primary tumor is located using standard white light procedures, the tumor will be imaged 
using the NIR camera and removed as per standard of care (i.e., wedge, lobectomy, etc.). The 
primary resection will then be imaged (front, back and margins) on a piece of normal tissue 
within the surgical field at a distance from the specimen equal to the working distance of the 
camera system. Inspection and imaging of the resection margins will be performed using the NIR 
camera system. If an area of suspicion is observed, images will be captured of the area, at the 
working distance of the camera away from the margin and documented for future correlation to 
pathological findings. Similarly, the in situ resection margin on the lung will be imaged for signs 
or residual fluorescence that may correspond to disease left behind. If found, images will be 
captured prior to the excision of the margin (in situ) and following its resection on a piece of 
normal tissue (ex situ). Resection of the suspicious or fluorescent margin is at the discretion of 
the surgeon. This procedure is to be repeated if additional SOC nodules are planned for 
resection. An identical approach will be followed for any SOC lymph nodes collected during the 
procedure. Lymph node imaging will occur prior to and following the removal of the suspected 
lymph nodes. 
Once SOC surgical resection is complete, the surgeon will use the NIR camera system to 
evaluate the thoracic cavity (i.e., chest wall, contralateral lung, bronchi, etc.) for indications of 
residual fluorescence. If additional fluorescent foci are observed, the area may be resected, at the 
discretion of the surgeon, for pathological confirmation of disease. Similar to the imaging of 
margin resections, the suspected area will be imaged prior to removal (in situ) and following 
resection (ex situ) on a piece of normal tissue within the thoracic cavity. Additional fluorescent 
lymph nodes, detected during the evaluation of the thoracic cavity, may also be collected and 
imaged both in situ and ex situ prior to being evaluated in pathology. Thus, each subject is their 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 


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Collected surgical specimens will be documented as called out by the surgeon. The fluorescence 
status of each specimen will then be correlated to the final pathology for each and the 
performance metrics of pegsitacianine will be tabulated. 
 
3.2 Study Treatment 
Each patient will be administered a  mg/kg dose of pegsitacianine 
 
The volume of drug and total dose will vary by patient and 
weight. 
4. SAMPLE SIZE AND POWER CALCULATION 
The sample size for this study will be 40 subjects. This sample size is based on historical 
experience with the proposed analysis. A sample size of 40 subjects will be sufficient for 
meeting the endpoints described herein.  
5. ANALYSIS ENDPOINTS 
5.1 Efficacy Endpoints 
5.1.1 Clinically Significant Event Endpoints 
Performance metrics will include the identification of a CSE detected by pegsitacianine. CSEs 
will include pegsitacianine detection of primary tumors otherwise undetectable using SOC 
procedures, detection of occult disease not otherwise known by the surgeon to exist (as 
synchronous disease), the detection of a positive surgical margin, and accurate identification of 
tumor negative SOC biopsies. A list of CSEs and their definitions is provided below. 
 Detection of Synchronous Disease  identification of tumor-containing tissue due to 
pegsitacianine fluorescence, confirmed via histopathological analysis, that otherwise 
went undetected during pre-operative imaging (PET, CT, MRI) or by the surgeon during 
SOC surgery. 
o This occurs when a specimen that comes from the thoracic cavity or lymph nodes, 
is (1) collected via fluorescence and (2) is pathologically-confirmed as a tumor. 
Note that any specimen collected from the thoracic cavity following SOC will be 
considered to be collected by fluorescence. 
 
 
 
 
 
 
 


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 Detection of Positive Surgical Margin  following the completion of SOC surgery, the 
surgical fields will be examined for residual fluorescence. Detection of a positive surgical 
margin would be those specimens collected as a result of residual pegsitacianine 
fluorescence following the completion of SOC surgery that return a histologically 
positive for tumor outcome.  
o This occurs when a margin specimen (either from the primary tumor or a lung 
resection margin) (1) is collected via fluorescence and (2) is pathologically-
confirmed as a tumor. Note that any lung resection margin collected will be 
considered to be collected by fluorescence.  
 Identification/Localization of the Primary Tumor  during SOC surgery, the surgeon will 
examine the affected lung to locate the primary tumor. If the primary tumor is not 
visually apparent using standard imaging methods the surgeon may use the NIR imaging 
device to aid in localization. If the NIR imaging identifies and/or locates the primary 
tumor, which was undetectable using SOC approaches, this will be deemed a CSE. 
o This occurs when the primary tumor is not located using SOC approaches, and is 
instead identified using NIR imaging. 
 Tumor Negative SOC Biopsies  during SOC surgery, the surgeon will collect tissue 
specimens that are suspicious for disease. If those samples do not demonstrate 
pegsitacianine fluorescence and are found to be devoid of tumor pathologically, those 
specimens would be included as a tumor negative SOC biopsy. 
o This occurs when any SOC sample (primary tumor, additional SOC specimen, or 
lymph node) that is collected that is found to be negative for tumor, and did not 
fluoresce during ex situ imaging.  
A subject with any CSE will be considered a CSE responder. 
5.1.2 Diagnostic Imaging Endpoints 
Diagnostic performance of pegsitacianine will be assessed by calculating sensitivity, specificity, 
NPV, and PPV of pegsitacianine in detecting tumor containing tissue. The ex situ fluorescence 
status (any fluorescence vs. no fluorescence) of collected SOC and pegsitacianine guided 
specimens, as well as lymph nodes, will be compared to histological analyses of the collected 
specimens. Sensitivity and specificity for detecting tumor-containing tissue versus normal tissue 
 
 
 


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will be evaluated using tissue specimen fluorescence and the corresponding pathological 
outcomes. 
True/false negative/positive definitions are as follows: 
 False positive is defined as fluorescence was observed on the sample but the sample was 
not found to have tumor via pathology.  
 False negative is defined as no fluorescence was observed on the sample but the sample 
was found to have tumor via pathology. 
 True positive is defined as fluorescence was observed on the sample and the sample was 
found to have tumor via patholog1y 
 True negative is defined as no fluorescence was observed on the sample and the sample 
was found not to have tumor via pathology. 
Pegsitacianine sensitivity and PPV will be calculated using the following equations:        
 
 
Pegsitacianine specificity values and NPV will be calculated using the following equations: 
 
 
5.1.3 Fluorescence Endpoints 
Specimen-to-background (SBR) ratios will also be calculated using white light and NIR images 
of both SOC and pegsitacianine guided specimens. SBRs will be calculated using ex situ images 
of collected specimens. The equation for calculating SBR is listed below.   
 
 
 
 
 
  
 
 
 
 
 
 
     
 
  
 
 
    
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   
 
 
 
 
   
  
 
 
 
 
 
  
 
  
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 


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Note: tumor-to-background (TBR) ratios are equivalent to SBR, but only relevant for specimens 
with tumors. 
5.2 Safety Endpoints 
- 
TEAE incidence, severity, and relationship to study drug 
- 
Physical examination results 
- 
Vital signs results 
- 
ECG results 
- 
Clinical laboratory values (hematology and serum chemistry) 
6. ANALYSIS POPULATIONS 
6.1 The Safety Population 
All patients who receive at least one dose of pegsitacianine.  
6.2 The Intent-to-Treat (ITT) Population 
Identical to the Safety Population.  
6.3 The Efficacy Population 
All patients who receive >75% intended dose of pegsitacianine, had a minimum of one (1) image 
collected during their procedure and had the opportunity for post-SOC evaluation of the thoracic 
cavity. 
7. ANALYTICAL PLAN AND STATISTICAL METHODS 
7.1 General Considerations 
All computations for statistical analyses will be performed using SAS® software, Version 9.4 or 
later. All SAS programs used in the production of statistical summary outputs will be validated 
with independent programming prior to finalization. In addition, all program outputs will be 
independently reviewed. The validation process will be used to confirm that all data 
manipulations and calculations were accurately done. Once validation is complete, a senior 


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statistical reviewer will perform a final review of the documents to ensure the accuracy and 
consistency with this plan and consistency within tables. Upon completion of validation and 
quality review procedures, all documentation will be collected and filed by the project statistician 
or designee. 
If necessary, the statistical analytical plan and statistical methods section may be updated before 
the database lock. Any changes in statistical methods that may have an impact on the primary 
conclusions drawn from this clinical trial will be described in an amendment to the protocol. All 
other changes in the statistical plan will be described in the clinical study report (CSR). An 
explanation will be provided for deviations from the planned analysis. 
7.2 Reporting Conventions 
The following conventions will be applied to all data presentations and analyses:  
 Continuous variables will generally be summarized by the number of patients with 
missing and non-missing values, mean, standard deviation, median, Q1 (quartile 1), Q3 
(quartile 3), minimum, and maximum.  
 Categorical variables will be summarized by the number and percentage of patients 
within each category. If not specified otherwise, the number of patients with non-missing 
values will be the denominator for percentage calculations. The number of patients with 
missing values will be presented. 
 Confidence intervals will be reported as two sided at the 95% level. 
 All mean and median values will be formatted to one more decimal place than the 
measured value. 
 Standard deviation values will be formatted to two more decimal places than the 
measured value. 
 Minimum and maximum values will be presented with the same number of decimal 
places as the measured value. 
 The number and percent of responses will be presented in the form XX (XX %), where 
the percentage is in parentheses. Percentages will be rounded to the nearest percent. In 
 
 
 
 
 
 
 


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the case of a frequency of zero, the frequency and percentage will be presented as 0 
rather than 0 (0%). 
 Date variables will be formatted as DDMMMYYYY for presentation 
All the analyses will be run displaying the results from Part 1. All rates will be summarized using 
counts and percentages. 
7.3 Definition of Baseline 
Baseline is defined as the measurement closest to, but prior to, the administration of study drug. 
No reassignment of visits will be conducted. 
7.4 Handling of Dropouts and Missing, Unused and Spurious Data 
All data will be analyzed.  
No imputation of missing data will be performed, and analyses will use the observed cases only.  
Formal outlier testing will not be performed. Data identified as potential outliers after review 
will be reviewed to determine whether they are spurious. 
Spurious data are data determined to be incorrect upon review of site documents. If data is 
deemed to be spurious, the spurious data and the reason for it being spurious will be documented 
by the Sponsor in a Note-to-file. Data identified as spurious will be treated as missing unless 
they can be definitively corrected. If spurious data is identified, sensitivity analysis may be 
performed to assess the potential impact of any missing data or outliers on trial results by 
including the spurious data. 
7.4.1 Dates 
Imputation of missing or partial dates is not expected, but if a complete date is required for 
calculations, the following algorithms will be applied:  
 For the start date:  
o If year, month, and day are missing then use the minimum of the patient's first 
visit date or the consent date.  
o If either only month or month and day are missing then use January 1.  
 
 


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o If only day is missing, impute the first day of the month.  
 For the end date:  
o If year, month, and day are missing then use the patient's last visit date.  
o If either only month or month and day are missing then use December 31.  
o If only day is missing then use the last day of the month.  
o Do not expand the record past the patient's last visit.  
o The original missing or partial date, the imputed complete date, and the indicator 
variable that indicates which dates were imputed will be retained in the database. 
7.5 Adjustment for Multiplicity 
There are no planned adjustments for multiple endpoints or analyses. 
7.6 Adjustment for Multiple Centers 
Differences between study centers will be not be incorporated into the statistical analyses for this 
study. There are no plans to analyze data within centers. 
7.7 Patient Disposition 
The number of screened patients will be summarized.  
The number of enrolled patients will be summarized, along with the number and percentage of 
enrolled patients in all of the analysis populations, patients completing the study, patients 
withdrawing from the study and their primary reason for withdrawal, and the number of deaths, 
by Part and overall.  
Data listings of patient disposition, screening failures, and patient assignment by Part will be 
created. 
7.8 Protocol Deviations 
A deviation from the protocol is an unintended and/or unanticipated departure from the 
procedures and/or processes approved by OncoNano Medicine and the IRB and agreed to by the 
 


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principal investigator. Deviations usually have an impact on individual patients or a small group 
of patients and do not involve inclusion/exclusion or primary endpoint criteria. Deviations which 
 
The number and percentage of patients with at least one major protocol deviation and the 
categories of the major protocol deviations will be summarized for the Safety population.  
All protocol deviations will be provided in a listing. 
7.9 Patient Characteristics 
Unless otherwise noted, all patient characteristics will use the Safety population. 
7.9.1 Baseline and Demographic Characteristics 
All baseline characteristics will be summarized by each Part and overall for the Safety 
population. The following parameters will be summarized: 
- 
Age (years) 
- 
Race 
- 
Ethnicity  
- 
Sex 
- 
Child-bearing Potential (if female) 
- 
Height (cm) 
- 
Weight (kg) 
- 
BMI (kg/m2) 
- 
Karnofsky Performance Status 
No hypothesis testing is planned for baseline characteristics, so the analysis will be purely 
descriptive. 
Data listings of baseline characteristics and demographics including Karnofsky Status will be 
created. 
 
  
 
 
 
 
 
 
 
 
 
 


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7.9.2 Medical History 
Medical history will be summarized for all non-cancer related medical events, previous cancers, 
and previous anti-cancer therapies. Medical conditions will be coded using Medical Dictionary 
of Regulatory Activities (MedDRA) 23.0 dictionary or higher and summarized by System Organ 
Class (SOC) and preferred term (PT) in the Safety population. A listing for the variables above 
will also be generated. 
A summary and listing of previous alcohol, drug, and tobacco use will be provided. 
7.9.3 Prior and Concomitant Medications and Procedures/Treatments 
Prior and concomitant medication (as well as concomitant procedures/treatments) will be 
summarized by Part and overall in the Safety population. Medications will be coded with the 
World Health Organization (WHO) Drug Dictionary (B3 2021-09-01 DDE). Concomitant 
medications will be summarized by Anatomical Therapeutic Chemical (ATC) Classification 
System level 2 and 4 codes (ATC2 and ATC4). Procedures/treatments will be coded using 
MedDRA 23.0 or higher and summarized by SOC and PT. A medication, procedure, or treatment 
will be considered to be concomitant if its end date is after the date of study drug administration. 
Medications with a missing end date will also be considered concomitant. A medication with an 
end date prior to the date of study drug administration will be considered prior medications. 
Listings for prior and concomitant medications / procedures will be provided.  
7.9.4 Administration and Exposure of Study Drug 
Administration of study drug (planned dose level, total dose administered, total volume 
administered, infusion flow rate, infusion duration, subjects with infusion reactions, and subjects 
with dose interruptions) will be summarized using descriptive statistics. Infusion flow rate will 
be calculated as total volume administered infusion duration. A listing will also be generated. 
7.9.5 Surgery Details and Specimen Collection 
Surgery details including the following will be summarized on a patient level using descriptive 
statistics in the Safety population: 
 Duration of surgery 
 Location of tumor 
 
 


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 Method of tumor localization, including number of subjects with primary tumor(s) 
identified using NIR imaging 
 Number of SOC specimens collected 
 Number of additional lung nodules/areas found as a result of NIR imaging following 
SOC procedures and confirmed to be cancer 
 Number of SOC specimens collected, found to be negative for tumor, and did not 
fluoresce during ex situ imaging 
 Number of areas with fluorescence removed on: 
o Lung resection margins 
o Thoracic cavity  
o Lymph nodes  
o Total (any area) 
 Number of areas with fluorescence on lung resection margins 
 Subjects with fluorescence observed anywhere in thoracic cavity 
 Number of lymph nodes collected as SOC surgery 
 Time spent imaging 
 Camera system used 
 A listing will also be generated. 
7.10 Efficacy Endpoint Analyses 
Imaging will be assessed on the Efficacy population.  
 
 
 
 
 
 
 
 
 
 


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7.10.1 Primary Analyses 
7.10.1.1 Analysis of Clinically Significant Events 
The number and proportion of subjects with CSEs will be summarized by Part and overall. CSE 
responders will be summarized, as well as subjects with each type of CSE. These summaries will 
include 95% 2-sided confidence intervals for the proportion of CSEs calculated via Clopper-
Pearson method.  
A listing of all CSEs by patient will be provided. 
7.10.2 Secondary Analyses 
7.10.2.1 Analysis of Diagnostic Imaging  
The positive predictive value, negative predictive value, sensitivity, and specificity (specimen 
level) will be calculated and summarized for each Part and overall. The summary will include the 
true/false negative/positive rate and number of specimens. Two-sided 95% confidence intervals 
will be calculated for each rate via Clopper-Pearson method. 
A listing by subject and specimen showing the specimen type (primary tumor/ additional SOC 
specimen, lung resection margin, thoracic cavity, lymph node), attainment by SOC or 
fluorescence (if primary/ additional SOC/ lymph), pathology result, fluorescence in situ and ex 
situ, and fluorescence measures (MFI and TBR) will be provided. 
7.10.3 Exploratory Analyses 
7.10.3.1 Analysis of Fluorescence Intensity 
A summary of MFI for the tumor (or suspected tumor), MFI for the normal background tissue, 
and TBR will be provided by Part and overall for each pathology result (tumor vs. no tumor). 
This summary will be at the specimen level, meaning each subject may have multiple specimens. 
7.10.3.2 ROC Analysis 
In this pilot study we are assuming that any fluorescence is indicative of tumor presence. To assess 
how the amount of observed fluorescence (e.g., the SBR or TBR) may affect the diagnostic 
accuracy of pegsitacianine, a receiver operating characteristic (ROC) analysis will be performed 
on all specimens using the observed thresholds of SBR and TBR. The corresponding ROC curve 


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will be provided along with the area under the curve (AUC) and the 95% confidence interval of 
the AUC.  
There is potential for within-subject correlation to bias 
diagnostic accuracy. To assess this issue, an ROC curve will be constructed from a mixed model 
that clusters specimens by the subject they originated from. We will compare the AUC of the 
mixed effects ROC curve to the standard ROC curve to assess if within-subject correlation is 
causing significant bias in our analyses. If it is determined that significant bias has been 
introduced, additional analyses on secondary endpoints may be performed to assess the 
diagnostic accuracy of pegsitacianine. 
7.10.3.3 Camera System Analysis 
An analysis on diagnostic results by camera system will be performed. 2x2 incidence tables 
comparing the true/false negative/positive results will be constructed for each camera system, 
and compared via the Breslow-Day  test to assess if there is any effect of camera system on 
diagnostic results (e.g. to test if the common odds-ratio of each incidence table is equivalent 
across camera types). A summary including the diagnostic true/false negative/positive numbers 
and rates for each camera system, sensitivity, specificity, and p-value of this analysis will be 
provided.  
7.10.3.4 Other Exploratory Analyses 
Relationships of sensitivity and specificity may be evaluated post-hoc with respect to additional 
covariates including, but not limited to, tumor size, tumor type, and tumor location. 
7.11 Safety Endpoint Analyses 
All the analyses in this section will be performed on the Safety population by Part and overall. 
Patients will be analyzed according to the actual treatment they received.  
Repeated or unscheduled results will not be included in the summary statistics but will be 
included in the individual data listings. 
7.11.1 Adverse Events 
All adverse events (AEs) will be coded using the Medical Dictionary for Regulatory Activities 
(MedDRA). A TEAE is defined as an AE that occurs (or worsens) during or after study drug 
administration and through Day 21 after administration of study drug.  
 
 
 
 


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An overview of all AEs will be provided that will include: 
 Number of AEs, TEAEs, non-serious TEAEs, and Serious Adverse Events (SAEs) 
 Number of TEAEs and SAEs related to study drug 
 Number of TEAEs related to study drug with CTCAE grade >= 3 
 Number of AEs and study drug related SAEs resulting in: 
o Death 
o Subject discontinuation 
o Study drug discontinuation 
TEAEs, treatment-related TEAEs, TEAEs by CTCAE grade, TEAEs that lead to discontinuation 
of study drug, SAEs, and SAEs that lead to discontinuation of study drug, will be summarized by 
system organ class (SOC) and preferred term (PT).  
Infusion related reactions (IRR) will be summarized by PT, and a descriptive summary of the 
duration of IRR will be provided. 
A listing of all AEs, including a separate listing for IRR, will also be generated. 
7.11.2 Laboratory Data 
The clinical laboratory data will be summarized by scheduled time point, Part, and change from 
baseline. Any laboratory data below/above the limit of quantification will be summarized using 
the value of the limit of quantification (upper or lower, respectively). A listing will be provided 
where the values that are below the lower limit or above the upper limit of the reference range 
will be flagged. Those values or changes in values that are considered clinically significant by 
the investigator will also be flagged in the listing.  
Repeated or unscheduled results will not be included in the by timepoint summaries but will be 
included when looking for extreme values and displayed in the individual data listings. 
 
 
 
 


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7.11.3 Vital Signs 
Vital signs data will be summarized by time point, Part, and change from baseline. A listing will 
also be provided. 
Repeated or unscheduled results will not be included in the summary statistics but will be 
included in the individual data listings. 
7.11.4 Physical Exam 
Physical examination results will be provided as a listing. 
7.11.5 ECG 
ECG results will be provided as a listing. 
8. DEVIATIONS FROM ANALYSIS AS DESCRIBED IN THE PROTOCOL 
No deviations from the protocol are planned.




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Footnotes 
a For female subjects, a serum sample or urine dipstick for a pregnancy test will be collected at screening and on Day 0 and Day 21. 
b Body mass index will be calculated at Screening only. 
c Vital sign measurements will include systolic and diastolic blood pressure, pulse, respiratory rate, and temperature (in degrees Celsius). Blood pressure 
and pulse will be measured after a resting period of at least 5 minutes in the supine position. 
d 
30 minutes afterward 
e The Sponsor recommends the use of prophylactic diphenhydramine prior to study drug administration, in an effort to reduce the prevalence of infusion-
related reactions.   
f Patients will be assessed for AEs occurring from the time of dosing through Day 21 (±10 days)  
g Hematology assessments may include hemoglobin, hematocrit, erythrocyte count (red blood cells), differential leukocytes, platelet count, and total 
leukocytes (white blood cells). 
h 
Pathologic analysis of surgical specimens will take place from the conclusion of surgery and up to 10 days after 
 
Serum chemistry  
X 
X 
 
X 
X 
X 
Hematologyg  
X 
X 
 
X 
X 
X 
Surgery 
 
 
 
X 
 
 
Intraoperative Imaging 
 
 
 
X 
 
 
Pathology evaluation of surgical specimens 
 
 
 
Xh