Clinical trial NCT05457842
Conditions: Sentinel Lymph Node Biopsy Breast Cancer Melanoma
Keywords: ASP5354 pudexacianinium chloride SLN Breast Cancer Melanoma
Sponsor: Astellas Pharma Inc
Start date: 2022-08-10; Completion date: 2022-09-20
Enrollment: 1 participants; Allocation: NA; Intervention model: SEQUENTIAL

Brief summary:
This study was for women with breast cancer and for adults with melanoma. Breast cancer was a type of cancer when cells in the breast turn into cancer cells, which might grow out of control. Melanoma was a type of skin cancer that starts in cells called melanocytes. These cells made a substance called melanin which gives the skin its color. In this study, people had surgery to remove the lymph node closest to the site of their cancer. This lymph node was called the sentinel node. This was done to check if the cancer had spread from the original site to the sentinel node. This procedure was called a sentinel node biopsy.

This study provided more information on a potential new dye, called ASP5354, used in sentinel node biopsies. ASP5354 helped to show the lymph nodes more clearly during surgery. This helped the surgeon find the lymph node closest to the site of the cancer (sentinel node).

The main aim of the study was to find the best dose of ASP5354 that clearly showed the lymph nodes during surgery. This was an open-label study. That means each person in the study and the study doctors knew that person received ASP5354.

Each person only received 1 dose of ASP5354.

People that wanted to take part in the study were checked by a study doctor. This was on a separate visit before their surgery. Before surgery, people who took part in the study were asked if they had any other medical problems. They had a physical exam, an ECG to check their heart rhythm, and had their vital signs checked (blood pressure, pulse rate, and breathing rate). Other checks included some blood and urine samples taken for laboratory tests.

During surgery, a study surgeon injected ASP5354 near the cancer site. They recorded how clearly they could see the lymph nodes. Some blood samples were taken for laboratory tests and an ECG was done. After their surgery, people were asked if they have any other medical problems.

People returned to the hospital 9 days later for a check-up. The check-up included a physical exam, an ECG to check their heart rhythm, and a check of their vital signs (blood pressure, pulse rate, and breathing rate). Other checks included some blood samples taken for laboratory tests. People were asked if they had any medical problems and asked to complete a feedback survey.

Detailed description:

Arms: arm 1: Participant received a single dose of 0.2 milligrams (mg) intradermal injection of ASP5354 on day 1.
Interventions: intervention 1: pudexacianinium chloride
Intervention descriptions: intervention 1: Intradermal injection

Protocol documents:
Prot_000.pdf: 
Sponsor: API 
ISN/Protocol 5354-CL-1201 
- CONFIDENTIAL - 
Clinical Study Protocol 
11 MAY 2023 
Astellas 
Page 1 of 98 
 
A Phase 2 Open-label, Dose-finding Study to Determine  
the Optimal Dose for Lymph Node Visualization using ASP5354 
in Participants with Breast Cancer or Melanoma Undergoing 
Sentinel Lymph Node Biopsy 
ISN/Protocol 5354-CL-1201 
 
Amendment 3 [Nonsubstantial] 
 
11 MAY 2023 
 
IND 154703 
 
Sponsor: 
Astellas Pharma Inc. 
2-5-1, Nihonbashi-Honcho, Chuo-Ku, 
Tokyo 103-8411, Japan 
 
 
 
 
Protocol history: 
Original protocol [26 APR 2021] 
Amendment 1 [Nonsubstantial] [30 AUG 2021] 
Amendment 2 [Nonsubstantial] [01 AUG 2022] 
 
 
 
The information contained in this document is supplied as a background for clinical investigations. This document contains 
confidential information, which is the intellectual property of Astellas. By accepting or reviewing this document, you agree 
to hold this information in confidence and not copy or disclose it to others or use it for unauthorized purposes except (1) as 
otherwise agreed to in writing; (2) where required by applicable law; (3) where disclosure is directly related to the care and 
safety of the research participant; and (4) where disclosure of such information is made to a member of the investigator’s 
team who agrees to hold this information in confidence.  
 
 


Sponsor: API 
ISN/Protocol 5354-CL-1201 
- CONFIDENTIAL - 
Clinical Study Protocol 
11 MAY 2023 
Astellas 
Page 2 of 98 
Table of Contents  
CONTACT DETAILS OF SPONSOR’S KEY PERSONNEL ································
······  7 
PROTOCOL AMENDMENT SUMMARY OF CHANGES ································
········  8 
1 
PROTOCOL SUMMARY ································
································
·······  18 
1.1 
Synopsis ································
································
························
 18 
1.2 
Study Schema ································
································
··················
 25 
1.3 
Schedule of Assessments ································
································
·····  28 
1.3.1 
Sample Collection and Timed Procedure Schedule ································
· 31 
2 
INTRODUCTION ································
································
·················
 32 
2.1 
Study Rationale ································
································
················
 32 
2.2 
Background ································
································
····················
 32 
2.2.1 
Nonclinical and Clinical Data ································
··························
 33 
2.2.1.1 
Summary of Nonclinical Data ································
····················
 33 
2.2.1.2 
Summary of Clinical Data ································
························
 34 
2.2.2 
Summary of Key Safety Information for Investigational Product(s) ··············
 36 
2.3 
Risk/Benefit Assessment ································
································
·····  37 
2.3.1 
Risk Assessment ································
································
·········  37 
2.3.2 
Benefit Assessment ································
································
·····  37 
2.3.3 
Overall Risk-Benefit Conclusion ································
······················
 37 
3 
OBJECTIVES, ENDPOINTS AND ESTIMANDS ································
·········  38 
4 
STUDY DESIGN AND DOSE RATIONALE ································
················
 39 
4.1 
Overall Study Design ································
································
·········  39 
4.2 
Scientific Rationale for Study Design ································
······················
 42 
4.3 
Dose Rationale ································
································
················
 43 
4.4 
End of Study Definition ································
································
······  43 
5 
STUDY POPULATION ································
································
··········
 43 
5.1 
Inclusion Criteria ································
································
··············
 43 
5.2 
Exclusion Criteria ································
································
·············
 44 
5.3 
Lifestyle Considerations ································
································
······  45 
5.4 
Screen Failures ································
································
················
 46 
5.4.1 
Rescreening ································
································
··············
 46 
6 
INVESTIGATIONAL PRODUCT ································
·····························
 46 


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11 MAY 2023 
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Page 3 of 98 
6.1 
Investigational Product Administered ································
······················
 46 
6.1.1 
Investigational Product Administration ································
···············
 46 
6.1.2 
Other Therapy ································
································
············
 49 
6.2 
Preparation/Handling/Storage/Accountability ································
·············
 49 
6.2.1 
Packaging and Labeling ································
································
 49 
6.2.2 
Handling, Storage and Accountability ································
················
 49 
6.3 
Randomization and Blinding ································
································
 49 
6.4 
Investigational Product Compliance ································
························
 50 
6.5 
Dose Modification ································
································
············
 50 
6.6 
Continued Access to Investigational Product After the End of the Study ··············
 50 
6.7 
Treatment of Overdose ································
································
·······  50 
6.8 
Concomitant Therapy ································
································
·········  50 
6.8.1 
Previous Treatment (Medication) ································
·····················
 50 
6.8.2 
Concomitant Treatment (Medication and Non-Medication Therapy) ·············
 51 
6.8.3 
Prohibited Medication ································
································
··  51 
7 
STUDY PROCEDURES AND ASSESSMENTS ································
············
 51 
7.1 
Efficacy Assessments ································
································
·········  52 
7.1.1 
Primary and Secondary Efficacy Assessments································
·······  52 
7.1.2 
Signal Background Ratio ································
·······························
 53 
7.2 
Safety Assessments ································
································
···········
 53 
7.2.1 
Laboratory Assessments ································
································
 53 
7.2.2 
Vital Signs ································
································
················
 54 
7.2.3 
Physical Examination ································
································
···  54 
7.2.4 
Electrocardiogram ································
································
·······  54 
7.2.5 
Order of Assessments ································
································
···  54 
7.2.6 
Other Assessments ································
································
······  55 
7.2.6.1 
Green Coloration of Urine or Skin································
···············
 55 
7.3 
Adverse Events and Other Safety Aspects ································
·················
 55 
7.3.1 
Time Period and Frequency for Collecting Adverse Event and Serious 
Adverse Event Information································
·····························
 55 
7.3.1.1 
Adverse Device Effects ································
···························
 56 
7.3.2 
Method of Detecting Adverse Events and Serious Adverse Events ···············
 56 
7.3.3 
Follow-up of Adverse Events and Serious Adverse Events ························
 56 


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ISN/Protocol 5354-CL-1201 
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11 MAY 2023 
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Page 4 of 98 
7.3.4 
Regulatory Reporting Requirements for Serious Adverse Events/Adverse 
Device Effects ································
································
···········
 57 
7.3.5 
Disease-related Events and/or Disease-related Outcomes Not Qualifying as 
Adverse Events or Serious Adverse Events ································
··········
 57 
7.3.6 
Special Situations ································
································
········  57 
7.4 
Pharmacokinetics ································
································
··············
 58 
7.4.1 
Analysis of ASP5354 in Plasma ································
·······················
 58 
7.5 
Total Amount of Blood ································
································
·······  58 
7.6 
Study Participant Feedback ································
································
··  58 
8 
PARTICIPANT DISCONTINUATION ································
······················
 59 
8.1 
Discontinuation of Individual Participant(s) from Study Treatment ····················
 59 
8.2 
Discontinuation of Individual Participant(s) from Study ································
· 59 
8.3 
Lost to Follow-up ································
································
·············
 59 
9 
STATISTICAL CONSIDERATIONS ································
·························
 59 
9.1 
Statistical Hypotheses ································
································
········  59 
9.2 
Analysis Sets ································
································
···················
 60 
9.3 
Statistical Analyses ································
································
···········
 60 
9.3.1 
General Considerations ································
································
· 60 
9.3.2 
Primary Endpoint(s)/Estimand(s) Analysis ································
··········
 60 
9.3.2.1 
Primary Analysis ································
································
···  61 
9.3.3 
Secondary Endpoint(s)/Estimand(s) Analysis ································
·······  61 
9.3.4 
Exploratory Endpoint(s)/Estimand(s) Analysis ································
······  61 
9.3.5 
Safety Analyses ································
································
··········
 61 
9.3.5.1 
Adverse Events································
································
·····  61 
9.3.5.2 
Laboratory Assessments ································
··························
 62 
9.3.5.3 
Vital Signs ································
································
··········
 62 
9.3.5.4 
Electrocardiogram ································
································
· 62 
9.3.6 
Pharmacokinetics Analysis ································
·····························
 62 
9.3.7 
Other Analyses ································
································
···········
 62 
9.4 
Interim Analysis ································
································
···············
 62 
9.5 
Sample Size Determination ································
································
··  63 
9.6 
Additional Conventions ································
································
······  63 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS ································
································
·············
 63 


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10.1 
Appendix 1 Ethical, Regulatory and Study Oversight Considerations ·················
 63 
10.1.1 
Regulatory and Ethical Considerations ································
···············
 63 
10.1.2 
Financial Disclosure ································
································
····  64 
10.1.3 
Informed Consent of Participants································
······················
 64 
10.1.3.1 
Informed Consent Process ································
························
 64 
10.1.3.2 
Supply of New and Important Information Influencing the 
Participant’s Consent and Revision of the Written Information ·············
 65 
10.1.4 
Data Protection ································
································
··········
 65 
10.1.5 
Committee Structure ································
································
····  66 
10.1.5.1 
Visualization Review Committee ································
················
 66 
10.1.6 
Dissemination of Clinical Study Data································
·················
 67 
10.1.7 
Data Quality Assurance ································
································
· 67 
10.1.8 
Source Documents ································
································
······  68 
10.1.9 
Study and Site Start and Closure ································
······················
 68 
10.1.10 
Arrangement for Use of Information and Publication of the Study ···············
 69 
10.1.11 
Quality Assurance ································
································
·······  69 
10.2 
Appendix 2 Contraception Requirements ································
··················
 71 
10.3 
Appendix 3 Adverse Events/Adverse Device Effects: Definitions and 
Procedures for Recording, Evaluating, Follow-up and Reporting·······················
 73 
10.3.1 
Definition of Adverse Events ································
··························
 73 
10.3.1.1 
Abnormal Laboratory Findings ································
··················
 73 
10.3.1.2 
Potential Cases of Drug-induced Liver Injury ································
··  74 
10.3.2 
Definition of Serious Adverse Events································
·················
 74 
10.3.3 
Adverse Device Effect (ADE) ································
·························
 75 
10.3.3.1 
Definition of ADE ································
································
· 75 
10.3.3.2 
Definition of Unanticipated adverse device effect (UADE) ··················
 75 
10.3.3.3 
Definitions of Technical Complaints and Device Deficiencies ··············
 75 
10.3.3.4 
Risks/Precautions ································
································
··  75 
10.3.4 
Assessment of Causality ································
································
 76 
10.3.5 
Assessment of Severity ································
································
· 78 
10.3.6 
Recording and Follow-up of AEs, SAEs and/or ADEs ·····························
 78 
10.3.7 
Reporting Procedures for Serious Adverse Events/Adverse Device Effects ·····  79 
10.3.8 
Reporting Procedures for Pregnancy and Special Situations ·······················
 80 
10.3.8.1 
Contraceptive Guidance and Collection of Pregnancy Information ·········  80 
10.3.8.2 
Medication Error, Overdose and “Off-label Use” ·····························
 81 
10.3.8.3 
Misuse/Abuse ································
································
······  81 


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10.3.8.4 
Occupational Exposure ································
···························
 81 
10.3.8.5 
Suspected Drug-drug Interaction ································
················
 82 
10.3.8.6 
(Suspicion of) Transmission of Infectious Agent ······························
 82 
10.3.9 
Supply of New Information Affecting the Conduct of the Study ··················
 82 
10.3.10 
Urgent Safety Measures ································
································
 82 
10.3.11 
Reporting Urgent Safety Measures ································
····················
 82 
10.4 
Appendix 4 Liver Safety Monitoring and Assessment ································
···  83 
10.5 
Appendix 5 List of Excluded Concomitant Medications ································
· 85 
10.6 
Appendix 6 Clinical Laboratory Assessments ································
·············
 86 
10.7 
Appendix 7 Clinical Study Continuity ································
······················
 87 
10.8 
List of Abbreviations and Definition of Key Study Terms ·······························
 92 
11 
REFERENCES ································
································
····················
 95 
12 
INVESTIGATOR’S SIGNATURE ································
····························
 97 
13 
PROTOCOL ATTACHMENTS ································
·······························
 98 
 
List of In-text Tables 
Table 1 
Schedule of Assessments ································
································
·····  28 
Table 2 
Sample Collection and Timed Procedure Schedule ································
·······  31 
Table 3 
Study Objectives and Endpoints ································
·····························
 38 
Table 4 
Investigational Product ································
································
·······  46 
Table 5 
Blood Sampling ································
································
···············
 58 
Table 6 
Quality Tolerance Limits ································
································
·····  70 
Table 7 
Grading Scale Defining the Severity of an Adverse Event ·······························
 78 
Table 8 
Moderate and Severe Liver Abnormalities ································
·················
 83 
Table 9 
Clinical Laboratory Tests ································
································
····  86 
Table 10 
Alternative Schedule of Assessments in Response to a Crisis ···························
 89 
 
List of In-text Figures 
Figure 1 
Study Schema ································
································
··················
 25 
Figure 2 
Dose Cohort Schema (Enrollment for breast cancer and melanoma population 
will be performed independently and simultaneously) ································
···  26 
 


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CONTACT DETAILS OF SPONSOR’S KEY PERSONNEL 
24-hour Contact for 
Serious Adverse Events 
See [Section 10.3.7 
Reporting Procedures for 
Serious Adverse Events] 
Please fax or email the serious adverse events/special 
situations worksheet to: 
Astellas Pharma Global Development Inc.  
US Pharmacovigilance 
North America fax number: +1-888-396-3750 
North America alternate fax number: +1-847-317-1241 
Email: safety-us@astellas.com 
Medical Monitor/Study 
Physician 
 
 
ICON Clinical Research, US 
  
 
 
 
PPD
PPD


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PROTOCOL AMENDMENT SUMMARY OF CHANGES 
DOCUMENT HISTORY 
Document 
Date 
Amendment 2 [Nonsubstantial] 
01 AUG 2022 
Amendment 1 [Nonsubstantial] 
30 AUG 2021 
Original Protocol 
26 APR 2021 
Amendment 3 [Nonsubstantial] 11 MAY 2023 
This amendment is considered to be nonsubstantial based on the criteria set forth in Article 10(a) of Directive 2001/20/EC of the European Parliament 
and the Council of the European Union and EU Clinical Trial Regulation because it neither significantly impacts the safety or physical/mental integrity 
of participants nor the scientific value of the study. 
Overall Rationale for the Amendment: 
The primary rationale for this amendment is to add the clarification to the study procedure throughout the protocol, and to provide the latest study status. 
Summary of Changes 
Nonsubstantial Changes 
Section Number 
Description of Change 
Brief Rationale 
1.1 
The planned study period end date is updated from 4Q2023 to 1Q2025 
To align with current planned study period 
1.1 
The planned total number of study sites is updated from 8 to 4 
To align with the current planned total number of study sites 


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Section Number 
Description of Change 
Brief Rationale 
1.1, 1.3 (Table 1, 
footnote no. 11), 3 
(Table 3), 9.3.5.4, 
10.7 (Table 10, 
footnote no. 6) 
Throughout 
Routine 12-lead ECGs is updated from “routine” to “standard” 
1.3 (Table 1, footnote no. 11) and10.7 (Table 10, footnote no. 6) 
The following text is added: 
“..as per the site’s standard of care routine for cardiac monitoring.” 
1.3 (Table 1, footnote no. 11) and 7.2.4 
Text is updated to the following: 
Routine Standard 12-lead ECGs will be taken per site’s standard 
procedure at screening, preoperative and safety follow-up period. 
Intraoperative ECG monitoring will occur at 30 minutes postdose (15 
min ≤ and < 45 min). During the surgery,A 12-lead ECG is not 
mandatory for intraoperative and postoperative ECG and assessment 
can be based on the rhythm strip or other site standard ECG as per the 
site’s standard of care routine for cardiac monitoring. 
For further clarification 


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1.1, 4.1 
Language for the visualization data to be collected is updated: 
● 
The visualization of the LN(s) will be assessed by the investigator 
intraoperatively using a binary “Yes or No” question.  
o If “Yes,” the time of surgical identification of LN(s) will be 
collected by the investigator. 
o If “Yes,” the number of LNs visualized and the number of 
LNs visualized with histopathology confirmed LN will be also 
assessed by the investigator. 
Note: Histopathology review will be conducted per 
standard practice at each site. 
● 
Transdermal visualization of lymphatic spread of ASP5354 after 
injection of the drug will also be assessed using a binary “Yes or 
No” question.  
● 
The fluorescence intensity of the LN will be qualitatively assessed 
using a Likert Scale (0 = None; 1 = Mild; 2 = Moderate; and 
3 = Strong). 
● 
The radioactivity count will be collected for all the LNs 
identified by ASP5354 and/or SoC.Recorded images will be used 
to determine the Signal Background Ratio (SBR). 
● 
The number of LN(s) with metastatic spread will be assessed based 
on histopathologic review. 
Note: Histopathology review will be conducted per standard 
practice at each site. 
● 
Following proportions will be compared between ASP5354 and 
SoC treatment with either Tc 99mSC or Lymphoseek. 
o Proportion of participants with at least 1 LN detected by 
visualization with histopathologic confirmation of LN tissue 
o Proportion of identified LNs with histopathologic 
confirmation of LN tissue 
o Proportion of LNs with metastatic spread, based on 
histopathologic review 
During the surgery, video and/or image will be recorded to capture 
the lymphatic flow of ASP5354 and the signal intensity of each LN 
To clarify assessments that are done by the investigator 


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Section Number 
Description of Change 
Brief Rationale 
visualized. These images are used for Signal Background Ratio 
Analysis done by external reviewer (see [Section 7.1.2]). 
The visualization data of the LNs will be assessed by a VRC and will 
be compared with the performance of SoC. 
1.1, 4.1, 10.1.5.1 
The following text is updated: 
The data for the participants, at each subsequent dose level, who 
completed the SLN biopsy will be assessed by the VRC to decide 
whether to:  
● 
Expand a dose level if additional participants are needed to increase 
confidence for the optimal dose selection; 
● 
Stop a dose level if 100% of participants lack visualization or all 
participants have a fluorescence intensity of 0 or 1 on the 
qualitative Likert Scale, in which case additional participants 
would not be added to a dose level; or 
● 
Define an optimal dose (in the case of 2 doses performing equally, 
the lower dose will be selected). 
Sections 1.1 and 4.1 
The VRC assessment for optimal dose determination will be based on 
the totality of the collected data, including but not limited to: 
• 
Cummulative LN visualization (Yes/No) 
For further clarification  
1.1 
The following text is added: 
The optimal dose for breast cancer and melanoma will be selected 
separately. 
To align with text in Section 4.1 


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Section Number 
Description of Change 
Brief Rationale 
1.1, 1.3 (Table 1, 
footnote no. 15), 4.1, 
6.1.1 
The following text is removed throughout: 
“..NIR-F imaging system (with FDA 510[k] cleared optical device 
system) followed by confirmation with the gamma probe.” 
 
The following text in bold is added: 
Once lymphatic mapping is completed, gamma probe will be used 
per SoC. For all the LNs observed by ASP5354, gamma probe will 
be attempted to measure radioactivity. If the LN is not observed with 
NIR-F after approximately 15 minutes following injection for breast 
cancer participants and approximately 15 to 30 minutes for 
melanoma participants depending on the location of the tumor and 
LNs, the investigator can proceed to use the gamma probe for LN 
detection. When the LN is detected with the gamma probe first, 
radioactivity will be measured and then NIR-F imaging should be 
attempted again for LN visualization. 
To add clarity to the study procedure relating to the biopsy. 
1.2 (Figure 2) 
● 
Bullet no. 2 is updated to provide clearer language. 
● 
Bullet no. 3 is updated from: 
 “The second interim analysis will be performed by the VRC across 
initial dose levels….” 
To: 
“The second VRC review will be performed for the initial dose 
levels….” 
For further clarification 
1.3 (Table 1, new 
note) 
The following text in bold is added as a new note: 
†† The assessments scheduled for the screening period can be done 
on day 1 if all eligibility criteria can be confirmed prior to 
registration in the IRT and prior to treatment. If screening 
procedures are performed on day 1, physical examination, vital 
signs, clinical laboratory tests, ECG, previous/concomitant 
medications and AE assessment will be done once. 
To align with surgery procedures at clinical sites 
1.3 (Table 1, 
footnote no. 4) 
Body mass index is removed 
Body mass index is not a procedure/assessment, but rather a 
calculated value derived from height and weight 


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Section Number 
Description of Change 
Brief Rationale 
1.3 (Table 1, 
footnote no. 6), 7.2.3, 
10.7 (Table 10, 
footnote no. 2) 
The following text in bold is added for clarification: 
At visit 2 and follow-up (visit 3), a full physical examination is not 
mandatory, but a symptom-directed physical examination will be 
performed. 
To provide explicit instruction about the required process of 
physical examination 
1.3 (Table 1, 
footnote no. 7) 
The following text is added to footnote no. 7: 
Vital signs include blood pressure, pulse and respiratory rate. All vital 
signs should be measured within 1 week prior to IP administration at the 
screening visit. All vital signs will be measured with the participant in 
the sitting or supine position. Height and weight will be measured using 
standard institution practice and equipment.  
To provide clear instruction on how to perform and record vital 
signs 
1.3 (Table 1, 
footnote no. 8) 
Text is updated to the following: 
Postoperative vital signs and clinical laboratory tests will be collected 
within 2 hours afterfollowing the end of surgery 
For further clarification 


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1.3 (Table 1, 
footnote nos. 9 and 
14), 7.2.6.1, 7.3.5, 
9.3.5.1, 9.3.7.1, 10.7 
(Table 10, 
footnote no. 7) 
The following text is removed: 
Section 1.3 (Table 1, footnote no. 9) 
Clinical laboratory tests include blood collection for hematology 
(complete blood count) and serum chemistry and urine samples for 
urinalysis and urine color evaluation. 
The following text in bold is added: 
Section 1.3 (Table 1, footnote no. 14), 7.2.6.1 and 10.7 (Table 10, 
footnote no. 7) 
The occurrence of green coloration of the urine or skin will be 
monitored throughout the study.  If observed, any visual green 
coloration abnormalities will be recorded as an AE… 
The occurrence of green coloration of the skin will be recorded as an 
AE any time after dose administration 
The following text is removed: 
Section 7.3.5 
Green coloration of the urine or skin will not be considered an AE as this 
is an expected, known effect of short duration without any expected 
untoward clinical symptoms; however, this still needs to be assessed and 
recorded.  The assessment details are described in [Section 7.2.6.1 Green 
Coloration of Urine or Skin]. 
The following text in bold is added: 
Section 9.3.5.1 
The number and percentage of participants with TEAEs, IP-related 
TEAEs, serious TEAEs, and IP-related serious TEAEs and green 
coloration of urine or skin will be summarized by system organ class, 
preferred term and dose group. 
The following text is removed: 
Section 9.3.7 
9.3.7.1 Green Coloration of Urine or Skin  
The frequency and percentage of participants who experience green 
coloration of urine or skin will be summarized by dose group. The 
duration of green coloration will be summarized using descriptive 
statistics by dose group. 
To align with other ASP5354 studies to handle "Green 
Coloration" as an AE 


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Section Number 
Description of Change 
Brief Rationale 
2.2 
The reference details for NCCN guidelines are added 
Reference is missing 
5.1 (Inclusion 
criterion no. 2) 
Text is updated from: 
“Participant has been diagnosed with localized breast cancer (female 
only, stage 1 to 2, N0 and M0) or…..”  
To: 
“Participant has been diagnosed with localized breast cancer (female 
only, stage 0 to 2, N0 and M0) or…..” 
To allow participants with stage 0 breast cancer to participate in 
the study based on study site feedback that lymphatic mapping 
is a part of Standard Care in this population. 
5.2 (Exclusion 
criterion no. 15) 
A new exclusion criterion is added: 
15. 
Participant has been diagnosed with bilateral breast cancer 
To exclude participants with bilateral breast cancer, because 
surgical removal of both breasts is expected and lymphatic 
mapping is also conducted to both sides. The dose setting for 
this study is planned for lymphatic mapping for one side of the 
body, and additional dosing is not allowed. Thus study team 
determined that participants with bilateral breast cancer is 
deemed ineligible. 
6.1.1 
The following text in bold is added for breast cancer and melanoma: 
Please refrain from massaging the area of administration after 
injection of IP.  
To clarify the intraoperative procedure for the site to refrain 
from massaging the area of administration after the injection of 
study drug. 
6.1.1 
The following text in bold is added for melanoma: 
Please note that the time from injection of IP to the first 
visualization may vary due to tumor and LN location. In the event 
that NIR-F imaging does not detect LN after approximately 15 to 
30 minutes, the gamma probe should be used for LN identification.. 
The following text is removed: 
Once identified with the gamma probe, NIR F should again assess the 
LNs for fluorescence. 
To clarify the intraoperative procedure for the site to understand 
the expected time for the visualization in the each indication 
7.1 
Text is updated to clarify the data collected in the eCRF for Sentinel LN 
or Non-sentinel LN 
To correct the clerical error for the data points to be collected in 
the eCRF 


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Section Number 
Description of Change 
Brief Rationale 
7.1.2 
The following text in bold is added: 
7.1.2 
Signal Background Ratio 
Video recording by NIR-F device is also required to evaluate the 
visualization of LN by ASP5354. Start recording a video at the time 
of injection of ASP5354 and continue until the first lymph node is 
visualized. For the remaining lymph nodes visualized, it is 
recommended to use video recording, but capturing images with the 
device is also acceptable. For further details, please refer to the 
Imaging Charter. 
The following text in bold is added and strikethrough text is removed: 
Images will be captured on a NIR-F and overlay view (if available) for 
all the LNs identified by ASP5354. SBR will be calculated using 
fluorescence images at the time point when ASP5354 detect the first LN 
as follows [Hoogstins et al, 2019]: 
For futher clarification 
7.2 
The following strikethrough text is removed: 
Procedures conducted as part of a participant’s routine clinical 
management (i.e., SoC) obtained before signing the ICF may be utilized 
for screening or baseline purposes, provided the procedures met the 
protocol specified criteria and were performed within the time frame, as 
defined in the Schedule of Assessments [Table 1]. 
To remove duplication 
7.2.4 
The following strikethrough text is removed: 
The anonymized ECG recordings will be printed, signed and saved in 
the source. Alternatively, per time point, the ECGs can be stored 
electronically and reviewed in a timely manner by the investigator. 
For futher clarification 
7.3, 7.3.1.1, 7.3.4, 
10.3, 10.3.3, 10.3.3.1, 
10.3.3.2, 10.3.3.3, 
10.3.3.4, 10.3.4, 
10.3.6, 10.3.7 
Language for the definition of an adverse device effect, along with 
collection and reporting details are added throughout 
To add the details and requirements of adverse event relating to 
device 


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Section Number 
Description of Change 
Brief Rationale 
9.2, 9.3.3, 9.3.4, 9.3.6 The pharmacokinetic analysis set population is removed as an analysis 
set and the analysis set population is clarified throughout. 
In the following sections, the text in bold is added: 
Section 9.3.3 
The secondary endpoint analysis will be performed using the FAS. 
Section 9.3.4 
The exploratory analysis will be performed using the FAS. 
Section 9.3.6 
The pharmacokinetics analysis will be performed using the SAF. 
Pharmacokinetic analysis will be conducted on the SAF and 
PKAS will be removed from the analysis sets. Considering that 
PK blood sampling is designed in this study to confirm the 
exposure to systemic circulation and overall safety of ASP5354 
in case of local administration, analysis based on SAF would be 
more appropriate for this study. 
9.3.7 
The following text in bold is added: 
Other analyses such as the relationship between LNs visualization 
and pharmacokinetics or other exploratory endpoints may be 
explored when deemed appropriate. All details will be described in a 
separate analysis plan and results will be described in a separate 
report and will not be incorporated in the integrated CSR. 
To add the details of other analysis considered in the future 
9.4 
The following text in bold is added: 
During the VRC review, enrollment at that indication will be closed 
until the determination of the next dose level to be made. This 
process will continue for every additional 3 participants assigned to each 
dose level until reaching the maximum of 12 participants. A new dose 
level might be added 
For further clarification 
8.2 
The following strikethrough text is removed: 
A discontinuation from the study is defined as a participant who enrolled 
in the study and for whom the study is permanently discontinued for any 
reason. 
To remove duplication of text in Section 10.1.9 
10.1.9 
The following strikethrough text is removed: 
The study start date is the date the first participant signs the ICF for the 
study (excluding screen failures). 
To remove duplication of text in Section 4.4 
Throughout 
Minor administrative-type changes were made, e.g., consistency issues, 
typos, formatting, section and footnote numbering, and continuity 
throughout the protocol. 
To provide clarifications to the protocol and ensure complete 
understanding of study procedures. 
 


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1 
PROTOCOL SUMMARY 
1.1 
Synopsis 
Title of Study:  
A Phase 2 Open-label, Dose-finding Study to Determine the Optimal Dose for Lymph Node 
Visualization using ASP5354 in Participants with Breast Cancer or Melanoma Undergoing Sentinel 
Lymph Node Biopsy 
Planned Study Period:  
From approximately 3Q2021 to 1Q2025. 
Planned Total Number of Study Sites and Location(s): 
Approximately 4 study sites in the US 
Study Objectives, Endpoints and Estimands: 
Objectives 
Endpoints 
Primary 
● 
To determine the optimal dose of locally 
administered ASP5354 for LN visualization in 
participants undergoing SLN biopsy 
● 
Optimal dose determination by the VRC based 
on: 
o 
LN tissue visualized (Yes/No) 
o 
Visualized tissue is lymphatic in origin 
based on pathologic confirmation 
o 
The Likert Scale determination of the 
intensity of fluorescence (0 to 3) 
o 
Proportion of identified LN with 
histopathologic confirmation of LN 
tissue by ASP5354 compared with SoC 
treatment with either Tc-99mSC 
(approved drug) or Lymphoseek® 
(generic name tilmanocept) 
Secondary 
● 
To evaluate LN visualization of locally 
administered ASP5354 in participants 
undergoing SLN biopsy 
● 
Proportion of participants with at least 1 LN 
detected by visualization (Yes/No) with 
histopathologic confirmation of LN tissue 
using ASP5354 
● 
To compare LN detection for ASP5354 with 
the technetium-based treatment in breast 
cancer and melanoma 
● 
Proportion of identified LN with 
histopathologic confirmation of LN tissue by 
ASP5354 compared with SoC treatment with 
either Tc-99mSC or Lymphoseek  
Table continued on next page 


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Objectives 
Endpoints 
Secondary (continued) 
 
● 
To investigate the safety and tolerability of 
locally administered ASP5354 in participants 
undergoing SLN biopsy  
● 
Vital signs (blood pressure, pulse and 
respiratory rate) 
● 
Standard 12-lead ECGs 
● 
Clinical laboratory tests (hematology 
[complete blood count], serum chemistry and 
urinalysis) 
● 
Nature, frequency and severity of TEAEs and 
SAEs 
● 
To investigate the pharmacokinetics of locally 
administered ASP5354 in participants 
undergoing SLN biopsy 
● 
Plasma concentrations of ASP5354  
Exploratory 
 
● 
To explore the visualization characteristics of 
ASP5354 
● 
The Likert Scale for qualitative response 
about the intensity of fluorescence 
● 
Signal background ratio 
● 
Transdermal visualization of lymphatic spread 
of ASP5354 after injection (Yes/No) 
● 
Time from injection to surgical identification 
of LN(s) 
● 
Number of LNs visualized by ASP5354 
● 
To compare LNs detected with metastatic 
spread for ASP5354 with the technetium-
based treatment 
● 
Proportion of LN with metastatic spread, 
based on histopathologic review, detected by 
ASP5354 compared with SoC treatment with 
either Tc-99mSC or Lymphoseek  
Abbreviations from table above: ECG: electrocardiogram; LN: lymph node; SAE: serious adverse event; 
SLN: sentinel lymph node; SoC: standard of care; Tc-99mSC: technetium-99m sulfur colloid; 
TEAE: treatment-emergent adverse event; VRC: Visualization Review Committee. 
Estimands 
Estimand is not defined for any endpoint.  
Study Population: 
Male or female participants ≥ 18 years of age with either breast cancer (female only) or melanoma 
scheduled to undergo sentinel lymph node (SLN) biopsy.  
 


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Number of Participants: 
Enrollment for breast cancer and melanoma population will be performed independently and 
simultaneously.  
The sample size is not based on statistical power calculation. The sample size is expected to 
provide adequate information to determine the optimal dose of ASP5354 for lymph node (LN) 
visualization in participants with breast cancer and melanoma. Approximately up to 12 participants 
will be assigned to each of the initial 3 dose levels of ASP5354 in each tumor type, and an 
estimated total of up to 36 participants will be required in each tumor type. In case some dose 
levels are stopped based on Visualization Review Committee (VRC) review, additional dose levels 
might be added. 
Breast Cancer
Dose Arm/IP Name 
ASP5354 Dose Level 
ASP5354 Dose Conc 
and Volume 
Number of 
Participants 
Breast Cancer 0.05 mg/ 
ASP5354  
0.05 mg 
1.0 mg/mL, 
0.05 mL in total 
Up to 12 
Breast Cancer 0.2 mg/ 
ASP5354  
0.2 mg 
1.0 mg/mL, 
0.2 mL in total 
Up to 12 
Breast Cancer 0.6 mg/ 
ASP5354  
0.6 mg 
1.0 mg/mL, 
0.6 mL in total 
Up to 12 
Breast Cancer 1 mg/ 
ASP5354  
1 mg 
1.0 mg/mL,  
1 mL in total 
Up to 12 
Breast Cancer 2 mg/ 
ASP5354  
2 mg 
1.0 mg/mL,  
2 mL in total 
Up to 12 
Breast Cancer 4 mg/ 
ASP5354  
4 mg* 
1.0 mg/mL,  
4 mL in total 
Up to 12 
IP: investigational product 
* The 4 mg dose level will be added only for breast cancer participants.   
Melanoma 
Dose Arm/IP Name 
ASP5354 Dose Level 
ASP5354 Dose Conc 
and Volume 
Number of 
Participants 
Melanoma 0.05 mg/ 
ASP5354  
0.05 mg 
1.0 mg/mL, 
0.05 mL in total 
Up to 12 
Melanoma 0.2 mg/ 
ASP5354  
0.2 mg 
1.0 mg/mL, 
0.2 mL in total 
Up to 12 
Melanoma 0.6 mg/ 
ASP5354  
0.6 mg 
1.0 mg/mL, 
0.6 mL in total 
Up to 12 
Melanoma 1 mg/ 
ASP5354  
1 mg 
1.0 mg/mL,  
1 mL in total 
Up to 12 
Melanoma 2 mg/ 
ASP5354  
2 mg 
1.0 mg/mL,  
2 mL in total 
Up to 12 
IP: investigational product 
 


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Study Design Overview: 
The study is an open-label, dose-finding study to determine the optimal dose for LN visualization, 
as part of lymphatic mapping, in adult participants with breast cancer and melanoma undergoing 
SLN biopsy as part of Standard of Care (SoC).  
Participants in each tumor type may be assigned to receive a single dose of ASP5354 (0.05, 0.2 or 
0.6 mg or 0.2, 0.6 or 1 mg) with the first 3 participants starting at 0.2 mg, which will be locally 
administered as subcutaneous (breast cancer) or intradermal (melanoma) injection to evaluate the 
visualization of at least 1 LN, with histopathologic confirmation of LN tissue for each tumor type. 
Three participants will be assigned to each dose in sequential order after the first 3 participants are 
evaluated by the VRC. An interim analysis will then be performed across the initial three dose 
levels (0.05, 0.2 and 0.6 mg or 0.2, 0.6 and 1 mg) for breast cancer and melanoma by the VRC 
prior to further enrollment of participants.  
Participants will receive technetium-99m sulfur colloid (Tc-99mSC) or Lymphoseek as part of SoC 
for SLN biopsy. In case the participant needs other surgery (e.g., lumpectomy, mastectomy etc.) on 
the same day of study drug administration, the other surgery is allowed once the SLN biopsy has 
completed. In the operating room, participants will receive ASP5354. After administration of 
ASP5354, visualization of the LN(s) will be assessed using a near-infrared fluorescence (NIR-F) 
imaging system followed by confirmation with the gamma probe. Once lymphatic mapping is 
completed, gamma probe will be used per SoC. For all the LNs observed by ASP5354, gamma 
probe will be attempted to measure radioactivity. If the LN is not observed with NIR-F after 
approximately 15 minutes following injection for breast cancer participants and approximately 
15 to 30 minutes for melanoma participants depending on the location of the tumor and LNs, the 
investigator can proceed to use the gamma probe for LN detection. When the LN is detected with 
the gamma probe first, radioactivity will be measured and then NIR-F imaging should be attempted 
again for LN visualization. For participants with melanoma, usage of lymphoscintigraphy or single 
photon emission computed tomography (SPECT) as part of SoC is also allowed. 
The visualization data to be collected will be consist of following: 
● 
The visualization of the LN(s) will be assessed by the investigator intraoperatively using a 
binary “Yes or No” question.  
o 
If “Yes,” the time of surgical identification of LN(s) will be collected by the investigator. 
o 
If “Yes,” the number of LNs visualized and the number of LNs visualized with 
histopathology confirmed LN will be also assessed by the investigator. 
Note: Histopathology review will be conducted per standard practice at each site. 
● 
Transdermal visualization of lymphatic spread of ASP5354 after injection of the drug will also 
be assessed using a binary “Yes or No” question.  
● 
The fluorescence intensity of the LN will be qualitatively assessed using a Likert Scale 
(0 = None; 1 = Mild; 2 = Moderate; and 3 = Strong). 
● 
The radioactivity count will be collected for all the LNs identified by ASP5354 and/or SoC. 
● 
The number of LN(s) with metastatic spread will be assessed by the investigator based on 
histopathologic review. 
Note: Histopathology review will be conducted per standard practice at each site. 
During the surgery, video and/or image will be recorded to capture the lymphatic flow of ASP5354 
and the signal intensity of each LN visualized. These images are used for Signal Background Ratio 
Analysis done by external reviewer. 
The visualization data of the LNs will be assessed by a VRC and will be compared with the 
performance of SoC. The VRC will be held at the following time points for each dose:  


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● 
Initial 3 participants at 0.2 mg in each tumor type have completed the SLN biopsies; 
● 
Initial 3 participants at each subsequent dose levels (0.05 and 0.6 mg or 0.6 and 1 mg) in each 
tumor type have completed the SLN biopsies (total of 6 participants); and 
● 
Additional 3 participants have completed SLN biopsies at the expanded or additional dose 
levels. Repeat this process until the optimal dose is defined but maximum participants for each 
cohort (i.e., 12) has not been reached. 
The data for the participants, at each subsequent dose level, who completed the SLN biopsy will be 
assessed by the VRC to decide whether to:  
● 
Expand a dose level if additional participants are needed to increase confidence for the optimal 
dose selection; 
● 
Stop a dose level if 100% of participants lack visualization or all participants have a 
fluorescence intensity of 0 or 1 on the qualitative Likert Scale, in which case additional 
participants would not be added to a dose level; or 
● 
Define an optimal dose (in the case of 2 doses performing equally, the lower dose will be 
selected). 
The VRC assessment for optimal dose determination will be based on the totality of the collected 
data, including but not limited to: 
• 
Cummulative LN visualization (Yes/No) 
• 
Histopathologic confirmation of LN tissue visualized with ASP5354  
• 
Fluorescence intensity based on the Likert Scale (0 to 3) 
• 
Proportion of identified LN with histopathologic confirmation of LN tissue by ASP5354 
compared with SoC treatment with either Tc-99mSC or Lymphoseek 
• 
Safety data 
The optimal dose is defined as the dose that provides a better visualization compared with lower 
doses and a comparable visualization to the next higher dose. In the case where 2 doses performed 
equally, the lower dose would be selected. Cumulative visualization data of the LNs from all 
treated participants will be used for optimal dose determination.The optimal dose for breast cancer 
and melanoma will be selected separately. 
Based on VRC review of the initial 3 dose levels, if none of the first 9 participants have LN 
visualization, then all 3 initial dose levels are stopped. The 1 and 2 mg dose levels or the 2 and 
4 mg (breast cancer only) dose levels will be added dependant on the highest dose used in the 
3 prior dose levels. If 2 of the initial 3 doses are stopped due to lack of visualization in the 
6 participants in the dose levels, then the dose level will be expanded to the 1 and/or 2 mg dose 
level. The 4 mg dose level for breast cancer will only be added if the 2 mg dose level remained 
open. For further details of the VRC, refer to the VRC Charter. 
If the investigator determines visualization of ASP5354 is not sufficient for LN imaging after local 
administration, the investigator can proceed with the use of other NIR-F imaging modalities or 
Blue dye for LN detection if indicated. 
Safety and tolerability will be assessed by recording treatment-emergent adverse events and 
adverse reactions associated with the use of an investigational product, clinical laboratory 
evaluations (hematology, serum chemistry and urinalysis), 12-lead electrocardiograms, vital signs 
measurements and physical examination. Blood samples for pharmacokinetic assessment will be 
collected pre- and post-administration of ASP5354 at defined time points up to the end of surgery. 


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Treatment Groups and Duration: 
ASP5354 lyophilization powder will be supplied by the sponsor or designee.  
Breast Cancer 
Arm/ 
IP Name 
Breast Cancer 0.05 mg/ 
ASP5354  
Breast Cancer 0.2 mg/ 
ASP5354  
Breast Cancer 0.6 mg/ 
ASP5354   
Use 
test product 
test product 
test product 
Dose 
0.05 mg 
0.2 mg 
0.6 mg 
Concentration 
1.0 mg/mL 
1.0 mg/mL 
1.0 mg/mL 
Volume 
0.05 mL in total 
0.2 mL in total 
0.6 mL in total 
Frequency 
single dose 
single dose 
single dose 
Route 
subcutaneous injection 
subcutaneous injection 
subcutaneous injection 
Location 
subareolar 
subareolar 
subareolar 
Injection site 
minimum of 2, up to 4, 
equally divided injections 
minimum of 2, up to 4, 
equally divided injections 
minimum of 2, up to 4, 
equally divided injections 
Duration 
single dose 
single dose 
single dose 
IP: investigational product 
 
Arm/ 
IP Name 
Breast Cancer 1 mg/ 
ASP5354  
Breast Cancer 2 mg/ 
ASP5354  
Breast Cancer 4 mg/ 
ASP5354  
Use 
test product 
test product 
test product 
Dose 
1 mg 
2 mg 
4 mg 
Concentration 
1.0 mg/mL 
1.0 mg/mL 
1.0 mg/mL 
Volume 
1 mL in total 
2 mL in total 
4 mL in total 
Frequency 
single dose 
single dose 
single dose 
Route 
subcutaneous injection 
subcutaneous injection 
subcutaneous injection 
Location 
subareolar 
subareolar 
subareolar 
Injection site 
minimum of 2, up to 4, 
equally divided injections 
minimum of 2, up to 4, 
equally divided injections 
minimum of 2, up to 4, 
equally divided injections 
Duration 
single dose 
single dose 
single dose 
IP: investigational product 


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Melanoma 
 
Arm/ 
IP Name 
Melanoma 0.05 mg/ 
ASP5354  
Melanoma 0.2  mg/ ASP5354 Melanoma 0.6  mg/ ASP5354 
Use 
test product 
test product 
test product 
Dose 
0.05 mg 
0.2 mg 
0.6 mg 
Concentration 
1.0 mg/mL 
1.0 mg/mL 
1.0 mg/mL 
Volume 
0.05 mL in total 
0.2 mL in total 
0.6 mL in total 
Frequency 
single dose 
single dose 
single dose 
Route 
intradermal injection 
intradermal injection 
intradermal injection 
Location 
peritumoral 
peritumoral 
peritumoral 
Injection site minimum of 2, up to 4, equally 
divided injections 
minimum of 2, up to 4, equally 
divided injections 
minimum of 2, up to 4, equally 
divided injections 
Duration 
single dose 
single dose 
single dose 
 
Arm/ 
IP Name 
Melanoma 1 mg/ ASP5354  
Melanoma 2 mg/ ASP5354  
Use 
test product 
test product 
Dose 
1 mg 
2 mg 
Frequency 
single dose 
single dose 
Concentration 
1.0 mg/mL 
1.0 mg/mL 
Volume 
1.0 mL in total 
2.0 mL in total 
Frequency 
single dose 
single dose 
Route 
intradermal injection 
intradermal injection 
Location 
peritumoral 
peritumoral 
Injection site 
minimum of 2, up to 4, equally divided 
injections 
minimum of 2, up to 4, equally divided 
injections 
Duration 
single dose 
single dose 
IP: investigational product 
The anticipated duration of the study for each participant, including screening and follow-up, is 
approximately 10 days. 
 






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● 
Enrollment of breast cancer and melanoma participants will be performed independently and simultaneously. Starting dose will be 0.2 mg for the initial 3 participants in each 
tumor type. 
● 
VRC review will be held when initial 3 participants have completed the surgical procedures at the 0.2 mg dose level to decide whether to enroll participants at the 0.05 mg and 
0.6 mg dose levels or the 0.6 mg and 1 mg dose levels. If LN visualization with histopathologic confirmation occurs in the first 3 participants in each indication, then dose level 
expansion will occur and an additional 3 participants in each indication will be enrolled first with the 0.05 mg dose, followed by another 3 participants with the 0.6 mg dose.  
If < 3 participants at 0.2 mg dose level have LN visualization with histopathologic confirmation on VRC review, 3 participants will be enrolled sequentially in the 0.6 mg and 
1 mg dose levels.   
● 
The second VRC review will be performed for the initial dose levels (0.05, 0.2 and 0.6 mg or 0.2, 0.6 and 1 mg) for breast cancer and melanoma prior to further enrollment of 
participants. Data for the 9 participants in the first 3 dose levels who completed the SLN biopsy will be assessed to decide whether to: 1) expand a dose under evaluation in 
3 participants increments up to 12 participants; 2) stop a dose in which case additional participants would not be added to a dose level; 3) add a new dose level(s); or 4) define a 
dose as optimal. 
● 
Based on VRC review of the initial 3 doses levels of 3 participants: 
o 
If 0 doses remain, then a 1 and 2 mg or 2 and 4 mg (breast cancer only) dose will be added 
o 
If 1 dose remains, then the 1 and/or 2 mg dose will be added.  
o 
The 4 mg dose level will be added for breast cancer participants if only the 2 mg dose level has visualization. 
● 
Up to 12 participants will be assigned to receive single doses of ASP5354 at each initial dose levels (0.05, 0.2 and 0.6 mg or 0.2, 0.6 and 1 mg) or added dose levels (1, 2 or 4 mg 
[breast cancer only]) for breast cancer or melanoma.  
 


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1.3 
Schedule of Assessments 
Table 1 
Schedule of Assessments 
Assessments 
Screening Period†† 
Treatment Period 
Safety Follow-up 
Period 
Day 
-28 to -1 
1 (Operation) 
10 (Outpatient) 1 
Preoperative 
Intraoperative 
Postoperative 
Window  
- 
- 
- 
- 
±4 
Visit Number 
1 
2 
3 
Informed Consent 
X 
 
 
 
 
Inclusion/Exclusion Criteria 
X 
 
 
 
 
Primary Diagnosis of Target Disease 2 
X 
 
 
 
 
Medical History3 
X 
 
 
 
 
Demographics 4 
X 
 
 
 
 
Drug and Alcohol Screen 5 
X† 
 
 
 
 
Physical Examination 6 
X 
X 
 
X 
X 
Vital Signs 7 
X 
X 
 
    X 8 
X 
Clinical Laboratory Tests 9 
X† 
X 
 
    X 8 
X 
Height 7 
X 
 
 
 
 
Body Weight 7 
X 
 
 
 
X 
Serum or Urine Pregnancy Test 10 
X† 
 
 
 
X 
ECG 11 
X 
X 
X 
X 
X 
Dosing ASP5354 12 
 
 
X 
 
 
Blood Sampling for ASP5354 Pharmacokinetics 13 
 
X 
X 
 
 
Confirmation of Urine or Skin Coloration 14 
 
 
→ 
→ 
→ 
Investigator Evaluation for LN Visualization 15 
 
 
X 
 
 
Record Surgery 16 
 
 
    X  
 
 
Previous/Concomitant Medications 
X 
X 
X 
X 
   X 17 
AE Assessment 
X 
X 
X 
X 
   X 17 
Study Participant Feedback Questionnaire 
X 
 
 
 
X 
Footnotes and abbreviations on next page 
 


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AE: adverse event, ECG: electrocardiogram; IP: investigational product; IRT: interactive response technology; LN: lymph node; NIR-F: near-infrared fluorescence; TNM: tumor, 
nodes and metastases 
† These assessments will be performed by a local laboratory and the results of these assessments must be obtained prior to participant enrollment. 
†† The assessments scheduled for the screening period can be done on day 1 if all eligibility criteria can be confirmed prior to registration in the IRT and prior to treatment. If screening 
procedures are performed on day 1, physical examination, vital signs, clinical laboratory tests, ECG, previous/concomitant medications and AE assessment will be done once. 
1. If a participant discontinues early from the study, day 10 outpatient visit procedures will be performed upon study discontinuation. 
2. Primary diagnosis of the target disease will include:  
• 
Breast cancer: T categorization in TNM classification, location of tumor (left breast, right breast or other)  
• 
Melanoma: T categorization in TNM classification and location of tumor (Extremities [upper/lower, left/right], Trunk, Head and Neck, Other), Tumor Thickness and 
Ulceration status. 
3. Medical History includes all significant medical conditions that have occurred or are currently ongoing. The medical condition, onset date, and recovery date will be collected. 
4. Demographic information will be collected for all participants at screening and will include age, sex, race, ethnicity, weight  and height. 
5. Drug screen and alcohol screen will be performed by the study site’s preferred method. 
6. A full physical examination will be performed at screening (visit 1). Body systems to be evaluated include general appearance, skin, lymphatic, head and neck, ears, nose and 
throat, chest and lungs, cardiovascular, abdomen, extremities, musculoskeletal and neuromuscular. At visit 2 and follow-up (visit 3), a full physical examination is not mandatory, 
but a symptom-directed physical examination will be performed. 
7. Vital signs include blood pressure, pulse and respiratory rate. All vital signs should be measured within 1 week prior to IP administration at the screening visit. All vital signs will 
be measured with the participant in the sitting or supine position. Height and weight will be measured using standard institution practice and equipment.  
8. Postoperative vital signs and clinical laboratory tests will be collected within 2 hours following the end of surgery. 
9. Clinical laboratory tests include blood collection for hematology (complete blood count) and serum chemistry and urine samples for urinalysis. These assessments will be 
performed by a local laboratory. 
10. This assessment will be required only for participants who are women of childbearing potential (see [Section 10.2, Appendix 2 Contraception Requirements]). 
11. Standard 12-lead ECGs will be taken per site’s standard procedure at screening, preoperative and safety follow-up period. Intraoperative ECG monitoring will occur at 30 minutes 
postdose (15 min ≤ and < 45 min).A 12-lead ECG is not mandatory for intraoperative and postoperative ECG and assessment can be based on the rhythm strip or other site 
standard ECG as per the site’s standard of care routine for cardiac monitoring. 
12. ASP5354 will be injected at a minimum of 2 and up to 4 sites of equally divided injections. The exact date and time and location (i.e., 3 and 9 o’clock position of tumor) of IP 
administration will be documented. 
13. Blood samples for pharmacokinetics of IP and possible metabolite(s) (if applicable) will be collected from every participant. A single pharmacokinetic sample will be collected at 
each of the following time points: prior to dose, approximately 10 minutes, 30 minutes, 60 minutes and 90 minutes postdose, and at the end of surgery.  
14. The occurrence of green coloration of the urine or skin will be monitored throughout the study. If observed, any visual green coloration abnormalities will be recorded as an AE. If 
it occurs for participants who have an urethral catheter in place, the color of urine will be assessed during surgery and every 120 minutes (± 30 minutes window) after the end of 
surgery until it resolves or the participant is discharged, whichever is earlier. If no urethral catheter is in place, voided urine will be assessed for green coloration and the time 
collected until it resolves or the participant is discharged, whichever is earlier.  The start and stop date and time will also be recorded. The occurrence of green coloration of the 
skin will be recorded as an AE any time after dose administration. The start and stop date and time will be recorded.  Skin coloration will be followed up until it resolves or the 
participant is discharged, whichever is earlier. 
Footnotes continued on next page 


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15. Surgeon evaluation for LN(s) visualization includes incidence of visualization of the LN(s) and distal or second tier LN(s) and the Likert Scale for qualitative response of the 
intensity of fluorescence. After administration of ASP5354, visualization of the LN(s) will be assessed first using a NIR-F imaging system followed by confirmation with the 
gamma probe. Once lymphatic mapping is completed, gamma probe will be used per SoC. For all the LNs observed by ASP5354, gamma probe will be attempted to measure 
radioactivity.If the LN is not observed with NIR-F after approximately 15 minutes following injection for breast cancer participants and approximately 15 to 30 minutes for 
melanoma participants depending on the location of the tumor and LNs, the investigator can proceed to use the gamma probe for LN detection. When the LN is detected with the 
gamma probe first, radioactivity will be measured and then NIR-F imaging should be attempted again for LN visualization. During surgery, parameters will be recorded such as 
time of incision and number of LN(s). 
16. Fluorescence images will be captured and recorded during the entire surgery and archived.  
17. If a participant experiences an AE or change in concomitant medications during the follow up period, the participant should call the study site to inform any of these changes. 
Should the participant develop a hypersensitivity reaction, an additional blood sample for determination of histamine and tryptase concentrations should be taken as soon as 
possible after the onset of the hypersensitivity reaction. 


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1.3.1 
Sample Collection and Timed Procedure Schedule 
Table 2 
Sample Collection and Timed Procedure Schedule 
Day 
Operation 
Time Point 
Sample Collection 
Collection Window 
Blood Sampling for Pharmacokinetics 
Day 1 
Preoperative 
Predose 
X 
Within 1 h before dosing 
Intraoperative 
10 min a 
X 
5 min ≤ and < 15 min 
30 min a 
X 
15 min ≤ and < 45 min 
60 min a 
X 
45 min ≤ and < 75 min 
90 min a  
X 
75 min ≤ and < 105 min 
End of surgery b 
- 
X 
- 
h: hour 
a. 
If the end of surgery is earlier than this time point, this time point(s) will not need to be collected. 
b. In case the participant needs other surgery following sentinel lymph node biopsy, this time point will be at the time of all surgeries end.  
 


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2 
INTRODUCTION 
2.1 
Study Rationale 
The currently approved agents for lymphatic mapping, while effective, have negative 
consequences such as allergic reaction, tissue staining, radiation exposure and/or require 
special handling and disposal.  An agent that is safe, easy to use and may shorten operative 
times without negative effects would be beneficial for both patients and surgeons.   
ASP5354 is a small-molecule, near-infrared fluorescence (NIR-F) imaging agent that is an 
indocyanine green (ICG)-derivative conjugated with cyclodextrin. The physical and chemical 
properties differ from ICG and allow shorter transit times from injection to lymph node (LN) 
uptake than ICG in non-clinical studies.   
ASP5354 will be evaluated in patients undergoing Standard of Care (SoC) lymphatic 
mapping to determine safety, optimal dose and preliminary efficacy in lymphatic mapping. 
2.2 
Background 
Lymphatic mapping is important in many cancer surgeries where detection of sentinel lymph 
nodes (SLNs) for removal is SoC and also for understanding lymphatic drainage in 
extremities post lymphadenectomy [Jeremiasse et al, 2020; Suami et al, 2018]. 
SLN resection has become an integral part of surgery for many localized cancers to diagnose 
the presence of lymphatic spread and secondarily to avoid the complications related to 
extensive removal of the lymphatics.  It has been shown that the degree of lymphatic 
complications is related to the number of LNs removed [Herd-Smith et al, 2001]. For patients 
with localized breast cancer, melanoma and cervical cancer, it has become SoC [National 
Comprehensive Cancer Network (NCCN) Guidelines® for cutaneous melanoma, 2019; 
NCCN) Guidelines® for breast cancer, 2021; Dessources et al, 2020; Giuliano et al, 2011].  
SLN removal has also been used in Head and Neck and gastrointestinal (GI) malignancies, 
although not uniformly [de Bree et al, 2020; Mueller et al, 2019].   
Intraoperatively, identification of the lymphatics can be performed visually; however, 
improvements to optimize the detection of the lymphatics, especially for SLNs, can be done 
using NIR-F imaging with ICG, blue dyes or radioisotopes.  
For breast cancer, surgeons primarily use radioisotope, blue dye or both for LN detection 
with rates of detection > 90%; both of which are FDA approved [Lymphazurin™ prescribing 
information, January 2012 and Lymphoseek prescribing information, June 2017].  For 
Melanoma, the FDA approved radioisotope that is typically used.  This preference may be 
partly due to variability of lymphatic drainage for truncal lesions.  For cervical or endometrial 
cancer, ICG is the preferred FDA approved modality for detection [Spy Agent™ Green 
prescribing information, November 2018]. In Head and Neck cancer, radioisotope has also 
been approved for use [Lymphoseek prescribing information, June 2017].  ICG use has also 
been reported for GI malignancies [Chen et al, 2020].   


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While each modality has high rates of SLN detection, they have some limitations. Radio-
isotope cannot be used intraoperatively, is associated with radiation and requires special 
equipment to prepare and handle and a gamma counter to detect the radiation emitted from 
the isotope [Lymphoseek prescribing information, June 2017; Wallace et al, 2013; Sondak et 
al, 2013]. Blue dye can lead to interactions and adverse reactions, mainly allergic, and also 
can stain the tissue making determination of the LN harder and may lead to cosmetic issues 
[Isosulfan Blue prescribing information, September 2020] [Thevarajah et al, 2005; Saha et al, 
2015].  ICG is considered safe but does have a 1 in 42,000 risk of allergic reaction, for which 
fatal outcomes have been reported [Spy Agent™ Green (indocyanine green) prescribing 
information, November 2018].  
ASP5354 is a small-molecule NIR-F imaging agent that is an ICG-derivative conjugated with 
cyclodextrin. ASP5354 is currently being developed by Astellas Pharma Global 
Development Inc. as an imaging agent to be administered intravenously for intraoperative 
NIR-F guided identification of the ureters. The physical and chemical properties of ASP5354 
are sufficiently different from ICG that non-clinical studies were performed to assess its 
utility in lymphatic mapping.  Non-clinical studies have determined the transit time from 
injection to LN uptake is shorter than comparable doses of ICG; in addition, no local toxicity 
was seen.  This data suggests that it might be advantageous for lymphatic mapping during 
breast cancer or melanoma surgery. The purpose of this phase 2 dose-range study is to 
characterize the safety, optimal dose and preliminary efficacy of ASP5354 in patients 
undergoing SLN biopsy as part of cancer treatment. 
2.2.1 
Nonclinical and Clinical Data 
Nonclinical and clinical data for ASP5354 available as of the writing of this protocol are 
summarized below.  Please refer to the current version of the ASP5354 Investigator’s 
Brochure (IB) for the most recent data. 
2.2.1.1 Summary of Nonclinical Data  
ASP5354, ICG-derivative conjugated with cyclodextrins, is a NIR-F imaging agent with 
hydrophilic properties. 
ASP5354 emitted NIR-F at wavelength 820 nm when irradiated with near-infrared excitation 
light at wavelength 780 nm in vitro.  Moreover, results of an in vivo imaging study using 
Göttingen minipigs suggested that subcutaneous administration of ASP5354 at 0.01 mg/body 
or greater allows for sufficient intraoperative imaging of the lymphatic system. 
ASP5354 at a concentration of 10 μmol/L did not have > 50% inhibition on radioligand 
binding to any of the 66 receptors, ion channels, transporters tested, nor did it affect the 
activity of enzyme reactions of 7 enzymes, suggesting that the potential off target activities of 
ASP5354 is low. 
The plasma protein binding of ASP5354 at concentrations of 1 to 1000 µg/mL was 76.11% to 
78.90% in rats, 68.50% to 73.84% in rabbits, 73.88% to 75.42% in Göttingen minipigs, 
72.98% to 74.90% in cynomolgus monkeys and 74.17% to 77.25% in humans. 


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No ASP5354 metabolites were detected after incubation with cryopreserved hepatocytes or 
liver microsomes from rats, dogs, cynomolgus monkeys and humans.  No ASP5354 
metabolites were also detected in rat, cynomolgus monkey and human plasma and in rat, 
Göttingen minipig, cynomolgus monkey and human urine obtained after intravenous 
administration of ASP5354.  These results suggest that ASP5354 is metabolically stable in 
these species. 
After intravenous administration ASP5354 was mainly excreted into urine in rats and 
Göttingen minipigs. 
ASP5354 up to 100 μmol/L have no inhibitory effects on cytochrome P450 (CYP) 1A2, 
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, P-glycoprotein (P-gp), breast 
cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, 
OATP1B3, organic anion transporter (OAT) 1, OAT3, organic cation transporter 2 (OCT2), 
multidrug and toxin extrusion (MATE) 1 and MATE2-K.  ASP5354 is not a substrate of 
P-gp, BCRP, OAT1, OAT3, OCT2, MATE1 and MATE2-K. 
ASP5354 had no genotoxic, teratogenic, local irritative or phototoxic effects, nor did it effect 
the central nervous, cardiovascular or respiratory systems. 
Subcutaneous or intramuscular administration of ASP5354 to rabbits resulted in no test 
article-related discernible gross or histopathological findings.  Systemic exposure following 
subcutaneous to Göttingen minipigs at minimum effective dose (0.01 mg/body) was below 
the lower limit of quantification, and below the levels seen following intravenous dosing to 
rats and cynomolgus monkeys.  Intravenous administration to rats or cynomolgus monkeys, 
showed no systemic toxicities at levels 15411-fold and 76712-fold the Cmax observed 
following subcutaneous administration to Göttingen minipigs at pharmacologically sufficient 
dose (0.1 mg/body), respectively. 
The intravenous pharmacokinetic and toxicity studies show that systemic exposure to 
ASP5354 is short and that high systemic exposure of the imaging agent had little or no 
discernible toxicities.  These data, coupled with the absence of local irritation following 
subcutaneous or intramuscular administration, support expanding the clinical use of this 
product for lymphatic mapping in breast cancer or melanoma surgeries.   
Administration of ASP5354 to pregnant rats or rabbits had no adverse effect on the pregnant 
animals nor did it result in embryofetal loss or fetal variations or malformations.  Despite 
these findings, appropriate restrictions on administration of ASP5354 in clinical studies are 
recommended. 
Please refer to the current ASP5354 IB for the most recent nonclinical data.   
2.2.1.2 Summary of Clinical Data 
2.2.1.2.1 
Phase 1 Results (Study 5354-CL-0001) 
The study, 5354-CL-0001, was a phase 1, double-blind, randomized, placebo controlled, 
single ascending dose study assessing the safety and pharmacokinetics of ASP5354 in 


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healthy adult volunteers. Investigational product (IP) was administered as a single 
intravenous bolus injection under fasting conditions. 
2.2.1.2.1.1 Pharmacokinetics and Product Metabolism in Humans 
Following a single intravenous administration, ASP5354 was rapidly eliminated. The 
observed pharmacokinetic parameters suggested a dose proportional increase in the 
investigated dose range from 0.1 to 24 mg. Almost all fraction of ASP5354 administered was 
excreted unchanged in urine. Renal clearance (CLR) was nearly equivalent to the total body 
clearance. 
2.2.1.2.1.2 Safety 
Final safety data from Study 5354-CL-0001 show that of the 30 healthy participants enrolled, 
2 (20%) participants who received placebo and 3 (15%) participants who received ASP5354 
experienced at least 1 treatment-emergent adverse event (TEAE) (dysuria, headache, 
incontinence, nausea, oral herpes, presyncope, pyelonephritis, urinary tract infection and 
vomiting [each in 1 participant]). The TEAEs reported in participants who received placebo 
were nausea, vomiting and incontinence (each in 1 participant). The TEAEs reported in 
participants who received ASP5354 were oral herpes, pyelonephritis, urinary tract infection, 
headache, presyncope and dysuria (each in 1 participant). None of the participants 
experienced TEAEs considered to be causally related to ASP5354. One participant who 
received 24 mg ASP5354 experienced a ≥ grade 3 TEAE that began on day 2 following 
dosing; this grade 3 event of pyelonephritis was also a serious adverse event (SAE) that was 
considered by the investigator to be not related to ASP5354; rather, it was assessed as related 
to the use of a urinary catheter. At doses of 2, 8 and 24 mg, participants experienced a 
transient green coloration of urine, due to the color of the dye. This observation was not 
considered as an adverse event (AE). 
2.2.1.2.2 
Phase 1 Results (Study 5354-CL-0002) 
The Japanese Phase 1 study (Study 5354-CL-0002) was a randomized participant- and 
investigator-blinded, placebo-controlled, single intravenous dose study in healthy adult 
Japanese participants. 
2.2.1.2.2.1 
Pharmacokinetics and Product Metabolism in Humans 
Plasma exposure (AUCinf, AUClast and Cmax) of ASP5354 following single intravenous doses 
increased proportional to increases in dose across the 0.5 to 8 mg dose range. 
Pharmacokinetics of ASP5354 appeared linear across the evaluated dose range. Urinary 
excretion of ASP5354 was rapid and complete urinary excretion was observed across the 
0.5 to 8 mg dose range. CLR estimates were nearly equivalent to observed total body 
clearance estimates and almost constant across the 0.5 to 8 mg dose range.  
2.2.1.2.2.2 Safety 
Single intravenous doses of ASP5354 0.5 mg, 2 mg or 8 mg in healthy adult Japanese male 
participants appeared to be safe and well tolerated. No TEAEs were reported in any of the 
12 participants. There were no clinically significant changes in vital signs, 12-lead ECG, 


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clinical laboratory evaluation or physical examination. Green coloration of urine was 
observed in all participants in the ASP5354 2 mg and 8 mg groups, but was not observed in 
the ASP5354 0.5 mg group or the placebo group. 
2.2.1.2.3 
Phase 2 Results (5354-CL-0201) 
The study, 5354-CL-0201, was a phase 2, randomized, open-label, dose-ranging study for 
ureter visualization, using ASP5354 in participants undergoing laparoscopic/minimally 
invasive colorectal surgery. Participants were randomly assigned to receive single doses of 
ASP5354 (0.3, 1, or 3 mg), which were administered as an intravenous bolus. 
Safety data from Study 5354-CL-0201 revealed 1 nonserious TEAE (proteinuria grade 1 with 
a duration of 11 days in the 1 mg dose expansion group) reported by the investigator as 
having a reasonable possibility of being related to ASP5354. There were 2 grade 3 SAEs of 
rectal perforation and colon cancer, which were both in the same participant (0.3 mg group) 
and not considered related to study treatment. Another participant (0.3 mg group) 
experienced a grade 3 SAE of blood loss anemia that was not considered related to study 
treatment. 
2.2.2 
Summary of Key Safety Information for Investigational Product(s)  
The safety profile of ASP5354 is based on the results of the nonclinical studies, clinical 
phase 1 studies (Study 5354-CL-0001 and Study 5354-CL-0002) and a clinical phase 2 study 
(5354-CL-0201). Please refer to the current version of the ASP5354 IB for the most recent 
information. 
In nonclinical studies, ASP5354 caused very slight macrophage accumulation in the alveolus 
in the lungs of rats and increased plasma histamine levels in dogs.  Abnormal urine or skin 
color (pale green, greenish yellow or green) and green coloration of the kidneys, 
epididymides and/or LNs in gross pathology were also observed in rats and monkeys.  These 
findings disappeared with discontinuation of drug administration.  These findings were 
considered to be caused due to the green color of ASP5354 and were not considered 
toxicologically significant. 
ASP5354 has shown no local irritation, discernible genotoxicity, phototoxicity or 
embryofetal toxicity potential in the nonclinical species tested. 
Since all the phase 1 and 2 studies were single-dose studies, TEAEs leading to withdrawal of 
study drug were not applicable. No deaths were reported in any of the studies. ASP5354 was 
well tolerated and had no clinically significant effect on vital signs, ECG, blood chemistry, 
hematology or urinalysis. 
In conclusion, the nonclinical and phase 1 and 2 clinical safety data support the initiation of a 
phase 2 dose-finding study in patients with either breast cancer or melanoma scheduled to 
undergo SLN biopsy. 
For full details please refer to the current version of the ASP5354 IB. 


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2.3 
Risk/Benefit Assessment 
2.3.1 
Risk Assessment 
Hypersensitivity is monitored as an important potential risk based on the safety profile of 
ICG, of which ASP5354 is a derivative.  Hypersensitivity reactions, including anaphylactic 
reactions with fatal outcome, are reported to have occurred after treatment with ICG.  The 
reported incidence for ICG-related anaphylactic reactions is 1:42000 administrations or 
lower.  The risk with ASP5354 is estimated to be lower, as ASP5354 is not iodinated, 
whereas ICG is.  
Hypersensitivity reactions are well known to occur, in varying frequencies, with all kinds of 
contrast agents (iodinated contrast agents for computed tomography, gadolinium-based 
contrast agents for magnetic resonance imaging and contrast agents for image enhancement 
during ultrasound).  No such reactions have been observed for ASP5354 during the 
completed phase 1 studies (Study 5354-CL-0001 and Study 5354-CL-0002) or during the 
phase 2 study (Study 5354-CL-0201).   
2.3.2 
Benefit Assessment 
FDA approved imaging agents for lymphatic mapping are available; however, they have been 
associated with undesirable effect such as allergic reactions, skin staining or radiation 
exposure.  ASP5354 provides a way to visualize LNs intraoperatively. ASP5354 has been 
safely administered up to 24 mg intravenously. ASP5354 was well tolerated, with no 
IP-related TEAEs reported.  As such, there are no safety concerns for its use in participants 
undergoing SLN biopsy.  The benefits of using ASP5354 intraoperatively may decrease 
operating times, avoid radiation exposure. 
2.3.3 
Overall Risk-Benefit Conclusion 
The benefit of a single administration of ASP5354 outweighs the anticipated risk.  No 
toxicities have been seen in intravenous doses up to 24 mg and no local toxicity was observed 
in non-clinical evaluations. Safety will be monitored during the study for all enrolled 
participants. 
 
 


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3 
OBJECTIVES, ENDPOINTS AND ESTIMANDS 
Table 3 
Study Objectives and Endpoints 
Objectives 
Endpoints 
Primary 
● 
To determine the optimal dose of locally 
administered ASP5354 for LN visualization in 
participants undergoing SLN biopsy 
● 
Optimal dose determination by the VRC based 
on: 
o 
LN tissue visualized (Yes/No) 
o 
Visualized tissue is lymphatic in origin 
based on pathologic confirmation 
o 
The Likert Scale determination of the 
intensity of fluorescence (0 to 3) 
o 
Proportion of identified LN with 
histopathologic confirmation of LN tissue 
by ASP5354 compared with SoC 
treatment with either Tc-99mSC or 
Lymphoseek  
Secondary 
● 
To evaluate LN visualization of locally 
administered ASP5354 in participants 
undergoing SLN biopsy 
● 
Proportion of participants with at least 1 LN 
detected by visualization (Yes/No) with 
histopathologic confirmation of LN tissue using 
ASP5354 
● 
To compare LN detection for ASP5354 with 
the technetium-based treatment in breast cancer 
and melanoma 
● 
Proportion of identified LN with 
histopathologic confirmation of LN tissue by 
ASP5354 compared with SoC treatment with 
either Tc-99mSC or Lymphoseek  
● 
 To investigate the safety and tolerability of 
locally administered ASP5354 in participants 
undergoing SLN biopsy 
● 
Vital signs (blood pressure, pulse and 
respiratory rate) 
● 
Standard 12-lead ECGs 
● 
Clinical laboratory tests (hematology [complete 
blood count], serum chemistry and urinalysis) 
● 
Nature, frequency and severity of TEAEs and 
SAEs 
● 
To investigate the pharmacokinetics of locally 
administered ASP5354 in participants 
undergoing SLN biopsy 
● 
Plasma concentrations of ASP5354  
Table continued on next page 


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Objectives 
Endpoints 
Exploratory 
 
● 
To explore the visualization characteristics of 
ASP5354 
● 
The Likert Scale for qualitative response about 
the intensity of fluorescence 
● 
Signal background ratio 
● 
Transdermal visualization of lymphatic spread 
of ASP5354 after injection (Yes/No) 
● 
Time from injection to surgical identification of 
LN(s) 
● 
Number of LNs visualized by ASP5354 
● 
To compare LNs detected with metastatic 
spread for ASP5354 with the technetium-based 
treatment 
● 
Proportion of LN with metastatic spread, based 
on histopathologic review, detected by 
ASP5354 compared with SoC treatment with 
either Tc-99mSC or Lymphoseek 
Abbreviations from table above: ECG: electrocardiogram; LN: lymph node; SAE: serious adverse event; 
SLN: sentinel lymph node; SoC: standard of care; Tc-99mSC: technetium-99m sulfur colloid;  
TEAE: treatment-emergent adverse event; VRC: Visualization Review Committee. 
Estimands 
Estimand is not defined for any endpoint.  
4 
STUDY DESIGN AND DOSE RATIONALE 
4.1 
Overall Study Design 
The study is an open-label, dose-finding study to determine the optimal dose for LN 
visualization, as part of lymphatic mapping, in adult participants with breast cancer and 
melanoma undergoing SLN biopsy as part of Standard of Care (SoC).  
Enrollment of breast cancer and melanoma participants will be performed independently and 
simultaneously. Dose level 2 (0.2 mg) will enroll the first 3 participants in each indication 
and then the Visualization Review Committee (VRC) will review the data for the breast 
cancer group and the melanoma group separately.  If LN visualization with histopathologic 
confirmation occurs in the first 3 participants in each indication, then dose level expansion 
will occur and an additional 3 participants in each indication will be enrolled first with  the 
0.05 mg dose, followed by another 3 participants  with the 0.6 mg dose. An interim analysis 
will be performed across initial dose levels (0.05, 0.2 and 0.6 mg) separately for breast cancer 
and melanoma by the VRC prior to further enrollment of participants.   
If < 3 participants at 0.2 mg dose level have LN visualization with histopathologic 
confirmation on VRC review, 3 participants will be enrolled sequentially in the 0.6 mg and 
1 mg dose levels.  An interim analysis will then be performed across initial dose levels (0.2, 
0.6 and 1 mg) separately for breast cancer and melanoma by the VRC prior to further 
enrollment of participants.   


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Dose Arm/IP Name 
ASP5354 Dose Level 
ASP5354 Dose Conc and 
Volume 
Number of Participants 
Breast Cancer and 
Melanoma 0.05 mg/ 
ASP5354 
0.05 mg 
1.0 mg/mL, 
0.05 mL in total 
Up to 12 
Breast Cancer and 
Melanoma 0.2 mg/ 
ASP5354 
0.2 mg 
1.0 mg/mL, 
0.2mL in total 
Up to 12 
Breast Cancer and 
Melanoma 0.6 mg/ 
ASP5354 
0.6 mg 
1.0 mg/mL, 
0.6 mL in total 
Up to 12 
Breast Cancer and 
Melanoma 1 mg/ 
ASP5354 
1 mg 
1.0 mg/mL,  
1 mL in total 
Up to 12 
Breast Cancer and 
Melanoma 2 mg/ 
ASP5354 
2 mg 
1.0 mg/mL,  
2 mL in total 
Up to 12 
Breast Cancer 4 mg/ 
ASP5354 
4 mg* 
1.0 mg/mL,  
4 mL in total 
Up to 12 
Conc: concentration  
* The 4 mg dose level will be added only for breast cancer participants.   
Depending on the outcome of the interim VRC analysis, additional participants may be added 
to the same dose level, a dose level may be closed, or a new dose level (1 mg, 2 mg or 4 mg 
[breast cancer only]) will begin enrollment. Up to 12 participants will be assigned at each 
dose level for both breast cancer and melanoma. 
Participants will receive Tc-99mSC or Lymphoseek as part of SoC for SLN biopsy. In case 
the participant needs other surgery (e.g., lumpectomy, mastectomy etc.) on the same day of 
study drug administration, the other surgery is allowed once the SLN biopsy has completed. 
In the operating room, participants will receive ASP5354. After administration of ASP5354, 
visualization of the LN(s) will be assessed  using a NIR-F imaging system  followed by 
confirmation with the gamma probe. Once lymphatic mapping is completed, gamma probe 
will be used per SoC. For all the LNs observed by ASP5354, gamma probe will be attempted 
to measure radioactivity. If the LN is not observed with NIR-F after approximately 
15 minutes following injection for breast cancer participants and approximately 15 to 
30 minutes for melanoma participants depending on the location of the tumor and LNs, the 
investigator can proceed to use the gamma probe for LN detection. When the LN is detected 
with the gamma probe first, radioactivity will be measured and then NIR-F imaging should 
be attempted again for LN visualization. For participants with melanoma, usage of 
lymphoscintigraphy or SPECT as part of SoC is also allowed. 


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The visualization data to be collected will consist of the following: 
● 
The visualization of the LN(s) will be assessed by the investigator intraoperatively using 
a binary “Yes or No” question.  
o If “Yes,” the time of surgical identification of LN(s) will be collected by the 
investigator. 
o If “Yes,” the number of LNs visualized and the number of LNs visualized with 
histopathology confirmed LN will be also assessed by the investigator. 
Note: Histopathology review will be conducted per standard practice at each site. 
● 
Transdermal visualization of lymphatic spread of ASP5354 after injection of the drug 
will also be assessed using a binary “Yes or No” question.  
● 
The fluorescence intensity of the LN will be qualitatively assessed using a Likert Scale 
(0 = None; 1 = Mild; 2 = Moderate; and 3 = Strong). 
● 
The radioactivity count will be collected for all the LNs identified by ASP5354 and/or 
SoC. 
● 
The number of LN(s) with metastatic spread will be assessed based on histopathologic 
review. 
Note: Histopathology review will be conducted per standard practice at each site. 
During the surgery, video and/or image will be recorded to capture the lymphatic flow of 
ASP5354 and the signal intensity of each LN visualized. These images are used for Signal 
Background Ratio Analysis done by external reviewer (see [Section 7.1.2]). 
The visualization data of the LNs will be assessed by the VRC and will be compared with the 
performance of SoC. For further details of the VRC, refer to [Section 10.1.5 Committee(s) 
Structure and the VRC Charter]. The VRC will be held at the following time points for each 
dose:  
● 
Initial 3 participants at 0.2 mg in each tumor type have completed the SLN biopsies; 
● 
Initial 3 participants at each subsequent dose levels (0.05 and 0.6 mg or 0.6 and 1 mg) in 
each tumor type have completed the SLN biopsies (total of 6 participants); and 
● 
Additional 3 participants have completed SLN biopsies at the expanded or additional 
dose levels. The review of an additional 3 participants will continue until the optimal 
dose is defined. Maximum participants for each cohort  (i.e., 12) has not been reached.  
The data for the participants, at each subsequent dose level, who complete the SLN biopsy 
will be assessed by the VRC to decide whether to:  
● 
Expand a dose level if additional participants are needed to increase confidence for the 
optimal dose selection; 
● 
Stop a dose level when 100% of participants lack visualization or all participants have a 
fluorescence intensity of 0 or 1 on the qualitative Likert Scale, in which case additional 
participants would not be added to the dose level; or 
● 
Define an optimal dose (in the case of 2 doses performing equally, the lower dose will be 
selected). 


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The VRC assessment for optimal dose determination will be based on the totality of the 
collected data, including but not limited to: 
● 
Cummulative LN visualization (Yes/No)  
● 
Histopathologic confirmation of LN tissue visualized with ASP5354  
● 
Fluorescence intensity based on the Likert Scale (0 to 3) 
● 
Proportion of identified LN with histopathologic confirmation of LN tissue by ASP5354 
compared with SoC treatment with either Tc-99mSC or Lymphoseek 
● 
Safety data  
The optimal dose is defined as the dose that provides  better visualization compared with 
lower doses and a comparable visualization to the next higher dose. In the case where 2 doses 
performed equally, the lower dose would be selected. Cumulative visualization data of the 
LNs from all treated participants will be used for optimal dose determination. The optimal 
dose for breast cancer and melanoma will be selected separately. 
Based on VRC review of the initial 3 dose levels, if none of the first 9 participants have LN 
visualization, then all 3 initial  dose levels are stopped.The 1 and 2 mg dose levels or the 
2 and 4 mg (breast cancer only) dose levels will be added dependant on the highest dose used 
in the 3 prior dose levels. If 2 of the initial 3 doses are stopped due to lack of visualization in 
the 6 participants in the dose levels, then the dose level will be expanded to the 1 and/or 2 mg 
dose level. The 4 mg dose level for breast cancer will only be added if the 2 mg dose level 
remained open. For further details of the VRC, refer to the VRC Charter. 
If the investigator determines visualization of ASP5354 is not sufficient for LN imaging after 
local administration, the investigator can proceed with the  use of other NIR-F imaging 
modalities or Blue dye for LN detection if indicated. 
Safety and tolerability will be assessed by recording TEAEs and adverse reactions associated 
with the use of an IP, clinical laboratory evaluations (hematology, serum chemistry and 
urinalysis), 12-lead ECGs, vital signs measurements and physical examination. Blood 
samples for pharmacokinetic assessment will be collected pre- and post-administration of 
ASP5354 at defined time points up to the end of surgery.  
4.2 
Scientific Rationale for Study Design 
This is an open label dose finding study to determine the optimal dose for lymphatic mapping 
in participants undergoing lymphatic mapping and SLN biopsy with a radioisotope as part of 
SoC. Radioisotope is used routinely for lymphatic mapping in localized breast cancer (blue 
dye may also be used) and melanoma and provides a unique opportunity to investigate 
ASP5354 as a lymphatic mapping agent in participants already undergoing this procedure. 
Based on the nonclinical data of fastest transit time and strength of fluorescence of 0.1 mg in 
minipigs, the starting dose is 0.2 mg was selected. This will decrease the possibility of 
starting with a dose that may not lead to visualization in human participants.  Treatment will 
be staggered so the data in the first 3 participants treated can be analyzed to determine 
whether to dose above and below 0.2 mg or use 2 higher doses.  Optimal dose determination 
will require an inferior dose and a comparable higher dose.  Important characteristics of an 


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optimal dose will include fluorescence of lymphatic tissue based on histopathologic 
confirmation and intensity of fluorescence.  
The current study design will allow sequential assessment for safety, tolerability and ability 
for a given dose to provide LN visualization before exposing additional subjects to ASP5354. 
LN identification is binomial (Yes or No), as such descriptive statistics will be used for 
analysis. 
4.3 
Dose Rationale 
The starting dose is 0.2 mg (0.2 mL of a 1 mg/mL ASP5354 solution) per participant, which 
will be locally administered as subcutaneous (breast cancer) or intradermal (melanoma) 
injection.  The rationale for the starting dose is based on the results of the toxicology studies 
in rabbits and the phase 1 study after intravenous administration (Study 5354-CL-0001) and 
the estimated clinical efficacious dose in humans.  ASP5354 solution at up to 5 mg/mL 
showed no local irritation after subcutaneous and intramuscular injection in rabbits (Studies 
5354-TX-0101 and 5354-TX-0102), indicating 1 mg/mL ASP5354 solution can be used in 
this study.  ASP5354 is confirmed safe and well tolerated up to 24 mg after intravenous 
administration in the phase 1 study (Study 5354-CL-0001).  So, the starting dose of 0.2 mg is 
considered to be safe.  The estimated clinical efficacious dose of 0.2 mg (0.2 mL of a 
1 mg/mL ASP5354 solution) per participant is proposed because 0.1 mL of a 1 mg/mL 
ASP5354 solution provided comparable LN visualization after injection as compared with 
0.1 mL of a 2.5 or 5 mg/mL ICG solution in the nonclinical study in Göttingen minipigs 
(Study 5354-PH-0004).   
The potential highest dose tested in this study will be 4 mg (4 mL of a 1 mg/mL ASP5354 
solution) per participant, which is well below the highest intravenous dose of 24 mg tested in 
the phase 1 study (Study 5354-CL-0001). 
4.4 
End of Study Definition  
The study start is defined as the date the first participant signs informed consent.  The end of 
the study is defined as the last visit or scheduled procedure shown in the Schedule of 
Assessments for the last participant in the study.  
5 
STUDY POPULATION 
All screening assessments must be completed and reviewed to confirm the potential 
participant meets all eligibility criteria. Prospective approval of protocol deviations to 
eligibility criteria (also known as protocol waivers or exemptions) is not permitted. 
5.1 
Inclusion Criteria 
Participant is eligible for participation in the study if all of the following apply: 
1. Institutional Review Board (IRB)/Independent Ethics Committee (IEC) approved written 
informed consent and privacy language as per national regulations (e.g., Health 
Insurance Portability and Accountability Act authorization for US study sites) must be 


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obtained from the participant prior to any study-related procedures (including withdrawal 
of prohibited medication, if applicable). 
2. Participant has been diagnosed with localized breast cancer (female only, stage 0 to 2, 
N0 and M0) or melanoma (stage 1 to 2, N0 and M0) and is scheduled to undergo surgical 
intervention for SLN detection and removal using Tc-99mSC or Lymphoseek as part of 
SoC. 
3. Participant is considered an adult (≥ 18 years of age) according to local regulation at the 
time of signing the informed consent form (ICF). 
4. Female participant is not pregnant (see [Section 10.2 Appendix 2 Contraception 
Requirements]) and at least 1 of the following conditions apply: 
a. Not a woman of childbearing potential (WOCBP) (see [Section 10.2 Appendix 2 
Contraception Requirements])  
b. WOCBP who agrees to follow the contraceptive guidance (see [Section 10.2 
Appendix 2 Contraception Requirements]) from the time of informed consent 
through at least 30 days after final study treatment administration. 
5. Female participant must agree not to breastfeed starting at screening and throughout the 
study period and for 30 days after final study treatment administration. 
6. Female participant must not donate ova starting at first dose of IP and throughout the 
study period and for 30 days after final study treatment administration. 
7. Male participant with female partner(s) of childbearing potential (including breastfeeding 
partner) must agree to use contraception (see [Section 10.2 Appendix 2 Contraception 
Requirements]) throughout the treatment period and for 30 days after final study 
treatment administration. 
8. Male participant must not donate sperm during the treatment period and for 30 days after 
final study treatment administration. 
9. Male participant with pregnant partner(s) must agree to remain abstinent or use a 
condom for the duration of the pregnancy throughout the study period and for 30 days 
after final study treatment administration.  
10. Participant agrees not to participate in another interventional study while participating in 
the present study. 
Waivers to the inclusion criteria will NOT be allowed.  
5.2 
Exclusion Criteria 
Participant will be excluded from participation in the study if any of the following apply: 
1. Participant has had prior LN surgery in the area where LN detection is needed.  
2. Participant has had prior LN radiation in the area where LN detection is needed. 
3. Participant has had prior neo-adjuvant chemotherapy. 
4. Participant has definitive LN metastases. 
5. Participant has metastatic cancer. 
6. Participant has received any investigational therapy within 28 days or 5 half-lives, 
whichever is longer, prior to screening.  


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7. Participant has any condition, which, in the investigator’s opinion, makes the participant 
unsuitable for study participation. 
8. Participant has a known or suspected hypersensitivity to ASP5354, ICG or any 
components of the formulation used.  
9. Participant has had previous exposure to ASP5354.  
10. Participant has moderate to severe cardiac disease that limits daily functioning (New 
York Heart Association Class III-IV) or other medical conditions that the investigator 
feels would impact safety or study compliance. 
11. Participant has a resting heart rate ≤ 45 bpm or ≥ 115 bpm, systolic blood pressure 
≥ 160 mmHg or diastolic blood pressure ≥ 100 mmHg within 1 week prior to IP 
administration. If the blood pressure exceeds the limits above, repeat readings can be 
taken. Participant who has adequately controlled blood pressure is eligible. 
12. Participant has any of the following screening laboratory values: 
● 
Hemoglobin ≤ 9 g/dL 
● 
Absolute neutrophil count ≤ 1500/µL 
● 
Platelet count ≤ 100000/µL 
● 
Estimated glomerular filtration rate < 60 mL/min/1.73 m2 based on the Chronic 
Kidney Disease Epidemiology Collaboration equation* 
● 
Serum bilirubin ≥ 1.5 × upper limit of normal (ULN) 
● 
Aspartate aminotransferase (AST) ≥ 2.5 × ULN 
● 
Alanine aminotransferase (ALT) ≥ 2.5 × ULN 
*eGFR = 141 × min(Scr/κ, 1)α × max(Scr/κ, 1)-1.209 × 0.993Age × 1.018 [if 
female] × 1.159 [if African American] 
where: 
Scr is serum creatinine in mg/dL, 
κ is 0.7 for females and 0.9 for males, 
α is -0.329 for females and -0.411 for males, 
min indicates the minimum of Scr/κ or 1, and 
max indicates the maximum of Scr/κ or 1 
13. Participant has received ICG, other NIR-F imaging agents, Blue dye (e.g., methylene 
blue or isosulfan blue) or Magtrace within 2 weeks prior to IP administration. 
14. Participant has active alcohol abuse or illicit drug abuse (non-prescription or over-the-
counter medications). Participant should not have consumed any alcohol or illicit drug 
(excluding cannabidiol products) within 24 hours of surgery. 
15. Participant has been diagnosed with bilateral breast cancer 
Waivers to the exclusion criteria will NOT be allowed. 
5.3 
Lifestyle Considerations 
Not applicable. 


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5.4 
Screen Failures 
A screen failure is defined as a potential participant who signed the ICF but did not meet 1 or 
more criteria required for participation in the study. 
For screen failures, the demographic data, date of signing the ICF, inclusion and exclusion 
criteria, AEs up to the time of screen failure and reason for screen failure will be collected in 
the electronic case report form (eCRF). 
5.4.1 
Rescreening 
Rescreening is not allowed. However, in case that the results of screening assessments do not 
meet the parameters required by the eligibility criteria (e.g., clinical laboratory tests, vital 
signs, physical examination, ECGs, etc.) screening assessments may be repeated once within 
the screening period without the need to register the participant as a screen failure. 
6 
INVESTIGATIONAL PRODUCT 
6.1 
Investigational Product Administered 
The assigned doses of ASP5354 will be administered as a single dose intraoperatively. 
Table 4 
Investigational Product 
Name 
ASP5354 
Use 
Test product 
Dosage Formulation 
Sterile powder for solution 
Physical Description 
Green cake 
Unit Dose Strength 
Each vial contains 1.3 mg of ASP5354 and has to be reconstituted with 
1.2 mL sterile water for injection, to a concentration of 1 mg/mL. 
Packaging and Labeling 
ASP5354 sterile lyophilized powder will be provided in 2R amber glass 
vials and stored according to the instructions on the label 
Route of Administration 
Subcutaneous (breast cancer) or intradermal (melanoma) administration 
Frequency/Duration 
Single dose 
Administration Instruction 
Administered under fasting conditions intraoperatively 
IMP or Non-IMP 
IMP 
Sourcing 
Provided by sponsor 
IMP: Investigational Medicinal Product 
Refer to the pharmacy manual for detailed information regarding preparation, handling and 
storage of the IP. 
6.1.1 
Investigational Product Administration 
ASP5354 sterile lyophilized powder for solution will be supplied by the sponsor or designee 
for subcutaneous (breast cancer) or intradermal (melanoma) administration.  
Each participant will be administered a single dose of the assigned dose level of ASP5354. 
ASP5354 will be injected at a minimum of 2 and up to 4 sites of equally divided injections. 


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The exact date and time and location (i.e., 3 and 9 o’clock position of tumor) of IP 
administration will be documented.  
Breast Cancer 
Arm/ 
IP Name 
Breast Cancer 0.05 mg/ 
ASP5354 
Breast Cancer 0.2 mg/ 
ASP5354 
Breast Cancer 0.6 mg/ 
ASP5354 
Use 
test product 
test product 
test product 
Dose 
0.05 mg 
0.2 mg 
0.6 mg 
Concentration 
1.0 mg/mL 
1.0 mg/mL 
1.0 mg/mL 
Volume 
0.05 mL in total 
0.2 mL in total 
0.6 mL in total 
Frequency 
single dose 
single dose 
single dose 
Route 
subcutaneous injection 
subcutaneous  injection 
subcutaneous injection 
Location 
subareolar 
subareolar 
subareolar 
Injection site 
Minimum of 2, up to 4, 
sites equally divided 
injections 
Minimum of 2, up to 4, 
sites equally divided 
injections 
Minimum of 2, up to 4, 
sites equally 
dividedinjections 
Duration 
single dose 
single dose 
single dose 
 
Arm/ 
IP Name 
Breast Cancer 1 mg/ 
ASP5354 
Breast Cancer 2 mg/ 
ASP5354 
Breast Cancer 4 mg/ 
ASP5354 
Use 
test product 
test product 
test product 
Dose 
1 mg 
2 mg 
4 mg 
Concentration 
1.0 mg/mL 
1.0 mg/mL 
1.0 mg/mL 
Volume 
1 mL in total 
2 mL in total 
4 mL in total 
Frequency 
single dose 
single dose 
single dose 
Route 
subcutaneous injection 
subcutaneous injection 
subcutaneous injection 
Location 
subareolar 
subareolar 
subareolar 
Injection site 
Minimum of 2, up to 4 
sites equally divided 
injections 
Minimum of 2, up to 4 
sites equally divided 
injections 
Minimum of 2, up to 4 
sites equally divided 
injections 
Duration 
single dose 
single dose 
single dose 
 
ASP5354 will be injected subcutaneously at a minimum of 2, and up to 4. sites of equally 
divided subareolar injections. Please refrain from massaging the area of drug administration.  
After approximately 5 to 10 minutes, lymphatic mapping will then occur based on 
intraoperative fluorescence visualization using ASP5354. LNs will be identified by NIR-F 
imaging, followed by identification with the gamma probe. In the event that NIR-F imaging 
does not detect LN after approximately 15 minutes, the gamma probe should be used for LN 
identification. When the LN is detected with the gamma probe first, NIR-F imaging should 
be attempted again for LN visualization. Dissection of the LN will be conducted following 
identification by NIR-F imaging and/or the gamma probe. Before completing the surgery, 
NIR-F imaging and the gamma probe will be used to confirm further presence of other LNs, 
with further dissection and LN removal if indicated.  


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Melanoma
Arm/ 
IP Name 
Melanoma 0.05 mg/ 
ASP5354 
Melanoma 0.2 mg/ 
ASP5354 
Melanoma 0.6 mg/ 
ASP5354 
Use 
test product 
test product 
test product 
Dose 
0.05 mg 
0.2 mg 
0.6 mg 
Concentration 
1.0 mg/mL 
1.0 mg/mL 
1.0 mg/mL 
Volume 
0.05 ml in total 
0.2 ml in total 
0.6 ml in total 
Frequency 
single dose 
single dose 
single dose 
Route 
intradermal injection 
intradermal injection 
intradermal injection 
Location 
peritumoral 
peritumoral 
peritumoral 
Injection site 
Minimum of 2, up to 4, 
sites equally divided 
injections 
Minimum of 2, up to 4, 
sites equally divided 
injections 
Minimum of 2, up to 4, 
sites equally divided 
injections 
Duration 
single dose 
single dose 
single dose 
 
Arm/ 
IP Name 
Melanoma 1 mg/ ASP5354 
Melanoma 2 mg/ ASP5354 
Use 
test product 
test product 
Dose 
1 mg 
2 mg 
Frequency 
single dose 
single dose 
Concentration 
1.0 mg/mL 
1.0 mg/mL 
Volume 
1.0 mL in Total 
2.0 mL in total 
Frequency 
single dose 
single dose 
Route 
intradermal injection 
intradermal injection 
Location 
peritumoral 
peritumoral 
Injection site 
Minimum of 2, up to 4, sites 
equally divided injections 
Minimum of 2, up to 4, sites 
equally divided injections 
Duration 
single dose 
single dose 
 
ASP5354 will be injected intradermally at a minimum of 2, and up to 4, sites of equally 
divided peritumoral injections. Please refrain from massaging the area of drug administration. 
After approximately 5 to 10 minutes, lymphatic mapping will occur based on intraoperative 
fluorescence visualization using ASP5354. LNs will be identified by NIR-F imaging, 
followed by identification with the gamma probe. Please note that the time from injection of 
IP to the first visualization may vary due to tumor and LN location. In the event that NIR-F 
imaging does not detect LN after approximately 15 to 30 minutes, the gamma probe should 
be used for LN identification.  When the LN is detected with the gamma probe first, NIR-F 
imaging should be attempted again for LN visualization. Dissection of the LN will be 
conducted following identification by NIR-F imaging and/or the gamma probe. Before 
completing the surgery, NIR-F imaging and the gamma probe will be used to confirm further 
presence of other LNs with further dissection and LN removal if indicated.  


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6.1.2 
Other Therapy 
Tc-99mSC or Lymphoseek will be administered per SoC. Blue dye or Magtrace may be used 
for breast cancer only if ASP5354 does not detect LN and an additional modality is indicated 
per the surgeon’s judgement. 
6.2 
Preparation/Handling/Storage/Accountability 
6.2.1 
Packaging and Labeling 
All study drug(s) used in this study will be prepared, packaged and labeled under the 
responsibility of qualified staff at Astellas Pharma Inc. (API), or sponsor’s designee in 
accordance with API or sponsor’s designee standard operating procedures (SOPs), Good 
Manufacturing Practice (GMP) guidelines, International Council for Harmonisation of 
Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice 
(GCP) guidelines and applicable local laws/regulations. 
Each carton and vial will bear a label conforming to regulatory guidelines, GMP and local 
laws and regulations that identifies the contents as an investigational drug. 
A qualified person of Astellas Pharma Europe BV or sponsor’s designee will perform the 
final release of the IP according to the requirements of the EU Directive 2003/94/EC Annex 
13. Refer to the pharmacy manual for detailed information regarding packaging and labeling 
of the IP. 
6.2.2 
Handling, Storage and Accountability 
● 
The investigator or designee must confirm appropriate temperature conditions have been 
maintained during transit for all IP received and any discrepancies are reported and 
resolved before use of the IP. 
● 
Only participants enrolled in the study may receive IP and only authorized study site 
personnel may supply or administer IP. Only IP with appropriate expiry/retest dating 
may be dispensed. 
● 
All IP must be stored in a secure, environmentally controlled and monitored (manual or 
automated) area in accordance with the labeled storage conditions and access must be 
limited to the investigator and authorized study site personnel. 
● 
The investigator, institution or the head of the medical institution (where applicable) is 
responsible for accountability, reconciliation and record maintenance (i.e., receipt, 
reconciliation and final disposition records).  
● 
Further guidance and instruction on final disposition of used and unused IP is provided 
in the pharmacy manual or other document. 
Refer to the pharmacy manual for detailed information regarding handling, storage and 
accountability of the IP. 
6.3 
Randomization and Blinding 
This is an open-label study, and no randomization is planned. Participant enrollment and 
dispensation of IP will be performed via the interactive response technology (IRT) system. 
Prior to initiation of treatment, study site personnel will obtain the participant number and 


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treatment assignment from the IRT system. Specific IRT procedures will be described in the 
respective study manual. 
6.4 
Investigational Product Compliance 
Not applicable, as ASP5354 is a single dose imaging agent.  
6.5 
Dose Modification 
Since this is a single dose study, dose modification is not applicable.  
6.6 
Continued Access to Investigational Product After the End of the 
Study  
Not applicable, as ASP5354 is a single dose anatomical imaging agent and has no therapeutic 
indications.  
6.7 
Treatment of Overdose  
In the event of suspected ASP5354 overdose, the participant should receive supportive care 
and monitoring. The medical monitor/expert should be contacted as applicable. 
Refer to [Section 10.3.8.2 Medication Error, Overdose and “Off-label Use”] for reporting 
requirements for suspected overdose or other medication error.  
6.8 
Concomitant Therapy 
6.8.1 
Previous Treatment (Medication) 
Investigate the following survey items and record in eCRF. 
Previous treatment 
Investigational period 
Investigational items 
ICG 
Blue Dye 
Magtrace 
Other NIR-F imaging agent 
Tc-99mSC  
Lymphoseek (generic name tilmanocept) 
2 weeks prior to study 
treatment 
administration to end 
of screening period  
Name of drug, dose, administration 
route, administration period, 
administration timing, and reason for 
use 
ICG: indocyanine green; NIR-F: near-infrared fluorescence; Tc-99mSC: technetium-99m sulfur colloid 
 


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6.8.2 
Concomitant Treatment (Medication and Non-Medication Therapy) 
Investigate the following survey items and record in the eCRF. 
Concomitant treatments 
Investigational period 
Investigational items 
Concomitant 
medication 
ICG 
Blue Dye 
Magtrace 
Other NIR-F imaging agent 
Tc-99mSC 
Lymphoseek (generic name 
tilmanocept) 
Start of treatment 
period to end of follow 
up period 
Name of drug, dose, administration 
route, administration period, 
administration timing, and reason 
for use 
All medication other than 
the above† 
Name of drug, administration 
route, administration period, 
administration timing, and reason 
for use 
Concomitant 
therapy 
All non-medication therapy 
including dietary therapy 
Name of therapy, treatment period, 
treatment timing, and reason for 
treatment 
ICG: indocyanine green; NIR-F: near-infrared fluorescence; Tc-99mSC: technetium-99m sulfur colloid  
† The following items are not the target of this investigation: 
Dissolution/dilution solution (injection water, physiological saline, etc.), treatment prior to assessment, 
sterilization solution, washing solution and topical anesthesia (xylocaine gel, etc.).  
6.8.3 
Prohibited Medication 
The following concomitant medications will not be allowed within 2 weeks prior to study 
treatment administration until the completion of the surgical procedure unless it is determined 
that LN detection with ASP5354, and radioisotope, is not sufficient. 
● 
ICG* 
● 
Other NIR-F imaging agent 
● 
Blue dye 
* ICG administration for other perspectives than LN visualization (e.g., vascular perfusion) is allowed after the 
completion of SLN biopsy. 
A list of excluded concomitant medications is provided in [Section 10.5 Appendix 5 List of 
Excluded Concomitant Medications].  
7 
STUDY PROCEDURES AND ASSESSMENTS 
● 
Study procedures and their timing are summarized in the Schedule of Assessments 
[Table 1]. Adherence to the study design requirements, including those specified in the 
schedule of assessments, is essential and required for study conduct. Prospective protocol 
waivers or exemptions are not allowed. 
● 
Refer to the Alternative Schedule of Assessments [Table 10] in [Section 10.7 
Appendix 7: Clinical Study Continuity] for acceptable alternate methods to assess safety 
and efficacy parameters in the event the study is interrupted due to a crisis (e.g., natural 
disaster, pandemic). 


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● 
Any change, divergence or departure from the study design or procedures identified in 
the protocol is considered a protocol deviation. All deviations from the protocol are to be 
recorded. 
● 
Immediate safety concerns should be discussed with the sponsor immediately upon 
occurrence or awareness to determine if the participant should continue or discontinue 
study treatment. 
● 
All screening evaluations must be completed and reviewed to confirm that potential 
participants meet all eligibility criteria. The investigator will maintain a screening log to 
record details of all participants screened and to confirm eligibility or record reasons for 
screening failure, as applicable. 
● 
Procedures conducted as part of the participant’s routine clinical management (e.g., 
imaging, blood count) and obtained before signing of the ICF may be utilized for 
screening or baseline purposes provided the procedures met the protocol-specified 
criteria and were performed within the time frame defined in the Schedule of 
Assessments [Table 1]. 
● 
Refer to the Alternative Schedule of Assessments in Response to a Crisis [Table 10] for 
acceptable alternate methods to assess safety and efficacy parameters in the event the 
study is interrupted due to a crisis (e.g., natural disaster, pandemic). 
7.1 
Efficacy Assessments 
The efficacy endpoint for this study is described in Study Objectives and Endpoints 
[Table 3]. 
7.1.1 
Primary and Secondary Efficacy Assessments 
Either during or post-operatively, the investigator will evaluate the details of all the LNs 
identified either by ASP5354 or Gamma Probe. These data will be captured to eCRF. 
Following data is captured in eCRF per participant. 
● Identification of at least 1 LN detected by ASP5354 and/or Tc-99mSC or Lymphoseek  
● Times of injection of ASP5354 and Tc-99mSC or Lymphoseek  
● Location (i.e., 3 and 9 o’clock) of ASP5354 administration 
● Time of start of surgery 
● Time of surgical identification of the LN(s) by ASP5354 
● Time of incision for lymphatic mapping 
● Time of removal of first LN 
● Time of end of surgery 
● Transdermal visualization of lymphatic spread of ASP5354 after injection  
Following data is captured in eCRF per LN. 
● 
“Sentinel LN” or “Non-sentinel LN”   
● 
Method of identification (ASP5354 and/or Tc-99mSC or Lymphoseek) 
● 
Dissection of LN tissue identified* 
● 
Histopathologic confirmation of LN tissue by pathologist* 


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● 
Histopathologic confirmation of metastatic spread by pathologist* 
● 
Likert scale: 
The intensity of fluorescence will be qualitatively assessed during surgery by the 
investigator, by direct examination of the LN. Likert Scale (0 = None, 1 = Mild, 
2 = Moderate and 3 = Strong) will be used to determine the intensity of fluorescence at 
the time point of the LN identification. 
● 
Radioactivity count: 
The radioactivity count of all the LNs identified by ASP5354 and /or Tc-99mSC or 
Lymphoseek will be counted with gamma probe.   
* may not be collected in case of Non-Sentinel LN 
7.1.2 
Signal Background Ratio 
Video recording by NIR-F device is also required to evaluate the visualization of LN by 
ASP5354. Start recording a video at the time of injection of ASP5354 and continue until the 
first lymph node is visualized. For the remaining lymph nodes visualized, it is recommended 
to use video recording, but capturing images with the device is also acceptable. For further 
details, please refer to the Imaging Charter. 
SBR is calculated by external reviewer based on the video recording by NIR-F device. 
Fluorescence images will be captured and recorded from injection time of ASP5354 until last 
LN is dissected. Images will be captured on a NIR-F and overlay view (if available) for all 
the LNs identified by ASP5354. SBR will be calculated as follows [Hoogstins et al, 2019]: 
SBR =  
mean region of interest 
mean signal background 
Additional detail will be provided in the Imaging Charter. 
7.2 
Safety Assessments 
Study procedures and their timing are summarized in the Schedule of Assessments [Table 1].  
Protocol waivers or exemptions are not allowed. 
7.2.1 
Laboratory Assessments 
● 
See [Section 10.6 Appendix 6 Laboratory Assessments] for the list of clinical laboratory 
tests to be performed and refer to Schedule of Assessments [Table 1] for timing and 
frequency. 
● 
The investigator must review the laboratory report, document this review, and record any 
clinically significant changes occurring during the study as an AE. The laboratory 
reports must be filed with the source documents.  
● 
Clinical significance of out-of-range laboratory findings is to be determined and 
documented by the investigator or subinvestigator who is a qualified physician. 
Abnormal laboratory findings associated with the underlying disease are not considered 
clinically significant unless judged by the investigator to be more severe than expected 
for the participant's condition. 


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7.2.2 
Vital Signs 
● 
Blood pressure, pulse rate and respiratory rate will be assessed. 
● 
Blood pressure and pulse measurements will be assessed with the participant in the 
sitting or supine position with a completely automated device. Manual techniques will be 
used only if an automated device is not available. 
● 
Blood pressure and pulse measurements should be preceded by at least 5 minutes of rest 
for the participant in a quiet setting without distractions (e.g., television, cell phones). 
● 
Vital signs will be taken before blood collection for laboratory tests. 
7.2.3 
Physical Examination  
Physical examinations will be performed as indicated in the Schedule of Assessments 
[Table 1] and whenever there is a medical indication. 
A full physical examination will be performed at screening (visit 1). Body systems to be 
evaluated include general appearance, skin, lymphatic, head and neck, ears, nose and throat, 
chest and lungs, cardiovascular, abdomen, extremities, musculoskeletal and neuromuscular. 
At visit 2 and follow-up (visit 3), a full physical examination is not mandatory, but a 
symptom-directed physical examination will be performed.   
Height and weight will be measured using standard institution practice and equipment. 
7.2.4 
Electrocardiogram  
Standard 12-lead ECGs will be collected per site’s standard procedure at screening, 
preoperative and safety follow-up period.  A single 12-lead ECGs will be collected after the 
participant has been resting in the supine position. Intraoperative ECG monitoring will occur 
at 30 minutes post dose (15 min ≤ and < 45 min).A 12-lead ECG is not mandatory for 
intraoperative and postoperative ECG, and assessment can be based on the rhythm strip or 
other site standard ECG as per the site’s standard of care routine for cardiac monitoring. 
The investigator will review, sign and date the ECG after recording to ensure participant 
safety. The time of the ECG, the interval measurements, as well as an overall conclusion, will 
be documented. This overall conclusion will be recorded as normal, abnormal not clinically 
significant or abnormal clinically significant in the eCRF. Any clinically significant ECG 
abnormalities should be recorded as an AE. 
The ECG recordings will be printed, signed and saved in the source.  
7.2.5 
Order of Assessments  
The following order should be followed when more than one assessment is required at a time 
point with blood sampling being collected nearest to the scheduled time point: 
● 
12-lead ECG 
● 
Vital signs 
● 
Blood collection for pharmacokinetics and clinical laboratory tests 


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7.2.6 
Other Assessments 
7.2.6.1 Green Coloration of Urine or Skin 
The occurrence of green coloration of the urine or skin will be monitored throughout the 
study. If observed, any visual green coloration abnormalities will be recorded as an AE. If 
urine coloration occurs for participants who have a urethral catheter in place, the color of 
urine will be assessed during surgery and every 120 minutes (± 30 minutes window) after the 
end of surgery until it resolves or the participant is discharged, whichever is earlier. If no 
urethral catheter is in place, voided urine will be assessed for green coloration and the time 
collected until it resolves or the participant is discharged, whichever is earlier.  The start and 
stop date and time will also be recorded.  The occurrence of green coloration of the skin will 
be recorded as an AE any time after dose administration.  The start and stop date and time 
will be recorded.  Skin coloration will be followed up until it resolves or the participant is 
discharged, whichever is earlier. 
7.3 
Adverse Events and Other Safety Aspects 
The definitions of an AE, SAE or ADE can be found in [Sections 10.3.1 Definition of 
Adverse Events, 10.3.2 Definition of Serious Adverse Events and 10.3.3 Adverse Device 
Effect], respectively. 
AEs will be reported by the participant (or, when appropriate, by a caregiver, surrogate, or 
the participant's legally authorized representative), investigator (surgeon) or anesthesiologist. 
The investigator and any qualified designees are responsible for detecting, documenting and 
recording events that meet the definition of an AE, SAE or ADE and remain responsible for 
following up AEs that are serious, considered related to the study, or that caused the 
participant to discontinue the IP and/or study [see Section 10.3 Appendix 3 Adverse 
Events/Adverse Device Effects: Definitions and Procedures for Recording, Evaluating, 
Follow-up and Reporting].  
The method of recording, evaluating, and assessing causality of AE, SAE and ADE and the 
procedures for completing and transmitting SAE/ADE reports are provided in [Section 10.3 
Appendix 3 Adverse Events/Adverse Device Effects: Definitions and Procedures for 
Recording, Evaluating, Follow-up and Reporting]. 
7.3.1 
Time Period and Frequency for Collecting Adverse Event and Serious Adverse 
Event Information 
All SAEs will be collected from the signing of the ICF until the follow-up visit at the time 
points specified in the Schedule of Assessments [Table 1] and reported on the eCRF. 
All AEs will be collected from the signing of the ICF until the follow-up visit at the time 
points specified in the Schedule of Assessments [Table 1] and reported on the eCRF.  
If the NCI CTCAE grade of an SAE/AE changes, the event should be relisted on the eCRF 
with the new NCI CTCAE grade and new onset date. 


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If the NCI CTCAE grade decreases, the SAE/AE should be relisted on the eCRF with the 
new NCI CTCAE grade and new onset date. The exception is ongoing pre-dose events that 
continue post-dose and improve post-dose. Such events should not be re-listed. 
If the NCI CTCAE grade of an SAE reduces, the details of the AE should be provided on the 
SAE worksheet for the medical assessor to be able to assess the course of the event. 
All SAEs will be recorded and reported to the sponsor or designee immediately and under no 
circumstance should this exceed 24 hours, as indicated in [Section 10.3 Appendix 3 Adverse 
Events/Adverse Device Effects: Definitions and Procedures for Recording, Evaluating, 
Follow-up and Reporting]. The investigator will submit any updated SAE data to the sponsor 
within 24 hours of it being available. 
Investigators are not obligated to actively seek AE or SAE after conclusion of the study 
participation. However, if the investigator learns of any SAE, including a death, at any time 
after a participant has been discharged from the study, and he/she considers the event to be 
reasonably related to the study IP or study participation, the investigator must promptly 
notify the sponsor. 
The Reference Safety Information that will be used to determine expectedness of an SAE for 
this study is in the IB. 
7.3.1.1 Adverse Device Effects 
● 
ADEs are to be collected via the ADE worksheet and reported to the sponsor within 
24 hours of awareness (see [Section 10.3.3.1]). 
● 
Any complaint about a device must be reported regardless of whether the defect or 
deficiency had any effect on a participant or on study personnel (see [Section 10.3.3.3]). 
7.3.2 
Method of Detecting Adverse Events and Serious Adverse Events 
Care will be taken not to introduce bias when detecting AEs and/or SAEs. Open-ended and 
non-leading verbal questioning of the participant is the preferred method to inquire about AE 
occurrences. 
7.3.3 
Follow-up of Adverse Events and Serious Adverse Events 
After the initial AE/SAE report, the investigator is required to proactively follow each 
participant at subsequent visits/contacts. All SAEs will be followed until resolution, 
stabilization, the event is otherwise explained, or the participant is lost to follow-up (as 
defined in [Section 8.3 Lost to Follow-up]). Further information on follow-up procedures is 
provided in [Section 10.3 Appendix 3 Adverse Events/Adverse Device Effects: Definitions 
and Procedures for Recording, Evaluating, Follow-up and Reporting].  
If after the protocol-defined AE collection period (see [Section 7.3.1 Time Period and 
Frequency for Collecting Adverse Event and Serious Adverse Event Information]), an AE 
progresses to an SAE, or the investigator learns of any (S)AE (serious adverse event or 
adverse event) including death, where he/she considers there is reasonable possibility it is 
related to the IP or study participation, the investigator must promptly notify the sponsor. 


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7.3.4 
Regulatory Reporting Requirements for Serious Adverse Events/Adverse 
Device Effects 
● 
Prompt notification by the investigator to the sponsor of a SAE/ADE is essential so that 
legal obligations and ethical responsibilities towards the safety of participants and the 
safety of a study IP under clinical investigation are met. 
● 
The sponsor has a legal responsibility to notify both the local regulatory authority and 
other regulatory agencies and/or device manufacturer about the safety of a study IP under 
clinical investigation. The sponsor will comply with country-specific regulatory 
requirements relating to safety reporting to the regulatory authority, IRB/IEC and 
investigators. 
● 
Investigator safety reports must be prepared for suspected unexpected serious adverse 
reactions (SUSAR) according to local regulatory requirements and sponsor policy and 
forwarded to investigators as necessary. 
● 
An investigator who receives an investigator safety report describing a SAE or other 
specific safety information (e.g., summary or listing of SAEs) from the sponsor will 
review and then file it along with the IB and will notify the IRB/IEC, if appropriate 
according to local requirements. 
7.3.5 
Disease-related Events and/or Disease-related Outcomes Not Qualifying as 
Adverse Events or Serious Adverse Events 
Under this protocol, the following event will not be considered as an (S)AE: 
● 
Preplanned and elective hospital/clinical procedures/interventions or procedures for 
diagnostic, therapeutic or surgical procedures for a preexisting condition that did not 
worsen during the course of the study. These procedures are collected in the eCRF. 
7.3.6 
Special Situations  
Certain special situations observed in association with the IP, such as incorrect administration 
(e.g., wrong dose of IP or background therapy) are reported as protocol deviations and/or 
may require special reporting, as described below. These special situations are not considered 
AEs but do require to be communicated to Astellas as per the timelines defined below. 
If a special situation is associated with, or results in, an AE, the AE is to be assessed 
separately from the special situation and captured as an AE in the eCRF. If the AE meets the 
definition of an SAE, the SAE is to be reported as described in [Section 10.3.7 Reporting 
Procedures for Serious Adverse Events/Adverse Device Effects] and the details of the 
associated special situation are to be included in the clinical description on the special 
situation worksheet or pregnancy reporting form. 
The special situations are: 
● 
Pregnancy 
● 
Medication error, overdose and use outside protocol 
● 
Misuse/abuse 
● 
Occupational exposure 


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● 
(Suspicion of) Transmission of infectious agent 
● 
Suspected drug-drug interaction 
Instructions and procedures for reporting special situations are provided in [Section 10.3.8 
Reporting Procedures for Pregnancy and Special Situations].  
7.4 
Pharmacokinetics 
7.4.1 
Analysis of ASP5354 in Plasma 
Blood samples for the analysis of ASP5354 in plasma will be collected as indicated in the 
Schedule of Assessments [Table 1] and Sample Collection and Timed Procedure Schedule 
[Table 2]. 
Blood samples will be collected via a peripherally placed intravenous cannula or by direct 
venipuncture in a suitable vein. Blood sampling, processing, storage and shipment 
instructions are provided in a Sample Handling and Processing Manual. 
When deemed appropriate at a later date, plasma samples remaining after the 
pharmacokinetic analysis may be used for exploratory metabolic profiling after the study. 
This test will be described in a separate report and will not be incorporated in the integrated 
clinical study report (CSR). 
7.5 
Total Amount of Blood 
The total amount of blood drawn for each participant will vary depending on the length of the 
surgery. At any time during the study, if any laboratory abnormalities are found for a 
participant, additional blood may be drawn for safety monitoring. The average expected 
blood draw is shown in Blood Sampling [Table 5]. 
Table 5 
Blood Sampling 
Sample Type 
Number of Samples 
Sample Volume (mL) 
Total Volume (mL) 
Serum chemistry  
4 
8.5 
34.0 
Hematology 
4 
4.0 
16.0 
ASP5354 pharmacokinetics 
6 
4.0 
24.0 
Total 
74.0 
 
7.6 
Study Participant Feedback 
This study will include an option for participants to complete an anonymized questionnaire in 
order to provide feedback on their clinical study experience. Individual participant  responses 
will not be reviewed by investigators. Responses will be used by the sponsor to understand 
where improvements can be made in the clinical study process. This questionnaire will not 
collect data about the participant’s condition, symptoms, treatment effect or AEs and 
therefore is not considered study data. Should any spontaneous information be collected 
about AEs, this would be reported and transferred to the safety database. 


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8 
PARTICIPANT DISCONTINUATION 
Refer to [Section 10.1.9 Study and Site Start and Closure] regarding discontinuation of study 
sites or of the study as a whole.  
8.1 
Discontinuation of Individual Participant(s) from Study Treatment  
Since this is a single dose study, treatment discontinuation is not applicable. 
8.2 
Discontinuation of Individual Participant(s) from Study 
The participant is free to withdraw from the study for any reason and at any time without 
giving reason for doing so and without penalty or prejudice. The investigator is also free to 
terminate a participant’s involvement in the study at any time if the participant’s clinical 
condition warrants it.  
The reason for discontinuation from study must be documented in the participant’s medical 
records.  
A participant must discontinue the study for any of the following reasons: 
● 
Participant requests to stop the study.  
● 
Any clinical AE, laboratory abnormality or intercurrent illness that, in the opinion of the 
investigator, indicates to continue in the study is not in the best interest of the participant. 
● 
Participant signs informed consent, but surgery is performed without administration of 
ASP5354. (Screening failure participants are excluded.) 
8.3 
Lost to Follow-up 
Every reasonable effort is to be made to contact any participant lost to follow-up during the 
course of the study to complete study-related assessments and record outstanding data. These 
contact attempts should be documented in the participant’s medical record. 
9 
STATISTICAL CONSIDERATIONS 
9.1 
Statistical Hypotheses 
No statistical hypothesis is planned.  


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9.2 
Analysis Sets 
The allocation of participants to analysis sets will be determined prior to database hard-lock.  
The following populations are defined: 
Population 
Description 
Enrolled  
All participants who sign the ICF. 
Full Analysis Set (FAS) 
All participants who are enrolled and receive study IP. 
Participants will be analyzed according to the IP they actually 
received.  
Safety Analysis Set 
(SAF) 
All participants who are enrolled and receive study IP. 
Participants will be analyzed according to the IP they actually 
received. SAF is the same as FAS for this study.  
IP: investigational product 
 
9.3 
Statistical Analyses 
A Statistical Analysis Plan (SAP) will be written to provide details of the analysis, along with 
specifications for tables, listings and figures to be produced. Changes from the planned 
analyses in the final SAP that impact the statistical analyses will be justified in the CSR. 
9.3.1 
General Considerations 
In general, data will be summarized with descriptive statistics for continuous endpoints, and 
frequency and percentage for categorical endpoints, unless otherwise specified. Percentages 
by categories will be based on the number of participants with no missing data (i.e., will add 
up to 100%). 
All the analyses will be performed by tumor type, unless otherwise specified. 
Baseline will be defined as the last non missing observation prior to administration of IP, 
unless otherwise specified. 
Demographics and baseline characteristics will be summarized by dose group and overall, for 
the full analysis set (FAS). 
The number and percentage of participants who completed and discontinued screening period 
and reasons for discontinuation will be presented for all participants who signed the ICF. A 
similar table for follow-up disposition will also be presented for the FAS by dose group and 
overall. All disposition details and dates of first and last evaluations for each participant will 
be listed.  
Previous and concomitant treatment and medical history will be listed. IP exposure will be 
summarized by descriptive statistics and will be listed. 
9.3.2 
Primary Endpoint(s)/Estimand(s) Analysis  
The primary analysis will be conducted on the FAS. No statistical testing will be performed.  


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9.3.2.1 Primary Analysis  
The optimal dose will be determined by VRC based on the following assessments: LN tissue 
visualized (Y/N), visualized tissue is lymphatic in origin based on pathologic confirmation, 
the Likert scale determination of the intensity of fluorescence, and proportion of identified 
LN with histopathologic confirmation of LN tissue by ASP5354 compared with SoC 
treatment with either Tc-99mSC or Lymphoseek. 
9.3.3 
 Secondary Endpoint(s)/Estimand(s) Analysis 
The proportion of participants with at least 1 LN detected by visualization with 
histopathologic confirmation of LN tissue using ASP5354 will be estimated with exact 95% 
confidence interval (CI) by dose group.  
The proportion of identified LNs with histopathologic confirmation of LN tissue will be 
estimated with exact 95% CI for ASP5354 by dose group and SoC treatment with either 
Tc-99mSC or Lymphoseek.  
The secondary endpoint analysis will be performed using the FAS. 
9.3.4 
Exploratory Endpoint(s)/Estimand(s) Analysis  
The Likert Scale of the intensity of the fluorescence will be summarized by frequency and 
percentage by dose group. 
The SBR will be summarized using descriptive statistics by dose group. 
The transdermal visualization of lymphatic spread of ASP5354 after injection will be 
summarized by frequency and percentage by dose group. 
The time from injection to surgical identification of LN(s) will be summarized by descriptive 
statistics by dose group.  
The frequency and percentage of participants with visualization of LNs using ASP5354 will 
be summarized by dose group. In addition, the number of LNs visualized using ASP5354 will 
be summarized by descriptive statistics by dose group.  
The exploratory analysis will be performed using the FAS. 
9.3.5 
Safety Analyses 
The safety and tolerability will be summarized by descriptive statistics for the safety analysis 
set (SAF) population. The SAF population consists of all participants who received 
ASP5354. 
9.3.5.1 Adverse Events  
AEs will be coded using MedDRA and graded using the National Cancer Institute-Common 
Terminology Criteria for Adverse Events (NCI-CTCAE version 5.0).  
A TEAE is defined as an AE observed after administration of the IP and up to the follow-up 
period. An IP-related TEAE is defined as any TEAE with a causal relationship assessed as 
“yes” by the investigator. 


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The number and percentage of participants with TEAEs, IP-related TEAEs, serious TEAEs, 
IP-related serious TEAEs and green coloration of urine or skin will be summarized by system 
organ class, preferred term and dose group. The worst grade will be summarized if the same 
AE is recorded more than once for a participant.  
AE data will be listed. 
9.3.5.2 Laboratory Assessments  
For quantitative clinical laboratory measurements, descriptive statistics will be used to 
summarize results and change from baseline by dose group and time point.  
Shifts from baseline to the postbaseline worst grade based on NCI-CTCAE until the 
follow-up period in clinical laboratory tests will be tabulated.  
Laboratory data will be listed. 
9.3.5.3 Vital Signs  
Descriptive statistics will be used to summarize vital sign results and changes from baseline 
by dose group and time point.  
Vital signs data will be listed. 
9.3.5.4 Electrocardiogram  
The standard 12-lead ECG results will be summarized by dose group and time point. 
12-lead ECG data and interpretations will be listed. 
9.3.6 
Pharmacokinetics Analysis 
Descriptive statistics will be used to summarize the plasma concentrations of ASP5354 by 
dose group and time point. Individual plasma concentrations of ASP5354 and the sampling 
times will be listed. 
The pharmacokinetics analysis will be performed using the SAF. 
9.3.7 
Other Analyses 
Other analyses such as the relationship between LNs visualization and pharmacokinetics or 
other exploratory endpoints may be explored when deemed appropriate. All details will be 
described in a separate analysis plan and results will be described in a separate report and will 
not be incorporated in the integrated CSR. 
9.4 
Interim Analysis 
VRC review will be held after 3 participants in 0.2 mg dose level in each tumor type have 
data for review. Three participants each will then be enrolled in the 2 additional dose levels in 
each tumor type. The determination of dose(s) to stop, continue or define as optimal dose will 
start when 3 participants assigned to each dose level have their data for visualization 
reviewed by the VRC. During the VRC review, enrollment at that indication will be closed 
until the determination of the next dose level to be made. This process will continue for every 


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additional 3 participants assigned to each dose level until reaching the maximum of 
12 participants. A new dose level might be added.  
9.5 
Sample Size Determination 
The sample size is not based on statistical power calculation. The sample size is expected to 
provide adequate information to determine the optimal dose of ASP5354 for LN visualization 
in breast cancer and melanoma. Approximately up to 12 participants will be assigned to each 
of the initial 3 dose levels of ASP5354 in each tumor type, and then an estimated total of up 
to 36 participants will be required in each tumor type. In case some dose levels are stopped 
based on VRC, additional dose levels might be added.  
9.6 
Additional Conventions 
As a general principle, no imputation of missing data will be done. Exceptions are the start 
and stop dates of AEs and concomitant medications if they are missing. 
See the SAP for details of the definitions for analysis windows to be used for analyses by 
time point. 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
10.1 
Appendix 1 
Ethical, Regulatory and Study Oversight 
Considerations 
10.1.1 Regulatory and Ethical Considerations 
● 
This study will be conducted in accordance with the protocol and with the following: 
o Consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical 
Sciences (CIOMS) international ethical guidelines 
o Applicable ICH GCP guidelines 
o Applicable laws and regulations 
● 
The protocol, protocol amendments, ICF, IB, and other relevant documents (e.g., 
advertisements) must be submitted to an IRB/IEC by the investigator and reviewed and 
approved by the IRB/IEC before the study is initiated. 
● 
Any amendments to the protocol will require IRB/IEC approval before implementation 
of changes made to the study design, except for changes necessary to eliminate an 
immediate hazard to study participants. 
● 
The investigator will be responsible for the following: 
o Providing written summaries of the status of the study to the IRB/IEC annually or 
more frequently in accordance with the requirements, policies, and procedures 
established by the IRB/IEC 
o Notifying the IRB/IEC of SAEs or other significant safety findings as required by 
IRB/IEC procedures 
o Providing oversight of the conduct of the study at the site and adherence to 
requirements of Title 21 of the US Code of Federal Regulations, ICH guidelines, the 


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IRB/IEC, European regulation 536/2014 for clinical studies (if applicable), and all 
other applicable local regulations 
10.1.2 Financial Disclosure 
Investigators and subinvestigators will provide the sponsor with sufficient, accurate financial 
information as requested to allow the sponsor to submit complete and accurate financial 
certification or disclosure statements to the appropriate regulatory authorities. Investigators 
are responsible for providing information on financial interests during the course of the study 
and for 1 year after completion of the study. 
10.1.3 Informed Consent of Participants 
10.1.3.1 Informed Consent Process 
● 
The investigator or his/her representative will explain the nature of the study to the 
participant and answer all questions regarding the study. 
● 
The information provided shall be provided in writing and shall: 
o enable the participant to understand:  
▪ the nature, objectives, benefits, implications, risks and inconveniences of the 
clinical study; 
▪ the conditions under which the clinical study is to be conducted, including the 
expected duration of the participant's participation in the clinical study;  
▪ and the possible treatment alternatives, including the follow-up measures if the 
participation of the participant in the clinical study is discontinued; 
o be kept comprehensive, concise, clear, relevant, and understandable to a layperson; 
o be provided in a prior interview with an appropriately qualified member of the study 
team. Special attention shall be paid to the information needs of specific patient 
populations and of individual participants, as well as to the methods used to give the 
information. Care should be taken to verify that the participant has understood the 
information; 
o include information about the applicable damage compensation system; 
o include the study ISN number and information about the future availability of the 
clinical study results in terms understandable to a layperson; 
● 
Participants must be informed that their participation is voluntary and shall have their 
protective rights and guarantees explained. In particular their right to refuse to participate 
and the right to withdraw from the clinical study at any time without any resulting 
detriment and without having to provide any justification shall be explained.  
● 
Participants will be required to sign a statement of informed consent that meets the 
requirements of 21 CFR 50, local regulations, ICH guidelines, Health Insurance 
Portability and Accountability Act requirements, where applicable, and the IRB/IEC or 
study center. 
● 
The medical record must include a statement that written informed consent was obtained 
before the participant was enrolled in the study and the date the written consent was 
obtained. The authorized person obtaining the informed consent must also sign the ICF. 


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● 
Participants must be reconsented to the most current version of the ICF(s) during their 
participation in the study. 
● 
A copy of the ICF(s) must be provided to the participant. 
10.1.3.2 Supply of New and Important Information Influencing the Participant’s 
Consent and Revision of the Written Information 
● 
The investigator or his/her representative will immediately inform the participant 
verbally whenever new information becomes available that may be relevant to the 
participant’s consent or may influence the participant’s willingness to continue 
participating in the study (e.g., report of serious adverse drug reaction). The 
communication must be documented in the participant’s medical records and whether the 
participant is willing to remain in the study or not must be confirmed and documented.  
● 
The investigator must update the participant’s ICF and submit it for approval to the 
IRB/IEC. The investigator or his/her representative must obtain written informed consent 
from the participant on all updated ICFs throughout their participation in the study. The 
investigator or his/her designee must reconsent participants with the updated ICF even if 
relevant information was provided verbally. The investigator or his/her representative 
who obtained the written informed consent and the participant should sign and date the 
ICF. A copy of the signed ICF will be given to the participant and the original will be 
placed in the participant’s medical record. An entry must be made in the participant’s 
records documenting the reconsent process. 
10.1.4 Data Protection 
The sponsor will use the personal data collected from participants in order to run the study 
and to use and publish the results of the study.  
Astellas relies on the permission (or “consent”) in order to use the data of participants and 
their permission is obtained by signing the ICF. 
The investigator and/or the site personnel will record information from the medical file of 
study participants in eCRF and in an external (electronic) data file (e.g., central laboratory 
data). These records will identify the participants with a code instead of their name or other 
personal data. Only the investigator and the site personnel can match the code with the name 
of the study participant. 
The sponsor collects information from the participants during the study that may be used for: 
● 
submission to government regulatory authorities and IRB/IEC  
● 
use in reports or public scientific presentations, and  
● 
use in research, now or in the future. 
However, the identity of participants will not be revealed if study participants’ personal data 
is shared for these purposes.  


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The personal data of participants may be shared with: 
● 
Astellas and its present or future affiliates, 
● 
research, collaboration and licensing partners and/or service providers (such as 
laboratories conducting tests on behalf of Astellas) and authorized representatives of 
Astellas, 
● 
study monitors appointed by Astellas or Astellas’ service providers, and  
● 
auditors/inspectors appointed by Astellas or Astellas’ service providers or by health and 
regulatory authorities for GCP inspections. 
The sponsor will inform the participants about their privacy rights and how to exercise them 
under the ICF which participants will sign in order to participate in the study. The sponsor 
will provide the investigator and study staff with a privacy notice explaining how their 
personal data will be used and how to exercise their privacy rights.  
The sponsor will comply and process personal data in accordance with all applicable privacy 
laws and regulations. 
The Sponsor has performed a privacy impact assessment to describe the data processing 
activities, assess their necessity and proportionality and manage the risks to the rights and 
freedoms of data participants resulting from the processing of personal information by 
assessing them and determining the measures or mitigation actions to address them. The 
Sponsor has assessed, among others, how the data is collected and the purposes they are used, 
the impact on data participant rights, the security mechanisms including the safeguards of 
potential transfer of data outside the European Union and the access rights to data. The 
Sponsor has taken actions to mitigate the risks to the rights and freedoms of data participants 
resulting from the processing of their data as part of their participation in the study.  
10.1.5 Committee Structure 
10.1.5.1 Visualization Review Committee 
The visualization data of the LNs will be assessed by the VRC and will be compared with the 
performance of SoC. Members of the VRC include the sponsor’s medical monitor, 
pharmacovigilance physician, and the investigator(s) who have treated participants. The VRC 
will be held at the following time points, but ad-hoc meetings may occur:  
● 
Initial 3 participants at 0.2 mg in each tumor type have completed the SLN biopsies; 
● 
Initial 3 participants at each subsequent dose levels (0.05 mg and 0.6 mg or 0.6 mg and 
1 mg) in each tumor type have completed the SLN biopsies (total of 6 participants; and 
● 
Additional 3 participants have completed SLN biopsies at the expanded or additional 
dose levels.  The review of an additional 3 participants will continue until the optimal 
dose is defined.Maximum participants for each cohort (i.e., 12) has not been reached.  
The data for the participants, at each subsequent dose level, who completed the SLN biopsy 
will be assessed by the VRC to decide whether to:  
● 
Expand a dose level if  additional participants are needed to increase confidence for the 
optimal dose selection; 


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● 
Stop a dose level when 100% of participants lack visualization or have a fluorescence 
intensity of 0 or 1 on the qualitative Likert Scale), in which case additional participants 
would not be added to the dose level; or 
● 
Define an optimal dose (in the case of 2 doses performing equally, the lower dose will be 
selected). 
For further details on the VRC, refer to the VRC charter. 
10.1.6 Dissemination of Clinical Study Data 
ICH E3 guidelines recommend and EU Directive 2001/83/EC requires that a final CSR that 
forms part of a marketing authorization application, be signed by the representative for the 
coordinating investigator(s) or the principal investigator(s). The representative for the 
coordinating investigator(s) or the principal investigator(s) will have the responsibility to 
review the final study results to confirm to the best of his/her knowledge it accurately 
describes the conduct and results of the study. The representative for the coordinating 
investigator(s) or the principal investigator(s) will be selected from the participating 
investigators by the sponsor prior to database lock. 
10.1.7 Data Quality Assurance 
● 
All participant data relating to the study will be recorded on the eCRF unless transmitted 
to the sponsor or designee electronically in an external data file. The investigator is 
responsible for verifying that data entries on the eCRF are accurate and correct by 
physically or electronically signing the eCRF. 
● 
Guidance on completion of CRFs will be provided in a separate eCRF Completion 
Guideline. 
● 
The investigator must permit study-related monitoring, audits, IRB/IEC review, and 
regulatory agency inspections and provide direct access to source data documents. 
● 
Monitoring details describing strategy, including definition of study critical data items 
and processes (e.g., risk-based initiatives in operations and quality such as risk 
management and mitigation strategies and analytical risk-based monitoring), methods, 
responsibilities and requirements, including handling of noncompliance issues and 
monitoring techniques (central, remote or on-site monitoring) are provided in the 
Monitoring Plan. 
● 
The sponsor or designee is responsible for the data management of this study including 
quality checking of the data. 
● 
The sponsor assumes accountability for actions delegated to other individuals (e.g., 
contract research organizations [CROs]). 
● 
Records and documents, including signed ICFs, pertaining to the conduct of this study 
must be retained by the investigator according to ICH or applicable local regulatory 
requirements, whichever is longer, after study completion. No records may be destroyed 
during the retention period without the written approval of the sponsor. No records may 
be transferred to another location or party without written notification to the sponsor. 


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10.1.8 Source Documents 
1. Source data must be available at the study site to document the existence of the 
participants and to substantiate the integrity of study data collected. Source data must 
include the original documents relating to the study, as well as the medical treatment and 
medical history of the participant.  
2. The investigator must maintain accurate documentation (source data) that supports the 
information entered in the CRF. 
3. The investigator is responsible for ensuring the source data are attributable, legible, 
contemporaneous, original, accurate and complete whether the data are handwritten on 
paper or entered electronically. If source data are created (first entered), modified, 
maintained, archived, retrieved or transmitted electronically via computerized systems 
(and/or other kind of electronic devices) as part of regulated study activities, such 
systems must be compliant with all applicable laws and regulations governing use of 
electronic records and/or electronic signatures. Such systems may include, but are not 
limited to, electronic medical/health records, protocol-related assessments, AE tracking, 
electronic clinical outcome assessment and/or drug accountability.  
4. Paper records from electronic systems used in place of electronic format must be 
certified copies. A certified copy must be an exact copy and must have all the same 
attributes and information as the original. Certified copies must include signature and 
date of the individual completing the certification. Certified copies must be a complete 
and chronological set of study records (including notes, attachments, and audit trail 
information, if applicable). All printed records must be kept in the participant file and be 
available for archiving.  
5. Study monitors will perform ongoing source data review to confirm that the safety and 
rights of participants are being protected; and that the study is being conducted in 
accordance with the currently approved protocol and any other study agreements, ICH 
GCP, and all applicable regulatory requirements. 
10.1.9 Study and Site Start and Closure 
The sponsor or designee reserves the right to close the study site or terminate the study at any 
time for any reason at the sole discretion of the sponsor. Study sites will be closed upon study 
completion. A study site is considered closed when all required documents and study supplies 
have been collected and a study-site closure visit has been performed. 
The investigator may initiate study-site closure at any time, provided there is reasonable 
cause and sufficient notice is given in advance of the intended termination. 
Reasons for the early closure of a study site by the sponsor or investigator may include but 
are not limited to: 
For study termination: 
● 
Discontinuation of further investigational study intervention development 


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For site termination: 
● 
Failure of the investigator to comply with the protocol, the requirements of the IRB/IEC 
or local health authorities, the sponsor's procedures or GCP guidelines 
● 
Inadequate or no recruitment (evaluated after a reasonable amount of time) of 
participants by the investigator 
● 
Total number of participants included earlier than expected 
If the study is prematurely terminated or suspended, the sponsor or designee shall promptly 
inform the investigators, the IRBs/ IECs, the regulatory authorities, and any CRO(s) used in 
the study of the reason for termination or suspension, as specified by the applicable 
regulatory requirements. The investigator shall promptly inform the participant and should 
assure appropriate participant therapy and/or follow-up. 
10.1.10 Arrangement for Use of Information and Publication of the Study 
Information concerning the test product, patent applications, processes, unpublished scientific 
data, the IB and other pertinent information is confidential and remains the property of the 
sponsor. Details should be disclosed only to the persons involved in the approval or conduct 
of the study. The investigator may use this information for the purpose of the study only. It is 
understood by the investigator that the sponsor will use the information obtained during the 
study in connection with the development of the product and therefore may disclose it as 
required to other clinical investigators or to regulatory agencies. In order to allow for the use 
of the information derived from this study, the investigator understands that he/she has an 
obligation to provide the sponsor with all data obtained during the study. 
Publication of the study results is discussed in the study agreement. 
10.1.11 Quality Assurance 
The sponsor is implementing and maintaining quality assurance (QA) and quality control 
(QC) systems with written SOPs to ensure that studies are conducted and data are generated, 
documented, recorded, and reported in compliance with the protocol, GCP and applicable 
regulatory requirement(s). Where applicable, the QA and QC systems and written SOPs of 
the CRO will be applied. 
The sponsor or sponsor’s designee may arrange to audit the study at any or all study sites and 
facilities. The audit may include on-site review of regulatory documents, CRFs and source 
documents. Direct access to these documents will be required by the auditors. 
To support quality around participant safety and reliability of study results, quality tolerance 
limits (QTLs) are defined and monitored. QTLs represent the acceptable variation of study 
data, taking into consideration the current state of medical and statistical knowledge about the 
variables to be analyzed, as well as the statistical design of the study. It is a level, point, or 
value associated with a parameter that should trigger an evaluation if a deviation is detected 
to determine if there is a possible systematic issue (i.e., a trend has occurred). The QTLs 
defined for this study are provided below. 


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Table 6 
Quality Tolerance Limits 
QTL #: Name and Parameter 
Definition 
Parameter Justification 
QTL1: Sufficient data for 
assessment to decide optimal dose 
of ASP5354 for LN visualization: 
% missing at least 1 of all below 
data of anatomical visualization of 
LN(s) after dosing ASP5354. 
• 
LN tissue visualized (Y/N) 
• 
Visualized tissue is lymphatic 
in origin based on pathologic 
confirmation 
Number of participants who 
missed at least 1 of all below data 
of anatomical visualization of 
LN(s) after dosing ASP5354 per 
total number of enrolled 
participants (%). 
• 
LN tissue visualized (Y/N) 
• 
Visualized tissue is lymphatic 
in origin based on pathologic 
confirmation 
Since all of the data of anatomical 
visualization of the LN(s) after 
dosing ASP5354 is the primary 
endpoint to determine the optimal 
dose of ASP5354, missing data 
points would hinder evaluation of 
efficacy. 
QTL2: Reliable data for 
assessment of LN visualization by 
ASP5354: % receiving excluded 
concomitant medications, listed in 
[Section 10.5 Appendix 5 List of 
Excluded Concomitant 
Medications] in protocol deviated 
manner  
Number of participants who 
received excluded concomitant 
medications, listed in [Section 
10.5 Appendix 5 List of Excluded 
Concomitant Medications] in 
protocol deviated manner per total 
number of enrolled participants 
(%). 
Since excluded concomitant 
medication might affect 
visualization of the LN(s) by 
ASP5354. 
QTL: quality tolerance limit, LN: lymph node 
QTL Management Activities: 
● 
For control of risks associated with QTL1: Sufficient data for assessment of anatomical 
visualization of the LN, refer to [Section 7.1 Efficacy Assessments]. 
● 
For control of risks associated with QTL2: Reliable data for assessment of LN 
visualization by ASP5354, refer to [Section 10.5 Appendix 5 List of Excluded 
Concomitant Medications]. 


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10.2 
Appendix 2 
Contraception Requirements 
WOCBP, who are eligible for participation in the study, including those who choose 
complete abstinence, must have pregnancy tests as specified in the Schedule of Assessments 
[Table 1]. Pregnancy test results must confirm that the participant is not pregnant. 
WOMEN OF CHILDBEARING POTENTIAL DEFINITIONS AND METHODS OF 
CONTRACEPTION DEFINITIONS 
A female is considered fertile (i.e., WOCBP) following menarche and until becoming 
postmenopausal unless permanently sterile.  
Females in the following categories are not considered WOCBP 
● 
Premenarchal 
● 
Premenopausal with one of the following (i.e., permanently sterile): 
o Documented hysterectomy 
o Documented bilateral salpingectomy 
o Documented bilateral oophorectomy 
● 
Postmenopausal 
A postmenopausal state is defined as at least 12 months after last menstrual bleeding without 
an alternative medical cause.  
In case the last menstrual bleeding cannot be clearly determined, confirmation with more 
than 1 follicle-stimulating hormone (FSH) measurement of at least > 40 IU/L (or higher per 
local institutional guidelines) is required.  
Females on hormone replacement therapy (HRT) and whose menopausal status is in doubt 
will be required to use one of the nonestrogen hormonal highly effective contraception 
methods if they wish to continue their HRT during the study. Otherwise, they must 
discontinue HRT to allow confirmation of postmenopausal status by repeated FSH 
measurements before study enrollment. 
Documentation of any of these categories can come from the study site personnel’s review of 
the female participant’s medical records, medical examination or medical history interview. 
CONTRACEPTION GUIDANCE FOR FEMALE PARTICIPANTS OF 
CHILDBEARING POTENTIAL 
Female participants of childbearing potential are eligible for participation in the study if they 
agree to use one of the highly effective methods of contraception listed below from the time 
of signing the ICF and until the end of relevant systemic exposure, defined as at least 30 days 
after final study treatment administration.a 
Highly effective methods of contraception (failure rate of < 1% per year when used 
consistently and correctly)b: 
● 
Combined (estrogen- and progestogen-containing) hormonal contraception associated 
with inhibition of ovulation 
o Oral 


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o Intravaginal 
o Transdermal 
● 
Progestogen-only hormonal contraception associated with inhibition of ovulation 
o Oral 
o Injectable 
o Implantable 
● 
Other combined (estrogen- and progesterone-containing) methods 
o Vaginal ring 
o Injectable 
o Implantable 
o Intrauterine hormone-releasing system or intrauterine device 
o Bilateral tubal occlusion 
● 
Vasectomized partner 
A vasectomized partner is a highly effective contraception method provided that the 
partner is the sole male sexual partner of the WOCBP and the absence of sperm has been 
confirmed. If not, an additional highly effective method of contraception should be used. 
● 
Sexual abstinence 
Sexual abstinence is considered a highly effective method only if defined as refraining 
from heterosexual intercourse during the entire period of risk associated with the test 
product. The reliability of sexual abstinence needs to be evaluated in relation to the 
duration of the study and the preferred and usual lifestyle of the participant. It is not 
necessary to use any other method of contraception when complete abstinence is elected. 
a Local laws and regulations may require use of alternative and/or additional contraception 
methods. 
b Typical use failure rates may differ from those when used consistently and correctly. Use 
should be consistent with local regulations regarding the use of contraceptive methods for 
participants participating in clinical studies. 
CONTRACEPTION GUIDANCE FOR MALE PARTICIPANTS WITH PARTNER(S) 
OF CHILDBEARING POTENTIAL 
Male participants with female partners of childbearing potential are eligible for participation 
in the study if they agree to the following during treatment and until the end of relevant 
systemic exposure defined as at least 30 days after final study treatment administration.a 
● 
Inform any and all partner(s) of their participation in a clinical drug study and the need to 
comply with contraception instructions as directed by the investigator 
● 
Use a condom 
● 
Female partners of male participants who have not undergone a vasectomy with the 
absence of sperm confirmed or a bilateral orchiectomy should consider use of effective 
methods of contraception 
a Local laws and regulations may require use of alternative and/or additional contraception 
methods. 


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10.3 
Appendix 3 
Adverse Events/Adverse Device Effects: 
Definitions and Procedures for Recording, Evaluating, Follow-up 
and Reporting 
10.3.1 Definition of Adverse Events 
AE Definition: 
An AE is any untoward medical occurrence in a patient or clinical study participant, 
temporally associated with the use of study IP, whether or not considered related to the study 
IP.  
NOTE: An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the 
use of study IP. This includes events related to the comparator and events related to the 
(study) procedures. 
Events Meeting the AE Definition 
● 
Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or 
other safety assessments (e.g., ECG, radiological scans, vital signs measurements), 
including those that worsen from baseline, considered clinically significant in the 
medical and scientific judgment of the investigator (i.e., not related to progression of 
underlying disease). 
● 
Exacerbation of a chronic or intermittent pre-existing condition including either an 
increase in frequency and/or intensity of the condition. 
● 
New conditions detected or diagnosed after study IP administration even though it may 
have been present before the start of the study. 
Events NOT Meeting the AE Definition 
● 
Any clinically significant abnormal laboratory findings or other abnormal safety 
assessments which are associated with the underlying disease, unless judged by the 
investigator to be more severe than expected for the participant’s condition. 
● 
The disease/disorder being studied or expected progression, signs, or symptoms of the 
disease/disorder being studied, unless more severe than expected for the participant’s 
condition. 
● 
Medical or surgical procedure (e.g., endoscopy, appendectomy): the condition that leads 
to the procedure is the AE. 
● 
Situations in which an untoward medical occurrence did not occur (social and/or 
convenience admission to a hospital). 
● 
Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or 
detected at the start of the study that do not worsen. 
10.3.1.1 Abnormal Laboratory Findings 
Any abnormal laboratory test result (e.g., hematology, biochemistry or urinalysis) or other 
safety assessment (e.g., vital signs, physical examination or ECGs), including those that 
worsen from baseline, that is considered to be clinically significant in the medical and 


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scientific judgment of the investigator and not related to underlying disease, is to be reported 
as an (S)AE. 
Any clinically significant abnormal laboratory finding or other abnormal safety assessment, 
which is associated with the underlying disease, does not require reporting as an (S)AE, 
unless judged by the investigator to be more severe than expected for the participant’s 
condition. 
Repeating an abnormal laboratory test or other safety assessment, in the absence of any of the 
above criteria, does not constitute an AE. Any abnormal test result that is determined to be an 
error does not require reporting as an AE. 
10.3.1.2 Potential Cases of Drug-induced Liver Injury 
Refer to [Section 10.4 Appendix 4 Liver Safety Monitoring and Assessment] for detailed 
instructions on drug induced liver injury (DILI). Abnormal values in AST and/or ALT 
concurrent or with abnormal elevations in total bilirubin (TBL) that meet the criteria outlined 
in [Section 10.4 Appendix 4 Liver Safety Monitoring and Assessment] in the absence of 
other causes of liver injury, are considered potential cases of DILI (potential Hy’s Law cases; 
[Temple, 2006]) and are always to be considered important medical events and reported per 
[Section 10.3.7 Reporting Procedures for Serious Adverse Events/Adverse Device Effects]. 
10.3.2 Definition of Serious Adverse Events 
An SAE is defined as any untoward medical occurrence that, at any dose: 
● 
Results in death 
● 
Is life-threatening 
The term “life-threatening” in the definition of “serious” refers to an event in which the 
participant was at risk of death at the time of the event. It does not refer to an event, which 
hypothetically might have caused death, if it were more severe. 
● 
Requires inpatient hospitalization or prolongation of existing hospitalization 
o 
In general, hospitalization signifies that the participant has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for 
observation and/or treatment that would not have been appropriate in the physician’s 
office or outpatient setting. Complications that occur during hospitalization are AEs. 
If a complication prolongs hospitalization or fulfills any other serious criteria, the 
event is serious. When in doubt as to whether “hospitalization” occurred or was 
necessary, the AE should be considered serious. 
o 
Hospitalization for elective treatment of a pre-existing condition that did not worsen 
from baseline is not considered an AE. 
● 
Results in persistent or significant disability/incapacity 
o 
The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions. 
o 
This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 


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and accidental trauma (e.g., sprained ankle), which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption. 
● 
Is a congenital anomaly/birth defect 
● 
Other situations: 
o 
Medical or scientific judgment should be exercised in deciding whether SAE 
reporting is appropriate in other situations such as important medical events that 
may not be immediately life-threatening or result in death or hospitalization but may 
jeopardize the participant or may require medical or surgical intervention to prevent 
one of the other outcomes listed in the above definition. These events should usually 
be considered serious. 
o 
Examples of such events include invasive or malignant cancers, intensive treatment 
in an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency 
or drug abuse. 
If an event is not an AE per definition in [Section 10.3.1 Definition of Adverse Events], then 
it cannot be an SAE even if serious conditions are met (e.g., hospitalization for 
signs/symptoms of the disease under study, death due to progression of disease).  
10.3.3 Adverse Device Effect (ADE) 
10.3.3.1 Definition of ADE 
● 
Any adverse event related to the use of device (includes all hardware components). 
o Includes any event that is a result of a user error or intentional misuse. 
10.3.3.2 Definition of Unanticipated adverse device effect (UADE)  
● 
UADE is any serious adverse effect on health or safety, any life-threatening problem or 
death caused by, or associated with a device, if that effect, problem, or death was not 
previously identified in nature, severity, or degree of incidence in the application; or any 
other unanticipated serious problem associated with a device that relates to the rights, 
safety, or welfare of participants. Note the reporting of UADEs should follow reporting 
requirements for SAEs/SADEs  
10.3.3.3 Definitions of Technical Complaints and Device Deficiencies 
● 
Device Complaint: A quality complaint received in writing, electronically, or orally that 
alleges deficiencies related to the identity, quality, durability, reliability, safety, 
effectiveness, or performance of a device product. (In this definition, "effectiveness" 
refers to the actual function of the device, not to how the participant responds to the 
action of the device 
● 
Device Deficiency: Inadequacy of a medical device with respect to its identity, quality, 
durability, reliability, safety or performance. Device deficiencies include malfunctions, 
use errors and inadequate labelling.   
10.3.3.4 Risks/Precautions 
Refer to the SPY Portable Handheld Imager (SPY-PHI) and 1688 Advanced Imaging 
Modalities (AIM) 4k Platform Manuals for a full description of the risks and precautions 


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associated with all components of the SPY-PHI and 1688 AIM systems, respectively. The 
entirety of the Operator Manuals should be read before using the device(s). Failure to follow 
the instructions and warnings in the manual may result in unsafe operation of the system 
and/or injury to the subject or operator. 
10.3.4 Assessment of Causality 
● 
The investigator is obligated to assess the relationship between study IP, imaging 
device(s), study procedures and each occurrence of each AE/SAE/ADE. 
● 
A “reasonable possibility” of a relationship conveys that there are facts, evidence, and/or 
arguments to suggest a causal relationship, rather than a relationship cannot be ruled out. 
● 
The investigator will use clinical judgment to determine the relationship. 
● 
Alternative causes, such as underlying disease(s), concomitant therapy, and other risk 
factors, as well as the temporal relationship of the event to study IP administration will 
be considered and investigated. 
● 
The investigator will also consult the IB and/or product information, for marketed 
products, in his/her assessment. 
● 
For each AE/SAE/ADE, the investigator must document in the medical notes that he/she 
has reviewed the AE/SAE/ADE and has provided an assessment of causality. 
● 
There may be situations in which an SAE/ADE has occurred and the investigator has 
minimal information to include in the initial report to the sponsor. However, it is very 
important that the investigator always make an assessment of causality for every event 
before the initial transmission of the SAE/ADE data to the sponsor. 
● 
The investigator may change his/her opinion of causality in light of follow-up 
information and send a SAE/ADE follow-up report with the updated causality 
assessment. 
● 
The causality assessment between study IP is one of the criteria used when determining 
regulatory reporting requirements. 
Following a review of the relevant data, the causal relationship between the IP and each 
(S)AE/ADE will be assessed by answering “yes” or “no” to the question “Do you consider 
that there is a reasonable possibility that the event may have been caused by the IP?” 
When making an assessment of causality, the following factors are to be considered when 
deciding if there is evidence and/or arguments to suggest there is a “reasonable possibility” 
that an (S)AE/ADE may have been caused by the IP (rather than a relationship cannot be 
ruled out) or if there is evidence to reasonably deny a causal relationship: 
● 
Has the participant been administered IP?  
● 
Plausibility (i.e., could the event have been caused by the suspect IP? Consider biologic 
and/or pharmacologic mechanism, half-life, literature evidence, drug class, preclinical 
and study data, etc.) 
● 
Dechallenge/dose reduction/rechallenge:  
o Dechallenge: Did the (S)AE/ADE resolve or improve after only stopping the dose of 
the suspect drug without any treatment? 


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o Dose reduction: Did the (S)AE/ADE resolve or improve after reducing the dose of 
the suspect drug? 
o Rechallenge: Did the (S)AE/ADE reoccur if the suspected drug was reintroduced 
after having been stopped?  
● 
Laboratory or other test results: a specific lab investigation supports the assessment of 
the relationship between the (S)AE/ADE and the IP (e.g., based on values pre-, during 
and post-treatment) 
● 
Available alternative explanations independent of IP exposure; such as other 
concomitant drugs, device malfunctions, past medical history, concurrent or underlying 
disease, risk factors including medical and family history, season, location, etc., and 
strength of the alternative explanation  
● 
Temporal relationship between exposure to the IP and (S)AE/ADE onset and/or 
resolution. Did the (S)AE/ADE occur in a reasonable temporal relationship to the 
administration of the IP?  
● 
Finally, judging which are more likely based on all the above contents, factors of 
reasonable possibility or confounding factors, comprehensive judgment of plausible will 
be provided.  
There may be situations in which an SAE/ADE has occurred and the investigator has 
minimal information to include in the initial report to the sponsor. While it is very important 
that the investigator always assesses causality for every event before the initial transmission 
of the SAE/ADE data to the sponsor, the initial report should be submitted without delay 
(i.e., within 24 hours of awareness). With limited or insufficient information about the event 
to make an informed medical judgment and in absence of any indication or evidence to 
establish a causal relationship, a causality assessment of “no” is to be considered. In such 
instance, the investigator is expected to obtain additional information regarding the event as 
soon as possible and to re-evaluate the causality upon receipt of additional information. The 
medically qualified investigator may revise his/her assessment of causality in light of new 
information regarding the SAE/ADE and shall send an SAE/ADE follow-up report and 
update the eCRF with the new information and updated causality assessment.  
Device Relatedness 
Events will be classified as device related if they meet the following criteria: 
• Not Related: Any reaction that does not follow a reasonable temporal sequence from 
administration of the device or surgical procedure, or study intervention AND that is 
likely to have been produced by the participant’s clinical state or other modes of 
therapy administered to the participant. 
• Related: A reaction that follows or may follow a reasonable temporal sequence from 
administration of the device, surgical procedure, or study intervention AND that 
follows a known response pattern to the suspected device/surgical procedure/study 
intervention. 


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10.3.5 Assessment of Severity 
AEs, including abnormal clinical laboratory values, will be graded using the NCI-CTCAE 
guidelines (version 6.0). The items that are not stipulated in the NCI-CTCAE version 6.0 will 
be assessed according to the criteria below and entered into the eCRF: 
Table 7 
Grading Scale Defining the Severity of an Adverse Event 
Grade 
Assessment Standard 
1 - Mild 
Asymptomatic or mild symptoms, clinical or diagnostic observations only; 
intervention not indicated 
2 - Moderate 
Minimal local or noninvasive intervention indicated; limiting age-appropriate 
instrumental ADL† 
3 - Severe 
Medically significant but not immediately life threatening, hospitalization or 
prolonged hospitalization indicated; disabling; limiting self-care ADL‡ 
4 - Life-threatening 
Life threatening consequences, urgent intervention indicated 
5 - Death 
Death related to AE 
ADL: activities of daily living; AE: adverse event 
† Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, managing 
money, etc. 
‡ Self-care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking medications and 
not bedridden. 
10.3.6 Recording and Follow-up of AEs, SAEs and/or ADEs 
AE, SAE and ADE Recording 
● 
When an AE/SAE/ADE occurs, it is the responsibility of the investigator to review all 
documentation (e.g., hospital progress notes, laboratory reports, and diagnostics reports) 
related to the event. 
● 
The investigator will then record all relevant AE/SAE/ADE information in the eCRF. 
● 
It is not acceptable for the investigator to send photocopies of the participant’s medical 
records to the sponsor in lieu of completion of the eCRF. 
● 
There may be instances when copies of medical records for certain cases are requested 
by the sponsor. In this case, all participant identifiers, with the exception of the 
participant number, will be redacted on the copies of the medical records before 
submission to the sponsor. 
● 
The investigator will attempt to establish a diagnosis of the event based on signs, 
symptoms, and/or other clinical information. Whenever possible, the diagnosis (not the 
individual signs/symptoms) will be documented as the AE/SAE/ADE. 
Follow-up of AEs, SAEs and ADEs 
● 
The investigator is obligated to perform or arrange for the conduct of supplemental 
measurements and/or evaluations as medically indicated or as requested by the sponsor 
to elucidate the nature and/or causality of the AE, SAE or ADE as fully as possible. This 
may include additional laboratory tests or investigations, histopathological examinations, 
or consultation with other health care professionals. 


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● 
If a participant dies during participation in the study or during a recognized follow-up 
period, the investigator will provide the sponsor with a copy of any postmortem findings 
including histopathology. New or updated information will be recorded in the originally 
completed eCRF. 
● 
The investigator will submit any updated SAE/ADE data to the sponsor within 24 hours 
of receipt of the information. 
10.3.7 Reporting Procedures for Serious Adverse Events/Adverse Device Effects 
The investigator must complete and submit an SAE or ADE worksheet containing all 
information that is required by local and/or regional regulations to the sponsor by fax or 
email immediately (within 24 hours of awareness).  
The SAE or ADE worksheet must be signed by a medically qualified investigator (as 
identified on delegation of authority log). Signature confirms accuracy and completeness of 
the SAE/ADE data, as well as the investigator causality assessment including the explanation 
for the causality assessment. 
For contact details, see [Contact Details of Sponsor's Key Personnel]. Fax or email the 
SAE/special situations/ADE worksheet to: 
Astellas Pharma Global Development Inc. 
Global Pharmacovigilance  
North America fax number: +1-888-396-3750 
North America alternate fax number: +1-847-317-1241 
Email: safety-us@astellas.com 
If there are any questions, or if clarification is needed regarding the SAE/ADE, please contact 
the sponsor’s medical monitor/study physician or their designee [Contact Details of 
Sponsor’s Key Personnel].  
Follow-up information for the event should be sent promptly (as soon as available, but no 
longer than within 7 days of the initial notification). 
Full details of the SAE/ADE should be recorded on the medical records, SAE/special 
situation or ADE worksheet and on the eCRF.  
The following minimum information is required: 
● 
International study number/study number  
● 
Participant number, sex and age 
● 
Date of report 
● 
Description of the SAE/ADE (event and seriousness criteria)  
● 
Causal relationship to the IP/device (including reason) 
● 
Drug provided (if any) 
The sponsor or sponsor’s designee will medically evaluate the SAE/ADE and determine if 
the report meets the requirements for expedited reporting based on seriousness, causality, and 
expectedness of the events (e.g., SUSAR reporting) according to current local/regional 
regulatory requirements. The sponsor or sponsor's designee will submit expedited safety 


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reports to competent authorities (CAs) and concerned ethics committee per current local 
regulations, and will inform the investigators of such regulatory reports as required. 
Investigators must submit safety reports as required by their IRB/IEC within timelines set by 
regional regulations (e.g., EMA, FDA) where required. Documentation of the submission to 
and receipt by the IRB/IEC of expedited safety reports should be retained by the study site. In 
the US, FDA expedited IND reporting guidelines will be followed. 
The sponsor or sponsor’s designee will also evaluate any ADE and determine if the report 
meets the requirements for expedited reporting. 
The sponsor will notify all investigators responsible for ongoing clinical studies with the test 
product of all SUSARs, which require submission per local requirements to the IRB. 
The investigators should provide written documentation of IRB notification for each report to 
the sponsor. 
The investigator may contact the sponsor’s medical monitor/study physician for any other 
problem related to the rights, safety or well-being of the participant. 
10.3.8 Reporting Procedures for Pregnancy and Special Situations 
10.3.8.1 Contraceptive Guidance and Collection of Pregnancy Information 
If a female participant becomes pregnant during the study dosing period or within 30 days 
from the final study treatment administration, the investigator is to report the information to 
the sponsor according to the timelines in [Section 10.3.7 Reporting Procedures for Serious 
Adverse Events] using the pregnancy form and in the eCRF.  
The investigator will attempt to collect pregnancy information on any female partner of a 
male participant who becomes pregnant during the study dosing period or within 30 days 
from the final study treatment administration and report the information to the sponsor 
according to the timelines in [Section 10.3.7 Reporting Procedures for Serious Adverse 
Events/Adverse Device Effects] using the pregnancy form.  
The expected date of delivery or expected date of the end of the pregnancy, last menstruation, 
estimated conception date, pregnancy result and neonatal data, etc., should be included in this 
information. 
While pregnancy itself is not considered to be an AE or SAE, any pregnancy complication or 
termination (including elective termination) of a pregnancy is to be reported for a female 
participant as an AE in the eCRF or SAE per [Section 10.3.7 Reporting Procedures for 
Serious Adverse Events/Adverse Device Effects]. Participant pregnancy outcomes listed 
below are to be reported as SAEs: 
● 
Spontaneous abortion/miscarriage, abortion and missed abortion 
● 
Death of a newborn or infant within 1 month after birth is to be reported as an SAE 
regardless of its relationship with the IP. 


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● 
If an infant dies more than 1 month after the birth, it is to be reported if a relationship 
between the death and intrauterine exposure to the IP is judged as “possible” by the 
investigator. 
● 
Congenital anomaly (including anomaly in miscarried fetus) 
● 
Benign hydatidiform mole 
● 
Blighted ovum 
Unless a congenital anomaly is identified prior to spontaneous abortion or miscarriage, the 
embryo or fetus should be assessed for congenital defects by visual examination or other 
means as appropriate. (S)AEs experienced by the newborn/infant should be reported via the 
pregnancy form. Pregnancy needs to be followed up until final pregnancy outcome is 
reported. 
10.3.8.2 Medication Error, Overdose and “Off-label Use” 
If a medication error (defined as an unintended failure in the treatment process that leads to, 
or has the potential to lead to, harm to the participant), overdose or “off-label use” (i.e., use 
outside of the target disease defined in the protocol) is suspected, refer to [Section 6.7 
Treatment of Overdose]. Any associated (S)AEs are to be reported in the eCRF. If the AE 
meets the definition of an SAE, the SAE is also to be reported as described in [Section 10.3.7 
Reporting Procedures for Serious Adverse Events/Adverse Device Effects] together with the 
details of the medication error, overdose and/or “off-label use.” 
10.3.8.3 Misuse/Abuse 
Definition of misuse: Situations where the IP is/are intentionally and inappropriately used not 
in accordance with the intended use as defined in the protocol. 
Definition of abuse: Persistent or sporadic, intentional excessive use of medicinal products 
which is accompanied by harmful physical or psychological effects. 
If misuse or abuse of the IP is suspected, the investigator must forward the special situation 
worksheet to the sponsor by fax or email immediately (within 24 hours of awareness). Any 
associated (S)AEs are to be reported in the eCRF. If the AE meets the definition of an SAE, 
the SAE is also to be reported as described in [Section 10.3.7 Reporting Procedures for 
Serious Adverse Events/Adverse Device Effects] together with details of the misuse or abuse 
of the IP.  
10.3.8.4 Occupational Exposure 
If occupational exposure (e.g., inadvertent exposure to the IP of study site personnel while 
preparing it for administration to the participant) to the IP occurs, the investigator must 
forward the special situation worksheet to the sponsor by fax or email immediately (within 
24 hours of awareness). Any associated (S)AEs occurring to the individual associated with or 
resulting from the special situation are to be reported on the special situations worksheet. 


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10.3.8.5 Suspected Drug-drug Interaction 
If a drug-drug interaction associated with the IP is suspected, the investigator must forward 
the special situation worksheet to the sponsor by fax or email immediately (within 24 hours 
of awareness). Any associated (S)AEs are to be reported in the eCRF. If the AE meets the 
definition of an SAE, the SAE is also to be reported as described in [Section 10.3.7 Reporting 
Procedures for Serious Adverse Events/Adverse Device Effects] together with details of the 
suspected drug-drug interaction. 
10.3.8.6 (Suspicion of) Transmission of Infectious Agent 
If transmission of an infectious agent associated with the study intervention is suspected, the 
investigator must forward the special situation worksheet to the sponsor by fax or email 
immediately (within 24 hours of awareness) and any associated (S)AEs are to be reported in 
the eCRF. If the AE meets the definition of an SAE, the SAE is also to be reported as 
described in [Section 10.3.7] together with the details of the suspected transmission of 
infectious agent. 
10.3.9 Supply of New Information Affecting the Conduct of the Study 
When new information becomes available that is necessary for conducting the study properly, 
the sponsor will inform all investigators involved in the study, as well as the appropriate 
regulatory authorities. Investigators should inform the IRB of such information when needed.  
The investigator will also inform the participants, who will be required to sign an updated 
ICF in order to continue in the study. 
10.3.10 Urgent Safety Measures  
An urgent safety measure (USM) is an intervention that is not defined by the protocol and 
can be put in place with immediate effect without needing to gain prior approval by the 
sponsor, relevant CA, IRB/IEC, where applicable, in order to protect participants from any 
immediate hazard to their health and/or safety. Either the investigator or the sponsor can 
initiate a USM. The cause of a USM can be safety-, product- or procedure-related.  
10.3.11 Reporting Urgent Safety Measures 
In the event of a potential USM, the investigator must contact the study physician (within 
24 hours of awareness). Full details of the potential USM are to be recorded in the 
participant’s medical records. The sponsor may request additional information related to the 
event to support their evaluation.  
If the event is confirmed to be a USM, the sponsor will take appropriate action to ensure the 
safety and welfare of the participants. These actions may include but are not limited to a 
change in study procedures or study treatment, halting further enrollment in the study, or 
stopping the study in its entirety. The sponsor or sponsor's designee will notify the relevant 
CAs and concerned ethics committee within the timelines required per current local 
regulations, and will inform the investigators, as required. When required, investigators must 
notify their IRB/IEC within timelines set by regional regulations.  


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10.4 
Appendix 4 
Liver Safety Monitoring and Assessment 
The purpose of this appendix is to provide guidance for the monitoring of DILI during the 
course of the study. It should be noted that this section does not specify the end-of-study 
analyses of liver enzymes. The end-of-study liver enzymes analyses will be described in the 
SAP. Any participant enrolled in a study with active drug therapy and reveals an increase of 
serum aminotransferases (AT) to > 3 × ULN or bilirubin > 2 × ULN should undergo detailed 
testing for liver enzymes (including at least alkaline phosphatase (ALP), ALT, AST and 
TBL). Testing should be repeated within 72 hours of notification of the test results. For 
studies for which a central laboratory is used, alerts will be generated by the central 
laboratory regarding moderate and severe liver abnormality to inform the investigator and 
study team. Participants should be asked if they have any symptoms suggestive of 
hepatobiliary dysfunction. 
Definition of Liver Abnormalities 
Confirmed abnormalities will be characterized as moderate and severe where ULN is as 
shown below. 
Table 8 
Moderate and Severe Liver Abnormalities 
 
ALT or AST 
 
TBL 
Moderate 
> 3 × ULN 
or 
> 2 × ULN 
Severe 
> 3 × ULN 
and† 
> 2 × ULN 
ALT: alanine aminotransferase; AST: aspartate aminotransferase; TBL: total bilirubin; ULN: upper limit of 
normal 
†Samples taken simultaneously or within maximum 24 hours. 
In addition, the participant should be considered to have severe hepatic abnormalities for any 
of the following:  
● 
ALT or AST > 8 × ULN 
● 
ALT or AST > 5 × ULN for more than 2 weeks 
● 
ALT or AST > 3 × ULN and† TBL > 2 × ULN or international normalized ratio (INR) 
> 1.5 (if INR testing is applicable/evaluated) 
● 
ALT or AST > 5 × ULN and† (TBL > 2 × ULN in participants with liver metastases) 
● 
ALT or AST > 3 × ULN with the appearance of fatigue, nausea, vomiting, right upper 
quadrant pain or tenderness, fever, rash and/or eosinophilia (> 5%)  
† Samples taken simultaneously or within a maximum of 24 hours. 
The investigator may determine that abnormal liver function results, other than as described 
above, may qualify as moderate or severe abnormalities and require additional monitoring 
and follow-up. 
Follow-up Procedures 
Confirmed moderate and severe abnormalities in hepatic functions should be thoroughly 
characterized by obtaining appropriate expert consultations, detailed pertinent history, 
physical examination and clinical laboratory tests. The study site personnel are to complete 


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the liver abnormality case report form (LA-CRF). Participants with confirmed abnormal liver 
function testing should be followed as described below. 
Confirmed moderately abnormal liver function tests should be repeated 2 to 3 times weekly, 
and then weekly or less if abnormalities stabilize or the IP has been discontinued and the 
participant is asymptomatic.  
Severe hepatic liver function abnormalities as defined above, in the absence of another 
etiology, may be considered an important medical event and may be reported as a SAE. The 
sponsor should be contacted and informed of all participants for whom severe hepatic liver 
function abnormalities possibly attributable to IP are observed. 
To further assess abnormal hepatic laboratory findings, the investigator is expected to: 
● 
Obtain a more detailed history of symptoms and prior or concurrent diseases. Symptoms 
and new-onset diseases are to be recorded as “AEs” within the eCRF. Illnesses and 
conditions such as hypotensive events, and decompensated cardiac disease that may lead 
to secondary liver abnormalities should be noted. Nonalcoholic steatohepatitis is seen in 
obese hyperlipoproteinemic and/or diabetic participants, and may be associated with 
fluctuating AT levels. The investigator should ensure that the medical history form 
captures any illness that predates study enrollment that may be relevant in assessing 
hepatic function.  
● 
Obtain a history of concomitant drug use (including nonprescription medication, 
complementary and alternative medications), alcohol use, recreational drug use and 
special diets. Medications are to be entered in the eCRF. Information on alcohol, other 
substance use and diet should be entered on the LA-CRF or an appropriate document. 
● 
Obtain a history of exposure to environmental chemical agents. 
● 
Based on the participant’s history, other testing may be appropriate including:  
o Acute viral hepatitis (A, B, C, D, E or other infectious agents) 
o Ultrasound or other imaging to assess biliary tract disease 
o Other clinical laboratory tests, including INR and direct bilirubin 
● 
Consider gastroenterology or hepatology consultations. 
● 
Submit results for any additional testing and possible etiology on the LA-CRF or an 
appropriate document. 


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10.5 
Appendix 5 
List of Excluded Concomitant Medications 
The following concomitant medications will not be allowed from 2 weeks prior to study 
treatment administration until the completion of the surgical procedure unless it is determined 
that LN detection is not sufficient. 
Please note that this is not an exhaustive list. Investigators should verify the participant’s 
concomitant medications for all prohibited and restricted drug classes.  
Prohibited Medications 
Class 
Drug 
ICG† 
Indocyanine green 
Other NIR-F imaging agent 
Other agents which emit fluorescent when excited by NIR-F spectrum 
Blue Dye 
Indigocarmine 
Isosulfan blue 
Methylene blue 
Sulfan blue 
Magtrace 
Magtrace 
† ICG administration for reasons other than LN visualization (e.g., vascular perfusion) is allowed after the 
completion of SLN biopsy. 
 


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10.6 
Appendix 6 
Clinical Laboratory Assessments 
Laboratory tests will be performed according to the Schedule of Assessments [Table 1] and 
sent to the local laboratory for analysis.  
Table 9 
Clinical Laboratory Tests 
Panel/Assessments 
Parameters to be Analyzed 
Hematology 
Hemoglobin 
Hematocrit 
Erythrocytes 
Leukocytes 
Differential leukocytes 
Platelets 
Biochemistry 
Albumin  
Alanine aminotransferase 
Alkaline phosphatase 
Aspartate aminotransferase 
Blood urea nitrogen 
Calcium 
Chloride 
Creatinine 
Glucose 
Inorganic phosphorus 
Lactate dehydrogenase 
Magnesium 
Potassium 
Sodium  
Total bilirubin 
Total protein 
 
 
Histamine and tryptase (only in participants with hypersensitivity reaction) 
Urinalysis 
Urine, dipstick as applicable 
(if protein, blood, leukocytes 
or nitrites are abnormal, 
microscopy will be 
performed) 
Protein 
Glucose 
pH 
Blood 
Leukocytes 
Urobilinogen 
Bilirubin 
Ketones 
Nitrite 
Color 
 
Microscopy (optional) 
Casts 
Crystals 
Epithelial cells 
Leucocytes 
Erythrocytes 
Bacteria 
Pregnancy – Serum or Urine 
Human chorionic gonadotropin (female participants only) 
 


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10.7 
Appendix 7 
Clinical Study Continuity 
INTRODUCTION 
The purpose of this appendix is to provide acceptable alternate methods to assess safety and 
efficacy parameters, as appropriate, in the event the clinical study is interrupted at the 
country, state, site or participant level during any crisis (e.g., natural disaster, pandemic).  
BENEFIT-RISK RATIONALE 
Maintaining the safety of clinical study participants and delivering continuity of care in the 
clinical study setting is paramount during any crisis. The site is expected to follow the 
protocol and associated Schedule of Assessments [Table 1] unless the site principal 
investigator discusses the need with the Astellas medical monitor to implement the alternate 
measures.  
The approach outlined within this appendix defines which assessments are required to 
maintain a favorable benefit/risk to the participant, to maintain overall study integrity and to 
provide acceptable alternate methods to complete the study required assessments and 
procedures if study activities are unable to be performed as described in [Section 7 Study 
Procedures and Assessments] due to a crisis. 
INFORMED CONSENT 
Participants who need to follow any or all of the alternate measures outlined in this Appendix 
will be required to provide informed consent, which explicitly informs them of the nature of 
and rationale for these changes, and gain their agreement to continue participation in the 
study prior to the implementation of any of these changes. In the event the urgency of 
implementing the alternate measures does not allow for the participant to provide written 
consent prior to implementation, the principal investigator or designee will obtain oral 
agreement from the participant followed by written documentation as soon as is feasible. A 
separate addendum to the study informed consent will be provided to document the 
participant’s consent of the changes. 
PARTICIPANT PROCEDURES ASSESSMENT 
Sites with participants who are currently enrolled into this clinical study may consider 
implementing the alternate methods outlined below if one or more of the following 
conditions are met due to the crisis: 
● 
Regional or local travel has been restricted, inclusive of mandatory shelter in place 
measures, which makes participant travel to/from the study site nearly impossible. 
● 
Site facilities have been closed for clinical study conduct.  
● 
Site has been restricted to treating patients with conditions outside of the scope of the 
study. 
● 
Site personnel have temporarily relocated the conduct of the study to a location that 
place a burden on the participant with respect to time and travel. 
● 
Participant(s) have temporarily relocated from the current study site to an alternate study 
site to avoid placing a burden on the participant with respect to travel. 


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● 
Participant(s) have temporarily relocated from their home location and the new distances 
from the site would cause undue burden with respect to time and travel. 
● 
Participant has risk factors for which traveling to the site poses an additional risk to the 
participant’s health and safety. 
Adherence to the original protocol as reflected in the Schedule of Assessments [Table 1] is 
expected, where plausible, in the case of a crisis. The alternate measures as noted in 
Alternative Schedule of Assessments in Response to a Crisis [Table 10] below are only 
permissible in the event of a crisis, and after discussing the need with the Astellas medical 
monitor to implement the alternate measures. This is to allow for continuity of receiving 
investigational medicinal product and maintaining critical safety and efficacy assessments for 
participants in the study at a time of crisis. 
If one or more of the alternate measures noted below is implemented for a participant, the site 
should document in the participant’s source document the justification for implementing the 
alternate measure and the actual alternate measures that were implemented, along with the 
corresponding time point(s).  
 


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Table 10 
Alternative Schedule of Assessments in Response to a Crisis 
Assessments 
Alternate Approaches 
Safety Follow-up Period 
Day 
 
10 (Outpatient) 
 1 
Window  
 
±4 
Visit Number 
 
3 
Physical Examination 2 
Can be obtained at local clinic. 
X 
Vital Signs 3 
Can be obtained at local clinic. 
X 
Clinical Laboratory Tests 4 
Visit collection of samples at local facility acceptable if results can be made available to investigative site. 
X 
Body Weight 3 
Can be obtained at local clinic. 
X 
Serum or Urine Pregnancy Test 5 
Visit collection of samples at local facility acceptable if results can be made available to investigative site. 
X 
ECG 6 
Can be obtained at local clinic. 
X 
Confirmation of Urine or Skin Coloration 7 
Visit collection of samples at local facility acceptable if results can be made available to investigative site. 
→ 
Previous/Concomitant Medications 
Remote/Virtual/Telemedicine Visits allowed. 
X 8 
AE Assessment 
Remote/Virtual/Telemedicine Visits allowed. 
X 8 
Study Participant Feedback Questionnaire 
Not need to be collected. 
X 
AE: adverse event, ECG: electrocardiogram; LN: lymph node 
1. In the event of crisis, the alternate measures may be implemented for study procedures and assessments only for visits 3 after discussion with the Astellas Medical Monitor and/or 
designee. 
2. A full physical examination will be performed at screening (visit 1). Body systems to be evaluated include general appearance, skin, lymphatic, head and neck, ears, nose and 
throat, chest and lungs, cardiovascular, abdomen, extremities, musculoskeletal and neuromuscular. At visit 2 and follow-up (visit 3), a full physical examination is not mandatory, 
but a symptom-directed physical examination will be performed. 
3. Vital signs include blood pressure, pulse and respiratory rate. All vital signs will be measured with the participant in the sitting or supine position. Height and weight will be 
measured using standard institution practice and equipment. 
4. Clinical laboratory tests include blood collection for hematology (complete blood count) and serum chemistry and urine samples for urinalysis and urine color evaluation. These 
assessments will be performed by a local laboratory 
5. This assessment will be required only for participants who are women of childbearing potential (see [Section 10.2, Appendix 2 Contraception Requirements]). 
6. Standard 12-lead ECGs will be taken per site’s standard of care for cardiac monitoring at safety follow-up period.  
7. The occurrence of green coloration of the urine or skin will be monitored throughout the study. If observed, any visual green coloration abnormalities will be recorded as an AE. If 
it occurs for participants who have an urethral catheter in place, the color of urine will be assessed during surgery and every 120 minutes (± 30 minutes window) after the end of 
surgery until it resolves or the participant is discharged, whichever is earlier. If no urethral catheter is in place, voided urine will be assessed for green coloration and the time 
collected until it resolves or the participant is discharged, whichever is earlier.  The start and stop date and time will also be recorded. The occurrence of green coloration of the 
skin will be recorded as an AE any time after dose administration.  The start and stop date and time will be recorded.  Skin coloration will be followed up until it resolves or the 
participant is discharged, whichever is earlier. 
Footnotes continued on next page 


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8. If a participant experiences an AE or change in concomitant medications during the follow up period, the participant should call the study site to inform any of these changes. 
Should the participant develop a hypersensitivity reaction, an additional blood sample for determination of histamine and tryptase concentrations should be taken as soon as 
possible after the onset of the hypersensitivity reaction. 
 


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INVESTIGATIONAL PRODUCT SUPPLY 
If any of the conditions outlined above in the Participants Procedures Assessment are met, 
one or all of the following mitigating strategies will be employed, as needed, to ensure 
continuity of IP supply to the participants: 
● 
Increase stock of IP on site to reduce number of shipments required, if site space will 
allow. 
DATA COLLECTION REQUIREMENTS 
Additional data may be collected in order to indicate how participation in the study may have 
been affected by a crisis and to accommodate data collection resulting from alternate 
measures implemented to manage the conduct of the study and participant safety.  
● 
Critical assessments for safety and efficacy based on study endpoints to be identified as 
missing or altered (performed virtually, at alternative locations, out of window or other 
modifications) due to the crisis.  
 


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10.8 
List of Abbreviations and Definition of Key Study Terms 
List of Abbreviations 
Abbreviations 
Description of abbreviations 
ADE 
adverse device effect 
ADL 
activities of daily living 
AE 
adverse event 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
API 
Astellas Pharma Inc. 
AST 
aspartate aminotransferase 
AT 
aminotransferases 
BCRP 
breast cancer resistance protein 
CA 
Competent Authorities 
CBD 
cannabidiol 
CFR 
Code of Federal Regulations 
CI 
confidence interval 
CIOMS 
Council for International Organizations of Medical Sciences 
CLR 
renal clearance 
CRO 
contract research organization 
CSR 
clinical study report 
CYP 
cytochrome P450 
DILI 
drug-induced liver injury 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
EMA 
European Medicines Agency 
FAS 
full analysis set 
FDA 
Food and Drug Administration 
FSH 
follicle-stimulating hormone 
GCP 
Good Clinical Practice 
GI 
gastrointestinal 
GMP 
Good Manufacturing Practices 
HRT 
hormone replacement therapy 
IB 
Investigator’s Brochure 
ICF 
informed consent form 
ICG 
indocyanine green 
ICH  
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IP 
investigational product 
IRB 
Institutional Review Board 
IRT 
interactive response technology 
ISN 
international study number 


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Abbreviations 
Description of abbreviations 
LA-CRF 
liver abnormality case report form 
LN 
lymph node 
MATE 
multidrug and toxin extrusion 
NCI-CTCAE 
National Cancer Institute - Common Terminology Criteria for Adverse Events 
NIR-F 
near-infrared fluorescence 
OAT 
organic anion transporter 
OATP 
organic anion transporting polypeptide 
OCT2 
organic cation transporter 2 
P-gp 
P-glycoprotein 
QA 
quality assurance 
QC 
quality control 
QTL 
quality tolerance limit 
SAE 
serious adverse event 
SAF 
safety analysis set 
SAP 
statistical analysis plan 
SBR 
signal background ratio 
SLN 
sentinel lymph node 
SoC 
Standard of Care 
SOP 
standard operating procedure 
SPECT 
single photon emission computed tomography 
SUSAR 
suspected unexpected serious adverse reaction 
TBL 
total bilirubin 
Tc-99mSC 
technetium-99m sulfur colloid 
TEAE 
treatment-emergent adverse event 
TNM 
tumor, nodes and metastases 
UADE 
unanticipated adverse device effect 
ULN 
upper limit of normal 
USM 
urgent safety measure 
VRC 
Visualization Review Committee 
WOCBP 
women of childbearing potential 
 


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Definition of Key Study Terms 
Terms 
Definition of Terms 
Baseline 
Assessment 
Assessments of participants as they enter a study before they receive any 
treatment.  
Endpoint  
Variable that pertains to the efficacy or safety evaluations of a study. 
Note: Not all endpoints are themselves assessments since certain endpoints 
might apply to populations or emerge from analysis of results. That is, 
endpoints might be facts about assessments (e.g., prolongation of survival). 
Enroll 
To register or enter a participant into a study. 
Screen failure 
Potential participant who signed the ICF, but did not meet 1 or more criteria 
required for participation in the study and was not enrolled. 
Screening 
A process of active consideration of potential participants for enrollment in a 
study. 
Screening 
period 
Period of time before entering the investigational period, usually from the time 
when a participant signs the consent form until just before the test product or 
comparative drug ( without randomization) is given to a participant.  
Study period 
Period of time from the first study site initiation date to the last study site 
completing the study. 
Variable 
Any quantity that varies; any attribute, phenomenon or event that can have 
different qualitative or quantitative values. 
 


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11 
REFERENCES 
Chen QY, Xie JW, Zhong Q, Wang JB, Lin JX, Lu J, et al. Safety and efficacy of indocyanine green 
tracer-guided lymph node dissection during laparoscopic radical gastrectomy in patients with 
gastric cancer: a randomized clinical trial. JAMA Surg. 2020;155(4):300-11.  
de Bree R, de Keizer B, Civantos FJ, Takes RP, Rodrigo JP, et al.  What is the role of sentinel lymph 
node biopsy in the management of oral cancer in 2020? Eur Arch Otorhinolaryngol. 
28 Dec 2020. doi: 10.1007/s00405-020-06538-y. Epub ahead of print. PMID: 33369691. 
Dessources K, Aviki E, Leitao MM Jr. Lower extremity lymphedema in patients with gynecologic 
malignancies. Int J Gynecol Cancer. 2020;30(2):252-60.  
Giuliano AE, Hunt KK, Ballman KV, Beitsch PD, Whitworth PW, et al. Axillary dissection vs no 
axillary dissection in women with invasive breast cancer and sentinel node metastasis: a 
randomized clinical trial. JAMA. 2011;305(6):569-75.   
Herd-Smith A, Russo A, Muraca MG, Del Turco MR, Cardona G. Prognostic factors for lymphedema 
after primary treatment of breast carcinoma. Cancer. 2001;92(7):1783-7.    
Hoogstins C, Burggraaf JJ, Koller M, Handgraaf H, Boogerd L, van Dam G, et al. Setting standards 
for reporting and quantification in fluorescence-guided surgery. Mol Imaging Biol. 
2019;21(1):11-8. 
Isosulfan Blue (prescribing information). Morgantown, WV: Mylan Institutional LLC; September 
2020. 
Jeremiasse B, van den Bosch CH, Wijnen MWHA, Terwisscha van Scheltinga CEJ, Fiocco MF, van 
der Steeg AFW.  Systematic review and meta-analysis concerning near-infrared imaging with 
fluorescent agents to identify the sentinel lymph node in oncology patients. Eur J Surg Oncol. 
2020;46(11):2011-22. 
Lymphazurin (prescribing information). Norwalk, CT: United States Surgical Corporation, a division 
of Tyco Healthcare Group LP; January 2012. 
Lymphoseek (prescribing information). Dublin, OH: Cardinal Health 414, LLC; June 2017. 
Mueller CL, Lisbona R, Sorial R, Siblini A, Ferri LE.  Sentinel lymph node sampling for early gastric 
cancer-preliminary results of a North American prospective study. J Gastrointest Surg. 
2019;23(6):1113-21.   
National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology (NCCN 
Guidelines®) for melanoma: cutaneous. 2019; Version 1. 2020 
National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology (NCCN 
Guidelines®) for breast cancer. 2021; Version 1. 2021 
Saha S, Berbiglia L, Patel V, Paez G, Gernand J, Lawrence L, et al. Comparative analysis of 1% 
lymphazurin vs 1% methylene blue for sentinel lymph node mapping in early stage breast 
cancer.  JACS:Scientific Forum Breast. 2015;221(4), supplement 2, e4. doi: 
10.1016/j.jamcollsurg.2015.08.311. 


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Sondak VK, King DW, Zager JS, Schneebaum S, Kim J, Leong SP, et al. Combined analysis of phase 
III trials evaluating [(99m)Tc]tilmanocept and vital blue dye for identification of sentinel 
lymph nodes in clinically node-negative cutaneous melanoma. Ann Surg Oncol. 
2013;20(2):680-8.   
Spy Agent Green (indocyanine green) (prescribing information). Ferentino (FR), Italy: Patheon Italia 
S.p.A; November 2018. 
Suami H, Koelmeyer L, Mackie H, Boyages J.  Patterns of lymphatic drainage after axillary node 
dissection impact arm lymphoedema severity: A review of animal and clinical imaging studies. 
Surg Oncol. 2018; 27(4):743-50.   
Temple R. Hy’s Law: Predicting Serious Hepatotoxicity. Pharmacoepidemiol Drug Saf. 
2006;15(4):241-3. 
Thevarajah S, Huston TL, Simmons RM. A comparison of the adverse reactions associated with 
isosulfan blue versus methylene blue dye in sentinel lymph node biopsy for breast cancer. Am J 
Surg. 2005;189(2):236-9.  
Wallace AM, Han LK, Povoski SP, Deck K, Schneebaum S, Hall NC, et al. Comparative evaluation 
of [(99m)tc]tilmanocept for sentinel lymph node mapping in breast cancer patients: results of 
two phase 3 trials. Ann Surg Oncol. 2013;20(8):2590-9.  
 
 


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12 
INVESTIGATOR’S SIGNATURE 
A Phase 2 Open-label, Dose-finding Study to Determine the Optimal Dose for Lymph 
Node Visualization using ASP5354 in Participants with Breast Cancer or Melanoma 
Undergoing Sentinel Lymph Node Biopsy  
ISN/Protocol 5354-CL-1201, Amendment 3 [Nonsubstantial] 
11 MAY 2023 
I have read all pages of this protocol for which Astellas is the sponsor. I agree to conduct 
the study as outlined in the protocol and to comply with all the terms and conditions set out 
therein. I confirm that I will conduct the study in accordance with International Council for 
Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good 
Clinical Practice (GCP) guidelines and applicable local regulations. I will also ensure that 
subinvestigator(s) and other relevant members of my personnel have access to copies of this 
protocol and the ICH GCP guidelines to enable them to work in accordance with the 
provisions of these documents. 
Principal Investigator: 
Signature: 
 
 
 
 
Date (DD-MMM-YYYY) 
Printed 
Name: 
 
  
  Insert name and qualification of the investigator 
Address of 
study site: 
 
 
 
 
 
 
 
 
 


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13 
PROTOCOL ATTACHMENTS 
 
Attachment 1 
Electronic Sponsor Signatures 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Global Protocol Format V11.0 

SAP_001.pdf: 
Sponsor: API 
SAP Version 3.0 
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Page 1 of 21 
 
STATISTICAL ANALYSIS PLAN 
Version 3.0 
30-Aug-2022 
 
 
A Phase 2 Open-label, Dose-finding Study to Determine 
the Optimal dose for Lymph Node Visualization using ASP5354 
in Participants with Breast Cancer or Melanoma Undergoing 
Sentinel Lymph Node Biopsy 
 
 
ISN/Protocol 5354-CL-1201 
 
 
IND 154703 
 
 
Sponsor: 
Astellas Pharma Inc. 
2-5-1, Nihonbashi-Honcho, Chuo-Ku, 
Tokyo 103-8411, Japan 
 
 
This document contains confidential information which is the intellectual property of Astellas. By accepting or 
reviewing this document, you agree to hold this information in confidence and not copy or disclose it to others 
or use it for unauthorized purposes except (1) as otherwise agreed to in writing; (2) where required by 
applicable law; (3) where disclosure is directly related to the care and safety of the research participant; and (4) 
where disclosure of such information is made to a member of the investigator’s team who agrees to hold this 
information in confidence. 
 


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Table of Contents 
I. 
LIST OF ABBREVIATIONS AND KEY TERMS ································
···········
 4 
1 
INTRODUCTION ································
································
··················
 6 
1.1 
Objectives and Endpoints ································
································
·····  6 
1.2 
Study Design ································
································
····················
 7 
1.3 
Randomization ································
································
··················
 9 
2 
STATISTICAL HYPOTHESES ································
································
· 9 
3 
ANALYSIS SETS ································
································
··················
 9 
4 
STATISTICAL ANALYSES ································
································
····  10 
4.1 
General Considerations ································
································
·······  10 
4.2 
Study Participants ································
································
·············
 10 
4.2.1 
Participant Disposition ································
································
··  10 
4.2.2 
Protocol Deviations ································
································
·····  10 
4.2.3 
Demographic and Other Baseline Characteristics ································
···  11 
4.2.4 
Previous and Concomitant Medications ································
··············
 11 
4.2.5 
Non-medication therapies ································
······························
 12 
4.2.6 
Prior history imaging medication treatment ································
··········
 12 
4.2.7 
Extent of Exposure ································
································
······  12 
4.2.8 
SoC Visualization Agent used during biopsy ································
········  12 
4.3 
Primary Endpoint Analysis ································
································
··  12 
4.3.1 
Definition of Endpoints ································
································
· 12 
4.3.2 
Main Analytical Approach ································
·····························
 12 
4.4 
Secondary Endpoints Analysis ································
······························
 13 
4.4.1 
Definition of Endpoints ································
································
· 13 
4.4.2 
Main Analytical Approach ································
·····························
 15 
4.5 
Exploratory Endpoints Analysis ································
·····························
 15 
4.5.1 
Patient Level ································
································
·············
 15 
4.5.2 
Lymph Node Level ································
································
······  15 
4.6 
Other Endpoints ································
································
···············
 16 
4.6.1 
Green Coloration of Urine or Skin ································
····················
 16 
4.7 
Safety Analyses ································
································
···············
 16 
4.7.1 
Adverse Events ································
································
··········
 16 
4.7.2 
Clinical Laboratory Evaluation ································
························
 17 


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4.7.3 
Liver Safety Assessment ································
·······························
 17 
4.7.4 
Vital Signs ································
································
················
 18 
4.7.5 
Electrocardiograms ································
································
······  18 
4.7.6 
COVID-19 Impact Assessment ································
························
 18 
4.8 
Analysis of PK ································
································
·················
 18 
4.8.1 
Estimation of PK parameters ································
···························
 18 
4.8.2 
Plasma Concentration and PK parameters ································
············
 18 
4.8.3 
Analysis of Pharmacokinetics ································
··························
 19 
4.9 
Interim Analysis ································
································
···············
 19 
4.10 
Sample Size Determination ································
································
··  19 
4.11 
Additional Conventions ································
································
······  19 
4.11.1 
Missing Data ································
································
·············
 19 
4.11.2 
Analysis Windows ································
································
······  20 
5 
SUPPORTING DOCUMENTATION ································
·························
 20 
6 
SIGNATURE ································
································
·······················
 21 
 


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I. 
LIST OF ABBREVIATIONS AND KEY TERMS 
List of Abbreviations 
Abbreviations 
Description of abbreviations 
AE 
Adverse Event 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Transaminase 
ASP5354 
Astellas Development Code for Pudexacianinium Chloride 
AST 
Aspartate Transaminase 
ATC 
Anatomical Therapeutic Chemical classification system 
CI 
Confidence Intervals 
CRF 
Case Report Form 
CSR 
Clinical Study Report 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
FAS 
Full Analysis Set 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
ISN 
International Study Number 
MedDRA 
Medical Dictionary for Regulatory Activities 
LN 
Lymph node 
mg 
Milligrams 
mL 
Milliliter 
PD 
Protocol Deviation 
PK 
Pharmacokinetic 
PKAS 
Pharmacokinetic Analysis Set 
SAE 
Serious Adverse Event 
SAF 
Safety Analysis Set 
SAP 
Statistical Analysis Plan 
SAS 
Statistical Analysis System 
SBR 
Signal Background Ratio 
SLN 
Sentinel Lymph Node 
TEAE 
Treatment Emergent Adverse Event 
TLF 
Tables, Listings and Figures 
WHO 
World Health Organization  
 


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Version History 
SAP Version History Summary 
The changes from the prior approved SAP that impact analyses are listed with the rationale in 
the table below. 
SAP 
Version 
Approval 
Date 
SAP 
Section(s) 
Change 
Rationale 
1.0 
03-Dec-21 
 
Not Applicable 
Original Version 
1.1 
07-Feb-22 
4.2.6  
4.2.8 
 
 
 
4.4.1.2  
 
 
 
 
4.5.2 
Sections 4.2.6 and 
4.2.8 added. 
 
 
 
Histopathologic 
confirmation 
information added at 
patient level 
 
Additional details for 
metastatic spread 
defined in 4.5.2 
Prior history imaging 
medication treatment and SoC 
Visualization Agent used during 
biopsy details added in the 
document. 
 
Histopathologic confirmation 
aspects added at patient level as 
secondary objective. 
 
Details for comparing LNs 
detected with metastatic spread 
at lymph node level added as 
exploratory objective. 
2.0 
07-Mar-22 
4.2.7 
 
 
 
 
4.7.1 
Additional details for 
study drug dosing 
defined in section 
4.2.7. 
 
TEAE of Interest: 
Hypersensitivity SMQ 
by NCI-CTC Grade 
added. 
Details for drug dosing 
information for volume 
prepared and administered from 
syringe added. 
 
Hypersensitivity SMQ by NCI-
CTC Grade details will be 
summarized as TEAE of 
Interest. 
2.1 
09-Aug-22 
1.2 
Dose Arm/IP Name 
Dose Arm/IP Name updated 
according to Protocol 
Amendment 2.  
4.5.1 
SBR added 
SBR definition and imputation 
in case of ‘Not Visualized’ 
added. 
4.6.1 
Green Coloration of 
Urine or Skin 
Green Discoloration was 
replaced by Green Coloration of 
urine or skin according to 
Protocol Amendment 2. 


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1 
INTRODUCTION 
This Statistical Analysis Plan (SAP) contains technical and detailed elaboration of the 
principal features of the analysis described in the protocol, and includes procedures for 
executing the statistical analysis to fulfil the objectives of the study.  
The final SAP will be approved before first subject screened.  
If there are any changes from the planned analyses in the final version of the SAP that impact 
the statistical analyses, then it will be documented in the Clinical Study Report (CSR).  
1.1 
Objectives and Endpoints  
Objectives 
Endpoints 
Primary 
• To determine the optimal dose of 
locally administered ASP5354 for 
LN visualization in participants 
undergoing SLN biopsy. 
• Optimal dose determination by the 
VRC based on: 
o LN tissue visualized (Yes/No) 
o Visualized tissue is lymphatic in 
origin based on pathologic 
confirmation 
o The Likert Scale determination 
of the intensity of fluorescence 
(0 to 3) 
o Proportion of identified LN with 
histopathologic confirmation of 
LN tissue by ASP5354 
compared with SoC treatment 
with either Tc-99mSC 
(approved drug) or Lymphoseek 
(generic name tilmanocept) 
Secondary 
 
• To evaluate LN visualization of 
locally administered ASP5354 in 
participants undergoing SLN biopsy 
• Proportion of participants with at 
least 1 LN detected by visualization 
(Yes/No) with histopathologic 
confirmation of LN tissue using 
ASP5354 
• To compare LN detection for 
ASP5354 with the technetium-based 
treatment in breast cancer and 
melanoma 
• Proportion of identified LN with 
histopathologic confirmation of LN 
tissue by ASP5354 compared with 
SoC treatment with either Tc-
99mSC or Lymphoseek 


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Objectives 
Endpoints 
• To investigate the safety and 
tolerability of locally administered 
ASP5354 in participants undergoing 
SLN biopsy 
• Vital signs (blood pressure, pulse 
and respiratory rate) 
• Routine 12-lead ECGs 
• Clinical laboratory tests 
(hematology [complete blood 
count], serum chemistry and 
urinalysis) 
• Nature, frequency and severity of 
TEAEs and SAEs 
• To investigate the pharmacokinetics 
of locally administered ASP5354 in 
participants undergoing SLN biopsy 
• Plasma concentrations of ASP5354 
Exploratory 
• To explore the visualization 
characteristics of ASP5354 
• The Likert Scale for qualitative 
response about the intensity of 
fluorescence 
• Signal background ratio 
• Transdermal visualization of 
lymphatic spread of ASP5354 after 
injection (Yes/No) 
• Time from injection to surgical 
identification of LN(s) 
• Number of LNs visualized by 
ASP5354 
• To compare LNs detected with 
metastatic spread for ASP5354 with 
the technetium-based treatment 
• Proportion of LN with metastatic 
spread, based on histopathologic 
review, detected by ASP5354 
compared with SoC treatment with 
either Tc-99mSC or Lymphoseek 
Abbreviations from table above: ECG: electrocardiogram; LN: lymph node; SAE: serious adverse event; SLN: sentinel 
lymph node; SoC: standard of care; Tc-99mSC: Technetium-99m sulfur colloid; TEAE: treatment-emergent adverse event; 
VRC: Visualization Review Committee. 
1.2 
Study Design 
The study is an open-label, dose-finding study to determine the optimal dose for LN 
visualization, as part of lymphatic mapping, in adult participants with breast cancer and 
melanoma undergoing SLN biopsy as part of cancer treatment. 


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Enrollment of breast cancer and melanoma participants will be performed independently and 
simultaneously. Dose level 2 (0.2 mg) will enroll the first 3 participants and then the VRC 
will review the data for the breast cancer group and the melanoma group separately. If LN 
visualization, with histopathologic confirmation, occurs in 3 participants, then 3 participants 
each will be enrolled first in 0.05 mg, and then in 0.6 mg dose levels. An interim analysis will 
then be performed across initial dose levels (0.05, 0.2 and 0.6 mg) in each cohort for breast 
cancer and melanoma by the VRC prior to further enrollment of participants. If 
< 3 participants at 0.2 mg have LN visualization with histopathologic confirmation on VRC 
review, then 3 participants will be enrolled sequentially in 0.6 mg and 1 mg. An interim 
analysis will then be performed across initial dose levels (0.2, 0.6 and 1 mg) in each cohort 
for breast cancer and melanoma by the VRC prior to further enrollment of participants. 
Breast Cancer
Dose Arm/IP Name 
ASP5354 Dose Level 
ASP5354 Dose Conc 
and Volume 
Number of Participants 
Breast Cancer 0.05 mg/ 
ASP5354  
0.05 mg 
1.0 mg/mL, 
0.05 mL in total 
Up to 12 
Breast Cancer 0.2 mg/ 
ASP5354  
0.2 mg 
1.0 mg/mL, 
0.2 mL in total 
Up to 12 
Breast Cancer 0.6 mg/ 
ASP5354  
0.6 mg 
1.0 mg/mL, 
0.6 mL in total 
Up to 12 
Breast Cancer 1 mg/ 
ASP5354  
1 mg 
1.0 mg/mL,  
1 mL in total 
Up to 12 
Breast Cancer 2 mg/ 
ASP5354  
2 mg 
1.0 mg/mL,  
2 mL in total 
Up to 12 
Breast Cancer 4 mg/ 
ASP5354  
4 mg* 
1.0 mg/mL,  
4 mL in total 
Up to 12 
IP: investigational product 
* The 4 mg dose level will be added only for breast cancer participants.   
Melanoma 
Dose Arm/IP Name 
ASP5354 Dose Level 
ASP5354 Dose Conc 
and Volume 
Number of Participants 
Melanoma 0.05 mg/ 
ASP5354  
0.05 mg 
1.0 mg/mL, 
0.05 mL in total 
Up to 12 
Melanoma 0.2 mg/ 
ASP5354  
0.2 mg 
1.0 mg/mL, 
0.2 mL in total 
Up to 12 
Melanoma 0.6 mg/ 
ASP5354  
0.6 mg 
1.0 mg/mL, 
0.6 mL in total 
Up to 12 
Melanoma 1 mg/ ASP5354  
1 mg 
1.0 mg/mL,  
1 mL in total 
Up to 12 
Melanoma 2 mg/ ASP5354  
2 mg 
1.0 mg/mL,  
2 mL in total 
Up to 12 
IP: investigational product 
Conc: concentration 
* The 4 mg dose level will be added only for breast cancer participants 


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Depending on the outcome of the interim VRC data reviews, additional participants may be 
added to the same dose level, a dose may be closed, or a new dose (1 mg, 2 mg or 4 mg) will 
begin enrollment. Up to 12 participants will be assigned at each dose level for breast cancer 
and melanoma. 
Details of the schedule of clinical assessments are available in the protocol (section 1.3). 
1.3 
Randomization 
This is an open-label study. Participant enrollment and dispensation of IP will be performed 
via the interactive response technology (IRT) system. Prior to initiation of treatment, study 
site personnel will obtain the participant number and treatment assignment from the IRT 
system. Specific IRT procedures will be described in the respective study manual. 
2 
STATISTICAL HYPOTHESES 
No statistical hypothesis is planned. 
3 
ANALYSIS SETS 
In accordance with International Conference on Harmonization (ICH) recommendations in 
guidelines E3 and E9, the following analysis sets will be used for the analyses. 
The determination of whether participants are included or excluded from the safety analysis 
sets will be made prior to database lock, except the pharmacokinetic analysis set. 
Population 
Description 
Enrolled 
All participants who sign the ICF 
Full Analysis Set (FAS) 
All participants who are enrolled and 
receive study IP. Participants will be 
analyzed according to the IP they actually 
received. 
Safety Analysis Set (SAF) 
All participants who are enrolled and 
receive study IP. Participants will be 
analyzed according to the IP they actually 
received. SAF is the same as FAS for this 
study. 
Pharmacokinetic analysis 
set (PKAS) 
All participants who receive at least 1 dose 
of IP for which at least 1 plasma 
concentration data are available with the 
time of dosing and sampling. Inclusion of 
participants in the PKAS with missing data 
or important protocol deviations will be 
considered by the pharmacokineticist on a 
case-by-case basis. 
 


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4 
STATISTICAL ANALYSES 
A Statistical Analysis Plan (SAP) will be written to provide details of the analysis, along with 
specifications for tables, listings and figures to be produced. Changes from the planned 
analyses in the final SAP that impact the statistical analyses will be justified in the Clinical 
Study Report (CSR). 
4.1 
General Considerations 
For continuous variables, descriptive statistics will include the number of subjects (n), mean, 
standard deviation, median, minimum and maximum. When needed, the use of other 
percentiles (e.g.10%, 25%, 75% and 90%) will be mentioned in the relevant section.  
Summaries based on FAS (e.g. efficacy data) will be presented by planned dose level, unless 
specifically stated otherwise. Safety analysis and other summaries based on SAF will be 
presented by actual dose level received.  
The 95% confidence interval (95% CI) in proportion is based on exact method unless 
specifically stated otherwise.  
Pharmacokinetic summaries based on PKAS will be presented by actual dose level received.  
All data processing, summarization, and analyses will be performed using SAS® Version 9.3 
or higher on Unix. Specifications for table, figure, and data listing formats can be found in 
the TLF specifications for this study. 
Unless otherwise specified, all summaries will be presented by tumor type and dose level. 
4.2 
Study Participants 
Participant disposition, demographics and baseline characteristics will be summarized by 
tumor type, dose level and overall for FAS population. 
Baseline will be defined as the last no missing observation prior to administration of IP, 
unless otherwise specified.  
4.2.1 
Participant Disposition 
The number and percentage of participants who completed and discontinued screening period 
and reasons for discontinuation will be presented for all participants who signed the ICF. 
A similar table for treatment and follow-up disposition will also be presented for the FAS by 
dose level and overall.  
All disposition details and dates of first and last evaluations for each participant will be listed. 
Number and percentage of participants for each analysis set will be summarized by dose level 
and overall.  
4.2.2 
Protocol Deviations  
The number and percentage of participants with the following important protocol deviation 
criteria will be summarized for each criterion and overall, by dose level and overall, and 


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tumor type. Participants deviating from a criterion more than once will be counted once for 
the corresponding criterion. 
The unique identifiers will be as follows: 
PD 1: Inclusion/Exclusion 
PD 2: Withdrawal Criteria 
PD 3: Study Intervention 
PD 4: Excluded Concomitant Medications 
PD 5: Informed Consent 
PD 6: Safety Reporting 
PD 7: Procedures/Tests 
4.2.3 
Demographic and Other Baseline Characteristics 
Demographic, and other baseline characteristics will be summarized descriptively by tumor 
type, dose level, and overall for the FAS.  
Descriptive statistics for age, age categories (<65, >=65 - <75, >=75), body mass index 
(BMI), weight and height will be presented. Frequency tabulations for sex, ethnicity and race 
will be presented.  
Medical history is coded in MedDRA, and will be summarized by System Organ Class (SOC) 
and Preferred Term (PT), by tumor type, dose level, and overall for the FAS. 
Primary diagnosis of the target disease will be listed including T categorization in TNM 
classification by tumor type and dose level. For breast cancer laterality (left/right) will be 
presented by dose level. The location of tumor (extremities: upper/lower, left/right), trunk 
(front, back or side [L or R]), head, neck, other, tumor thickness, and ulceration status will be 
presented by melanoma and dose level.  
4.2.4 
Previous and Concomitant Medications 
Previous and concomitant medications will be summarized by therapeutic subgroup (ATC 
2nd level), chemical subgroup (ATC 4th level) and preferred WHO name (active ingredients 
for combination drugs) by tumor type and dose level for the SAF.  
Participants taking the same medication multiple times will be counted once per medication 
and investigational period. A medication which can be classified into several chemical and/or 
therapeutic subgroups is presented in all chemical and therapeutic subgroups. 
Previous medications are defined as medications that participants started prior to IP 
administration period. Concomitant medications are defined as any medications that 
participants took after IP administration until the end of follow up period. Medications that 
started prior to and continued after IP administration will be counted in both previous and 
concomitant medications. 


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4.2.5 
Non-medication therapies 
Frequency tabulations of subjects with non-medication therapy and reason for use will be 
presented by tumor type and dose level and overall for SAF.  
4.2.6 
Prior history imaging medication treatment 
Frequency tabulations of subjects with prior history imaging medication treatment will be 
presented by tumor type and dose level and overall for SAF.   
4.2.7 
Extent of Exposure  
Since this is a single dose study, the analysis of duration of exposure is not applicable. Study 
drug dosing information which include dosing date and time, volume prepared and 
administered from syringe, total drug used (derived following the pharmacy manual), 
compliance, number of injection sites, route of administration, directionality of the 
anatomical location, and location of administration will be listed.  
4.2.8 
SoC Visualization Agent used during biopsy 
SoC visualization agent used during biopsy at Day 1 intraoperative will be summarized. 
4.3 
Primary Endpoint Analysis 
The primary analysis will be conducted on the FAS. No statistical testing will be performed. 
4.3.1 
Definition of Endpoints 
Optimal dose determination will be assessed based on the totality of collected data by the 
VRC based on:  
- 
LN tissue visualized (Yes/No) 
- 
Visualized tissue is lymphatic in origin based on pathologic confirmation  
- 
The Likert Scale determination of the intensity of fluorescence (0 to 3)  
- 
Proportion of identified LN with histopathologic confirmation of LN tissue by 
ASP5354 compared with SoC treatment with either Tc-99mSC or Lymphoseek 
The optimal dose is defined as the dose that provides a better visualization compared with 
lower doses and a comparable visualization to the next higher dose. In the case where 2 doses 
performed equally, the lower dose would be selected. Cumulative visualization data from all 
treated participants will be used for optimal dose determination. 
4.3.2 
Main Analytical Approach 
The optimal dose will be determined by VRC based on the totality of the data and including 
the following assessments: LN tissue visualized (Y/N), visualized tissue is lymphatic in 
origin based on pathologic confirmation, the Likert scale determination of the intensity of 
fluorescence, and proportion of identified LN with histopathologic confirmation of LN tissue 
by ASP5354 compared with SoC treatment with either Tc-99mSC or Lymphoseek. The 
individual listings for the visualization parameters not limited to the parameters above are 
created.  


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4.4 
Secondary Endpoints Analysis 
The secondary analysis will be conducted on the FAS. No statistical testing will be 
performed. 
4.4.1 
Definition of Endpoints 
4.4.1.1 Lymph Node Level 
- 
The number of LN identified intraoperatively. 
- 
The number and percentage a) of LN identified intraoperatively by histopathologic 
confirmation.  
- 
The number of sentinel LN identified intraoperatively. 
- 
The number and percentage b) of sentinel LN identified intraoperatively by 
histopathologic confirmation. 
a) The denominator is the number of LN identified intraoperatively. 
b) The denominator is the number of sentinel LN identified intraoperatively. 
Sentinel LN detailed: 
- 
The number of sentinel LN identified by ASP5354 or SoC treatment with either 
Tc-99mSC or Lymphoseek. 
- 
The number of sentinel LN identified by ASP5354 and SoC treatment with either 
Tc-99mSC or Lymphoseek. 
- 
The number and percentage c) of sentinel LN identified by ASP5354 or SoC treatment 
with either Tc-99mSC or Lymphoseek with histopathologic confirmation. 
- 
The number of sentinel LN identified by ASP5354. 
- 
The number and percentage c) of sentinel LN identified by ASP5354 with 
histopathologic confirmation and 95% CI.  
- 
The number of sentinel LN identified by SoC treatment with either Tc-99mSC or 
Lymphoseek. 
- 
The number and percentage c) of sentinel LN identified by SoC treatment with either 
Tc-99mSC or Lymphoseek with histopathologic confirmation and 95% CI. 
- 
The difference in proportion of identified sentinel LN with histopathologic 
confirmation of LN tissue between ASP5354 and SoC treatment with either 
Tc-99mSC or Lymphoseek and 95% CI. 
c) The denominator is the number of sentinel LN identified intraoperatively by 
histopathologic confirmation. 


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LN detailed: 
- 
The number of LN identified by ASP5354 or SoC treatment with either Tc-99mSC or 
Lymphoseek. 
- 
The number of LN identified by ASP5354 and SoC treatment with either Tc-99mSC 
or Lymphoseek. 
- 
The number and percentage d) of LN identified by ASP5354 or SoC treatment with 
either Tc-99mSC or Lymphoseek with histopathologic confirmation. 
- 
The number of LN identified by ASP5354. 
- 
The number and percentage d) of LN identified by ASP5354 with histopathologic 
confirmation* and 95% CI.  
- 
The number of LN identified by SoC treatment with either Tc-99mSC or 
Lymphoseek. 
- 
The number and percentage d) of LN identified by SoC treatment with either 
Tc-99mSC or Lymphoseek with histopathologic confirmation* and 95% CI. 
- 
The difference in proportion of identified LN with histopathologic confirmation of 
LN tissue between ASP5354 and SoC treatment with either Tc-99mSC or 
Lymphoseek and 95% CI.  
d) The denominator is the number of LN identified intraoperatively by 
histopathologic confirmation 
4.4.1.2 Patient Level 
- 
The number of LN identified intraoperatively per patient. 
- 
The number of LN identified by histopathologic confirmation per patient. 
- 
The number of LN identified by ASP5354 with histopathologic confirmation per 
patient. 
- 
The number of LN identified by SoC treatment with either Tc-99mSC or Lymphoseek 
with histopathologic confirmation per patient. 
- 
The number of LN identified by ASP5354 or SoC treatment with either Tc-99mSC or 
Lymphoseek with histopathologic confirmation per patient. 
- 
The number of LN identified by ASP5354 and SoC treatment with either Tc-99mSC 
or Lymphoseek with histopathologic confirmation per patient. 
- 
Proportion of participants with at least 1 LN detected with histopathologic 
confirmation of LN tissue using ASP5354 and 95% CI. 
- 
Proportion of participants with at least 1 LN detected with histopathologic 
confirmation of LN tissue using SoC treatment with either Tc-99mSC or Lymphoseek 
and 95% CI. 


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- 
The difference in proportion of identified LN with histopathologic confirmation of LN 
tissue between ASP5354 and SoC treatment with either Tc-99mSC or Lymphoseek and 95% 
CI.  
- 
Proportion of participants with at least 1 LN with histopathologic confirmation 
detected by the method other than ASP5354 or SoC treatment with either Tc-99mSC 
or Lymphoseek and 95% CI. 
4.4.2 
Main Analytical Approach 
4.4.2.1 Lymph Node Level 
The number of and percentage for lymph nodes or sentinel lymph nodes will be summarized 
per the specified denominator. 
4.4.2.2  Patient Level 
The number of and percentage for the detection of lymph nodes or sentinel lymph nodes per 
patient will be summarized with specified statistics. 
4.5 
Exploratory Endpoints Analysis 
4.5.1 
Patient Level 
- 
The Maximum of Likert Scale of the intensity of the fluorescence in ASP5354 will be 
summarized by frequency and percentage.   
- 
The average score of Likert Scale (0- None, 1- Mild, 2- Moderate, 3- Strong) in 
sentinel lymph nodes per patients for the intensity of the fluorescence in ASP5354 
will be summarized by descriptive statistics.   
- 
The Signal Background Ratio (SBR) in ASP5354 will be summarized using 
descriptive statistics. 
The SBR will be defined as follows: 
𝑆𝐵𝑅= 𝑀𝑒𝑎𝑛 𝑟𝑒𝑔𝑖𝑜𝑛 𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡
𝑀𝑒𝑎𝑛 𝑠𝑖𝑔𝑛𝑎𝑙 𝑏𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑 
based on Hoogstins et al, 2019. For SBR records where images are reviewed and no 
visualization occurs, the SBR will be imputed as 1 for the summary tables. 
- 
The transdermal visualization of lymphatic spread of ASP5354 after injection will be 
summarized by frequency and percentage. 
- 
The time from injection to surgical identification of LN(s) will be summarized by 
descriptive statistics. 
4.5.2 
Lymph Node Level 
- 
The number and percentage e) of LN with metastatic spread, based on histopathologic 
review, detected by ASP5354 


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- 
The number and percentage e) of LN with metastatic spread, based on histopathologic 
review, detected by SoC treatment with either Tc-99mSC or Lymphoseek 
- 
The difference in proportion of LN with metastatic spread, based on histopathologic 
confirmation of LN tissue between ASP5354 and SoC treatment with either Tc-99mSC or 
Lymphoseek and 95% CI 
e) The denominator is the number of LN identified intraoperatively by 
histopathologic confirmation 
4.6 
Other Endpoints 
4.6.1 
Green Coloration of Urine or Skin 
The frequency and percentage of patients who experience green coloration of urine will be 
summarized by dose level and tumor type only for subjects who used urethral catheter. The 
duration of green coloration of urine or skin (in hours) will be summarized using descriptive 
statistics by dose level and tumor type. The duration is calculated from first date/time point of 
green coloration of urine or skin to last date/time point of green skin coloration. If the green 
coloration of urine or skin is still present at discharge, the duration is from first date/time 
point of green coloration to the date/time of discharge. 
Additional to descriptive statistics, if applicable, the duration will be analyzed using Kaplan 
Meier estimation. For this purpose, subjects with green coloration of urine or skin at 
discharge are censored at the time point of discharge. 
4.7 
Safety Analyses 
The safety and tolerability will be summarized by descriptive statistics for the SAF 
population. 
4.7.1 
Adverse Events 
AEs will be coded using MedDRA and graded using the National Cancer Institute-Common 
Terminology Criteria for Adverse Events (NCI-CTCAE version 5.0). 
A TEAE is defined as an AE observed after administration of the IP and up to the follow-up 
period. An IP-related TEAE is defined as any TEAE with a causal relationship assessed as 
“yes” by the investigator. 
The number and percentage of participants with TEAEs, IP-related TEAEs, serious TEAEs 
and IP-related serious TEAEs will be summarized by system organ class, preferred term and 
dose level. The worst grade will be summarized if the same AE is recorded more than once 
for a participant. 
AE data will be listed. 
An overview table will include the following by tumor type and dose level: 
● 
TEAEs 
● 
Drug related TEAEs 
● 
Serious TEAEs 


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● 
Drug related serious TEAEs 
● 
TEAEs leading to death 
● 
TEAEs of Interest: Hypersensitivity SMQ by NCI-CTC Grade 
● 
Drug-related TEAEs leading to death 
● 
Deaths 
● 
Drug-related Grade 3 or higher TEAE 
● 
Drug-related Grade 3 or higher: Local Site Reaction 
● 
Drug-related Grade 3 or higher: Infusion Related Reactions 
● 
Drug-related Grade 3 or higher: Hypersensitivity SMQ 
4.7.2 
Clinical Laboratory Evaluation 
For quantitative clinical laboratory measurements, descriptive statistics will be used to 
summarize results and change from baseline by dose level and time point.  
Shifts from baseline to the post baseline worst grade based on NCI-CTCAE until the follow 
up period in clinical laboratory tests will be tabulated. Quantitative laboratory test results by 
NCI-CTCAE Grade (version 5.0) will be summarized with frequencies and percentages by 
dose level and time point. 
Laboratory data will be listed. 
4.7.3 
Liver Safety Assessment 
The liver safety assessments will be summarized by the categories below based on the 
measurements from Alkaline Phosphatase (ALP), Alanine Transaminase (ALT), Total 
Bilirubin, Aspartate Transaminase (AST) and their combination. These parameters will be 
based on measurements from a local laboratory. 
The participant’s highest value during the course of the study will be used; upper limit of 
normal (ULN).  
● ALT: > 3xULN, > 5xULN, > 10xULN, > 20xULN  
● AST: > 3xULN, > 5xULN, > 10xULN, > 20xULN  
● ALT or AST: > 3xULN, > 5xULN, > 10xULN, > 20xULN  
● ALP: > 1.5xULN  
● Total Bilirubin: > 2xULN  
● (ALT or AST > 3xULN) and Total Bilirubin > 2xULN  
● (ALT or AST > 3xULN) and Total Bilirubin > 2xULN and ALP < 2xULN  
The last 2 criteria where 2 or more parameters are evaluated will use the measurements on 
the same day or up to 1 day apart. The number and percentage of subjects meeting the criteria 
post-baseline will be summarized. 
Information on alcohol, other substance use will be listed by dose level and tumor type. 


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4.7.4 
Vital Signs 
Descriptive statistics will be used to summarize vital sign results and changes from baseline 
by dose level and time point. Vital signs data will be listed. 
A table for potentially clinically significant vital signs (blood pressure >150/90 mmHg or 
< 70/60 mmHg; pulse > 100 per min or <60 per min; respiratory rate <12 and >16) will be 
generated by tumor type and dose level. 
4.7.5 
Electrocardiograms 
The routine 12-lead ECG results and intraoperative ECG monitoring will be summarized by 
dose level and time point.  
12-lead ECG and intraoperative ECG data and interpretations will be listed. 
4.7.6 
COVID-19 Impact Assessment 
Assessments affected by the COVID-19 pandemic will be listed for visit-based assessments 
and for non-visit-based assessments.  
For visit-based assessments affected by COVID-19, the listing shows if an assessment was 
not performed due to COVID-19, if it was out of window, if the assessment was performed at 
an alternative location or if it was a virtual assessment. Other information and comments 
reported on assessments affected by COVID-19 are also included. 
For non-visit-based assessments affected by COVID-19, subjects who experience any of the 
following items: COVID-19 adverse event, hospitalization due to COVID-19, or COVID-19 
death, will be flagged in a listing.   
Any events like adverse events, hospitalization, or death, which are related to COVID-19, 
will be flagged in the corresponding listing. 
4.8 
Analysis of PK 
PK analysis will be conducted on the PKAS.  
4.8.1 
Estimation of PK parameters  
The plasma PK parameter calculation will be performed using noncompartmental method in 
Phoenix® (Certara St. Louise, MO, USA, version 6.3 or higher) software. For AUC 
determination, linear up and log down analysis will be used. 
4.8.2 
Plasma Concentration and PK parameters  
Plasma concentrations and PK parameters of ASP5354 will be summarized by tumor type  
and dose level and, where appropriate, by nominal time points and days using descriptive 
statistics, including n, mean, SD, coefficient of variation (CV), geometric mean, geometric 
CV, median, minimum and maximum.  For the pharmacokinetic parameters tmax only n, 
median, minimum and maximum will be calculated.  For induction phase nominal days for 
Ctrough concentration based on start of ASP5354 administration will be used. 


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4.8.3 
Analysis of Pharmacokinetics  
A listing and summary statistics of the individual plasma concentrations of ASP5354 and 
sampling times by treatment and assessment time interval will be prepared.  
4.9 
Interim Analysis  
VRC review will be held after the initial 3 participants in 0.2 mg in each tumor type have 
data for review. Three participants each will then be enrolled in the 2 additional dose levels 
in each tumor type. The determination of dose(s) to stop, continue or define as optimal dose 
will start when 3 participants assigned to each dose level have their data for visualization 
reviewed by the VRC. This process will continue for every additional 3 participants assigned 
to each dose level until reaching the maximum of 12 participants. A new dose level might be 
added. 
4.10 
Sample Size Determination  
The sample size is not based on statistical power calculation. The sample size is expected to 
provide adequate information to determine the optimal dose ASP5354 for LN visualization in 
breast cancer and melanoma. Approximately up to 12 participants will be assigned to each 
3 dose levels of ASP5354 in each tumor type, and then an estimated total of up to 
36 participants will be required in each tumor type. In case some dose levels are stopped 
based on VRC, additional dose levels might be added. 
4.11 
Additional Conventions  
4.11.1 Missing Data 
As a general principle, no imputation of missing data will be done. Exceptions are the start 
and stop dates of AEs and concomitant medications if they are missing 
In case of missing or partial start and stop dates for concomitant medications, the following 
rules will be used: 
If the start date is missing or partial: 
● 
if the month is missing, use January 
● 
if the day is missing, use the first day of the month under consideration 
● 
if the year is missing, use year of the informed consent date 
● 
if the entire date is missing, use informed consent date 
If the stop date is missing or partial: 
● 
if the month is missing, use December 
● 
if the day is missing, use the last day of the month under consideration 
● 
if the year or the entire date is missing, set the stop date to December 31st, 2099 
If the imputed start date is after the stop date, then the imputed start date will be 1 day prior 
to the stop date. 
For AEs, a missing or incomplete onset date will be imputed according to the following 
conventions. 


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If only the year is known for the AE onset date, the imputed onset date will be the latest of 
the following non-missing dates:  
● Date of first dose of study drug  
● January 1 of the year of AE onset date  
If only the month and year is known for the onset date, set the surrogate onset date to the first 
day of that month and then apply the following rules.  
● If the month and year of the onset date is prior to the month and year of the first dose of 
study drug, then the surrogate onset date will be the imputed onset date.  
● If the month and year of the onset date is on or after the month and year of the first dose of 
study drug, then the imputed onset date will be the latest of the following non-missing dates:  
○ Date of first dose of study drug  
○ Surrogate onset date  
If the imputed onset date is after the adverse event end date, the imputed onset date will be 
the same as the adverse event end date. 
4.11.2 Analysis Windows 
Nominal visits will be used by visit summary.  
5 
SUPPORTING DOCUMENTATION 
Please refer to protocol 5354-CL-1201 for all supporting documents and references. 


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6 
SIGNATURE 
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Approved by: 
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PPD
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