Conditions: Parotid Neoplasm Thyroid Neoplasms Head and Neck Neoplasms Surgery Nerve Injury Imaging
Keywords: Nerve Fluorescence Surgery Imaging Real-time Intraoperative Highlighting
Allocation: NON_RANDOMIZED | Intervention model: SEQUENTIAL
Enrollment: 27
Healthy volunteers: no

Brief summary:
Bevonescein to Highlight Nerves in Patients Undergoing Head \& Neck Surgery

Detailed description:
This study will evaluate the safety, tolerability, and efficacy of Bevonescein (ALM-488) administered as an intravenous (IV) infusion to patients undergoing head \& neck surgery. The study will also characterize the pharmacokinetics of Bevonescein (ALM-488) in this subject population and determine the dose of Bevonescein (ALM-488) needed to generate a fluorescence signal in nerve tissue to enable fluorescence recordings and image analysis with an imaging system. The study will also evaluate the effect of timing of Bevonescein (ALM-488) administration, relative to surgery, on fluorescence characteristics.

Arms:
arm 1: This arm of the study will include Dose Escalation/De-Escalation cohorts of Bevonescein (ALM-488). arm 2: This arm of the study will include Dose Timing cohorts of Bevonescein (ALM-488).

Interventions: intervention 1: Bevonescein
Intervention descriptions: intervention 1: Bevonescein Sterile Solution is an intravenously administered, synthetic, peptide dye conjugate indicated for the real-time intraoperative fluorescence detection and localization of nerve tissue.

Protocol document:
SAP_002.pdf: 
 
Protocol No.: ALM-488-001 
CP Project ID: ALU19001   
Version: 2.0 
Date: 25JAN2021  
 
 
QMS Document Name: Statistical Analysis Plan 
Page 1 of 29 
QMS No (include version ref): STF-015-GL.02 
Document date: 09JUL2019 
CONFIDENTIAL 
 
 
 
 
 
 
 
 
 
 
Protocol No: ALM-488-001 
CP Project ID: ALU19001 
 
Phase 1/2 Trial of ALM-488 in Subjects Undergoing Head & Neck Surgery  
 
 
 
 
 
Statistical Analysis Plan 
Draft Version: 2.0  
Date: 25JAN2021 
 
 
 
 
 
 
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Protocol No.: ALM-488-001 
CP Project ID: ALU19001   
Version: 2.0 
Date: 25JAN2021  
 
 
QMS Document Name: Statistical Analysis Plan 
Page 3 of 29 
QMS No (include version ref): STF-015-GL.02 
Document date: 09JUL2019 
CONFIDENTIAL 
 
 
 
Table of Contents 
1 
Abbreviations ................................................................................................................................. 6 
2 
Introduction .................................................................................................................................... 7 
2.1 
Preface ................................................................................................................................... 7 
2.2 
Timing of Statistical Analyses ................................................................................................ 7 
3 
Modification History ....................................................................................................................... 7 
3.1 
Changes to the Planned Analyses ......................................................................................... 7 
4 
Study Design.................................................................................................................................... 7 
4.1 
Sample Size Estimation ......................................................................................................... 9 
4.2 
Randomization, blinding and unblinding procedures ........................................................... 9 
5 
Analysis Sets and Cohorts ............................................................................................................... 9 
6 
Objectives...................................................................................................................................... 10 
6.1 
Primary Objective ................................................................................................................ 11 
6.2 
Secondary Objectives .......................................................................................................... 11 
6.3 
Exploratory Objectives ........................................................................................................ 11 
7 
Endpoints ...................................................................................................................................... 11 
7.1 
Safety Endpoints .................................................................................................................. 11 
7.2 
Secondary Endpoints ........................................................................................................... 11 
7.3 
Exploratory Endpoints ......................................................................................................... 12 
8 
General Statistical Methods and Definitions ................................................................................ 13 
8.1 
General statistical methods ................................................................................................ 13 
8.2 
Covariates and strata .......................................................................................................... 14 
8.3 
Subgroups ............................................................................................................................ 14 
8.4 
Missing data ........................................................................................................................ 14 
8.5 
Rescreening ......................................................................................................................... 15 
8.6 
Baseline Definition .............................................................................................................. 15 
8.7 
Study Day, Visit Window, and COVID-19 ............................................................................. 15 
9 
Patient Accounting and Disposition .............................................................................................. 16 
9.1 
Patient accounting .............................................................................................................. 16 
9.2 
Disposition and withdrawals ............................................................................................... 16 
9.3 
Minor and Major Protocol deviations ................................................................................. 16 
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Protocol No.: ALM-488-001 
CP Project ID: ALU19001   
Version: 2.0 
Date: 25JAN2021  
 
 
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Document date: 09JUL2019 
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10 Demographics and Baseline Characteristics ................................................................................. 16 
10.1 Demographics...................................................................................................................... 16 
10.2 Surgery Type ........................................................................................................................ 17 
10.3 Medical history .................................................................................................................... 17 
10.4 Exposure and Compliance ................................................................................................... 17 
10.5 Dosage and Timing of Administration ................................................................................. 18 
11 Previous and Concomitant Therapies ........................................................................................... 18 
12 Determination of Optimal Dose .................................................................................................... 18 
13 Efficacy .......................................................................................................................................... 18 
13.1 Timing, Dose, Size and Intensity .......................................................................................... 19 
13.2 Surgical Imaging Worksheet/ Rating Scale .......................................................................... 20 
13.3 Clinical Benefit ALM-488 Questionnaire ............................................................................. 20 
13.4 Nerve Dissecting Time ......................................................................................................... 20 
14 Safety ............................................................................................................................................ 20 
14.1 Adverse Events .................................................................................................................... 20 
14.2 Vital Signs ............................................................................................................................ 22 
14.3 Clinical Laboratory evaluations ........................................................................................... 22 
14.4 Echocardiograms (ECGs) ..................................................................................................... 23 
14.5 Physical Examinations ......................................................................................................... 24 
14.6 Pregnancy Tests ................................................................................................................... 24 
14.7 Serum Samples .................................................................................................................... 24 
14.8 Chest X-Ray/CT .................................................................................................................... 24 
15 Pharmacokinetic Analyses (PK) ..................................................................................................... 24 
16 Interim Analyses............................................................................................................................ 24 
17 Statistical Analyses for Safety Monitoring .................................................................................... 24 
18 Statistical Analyses for Clinical Trial Registries ............................................................................. 25 
19 Software ........................................................................................................................................ 25 
20 Tables, Listings, and Figures .......................................................................................................... 26 
20.1 Tables .................................................................................................................................. 26 
20.2 Listings ................................................................................................................................. 27 
20.3 Figures ................................................................................................................................. 28 
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Protocol No.: ALM-488-001 
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21 References .................................................................................................................................... 28 
22 Appendices .................................................................................................................................... 28 
Appendix I: Normal ranges for vital signs, oxygen saturation and body temperature ................. 29 
 
 
 
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Protocol No.: ALM-488-001 
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Document date: 09JUL2019 
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1 
Abbreviations 
AE 
Adverse Event 
ATC 
Anatomical Therapeutic Class 
DLT 
Dose Limiting Toxicity 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
ENR 
Enrolled analysis set 
FAS 
ALM-488 Activity Full Analysis Set 
FL 
Fluorescence 
ICF 
Informed consent form 
IDMC 
Independent Data Monitoring Committee 
IRR 
Infusion-related reaction 
IV 
Intravenous 
MedDRA 
Medical Dictionary for Regulatory Activities 
NCI-CTCAE 
National Cancer Institute – Common Terminology Criteria for Adverse events 
PAS 
PK Analysis Set 
PK 
Pharmacokinetics 
PT 
Preferred Term 
RMANOVA 
Repeated Measures Analysis of Variance 
SAE 
Serious Adverse Event 
SAF 
Safety Analysis Set 
SAP 
Statistical Analysis Plan 
SOC 
System Organ Class 
SRC 
Safety Review Committee 
TEAE 
Treatment Emergent Adverse Event 
TLF 
Tables, Listings, and Figures 
WHO 
World Health Organization 
WLR 
White Light Reflectance 
 
 
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Protocol No.: ALM-488-001 
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2 
Introduction 
2.1 
Preface 
The purpose of this document is to detail the statistical methodology to be used for the final 
statistical analysis of study ALM-488-001.  
This Statistical Analysis Plan (SAP) covers the statistical summaries, analyses and methods of all the 
non-pharmacokinetic (PK) data. All details of the statistical summaries, analyses and methods for the 
PK data will be specified in a separate PK SAP. This SAP should be used in conjunction with the 
protocol. If there are any discrepancies between the protocol and SAP, this SAP will prevail. Any 
deviations from this SAP that are implemented in the final analysis will be documented with sound 
clinical and statistical rationale in the Clinical Study Report (CSR). 
A separate document contains the table, listing and figure specifications and any example 
programming codes. 
2.2 
Timing of Statistical Analyses 
 
The following statistical analyses are planned for this study: 
 
Dose Escalation or De-Escalation 
 
Dose Timing Determination 
3 
Modification History 
3.1 
Changes to the Planned Analyses 
The statistical analyses as specified in this SAP are mostly consistent with the statistical analyses as 
specified in the study protocol version Amendment 1, 24 August 2020. 
We propose to include two additional specific analysis sets ENR, and DLT. They will allow to better 
describe the different steps of the study. They are not defined in the protocol. The additional 
analysis sets are defined below in section 5. 
 This SAP has been updated based on changes associated with protocol amendment 1.0. 
4 
Study Design 
This is a Phase 1/2, open-label study in subjects undergoing head and neck surgery. The study will 
include up to 6 Dose Escalation/De-Escalation cohorts, followed by 2 pre-planned additional Dose 
Timing cohorts. Dose cohorts will start at 100 mg and follow the escalation/de-escalation described 
in the protocol. 
Indication 
Parotid Neoplasm, Thyroid Neoplasms, Head and Neck Neoplasms, 
Head and Neck Surgery 
Design 
Open-label study: Dose Escalation/Dose De-Escalation and Dose Timing 
cohorts 
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Protocol No.: ALM-488-001 
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Phases 
Phase ½ 
Primary Objective 
To evaluate the safety of ALM-488 administered by intravenous (IV) 
infusion to subjects undergoing head and neck surgery. 
Secondary 
Objectives 
 
To characterize the pharmacokinetics (PK) of ALM-488 in subjects 
undergoing head and neck surgery. 
 
To determine the recommended dose of ALM-488 needed to generate 
an adequate fluorescence signal in nerves.  
 
To evaluate the effect of timing of administration of ALM-488 relative 
to surgery (e.g., up to 5 hours before surgery) on adequacy of 
fluorescence nerve labeling. 
Exploratory Efficacy 
Objectives 
 
To evaluate the ability of imaging techniques (i.e. camera systems) to 
distinguish nerve tissue from non-nerve tissue. 
 
To evaluate the intensity of signal of fluorescence relative to nerve 
diameter.  
 
To evaluate the intensity of signal of fluorescence when there is 
overlying tissue. 
 
To assess the depth of fluorescence penetration when there is 
overlying non-nerve tissue.  
 
To evaluate surgeon confidence and operating time with a surgeon 
rating scale. 
Treatment 
ALM-488 Sterile Solution is an intravenously administered, synthetic, 
peptide dye conjugate indicated for the real-time intraoperative 
fluorescence detection and localization of nerve tissue. 
Number of subjects 
9-36 subjects in the Dose Escalation/De-Escalation cohorts 
12 subjects in the Dose Timing Cohorts 
Total 21-48 subjects 
Interim analysis 
No formal interim analysis is planned for this study 
Planned enrolment 
Three sites in the United States.  
 
 
The aim is to determine the lowest possible dose that achieves adequate imaging performance 
(nerve identification) with an acceptable toxicity profile. The Safety Review Committee (SRC) will 
determine the Optimal Dose based on 1) the safety data and 2) adequacy of fluorescence observed 
in each of the dose groups. Following determination of the Optimal Dose, Dose Timing Cohorts will 
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Protocol No.: ALM-488-001 
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commence at this Optimal Dose. The Dose Timing Cohorts will compare fluorescence characteristics 
at different ALM-488 administration times before surgery (3-5 hours, 1-3 hours).   
 
Please refer to Section 5 of the protocol for full details on the study design. 
4.1 
Sample Size Estimation 
The primary objective of this study is to evaluate the safety of ALM-488 administered by 
intravenous (IV) infusion to subjects undergoing head and neck surgery. 
At each dose level, a cohort of 3 subjects will be enrolled for safety and tolerability evaluation. If 
there are no subjects with a DLT at a given dose level, new subjects will be enrolled at the next 
dose escalation level. If there are ≥1 subjects having any DLT, the enrollment to that specific 
dose and dose escalation will stop. New subjects will be enrolled at the next dose de-
escalation level. There will be a minimum of 3 cohorts. Given that there will be 3-6 subjects for 
each dose level, 9-36 subjects will be needed for the dose escalation/de-escalation cohorts. 
It is anticipated that the proposed dose range (100-1000mg) is expected to yield optimal 
imaging performance, with an initial starting dose of 100mg that has been selected based on 
supportive nonclinical testing. If the 100 mg dose is found to provide adequate fluorescence 
imaging of nerves, a dose de-escalation schedule will be followed to ensure that the lowest 
effective dose is determined. If imaging at starting first de-escalated dose (75 mg) becomes 
less adequate compared to 100mg as determined by the SRC, then imaging at dose escalation 
schedule will resume - beginning at 200mg. The Optimal Dose will be determined by the SRC as 
the lowest possible dose and toxicity profile that achieves adequate imaging performance of 
nerves using WLR combined with FL. 
The Dose Timing Cohort will use the Optimal Dose as defined by the SRC following completion of 
the Dose Escalation/De-Escalation cohorts. Subjects in the Dose Timing phase will be administered 
ALM-488 at two planned operative times before surgery to assess the impact of timing of 
administration on fluorescence image quality. This phase will consist of 2 cohorts, 6 
subjects/cohort: (1) dose administration 3-5 hours in advance of surgery, (2) dose administration 
1-3 hours in advance of surgery. A total of 12 subjects will be needed for the Dose Timing phase. 
In summary, 9-36 subjects may be enrolled into the dose escalation/dose de-escalation cohorts and 
12 subjects may be enrolled into the dose timing cohort.  A total of 21-48 subjects may be enrolled 
into this study. 
4.2 
Randomization, blinding and unblinding procedures 
No randomization, blinding or unblinding procedures are planned for this study. 
5 
Analysis Sets and Cohorts 
 Analysis Sets are described as follows:    
 
Enrolled analysis set (ENR) defined as all subjects who provided informed consent. 
 
Safety Analysis Set (SAF), defined as all subjects who received at least one dose of ALM-488 
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Protocol No.: ALM-488-001 
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 
Dose-Limiting Toxicity (DLT) Evaluable Set, defined as subjects who receive ALM-488 and  either 
1) complete the safety follow-up through the DLT evaluation period (i.e., Day 15 ± 8)  or 2) 
experiences a DLT at any time during the DLT evaluation period. 
 
Activity Analysis or Full Analysis Set (FAS), defined as all subjects in SAF who also received at 
least one evaluable image.  
 
Pharmacokinetics (PK) Analysis Set defined as all subjects who completed a PK blood sampling 
period without any major protocol violations which would render the data unreliable. The PK will 
be the primary analysis set for the pharmacokinetic analyses. PK of analyses will be fully 
described in the PK SAP. 
SAF will be used for all safety analyses. FAS will be used for all efficacy analyses. The PK Analysis Set 
will be used for all PK analyses; ENR set will be the primary analysis set for disposition and listings. 
PP set will constitute the primary analysis set for early efficacy. DLT Evaluable Analysis Set will be 
used to assess the tolerability of ALM-488 in the dose escalation phase. 
 
The Dose Escalation/De-Escalation cohort (Cohort A) consists of all dose escalation/de-escalation 
cohorts: Cohort A1, A2, A3, A4, A5, A6. In case of DLT rate = 0 or special FDA requests additional 
cohorts may be added to the tables. 
 
The Dose Time Determination cohort (Cohort B) consists of: Cohort B1 - dose administration 3-5 
hours in advance of surgery, and Cohort B2 - dose administration 1-3 hours in advance of surgery. 
 
If not otherwise stated in the respective section, the statistical analyses will be performed for the 
following analysis sets: 
Analyses 
ENR 
FAS 
DLT 
SAF 
PK 
Disposition 
 
 
 
 
 
Demographics and baseline 
characteristics 
 
 
 
 
 
Medical history 
 
 
 
 
 
Exposure and compliance 
 
 
 
 
 
Previous and concomitant therapies 
 
 
 
 
 
Early Efficacy  
 
 
 
 
 
Pharmacokinetic 
 
 
 
 
 
Safety 
 
 
 
 
 
Dose-Limiting Toxicity 
 
 
 
 
 
Listings 
 
 
 
 
 
6 
Objectives 
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6.1 
Primary Objective 
To evaluate the safety of ALM-488 administered by intravenous (IV) infusion to subjects 
undergoing head and neck surgery. 
6.2 
Secondary Objectives 
• 
To characterize the PK of ALM-488 in subjects undergoing head and neck surgery. 
• 
To determine the dose of ALM-488 needed to generate an adequate fluorescence signal in 
nerves. 
• 
To evaluate the effect of timing of administration of ALM-488 relative to surgery (e.g., up to 5 
hours before surgery) on adequacy of fluorescence nerve labeling. 
6.3 
Exploratory Objectives 
Exploratory objectives will evaluate early evidence of efficacy. These are: 
• 
To evaluate the ability of imaging techniques (i.e. camera systems) to distinguishing nerve 
tissue from non-nerve tissue.  
• 
To evaluate the intensity of signal of fluorescence relative to nerve diameter.  
• 
To evaluate the intensity of signal of fluorescence when there is overlying tissue. 
• 
To assess the depth of fluorescence penetration when there is overlying non nerve tissue.  
• 
To evaluate surgeon confidence and operative time with a surgeon rating scale.   
7 
Endpoints 
7.1 
Safety Endpoints 
The endpoints to assess the safety of ALM-488 include: 
• 
Adverse Events (TEAEs) 
• 
Vital signs 
• 
Clinical laboratory evaluations 
• 
ECGs 
• 
Physical examinations 
Summaries of AEs will be based on Treatment Emergent Adverse Events (TEAEs) and will be 
summarized separately for each device and each of the Cohorts A1-A6, B1 and B2, as well as 
overall for Cohort A and Cohort B. 
Vital signs, labs, ECGs, and physical exams will be summarized separately for each of the Cohorts 
A1-A6, B1 and B2, as well as overall for Cohort A and Cohort B. 
7.2 
Secondary Endpoints 
The endpoints to characterize the PK of ALM-488 will be fully described in the PK SAP. 
The endpoints to determine the dose of ALM-488 needed to generate an adequate fluorescence 
signal in nerves include the following: 
 
Timing of administration of ALM-488 relative to surgery 
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Protocol No.: ALM-488-001 
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 
Dose 
 
Intensity (signal-to-background ratio- SBR) 
 
Surgeon rating scale 
The endpoints to determine the dose of ALM-488 needed to generate an adequate fluorescence 
signal in nerves will be summarized for each of Cohorts A1-A6, as well as overall for Cohort A. 
The endpoints to evaluate the effect of timing of administration of ALM-488 relative to surgery 
on adequacy of fluorescence nerve labeling include the following: 
 
Timing of administration of ALM-488 relative to surgery 
 
Time from injection to skin incision 
 
Intensity (SBR) 
 
Surgeon rating scale 
The endpoints to evaluate the effect of timing of administration of ALM-488 relative to surgery 
on adequacy of fluorescence nerve labeling will be summarized for each of Cohorts B1 and B2, 
as well as overall for Cohort B. 
7.3 
Exploratory Endpoints 
To evaluate the ability of imaging techniques (i.e. camera systems) to distinguishing nerve tissue 
from non-nerve tissue, the following endpoints will be evaluated: 
 
Ability to detect fluorescence will be studied by determing an optimal cut-off using Youden 
index and receiver operating characteristic ROC curve analysis. 
This endpoint will be summarized separately for each device and each of the Cohorts A1-A6, B1 
and B2, as well as overall for Cohort A and Cohort B. 
To evaluate the intensity of signal of fluorescence relative to nerve diameter, the following 
endpoints will be evaluated: 
 
Intensity (signal-to-background ratio SBR) 
 
nerve diameter 
These endpoints will be plotted against each other, separately for each device and each of the 
Cohorts A1-A6, B1 and B2, as well as overall for Cohort A and Cohort B. 
To evaluate the intensity of signal of fluorescence when there is overlying tissue, the following 
endpoints will be evaluated: 
 
Signal-to-background ratio) 
This endpoint will be summarized separately for images and each of the Cohorts A1-A6, B1 and 
B2 as well as overall for Cohort A and Cohort B. 
To evaluate surgeon confidence and operative time with a surgeon rating scale, the following 
endpoints will be evaluated: 
 
Surgeon confidence will be captured via the surgeon rating scale (1=do not see nerve 
compared to WLR, 2=see nerve less well compared to WLR, 3=See nerve equally well 
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compared to WLR, 4=See nerve  better compared to WLR, 5=See nerve much better 
compared to WLR) for each image. 
 
For each surgery, the following yes/no question will be asked: “Outside of a clinical trial 
setting with all the required extra documentation, would this technology have saved you 
operative nerve-dissecting time compared to WLR?” 
Surgeon confidence will be summarized by image, while time saving (yes/no) will be summarized 
by patient.   
Surgeon confidence will be summarized for each device and each of the Cohorts A1-A6, B1 and 
B2, as well as overall for Cohort A and Cohort B. 
Time saving (yes/no) will be for each of the Cohorts A1-A6, B1 and B2, as well as overall for 
Cohort A and Cohort B. 
8 
General Statistical Methods and Definitions 
8.1 
General statistical methods 
Summary tables will be structured with a column for each device and/or cohort. 
The statistical analysis will be based on separate analysis populations, defined in section 4 Analysis 
Sets and Cohorts. Summary tables will usually be structured with a column for each cohort and the 
dose associated  (ALM-488 100mg, ALM-488 XXXmg ,…, ALM-488 XXXmg for Dose Escalation/De-
Escalation, and as well as for Dose Timing. If dose modification occurs or if fluorescence signal has 
not been achieved after 6 dose cohorts the modified or the additional dose escalation cohorts will 
be presented on the tables. 
Additional cohorts beyond dose cohort 6 may be enrolled if an adequate fluorescence signal has not 
been achieved after 6 dose cohorts have been evaluated. Additional dose escalation cohorts may be 
added following discussion with FDA if further improvement of fluorescence is needed for nerve 
identification and if the DLT rate is 0  
In general, continuous variables will be summarised using descriptive statistics, i.e. generally 
displaying number of subjects in the respective analysis population, number of subjects with data, 
number of subjects with missing values, mean, standard deviation, minimum, lower quartile, 
median, upper quartile, and maximum.  
Categorical variables will be summarised by using frequency counts and percentages.  For events 
that can occur more than once, the number of events may also be summarized. In addition, the 
number of subjects with missing values will be displayed.  
Means and medians will be presented by 1 additional decimal place and standard deviation will be 
presented by 2 additional decimal places than the standard presentation level of the respective data. 
Minimum and maximum values will be presented using the same number of decimal places as the 
data. Percentages will be presented to 1 decimal place if not otherwise stated.  
If the number of subjects in a category is 0, then percentage will not be displayed, and only a count 
of 0 will be shown.  
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P-values will be reported to 4 decimal places at least. Values less than 0.0001 will be displayed as 
<0.0001. Values above 0.9995 will be displayed as 1.0000. 
In listings, data will be sorted by dose Cohort, site, and subject ID, and when appropriate by image, 
visit or other identifiers for sequence or type of observation.  
Factor 365.2425 is used for duration conversions between days and years. Factor 30.436875 is used 
for duration conversions between days and months. 
If not otherwise specified, all statistical tests will be two-sided, and confidence intervals will be two-
sided and will be at the 95% confidence level. 
8.2 
Covariates and strata 
No stratified analyses are planned for this study. 
Study sites, countries, and regions 
The three sites are in the Unites States.  
 
101 - UCSD  
 
102  Harvard/MEE 
 
103 - Stanford 
8.3 
Subgroups 
For the efficacy analysis, the subgroup of images with overlying tissue will be explored. In this 
subgroup, the depth will be measured via the amount of overlying tissue that there can be to still 
maintain the ratio of nerve to non-nerves. Other subgroup analyses will be presented by cohorts and 
by cameras (OnLume or Zeiss). Both cameras are able to capture WLR and FL lights. 
 
Results for the different subgroups will be presented descriptively.  
8.4 
Missing data 
Only observed data will be summarized for all Cohorts. Except for partial or missing dates, no 
imputation for missing data is planned for the summary or analyses of these study data. 
The missing component(s) of incomplete dates (e.g. start and/or stop dates of AE, concomitant 
medication, medical history) will be assumed as the most conservative value possible. For example, 
if the start date has a missing day value, the first day of the month will be imputed for study day 
computations, etc. If day is missing for an end date, the last day of the month will be imputed. If the 
start date has a missing month value, the first month of the year will be imputed for study day 
computations, etc. If month is missing for an end date, the last month of the year will be imputed. 
For determination of treatment-emergent status, the start date will be imputed as the date of the 
first dose of study drug, unless there is clear evidence (through comparison of partial dates/times) to 
suggest otherwise. 
Date imputation will only be used for computational purposes such as treatment-emergent status, 
etc. Actual data values, as they appear in the original eCRFs, will be presented in the subject data 
listings. 
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8.5 
Rescreening 
Note, for subjects who are rescreened, their Day 1 labs will be used as their Screening and Day 1 
values. 
8.6 
Baseline Definition 
For all variables, baseline will be considered to be the last value obtained prior to the date and time 
of incision. Change from baseline will be the difference between the value at a given time point and 
the baseline value. The baseline value corresponds to the screening visit in the study schedule. 
8.7 
Study Day, Visit Window, and COVID-19 
For each patient, the day of surgery will be considered Study Day 1. Each assessment will be 
assigned a study day. The calculation for study day is dependent on whether the actual date of 
assessment is before or after the date of study Day 1 and is calculated as follows:  
 
Before study Day 1:  
 
study day = (date of assessment – Date of first dose of study medication Day 1). 
 
On or after study Day 1:  
 
study day = (date of assessment – Date of first dose of study medication Day 1) +1. 
 
Days within the screening period will be numbered with negative numbers until day -1 defined as 
the day before AML-488 treatment (Day 1). 
If the full date of the assessment is not known or not imputable based on the guidelines in Section 
7.3, then no study day will be assigned. Study Day, and any corresponding durations will appear 
partial or missing in the listings. 
 For each patient, the end of the study is defined as the date of their last contact in this study. 
 
Data will be analyzed based on the categorized visit captured in the database. No dates will be used 
to reassign visits or define visit windows. Although Day 15 or Early Termination is defined with +/-8 
days, extensions to the visit window may be allowed due to impacts related COVID-19.  Although 
Day 28/End of Study is defined with +/- 5 days, extensions to the visit window may be allowed due 
to impacts related COVID-19.   
 
Any impacts due to COVID-19 will be noted in the study documentation. 
 
The visits captured in the database include: 
 
Screening (Day -28 to Day -1) 
 
Baseline (Day 1) (pre- and post-operative) 
 
Day 15 or Early Termination 
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 
Day 28/End of Study 
9 
Patient Accounting and Disposition 
9.1 
Patient accounting 
The number and relative frequencies of subjects in each Cohort will be presented overall, by Analysis 
Sets, and individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, and B2 ), including 
individual reasons of exclusion from the respective analysis set. 
A list of subjects’ first ALM-488 infusion date time start, date time end, time to discontinuation and 
reasons for discontinuation will be presented. 
9.2 
Disposition and withdrawals 
The number and relative frequency will be presented for 
 
Subjects completing the study 
 
Subjects who prematurely discontinued the study 
 
Reasons for study discontinuation (Adverse Event, death, lost to follow-up, physician decision, 
pregregnancy, progressive disease, site or study terminated by sponsor, technical problems, 
withdrawal by subject, adverse device effect or other) 
 
Subjects completing surgery 
 
Subjects completing treatment 
 
Completing the study is defined as having completed the infusion, surgery, and end of study telephone 
call. 
A list of subjects’ trial end date, time to trial discontinuation and reason for trial discontinuation will 
be created. 
 
9.3 
Minor and Major Protocol deviations 
The number and relative frequency of subjects with major protocol deviations will be presented by 
treatment cohort and study site for FAS. 
Listings of all subjects with protocol deviations as specified above violating in- and exclusion criteria 
and allocation to trial populations will be created. 
10 Demographics and Baseline Characteristics 
Demographic and baseline characteristics as specified in detail below will be presented descriptively 
overall, by Analysis Sets, and by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2).  
10.1 Demographics 
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The following demographic characteristics will be presented by individual Cohorts (i.e., Cohorts: A, 
A1, A2, A3, A4, A5, A6, B, B1, B2). 
 
Sex (male/female) 
 
Age (years) 
 
Age group (≥18 and <65, ≥65 and <85, ≥85 years) 
 
Race (American Indian or Alaska Native, Asian, Black or African American, Native Hawaiian or 
Other Pacific Islander, White, Unknown, Other) 
 
Ethnicity (Hispanic or Latino, Not Hispanic or Latino, Not Reported, Unknown) 
 
Childbearing potential or less than 2 years menopausal (yes/no);  
 
Acceptable contraception (yes/no) 
 
Surgery type (Thyroidectomy, Parotidectomy, Neck Dissection) 
 
Date of informed consent, protocol version, informed consent form (ICF) type and ICF version 
number will be listed. 
10.2 Surgery Type 
The type of surgery (Thyroidectomy, Parotidectomy, Neck Dissection) will be summarized   overall in 
the Demographics and Baseline Characteristics and further listed overall, and by individual Cohorts 
(i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2). 
10.3 Medical history 
The diseases are coded according to the latest available Medical Dictionary for Regulatory Activities 
(MedDRA® version 23.1). The frequency of diseases recorded from medical history will be presented 
after classification into previous and concomitant conditions by system organ class (SOC) as well as 
the frequencies of preferred terms (PT) within each SOC. If subjects have more than one disease 
within an SOC or PT they will be counted only once for the respective SOC or PT. 
A listing of medical history will be provided. 
10.4 Exposure and Compliance 
The number and percentage of subjects will be presented by individual Cohorts (i.e., Cohorts: A, A1, 
A2, A3, A4, A5, A6, B, B1, B2) on the SAF analysis set. 
The infusion duration will be calculated in minutes as:  
When there are no interruptions in infusions: 
Infusion duration (minutes) = (time of infusion end – time of infusion start) + 1  
When infusion interruptions will be reported the time of the interruption need to be subtracted 
from time of infusion. 
Descriptive statistics for the treatment duration will be presented. 
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The infusion completed per protocol, details if deviated as well as infusion reactions IR will be 
described.  
Study ALM-488 dose and timing of administration and the summary of exposure will be listed by 
individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2) for the SAF Analysis Set  
10.5 Dosage and Timing of Administration 
Descriptive statistics for the dose and timing will be presented overall and by individual Cohorts (i.e., 
Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2) for the SAF Analysis Set. 
11 Previous and Concomitant Therapies 
Previous and concomitant medications are coded according to the World Health Organization drug 
dictionary (WHO-Drug version B3 March 2020) and stored with Anatomical Therapeutic Class (ATC) 
codes and generic names.  
Therapies will be classified as previous if the stop date and/or time was before the date of first dose 
of study medication. All other medications are defined as concomitant. Missing or partly missing 
stop dates will be imputed using the rules defined in Section 7.3. 
The number and frequency of previous and concomitant medications will be given per ATC level 2. If 
a patient has received more than 1 drug within an ATC class, he/she will be counted only once for 
this ATC class. 
Previous and Concomitant Therapies will be listed overall, by Analysis Sets, and by individual Cohorts 
(i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2). 
12 Determination of Optimal Dose 
The optimal dose will be the lowest possible dose and toxicity profile that achieves adequate 
imaging performance: (1) DLT rate is 0 subjects for a 3-patient cohort (2) adequate fluorescence 
signal of nerves compared to background non-nerve tissue. A dot plot will be presented to display 
Cohorts A1 through A6.  Cohorts A1 through A6 will be displayed on the x-axis, with a cluster of 3 to 
6 dots for each cohort.  The dose will be displayed on the y-axis.  Dots will be open circles for 
subjects who do not experience a DLT. Solid red dots will be used for subjects who experience a DLT. 
A listing of the DLTs will also be provided. 
 
Image intensity ratios will also be plotted with time from injection to skin incision on the x-axis, 
image intensity ratio on the y-axis, and a separate color for each dose level. 
13 Efficacy 
The data on the signal-to-background ratio for FL and WLR will be provided by Stanford University 
for the different regions of interest ROIs. All efficacy analyses will be completed on the FAS Analysis 
Set and will be repeated in PP analysis set if the latest is different from FAS Analysis Set. The 
analyses using signal to background ratio SBR for cohort A will only use images produced by Zeiss 
camera wihle both Zeiss and OnLume devices will be used for timing cohort B.  
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13.1 Timing, Dose, Size and Intensity 
The efficacy endpoints pertaining to timing of administration, dose, and intensity (signal-to-
background ratio SBR), along with the surgeon rating scale will be assessed throughout enrollment 
into Cohorts A1-A6 to determine the final dose for use in Cohorts B1 and B2. These efficacy 
endpoints will also be used to evaluate the effect of timing of administration of ALM-488 by 
comparison of Cohort B1 and B2.  The following analyses are planned for these efficacy endpoints: 
 
For each Cohort, SBR will be averaged to get a single mean ratio per patient; then a paired t-
test or Wilcoxon signed rank test will be used, as appropriate, to compare SBR between FL 
and WLR. 
 
SBR will be also listed by cohort type, and surgery type and device for both FL with WLR. 
 
Image intensity ratios will also be analyzed with repeated measures analysis of variance 
(RMANOVA) with timing, image, method (FL /WLR), and device (Zeiss/OnLume) as the 
repeated factors in the model. 
 
SBR for FL and WLR measurements will be plotted. Values to the right of the line will indicate 
that there is improved visualization with FL images and values to the left of the line will 
indicate that there is improved visualization with WLR images(Equal performance of FL 
versus WLR will be indicated by dashed line (slope = 1)]. 
 
To further assess timing, Cohorts B1 and B2 will be compared. Image ratios will be averaged 
to get a single mean ratio per patient; then an unpaired t-test or Wilcoxon rank sum test will 
be used, as appropriate, separately for FL and WLR. 
Paired t-tests will be used unless markedly non-normal data is observed including bimodal or other 
severe assumption violations; minor skewness is robust to the paired t-test and RMANOVA 
assumptions. If extreme skewness with outliers, or bimodal distributions are observed, then a 
Wilcoxon signed rank test will be used instead of a paired t-test and RMANOVA will be performed on 
the ranked data. 
Similarly, unpaired t-tests will be used unless markedly non-normal data is observed including 
bimodal or other severe assumption violations; minor skewness is robust to the unpaired t-test 
assumptions. If extreme skewness with outliers, or bimodal distributions are observed, then a 
Wilcoxon rank sum test will be used instead. 
The optimal cut-off points of SBR and the ability to detect the nerve will be studied using ROC 
(Receiver Operating Characteristics) curves analyses through logistic regression. In the logistic 
regression the outcome variable will be the categorized surgeon rating scale 1 : I do not see the 
nerve  versus 4 to 5 : the nerve is visible. The Youden J index  given by J= Sensitivity - (1 - Specificity ) 
will be used to select the optimal predicted probability cut-off. It is the maximum vertical distance 
between ROC curve and diagonal line. This will allow to maximize the difference between True 
Positive and False Positive. All possible SBR values and the corresponding sensitivity and specificity 
will be obtained using proc logistic in SAS [1]. The optimal cut-off will be determined overall and by 
device and individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2). 
Image intensity ratios will be plotted against nerve diameter, with ratios on the y-axis and diameters 
on the x-axis. Plots will be displayed overall and by device and individual Cohorts (i.e., Cohorts: A, A1, 
A2, A3, A4, A5, A6, B, B1, B2).   
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Image intensity ratios will be summarized separately for images with and without overlying tissue, 
overall and by device and individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2).   
In the subset of images with overlying non-nerve tissue, depth will be measured via the amount of 
overlying tissue that there can be to still maintain the ratio of nerve to non-nerves. Depth will be 
summarized overall and by device and individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, 
B1, B2). 
13.2 Surgical Imaging Worksheet/ Rating Scale 
Surgeon confidence will be captured via the surgeon rating scale (1=do not see nerve , 2=see nerve 
less well compared to WLR, 3=see nerve equally well compared to WLR, , 4=see nerve  better 
compared to WLR, 5=see nerve much better compared to WLR ) for each image. Surgeon confidence 
will be summarized overall and by device, individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, 
B, B1, B2) as well surgery type. 
Image intensity ratios will also be correlated with the surgeon rating scale using Pearson correlation 
coefficients unless markedly non-normal data is observed including bimodal or other severe 
assumption violations; otherwise Spearman correlation coefficients will be used.  Correlation 
coefficients will be presented overall and by device and by individual Cohorts (i.e., Cohorts: A, A1, 
A2, A3, A4, A5, A6, B, B1, B2). 
A listing will be provided. 
13.3 Clinical Benefit ALM-488 Questionnaire 
Clinical benefit for intraoperative nerve visualization questionnaire will be listed by individual 
Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2) for each patients and type of surgery. 
13.4 Nerve Dissecting Time 
For each surgery, the following yes/no question will be asked: Outside of a clinical trial setting with 
all the required extra documentation, would this technology have saved you operative nerve-
dissecting time compared to WLR alone (yes/no)?  
Counts and percentages will be summarized overall and by device and individual Cohorts (i.e., 
Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2). 
14 Safety 
All safety analyses will be completed on the SAF Analysis Set. 
14.1 Adverse Events 
All information obtained on AEs will be displayed by treatment and patient. The number and 
percentage of subjects with AEs will be tabulated by System Organ Class (SOC) and Preferred Term 
(PT) with a breakdown by treatment. A patient with multiple AEs events within a SOC is only counted 
once towards the total of this SOC. 
AEs will be coded using MedDRA version 23.1 or higher and presented by SOC and PT. 
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The analysis will focus on the treatment-emergent AEs (TEAE), i.e., AEs which started or worsened 
on or after the study ALM-488 infusion and before the end of the study.  
Number and frequencies of subjects with TEAEs as well as the number of events will be given by SOC 
and by PT within each SOC overall and by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, 
B, B1, B2) for the following: 
 
Any AE 
 
AE type  
 
At least one TEAE 
 
Serious TEAEs 
 
TEAE by CTCAE  
 
Infusion reactions IR overall and by CTCAE 
 
Related TEAEs  
 
Adverse Device Effects 
 
All TEAEs by maximum intensity  
The counts and percentages of subjects with TEAEs as well as number of events will be presented by 
SOC and by PT and by decreasing frequency for:  
 
All TEAEs  
 
IR 
 
Serious TEAEs. 
 
Related TEAEs 
 
Serious Related. 
 
TEAEs leading to study discontinuation (if occurring in more than 5 subjects, otherwise a listing 
will be sufficient) 
 
Adverse Device Effects 
 
All TEAEs by intensity. 
 
All related TEAEs by intensity 
 
All TEAEs by outcome  
Additionally, number and frequencies of subjects with TEAEs and number of events will be 
presented by PT and by decreasing frequency for 
 
All TEAEs  
 
Serious TEAEs  
 
Related TEAEs. 
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Finally, a summary of Serious TEAEs and deaths by individual Cohorts and for all patients 
Listings will be created for 
 
All AEs  
 
Serious AEs (SAEs)  
 
AEs leading to discontinuation of study medication  
 
AEs leading to discontinuation from study  
 
AEs leading to death  
and will also include subjects screened but not infused with ALM-488. 
If severity is missing, the event will not be included in the frequency tables presenting events by 
intensity. If relationship to study drug is missing, the event will be assessed as unrelated if it started 
before start of study medication; in all other cases it will be assumed to be related. 
14.2 Vital Signs 
The actual values and the change from baseline in vital signs will be summarized over time with n, 
mean, standard deviation, and median, minimum, and maximum.  
Vital signs i.e. height, weight, systolic blood pressure, diastolic blood pressure, heart rate, and 
respiratory rate will be summarized descriptively over time by individual Cohorts (i.e., Cohorts: A, 
A1, A2, A3, A4, A5, A6, B, B1, B2). 
Furthermore, the number and percentage of subjects based on reference ranges will be described 
according to the derived categories (i.e. Low, Normal, High) for vital signs. 
A listing of vital signs will be presented for all measurements  
14.3 Clinical Laboratory evaluations 
Hematology, Clinical Chemistry, and Coagulation, Urinalysis 
All laboratory data will be listed by individual cohort, patient and time point and if normal ranges are 
available abnormalities will be flagged. Summary statistics will be provided by study dose and time 
point. 
The Hematology parameters include: red blood cells, hemoglobin, hematocrit, mean corpuscular 
volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood 
cells, neutrophils, eosinophils, basophils, lymphocytes, monocytes, %neutrophils, %eosinophils, 
%basophils, %lymphocytes, %monocytes. 
The clinical chemistry parameters include: alkaline phosphatase, AST/SGOT, ALT/SGPT, total 
bilirubin, calcium, phosphorus, blood urea nitrogen, creatinine, GFR, total protein, albumin, glucose, 
potassium, sodium, chloride, magnesium, bicarbonate, uric acid, lactate dehydrogenase. 
Coagulation parameters include: PT, aPTT, INR. 
The urinalysis analysis will include protein, blood, leukocytes, glucose, ketones and pH. 
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Quantitative data will be examined for trends using descriptive statistics (number of patients with 
data, number of patients with missing values, mean, standard deviation, median, minimum, and 
maximum) of actual values and changes from baseline to each visit/time point. This will be 
presented by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2). 
Quantitative data based on reference ranges will be described according to the NCI-CTCAE version 
5.0 grade at each visit over time and by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, 
B1, B2). 
Qualitative data will be described according to the categories (i.e. Normal; Abnormal, Not clinically 
significant; Abnormal, Clinically significant; Missing) and by individual Cohorts (i.e., Cohorts: A, A1, 
A2, A3, A4, A5, A6, B, B1, B2).  
Shift tables of quantitative data showing changes with respect to the normal range and clinical 
significance between baseline and endpoint post-baseline visits as well as the worst value at any 
post-baseline visit.  
Shift tables of qualitative data showing changes between baseline and post-baseline visits.  
Data from unplanned determinations, i.e. usually determinations where the investigator felt follow-
up (FU) was necessary, will be included in the number and frequency counts of clinically significant 
values. They will also be included in the data listings. 
A listing of laboratory values will be presented for all measurements and for abnormal findings, i.e. 
measurements outside of reference ranges. Laboratory values that are outside of reference ranges 
will be flagged in the data listings, along with corresponding reference ranges.  
Shift tables showing changes with respect to toxicity grade according to NCI-CTCAE version 5.0 will 
be listed using the worst grade. 
Laboratory values will be listed by timepoint (screening, prior to ALM-488 infusion (baseline), after 
ALM-488 infusion, Day 15, Day 28), overall and by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, 
A5, A6, B, B1, B2) and for all patients. 
Data from unplanned visits will not be summarized but will be included in the data listings. 
 
14.4 Echocardiograms (ECGs) 
Values of heart rate, PR, QRS, QT and QTcF, the interpretation and the clinical significance of the ECG 
will be summarized by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2) using 
descriptive statistics for 12-Lead ECG/ Lead II Rhythm Strip. In addition, QTcF will be summarized 
using categories as defined in ICH E14 (i.e., QTcF (ms): >450-480; >480-500; >=500). 
The Bazett’s Correction (QTcB) and Fridericia’s Correction (QTcF) are derived as follows:  
Bazett’s Correction (QTcB)  
RR
QT
QTc b 
 
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Fridericia’s Correction (QTcF)  
3 RR
QT
QTc f 
 
where: RR = RR-interval measured in seconds. 
ECG results will be listed including clinically significant data for the 12-lead ECG.  
14.5 Physical Examinations 
Results from physical examinations will be summarized by visit/timepoint (screening, prior to ALM-
488 infusion (baseline), after ALM-488 infusion, Day 15, Day 28), change from baseline by individual 
Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2). 
PE will be provided in a data listing by subject. 
14.6 Blood alergy 
Results from blood allergy parameters  will be summarized by visit/timepoint (pre-infusion, end of 
infusion, thirty minutes after end of infusion and 22 hours after the end of infusion), by individual 
Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, B2). 
Blood alergy will be provided in a data listing by subject. 
14.7 Pregnancy Tests 
All available data will be listed by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, 
B2). 
14.8 Serum Samples 
All available data will be listed by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, 
B2). 
14.9 Chest X-Ray/CT 
All available data will be listed by individual Cohorts (i.e., Cohorts: A, A1, A2, A3, A4, A5, A6, B, B1, 
B2). 
15 Pharmacokinetic Analyses (PK) 
The PK analyses will be described in a separate PK SAP and will use the PP and PK analysis set. 
16 Interim Analyses 
An interim analysis is not planned for this study. 
17 Statistical Analyses for Safety Monitoring 
Whenever a cohort is completed or when needed based on safety and toxicity information, selected 
outputs from the final analysis will be created for safety monitoring by the IDMC. The content of 
these safety analyses is specified in a separate IDMC Charter. 
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18 Statistical Analyses for Clinical Trial Registries 
The following additional analyses will be performed for the purpose of fulfilling the Clinical Trial 
Registry (CTR) requirements: 
Both Serious Adverse Events and Non-Serious Adverse Events will be summarized by MedDRA 
preferred term. 
 
An adverse event is considered ‘Serious’ whether or not it is a TEAE 
 
An adverse event is considered in the ‘Non-Serious’ category if it is both a TEAE and is not 
serious  
For each Serious AE and Non-Serious AE, for each term and treatment group, the following are 
provided: 
 
the number of subjects at risk of an event; 
 
the number of subjects who experienced each event term; 
 
the number of events experienced. 
 
19 Software  
The data will be analysed using SAS Version 9.4 or higher.  
 
 
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20 Tables, Listings, and Figures 
20.1 Tables 
Below is Table of Contents for the Tables. 
SUBJECT DISPOSITION 
Table 14.1.1 Reasons for Screen Failure, cohort A Analysis set: ENR 
Table 14.1.2 Summary of Analysis Sets and Cohorts Analysis Set: ENR 
Table 14.1.3 Disposition and Discontinuation Reasons [1], Cohort A  Analysis Set: ENR 
Table 14.1.3 Disposition and Discontinuation Reasons, Cohort B  Analysis Set: ENR 
Table 14.1.4 Disposition by Site, Cohort A Analysis Set: ENR 
Table 14.1.4 Disposition by Site, Cohort B  Analysis Set: ENR 
PROTOCOL DEVIATIONS 
Table 14.1.5 Minor and Major Protocol Deviations overall and by site, Cohort A Analysis Set: SAF 
Table 14.1.5 Minor and Major Protocol Deviations overall and by site, Cohort B  Analysis Set: SAF 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS 
Table 14.1.6 Demographics and Baseline Characteristics, Cohort A Analysis Set: SAF 
Table 14.1.6 Demographics and Baseline Characteristics, Cohort B  Analysis Set: SAF 
MEDICAL HISTORY AND CONCOMITANT DISEASES 
Table 14.1.7 Medical History, Cohort A  Analysis Set: SAF 
Table 14.1.7 Medical History, Cohort B  Analysis Set: SAF 
PRIOR AND CONCOMITANT MEDICATIONS 
Table 14.1.8 Prior and Concomitant Medications, Cohort A Analysis Set: SAF 
Table 14.1.8  Prior and Concomitant Medications, Cohort B  Analysis Set: SAF 
EXPOSURE AND COMPLIANCE 
Table 14.1.9 Exposure and Compliance, Cohort A Analysis Set: SAF 
Table 14.1.9 Exposure and Compliance, Cohort B  Analysis Set: SAF 
EFFICACY 
Table 14.2.2.1 SBR comparison between FL and WLR, Zeiss camera Cohort A Analysis Set: FAS 
Table 14.2.2.2 SBR comparison between FL and WLR, Cohort B  Analysis Set: FAS 
Table 14.2.3.1 SBR comparison between FL and WLR – Zeiss Image Analysis, Cohort A Analysis Set: FAS 
Table 14.2.3.2 SBR comparison between FL and WLR – Image Analysis, Cohort B  Analysis Set: FAS 
Table 14.2.4.1 SBR comparison between B1and B2 – FL  Analysis Set: FAS 
Table 14.2.4.2 SBR comparison between B1 and. B2– WLR  Analysis Set: FAS 
Table 14.2.5.1 Surgeon Rating Scale, Cohort A  Analysis Set: FAS 
Table 14.2.5.1 Surgeon Rating Scale, Cohort B Analysis Set: FAS 
Table 14.2.6.1 Correlation Between Surgeon Rating Scale and SBR for Zeiss device, Cohort A  -  Analysis Set: FAS 
Table 14.2.6.2 Correlation Between Surgeon Rating Scale and SBR, Cohort B  Analysis Set: FAS 
Table 14.2.7.1 SBR and Surgeon Rating Scale for Zeiss device Cohort A, FL Analysis Set: FAS 
Table 14.2.7.2 SBR and Surgeon Rating Scale Cohort B, FL Analysis Set: FAS 
Table 14.2.8 Optimal cut-off point of SBR Cohort A and B, FL-  Analysis Set: FAS 
Table 14.2.9.1 Surgeon nerve dissecting time saved, Cohort A Analysis Set: FAS 
Table 14.2.9.1 Surgeon nerve dissecting time saved, Cohort B Analysis Set: FAS 
SAFETY 
Table 14.3.1.1 Overview of Averse events and Treatment Emergent Adverse Events in Cohort  A  Analysis Set: SAF 
Table 14.3.1.1 Overview of Averse events and Treatment Emergent Adverse Events in Cohort  B (same table presented in 
separate page) Analysis Set: SAF 
Table 14.3.1.2 TEAEs incidence primary System Organ Class and Preferred Term Cohort A-  Analysis set: SAF 
Table 14.3.1.2 TEAEs incidence primary System Organ Class and Preferred Term Cohort B Analysis set: SAF 
Table 14.3.1.3 Infusion Related Reactions– by Primary SOC and PT, Cohort A  Analysis set: SAF 
Table 14.3.1.3 Infusion Related Reactions– by Primary SOC and PT, Cohort B   Analysis set: SAF 
Table 14.3.1.4 Serious TEAEs by SOC and PT,Cohort A Analysis set: SAF 
Table 14.3.1.4 Serious TEAEs by SOC and PT, Cohort B  Analysis set: SAF 
Table 14.3.1.5 Related TEAEs by MedDRA System Organ Class and Preferred Term, Cohort A   Analysis set: SAF 
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Table 14.3.1.5 Related TEAEs by MedDRA System Organ Class and Preferred Term, Cohort B-  Analysis set: SAF 
Table 14.3.1.6 Serious Related TEAEs by SOC and PT, Cohort A Analysis set: SAF 
Table 14.3.1.6 Serious Related TEAEs by SOC and PT, Cohort B  Analysis set: SAF 
Table 14.3.1.7 TEAEs Leading to Study Discontinuation by SOC and PT, Cohort A Analysis set: SAF 
Table 14.3.1.7 TEAEs Leading to Study Discontinuation by SOC and PT, Cohort B  Analysis set: SAF 
Table 14.3.1.8 Adverse Device Effects, Cohort A  Analysis set: SAF 
Table 14.3.1.8 Adverse Device Effects, Cohort B  Analysis set: SAF 
Table 14.3.1.9 TEAEs by Primary SOC and PT by intensity, Cohort A Analysis set: SAF 
Table 14.3.1.9 TEAEs by Primary SOC and PT by intensity, Cohort B Analysis set: SAF 
Table 14.3.1.10 Related TEAEs by Primary SOC and PT by intensity, Cohort A Analysis set: SAF 
Table 14.3.1.10 Related TEAEs by Primary SOC and PT by intensity, Cohort B  Analysis set: SAF 
Table 14.3.1.11 TEAEs by Primary SOC and PT by Outcome, Cohort A Analysis set: SAF 
Table 14.3.1.11 TEAEs by Primary SOC and PT by Outcome, Cohort B  Analysis set: SAF 
Table 14.3.1.12 TEAEs by PT, Cohort A Analysis set: SAF 
Table 14.3.1.12 TEAEs by PT, Cohort B Analysis set: SAF 
Table 14.3.1.13 Serious TEAEs by PT, Cohort A Analysis Set: SAF 
Table 14.3.1.13 Serious TEAEs by PT, Cohort B  Analysis Set: SAF 
Table 14.3.1.14 Incidence of Related TEAEs by PT, Cohort A Analysis Set: SAF 
Table 14.3.1.14 Incidence of Related TEAEs by PT, Cohort B  Analysis Set: SAF 
Table 14.3.1.15 Summary of Treatment-Emergent Serious adverse events and Deaths, Cohort A  Analysis Set: SAF 
Table 14.3.1.15 Summary of Treatment-Emergent Serious adverse events and Deaths, Cohort B  Analysis Set: SAF 
SAFETY: VITAL SIGNS AND OXYGEN SATURATION 
Table 14.3.2.1.1 Vital Signs and Oxygen Saturation, Cohort A Analysis set: SAF 
Table 14.3.2.1.2 Vital Signs and Oxygen Saturation, Cohort B Analysis set: SAF 
Table 14.3.2.2.1 Vital Signs and Oxygen Saturation - Categorized by Reference Ranges, Cohort A Analysis set: SAF 
Table 14.2.2.2.2 Vital Signs and Oxygen Saturation - Categorized by Reference Ranges, Cohort B Analysis set: SAF 
SAFETY: LABORATORY PARAMETERS 
Table 14.3.3.1.1 Laboratory Parameters (Quantitative), Cohort A Analysis set: SAF 
Table 14.3.3.1.2 Laboratory Parameters (Quantitative), Cohort B  Analysis set: SAF 
Table 14.3.3.2.1 Laboratory Parameters (Quantitative) by Derived Categories, Cohort A Analysis Set: SAF 
Table 14.3.3.2.2 Laboratory Parameters (Quantitative) by Derived Categories, Cohort B  Analysis Set: SAF 
Table 14.3.3.3.1 Laboratory Parameters (Qualitative), Cohort A Analysis Set: SAF 
Table 14.3.3.3.2 Laboratory Parameters (Qualitative), Cohort B  Analysis Set: SAF 
Table 14.3.3.4.1 Laboratory Parameters (Quantitative) – Shift Table Regarding Reference Ranges and Clinical Significance, 
Cohort A Analysis Set: SAF 
Table 14.3.3.4.2 Laboratory Parameters (Quantitative) – Shift Table Regarding Reference Ranges and Clinical Significance, 
Cohort B Analysis Set: SAF 
Table 14.3.3.5.1 Laboratory Parameters (Qualitative) – Shift Table, Cohort A Analysis Set: SAF 
Table 14.3.3.5.2 Laboratory Parameters (Qualitative) – Shift Table, Cohort B Analysis Set: SAF 
BLOOD ALERGY SAMPLES 
Table 14.3.3.6 Blood allergy samples Cohort A Analysis Set: SAF 
Table 14.3.3.6 Blood allergy samples Cohort B Analysis Set: SAF 
PHYSICAL EXAMINATIONS 
Table 14.3.4.1.1 Physical Examinations, Cohort A Analysis Set: SAF 
Table 14.3.4.1.2 Physical Examinations, Cohort B  Analysis Set: SAF 
ELECTROCARDIOGRAMS 
Table 14.3.5.1 12-Lead Electrocardiography/ Lead II Rhythm Strip, Cohort A Analysis Set: SAF 
Table 14.3.5.2 12-Lead Electrocardiography/ Lead II Rhythm Strip, Cohort B  Analysis Set: SAF 
20.2 Listings 
Below is Table of Contents for the Listings. 
Listing 16.2.1 Patient Completion/Discontinuation Analysis Set: SAF 
Listing 16.2.2 Minor and Major Protocol Deviations Analysis Set: SAF 
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Listing 16.2.3 Subject Allocation to Trial Populations And Eligibility Assessment Analysis Set: FAS 
Listing 16.2.4 Demographic Data and Baseline Characteristics Analysis Set: SAF 
Listing 16.2.5 Medical History and Concomitant Diseases Analysis Set: SAF 
Listing 16.2.6.1 Previous and Concomitant Medication (Part 1) Analysis Set: SAF 
Listing 16.2.6.2 Previous and Concomitant Medication (Part 2) Analysis Set: SAF 
Listing 16.2.7 Exposure and compliance Analysis Set: SAF 
Listing 16.2.8 Clinical benefit for intraoperative nerve visualization Analysis Set: FAS 
Listing 16.2.9 Dose Limiting Toxicity Analysis Set: FAS 
Listing 16.2.10.1.1 All Adverse Events (Part 1 ) Analysis Set: SAF 
Listing 16.2.10.1.2 All Adverse Events (Part 2 ) Analysis Set: SAF 
Listing 16.2.10.2 Serious Adverse Events Analysis Set: SAF 
Listing 16.2.10.3 Adverse Events Leading to Withdrawal of Study Medication Analysis Set: SAF 
Listing 16.2.10.4 Adverse Events Leading to Discontinuation from Trial Analysis Set: SAF 
Listing 16.2.10.5 Adverse Events Leading to Death Analysis Set: SAF 
Listing 16.2.11 Vital Signs, Oxygen Saturation and Body Temperature Values Analysis Set: SAF 
Listing 16.2.11 Vital Signs, Oxygen Saturation and Body Temperature – Values Outside of Reference Ranges Analysis Set: 
SAF 
Listing 16.2.12 Laboratory Values Analysis Set: SAF 
Listing 16.2.12 Laboratory Values Outside of Reference Ranges Analysis Set: SAF 
Listing 16.2.12.3 Urinalysis Values Analysis Set: SAF 
Listing 16.2.13 Childbearing Potential and Pregnancy Test Results Analysis Set: FAS 
Listing 16.2.14 Electrocardiography 12-Lead ECG/Lead II Rhythm Strip Analysis Set: SAF 
Listing 16.2.14 Electrocardiography 12-Lead ECG/Lead II Rhythm Strip – Clinically Significant Findings Analysis Set: SAF 
Listing 16.2.15 Chest X-Rays/CT Analysis Set: SAF 
Listing 16.2.16 Physical Examination Analysis Set: SAF 
Listing 16.2.17.1 Surgical Imaging Worksheet (Part 1) Analysis Set: FAS 
Listing 16.2.17.2  Surgical Imaging Worksheet (Part 2) Analysis Set: FAS 
Listing 16.2.18 SBR Analysis Set: FAS 
20.3 Figures 
Below is Table of Contents for the Figures. 
Figure 14.3.1  Dot Plot of Dose Limiting Toxicities by Dose Analysis Set: DLT 
Figure 14.3.2  Scatter Plot of Intensity Ratios by Time of Injection to Skin Incision and by Dose Analysis Set: FAS 
Figure 14.3.3  Scatter Plot of Intensity Ratios by Nerve Diameter and by Dose  Analysis Set: FAS 
Figure 14.3.4 SBR for WLR and FL Analysis Set: FAS 
21 References 
1. Manoj Pandey (2016) ROC Curve: Making way for correct diagnosis SP11 - PharmaSUG 2016 
Ephicacy Lifescience Analytics Pvt. Ltd., Bangalore, India Abhinav Jain, Ephicacy Consulting Group 
Inc., Iselin, NJ https://www.pharmasug.org/proceedings/2016/SP/PharmaSUG-2016-SP11.pdf 
2. Hassan T. et al. (2019): Vital signs in adults. National quality improvement project, national 
report 2018/19. The Royal College of Emergency Medicine. 
 
22 Appendices 
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Appendix I: Normal ranges for vital signs, oxygen saturation and body temperature 
Parameter 
LLN 
ULN 
Blood pressure (diastolic) 
50 mmHg 
90 mmHg 
Blood pressure (systolic) 
90 mmHg 
140 mmHg 
Pulse rate 
60 bpm 
100 bpm 
Respiratory rate 
10 bpm 
20 bpm 
Oxygen saturation 
92 % 
100 % 
Body temperature 
35.0 °C 
38.0 °C 
LLN = lower limit of normal, ULN = upper limit of normal, bpm = beats per minute. 
LLNs and ULNs for pulse rate, respiratory rate, oxygen saturation and body temperature are taken 
from [2]. 
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Prot_001.pdf: 
ALUME BIOSCIENCES, INC  
Protocol ALM-488-001 
Amendment 1, 24 August 2020  
 
  
 
 
 
 
 
 
PHASE 1/2 TRIAL OF ALM-488 IN PATIENTS UNDERGOING  
HEAD & NECK SURGERY 
 
PROTOCOL NUMBER: ALM-488-001 
 
 
 
 
Product:  
 
 
ALM-488 Sterile Solution  
 
Sponsor:  
 
 
Alume Biosciences, Inc. 
3210 Merryfield Row 
San Diego, CA 92121 
 
 
Date of Protocol:  
 
24 August 2020 
Protocol Version 
Issue Date 
Original Protocol 
26 April 2020 
Amendment 1 
24 August 2020 
 
 
 
 
 
 
 
 
 
Illuminating nerves, preserving  function.™ 
                  Precision Surgery.™  
  3210 Merryfield Row, San Diego, CA 92121 


ALUME BIOSCIENCES, INC  
Protocol ALM-488-001 
Amendment 1, 24 August 2020  
 
  
STUDY SUMMARY 
 
Name of Sponsor 
Alume Biosciences, Inc. 
Name of Investigational 
Product 
ALM-488 Sterile Solution  
Name of Active  
Ingredient 
ALM-488 
Protocol Title 
Phase 1/2 Trial Of ALM-488 In Patients Undergoing Head & Neck Surgery 
Protocol Number 
ALM-488-001 
Phase of Development 
Phase 1/2 
Study Duration 
18 months 
Study Center(s) 
Multi-center study (United States) 
Objectives 
Primary Objective: 
• 
To evaluate the safety of ALM-488 administered by intravenous (IV) 
infusion to patients undergoing head and neck surgery.  
 
Secondary Objectives: 
• 
To characterize the pharmacokinetics (PK) of ALM-488 in patients 
undergoing head and neck surgery. 
• 
To determine the recommended dose of ALM-488 needed to generate 
an adequate fluorescence signal in nerves.  
• 
To evaluate the effect of timing of administration of ALM-488 relative 
to surgery (e.g., up to 5 hours before surgery) on  adequacy of 
fluorescence nerve labeling. 
 
Diagnosis 
and 
Main 
Inclusion/Exclusion 
Criteria 
This study will be conducted in patients who are undergoing head and neck 
surgery. Each subject must meet the following criteria to be enrolled in this 
study: 
 
Inclusion: 
1. The subject has a neoplasm located in the head and neck including 
parotid neoplasm, thyroid neoplasm, squamous cell carcinoma or an 
unknown primary with metastases to the cervical lymph node(s) or is 
undergoing a neck dissection for head and neck neoplasm in an 
unknown location. 
2. The subject’s primary surgical treatment is by parotidectomy 
(superficial or total) or thyroidectomy (unilateral or bilateral) or 
cervical neck dissection, respectively. 
3. The subject can understand and is willing to sign a written informed 
consent document. 
4. The subject is a ≥18 years of age. 
5. The subject has a life expectancy of at least 6 months. 
6. The subject has a total bilirubin within institution’s normal laboratory 
limits. 
7. The subject has an aspartate aminotransferase (AST)/serum glutamic-
oxaloacetic transaminase (SGOT) and alanine aminotransferase 
(ALT)/serum 
glutamic-pyruvic 
transaminase 
(SGPT) 
within 
institution’s normal laboratory limits. 
8. The subject has adequate renal function, defined as glomerular 


ALUME BIOSCIENCES, INC  
Protocol ALM-488-001 
Amendment 1, 24 August 2020  
 
  
filtration rate (GFR) ≥ 60 mL/min/1.73 m2. 
9. The subject, if of childbearing potential, must have a negative urine 
or serum pregnancy test and be using a medically acceptable form of 
contraception (e.g., hormonal birth control, intrauterine devices, 
double-barrier method) or abstinence. The subject, if male, must 
use a medically acceptable form of contraception (e.g. condom) 
or abstinence. 
10. The subject is willing to remain on-site for approximately 23 hours 
after administration of ALM-488 or, if required, stay overnight after 
the surgical procedure. 
11. The subject plans to undergo head and neck surgery. 
 
Exclusion: 
1. The subject has had prior radiation or chemotherapy for any prior 
head and neck neoplasm. 
2. The subject has had open surgery in the ipsilateral head and neck 
within 1 year before administration of the investigational product (IP). 
3. The subject has abnormal cardiac rhythm not controlled with 
medication, history of stroke within 1 year, history of coronary events 
within 1 year, and/or heart failure within 1 year before administration 
of the IP. 
4. The subject has a history of drug-induced acute tubular necrosis. 
5. The subject has current evidence of renal disease defined as 
glomerular filtration rate (GFR) < 60 mL/min/1.73 m2. 
6. The subject has received a systemic investigational drug of any kind 
for any indication within 6 weeks before administration of the IP. 
7. The subject is pregnant or breastfeeding. 
8. The subject has unresolved acute toxicity from prior anti-cancer 
therapy, as graded by the National Cancer Institute (NCI) Common 
Terminology Criteria for Adverse Events (CTCAE) v5.0.  Alopecia, 
neuropathy ≤ Grade 2, and other non-acute toxicities are acceptable. 
9.  The subject has a history of fluorescein allergy. 
10.  The subject has a history of severe or steroid dependent asthma. 
11.  Any other criteria deemed by the PI that may prevent the patient from 
successfully completing the trial.