Clinical trial NCT03579602
Conditions: Pediatric Central Nervous System Tumor
Keywords: Tumor Paint tozuleristide Canvas imaging system BLZ-100 CNS tumor brain cancer pediatrics fluorescence guided surgery
Sponsor: Blaze Bioscience Inc.
Start date: 2018-11-26; Completion date: 2022-06-13
Enrollment: 118 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Many types of cancer are primarily treated with surgery and patient survival is directly related to the extent to which the tumor is able to be removed. It is often difficult for surgeons to distinguish tumor tissue from normal tissue or to detect tumor cells that have spread from the original tumor site, resulting in incomplete removal of the tumor and reduced patient survival. In some sites, such as the brain, it is critical to avoid damage to normal tissue around the tumor to prevent adverse effects of surgery on function. Tozuleristide is a drug that is thought to attach to tumor tissue and then fluoresces (glows) when a special light from the Canvas is shined on it. It is hypothesized that tozuleristide, when imaged with the Canvas, will improve surgical outcomes by allowing surgeons to visualize the edges of the tumor or other ambiguous tissue in real-time as they operate. The purpose of this study is to evaluate how well tozuleristide imaged with Canvas work at helping to distinguish between tumor and normal tissue during surgery in pediatric primary central nervous system tumors.

Detailed description:
Subjects who provide voluntary written informed consent, or have it provided by their legally acceptable representative, will be screened for eligibility. Subjects meeting all of the inclusion and none of the exclusion criteria will be eligible to participate.

Surgical excision will occur at least 1 hour and no more than 36 hours after tozuleristide administration. Surgery will be performed by a neurosurgeon and the Canvas will be operated by a designated Imaging Operator. Fluorescence of tumor and ambiguous tissue during surgery will be assessed and scored. Biopsy samples of these tumor and ambiguous tissues will be collected for pathology analysis.

All subjects will be monitored for safety during their participation in the study.

Arms: arm 1: Subjects randomized to Arm 1 (\~9% of subjects) will not receive tozuleristide but will undergo standard of care neurosurgery and will have imaging performed with the Canvas System. arm 2: Subjects randomized to Arm 2 (\~ 91% of subjects) will be administered tozuleristide at a dose of 15 mg/m\^2 at least 1 hour and no more than 36 hours prior to surgery. They will undergo standard of care neurosurgery and will have imaging performed with the Canvas System.
Interventions: intervention 1: tozuleristide intervention 2: Canvas System
Intervention descriptions: intervention 1: Tozuleristide will be administered at least 1 hour and no more than 36 hours prior to planned surgical excision of their tumor. intervention 2: All subjects enrolled in the study will have their tissue imaged with the Canvas System.

Protocol documents:
SAP_002.pdf: 
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Statistical Analysis Plan 
A randomized, blinded study of fluorescence detection of 
pediatric primary central nervous system tumors in subjects 
receiving tozuleristide and imaged with the Canvas System 
Protocol BB-006 
Sponsored by: 
Blaze Bioscience, Inc. 
Version: 5.1 
Version Date: October 17, 2022 
CONFIDENTIALITY STATEMENT 
The information contained in this document is confidential belonging to Blaze Bioscience. 
Acceptance of this document constitutes agreement by the recipient that no information contained 
herein will be published or disclosed without prior written authorization from an official of Blaze 
Bioscience.  However, this document may be disclosed to appropriate Institutional Review Board 
and Ethics Committees or duly authorized representatives of a national regulatory authority under 
the condition that they are requested to keep it confidential.  
NCT03579602


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Approval Signatures 
Product: 
Protocol Number: 
SAP Version: 
Version Date: 
Tozuleristide 
BB-006 
5.1 
The individuals signing below have reviewed and approve this statistical analysis plan. 
Date 
Date 


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Table of Contents 
1 
INTRODUCTION............................................................................................................................... 7 
2 
STUDY OBJECTIVES ....................................................................................................................... 7 
2.1 
PRIMARY OBJECTIVES ....................................................................................................................... 7 
2.2 
SECONDARY OBJECTIVES .................................................................................................................. 7 
2.3 
EXPLORATORY OBJECTIVES .............................................................................................................. 8 
3 
STUDY DESIGN ................................................................................................................................. 8 
3.1 
OVERALL STUDY DESIGN ................................................................................................................. 8 
3.2 
CENTRAL PATHOLOGY .....................................................................................................................10 
3.3 
CENTRAL RADIOLOGY .....................................................................................................................10 
 
 
3.5 
INDEPENDENT SAFETY MONITORING COMMITTEE ...........................................................................10 
3.6 
PROTOCOL ENDPOINT ASSESSMENT COMMITTEE ............................................................................10 
4 
STUDY ENDPOINTS ........................................................................................................................11 
4.1 
PRIMARY EFFICACY ENDPOINTS ......................................................................................................11 
4.2 
SECONDARY ENDPOINTS ..................................................................................................................12 
4.3 
EXPLORATORY ENDPOINTS ..............................................................................................................14 
5 
STATISTICAL HYPOTHESES AND CRITERIA OF SIGNIFICANCE ...................................15 
6 
DETERMINATION OF SAMPLE SIZE ........................................................................................15 
7 
ANALYSIS POPULATIONS ............................................................................................................16 
8 
PLANNED METHODS OF ANALYSIS .........................................................................................18 
8.1 
OVERALL PLAN ................................................................................................................................18 
8.2 
KEY DEFINITIONS ............................................................................................................................18 
8.3 
MISSING DATA HANDLING ..............................................................................................................19 
8.4 
VISIT WINDOWS ...............................................................................................................................19 
8.5 
POOLING OF CENTERS ......................................................................................................................19 
8.6 
SUBJECT ACCOUNTABILITY .............................................................................................................19 
8.7 
TISSUE BIOPSY ACCOUNTABILITY ...................................................................................................20 
8.8 
PROTOCOL DEVIATIONS ...................................................................................................................20 
8.9 
DEMOGRAPHIC AND BASELINE CHARACTERISTICS ..........................................................................20 
8.10 MEDICAL HISTORY ..........................................................................................................................21 
8.11 EFFICACY ANALYSES .......................................................................................................................22 
8.11.1 
Primary Efficacy Endpoints ..............................................................................................22 
8.11.2 
Secondary Endpoints ........................................................................................................25 
8.11.3 
Exploratory Analyses ........................................................................................................27 
8.12 SAFETY ANALYSES ..........................................................................................................................27 
8.12.1 
Exposure to Study Drug ....................................................................................................27 
8.12.2 
Adverse Events .................................................................................................................28 
8.12.3 
Laboratory Parameters ......................................................................................................29 
8.12.4 
Vital Signs ........................................................................................................................31 
8.12.5 
Electrocardiogram Parameters ..........................................................................................31 
8.12.6 
Prior and Concomitant Medications .................................................................................32 
8.12.7 
Physical Examination........................................................................................................32 
 
 
10 
INTERIM ANALYSIS ......................................................................................................................32 
 
 
 
 
 


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12 
REFERENCES ...................................................................................................................................34 
13 
APPENDICES ....................................................................................................................................34 
13.1 CALCULATION OF 95% CONFIDENCE INTERVAL FOR RATIO OF PAIRED PERCENTAGES ...................34 
13.2 DEFINITION OF TUMOR DIAGNOSIS ..................................................................................................35 
 
 
 
 
 
 
 
 


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Glossary of Abbreviations 
 
Abbreviation 
Description 
AE 
adverse event 
ASP 
all-subject population 
BSA 
body surface area 
CI 
confidence interval 
CNS 
central nervous system 
CRF 
case report form 
CTCAE 
Common Terminology Criteria for Adverse Events 
CSF 
cerebrospinal fluid 
DAP 
Data Access Plan 
DNA 
Deoxyribonucleic acid 
ECG 
Electrocardiogram 
eCRF 
electronic case report form 
EOR 
extent of resection 
FAP 
full analysis population 
FLAIR 
Fluid-Attenuated Inversion Recovery  
GEE 
general estimating equation 
GTR 
gross total resection 
ITT 
intent-to-treat 
Max 
Maximum 
MedDRA 
Medical Dictionary for Regulatory Activities 
Min 
Minimum 
MRI 
magnetic resonance imaging 
NCI 
National Cancer Institute 
NIR 
near infrared 
NPV 
negative predictive value 
NTR 
near total resection 
PEAC 
Protocol Endpoint Assessment Committee 
PK 
Pharmacokinetics 
PKAP 
pharmacokinetic analysis population 


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Abbreviation 
Description 
PPV 
positive predictive value 
PT 
preferred term 
QTc 
corrected QT interval 
SAE 
serious adverse event 
SAF 
safety analysis population 
SAP 
Statistical Analysis Plan 
SD 
standard deviation 
SI 
standard international system of units 
SMC 
Safety Monitoring Committee 
SOC 
system organ class 
STR 
subtotal resection 
T1 
Longitudinal relaxation time 
T2 
Transverse relaxation time 
TEAE 
treatment emergent adverse event 
WHO 
World Health Organization 
 
 
 


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1 
INTRODUCTION 
This document outlines the statistical methods to be implemented within the scope of 
Protocol BB-006, entitled “A randomized, blinded study of fluorescence detection of 
pediatric primary central nervous system tumors in subjects receiving tozuleristide and 
imaged with the Canvas System”.  Results of the proposed analyses will become the basis 
of the clinical study report for this protocol.  
The purpose of this plan is to provide general, and in some instances specific, guidelines 
from which the analysis will proceed.  Nevertheless, an efficient data analysis should 
allow modifications to the original analysis plan to provide a more reliable and valid 
analysis of the data.  Any such “post hoc” or “data driven” analyses will be identified in 
the final clinical study report.  All planned analyses specified in this document will be 
performed.  Any changes to this plan will either be reflected in amendments (to this plan) 
before the database lock or documented in the clinical study report. 
2 
STUDY OBJECTIVES 
2.1 
Primary Objectives 
The primary objectives of this study in subjects undergoing neurosurgical resection of 
pediatric primary central nervous system (CNS) tumors are: 
• To compare the sensitivity of tozuleristide and the Canvas near infrared (NIR) 
imaging system fluorescence to the sensitivity of surgical designation in equivocal 
tissue in situ; 
• To compare the specificity of tozuleristide and the Canvas system fluorescence to 
the specificity of surgical designation in equivocal tissue in situ; 
• To evaluate imaging efficacy as measured by sensitivity of tozuleristide and the 
Canvas system in facilitating intraoperative fluorescence detection and 
visualization of tumor in equivocal tissue in situ; 
• To evaluate imaging efficacy as measured by specificity of tozuleristide and the 
Canvas system in facilitating intraoperative fluorescence detection and 
visualization of tumor in equivocal tissue in situ. 
2.2 
Secondary Objectives 
The secondary objectives of this study are:  
•
 
 


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• 
 
 
 
• 
  
• 
 
 
2.3 
Exploratory Objectives 
The exploratory objectives of this study will evaluate the: 
• 
 
 
 
 
 
 
 
 
3 
STUDY DESIGN 
3.1 
Overall Study Design 
This is a randomized, blinded (central pathology, central fluorescence assessment and 
central radiology), multi-center Phase 2 study of a single dose of tozuleristide in subjects 
with pediatric primary CNS tumors; intraoperative imaging will be performed with the 
Canvas system.  
Eligible patients will be randomized into one of the two treatment arms in a 1:10 (no drug 
treatment tozuleristide) allocation: 
Arm 1: Approximately 9% of subjects will be randomized to receive standard of 
care neurosurgery without tozuleristide. 
Arm 2: Approximately 91% of subjects will be randomized to receive standard of 
care neurosurgery and tozuleristide. 
Within this Statistical Analysis Plan (SAP), study-related tissue collection may be 
referred to as specimens or biopsies.   
During the surgical procedure for each subject, and once the tumor has been accessed, the 
Canvas system, which is attached to the surgical microscope, will be used to capture and 
display intraoperative in situ images (white light and fluorescence) of the region from 
which surgical specimens for biopsy or excision will be collected. All clinical decision-


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making regarding tissue resection will be made based on the routine neurosurgical 
practices, and tissue resection will take place under normal light conditions in line with 
best clinical practices. 
In addition to primary tumor biopsy, regions of equivocal nature (uncertain if tumor or 
not tumor) will be identified before fluorescence in the operating room is determined. 
 
 
 
 
 
 
 
 
 
 
Tozuleristide and the Canvas system are intended as an aid to surgical decision-making. 
As such, when a region of interest in the brain/spine is noted to be fluorescent, it is 
always the surgeon’s decision whether to biopsy/resect or not, if safe to do so. 
 
 
 
  
luorescence assessment by the 
imaging operators (local assessment) will be used in the primary efficacy analyses. 
Subjects who are randomized to Arm 1 or Arm 2 and need to undergo an additional 
surgery for their brain tumor will be eligible to receive tozuleristide without being subject 
to randomization. Subjects must meet all study eligibility requirements and re-consent for 
study prior to re-enrollment for open-label treatment of tozuleristide (re-treatment). 
 
 
 
 


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3.2 
Central Pathology 
The pathology assessment of a tissue specimen (primary, equivocal or other) as tumor or 
not tumor, for both arms, will be performed by a centralized and independent panel of 
pathologists without knowledge of fluorescence status or treatment arm (blinded 
pathology). 
3.3 
Central Radiology 
Post-operative centralized and independent MRI assessment
 
3.4 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
3.5 
Independent Safety Monitoring Committee 
An Independent Safety Monitoring Committee (SMC) will have responsibility for 
oversight of safety on the study. The SMC members and its responsibilities and processes 
will be described in the SMC charter.   
3.6 
Protocol Endpoint Assessment Committee 
A Protocol Endpoint Assessment Committee (PEAC) will monitor the progress of the 
study 
 
 
 
 
 


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4 
STUDY ENDPOINTS  
4.1 
Primary Efficacy Endpoints 
The primary efficacy endpoints are: 
1. Ratio of sensitivity of tozuleristide fluorescence in equivocal tissue biopsies from 
subjects with fluorescence-positive tumors compared to the sensitivity of surgical 
designation from the same biopsies under white light conditions 
2. Ratio of specificity of tozuleristide fluorescence in equivocal tissue biopsies from 
subjects with fluorescence-positive tumors compared to the specificity of surgical 
designation from the same biopsies under white light conditions 
3. Sensitivity of tozuleristide fluorescence to detect tumor in equivocal tissue 
biopsies from subjects with fluorescence-positive tumor 
4. Specificity of tozuleristide fluorescence to detect tumor in equivocal tissue 
biopsies from subjects with fluorescence-positive tumor 
Fluorescence positivity for the primary endpoints is defined as weak or strong 
fluorescence as determined by the imaging operator fluorescence assessment based on 
displayed fluorescence signal and exposure setting. 
 
 
 
 
 
  
 
 
 
  


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4.2 
Secondary Endpoints 
Study endpoints related to the secondary efficacy objective: 
1. 
 
 
 
 
 
2. 
 
 
 
3. 
 
 
 
4. 
 
 
 
 
 
 
 
Study endpoints related to the safety objective: 
1. All adverse events (AE) will be graded according to the National Cancer 
Institute’s Common Terminology for Adverse Events (NCI CTCAE, Version 5.0)  
2. Serious adverse events (SAEs)  


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3. AE attribution of relatedness to tozuleristide and/or Canvas system (related vs. 
unrelated) 
4. Laboratory parameters 
5. Electrocardiogram (ECG) parameters 
6. Vital signs 
Study endpoints related 
: 
1. 
 
 
 
2. 
 
 
 
3. 
 
4. 
 
 
Study endpoints related 
 
• 
 
 
Study endpoints related 
: 
• 
 
1. 
  
 
 
 


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4.3 
Exploratory Endpoints 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 


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5 
STATISTICAL HYPOTHESES AND CRITERIA OF SIGNIFICANCE 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6 
DETERMINATION OF SAMPLE SIZE 
Approximately 114 subjects are planned for treatment in this study.  
 
 


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7 
ANALYSIS POPULATIONS 
The full analysis population (FAP) for efficacy analyses will consist of all subjects who  


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1. meet all eligibility criteria, 
2. undergo neurosurgery, and 
3. have central pathology tumor assessment and imaging operator fluorescence 
assessment of equivocal tissue specimens obtained from the first neurosurgery on 
study. 
Subjects in the FAP will be classified into 4 groups: 
• Group 1 (Arm 1): subjects who do not receive tozuleristide, regardless of 
fluorescence outcomes and tumor diagnosis   
• Group 2 (Arm 2 Fluorescent): subjects who receive tozuleristide, have at least one 
fluorescence-positive study tissue specimens of any type (primary, equivocal, and 
other) based on imaging operator fluorescence assessment, and the subject's 
central pathology consensus diagnosis is tumor, ambiguous, or not definitive 
• 
 
 
 
 
 
 
 
A subject is evaluable for efficacy if the subject meets the three criteria of the FAP 
 
 
 
 
 
 
The FAP will be the primary analysis population for all efficacy analyses, summaries, 
and listings. 
The safety analysis population (SAF) will consist of all subjects who receive any amount 
of tozuleristide or receive neurosurgery prior to any re-treatment. The SAF will be used 
for all safety data summaries and listings.   
 
 


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The all-subject population (ASP) is defined as all subjects who sign informed consent. 
The ASP will be used for screen failure summaries and listings. 
The intent-to-treat (ITT) population is defined as all subjects who are randomized. The 
ITT population will be used for disposition summaries, protocol deviations, and listings. 
No subjects will be excluded from any analysis populations due to protocol deviations. 
8 
PLANNED METHODS OF ANALYSIS 
8.1 
Overall Plan 
In general, descriptive statistics will be provided for all the study endpoints and will be 
presented in tables and/or graphs. Continuous variables will be summarized using the 
number of observations (n), mean, median, standard deviation, minimum and maximum. 
Categorical variables will be summarized with frequency counts and percentages. 
Percentages will be rounded to one decimal place, with the denominator being the 
number of subjects in the relevant population (N), unless otherwise stated. 
Statistical tests will be performed at either a one-sided significance level of 2.5% or a 
two-sided significance level of 5%. All confidence intervals (CIs) will be constructed at a 
two-sided 95% confidence level.  
In addition, data collected after re-treatment will be listed and will not be included in the 
primary efficacy and safety analyses. 
8.2 
Key Definitions 
Study Day is the day relative to the date of neurosurgery, where Day 1 is the day of the 
first neurosurgery performed in this study. 
Study Day will be calculated using the first neurosurgery date as the reference date. If the 
date of interest occurs on or after the first neurosurgery date, study day will be calculated 
as (date of interest – first neurosurgery date) + 1. If the date of interest occurs prior to the 
first neurosurgery date, study day will be calculated as (date of interest – first 
neurosurgery date). There will be no study Day 0. 
Unless otherwise specified, the baseline value for a measurement used in all statistical 
analyses will be the most recent non-missing measurement prior to the administration of 
the study drug for Arm 2 and prior to the first neurosurgery for Arm 1.  
A change from baseline value is calculated as the post-baseline value minus the baseline 
value. 


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Treatment-emergent adverse event (TEAE) is defined as an adverse event that is newly 
occurring or worsens following neurosurgery (Arm 1) or treatment with tozuleristide 
(Arm 2). 
8.3 
Missing Data Handling 
Missing data will not be imputed. Subjects with a missing variable at a particular time 
point (e.g., a laboratory variable at a visit week) will be excluded from the analysis of 
that variable at that time point unless otherwise specified. 
Section 8.12.2 describes how to deal with partial AE start dates in determining treatment 
emergence.  
Section 8.12.6 describes how to determine prior and concomitant medications with partial 
medication start dates. Every effort will be made to ensure that the attributes of an AE 
(seriousness, severity, relatedness to study drug/device/surgical procedure and outcome) 
are complete. Missing attributes will not be imputed and will be summarized as 
"unknown".  
8.4 
Visit Windows 
All assessments will be included in the listings. Analytic visit windows will be applied to 
the following assessments that do not have a visit on the CRF: 
 
Assessments 
Analytic Visit 
Study drug administration 
Day 1 
Central radiology 
Protocol specified visit windows1 
Central pathology 
Day 1 
 
 
 
 
 
 
 
Analytic visit will be set to the CRF visit for the assessments with a visit on the CRF. 
8.5 
Pooling of Centers 
Data from all centers will be pooled prior to analysis. 
8.6 
Subject Accountability 
Subject accountability will be summarized for the ITT population. Subjects who 
discontinue tozuleristide prematurely and the reason for tozuleristide discontinuation will 
be summarized for Arm 2. Subjects who withdraw from the study and the reason for 


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study discontinuation will be summarized by treatment arm. The number of subjects who 
do not undergo surgery will also be summarized by treatment arm. 
The number of screen failures and the reason for screen failure will be summarized as a 
percentage of the all-subject population.  
The number of subjects in each analysis population (Section 7) and the reason for 
excluding from the analysis population will be summarized as a percentage of the all-
subject population by treatment arm. 
Informed consent (including protocol version), screen failure, eligibility and 
randomization data will be listed. 
8.7 
Tissue Biopsy Accountability 
Tissue biopsy/specimen accountability will be summarized for the ITT population by 
treatment arm.
 
 
8.8 
Protocol Deviations 
Subject data will be examined for evidence of protocol deviations in order to assess how 
well the protocol was followed. This is described in the Protocol Deviations and Non-
compliance Management Plan. All protocol deviations will be listed using the ITT 
population. 
8.9 
Demographic and Baseline Characteristics 
Demographic and other baseline characteristics will be summarized with descriptive 
statistics by treatment arm for the full analysis population. Demographics include: 
• Age at informed consent calculated as Age (years) = integer value of ((Date of 
Informed Consent – Date of Birth + 1) / 365.25),  
• Gender,  
• Ethnicity, and  
• Race.  
Baseline characteristics include:  
• Baseline weight (kg), 


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• Baseline height (cm), 
• Baseline body surface area (BSA m2, using Mosteller formula),  
• Karnofsky performance status, 
• Lansky performance status, 
• Received prior CNS cancer surgery (Yes, No), 
• Time from the most recent prior CNS cancer surgery to current surgery, 
• Outcome of the most recent prior CNS cancer surgery (GTR, NTR, STR, Biopsy 
only, Other), 
• Received prior CNS cancer systemic therapy (Yes, No), 
• Time from the most recent prior CNS cancer systemic therapy to current surgery, 
• Received prior CNS cancer radiation therapy (Yes, No), 
• Time from the most recent prior CNS cancer radiation therapy to current surgery, 
• Location of current tumor (Brain – supratentorial, Brain – posterior fossa, Spinal 
cord, Other), 
• Recurrent or progressive disease for current tumor (Yes, No), 
• Evidence of metastatic disease (Yes, No) 
All baseline data will be listed.  
8.10 Medical History 
Medical history will be coded by using the Medical Dictionary for Regulatory Activities 
(MedDRA). Medical history frequencies will be summarized by treatment arm for the 
full analysis population and will be ordered by descending frequency for total by system 
organ class (SOC) then, within a SOC, by overall descending frequency of preferred term 
(PT). If incidence for more than one term is identical, they will be sorted alphabetically.  


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8.11 Efficacy Analyses 
8.11.1 Primary Efficacy Endpoints 
8.11.1.1 Primary Analyses of Sensitivity and Specificity 
The primary efficacy analyses will be based on Group 2 in the full analysis population 
(Section 7). Each of the 4 sets of statistical hypotheses will be tested at two-sided 5% 
level (Section 5) using the repeated measures log-linear model specified in this section.   
Each equivocal tissue specimen from Group 2 that is determined to be either tumor 
positive or tumor negative by central pathology, and has an image operator assessment 
and/or a surgeon assessment will be used in the analysis. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
8.11.1.2 Secondary Analyses of Sensitivity and Specificity 
1. 
 
 


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.  
2. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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13. 
 
 
8.11.2 Secondary Endpoints 
8.11.2.1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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8.11.2.2 
 
 
 
 
 
 
 
 
 
 
 
 
  
= 
 
 
 
 
 
 
= 
 
 
 
 
 


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8.11.2.3 
 
 
 
  
 
8.11.2.4 
 
 
 
8.11.3 
 
 
  
 
  
 
 
 
8.12 Safety Analyses 
All safety analyses will be based on the safety analysis population. Safety data collected 
after re-enrollment for open-label treatment of tozuleristide will be listed. 
8.12.1 Exposure to Study Drug 
The dose of tozuleristide administrated in volume (mL) and amount (mg) will be 
summarized as a continuous variable. Dose compliance as measured by the actual dose 


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received as the percentage of the intended dose [(Total Volume Infused/Scheduled 
Volume) x 100] will also be summarized. 
8.12.2 Adverse Events 
Adverse events will be summarized by the system organ class (SOC) and preferred term 
(PT) based on the MedDRA dictionary. The actual version will be documented in the 
CSR. 
Adverse events will be defined as treatment-emergent if they are newly occurring or 
worsen following neurosurgery (Arm 1) or treatment with tozuleristide (Arm 2). Adverse 
events with incomplete start dates will be considered treatment emergent if: 
• Day and month are missing and the year is equal to or after the year of the first 
neurosurgery for Arm 1 or the first dose date for Arm 2;  
• Day is missing and the year is after the year of the first neurosurgery for Arm 1 or 
the first dose date for Arm 2;  
• Day is missing and the year is equal to the year of the first neurosurgery for Arm 
1 or the first dose date for Arm 2 and the month is equal to or after the month of 
the first neurosurgery for Arm 1 or the first dose date for Arm 2; 
• Year is missing; or 
• Start date is completely missing. 
However, if the end date of the AE is before the first neurosurgery for Arm 1 or the first 
dose date for Arm 2, this AE will not be treatment emergent regardless of the 
completeness of its start date. 
Adverse events will be graded using the NCI CTCAE, version 5.0 if applicable, or the 
protocol specified severity stated in Section 7.1 of the protocol. 
Treatment-emergent AEs, SAEs, severity of AEs, tozuleristide-related AEs, Canvas-
related AEs, neurosurgical-related AEs and AEs leading to withdrawal from the study 
will be summarized by MedDRA preferred term, system organ class and treatment arm 
for the safety analysis population. In the event of multiple occurrences of the same AE 
with the same preferred term in the same subject, the AE will be counted once at the 
greatest severity/relatedness. 
The summaries presenting frequency of AEs by SOC and PT will be ordered by overall 
descending frequency of SOC and then, within a SOC, by overall descending frequency 
of PT for Arm 2. 


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Only the TEAEs from Arm 1 and Arm 2 will be included in the summary tables. All AEs 
will be included in the listings. Additional listings will be provided for SAEs, AEs 
leading to death and AEs leading to study withdrawal. 
In addition, treatment-emergent AEs for the re-treatment period are defined as newly 
occurring or worsen following the open-label treatment of tozuleristide. All AEs reported 
during the re-treatment period will be listed. 
8.12.3 Laboratory Parameters  
All summaries will be based on results in standard international (SI) system units. 
Reference ranges for all laboratory parameters collected throughout the study are 
provided by the respective laboratory. A laboratory value that is outside the reference 
range is considered either high abnormal (value above the upper limit of the reference 
range) or low abnormal (value below the lower limit of the reference range).  
The laboratory reference ranges will be provided on the listings of laboratory data with 
out of range values flagged (low or high). 
Hematology and chemistry laboratory samples are scheduled to be collected at Screening 
(all subjects) and Day 8 (Arm 2 and re-treatment only). Coagulation laboratory samples 
will be collected at Screening (all subjects). For hematology and clinical chemistry 
summary tables, the actual values and change from baseline for each laboratory 
parameter will be provided by scheduled visit.  Summaries will include data from 
scheduled assessments only, and all data will be reported according to the nominal visit 
date for which it was recorded (i.e., no visit windows will be applied).  
Toxicity grading will follow the NCI CTCAE grades version 5.0. NCI CTCAE toxicity 
grading will be derived programmatically for laboratory variables where possible. 
Toxicity grading that requires investigator/clinical input will not be considered and only 
the numeric criteria will be used for the programmed grade assignments. If this results in 
the criteria for more than one grade being met, the highest (worst) CTCAE grade will be 
assigned.  Where a result does not meet any of the CTCAE grading criteria, the record 
will be counted in the ‘No Grade’ category. For a parameter that can be low or high, e.g. 
‘hemoglobin increased’ or ‘anemia’, if CTCAE criteria for a grade for ‘anemia’ is 
satisfied, then ‘hemoglobin increased’ will be set to ‘No Grade’. 
 
 
 


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All laboratory results in original and SI units will be included in data listings.  Tests will 
be listed in CRF order.  
All abnormal laboratory values will be included in the listings. 
Pregnancy data will be listed. 
8.12.4 Vital Signs 
Vital signs will be collected at Day 1 pre-surgery (Arm 1)/pre-dose (Arm 2 and re-
treatment), Day 1 between 1-30 minutes post-dose and Day 8 (Arm 2 and re-treatment). 
Summary tables of the actual values and change from baseline for each vital sign will be 
provided by scheduled visit.  Summaries will include data from scheduled assessments 
only, and all data will be reported according to the nominal visit date for which it was 
recorded (i.e., no visit windows will be applied).  
8.12.5 Electrocardiogram Parameters 
Electrocardiogram (ECG) parameters (rhythm, ventricular rate, PR interval, QRS 
duration, QT interval and QTcF interval) will be collected pre-dose, between 1-30 
minutes post-dose and Day 8 and will be summarized with descriptive statistics of the 
change from baseline value by time point for Arm 2 and re-treatment arm. Overall ECG 
result (normal or abnormal) will be summarized by time point. Summaries will include 
data from scheduled assessments only, and all data will be reported according to the 
nominal visit for which it was recorded (i.e., no visit windows will be applied).  
 
 
 
 
 
 
 
  
 
 
 
 
 
 


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8.12.6 Prior and Concomitant Medications 
All medications will be coded using the latest version of the World Health Organization 
(WHO) Drug coding dictionary. The actual dictionary versions used will be presented in 
the CSR. 
Concomitant medications will be summarized by standardized medication name and 
treatment arm for the safety analysis population. Subject incidence will be presented. 
Subjects who take the same medication (in terms of the standardized medication name) 
more than once will only be counted once for that medication. 
Prior medications are those medications that were stopped before the first neurosurgery 
date for Arm 1 or the first dose date for Arm 2. Concomitant medications are medications 
taken at least once on or after the first neurosurgery date for Arm 1 or the first dose date 
for Arm 2.  
If partial dates exist for concomitant medication, the start dates will be imputed to the 
first of the month (for missing day) or first of the year (for missing day and month) for 
purposes of assigning the medication to a prior or concomitant medication.  
Prior medications will be listed only. Prior and concomitant medications will be listed 
separately, including derived study day. 
8.12.7 Physical Examination 
Physical examination will be listed for the safety analysis population. 
9 
 
 
 
 
 
 
 
 
 
  
10 INTERIM ANALYSIS 
Interim analysis is not planned. 
 


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12 REFERENCES 
Pepe MS. The Statistical Evaluation of Medical Tests for Classification and Prediction. 
Oxford University Press; 2003 
13 APPENDICES 
13.1 Calculation of 95% Confidence Interval for Ratio of Paired Percentages 
The 95% CI for the ratio of paired percentages (sensitivity and specificity) will be 
calculated using Pepe's formula (Pepe, 2003) as follows.  
The paired data of sensitivity between tozuleristide and surgical designation can be 
summarized in a 2 x 2 table of all tumor-positive biopsies by central pathology: 
 
 
 
Surgical Designation 
 
 
Tumor* 
Not Tumor* 
Tozuleristide 
Fluorescence 
Positive* 
a 
b 
Negative* 
c 
d 
*See Section 4.1 
 
  
 
 
 
The paired data of specificity between tozuleristide and surgical designation can be 
summarized in a 2 x 2 table of all tumor-negative biopsies by central pathology: 
 
 
 
Surgical Designation 
 
 
Tumor* 
Not Tumor* 
Tozuleristide 
Fluorescence 
Positive* 
e 
f 
Negative* 
g 
h 
*See Section 4.1 
 
  
 
 
 


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13.2 Definition of Tumor Diagnosis 
Subjects are eligible for enrollment based on MRI evidence consistent with a pediatric 
primary CNS tumor.  Study specimens are subjected to pathological analysis and along 
with local pathology assessments, a diagnosis is assigned by Central Pathology.  Tumor 
diagnosis is based on WHO Classification of Tumors of the Central Nervous System, 4th 
Edition, 2016.  Subjects with ambiguous pathological diagnosis of tumor or no definitive 
pathological diagnosis are considered to have a pediatric primary CNS tumor. Subjects 
with a pathological diagnosis indicative of non-tumorous abnormality are considered to 
not have a pediatric primary CNS tumor.  
13.3 Sample SAS Code for Repeated Measures Log-Linear Regression 
 
 
       
 
  
       
 
  
     
 
                               
     
 
                               
     
  
       
 
  
             
 
  
 
  
 
  
 
 
 
 
  
 
  
 
  
 
     
 
     
 
     
 
  
 
 
 
 
  
 
 
 
 
 
  
 
  
 
                               
 
                               
    
  
 
  
             
 
  
 
  
 
  
 
 
 
 
  
 
  
 
  
   
     
 
     


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13.4 

Prot_000.pdf: 
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Version Date: 28 Oct 2021 
 
 
 
A randomized, blinded study of fluorescence detection of pediatric primary central nervous 
system tumors in subjects receiving tozuleristide and imaged with the Canvas System 
 
Protocol #: BB-006 
(PNOC012) 
Version Number: 5.0 
 
 
Version Date: October 28, 2021 
IND: 122229 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CONFIDENTIALITY STATEMENT 
This document contains confidential information of Blaze Bioscience (“Blaze Bioscience”). 
The information contained in this document, particularly unpublished data, is the property of or under the control of 
Blaze Bioscience and is provided to you in confidence as an investigator, potential investigator, or consultant, for 
review by you, your staff, and an applicable Institutional Review Board/Human Research Ethics Committee. 
The information is only to be used by you in connection with authorized clinical studies of the investigational drug 
and medical device described in the protocol.  
Disclosure of any of the information to others requires written authorization from Blaze Bioscience except to the 
extent necessary to obtain informed consent from those persons to whom the investigational product(s) may be 
administered. 


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Study Chair  
Sarah Leary, MD MS 
Seattle Children’s Hospital 
 
 
 
 
 
 
Co- Chair  
 
 
 
 
 
 
 
 Project Leader 
 
 
   
 
 
 
 
 
Co-Chair 
 
 
 
 
 
 
Project Co-Leader 
 
   
 
 
 
Coordinating Center 
 
 
 
 
 
 
 
 
Revision History 
Version 1.0 
 
Version 1.1 
 
Version 2.0 
 
Version 3.0 
 
Version 4.0 
 
Version 5.0 
 
 


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Participating 
 Institutions 
Participating 
 Institutions 
Children’s Hospital of Michigan 
3901 Beaubien Street 
Detroit, MI 48201 
Children’s Hospital Los Angeles 
4650 Sunset Blvd., Mail Stop #54 
Los Angeles, CA 90027 
Children’s Minnesota 
Jim and Colleen Ryan Pediatric Cancer and Blood 
Disorders Center 
2525 Chicago Avenue South 
Minneapolis, MN 55404 
Children’s National Medical Center 
Department of Neurology 
111 Michigan Ave NW 
Washington, DC 20010 
Johns Hopkins Hospital 
Johns Hopkins University 
CMSC-800; 600 N. Wolfe St. 
Baltimore, MD 21287 
Seattle Children’s Hospital 
University of Washington 
4800 Sand Point Way NE 
Seattle, WA 98105 
St. Jude Children’s Research Hospital 
Research Hospital 
262 Danny Thomas Place, MS 260 
Memphis, TN 38105 
St. Louis Children’s Hospital 
Washington University in St. Louis 
 One Children’s Place, 4S10 
St. Louis, MO 63110 
UCSF Benioff Children’s Hospital  
University of California, San Francisco 
Pediatric Brain Center 
Ron Conway Family Gateway Medical Building 
1825 Fourth Street, 6th Floor 
San Francisco, CA 94158 
University of Florida 
UF Health Shands Hospital/Shands Children’s 
Hospital 
1600 SW Archer Road, Gainesville, Florida  
32608 
Texas Children’s Cancer 
and Hematology Centers 
6701 Fannin Street, Suite 1400 
Houston, TX 77030 


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PROTOCOL SIGNATURE PAGE 
 
Protocol No.:    BB-006 (PNOC012) Amendment 5.0 
Version Date: 28 October 2021 
Investigator Statement 
I have read and understood the protocol entitled: “A randomized, blinded study of fluorescence 
detection of pediatric primary central nervous system tumors in subjects receiving tozuleristide 
and imaged with the Canvas System” 
I will conduct this study in accordance with this protocol (subject to amendments) and the 
current principles of Good Clinical Practice (GCP), International Conference on Harmonization 
(ICH E6R2) guidelines and any additional local guidelines as applicable. Any changes in 
procedure will only be made if necessary to protect the safety, rights or welfare of subjects. I 
will make all reasonable efforts to complete the study within the time designated. I agree to 
disclose the financial interests of all investigators or sub-investigators who are directly involved 
in the treatment or evaluation of research subjects.  
This study will support an FDA submission and is subject to compliance with 21 CFR 312.60 
for the drug component and ISO 14155:2011, 21 CFR 812.100 and 812.110 for the device 
component in addition to 21 CFR 11, 50, 54 and 56. 
I agree to ensure that the confidential information contained in this document will not be used 
for any purpose other than the evaluation or conduct of the clinical investigation without the 
prior written consent of Blaze Bioscience. I will provide all study personnel under my 
supervision with copies of the protocol and access to all information provided by Blaze 
Bioscience. I will discuss this material with them to ensure that they are fully informed about 
the investigational product(s) and the study 
 
 
 
 
 
Principal Investigator’s Name (Printed) 
 
Signature 
 
 
 
 
 
Date (dd mmm yyyy) 


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TABLE OF CONTENTS 
PROTOCOL SIGNATURE PAGE .......................................................................................... 4 
ABSTRACT ................................................................................................................................ 9 
EXPERIMENTIAL DESIGN SCHEMA ............................................................................... 17 
LIST OF ABBREVIATIONS .................................................................................................. 18 
1 
OBJECTIVES .......................................................................................................... 21 
1.1 
Primary Objectives .................................................................................................... 21 
1.2 
Secondary Objectives ................................................................................................ 21 
1.3 
Exploratory Objectives .............................................................................................. 21 
2 
BACKGROUND ...................................................................................................... 22 
2.1 
Study Disease ............................................................................................................ 22 
2.2 
Study Agent and Device ............................................................................................ 24 
2.3 
Nonclinical Studies .................................................................................................... 25 
2.3.1 
Safety summary: Tozuleristide ..................................................................................... 25 
2.3.1.1 
Serious Adverse Events ............................................................................................. 26 
2.4 
Clinical Studies .......................................................................................................... 27 
2.4.1 
Overview of Clinical Studies ....................................................................................... 27 
 
 
 
 
 
 
 
 
 
 
2.5 
Rationale .................................................................................................................... 38 
2.5.1 
Study Rationale ............................................................................................................ 38 
2.5.2 
Dose Rationale ............................................................................................................. 39 
2.5.3 
Device Rationale .......................................................................................................... 39 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3 
STUDY DESIGN ..................................................................................................... 43 
3.1 
Characteristics ........................................................................................................... 43 
3.2 
Number of Subjects ................................................................................................... 43 
3.3 
Inclusion Criteria ....................................................................................................... 43 
3.4 
Exclusion Criteria ...................................................................................................... 44 
4 
REGISTRATION PROCEDURES ....................................................................... 46 
4.1 
General Guidelines .................................................................................................... 46 
4.1.1 
Treatment Allocation .................................................................................................... 46 
4.1.2 
Blinding ........................................................................................................................ 46 
4.1.3 
Re-treatment ................................................................................................................. 46 


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5 
AGENT ADMINISTRATION ............................................................................... 47 
5.1 
Investigational Product .............................................................................................. 47 
5.2 
Imaging Device ......................................................................................................... 47 
5.2.1 
Canvas System Data ..................................................................................................... 50 
5.3 
Regimen Description ................................................................................................. 51 
5.4 
Phase 0/1 Dose Escalation Scheme ........................................................................... 52 
5.5 
Phase 0/1 Definition of Dose-Limiting Toxicity ...................................................... 52 
5.6 
General Concomitant Medication and Supportive Care Guidelines ......................... 52 
5.6.1 
Special Dietary Requirements ...................................................................................... 52 
5.6.2 
Concurrent Medications / Treatments Not Permitted ................................................... 52 
5.6.2.1 
Prior to Study Entry .................................................................................................. 52 
5.6.2.2 
On Study .................................................................................................................... 52 
5.7 
Dosing Modifications and Delays ............................................................................. 53 
5.7.1 
Criteria for dose-modification in case of suspected tozuleristide toxicity and re-
initiation of treatment ................................................................................................... 53 
6 
TREATMENT PLAN ............................................................................................. 54 
6.1 
Study Calendar .......................................................................................................... 54 
6.2 
Observations and Procedures .................................................................................... 56 
6.2.1 
Informed Consent ......................................................................................................... 56 
6.2.2 
Screening Period (Day -14 to day of surgery) .............................................................. 56 
6.2.3 
1 to 36 Hours Pre-Surgery ............................................................................................ 58 
6.2.4 
Day 1 (Day of Surgery) ................................................................................................ 58 
6.2.5 
Day 8 (7 Days Post-Surgery ± 3 days) ......................................................................... 62 
6.2.6 
30 Days Post-Surgery (±7 days) ................................................................................... 62 
6.2.7 
2 Months Post-Surgery (±7 days) and 3 Months Post-Surgery (±7 days) .................... 62 
6.3 
Off-Treatment Criteria............................................................................................... 63 
6.4 
Off Study Criteria ...................................................................................................... 63 
7 
ADVERSE EVENTS ............................................................................................... 64 
7.1 
Adverse Event Characteristics ................................................................................... 64 
7.1.1 
Serious Adverse Events ................................................................................................ 65 
7.1.1.1 
Clarification of Serious Adverse Events .................................................................... 66 
7.1.2 
Life-threatening ............................................................................................................ 66 
7.2 
Adverse Event Reporting .......................................................................................... 66 
7.3 
SAEs and Expedited Reporting ................................................................................. 67 
7.4 
Toxicity Management ................................................................................................ 67 
7.4.1 
Definitions .................................................................................................................... 67 
7.4.2 
Guidance for Dose Modification or Discontinuation of Treatment ............................. 68 
7.4.3 
Warnings and Precautions ............................................................................................ 68 
8 
SPECIAL STUDIES ........................... 69 
8.1 
..................................................................................................... 69 
8.2 
Investigational Device Information ........................................................................... 69 
8.3 
 .................................................................................................... 69 
9 
EVALUATION CRITERIA ................................................................................... 70 
9.1 
Independent Safety Monitoring Committee (SMC) .................................................. 70 
9.2 
Central Radiology ...................................................................................................... 70 
9.3 
Central Pathology ...................................................................................................... 70 


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9.5 
Protocol Endpoint Assessment Committee (PEAC) ................................................. 71 
10 
STATISTICAL CONSIDERATIONS ................................................................... 72 
10.1 
Study Design/Endpoints ............................................................................................ 72 
10.1.1 
Primary Endpoints ........................................................................................................ 72 
10.1.2 
Secondary Endpoints .................................................................................................... 73 
10.1.3 
Exploratory Endpoints .................................................................................................. 74 
10.2 
Statistical Analysis Plan ............................................................................................ 74 
 
 
10.2.2 
Analysis Populations .................................................................................................... 75 
10.2.3 
Subject Disposition ....................................................................................................... 76 
10.2.4 
Demographics and Subject Characteristics .................................................................. 76 
10.2.5 
Analyses of Primary Efficacy Endpoints ..................................................................... 76 
 
 
10.2.7 
Study Product Exposure ............................................................................................... 77 
10.2.8 
Safety Analyses ............................................................................................................ 78 
10.2.8.1 Adverse Events .......................................................................................................... 78 
10.2.8.2 Clinical Laboratory Parameters, ECG and Vital Signs ............................................ 78 
 
 
 
 
 
 
 
10.2.12 
Interim Analysis ........................................................................................................... 79 
10.3 
Sample Size ............................................................................................................... 79 
10.4 
Stratification Factors ................................................................................................. 80 
11 
DATA REPORTING / REGULATORY REQUIREMENTS ............................. 81 
11.1 
Data Reporting .......................................................................................................... 81 
11.1.1 
Method .......................................................................................................................... 81 
11.2 
Record Keeping and Record Retention ..................................................................... 81 
11.3 
Regulatory Documentation ........................................................................................ 82 
11.3.1 
Institutional Review Board ........................................................................................... 82 
11.3.2 
Audits and Inspections ................................................................................................. 82 
11.3.3 
Training ........................................................................................................................ 83 
11.3.4 
Monitoring the Study .................................................................................................... 83 
11.3.5 
Conditions for Modifying the Protocol ........................................................................ 83 
11.3.6 
Conditions for Terminating the Study .......................................................................... 84 
11.3.7 
Confidentiality of Trial Documents and Subject Records ............................................ 84 
11.3.8 
Publication of Data and Protection of Trade Secrets.................................................... 84 
REFERENCES ......................................................................................................................... 86 
APPENDIX A: PERFORMANCE STATUS CRITERIA .................................................... 88 
 
 
 


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TABLE OF TABLES 
Table 1 
Survival in children with brain tumors according to extent of tumor resection
 ................................................................................................................................... 23 
 
 
 
 
Table 4 
Clinical studies with tozuleristide .......................................................................... 27 
 
 
 
 
 
 
 
 
 
 
Table 9 
CTCAE Grading Scale ............................................................................................ 65 
 
TABLE OF FIGURES 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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ABSTRACT 
Title 
A randomized, blinded study of fluorescence detection of pediatric 
primary central nervous system tumors in subjects receiving 
tozuleristide and imaged with the Canvas System 
Subject population 
Subjects >1 month and ≤30 years of age at the time of study 
enrollment who have an MRI obtained within 30 days of study 
enrollment documenting a measurable lesion consistent with a 
pediatric primary CNS tumor for which maximal safe resection is 
indicated. 
Rationale for Study 
There is an urgent unmet need for a tumor-targeted imaging agent to 
enable real-time intraoperative distinction between malignant and 
normal tissue to improve neurosurgical outcomes,
 
 
Primary Objective 
The primary objectives of this study in subjects undergoing 
neurosurgical resection of pediatric primary central nervous system 
(CNS) tumors are: 
• To compare the sensitivity of tozuleristide and the Canvas near 
infrared (NIR) imaging system fluorescence to the sensitivity 
of surgical designation in equivocal tissue in situ; 
• To compare the specificity of tozuleristide and the Canvas 
system fluorescence to the specificity of surgical designation in 
equivocal tissue in situ; 
• To evaluate imaging efficacy as measured by sensitivity of 
tozuleristide and the Canvas system in facilitating 
intraoperative fluorescence detection and visualization of 
tumor in equivocal tissue in situ; 
• To evaluate imaging efficacy as measured by specificity of 
tozuleristide and the Canvas system in facilitating 
intraoperative fluorescence detection and visualization of 
tumor in equivocal tissue in situ. 
Secondary/Exploratory 
Objectives 
The secondary objectives of this study are: 
• To evaluate the safety of tozuleristide administration and 
Canvas NIR imaging system during surgical resection of 
pediatric primary CNS tumors 
• 
 
 
 
 
 
 
 
 


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The exploratory objectives of this study will evaluate the: 
• 
 
 
 
 
 
 
 
Study Design 
Randomized, blinded (central pathology, central fluorescence 
assessment and central radiology), multi-center study of a single dose 
of tozuleristide in subjects with pediatric primary CNS tumors; 
intraoperative imaging will be performed with the Canvas system.  
Two Arms (randomized 1:10): 
• Arm 1 (no drug treatment): ~9% of subjects   
Subject not injected with tozuleristide and undergoes standard 
of care neurosurgery  
• Arm 2 (tozuleristide treated): ~91% of subjects 
Subject injected with tozuleristide and undergoes standard of 
care neurosurgery  
During the surgical procedure, and once the tumor has been accessed, 
the Canvas system, which is attached to the surgical microscope, will 
be used to capture and display intraoperative in situ images (white 
light and fluorescence) of the region from which surgical specimens 
for biopsy or excision will be collected. All clinical decision-making 
regarding tissue resection will be made based on the routine 
neurosurgical practices, and tissue resection will take place under 
normal light conditions in line with best clinical practices.  
In addition to primary tumor biopsy, regions of equivocal nature 
(uncertain if tumor or not tumor) will be identified before fluorescence 
in the operating room is determined. Each clinical site will have an 
imaging operator who is not operating nor involved with patient 
surgical management. The imaging operator will ensure white light 
and fluorescent images are captured and will assess and document 
fluorescence status (none, weak, or strong) by looking at the Canvas 
monitor. The operating surgeon will assign presumed status of 
equivocal tissue (tumor/not tumor) and indicate the region to be 
biopsied and imaged by pointing at it with an instrument under the 
microscope.  
 
 


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Tozuleristide and the Canvas system are intended as an aid to surgical 
decision-making. As such, when a region of interest in the brain/spine 
is noted to be fluorescent, it is always the surgeon’s decision whether 
to biopsy/resect if safe to do so. 
A 1:10 randomization scheme will be used to help reduce selection 
bias, and aid in the integrity of the blinded pathology, 
 
 and radiology assessments.  Subjects will be randomly 
assigned to either Arm 1 (no tozuleristide treatment) or Arm 2 
(tozuleristide treated) after enrollment.
 
 
 
 
 
 
 
 
 
 
  
 
Fluorescence 
assessment by the imaging operators will be used in the primary 
efficacy analyses. 
Subjects who are randomized to Arm 1 or Arm 2 and need to undergo 
an additional surgery for their brain tumor will be eligible to receive 
tozuleristide without being subject to randomization. Subjects must 
meet all study eligibility requirements and re-consent for study prior to 
re-enrollment for open-label treatment with tozuleristide (re-
treatment). 
The pathology assessment of tissue as tumor or not tumor, for both 
arms, will be done by a centralized and independent panel of 
pathologists without knowledge of fluorescence status or treatment 
arm (blinded pathology). 
Post-operative centralized and independent MR image assessment 
 
, for both arms, will be done without knowledge of 
fluorescence status, treatment assignment, or surgical assessment 
(blinded radiology). 
 


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Number of Subjects 
Approximately 114 subjects are planned. 
Duration of Therapy 
For subjects in Arm 2, tozuleristide will be administered as a single 
intravenous (IV) bolus dose at least one hour and no more than 36 
hours before planned start time of surgery. 
Duration of Follow up Three months post-surgery  
Duration of study 
Approximately 52 months 
Study Drug(s) 
 
 
 
 
 
Safety Assessments 
All adverse events (AEs) and SAEs for up to three months after 
surgery will be recorded and graded for evaluable subjects according 
to the National Cancer Institute’s Common Terminology for Adverse 
Events (NCI CTCAE, Version 5.0) along with attribution of 
relatedness to tozuleristide and/or Canvas system and neurosurgery 
(related vs. unrelated). After the Day 8 visit, all subjects will be 
followed by chart review and/or telephone contact monthly for three 
months, or until an off-study criterion is met, to collect survival, 
toxicity assessment, and disease status information. 
Any subject receiving additional therapy for their CNS tumor, 
including chemotherapy and/or radiation therapy within three months 
of surgery will be considered off study for safety observations at the 
time of starting additional therapy. Safety data in these subjects will be 
included up to the time the subject begins additional tumor-directed 
therapy. Quality of life assessments will be collected in all subjects 
through three months post-surgery. 
Primary endpoints 
Primary efficacy endpoints are: 
1. Ratio of sensitivity of tozuleristide fluorescence in equivocal 
tissue biopsies from subjects with fluorescence-positive tumors 
compared to the sensitivity of surgical designation from the 
same biopsies under white light conditions 
2. Ratio of specificity of tozuleristide fluorescence in equivocal 
tissue biopsies from subjects with fluorescence-positive tumors 
compared to the specificity of surgical designation from the 
same biopsies under white light conditions 


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3. Sensitivity of tozuleristide fluorescence to detect tumor in 
equivocal tissue biopsies from subjects with fluorescence-
positive tumor 
4. Specificity of tozuleristide fluorescence to detect tumor in 
equivocal tissue biopsies from subjects with fluorescence-
positive tumor 
Tozuleristide fluorescence is determined by the imaging operator 
fluorescence assessment.
 
 
 
  
 
 
Secondary endpoints 
Secondary efficacy endpoints are: 
1.
 
 
2. 
 
 
 
 
 
 
  
Secondary endpoints related to the safety objective: 
1. All reported adverse events (AEs) will be graded according to 
the National Cancer Institute’s Common. Terminology for 
Adverse Events (NCI CTCAE, Version 5.0) 
2. SAEs 
3. AE attribution of relatedness to tozuleristide and/or Canvas 
system (related vs. unrelated). 


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4. Laboratory parameters 
5. ECG parameters 
6. Vital signs 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Other study endpoints 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sample size 
determination 
The sample size is based on the four co-primary endpoints for the 
tozuleristide arm (Arm 2). 
The sample size for the ratio of sensitivity of tozuleristide fluorescence 
in biopsies compared to the sensitivity of surgical designation from the 
same biopsies under white light conditions is based on the equivocal 
tissue biopsies from the subjects with fluorescence-positive tumor 
assessed for fluorescence and standard of care/white light conditions. 
 
 
 
  
  
 


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The sample size for sensitivity and specificity of tozuleristide 
fluorescence is based on the equivocal tissue biopsies from the subjects 
with fluorescence-positive tumor. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Unique Aspects of this 
Study 
This is a novel and rapidly emerging technology that may help 
surgeons visualize the extent of tumor or residual disease more 
precisely during surgery. 
 


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EXPERIMENTIAL DESIGN SCHEMA 
 
 
 
Figure 1 
 
 
 
 


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LIST OF ABBREVIATIONS 
oC 
degrees Celsius 
AE 
adverse event 
ALA 
5-aminolevulinic acid 
ALT 
alanine aminotransferase 
AUC 
area under the curve 
AUC0-inf 
AUC from zero to infinity 
AUC0-t 
AUC from zero to the last measurable time point 
BSA 
body surface area 
BUN 
blood urea nitrogen 
cGMP 
current good manufacturing principles 
CI 
confidence interval(s) 
CL 
total body clearance 
Cmax 
maximum observed concentration 
CNS 
central nervous system 
C0 
concentration back-extrapolated to time 0 
CRF 
case report form 
CSF 
cerebrospinal fluid 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTX 
Chlorotoxin 
FDA 
Food and Drug Administration 
FI 
fluorescence intensity 
FIH 
first in human 
FN 
false negative 
FP 
false positive 
FPFV 
first patient first visit 
GCP 
good clinical practice 
GEE 
general estimating equation 
GFAP 
glial fibrillary acidic protein 
GLP 
good laboratory practice(s) 
GTR 
gross total resection 
H&E 
hematoxylin and eosin 
HDD 
hard-disk drive 


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hERG 
human ether-a-go-go-related gene 
HIV 
human immunodeficiency virus 
Hr 
Hour 
ICG 
indocyanine green 
ICH 
International Conference on Harmonization 
IHC 
Immunohistochemistry 
IND 
investigational new drug application 
IRB 
institutional review board 
ISF 
investigator site file 
IV 
Intravenous 
LC/MS 
liquid chromatography / mass spectrometry 
LLOQ 
lower limit of quantitation 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRI 
magnetic resonance imaging 
NCI 
National Cancer Institute 
NHP 
nonhuman primate 
NIR 
near infrared 
NOAEL 
no-observed-adverse-effect-level 
NPV 
negative predictive value 
NTR 
near total resection 
OS 
overall survival 
PEAC 
Protocol Endpoint Assessment Committee 
PFS 
progression-free survival 
PK 
pharmacokinetic(s) 
PPV 
positive predictive value 
ROI 
region of interest 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SD 
standard deviation 
SIRIS 
Synchronized Infrared Imaging System 
SMC 
Safety Monitoring Committee 
SOC 
standard of care 
SOP 
standard operating procedure 


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STR 
subtotal resection 
T 
Time 
TK 
Toxicokinetic 
Tmax 
time of maximum observed concentration 
TN 
true negative 
TP 
true positive 
t1/2 
terminal half-life 
ULN 
upper limit of normal 
V 
Volume 
Vss 
apparent volume of distribution at steady-state 
WHO 
World Health Organization 
 


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1 
OBJECTIVES 
1.1 
Primary Objectives 
The primary objectives of this study in subjects undergoing neurosurgical resection of pediatric 
primary CNS tumors are: 
• To compare the sensitivity of tozuleristide and the Canvas near infrared (NIR) imaging 
system fluorescence to the sensitivity of surgical designation in equivocal tissue in situ 
• To compare the specificity of tozuleristide and the Canvas system fluorescence to the 
specificity of surgical designation in equivocal tissue in situ 
• To evaluate imaging efficacy as measured by sensitivity of tozuleristide and the Canvas 
system in facilitating intraoperative fluorescence detection and visualization of tumor in 
equivocal tissue in situ 
• To evaluate imaging efficacy as measured by specificity of tozuleristide and the Canvas 
system in facilitating intraoperative fluorescence detection and visualization of tumor in 
equivocal tissue in situ. 
1.2 
Secondary Objectives 
The secondary objectives of this study are: 
• 
 
 
 
 
 
 
 
 
 
1.3 
Exploratory Objectives 
The exploratory objectives of this study will evaluate the: 
•
 
 
 
 
 
 
 
 


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2 
BACKGROUND 
2.1 
Study Disease 
Brain tumors are the leading cause of cancer death in children. Neurosurgical resection of gross 
disease remains an essential component of care and cure for the majority of pediatric brain 
tumors, including high-grade glioma (Yang 2013), ependymoma (Pollack 1995, Rogers 2005), 
medulloblastoma (Zeltzer 1999), supratentorial primitive neuroectodermal tumor (Jakacki 2015) 
and atypical teratoid/rhabdoid tumor (Chi 2009) as detailed in Table 1 below. Low-grade 
gliomas may be cured with surgery alone (Fisher 2008). Survival of patients with both low- and 
high-grade gliomas is enhanced with maximal tumor resection (Hervey-Jumper 2015). 
 


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Table 1 
Survival in children with brain tumors according to extent of tumor resection 
 
Tumor histology 
Survival 
measure-
ment time 
Survival for 
children with 
complete 
resection 
Survival for 
children 
with 
residual 
disease 
P value 
Estimated 
GTR rate 
Reference 
Glioblastoma 
5-year OS 
42% (n=17) 
0% (n=20) 
NA 
~45% 
Yang, 2013 
Ependymoma 
5-year OS 
80% (n=23) 
22% (n=14) 
< 0.0001 
~60% 
Pollack, 
1995 
Supratentorial 
primitive 
neuroectodermal 
tumor 
5-year OS 
(stage M0 
tumor) 
59% (n=17) 
10% (n=12) 
   0.017 
~60% 
Jakacki, 
2015 
Medulloblastoma 
5-year PFS 
(stage M0 
tumor and 
≥3 years 
age) 
78% (n=61, 
includes 
patients with 
<1.5cm2 
residual tumor) 
54% (n=22, 
includes 
patients 
with 
≥1.5cm2 
residual 
tumor) 
   0.023 
~75% 
(NTR+GTR) 
Zeltzer, 
1999 
Atypical 
teratoid/rhabdoid 
tumor 
2-year OS 
91% (n=11) 
NA 
   0.004 
~55% 
Chi, 2009 
Low grade glioma 
5-year PFS 
90% (n=52) 
36-48% 
(n=213) 
<0.001 
~20% 
Fisher, 2008 
OS: overall survival; PFS: progression-free survival; GTR: gross total resection; M0: no evidence of metastasis 
Even the most experienced neurosurgeons are challenged by the difficulty of distinguishing 
cancer tissue from healthy brain. 5-aminolevulinic acid (ALA) is an intraoperative imaging agent 
that was developed and approved for clinical use in Europe for adults with glioblastoma. In a 
randomized controlled trial of adults with glioblastoma, ALA was significantly more likely to 
result in gross total resection, with 65% in the ALA group achieving gross total resection 
compared to 36% in the control group (p<0.05). In addition, the ALA group achieved a 
significantly (p<0.05) greater 6-month survival of 41% compared to 21% in the control arm 
(Stummer 2006). The study of ALA in adults with glioblastoma demonstrates that intraoperative 
imaging may have a greater impact on patient survival (20% difference) than any medical 
therapy developed to date, including temozolomide which resulted in 15% improvement in 
survival in the initial landmark study leading to United States Food and Drug Administration 
(FDA) approval (Stupp 2005). A recent systematic review and meta-analysis concluded that the 
intraoperative imaging agent ALA was superior to neuronavigation for adults with high grade 
glioma, resulting in improved extent of resection, survival, and quality of life (Zhao 2013). 
However, ALA is not likely to be applicable to pediatric use. Several reports of off-label use of 
ALA in children in Europe have decreased enthusiasm for developing this particular agent for 
pediatric use, most significant of which is the limited or lack of binding to the most common 
pediatric histologic types of brain tumors, low-grade lesions and embryonal tumors such as 
medulloblastoma (Barbagallo 2014, Beez 2014). Furthermore, ALA does not bind to brain tumor 
cells, but instead localizes based on blood brain barrier disruption, leading to potential false-
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Tozuleristide (also known as BLZ-100) is a targeted fluorescent molecule designed by Blaze 
Bioscience to illuminate cancer foci to facilitate surgical resection. The peptide component of 
tozuleristide 
 
 
 
 
 
 
The fluorophore component of tozuleristide is a conjugatable form of indocyanine green (ICG). 
ICG is a cyanine dye that has been approved by the FDA and used as an in vivo fluorescent 
imaging agent since the 1950s. Following excitation, it has an emission wavelength higher than 
800 nm which is minimally absorbed by water or hemoglobin, making it well-suited for 
intraoperative imaging when used with an appropriate detection device. ICG is frequently 
employed in vascular and ophthalmology surgery as well as for sentinel lymph node mapping. 
Visualization of the near infrared (NIR) fluorescence from ICG is accomplished using an 
imaging system, typically composed of a light source for excitation, a camera to detect NIR 
fluorescence and a monitor to display the images from the camera. Commercially available NIR 
imaging systems for ICG detection, such as the Fluobeam® 800 (Fluoptics) imaging system or 
the Artemis Handheld Imaging System/Spectrum (Quest Medical Imaging), 
 
 
 
 
 
 
 
Preclinical data show that chlorotoxin-based dye conjugates bind specifically to a range of 
human and animal tumors and provide greatly improved image resolution compared with pre-
operative magnetic resonance imaging (MRI) or direct intraoperative observation, and greater 
imaging time window flexibility. Thus, they may enable surgeons to visualize and remove tumor 
cells that would otherwise have remained undetected and more precisely view tumor margins, 
resulting in more complete surgical resection. 
2.2 
Study Agent and Device 
Agent: Tozuleristide (also known as BLZ-100) supplied by Blaze Bioscience 
Investigational Agent  
IND# 
IND Sponsor  
Tozuleristide 
IND: 
 Blaze Bioscience 
 
Investigational Device  
IDE# (see 
note below) 
IDE Sponsor  
Canvas NIR imaging system 
IND: 
 
Blaze Bioscience 


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For more information on tozuleristide, please refer to the current Investigator’s Brochure(s): 
Tozuleristide Investigator’s Brochure. 
For Device information, please refer to the current Canvas Imaging System Investigator’s 
Brochure. 
2.3 
Nonclinical Studies 
All nonclinical studies are described and summarized in the Tozuleristide Investigator’s 
Brochure. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.3.1 
Safety summary: Tozuleristide 
 
 
 
For the most current safety information, refer to the Tozuleristide Investigator’s Brochure.  
 
 
 
 
 
 
Overall, the clinical studies in adults and pediatrics have shown that tozuleristide appears to be 
safe and well-tolerated. 


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2.3.1.1 
Serious Adverse Events 
To date, there have been no tozuleristide related serious adverse events reported. 


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2.4 
Clinical Studies 
2.4.1 
Overview of Clinical Studies 
Table 4 provides an overview of the Phase 1 clinical studies with tozuleristide. 
Table 4 
Clinical studies with tozuleristide 
Study No. 
Phase / Status 
Design  
Objectives 
Population 
Dosage of Tozuleristide 
and Frequency 
 
BB-001  
Phase 1 / 
Complete 
Open-label, FIH, dose-
escalation/expansion study 
Primary objectives: safety and 
tolerability 
Secondary objectives: PK, dose level 
for Phase 2 studies 
Adult subjects with 
skin cancer 
undergoing 
surgical excision of 
their tumor(s) 
21 subjects treated 
1 – 18 mg IV, single 
15 minute infusion 
48 hours prior to surgery 
 
BB-002  
Phase 1 / 
Complete 
Open-label, dose-escalation study 
evaluating doses up to 30 mg 
Primary objectives: safety and 
tolerability 
Secondary objectives: PK, fluorescent 
signal from excised brain tumor tissue, 
dose level for Phase 2 studies 
Adult subjects with 
glioma undergoing 
surgical excision of 
their tumor(s) 
17 subjects treated 
3 – 30 mg IV, single 
bolus administration at 
least two hours prior to 
surgery 
 
BB-004 
Phase 1 /  
Complete 
Open-label, dose-escalation and 
expansion study  
Primary objectives: safety and 
tolerability, PK, recommended Phase 2 
dose  
Pediatric subjects 
with primary CNS 
tumors 
 
 
 
 
BB-005 
Phase 1 / 
Complete 
Open-label, exploratory study 
Primary objective: safety and tolerability  
Secondary objectives: fluorescent signal 
intensity from excised tumor tissue, PK 
in subjects with breast cancer. 
Adult subjects with 
solid tumors 
(breast, lung, 
prostate, 
colorectal, and 
“other”) 
undergoing surgery 
30 subjects treated 
 
 
12 mg or 6 mg IV, single 
bolus administration at 
least two hours prior to 
surgery 
CNS = central nervous system; FIH = first in human; IV = intravenous; PK = pharmacokinetic 
 
 
 
The above clinical studies are presented in detail in Tozuleristide Investigator’s Brochure. 
In the clinical studies to date, visualization of tozuleristide has been performed using more than 
one imaging system, 
 
 
 
 
 
 
 


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2.4.2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 


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2.4.3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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2
1 
Q
nt 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.4.3.2 
 
 
 


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2.5 
Rationale 
2.5.1 
Study Rationale 
Brain tumors are the most common solid tumor type within the pediatric population, with about 
2,500 new cases of malignant brain tumors per year (ages 0-19) in the US (Gajjar 2013). 
Furthermore, pediatric central nervous system cancer is the leading cause of cancer death in 
children (U.S. Cancer Statistics Working Group). Surgery is first-line treatment for eligible 
pediatric brain tumor patients (Sievert 2009, Robertson 2006). Surgical resection significantly 
affects chance of cure in the majority of pediatric CNS tumor types, including medulloblastoma, 
atypical teratoid/rhabdoid tumor, ependymoma, high-grade glioma, and low-grade glioma 
(Fisher 2008). However, surgical damage to healthy brain can lead to devastating long-term side 
effects. 
Current detection techniques such as intraoperative MRI, ultrasound, positron emission 
tomography scanning, and magnetoencephalography are not precise enough and do not have 


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high surgical usefulness as these techniques are often not feasible during the time of surgery 
without significant disruption to the operative flow (Huang 2013). 
While primary tumor may be readily recognized by an experienced neurosurgeon using visual 
and tactile stimuli, there are frequently areas of equivocal tissue identified during the course of a 
prolonged tumor resection where the goal is maximal safe surgical resection. Equivocal tissue 
may be present for a number of reasons including tumor invasion into normal brain, retraction 
artifact, gliosis, local tissue reaction to the presence of tumor, or scar tissue from previous 
procedures. Equivocal tissue may represent marginal or ambiguous areas where the addition of 
neurosurgical imaging agent such as tozuleristide may provide the most utility. For this reason, 
this study is designed to evaluate efficacy in these equivocal areas. 
There are few other studies in pediatric neuro-oncology that have attempted to evaluate and 
quantitate routine neurosurgical experience with areas of equivocal tissue. In this study we will 
therefore focus the primary efficacy analysis on an assessment of tissue fluorescence in the 
operating room (local assessment) and in addition capture and compare to the routine 
neurosurgical assessment of equivocal tissue planned for resection.   
There is an urgent unmet need for a tumor-targeted imaging agent to enable real-time 
intraoperative distinction between tumor and non-tumor tissue to improve neurosurgical 
outcomes, either as increased chance of a complete total resection or decreased risk of 
inadvertent normal brain resection or both. 
2.5.2 
Dose Rationale 
Safety, pharmacokinetic, and imaging data from the Phase 1 clinical studies support a near-
optimal tozuleristide dose for pediatric central nervous system tumors of ~15 mg/m2 
administered at least 1 hour before the start of surgery. 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.5.3 
Device Rationale 
The NIR fluorescence signal from tozuleristide is not visible to the human eye. As such, an 
imaging device is needed to capture and display fluorescence. Additionally, while currently 
marketed surgical microscopes, 
 have features capable of detecting 


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ICG, they are not sensitive enough to detect fluorescence from tozuleristide. 
 
 
  
 
  
The Canvas system, an investigational device 
, will be used for intraoperative 
imaging in this study to allow the surgeon the ability to use a fluorescence detection camera that 
is mounted to the optical head of the surgical microscope. No alterations in ambient light 
conditions are needed for the Canvas system to capture NIR images. 
The Canvas system is designed to detect the NIR fluorescence of tozuleristide in the 
intraoperative setting of neurosurgery. 
 
 
 
 
  
 
 
 
 
 
2.5.4 
 
2.5.4.1 
 
2.5.4.2 
R
n 
 
 
 
 
 
 
2.5.4.3 
 
2.5.4.4 
 
 


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2.5.4.5 
 
 
 
 
 
 
 
 
2.5.4.6 
R
s 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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2.6 
 
 
• 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  


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3 
STUDY DESIGN 
3.1 
Characteristics 
This is a randomized, blinded (central pathology, central fluorescence assessment, and central 
radiology), multi-center Phase 2 study of a single dose of tozuleristide in subjects with pediatric 
primary CNS tumors; intraoperative imaging will be performed with the Canvas system. 
There will be 2 arms (randomized 1:10): 
• Arm 1 (no drug treatment): ~9% of subjects 
Subject not injected with tozuleristide and undergoes standard of care neurosurgery 
• Arm 2 (tozuleristide treated): ~91% of subjects 
Subject injected with tozuleristide and undergoes standard of care neurosurgery 
 
In addition, re-treatment with tozuleristide after the initial neurosurgery may be allowed if 
additional neurosurgery is needed (Section 4.1.3). Data collected after re-treatment will 
constitute the re-treatment arm.  
3.2 
Number of Subjects 
Approximately 114 subjects are planned. 
3.3 
Inclusion Criteria 
Subjects must meet all of the following criteria to be included in this study: 
1. Subjects must be >1 month and ≤30 years of age at the time of study enrollment 
2. Subjects must have MRI obtained within 30 days of study enrollment documenting a 
measurable lesion consistent with a pediatric primary CNS tumor for which maximal safe 
resection is indicated 
3. Adequate renal function defined as: 
a. Serum creatinine within normal limits for age or calculated creatinine clearance 
≥75 mL/min/1.73 m2 based on Schwartz equation or normal creatinine for age 
based on the following table: 
Age 
Maximum Serum 
Creatinine (mg/dL) 
 
Male 
Female 
1 to < 2 years 
0.6 
0.6 
2 to < 6 years 
0.8 
0.8 
6 to < 10 years 
1 
1 
10 to < 13 years 
1.2 
1.2 
13 to < 16 years 
1.5 
1.4 
≥ 16 years 
1.7 
1.4 
 
4. Adequate liver function defined as: 
a. Bilirubin <2 x upper limit of normal 
b. Alanine aminotransferase (ALT) < 3 x upper limit of normal 
c. Serum albumin >2 g/dL 


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5. Prior therapy: Subjects with prior therapy are eligible provided they have recovered from 
any acute toxic effects of prior therapy and have sufficient time interval prior to 
enrollment: 
a. Radiation therapy: subjects may not have had radiation therapy to the area of 
tumor planned to be resected within 28 days of study enrollment 
b. Chemotherapy: at least 14 days from any myelosuppressive chemotherapy 
(28 days if prior nitrosourea) and if prior chemotherapy, must have an absolute 
neutrophil count recovery of ≥ 1000/mm3 following count nadir 
c. Biologic: at least seven days from any anti-neoplastic biologic agent (at least 
three half-lives since last administration of monoclonal antibodies) 
d. Immunotherapy: at least 42 days after completion of any cellular immunotherapy, 
such as CAR-T cell therapy 
e. Prior surgery for CNS tumors is allowed 
f. Prior tozuleristide: at least one week after prior dose of tozuleristide if previously 
treated. NOTE: Subjects previously enrolled on PNOC012 study and randomized 
to either Arm 1 (control) or Arm 2 (treatment) will be eligible for re-enrollment 
for open-label treatment of tozuleristide (re-treatment) in the event another 
surger(ies) is clinically indicated (Section 4.1.3).  
 
6. Written informed consent must be obtained from the subject or parent or legal guardian 
prior to the conduct of study activities. Routine clinical tests, e.g., MRI, clinical 
laboratory studies, may be used for screening requirements. Assent, when appropriate, 
will be obtained according to institutional guidelines. 
7. The risks of treatment with tozuleristide during pregnancy have not been evaluated. 
Female subjects of child-bearing potential must agree not to attempt to become pregnant 
or undergo in vitro fertilization and, if participating in sexual activity that could lead to 
pregnancy, must use two reliable methods of contraception simultaneously for 30 days 
after surgery. Male subjects must agree not to attempt to father a child and, if 
participating in sexual activity that could lead to pregnancy, must use two reliable 
methods of contraception simultaneously for 30 days after surgery if their partner is of 
child-bearing potential. (Refer to Section 6.2.2 for reliable methods of contraception). 
3.4 
Exclusion Criteria 
Subjects meeting any of the following criteria will be considered ineligible for this study: 
1. Pregnancy and contraception: Subjects who are pregnant or breastfeeding or planning to 
conceive a child within 30 days are not eligible. Males and females of childbearing 
potential must agree to use two effective forms of contraception from the time of 
enrollment until 30 days post-surgery. 
2. Subjects with on-going serious medical conditions (poorly controlled asthma, diabetes, 
heart disease) such that participation in the study could put the subject at increased risk of 
worsening their condition. 
3. Subjects planned to undergo only a diagnostic biopsy procedure, without intent to resect 
tissue for therapeutic purposes (e.g., stereotactic pontine biopsy). 
4. Subjects who in the opinion of the investigator are not willing or able to comply with 
randomization procedures or other study-required study procedures and observations. 


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Subjects previously enrolled and randomized to Arm 1 (control) are not eligible for re-
enrollment unless a second surgery is required by standard of care. 
Important note: The eligibility criteria listed above are interpreted literally and cannot be 
waived. 


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4 
REGISTRATION PROCEDURES 
4.1 
General Guidelines 
Subjects must meet all inclusion criteria and no exclusion criteria should apply. The subject must 
have signed and dated an approved, current version of all applicable consent forms.  To allow 
non-English speaking subjects to participate in this study, bilingual health services will be 
provided in the appropriate language when feasible. 
Please see Study Manual for details of enrollment procedures in the Electronic System. 
Enrollment checklist and source documentation must be provided for Study Chair and/or 
 
Project Leader review and confirmation of eligibility prior to randomization.  
4.1.1 
Treatment Allocation 
After enrollment,
, subjects will be 
randomized 1:10 to Arm 1 (no drug treatment) or Arm 2 (tozuleristide treatment). 
Arm 1: Approximately ten subjects (~9% of subjects) will not receive tozuleristide and will 
undergo standard of care neurosurgery. 
Arm 2: Approximately 104 subjects (~91% of subjects) will receive tozuleristide at a dose of 
15 mg/m2 administered as a single IV bolus dose at least one hour and no more than 36 hours 
before the planned start time of surgery. 
Re-treatment arm: See Section 4.1.3 below regarding re-treatment. 
4.1.2 
Blinding 
This is a randomized, blinded (central pathology, central fluorescence assessment, and central 
radiology) study. A 1:10 randomization scheme will allocate ~9% of subjects to a no study drug 
standard of care neurosurgery arm. This will help to reduce subject selection bias and 
expectation of fluorescence in the operating room.
 
 
The pathology assessment of tissue as tumor or not tumor, for both arms, will be done by a 
centralized and independent panel of two pathologists without knowledge of fluorescence status, 
surgeon’s designation of tissue status or treatment arm (blinded pathology). 
 
 
 
Post-operative centralized MR image assessment 
, for both arms, will be 
performed by a central and independent radiologist without knowledge of fluorescence status, 
treatment assignment or surgical assessment (blinded radiology). 
4.1.3 
Re-treatment 
Subjects who are randomized to Arm 1 (no tozuleristide) or Arm 2 and need to undergo 
additional surgeries for their brain tumor will be eligible to receive tozuleristide without being 
subject to randomization. Subjects must meet all study eligibility requirements and re-consent for 
study prior to re-enrollment.  


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5 
AGENT ADMINISTRATION 
5.1 
Investigational Product 
 
 
 
 
 
ozuleristide will be administered by an IV 
bolus injection at least one hour (and no more than 36 hours) before the subject’s planned 
surgical tumor excision.  
   
 
  
Tozuleristide will be given at an IV bolus dose of 15 mg/m2 over approximately 1-5 minutes. 
5.2 
Imaging Device 
 
 
 
 
 
 
 
 
 
 
 
 
 
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5.2.1 
Canvas System Data 
For every imaging procedure, the Canvas system collects a variety of data.
 


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5.3 
Regimen Description 
Arm 1: Approximately ten subjects.  
Subjects randomized to standard of care neurosurgery arm (Arm 1) will not receive tozuleristide. 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
All clinical decision-making regarding tissue resection should be made based on the routine 
neurosurgical practices, and tissue resection will take place under normal light conditions in line 
with best clinical practices.  
 
Arm 2: Approximately 104 subjects. 
Tozuleristide will be administered as a single IV bolus dose at least one hour and no more than 
36 hours before the planned start time of surgery. Tozuleristide will be given at a dose of 15 
mg/m2. 
 
 
 
 
 
 


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All clinical decision-making regarding tissue resection should be made based on the routine 
neurosurgical practices, and tissue resection will take place under normal light conditions in line 
with best clinical practices. 
Tozuleristide and the Canvas system are intended as an aid to surgical decision-making. As such, 
when a region of interest is noted to be fluorescent, it is always the surgeon’s decision whether to 
biopsy/resect if safe to do so.   
5.4 
Phase 0/1 Dose Escalation Scheme 
Not applicable. 
5.5 
Phase 0/1 Definition of Dose-Limiting Toxicity 
Not applicable. 
5.6 
General Concomitant Medication and Supportive Care Guidelines 
Per routine preoperative and postoperative practices, subjects on study may receive steroids, 
anti-emetics, anti-epileptics, antibiotics or any other medications that are clinically indicated. 
Administration of tozuleristide is not anticipated to interfere with the standard of care for 
subjects on this trial. 
At each study visit or contact, the investigator should question the subject or their legal 
representative about any medication taken, including vitamin supplements and herbal remedies. 
Any concurrent medications from Screening (-14 days prior to surgery) until Day 8 will be 
recorded in the subject’s records and the case report form (CRF). Any changes in doses or 
introduction of new medications during this period will also be recorded. Any medications 
necessary for treatment of a SAE up to three months post-surgery should be recorded in the 
subject’s CRF. 
5.6.1 
Special Dietary Requirements 
There are no special dietary requirements. 
5.6.2 
Concurrent Medications / Treatments Not Permitted 
5.6.2.1 
Prior to Study Entry 
None 
5.6.2.2 
On Study 
• Concurrent Anticancer Therapy 
Subjects may begin other appropriate anti-cancer therapy including chemotherapy and/or 
radiation therapy, but they will be considered off study for safety endpoints on the day 
when further tumor-directed therapy is initiated. 
• Supportive Care 
No specific supportive care is recommended related to tozuleristide. Subjects should 
continue to receive all routine preoperative and postoperative supportive care, including 


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steroids, blood products, fluids, and prophylaxis or treatment of pain, nausea, vomiting, 
seizures, or infection. Allergic reaction may occur with any medication and should be 
managed per institutional guidelines. 
• Pregnancy 
If a subject on study is found to be pregnant or the partner of a male subject becomes 
pregnant within 30 days of study drug administration, then the possible prenatal exposure 
to study drug should be promptly reported. 
5.7 
Dosing Modifications and Delays 
No dosing modifications are expected. 
5.7.1 
Criteria for dose-modification in case of suspected tozuleristide toxicity and re-
initiation of treatment 
Since tozuleristide is given as a single IV bolus; no dose modifications are expected. 
 


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6 
TREATMENT PLAN 
6.1 
Study Calendar 
. 


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6.2 
Observations and Procedures 
6.2.1 
Informed Consent 
• Signed informed consent: Protocol and consent documents must be approved by the IRB 
prior to subject enrollment. The investigational nature and objectives of the trial, 
procedures involved, potential risks and discomforts, and alternative options will be 
carefully explained to the subject and/or the subject’s parents or guardian, and a signed 
informed consent and assent, if appropriate, will be obtained according to institutional 
guidelines. Informed consent must be obtained prior to obtaining any study specific 
observations that are outside of standard clinical care in order to determine study 
eligibility. 
6.2.2 
Screening Period (Day -14 to day of surgery) 
• Eligibility criteria: Before the subject is enrolled, a site investigator must sign and date a 
completed eligibility checklist verifying review and determination that the subject meets 
all eligibility requirements and does not meet any exclusion criteria. These criteria and 
supporting information will be reviewed by the study chair or back-up and confirmed by 
the 
 lead or back-up prior to randomization. 
Eligibility criteria (inclusion and exclusion criteria) cannot be waived. Clinical and 
laboratory data to determine eligibility must be available in the subject’s medical or 
research record which will serve as source documentation for verification at the time of 
audit. 
In order to assess any potential impact on subject eligibility with regard to safety, the 
investigator must refer to the relevant document(s) for detailed information regarding 
warnings, precautions, contraindications, AEs, and other significant data pertaining to the 


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tozuleristide study drug product and the Canvas system being used in this study. Such 
documents may include, but are not limited to, the Investigator’s Brochure or equivalent 
document(s) provided by Blaze Bioscience. 
Subjects will be eligible for enrollment once all screening tests and procedures are 
completed and results indicate that all eligibility criteria have been met. Once approved, 
subjects will be randomized 1:10 to Arm 1 (no drug treatment, standard of care 
neurosurgery) or Arm 2 (tozuleristide treatment). Subjects on re-treatment arm will be 
assigned a new subject number and not be subject to randomization, but still must go 
through the study eligibility and approval process. 
• Demographics 
• Medical history 
• Medication history 
• Body weight 
• Height 
• Performance status: See Appendix A 
• Physical examination 
• Concurrent medications 
• Measurement of clinical chemistry parameters: Sodium, Potassium, Chloride, CO2, blood 
urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), bilirubin, albumin. 
• Measurement of hematology parameters: Complete blood count (red blood cells, white 
blood cells with differential, platelets), hemoglobin and hematocrit. 
• Measurement of coagulation parameters: PT/INR and PTT. 
• Pregnancy test (for females of childbearing potential): All women of child bearing 
potential (defined as sexually mature female who has had menses within the preceding 24 
months and has not undergone hysterectomy, bilateral oophorectomy, or tubal ligation) 
must have a negative pregnancy test (urine or serum test with a sensitivity of at least 50 
IU/mL) performed at Screening/ Baseline. 
Women of childbearing potential must agree not to attempt to become pregnant or 
undergo in vitro fertilization and, if participating in sexual activity that could lead to 
pregnancy, must use two reliable methods of contraception simultaneously for 30 days 
after surgery. Male subjects must agree to use two reliable methods of contraception 
simultaneously for 30 days after surgery if their partner is of child-bearing potential. 
A combination of two of the following methods must be used: 
o Condoms (male or female) with spermicide 
o Diaphragm or cervical cap  
o Intrauterine Device 
o Hormonal-based contraception 
o Vasectomy 
 
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required to use contraception. 
 
• MRI of tumor: MRI with and without contrast prior to treatment should be obtained 
within 30 days of enrollment. MRI sequences should include T1 pre-contrast, T1 post-
contrast, T2, FLAIR, and Diffusion weighted images.
 
For recommended 
imaging protocol and sequence parameters, please see Appendix F.
 
 
 All MRI processes for central radiology will be 
detailed in the study reference manual.  
 
• 
 
6.2.3 
1 to 36 Hours Pre-Surgery 
• ECG (Arm 2 and re-treatment arm only):  On the day of tozuleristide dosing, a baseline 
ECG will be obtained prior to dosing (within 24 hours), and an additional ECG will be 
obtained 1-30 minutes post-end of dose.  ECG recordings will be performed once the 
subject should have been resting semi-supine for at least ten minutes and will be 
measured in triplicate over approximately 3 minutes (with wait time of approximately 
one minute between recordings). A 12-lead ECG recording will be performed in a 
standardized manner as outlined in the Study Reference Manual. Repeat measurements 
will be performed if any clinical abnormalities are observed or if artifacts are present.  
• Vital signs obtained pre-surgery (Arm 1) or pre-dose and 1-30 minutes post-end of dose 
(Arm 2 and re-treatment arm). 
• Concurrent medications: continuous assessment 
• Adverse events: continuous assessment 
• Study drug administration (Arm 2 and re-treatment arm only): Tozuleristide is to be 
administered at least 1 hour (and no more than 36 hours) before the planned start of the 
surgical excision of the tumor. 
• 
 
 
 
 
 
6.2.4 
Day 1 (Day of Surgery) 
• Imaging and pathology during surgery (for all subjects) 
• 
 
o Surgery will be conducted according to standard of care procedures, including for 
tumor approach and resection
 
All clinical decision making 
should be made based on routine neurosurgical practices and tissue resection will 


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take place under normal light conditions in line with best clinical practice 
o 
 
 
o 
 
 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1. 
 
 
 
 
 
 
 
 
  
 
 
  
 
 
 


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7.
 
 
 
 
 
 
o 
 
 
 
 
 
 
 
 
 
• Concurrent medications: continuous assessment 
• Adverse events: continuous assessment 


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• MRI of tumor: MRI with and without contrast should be obtained within 72 hours of 
surgery including the site of primary tumor. MRI sequences should include T1 pre-contrast, 
T1 post-contrast, T2, FLAIR, and Diffusion weighted images. For recommended imaging 
protocol and sequence parameters, please see Appendix F. Images will be evaluated by site 
radiology for patient management and local assessment 
. Images will be made 
available for central imaging review 
 
 All MRI processes for central radiology will be detailed in the 
study reference manual. 
• 
 
6.2.5 
Day 8 (7 Days Post-Surgery ± 3 days) 
• Performance status 
• Physical examination 
• ECG (Arm 2 and re-treatment arm only): An ECG will be obtained at seven days post-
surgery.  ECG recordings will be performed once the subject has been resting semi-
supine for at least ten minutes and will be measured in triplicate over approximately three 
minutes (with wait time of approximately 1 minute between recordings). A 12-lead ECG 
recording will be performed in a standardized manner as outlined in the Study Reference 
Manual. Repeat measurements will be performed if any clinical abnormalities are 
observed or if artifacts are present.  
• Vital signs 
• Concurrent medications: continuous assessment 
• Adverse events: continuous assessment 
• Measurement of clinical chemistry parameters (Arm 2 and re-treatment arm only) 
• Measurement of hematology parameters (Arm 2 and re-treatment arm only) 
 
 
6.2.6 
30 Days Post-Surgery (±7 days) 
• Adverse events 
o Chart review and/or telephone contact if no patient visit is documented to collect 
survival, 
, brain and spine MRIs, toxicity 
assessment and disease status information. 
• Urine or serum pregnancy test (for females of childbearing potential) 
6.2.7 
2 Months Post-Surgery (±7 days) and 3 Months Post-Surgery (±7 days) 
• Adverse events 
o Monthly chart review and/or telephone contact if no patient visit is documented to 
collect survival, 
, brain and spine MRIs, 
toxicity assessment and disease status information. Any subject receiving 
additional therapy for their CNS tumor, including chemotherapy and/or radiation 
therapy within 3 months of surgery will be considered off study for safety 


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observations at the time of starting additional therapy.  Safety data in these 
subjects will be included up to the time the subject begins additional tumor-
directed therapy. 
• 
 
 
 
 
6.3 
Off-Treatment Criteria 
A subject may be removed from protocol therapy after enrollment but prior to receiving study drug 
for any of the following reasons: 
• Withdrawal of consent for treatment 
• Subject is unable to receive study drug for any reason 
• Pregnancy 
The study may be terminated at any point in time at the discretion of the sponsor (See Section 
11.3.6). 
6.4 
Off Study Criteria 
Subjects will be considered Off Study for the following reasons: 
• Physician determines that study participation is not in subject’s best interest. 
• Study is terminated by the Sponsor. 
• Parent, subject, or guardian withdraws consent or assent for continued participation. 
• Subject death. 
• Completion of protocol specific follow up period, three months following surgery 
• Any subject receiving additional therapy for their CNS tumor, including chemotherapy 
and/or radiation therapy within three months of surgery will be considered off study for 
safety observations at the time of starting additional therapy.  Safety data in these subjects 
will be included up to the time the subject begins additional tumor-directed therapy. Any 
on-going adverse events (AEs) at the time of off-study will be considered unresolved. 
 
The date and reason for the subject coming off study must be documented in the case report form.  
Subjects coming off study due to start of additional therapy are considered to have completed study 
as planned.  
 
 
 


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7 
ADVERSE EVENTS 
An AE or adverse experience is any untoward medical occurrence in a subject who is 
administered an investigational product (whether it is the experimental product or the control) 
and which does not necessarily have a causal relationship with the investigational product. An 
AE can therefore be any unfavorable and unintended sign, symptom, or disease temporally 
associated with the use of a medicinal product, whether or not considered related to the 
medicinal product. Pre-existing events that increase in frequency or severity or change in nature 
during or as a consequence of use of a drug in human clinical trials, will also be considered as 
AEs. AEs may also include pre- or post-treatment complications that occur as a result of 
protocol-mandated procedures (e.g., invasive procedures such as biopsies). 
For investigational medical devices, adverse events are any untoward medical occurrence, 
unintended disease or injury, or untoward clinical signs (including an abnormal laboratory 
finding) in subjects, users or other persons, whether or not related to the investigational medical 
device.  An adverse device effect is an adverse event related to the use of the investigational 
medical device.  It can include adverse events from insufficient or inadequate instructions for 
use, deployment, implantation, installation or operation, or any malfunction of the investigational 
device. 
Any medical condition or laboratory abnormality with an onset date before randomization is 
considered pre-existing and should be recorded on the medical history CRF page(s).  
AEs will be collected from time of randomization through three months after surgery or until 
start of other tumor-directed therapy, whichever is earlier. After the Day 8 visit, all subjects will 
be followed by chart review and/or telephone contact if no patient visit is documented every 
month for three months, or until start of other tumor-directed therapy (chemotherapy, radiation 
therapy or other investigational therapy), whichever is sooner.  
If the patient experiences an AE considered to be related to study products, then the patient will 
be followed until resolution of that AE or until they are off study (whichever is earlier).  
An AE does not include: 
• Medical or surgical procedures (e.g., surgery, endoscopy, tooth extraction, transfusion); 
the condition that leads to the procedure is an AE 
• Pre-existing diseases or conditions present or detected prior to randomization that do not 
worsen 
• Situations where an untoward medical occurrence has not occurred (e.g., hospitalization 
for elective surgery, social and/or convenience admissions) 
• Overdose of either study product or concomitant medication without any signs or 
symptoms unless the subject is hospitalized for observation 
7.1 
Adverse Event Characteristics 
All AEs will be assessed by an investigator and recorded in the subject medical record, including 
the time and date of onset and resolution (where possible), severity, relationship to study product 
(tozuleristide, Canvas system, or both), relationship to neurosurgery, and outcome. 
Severity should be recorded and graded according to the CTCAE Version 5.0. In the absence of a 
specific toxicity grade, the following grading should be used: 


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Table 9 
CTCAE Grading Scale 
Grade 
Severity 
Comments 
1 
Mild 
Aware of sign or symptom, but easily tolerated 
2 
Moderate 
Discomfort enough to cause interference with usual 
activities 
3 
Severe 
Incapacitating with inability to work or perform usual 
activities 
4 
Life-threatening 
Participant is at immediate risk of death 
5 
Fatal 
Death 
 
The relationship to study product (tozuleristide, Canvas system, or both) and to neurosurgery 
should be assessed using the following definitions: 
1. Related: There is a reasonable causal relationship between study drug 
administration/device/procedure and the AE 
2. Not related: There is not a reasonable causal relationship between study drug 
administration/device/procedure and the AE 
The term "reasonable causal relationship" means there is evidence to suggest a causal 
relationship 
All AEs, regardless of severity, causality, or seriousness, will be reported from the date of 
randomization until three months after surgery or until start of tumor-directed therapy, whichever 
is earlier. After the Day 8 visit, all subjects will be followed by chart review and/or telephone 
contact if no patient visit is documented every month for three months, or until an off-study 
criterion is met, to collect survival, CSF cytology if clinically indicated, brain and spine MRIs, 
toxicity assessment and disease status information. 
7.1.1 
Serious Adverse Events 
A SAE is defined as follows: 
Any adverse drug or medical device experience that results in any of the following outcomes: 
• 
Death 
• 
Life-threatening situation (subject is at immediate risk of death) 
• 
Inpatient hospitalization or prolongation of existing hospitalization for an adverse event 
(excluding those for study therapy or placement of an indwelling catheter, unless 
associated with other serious events) 
• 
Persistent or significant disability/incapacity 
• 
Permanent impairment or a body structure or a body function  
• 
Congenital anomaly/birth defect in the offspring of a subject who received study product 
or led to fetal distress or fetal death 
• 
Other: Important medical events that may not result in death, be immediately life-
threatening, or require hospitalization, may be considered a 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 events are: 
• 
Intensive treatment in an emergency room or at home for allergic bronchospasm 


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• 
Resulted in medical or surgical intervention to prevent life threatening illness or 
injury or permanent impairment to a body structure or a body function  
• 
Development of drug dependency or drug abuse 
7.1.1.1 
Clarification of Serious Adverse Events 
Death is an outcome of an AE and not an AE in itself. 
All deaths of subjects on study, regardless of cause, must be reported to Blaze Bioscience. 
“Life-threatening” means that the subject was at immediate risk of death from the event as it 
occurred. This does not include an event that might have led to death, if it had occurred with 
greater severity. 
Complications that occur during hospitalizations are AEs. If a complication prolongs 
hospitalization, it is a SAE. 
The investigator should attempt to establish a diagnosis of the event based on signs, symptoms, 
and/or other clinical information. When available, a diagnosis should be documented as the AE 
and/or SAE and not the individual signs/symptoms. 
“In-patient hospitalization” means the subject has been formally admitted to a hospital for 
medical reasons, for any length of time. 
The following hospitalizations are not considered SAEs: 
• 
A visit to the emergency room or other hospital department <24 hours, that does not result 
in admission (unless considered an important medical or life-threatening event). 
• 
Hospital admission for observation only does not constitute an SAE 
• 
Elective surgery, planned prior to signing consent 
• 
Admissions as per protocol for a planned medical/surgical procedure 
• 
Routine health assessment requiring admission for baseline/trending of health status (e.g., 
routine colonoscopy) 
• 
Medical/surgical admission other than to remedy ill health and planned prior to entry into 
the study. Appropriate documentation is required in these cases 
• 
Admission encountered for another life circumstance that carries no bearing on health 
status and requires no medical/surgical intervention (e.g., lack of housing, economic 
inadequacy, caregiver respite, family circumstances, administrative reason) 
• 
Extended hospitalization due to recovery from surgery associated with study 
 
7.1.2 
Life-threatening 
An adverse event or suspected adverse reaction is considered life threatening if, in the view of 
either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of 
death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a 
more severe form, might have caused death. 
7.2 
Adverse Event Reporting 
The study period during which AEs must be reported begins after randomization and ends three 
months following surgery or until start of other tumor-directed therapy, whichever is earlier. For 
subjects on the re-treatment arm, AE reporting begins once the subject is confirmed eligible and 
is thus enrolled on the re-treatment arm.  


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After the Day 8 visit, all subjects will be followed by chart review and/or telephone contact 
every month for three months, or until an off-study criterion is met, to collect survival, CSF 
cytology if clinically indicated, brain and spine MRIs, toxicity assessment and disease status 
information. 
If the patient experiences an AE considered to be related to study products, then the patient will 
be followed until resolution of that AE or until the patient is off study (whichever is earlier). 
The Investigator will assign attribution of the possible association of the event with tozuleristide 
or the Canvas system or both, or to neurosurgery, and this information will be entered into the 
appropriate case report form. The Investigator must also comply with all reporting requirements 
to their institutional Data and Safety Monitoring Committee and Institutional Review Board 
(IRB). 
7.3 
SAEs and Expedited Reporting 
Blaze Bioscience has requirements for the expedited reporting of SAEs that meet specific criteria 
of the appropriate regulatory authorities; therefore, all appropriate parties must be notified as 
soon as possible and within 24 hours of the investigator becoming aware of the event. The 
procedures for reporting all SAEs, regardless of causal relationship, are as follows: 
• In the case of a SAE, information should be recorded on a SAE Report Form and faxed 
or emailed to the contacts below. 
• Initial SAE notification can be done by email but must be with 24 hours 
 
 
 
For fatal or life-threatening events, also send copies of hospital case reports, autopsy reports, and 
other documents when requested and applicable. 
Blaze Bioscience may request additional information from the investigator to ensure the timely 
completion of accurate safety reports. 
The investigator must take all therapeutic measures necessary for resolution of the SAE. Any 
medications necessary for treatment of the SAE must be recorded in the concomitant medication 
section of the subject’s CRF. 
An SAE may qualify for reporting to regulatory authorities if the SAE is considered to have a 
possible causal relationship to the study product and is unexpected/unlisted based upon the 
current Investigator’s Brochure. In this case, all investigators will receive a formal notification 
describing the SAE. 
Where this is required by local regulatory authorities, and in accordance with the local 
institutional policy, the investigator should notify (in writing) the IRB of SAEs according to 
local guidelines. 
7.4 
Toxicity Management 
7.4.1 
Definitions 
The grading of AEs and abnormal laboratory values will be based on the guidelines provided in 
the CTCAE Version 5.0. 


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For the purpose of monitoring toxicities, the baseline value is defined as the last value prior to 
randomization. This value must be obtained from the laboratory appointed to analyze study 
samples. All management is based on changes from this value. 
7.4.2 
Guidance for Dose Modification or Discontinuation of Treatment 
Dosing will be stopped if suspected adverse drug reactions, changes in vital signs, or clinical 
laboratory results are observed, and these changes pose a significant health risk. 
If the patient experiences an AE considered to be related to study products, then the patient will 
be followed until resolution of that AE or until they are off study (whichever is earlier). 
7.4.3 
Warnings and Precautions 
There are no specific warnings or precautions for tozuleristide. 
For further information regarding warnings and precautions with the study product, please refer 
to the current Tozuleristide Investigator’s Brochure. 


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8 
 SPECIAL STUDIES 
8.1 
 
 
 
 
 
 
 
8.2 
Investigational Device Information 
Refer to Section 5.2 for information of the Canvas system. Detailed information is also provided 
in the current Canvas Imaging System Investigator’s Brochure. 
8.3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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9 
EVALUATION CRITERIA 
9.1 
Independent Safety Monitoring Committee (SMC) 
The SMC’s responsibilities are to: 
• Review the protocol and plans for data safety and monitoring; 
• Conduct regular interim and final evaluation of study safety including aggregate and 
individual subject data related to safety, data integrity, and overall conduct of the study; 
• Consider factors external to the study when relevant information becomes available, such 
as scientific or therapeutic developments that may have an impact on participant safety, 
scientific integrity, or the ethics of conducting the study; 
• Protect the safety of the study subjects; 
• Review and evaluate ad hoc safety issues concerning the study at the request of Blaze 
Bioscience; 
• Make recommendations to Blaze Bioscience concerning continuation, termination, or other 
modifications of the study based on the observed beneficial or adverse effects of the study; 
and 
• Operate according to the procedures described in the 
 charter and 
all procedures of the SMC. 
9.2 
Central Radiology 
• 
 
 
• 
 
 
 
• 
 
 
 
• All MRI processes for central radiology are detailed in the study reference manual. 
9.3 
Central Pathology 
• Primary tumor tissue specimens and equivocal tissue specimens will be required for 
central pathology assessment of presence or absence of tumor. Central pathology samples 
will be requested after completion of site final pathology report for standard clinical 
management.  “Other biopsy” tissue specimens, if obtained, will also be evaluated by 
central pathology. 
• 
 
 


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• 
 
  
• Central pathology will be blinded to treatment allocation and fluorescence data but will 
have access to routine pathology data (gross and microscopic pathology reports), images 
and/or slides for microscopic evaluation, and molecular/genetic data. 
• 
 
9.4 
 
 
 
 
 
 
 
 
 
 
9.5 
Protocol Endpoint Assessment Committee (PEAC) 
The PEAC will monitor the progress of the study 
 
 
 
 
 
 
 
 
 
Enrollment will be halted once sufficient 
data (specimens and sub-groups) are available for analysis.  
 
 
   
Roles and responsibilities are outlined in the PEAC study charter. 


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10 
STATISTICAL CONSIDERATIONS 
10.1 
Study Design/Endpoints 
The study design is presented in Section 3. 
 
. 
10.1.1 
Primary Endpoints 
The primary efficacy endpoints are: 
1. Ratio of sensitivity of tozuleristide fluorescence in equivocal tissue biopsies from 
subjects with fluorescence-positive tumors compared to the sensitivity of surgical 
designation from the same biopsies under white light conditions 
2. Ratio of specificity of tozuleristide fluorescence in equivocal tissue biopsies from 
subjects with fluorescence-positive tumors compared to the specificity of surgical 
designation from the same biopsies under white light conditions 
3. Sensitivity of tozuleristide fluorescence to detect tumor in equivocal tissue biopsies from 
subjects with fluorescence-positive tumor 
4. Specificity of tozuleristide fluorescence to detect tumor in equivocal tissue biopsies from 
subjects with fluorescence-positive tumor 
 
 
   
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 


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10.1.2 
Secondary Endpoints 
The secondary efficacy endpoints are: 
1. 
 
 
 
2.
 
 
 
 
 
 
 
 
Study endpoints related to the safety objective: 
1. All reported adverse events (AEs) will be graded according to the National Cancer 
Institute’s Common Terminology for Adverse Events (NCI CTCAE, Version 5.0) 
2. SAEs 
3. AE attribution of relatedness to tozuleristide and/or Canvas system (related vs. 
unrelated). 
4. Laboratory parameters 
5. ECG parameters 
6. Vital signs 
 
 
 
 
 
 
 
 
 
 
 
 


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• 
 
 
 
 
 
 
 
 
 
 
10.1.3 
Exploratory Endpoints 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
10.2 
Statistical Analysis Plan 
In general, descriptive statistics will be provided for all study endpoints. Descriptive statistics 
will include number of subjects, mean, standard deviation, median, and range (minimum, 
maximum) for continuous variables, and frequencies and percentages for categorical variables.  
Statistical tests will be performed at either a one-sided significance level of 2.5% or a two-sided 


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significance level of 5%. All confidence intervals (CIs) will be constructed at a two-sided 95% 
confidence level. 
In addition, data collected after re-treatment will be listed and will not be included in the efficacy 
and safety analyses.  
10.2.1 
 
 
 
 
10.2.2 
Analysis Populations 
The full analysis population (FAP) for efficacy analyses will consist of all subjects who 
1. meet all eligibility criteria
2. undergo neurosurgery and


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3. have central pathology tumor assessment and imaging operator fluorescence assessment 
of equivocal tissue specimens obtained from the first neurosurgery on study.   
Study subjects in the full analysis population will be classified into four treatment groups: 
• Group 1 (Arm 1): subjects who do not receive tozuleristide, regardless of fluorescence 
outcomes and tumor diagnosis 
• Group 2 (Arm 2 Fluorescent): subjects who receive tozuleristide, have at least one 
fluorescence-positive study tissue specimens of any type (primary, equivocal and other) 
based on imaging operator fluorescence assessment, and the subject’s central pathology 
consensus diagnosis is tumor, ambiguous, or not definitive 
• 
 
 
 
 
 
A subject is evaluable for efficacy if the subject meets the three criteria of the FAP
 
 
   
The safety analysis population will consist of all subjects who receive any amount of 
tozuleristide or receive neurosurgery prior to any re-treatment. Safety data collected in the re-
treatment portion of the study will be listed separately.  
 
 
 
10.2.3 
Subject Disposition 
An accounting of subject disposition will be tabulated by treatment arm. The number of subjects 
in each analysis population will be summarized by treatment arm. Subjects who withdraw from 
the study prematurely will be summarized. The reason(s) for study discontinuation will be 
summarized. The number of subjects who do not undergo surgery will also be summarized by 
treatment arm. 
10.2.4 
Demographics and Subject Characteristics 
Demographic and other baseline characteristics will be summarized with descriptive statistics by 
treatment arm. 
10.2.5 
Analyses of Primary Efficacy Endpoints 
The primary efficacy analyses will be based on Group 2 in the full analysis population (Section 
10.2.2). The four sets of statistical hypotheses will be evaluated by assessing the statistical 
significance of the co-primary endpoints. 


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10.2.6 
 
 
 
 
  
 
 
 
 
 
 
 
10.2.7 
Study Product Exposure 
The dose of tozuleristide administrated in volume (mL) and amount (mg) will be summarized 
 
 


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10.2.8 
Safety Analyses 
All safety summaries will be tabulated for subjects who receive tozuleristide and subjects who 
do not receive tozuleristide in the safety population.  Safety data collected in the re-treatment 
portion of the study will be listed separately. 
10.2.8.1 Adverse Events 
Adverse events will be coded using MedDRA 21.0. Adverse events will be defined as treatment-
emergent if they are newly occurring or worsen following surgery (Arm 1) or treatment with 
tozuleristide (Arm 2 and re-treatment). Adverse events will be scored for severity using NCI 
CTCAE version 5.0 if applicable, or the protocol specified severity stated in Section 7.1. 
All adverse events (AEs) and SAEs collected during the study will be graded according to the 
National Cancer Institute’s Common Terminology for Adverse Events (NCI CTCAE, Version 
5.0) along with attribution of relatedness to tozuleristide and/or Canvas system and neurosurgery 
(related vs. unrelated). 
Treatment-emergent AEs, SAEs, severity of AEs, tozuleristide-related AEs, Canvas-related AEs, 
neurosurgical-related AEs, and AEs leading to withdrawal from study will be summarized by 
MedDRA preferred term and system organ class. In the event of multiple occurrences of the 
same AE with the same preferred term in the same subject, the AE will be counted once at the 
greatest severity/relatedness. 
10.2.8.2 Clinical Laboratory Parameters, ECG and Vital Signs 
Laboratory parameters will be summarized with descriptive statistics of the change from baseline 
value by time point. For laboratory parameters defined in CTCAE, shifts of baseline grade to the 
worse post-baseline grade will also be presented. Subject laboratory values will be listed with 
toxicity grades and out-of-range flags.  
ECG parameters, including QT and QTcF will be summarized with descriptive statistics per time 
point.  Changes from baseline for individual QT/QTcF will be reported as both absolute values 
(e.g., >450 ms) and relative changes (e.g., > 30 ms).   If available, out of range ECG parameters 
will be assessed for the presence of genetic factors that could contribute to the abnormal ECG 
findings.  ECG data will also be analyzed as a function of pharmacokinetic exposure measures. 
10.2.9 
 
 
 
 
 
10.2.10 
 
 


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10.2.11 
 
 
 
 
10.2.12 
Interim Analysis 
Interim analysis is not planned.
 
 
 
10.3 
Sample Size 
Approximately 114 subjects are planned for treatment in this study. 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 


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10.4 
Stratification Factors 
The randomization procedure will not be stratified.  Permuted block randomization will be used. 


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11 
DATA REPORTING / REGULATORY REQUIREMENTS 
11.1 
Data Reporting 
11.1.1 
Method 
The investigator will ensure that this study is conducted in full conformance with the protocol, 
the “Declaration of Helsinki” prior to 2000 and its amendments, and with the requirements of 
national drug and data protection laws of the countries in which the research is conducted. 
11.2 
Record Keeping and Record Retention 
It is the responsibility of the investigator to obtain written informed consent from each subject 
participating in this study or legally acceptable representative prior to undertaking any study 
related procedures. A legally acceptable representative is an individual or other body authorized 
under applicable law to consent, on behalf of a prospective subject, to the subject’s participation 
in the clinical study. Assent, when appropriate, will be obtained according to institutional 
guidelines. 
The investigator must utilize an IRB-approved consent form for documenting written informed 
consent. The original signed and dated informed consent form must be retained in accordance 
with institutional policy, and a copy of the signed consent form must be provided to the subject 
or legally acceptable representative. 
The investigator must maintain adequate and accurate records to enable the conduct of the study 
to be fully documented and the study data to be subsequently verified. 
These documents should be classified into two separate categories: 
• Investigator’s Site File (ISF), and 
• Subject clinical source documents 
The ISF will contain the protocol/amendments, CRFs, query forms, IRB approval with 
correspondence, informed consent, drug records, staff curriculum vitae, and authorization forms, 
and other appropriate documents and correspondence. 
Subject clinical source documents include subject hospital/clinic records, physician’s and nurse’s 
notes, appointment books, original laboratory reports, ECG, electroencephalogram, X-ray, MRI, 
pathology and special assessment reports, and consultant letters. All clinical study documents, 
including documents created or modiﬁed in electronic format, must be retained by the 
Investigator for at least 15 years following the completion of the study or until at least two years 
after the last approval of a marketing application in an ICH region (USA, Europe, or Japan) and 
until there are no pending or contemplated marketing applications in an ICH region; or if no 
application is filed or if the application is not approved for such indication, until two years after 
the investigation is discontinued and regulatory authorities have been notified. The investigator 
must notify Blaze Bioscience, prior to destroying any clinical study records. 
Should the investigator wish to assign the study records to another party or move them to another 
location, Blaze Bioscience must be notified in advance. 
If the investigator cannot guarantee this archiving requirement at the study site for any or all of 
the documents, special arrangements must be made between the investigator and Blaze 
Bioscience to store these in a sealed container(s) offsite so that they can be returned to the 


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investigator in case of a regulatory audit. Where source documents are required for the continued 
care of the subject, appropriate copies should be made for storage offsite. 
When requested in writing by the Sponsor, unused study medication supplies may be returned to 
the Sponsor or destroyed by the investigator. Records shall be maintained by the investigator of 
any such disposition of the study medication. These records must show the identification and 
quantity of each unit disposed of, the method of destruction (taking into account the 
requirements of local law), and the person who disposed of the test substance. Such records shall 
be submitted to the Sponsor. 
  
 
 
 
 
 
 
 
 
 
 
 
11.3 
Regulatory Documentation 
11.3.1 
Institutional Review Board 
A copy of the protocol, proposed informed consent form, other written subject information, and 
any proposed advertising material must be submitted to the IRB for written approval. A copy of 
the written approval of the protocol and informed consent form must be received by Blaze 
Bioscience before recruitment of subjects into the study and shipment of investigational 
products. Approval from the IRB must be obtained before starting the study and should be 
documented in a letter to the investigator specifying the protocol number and version and the 
date on which the IRB met and granted the approval. 
Any modifications made to the protocol and/or informed consent form after receipt of IRB 
approval must also be submitted by the investigator to the IRB in accordance with institutional 
procedures and regulatory requirements. 
11.3.2 
Audits and Inspections 
An audit is a systematic and independent examination of trial related activities and documents to 
determine whether the evaluated trial-related activities were conducted, and the data were 
recorded, analyzed, and accurately reported according to the protocol, standard operating 
procedures (SOPs) of Blaze Bioscience and/or its representatives or designees, GCP, and 
applicable regulatory requirement(s). 
Authorized representatives of Blaze Bioscience, its designee, a regulatory authority, or the IRB 
may visit the center to perform audits or inspections. The investigator should contact Blaze 
Bioscience immediately if they are contacted by a regulatory agency about an inspection at their 


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center.  If an audit or inspection occurs, the investigator and institution agree to allow the 
auditor/inspector direct access to all relevant documents and allocate their time and the time of 
their staff to the auditor/inspector to discuss findings and any relevant issues. 
11.3.3 
Training 
Study teams at the clinical sites will be trained on all aspects of the study. 
Training will be conducted at the study initiation visits with the study coordinators, pharmacists, 
and investigators as well as at a central investigator meeting and in pre-scheduled webinars. 
The study teams will be trained on the protocol evaluations, safety reporting, investigator brochure, 
informed consent, CRFs, pharmacy procedures, dosing, receipt and storage of tozuleristide, 
 
, inclusion and exclusion criteria, study manual, investigator responsibilities, 
GCP, 
 and data entry into the clinical database. 
Every site’s imaging operators and neurosurgeons will also receive specialized training in 
operating the Canvas system and image acquisition and interpretation including the following: 
• Instructions on specimen flow in the Operating room and labeling of imaged specimens 
• 
 
• Operation of the Canvas system including pitfalls and recognition of problems 
• 
 
• Assessment of fluorescence intraoperatively 
• Image and video capture and storage of data 
 
 
11.3.4 
Monitoring the Study 
In accordance with ICH GCP guidelines, the study monitor must have direct access to the 
investigator’s source documentation in order to verify the data recorded in the CRFs for 
consistency. 
The study monitor, as a Blaze Bioscience representative, will contact and visit the investigator 
regularly and that he/she will be allowed, on request, to inspect the various records of the trial 
(CRFs and other pertinent data) provided that subject confidentiality is maintained in accord with 
local requirements. 
It will be the monitor’s responsibility to monitor the CRFs at regular intervals throughout the 
study, to verify the adherence to the protocol and the completeness, consistency, and accuracy of 
the data being entered on them. The investigator agrees to allow the monitor direct access to all 
relevant documents and to allocate his/her time and the time of his/her staff to the monitor to 
discuss findings and any relevant issues. 
11.3.5 
Conditions for Modifying the Protocol 
Site-requested protocol modifications which alter the scope of the investigation, the scientific 
quality of the study, the experimental design, dosages, duration of therapy, assessment variables, 
the number of subjects treated, or subject selection criteria, may be made after the review and 
approval of Blaze Bioscience. 
All protocol modifications must be submitted to the IRB and responsible regulatory authority in 
accordance with local requirements. Approval must be awaited before significant changes can be 


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implemented except when necessary to eliminate immediate hazards to patients; i.e., if the risk 
benefit ratio is affected and/or the modification represents a change in basic trial definitions such 
as objectives, design, and sample size or outcome measures. 
In the event of a SAE or AE, the investigator may institute any medical procedures deemed 
appropriate. Administrative changes to the protocol are defined as minor corrections and/or 
clarifications that have no effect on the way the study is to be conducted or on the safety of the 
subjects. These administrative changes will be agreed upon by Blaze Bioscience and the 
investigator and submitted to the IRB.  
11.3.6 
Conditions for Terminating the Study 
The study may be prematurely terminated by the IRB or relevant regulatory authorities if the 
perception of the benefit/risk becomes unfavorable for continuation of the study. Additionally, 
Blaze Bioscience reserves the right to terminate the study at any time on the basis of new 
information regarding safety or efficacy, or if study progress is unsatisfactory, or for other 
administrative or commercial reasons. 
 
Should this be necessary, the procedures will be arranged on an individual study basis after 
review and consultation by all parties. In terminating the study, Blaze Bioscience and the 
investigators will ensure that adequate consideration is given to the protection of the subjects’ 
interests. The investigator should promptly inform the subjects and ensure appropriate therapy 
and follow-up and inform the relevant regulatory authorities and the IRB. All delivered study 
materials must be collected and all CRFs completed to the extent possible. 
11.3.7 
Confidentiality of Trial Documents and Subject Records 
The investigator must assure the subjects’ anonymity will be maintained and that their identities 
are protected from unauthorized parties. On CRFs or other documents submitted to the Sponsor, 
subjects should not be identified by their names, but by the subject’s initials and an identification 
code. The investigator should keep a subject identification log showing codes, names, and 
addresses. Documents not for submission to Blaze Bioscience (e.g., subject’s written consent 
forms, identification log), should be maintained by the investigator in strict confidence. 
All information concerning the study treatment and the Sponsor Company and its operation, such 
as patent applications, formulae, manufacturing processes, basic scientific data, and material not 
previously published are considered confidential and shall remain the sole property of the Sponsor. 
The investigator agrees to use this information only in accomplishing the study and will not use it 
for any other purposes without written consent from the Sponsor. 
11.3.8 
Publication of Data and Protection of Trade Secrets 
In accord with standard editorial and ethical practice, Blaze Bioscience will support publication 
of the data from this study. 
 
 
 
 
 


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33. Zeltzer PM, Boyett JM, Finlay JL, Albright AL, Rorke LB, Milstein JM, Allen JC, Stevens 
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RJ. Metastasis stage, adjuvant treatment, and residual tumor are prognostic factors for 
medulloblastoma in children: conclusions from the Children's Cancer Group 921 randomized 
phase III study. J Clin Oncol. 1999 Mar;17(3):832-45. 
34. Zhao S, Wu J, Wang C, Liu H, Dong X, Shi C, Shi C, Liu Y, Teng L, Han D, Chen X, Yang G, 
Wang L, Shen C, Li H. Intraoperative fluorescence-guided resection of high-grade malignant 
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APPENDIX A: PERFORMANCE STATUS CRITERIA 
Karnofsky 
Lansky 
Score 
Description 
Score 
Description 
100 
Normal, no complaints, no evidence 
of disease 
100 
Fully active, normal. 
90 
Able to carry on normal activity, 
minor signs or symptoms of disease. 
90 
Minor restrictions in physically strenuous 
activity. 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
80 
Active, but tires more quickly 
70 
Cares for self, unable to carry on 
normal activity or do active work. 
70 
Both greater restriction of and less time 
spent in play activity. 
60 
Required occasional assistance, but is 
able to care for most of his/her needs. 
60 
Up and around, but minimal active play; 
keeps busy with quieter activities. 
50 
Requires considerable assistance and 
frequent medical care. 
50 
Gets dressed, but lies around much of the 
day; no active play, able to participate in 
all quiet play and activities. 
40 
Disabled, requires special care and 
assistance. 
40 
Mostly in bed; participates in quiet 
activities. 
30 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
30 
In bed; needs assistance even for quiet 
play. 
20 
Very sick, hospitalization indicated.  
Death not imminent. 
20 
Often sleeping; play entirely limited to 
very passive activities. 
10 
Moribund, fatal processes 
progressing rapidly. 
10 
No play; does not get out of bed. 
 
 
 
 
 
 
 


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