Clinical trial NCT04950166
Conditions: Peritoneal Carcinomatosis
Sponsor: OncoNano Medicine, Inc.
Start date: 2021-11-30; Completion date: 2023-01-20
Enrollment: 51 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Peritoneal carcinomatosis (PC) results from the metastasis of a primary cancer of the peritoneum (e.g., appendiceal, ovarian, uterine, colorectal, and gastric cancers) that then disseminates throughout the abdominal cavity. Historically progression to PC was considered terminal and resulted in survival times on the scale of a few months with palliative care being the best option for patients. More recently, cytoreductive surgery (CS) has emerged as a means to prolong and improve patient lives with a median increase in survival of up to \~5 years. It has been reported that for every 10% increase in cytoreduction there is a 5.5% increase in median survival time. In addition to surgical tumor debulking within the peritoneal space, it has also been shown that coupling surgical intervention with hyperthermic intraperitoneal chemotherapy (HIPEC) can have an even greater impact on patient outcomes. Pegsitacianine, a micellar fluorescence agent, exploits the ubiquitous pH differences observed between cancerous and normal tissues. This in turn, provides a highly sensitive and specific fluorescence response after localizing within the tumor microenvironment, thus allowing the detection of primary tumors, their margins, metastatic disease, and tumor-containing lymph nodes.

Detailed description:

Arms: arm 1: 1 mg/kg of pegsitacianine administered IV 24-72 hours prior to surgery.
Interventions: intervention 1: pegsitacianine
Intervention descriptions: intervention 1: An intraoperative nanoparticle-based fluorescence imaging agent comprised of micelles covalently conjugated to indocyanine green (ICG).

Protocol documents:
Prot_002.pdf: 
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 PERITONEAL 
METASTASES, IN PATIENTS UNDERGOING CYTOREDUCTIVE SURGERY  
PROTOCOL NUMBER: ON-1003 
Name of Drug: 
Pegsitacianine (ONM-100) 
Phase of Development: 
Phase 2 
Sponsor: 
OncoNano 
Sponsor Contact: 
Project Manager: 
Medical Monitor: 
, MD 
Version: 
3 
Date of Protocol: 
09SEP2022 
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 
subjects 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 peritoneal 
metastases, in patients undergoing cytoreductive surgery 
 
 
PROTOCOL NUMBER: ON-1003  
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   (Original) 
23JUN2021 
Original 
2 
18JAN2022 
• Updated the Medical Monitor 
• Updated the Project Manager 
• Included use of prophylactic diphenhydramine 
prior to pegsitacianine dosing 
• Removal of urinalysis, oral temperature, 
triglycerides, uric acid and iron tests 
• Corrected section numbering 
• Defined mucinous tumors as those that contain 
≥50% mucin 
• Updated “Day 10” follow-up visit to “Day  10 
(±5 days) or Day of Discharge (Whatever 
event occurs first)” 
• Updated safety information for the completed 
ON-1002 study 
• Corrected secondary endpoints 
• Removed hypothesis testing from sample size 
calculations 
• Updated the requirements to open Group 2 to 
enrollment 
• Removed Child-Pugh scores from exclusion 
criteria and restriction on use of investigational 
products 30 days prior to, and throughout the 
duration of the study  
• Included description of site personnel protocol 
training  
• Updated information on the intent to treat, 
efficacy and safety populations 
• Updated the statistical analysis to account for 
correlation 
3 
09SEP2022 
• Increased study enrollment size 
• Updated efficacy population definition 
• Updated sponsor contact 


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• Updated dosing window to 24-72 (± 8) hours  
• Clarified Screening Timing 
• Updated consumption of alcohol prior to 
surgery requirements 
• Updated imaging and/or biopsy inclusion 
wording 
• Updated wording on infusion rate 
• Update wording on day of surgery labs 
 
 
 


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TABLE OF CONTENTS 
Signature Page ................................................................................................................................ 2 
Table Of Contents ........................................................................................................................... 5 
Protocol Summary .......................................................................................................................... 8 
Abbreviations ................................................................................................................................ 14 
1. 
Introduction ................................................................................................................... 16 
1.1 
Overview ....................................................................................................................... 16 
1.2 
Pegsitacianine Clincal Trial Background...................................................................... 17 
1.3 
Pharmacodynamics and Dose Selection ....................................................................... 18 
2. 
Study Objectives ........................................................................................................... 21 
2.1 
Primary Objective ......................................................................................................... 21 
2.2 
Secondary Objectives.................................................................................................... 21 
2.3 
Exploratory Objectives ................................................................................................. 21 
3. 
Study Plan ..................................................................................................................... 21 
3.1 
Overall Design .............................................................................................................. 21 
3.2 
Imaging Assessments .................................................................................................... 22 
3.3 
Safety Assessments ....................................................................................................... 22 
4. 
Study Drug Dosage and Administration ....................................................................... 22 
4.1 
Prohibited Medications and Restrictions ...................................................................... 22 
4.2 
Study Drug Description ................................................................................................ 23 
4.3 
Study Drug Safety ......................................................................................................... 23 
4.4 
Study Drug Packaging and Storage .............................................................................. 25 
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 


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5.5 
Breaking the Blind ........................................................................................................ 27 
5.6 
Subject Withdrawal ....................................................................................................... 27 
5.6.1 
Reasons for Withdrawal ................................................................................................ 27 
5.6.2 
Handling of Withdrawals .............................................................................................. 27 
5.6.3 
Replacements ................................................................................................................ 28 
5.6.4 
Termination of Study .................................................................................................... 28 
6. 
Study Visits ................................................................................................................... 28 
6.1 
Screening and Enrollment (Days –30 to 0) ................................................................... 28 
6.2 
Pegsitacianine Administration (Days 0) ....................................................................... 29 
6.3 
Surgery and Follow-up Visits (Group 1 and 2) ............................................................ 29 
6.4 
Early Withdrawal Procedures ....................................................................................... 31 
6.5 
Training ......................................................................................................................... 32 
7. 
Study assessments ......................................................................................................... 32 
7.1 
Demographic Data/Medical History ............................................................................. 32 
7.2 
Physical Examination.................................................................................................... 32 
7.3 
Weight and Height ........................................................................................................ 32 
7.4 
Vital Sign Measurements .............................................................................................. 32 
7.5 
12-lead Electrocardiogram ............................................................................................ 33 
7.6 
Clinical Laboratory Tests .............................................................................................. 33 
7.6.1 
Pregnancy Tests ............................................................................................................ 33 
7.6.2 
Laboratory Measurements ............................................................................................ 33 
8. 
Reporting Adverse Events ............................................................................................ 34 
8.1 
Definitions..................................................................................................................... 34 
8.2 
Adverse Event Reporting .............................................................................................. 35 
8.3 
Assessment of Causality ............................................................................................... 36 
8.4 
Assessment of Severity ................................................................................................. 36 
8.5 
Serious Adverse Event Reporting ................................................................................. 37 
8.5.1 
SAE Reporting Contact Information ............................................................................ 37 
9. 
Statistical Methods ........................................................................................................ 37 
9.1 
Sample Size ................................................................................................................... 37 


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9.2 
Populations .................................................................................................................... 37 
9.3 
Rate of Clinically Significant Events ............................................................................ 37 
9.4 
Imaging Analysis .......................................................................................................... 38 
9.5 
Quantification of Fluorescence Intensity ...................................................................... 39 
9.6 
Diagnostic Imaging Sensitivity Analyses ..................................................................... 40 
9.7 
Safety Analysis ............................................................................................................. 40 
9.8 
Interim Analyses ........................................................................................................... 40 
10. 
Data handling and quality assurance............................................................................. 40 
10.1 
Data Security ................................................................................................................. 40 
10.2 
Case Report Forms ........................................................................................................ 41 
10.3 
Monitoring of the Study ................................................................................................ 41 
10.4 
Inspection of Records ................................................................................................... 41 
10.5 
Study Record Retention ................................................................................................ 42 
11. 
Administrative Considerations...................................................................................... 42 
11.1 
Confidentiality .............................................................................................................. 42 
11.2 
Institutional Review Board/Ethics Committee Approval ............................................. 42 
11.3 
Modification of the Protocol ......................................................................................... 43 
11.4 
Informed Consent.......................................................................................................... 43 
11.5 
Protocol Violations and Deviations .............................................................................. 43 
11.6 
Study Reporting Requirements ..................................................................................... 44 
11.7 
Investigator Documentation .......................................................................................... 44 
11.8 
Study Conduct ............................................................................................................... 45 
11.9 
Publications ................................................................................................................... 45 
12. 
References ..................................................................................................................... 46 
13. 
Appendix 1: Schedule of Events (Groups 1 and 2)....................................................... 48 
14. 
Appendix 2: Completeness of Cyotoreduction Score ................................................... 50 
15. 
Appendix 3: Peritoneal Carcinomatosis Index ............................................................. 51 
 
 


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PROTOCOL SUMMARY 
Protocol 
Number 
ON-1003 
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 peritoneal metastases, in patients undergoing cytoreductive surgery 
Phase of Clinical 
Trial 
Phase 2 
Indications 
Pegsitacianine is indicated as an imaging agent for intraoperative detection of 
peritoneal metastases originating from intra-abdominal tumors such as ovarian, 
colorectal, and gastric cancers. Pegsitacianine is administered intravenously as a 
single dose prior to surgery. 
Study Centers 
up to 10 centers  
Trial Objectives 
Primary Objective: 
The primary ob ective of this study is to determine if administration of 
pegsitacianine 
mg/kg) results in the detection of metastatic disease left behind 
following standard of care surgical resection of peritoneal metastases. 
 
Secondary Objectives: 
Key secondary objectives are to demonstrate an acceptable safety profile, reliable 
sensitivity, specificity, negative predictive values, and positive predictive values of 
the imaging agent at the level of the individual specimens.  
Background 
Pegsitacianine is an intraoperative nanoparticle-based fluorescence imaging agent 
that was evaluated in the first-in-human Phase 1 study (ON-1001) completed in the 
Netherlands in patients with solid cancers undergoing surgical excision of their 
tumors. In addition, it was also evaluated in the 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 low-
pH tumor microenvironment. Pegsitacianine was demonstrated to be well tolerated 
and showed no dose-limiting toxicity (DLT).  
Trial Design 
This Phase 2 study will be a two Group interventional, open-label, single arm trial 
where each patient is his/her own “intra atient” control. All atients will receive a 
single  mg/kg dose of pegsitacianine 
prior to standard of care surgery. It is recommended by the sponsor to administer 
prophylactic diphenhydramine before administration of pegsitacianine to decrease 
the possibility of an infusion-related reaction. 
 
Patients enrolled in Group 1 of the study will have a biopsy confirmed diagnosis of 
non-mucinous (i.e., ≤50% mucin) peritoneal carcinomatosis with a suspected 
Peritoneal Carcinomatosis Index (PCI) greater than or equal to 10. Group 2 of the 
study will include patients with a suspected PCI greater than or equal to 10 and a 
biopsy confirmed diagnosis of mucinous (i.e., >50% mucin) peritoneal 
carcinomatosis. A total of approximately 60 patients will be enrolled across both 
Groups. Enrollment will be open first to Group 1 with the opportunity to open 
Group 2 for enrollment following the demonstration of satisfactory pegsitacianine 
sensitivity and specificity values of 70% or greater in Group 1. 
 
In both Groups 1 and 2, the surgeon will perform their SOC resection of visible 
metastases and image up to a total of 15 resected tumor (n = 10) and normal (n=5) 






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• 
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 
breathing rate) 
• 
Obtain a blood sample for routine laboratory tests, including a 
pregnancy test for females who are capable of becoming pregnant 
• 
Record medications they have been taking, including prescription, 
over-the-counter, and herbal medications 
• 
Confirm that the patient has met all the study criteria to participate 
 
Day 0 (Pegsitacianine administration) 
 
Predose procedures 
• 
Confirm eligibility for the study and that medical information is 
complete 
• 
Record weight and height and calculate BMI 
• 
Discuss how the patient is feeling and their everyday activities  
• 
Record vital signs 
• 
Perform a 12-lead electrocardiogram (ECG) to check the heart 
activity  
• 
Install an IV line  
• 
Obtain blood samples for routine laboratory tests, including a 
pregnancy test for females who are capable of becoming pregnant 
• 
Obtain a blood, urine or breath test to test for alcohol. This test 
may be performed if relevant to the patient’s medical history. If a 
breath/urine test is used and is positive, a blood test may be used 
to confirm the test results 
• 
Record all medications taken since the Screening visit, including 
prescription, over-the-counter, and herbal medications 
 
The Sponsor recommends the use of prophylactic diphenhydramine prior to 
study drug administration to decrease the possibility of an infusion-related 
reaction.  
 
Dosing and postdose procedures 
 
Procedures during and after dosing of pegsitacianine are summarized below: 
 
• 
Dosin  of e sitacianine will be administered 
  
. Total dosing time will be 
dependent on the the patient’s body weight and total volume to be 
administered 
• 
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 60 minutes afterward 
o 
Discuss any new or unexpected changes in how the 
patient is feeling 
o 
Record all medications they are taking, including 
prescription, over-the-counter, and herbal medications 


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Peritoneal Cytoreductive Surgery 
; Groups 1 and 
2)  
 
Procedures at the time of cytoreductive surgery 
 
The following procedures will be performed on the day of surgery: 
• 
Record the patient’s vital signs (only if they were abnormal on 
Day 0) 
• 
Obtain a blood sample for routine laboratory tests  
• 
Record all medications being taken, including prescription, over-
the-counter, and herbal medications 
• 
Discuss with the patient any new or unexpected changes in they 
are feeling 
 
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. 
• 
During SOC resection, up to 15 specimens (10 suspected tumor 
and 5 normal) specimens will be imaged using the intraoperative 
camera on a section of normal tissue  
• 
Once the SOC resection is complete, the surgeon will examine the 
peritoneal cavity with the intraoperative NIR camera in search of 
additional fluorescent metastatic disease that was missed during 
SOC using an assessment method similar to that used for 
establishing the PCI score (i.e., 13 regions) 
• 
Additional fluorescent lesions will be removed and imaged on a 
section of normal tissue in a manner analogous to the SOC 
specimens 
 
Day 10 (±5 days) or Day of Discharge (Whichever event occurs first) 
• 
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 
• 
Record all medications being taken, including prescription, over-
the-counter, and herbal medications 
• 
Discuss any new or unexpected changes or adverse events 
 
Day 28 (±5 days) after dosing 
• 
Obtain blood samples for routine laboratory tests, including a 
pregnancy test for females who are capable of becoming pregnant 
• 
Record all medications they are taking, including prescription, 
over-the-counter, and herbal medications 
• 
Discuss new or unexpected changes in they are feeling 
• 
Record vital signs (temperature, heart rate, blood pressure, and 
breathing rate) 


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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 occurs during initial surgical 
procedures with Sponsor Representatives present. Sponsor Representatives will 
ensure all study personnel are well 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 to the database. 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 
ARE 
AST 
BMI 
antioxidant response element 
aspartate aminotransferase 
body mass index 
BUN 
CC 
blood urea nitrogen 
Completeness of Cytoreduction Score 
CFR 
Code of Federal Regulations 
CS 
cytoreductive surgery  
CSE 
CT 
CV 
D5W 
DoH 
ECG 
clinically significant event 
computed tomography 
Curriculum Vitae 
5% Dextrose in water 
Declaration of Helsinki   
electrocardiogram 
eCRF 
EAFUS 
electronic case report form 
Everything added to food in the U.S. 
FDA 
United States Food and Drug Administration 
HIPEC 
Hyperthermic intraperitoneal chemotherapy 
HNSCC 
ICG 
head and neck squamous cell carcinoma  
indocyanine green 
ICH 
International Conference on Harmonisation 
IRB 
IV 
institutional review board 
intravenous 
MFI 
MRI 
NIR 
NSCLC 
NPV 
PC 
PCI 
PEG 
PET 
PK 
mean fluorescence imaging 
magnetic resonance imaging 
near-infrared 
non small cell lung carcinoma 
negative predictive value 
peritoneal carcinomatosis 
peritoneal carcinomatosis index 
polyethylene glycol 
positron emission tomography 
pharmacokinetics 


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ABBREVIATION 
TERM 
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 
TEAE 
WHO 
WMA 
treatment-emergent adverse event 
World Health Organization 
World Medical Association 
 
 
 


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1. INTRODUCTION
1.1 Overview 
Peritoneal carcinomatosis (PC) results from the metastasis of a primary cancer of the peritoneum 
(e.g., appendiceal, ovarian, uterine, colorectal, and gastric cancers) that then disseminates 
throughout the abdominal cavity.1 Historically, progression to PC was considered terminal and 
resulted in survival times on the scale of a few months with palliative care being the best option 
for patients.2 More recently, cytoreductive surgery (CS) has emerged as a means to prolong and 
improve patient lives with a median increase in survival of up to ~5 years.3 It has been reported 
that for every 10% increase in cytoreduction there is a 5.5% increase in median survival time.4,5 
In addition to surgical tumor debulking within the peritoneal space, it has also been shown that 
coupling surgical intervention with hyperthermic intraperitoneal chemotherapy (HIPEC) can 
have an even greater impact on patient outcomes.6  
The extent of survival benefit gained via CS is directly linked to the completeness of 
cytoreduction, or how much macroscopic disease (>2.5 mm deposits) has been removed.7,8 
During CS, surgeons examine the peritoneal cavity for evidence of disease with the intent of 
removing all visible tumor deposits, and thus providing a complete cytoreduction. Any tumor 
deposits left behind, smaller than 2.5 mm, are believed to be treated efficiently with the follow-
on HIPEC treatment.9 These surgical procedures are lengthy in duration, with total CS and 
HIPEC treatment times taking upwards of 14 hours to complete. Additional intraoperative tools 
for the identification of metastatic deposits may help to reduce total surgical time, as well as help 
identify disease that may be microscopic or unable to be detected with the naked eye (i.e., deep 
metastases). In an effort to aid surgeons in disease detection, focus has been placed on the 
development of fluorescent imaging agents that can accurately detect small deposits of disease 
that may be missed under standard white light surgical procedures.10,11  
One strategy to address the challenges that physicians face during surgery, and to overcome the 
complexity encountered due to the diversity in oncogenotypes and histologic phenotypes, is to 
target metabolic vulnerabilities that are ubiquitous in cancer. Aerobic glycolysis, known as the 
Warburg effect, in which cancer cells preferentially take up glucose and convert it into lactic 
acid, occurs in all solid cancers.12 Lactic acid, a by-product of cellular metabolism, accumulates 
in the extracellular space through excretion from tumor cells via the action of monocarboxylate 
transporters.13 The resulting acidification of the extra-cellular space promotes remodelling of the 
extracellular matrix for further tumor invasion and metastasis.14  
Pegsitacianine, a micellar fluorescence imaging agent, exploits the ubiquitous pH differences 
observed between cancerous and normal tissues. This in turn, provides a highly sensitive and 
specific fluorescence response after localizing within the tumor microenvironment, thus allowing 
the detection of primary tumors, their margins, metastatic disease, and tumor-containing lymph 
nodes.15-17 


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There exists a clear unmet clinical need for the intraoperative detection of tumor margins, 
metastatic disease and cancer-involved nodes. Solutions to these challenges would allow 
surgeons to more precisely identify and remove primary cancers or metastases, and selectively 
remove only those lymph nodes in which disease is present. In addition to enabling a more 
comprehensive removal of malignant disease, the use of technologies, such as pegsitacianine, 
would help to reduce the morbidity of radical surgery by reducing the excisional volume of 
normal tissue and overall surgical time.18 
 
1.2 Pegsitacianine Clincal Trial Background 
Three clinical studies have been performed using pegsitacianine. The study ON-1001: Image-
Guided Surgery for Tumor Detection in Solid Tumors Using the pH Activated Micellar Probe 
ONM-100: The SHINE Study, was conducted in the Netherlands at the University Medical 
Center Groningen and completed in 2019.19 The completed Phase 2 study, ON-1002: A Study to 
Evaluate ONM-100, an Intraoperative Fluorescent Imaging Agent for the Detection of Cancer 
(NCT03735680) was a multicenter trial in the United States. ON-1004 was a Phase 1 normal 
healthy volunteer study to further evaluate the pharmacokinetic profile of pegsitacianine.  
 
The purpose of  the ON-1001 study was to investigate the safety, pharmacokinetics and 
feasibility of pegsitacianine as an intraoperative optical tracer for the detection of tumors and 
metastatic lymph nodes in solid cancers. Additionally, the study investigated the optimal dose 
range of pegsitacianine for an adequate tumor-to-background/contrast-to-noise ratio of 
fluorescence obtained intraoperatively and with ex vivo specimens using indocyanine green 
(ICG) compatible cameras and imaging devices. A single pegsitacianine micelle dose was 
administered intravenously (IV) as a one to five-minute infusion to patients in five dose cohorts (0.1, 
0.3, 0.5, 0.8, and 1.2 mg/kg) with three patients per cohort in Phase 1A, and 15 patients at a dose of 
1.2 mg/kg in Phase 1B. Near-infrared (NIR) imaging was conducted intraoperatively, on the back 
table, and postoperatively of the primary tumor, lymph nodes, and pegsitacianine guided biopsies. 
Additionally, the bread loaf slices from the primary tumor were also imaged postoperatively. The 
median tumor-to-background (TBR) value from all bread loaf slice image analysis (n = 97 slices 
from 27 patients) was determined to be 4.5 with an interquartile range of 3.1. Additionally, 
pegsitacianine demonstrated a sensitivity of 100% across all tumor types tested with a specificity of 
75% and 57% in breast cancer and head and neck squamous cell carcinoma (HNSCC), respectively.19  
 
The purpose of the ON-1002 study was to investigate whether pegsitacianine can be used to 
image primary tumors and metastatic lymph nodes using an imaging schedule earlier than 24 ±8 
hours postdose in patients undergoing routine surgery of their solid cancers and whether the 
diagnostic performance to detect metastatic lymph nodes can be improved by optimizing the 
dose and the imaging schedule. The study was designed to be executed in three parts: 
 
Part 1 was designed to evaluate the dose(s) at which pegsitacianine fluorescence imaging is 
feasible at 3 ±2 hours postdose and, if needed, at an alternate postdose imaging schedule, for the 




















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5.5 Breaking the Blind 
Not applicable. 
5.6 Subject Withdrawal 
Subjects are free to withdraw from the study at any time for any reason, without affecting future 
medical management and treatment. 
5.6.1 
Reasons for Withdrawal 
A subject may be withdrawn from the study by the investigator or OncoNano Medicine Inc for 
any of the following reasons: 
• The  subject develops a disease or condition that, in the opinion of the investigator, 
would compromise the subject’s safety by continuing in the study 
• The subject violates the protocol. If, in the opinion of the investigator and OncoNano 
Medicine, the violation is not likely to impact safety or endpoint evaluations, the 
investigator may permit the subject to remain in the study at the investigator’s 
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 for any reason, study staff will make every 
effort to perform the Day 28 (±5 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. 


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5.6.3 
Replacements 
Withdrawn subjects 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 subjects’ well-being. 
If the study is terminated, all subjects 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. 
6. STUDY VISITS 
6.1 Screening and Enrollment (Days –30 to 0) 
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: 
 
• 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 breathing rate) 
• Obtain blood for routine laboratory tests, including a pregnancy test for females who 
are capable of becoming pregnant 
• Record medications they have been taking, including prescription, over-the-counter, 
and herbal medications 
• Determine Karnofsky Performance Status (to asses the ability to perform ordinary 
daily tasks) 
• Confirm that the patient has met all the study criteria to participate 


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6.2 Pegsitacianine Administration (Days 0) 
Predose procedures 
• Confirm eligibility for the study and that medical information is complete 
• Record weight and height and calculate BMI 
• Discuss how the patient is feeling and their everyday activities  
• Record vital signs 
• Perform a 12-lead electrocardiogram (ECG) to check the heart activity during 
pre-operative screening  
• Install an IV line  
• Obtain blood for routine laboratory tests, including a pregnancy test for females 
who are capable of becoming pregnant  
• Obtain blood, urine, or a breath test to test for alcohol. This test may be performed 
if relevant to the patient’s medical history. If a urine or breath test is used and is 
positive, a blood test may be used to confirm the test results 
• Record all medications taken since the Screening visit, including prescription, 
over-the-counter, and herbal medications 
 
The Sponsor recommends the use of prophylactic diphenhydramine prior to study drug 
administration to decrease the possibility of an infusion-related reaction.  
 
Dosing and postdose procedures 
Procedures during and after dosing of pegsitacianine are summarized below: 
• Dosing of pegsitacianine will be administered through an IV injection 
 Total dosing time will depend on the 
patient’s body weight and total volume to be administered 
• 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 
60 minutes afterward 
o Discuss any new or unexpected changes in how the patient is feeling 
o Record all medications they are taking, including prescription, over-the-
counter, and herbal medications 
6.3 Surgery and Follow-up Visits (Group 1 and 2) 
Procedures at the time of cytoreductive surgery 
 
The following procedures will be performed on the day of surgery: 
 
• Record the patient’s vital signs (only if they were abnormal at dosing) 


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• Obtain blood for routine laboratory tests  
• Record all medications being taken, 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 steps listed below. 
 
• During SOC resection, the surgeon will select up to 15 (10 suspected tumor and 5 
normal) specimens that will be imaged using the intraoperative camera on a 
section of normal tissue following excision (Note: NIR camera should not be used 
to examine the peritoneal cavity at this time) 
o A total of up to 15 specimens are to be imaged: 5 representative normal pieces 
of tissue and 10 individual suspected tumor specimens 
o The SOC specimens are to be resected upon the initiation of surgery and 
imaged on a section of normal tissue (i.e., mesentery) immediately following 
the collection of all SOC samples 
o The width of the camera’s field of view is to be confined to a distance of 10 
cm. A surgical ruler is to be placed in frame to control the width of the field of 
view 
 Note: while there are no specimen size stipulations provided in this 
protocol, the samples must be able to be imaged within the 10 cm wide 
imaging window.  
o For samples with multiple nodules, individual nodules should be dissected 
away from the bulk sample and imaged individually as single specimens.  
 Representative normal sections of tissue should be imaged in an 
analogous manner 
• Samples will be imaged using the NIR camera and fluorescence status and 
specimen name will dictated by the surgeon for documentation by the clinical 
research coordinator in the provided Imaging Workbook 
• Once the SOC resection is complete, the surgeon will dictate their completeness 
of cytoreduction score to the clinical research coordinator for documentation prior 
to examining the peritoneal cavity with the intraoperative NIR camera in search of 
additional fluorescent metastatic disease that was missed during SOC 
o The peritoneal cavity will be evaluated using a manner similar to that used for 


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the calculation of the Peritoneal Carcinomatosis Index. Specifically, the 
peritoneal cavity will be examined in a methodical manner using the 13 
described regions used for calculating the PCI score 
• If additional fluorescent areas are to be collected, an image of the additional 
fluorescent lesion(s) is to be captured prior to excision from the patient 
o The camera should be held 15 cm away from the area of tissue to be excised. 
The camera’s distance can be measured using an available surgical ruler 
• Additional fluorescent lesions will then be removed and imaged on a section of 
normal tissue in a manner analogous to the SOC specimens with the surgeon 
dictating fluorescence status and specimen name to the clinical research 
coordinator for documentation 
o The width of the camera’s field of view is to be confined to a distance of 10 
cm. A surgical ruler is to be placed in frame to control the width of the field of 
view 
 
Day  10 (±5 days) or Day of Discharge (Whichever event occurs first) 
• 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 
• Record all medications being taken, including prescription, over-the-counter, and 
herbal medications 
• Discuss any new or unexpected changes they are feeling.  
• Record any AEs 
 
Day 28 (±5 days) after dosing 
• Obtain blood samples for routine laboratory tests, including a pregnancy test for 
females who are capable of becoming pregnant 
• Record all medications they are taking, including prescription, over-the-counter, 
and herbal medications 
• Discuss new or unexpected changes they are feeling 
• Record the patient’s vital signs 
6.4 Early Withdrawal Procedures 
If a subject is withdrawn from the study before study completion, all procedures scheduled for 
the final visit (Day 28 ±5 days) should be performed if possible. 


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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 and on Day 10 (±5 days) or 
Day of Discharge. 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 at Day 0 prior to dosing. 
The subject’s body mass index (BMI) will be calculated in the eCRF at Screening using the 
following formula: 
ܤܯܫ= ܹ݄݁݅݃ݐ (݇݃)
(ܪ݄݁݅݃ݐ [݉])ଶ 
7.4 Vital Sign Measurements 
Vital sign measurements (systolic and diastolic blood pressure, pulse, respiratory rate, and oral 
temperature) will be obtained at Screening, Day 0 (Pre/Post dose), Day of Surgery (only if 
abnormal at dosing), Day 10 (±5 days) or Day of Discharge and Day 28.  
Oral temperature will be documented in degrees Celsius. 
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.  


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7.5 12-lead Electrocardiogram 
A standard safety 12-lead ECG will be conducted on Day 0 (Pre-Dose) and on Day 10 (±5 days) 
or Day of Discharge. 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 10 (±5 days) or Day of Discharge and on Day 28.  
7.6.1 
Pregnancy Tests 
For female subjects, a serum sample will be collected for a serum pregnancy test (β-human 
chorionic gonadotropin) at Screening and blood samples will be collected for pregnancy tests on 
Day 0, and on Day 28. Negative pregnancy test results will be required for subjects to enroll and 
continue study participation. 
7.6.2 
Laboratory Measurements 
The following clinical laboratory parameters will be measured: 


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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 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. Subjects 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. 


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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 
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. 


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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. 
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 as mild, moderate, or severe using the following criteria: 
• Mild: These events require minimal or no treatment and do not interfere with the 
subject’s daily activities 
• Moderate: These events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with functioning 
• Severe: These events interrupt a subject’s usual daily activity and may require systemic 
drug therapy or other treatment. Severe events are usually incapacitating 
Changes in the severity of an AE should be documented to allow an assessment of the duration 
of the event at each level of intensity to be performed. Adverse events characterized as 
intermittent require documentation of onset and duration of each episode. 


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8.5 Serious Adverse Event Reporting  
All SAEs must be reported to the 
email within 24 hours from the 
time site personnel first learn about the event. Additional follow-up information, when available, 
should also be sent to
. 
8.5.1 
SAE Reporting Contact Information 
Serious Adverse Event Report Forms must be submitted within 24 hours and should consist of 
the trial provided Serious Adverse Event Report Form. The following information should be 
entered into the EDC database at the time the SAE form is submitted: the demographics page(s), 
the medical history page(s), the AE page(s) and the concomitant medications page(s). If the 
subject is hospitalized because of or during the course of an SAE  then a co
 of the hospital 
discharge summary should be provided to the 
 email as soon as it 
becomes available. Withdrawal from the study and all therapeutic measures will be at the 
discretion of the principal investigator. All SAEs, regardless of relationship to the study drug, 
will be followed until satisfactory resolution or until the investigator deems the event to be 
chronic or the subject to be stable. 
9.STATISTICAL METHODS 
Additional statistical analyses, other than those described in this section, may be performed if 
deemed appropriate. Details of the analyses are described in the statistical analysis plan. 
9.1 Sample Size 
The sample size for this study was originally 40 subjects. This sample size is based on historical 
experience with similar pilot studies with the goal of informing power calculations for future 
studies. This study is not formally powered for hypothesis testing. While the original sample size 
of 40 subjects would be sufficient for developing further understanding of the endpoints 
described herein, increasing the amount of subjects to approximately 60 will provide superior 
estimates for endpoints to power future studies. Sample size calculations account for non-
evaluable and drop-out patients. 
9.2 Populations 
All subjects who receive any dose of the study drug will be included in the intent to treat 
population. The efficacy population will consist of all subjects who receive >75% of the intended 
dose of pegsitacianine, had a minimum of one (1) image collected during their procedure, and 
had the opportunity for post-SOC exploration of the peritoneal cavity. The safety population will 
include all subjects who were administered pegsitacianine whether it be a full or partial dose. 
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 


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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 Occult Disease or Positive Surgical Margins– identification of tumor-
containing tissue due to pegsitacianine fluorescence, confirmed via histopathological 
analysis, that otherwise went undetected during pre-operative imaging (PET, MRI) or by 
the surgeon during SOC surgery 
o This occurs when a sample that (1) exhibits fluorescence in situ and (2) is 
pathologically-confirmed as a tumor, is obtained by fluorescence during SOC 
surgery.  
 
• 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 a sample that (1) exhibits no fluorescence ex situ and (2) is 
pathologically-confirmed as normal tissue, is classified as suspicious tissue during 
SOC surgery.  
 
• 
Alteration to the Completeness of Cytoreduction (CC) Score – following the completion of 
the SOC surgery, the surgeon will document a CC score prior to fluorescence imaging using 
pegsitacianine. If the surgeon finds residual disease using fluorescence imaging that results in 
the increase of the CC score, this will be considered a CSE  
 
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”. 


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• 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: 
ࡿࢋ࢔࢙࢏࢚࢏࢜࢏࢚࢟=
# ݋݂ ܶݎݑ݁ ܲ݋ݏ݅ݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ
# ݋݂ ܶݎݑ݁ ܲ݋ݏ݅ݐ݅ݒ݁+  ܨ݈ܽݏ݁ ܰ݁݃ܽݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ 
ࡿ࢖ࢋࢉ࢏ࢌ࢏ࢉ࢏࢚࢟=
#݋݂ ܶݎݑ݁ ܰ݁݃ܽݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ
# ݋݂ ܶݎݑ݁ ܰ݁݃ܽݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ+ # ݋݂ܨ݈ܽݏ݁ ܲ݋ݏ݅ݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ  
 
ࡼࡼࢂ=
# ݋݂ ܶݎݑ݁ ܲ݋ݏ݅ݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ
# ݋݂ ܶݎݑ݁ ܲ݋ݏ݅ݐ݅ݒ݁+ ܨ݈ܽݏ݁ ܲ݋ݏ݅ݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ  
ࡺࡼࢂ=
# ݋݂ ܶݎݑ݁ ܰ݁݃ܽݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ
# ݋݂ ܶݑ݉݋ݎ ܰ݁݃ܽݐ݅ݒ݁+ ܨ݈ܽݏ݁ ܰ݁݃ܽݐ݅ݒ݁ ܵ݌݁ܿ݅݉݁݊ݏ 
 
These rates will be reported along with exact two-sided 95% confidence intervals. 
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.   
 
 
ࡿ࡮ࡾ=
ܯ݁ܽ݊ ܨ݈ݑ݋ݎ݁ݏܿ݁݊ܿ݁ ܫ݊ݐ݁݊ݏ݅ݐݕ (ܵ݌݁ܿ݅݉݁݊)
ܯ݁ܽ݊ ܨ݈ݑ݋ݎ݁ݏܿ݁݊ܿ݁ ܫ݊ݐ݁݊ݏ݅ݐݕ (ܰ݋ݎ݈݉ܽ ܶ݅ݏݏݑ݁) 
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”). 


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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 subjects, 
mean, standard deviation, median, minimum, and maximum values. Categorical data will be 
summarized by presenting the number (frequency) and percentage of subjects at each level of 
response. Subject disposition will be presented and summarized. 
 
Adverse events will be coded using MedDRA, Version 23.0 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 
None planned. 
10. DATA HANDLING AND QUALITY ASSURANCE 
10.1 
Data Security 


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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 subjects 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.  
OncoNano Medicine 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 in a timely manner. 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 
The clinical monitor, as a representative of 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 OncoNano Medicine or its 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 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 OncoNano Medicine of any audits scheduled 
by any regulatory authorities and promptly forward copies of any audit reports received to 
OncoNano Medicine. 


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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 OncoNano Medicine. It is the responsibility of 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 
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 subjects 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. 


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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 subjects 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 subjects must sign the IRB-approved 
revised form. 
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 
subjects 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 


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principal investigator. Deviations usually have an impact on individual subjects or a small group 
of subjects 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 
• 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 subinvestigators 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 


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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 
Jafari, M. D. et al. Surgical outcomes of hyperthermic intraperitoneal chemotherapy: 
analysis of the american college of surgeons national surgical quality improvement 
program. JAMA Surg 149, 170-175, doi:10.1001/jamasurg.2013.3640 (2014). 
2 
Lambert, L. A. & Wiseman, J. Palliative Management of Peritoneal Metastases. Annals of 
Surgical Oncology 25, 2165-2171, doi:10.1245/s10434-018-6335-7 (2018). 
3 
Sugarbaker, P. H. Peritonectomy procedures. Ann Surg 221, 29-42, 
doi:10.1097/00000658-199501000-00004 (1995). 
4 
Bristow, R. E., Tomacruz, R. S., Armstrong, D. K., Trimble, E. L. & Montz, F. J. 
Survival effect of maximal cytoreductive surgery for advanced ovarian carcinoma during 
the platinum era: a meta-analysis. J Clin Oncol 20, 1248-1259, 
doi:10.1200/jco.2002.20.5.1248 (2002). 
5 
Chang, S. J., Bristow, R. E. & Ryu, H. S. Impact of complete cytoreduction leaving no 
gross residual disease associated with radical cytoreductive surgical procedures on 
survival in advanced ovarian cancer. Ann Surg Oncol 19, 4059-4067, 
doi:10.1245/s10434-012-2446-8 (2012). 
6 
Simkens, G. A., Rovers, K. P., Nienhuijs, S. W. & de Hingh, I. H. Patient selection for 
cytoreductive surgery and HIPEC for the treatment of peritoneal metastases from 
colorectal cancer. Cancer Manag Res 9, 259-266, doi:10.2147/cmar.S119569 (2017). 
7 
Eisenkop, S. M., Friedman, R. L. & Wang, H.-J. Complete Cytoreductive Surgery Is 
Feasible and Maximizes Survival in Patients with Advanced Epithelial Ovarian Cancer: 
A Prospective Study. Gynecologic Oncology 69, 103-108, 
doi:https://doi.org/10.1006/gyno.1998.4955 (1998). 
8 
Gill, R. S. et al. Treatment of gastric cancer with peritoneal carcinomatosis by 
cytoreductive surgery and HIPEC: a systematic review of survival, mortality, and 
morbidity. J Surg Oncol 104, 692-698, doi:10.1002/jso.22017 (2011). 
9 
Kusamura, S., Dominique, E., Baratti, D., Younan, R. & Deraco, M. Drugs, carrier 
solutions and temperature in hyperthermic intraperitoneal chemotherapy. J Surg Oncol 
98, 247-252, doi:10.1002/jso.21051 (2008). 
10 
Asanuma, D. et al. 6HQVLWLYH ȕ-galactosidase-targeting fluorescence probe for visualizing 
small peritoneal metastatic tumours in vivo. Nat Commun 6, 6463, 
doi:10.1038/ncomms7463 (2015). 
11 
Boonstra, M. C. et al. uPAR-targeted multimodal tracer for pre- and intraoperative 
imaging in cancer surgery. Oncotarget 6, 14260-14273, doi:10.18632/oncotarget.3680 
(2015). 
12 
Warburg, O., Wind, F. & Negelein, E. THE METABOLISM OF TUMORS IN THE 
BODY. J Gen Physiol 8, 519-530, doi:10.1085/jgp.8.6.519 (1927). 
13 
Webb, B. A., Chimenti, M., Jacobson, M. P. & Barber, D. L. Dysregulated pH: a perfect 
storm for cancer progression. Nature Reviews Cancer 11, 671-677, doi:10.1038/nrc3110 
(2011). 


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14 
Estrella, V. et al. Acidity generated by the tumor microenvironment drives local invasion. 
Cancer Res 73, 1524-1535, doi:10.1158/0008-5472.CAN-12-2796 (2013). 
15 
Li, Y. et al. Molecular basis of cooperativity in pH-triggered supramolecular self-
assembly. Nat Commun 7, 13214-13214, doi:10.1038/ncomms13214 (2016). 
16 
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). 
17 
Bennett, Z. T. et al. Detection of Lymph Node Metastases by Ultra-pH-Sensitive 
Polymeric Nanoparticles. Theranostics 10, 3340-3350, doi:10.7150/thno.41239 (2020). 
18 
Nagaya, T., Nakamura, Y. A., Choyke, P. L. & Kobayashi, H. Fluorescence-Guided 
Surgery. Frontiers in Oncology 7, doi:10.3389/fonc.2017.00314 (2017). 
19 
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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13. APPENDIX 1: SCHEDULE OF EVENTS (GROUPS 1 AND 2) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening 
Treatment 
Evaluation 
Days 
–30 to 0 
Day 0 
Sur er   
 
postdose) 
Day  10 (±5 days) 
or Day of 
Discharge  
 
Day 28 
(± 5 
days) 
Informed consent & subject enrollment 
X 
 
 
 
 
Inclusion/exclusion criteria 
X 
X 
 
 
 
Blood/Urine/Breath alcohol 
 
X 
 
 
 
Pregnancy testa 
X 
X 
 
 
X 
Demographic data 
X 
 
 
 
 
Medical history 
X 
 
 
 
 
Physical examination 
X 
 
 
X 
 
Karnofsky Performance Status 
X 
 
 
 
 
Heightb 
X 
X 
 
 
 
Body weightb 
X 
X 
 
 
 
Vital signsc 
X 
X 
Xd 
X 
X 
12-lead electrocardiogram 
 
Xe 
 
X 
 
Concomitant medications 
X 
X 
X 
X 
X 
Study drug administration 
 
Xf 
 
 
 
Adverse events reviewg 
 
X 
X 
X 
X 
Serum chemistry  
X 
X 
X 
X 
X 
Hematologyh  
X 
X 
X 
X 
X 
Surgery 
 
 
X 
 
 
Intraoperative Imaging 
 
 
X 
 
 
Pathology evaluation of surgical specimens 
 
 
Xi 
 
 


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Footnotes 
a For female subjects, a serum sample for a serum pregnancy test will be collected at screening and on Day 0 and Day 28. 
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 Vitals are only to be collected if they are found to be abnormal at dosing 
e 12-lead ECG is to be performed prior to study drug administration 
f 
Prophylactic diphenhydramine may be administered prior to the administration of pegsitacianine 
g Patients will be assessed for AEs occurring from the time of dosing through Day 28 (±5 days)  
h Hematology assessments will include hemoglobin, hematocrit, erythrocyte count (red blood cells), differential leukocytes, platelet count, and total leukocytes 
(white blood cells). 
i 
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: COMPLETENESS OF CYOTOREDUCTION SCORE  
 
 
 
 
 
 


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15. APPENDIX 3: PERITONEAL CARCINOMATOSIS INDEX 
 
 

SAP_003.pdf: 




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Statistical Analysis Plan v3 
 
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SIGNATURES OF APPROVAL 
Study 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 
peritoneal metastases, in patients undergoing cytoreductive surgery 
Protocol Number: ON-1003 
Protocol Version:V3 09SEP2022 
 
 
Date: 
  
Senior Biostatistician 
 
Date: 
Approver: 
 
 
 
 
 
 
 


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Statistical Analysis Plan v3 
 
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TABLE OF CONTENTS 
Statistical Analysis Plan .................................................................................................................. 1 
Document History ........................................................................................................................... 2 
Signatures of Approval ................................................................................................................... 3 
List of Abbreviations ...................................................................................................................... 7 
1. Introduction ................................................................................................................................. 9 
2. Study Objectives ......................................................................................................................... 9 
2.1 Objectives and Endpoints ......................................................................................................... 9 
3. Study Description...................................................................................................................... 10 
3.1 Study Design ........................................................................................................................... 10 
3.2 Study Treatment ...................................................................................................................... 11 
4. Sample Size and Power Calculation ......................................................................................... 11 
5. Analysis Endpoints ................................................................................................................... 11 
5.1 Efficacy Endpoints .................................................................................................................. 11 
5.1.1 Clinically Significant Event Endpoints ................................................................................ 11 
5.1.2 Diagnostic Imaging Endpoints ............................................................................................. 12 
5.1.3 Fluorescence Endpoints ....................................................................................................... 13 
5.2 Safety Endpoints ..................................................................................................................... 14 
6. Analysis Populations ................................................................................................................. 14 
6.1 The Safety Population ............................................................................................................. 14 
6.2 The Intent-to-Treat Population ............................................................................................... 14 


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6.3 The Efficacy Population ......................................................................................................... 14 
7. Analytical Plan and Statistical Methods ................................................................................... 14 
7.1 General Considerations ........................................................................................................... 14 
7.2 Reporting Conventions ........................................................................................................... 15 
7.3 Definition of Baseline ............................................................................................................. 16 
7.4 Handling of Dropouts and Missing, Unused and Spurious Data ............................................ 16 
7.4.1 Dates .................................................................................................................................... 16 
7.5 Adjustment for Multiplicity .................................................................................................... 17 
7.6 Adjustment for Multiple Centers ............................................................................................ 17 
7.7 Patient Disposition .................................................................................................................. 17 
7.8 Protocol Deviations ................................................................................................................. 17 
7.9 Patient Characteristics ............................................................................................................. 18 
7.9.1 Baseline and Demographic Characteristics ......................................................................... 18 
7.9.2 Medical History ................................................................................................................... 19 
7.9.3 Prior and Concomitant Medications and Procedures/Treatments ........................................ 19 
7.9.4 Administration and Exposure of Study Drug....................................................................... 19 
7.9.5 Cytoreductive Surgery Details and Specimen Collection .................................................... 19 
7.10 Efficacy Endpoint Analyses .................................................................................................. 20 
7.10.1 Primary Analyses ............................................................................................................... 20 
7.10.2 Secondary Analyses ........................................................................................................... 20 
7.10.3 Exploratory Analyses ......................................................................................................... 21 


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7.11 Safety Endpoint Analyses ..................................................................................................... 22 
7.11.1 Adverse Events and Infusion Related Reactions ............................................................... 22 
7.11.2 Laboratory Data ................................................................................................................. 23 
7.11.3 Vital Signs .......................................................................................................................... 23 
7.11.4 Physical Exam .................................................................................................................... 23 
7.11.5 ECG.................................................................................................................................... 23 
8. Deviations from Analysis as Described in the Protocol ........................................................... 24 
9. Appendix 1: Schedule of Events (Groups 1 and 2)................................................................... 25 
 
 
 
 


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LIST OF ABBREVIATIONS 
ABBREVIATION 
TERM 
AE 
adverse event 
ALT 
ANOVA 
alanine aminotransferase 
analysis of variance 
ARE 
AST 
BMI 
antioxidant response element 
aspartate aminotransferase 
body mass index 
BUN
CC 
blood urea nitrogen 
Completeness of Cytoreduction Score 
CFR 
Code of Federal Regulations 
CS 
cytoreductive surgery  
CSE 
CT 
CV 
DoH 
ECG 
clinically significant event 
computed tomography 
Curriculum Vitae 
Declaration of Helsinki   
electrocardiogram 
eCRF 
electronic case report form 
FDA 
United States Food and Drug Administration 
HIPEC 
Hyperthermic intraperitoneal chemotherapy 
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 
MRI 
NIR 
NSCLC 
NPV 
PC 
PEG 
PET 
PK 
mean fluorescence imaging 
magnetic resonance imaging 
near-infrared 
non-small cell lung carcinoma 
negative predictive value 
peritoneal carcinomatosis 
polyethylene glycol 
positron emission tomography 
pharmacokinetics 


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ABBREVIATION 
TERM 
PMMA 
PPV 
ROC 
polymethylmethacrylate 
positive predictive value 
receiver operating characteristic 
SAE 
SBR 
Serious adverse event 
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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In both Groups 1 and 2, the surgeon will perform their SOC resection of visible metastases and 
image each resected specimen, and normal specimens on an area of normal tissue outside of the 
surgical field using the intraoperative NIR camera; no NIR imaging of the peritoneal cavity will 
be performed at this time. At the conclusion of SOC resection, the intraoperative NIR camera 
will be used to investigate the peritoneal cavity further for evidence of residual metastases 
highlighted by fluorescence using an approach similar to that performed for the calculation of the 
Peritoneal Carcinomatosis Index (i.e., 13 regions). Additional fluorescent specimens will also be 
imaged on an area of normal tissue within the surgical field prior to sending all surgical 
specimens to pathology for examination. The fluorescence status of each specimen will then be 
directly correlated to the histopathological outcomes of each specimen. This study design 
enables the calculation of pegsitacianine sensitivity, specificity, NPV, and PPV. Additionally, 
Completeness of Cytoreduction (CC) scores can be calculated following SOC resection and then 
adjusted following the additional resection of fluorescent metastases. The schedule of events for 
this study is presented in Appendix 1. 
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 will 
. 
4. SAMPLE SIZE AND POWER CALCULATION 
The sample size for this study will be approximately 60 total subjects. This sample size is based 
on historical experience with similar pilot studies with the goal of informing power calculations 
for future studies. This study is not formally powered for hypothesis testing. A sample size of at 
least 40 total subjects (the originally planned sample size) will be sufficient for developing 
further understanding of the endpoints described herein. Sample size calculations account for 
non-evaluable and drop-out patients 
5. ANALYSIS ENDPOINTS 
5.1 Efficacy Endpoints 
5.1.1 Clinically Significant Event Endpoints 
The primary endpoint will include the identification of a clinically significant event (CSE) 
detected by pegsitacianine. CSEs will include increase in the Completeness of Cytoreduction 


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score following standard of care surgery, pegsitacianine 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 Occult Disease or Positive Surgical Margins  identification of tumor-
containing tissue due to pegsitacianine fluorescence, confirmed via histopathological 
analysis, that otherwise went undetected during pre-operative imaging (PET, MRI) or by 
the surgeon during SOC surgery.  
o This occurs when a sample that (1) exhibits fluorescence in situ and (2) is 
pathologically-confirmed as a tumor, is obtained by fluorescence during SOC 
surgery. 
 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 (e.g. do not exhibit fluorescence ex situ) and are found to be 
devoid of tumor pathologically, those specimens would be included as a tumor negative 
SOC biopsy. 
o This occurs when a sample that (1) exhibits no fluorescence ex situ and (2) is 
pathologically-confirmed as normal tissue, is classified as suspicious tissue during 
SOC surgery.  
 Alteration to the Completeness of Cytoreduction (CC) Score  following the completion 
of the SOC surgery, the surgeon will document a CC score prior to fluorescence imaging 
using pegsitacianine. If the surgeon finds residual disease that results in the increase of 
the CC score, this will be considered a CSE. 
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 pathology 
 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:        
 
 
Specificity values and negative predictive values (NPV) calculated by the equation below: 
 
 
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 (CMP, hemoglobin, and CBC with differentials) 
6. ANALYSIS POPULATIONS 
6.1 The Safety Population 
All patients who receive at least one dose (either partial or full) of pegsitacianine.  
6.2 The Intent-to-Treat 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 
peritoneal 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. 
 For non-efficacy values 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 


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the nearest percent. In 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 Group (Group 1 vs. Group 2). 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 Group and overall.  
Data listings of patient disposition, screening failures, and patient assignment to analysis groups 
will be created. 
7.8 Protocol Deviations 
Protocol deviations are defined as any variation from the protocol, such as enrollment of a 
patient who did not meet all inclusion and exclusion criteria or failure to perform the assessments 


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and procedures within the required time frame, that are identified by the clinical research 
associate monitoring the study. 
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 Group 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 Rating 
No hypothesis testing is planned for baseline characteristics, so the analysis will be purely 
descriptive. 
Data listings of baseline characteristics and demographics including Karnofsky Score 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. 
7.9.3 Prior and Concomitant Medications and Procedures/Treatments 
Prior and concomitant medication (as well as concomitant procedures/treatments) will be 
summarized by Group 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 Cytoreductive Surgery Details and Specimen Collection 
Cytoreductive surgery details including the following will be summarized on a patient level 
using descriptive statistics in the Safety population: 
 Duration of surgery 
 Suspected PCI score and fluorescence imaging PCI score 
 Method of localization 


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 Number of SOC suspicious nodules collected 
 Number of SOC normal specimens collected 
 Additional fluorescent areas collected 
 Cytoreduction score 
 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.  
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 Group. CSE 
responders will be summarized, as well as 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 (biopsy level) 
will be calculated and summarized for each Group. The summary will include the true/false 
negatives/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 fluorescence status, pathology result, specimen 
type (SOC vs. fluorescence), suspicious vs. normal, fluorescence in in situ and ex situ, and 
fluorescence measures (MFI, SBR or TBR) will be provided. 


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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 Group and 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 
will be provided along with the area under the curve (AUC).  
7.10.3.3 Adjustment for Clustering 
There is potential for within-subject correlati
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 
The confidence intervals for diagnostic accuracy (e.g. the positive predictive value, negative 
predictive value, sensitivity, specificity, and true/false negative/positive rate) will also be 
constructed via a cluster-bootstrap to determine confidence intervals that account for the effects 
of within-subject clustering. Since the number of specimens each subject has is variable, in order 
to keep the same number of specimens in the bootstrapped sample, we will cluster initially by the 
number of specimens each subjects has, and then resampling will occur by first choosing a 
subject within that cluster and then resampling the same number of specimens they originally 
had.  


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7.10.3.4 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.  
7.10.3.5 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 Group. 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 and Infusion Related Reactions 
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 28 after administration of study drug.  
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 


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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).  
A listing of all AEs will also be generated. 
Infusion related reactions (IRR) will be summarized, 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, Group, and change 
from baseline. 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. 
7.11.3 Vital Signs 
Vital signs data will be summarized by time point, Group, 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. 


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8. DEVIATIONS FROM ANALYSIS AS DESCRIBED IN THE PROTOCOL 
No deviations from the protocol are planned.




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a For female subjects, a serum sample for a serum pregnancy test will be collected at screening and on Day 0 and Day 28. 
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 oral 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 Vitals are only to be collected if they are found to be abnormal at dosing 
e 12-lead ECG is to be performed prior to study drug administration 
f 
Patients will be assessed for AEs occurring from the time of dosing through Day 28 (±5 days)  
g Hematology assessments will 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