Conditions: Colo-rectal Cancer
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 490
Healthy volunteers: no

Brief summary:
This is a multicenter, randomized, parallel-group, Phase III study in at least 440 patients with advanced colorectal cancer to compare the efficacy of treatment with arfolitixorin versus Leucovorin in combination with 5-fluorouracil, oxaliplatin, and bevacizumab according to modified FOLFOX-6 until PD according to RECIST 1.1 criteria.

Detailed description:

Arms:
arm 1: ARFOX (Arfolitixorin and 5-FU and Oxaliplatin) and Bevacizumab arm 2: mFOLFOX-6 (Leucovorin and 5-FU and Oxaliplatin) and Bevacizumab

Interventions: intervention 1: Arfolitixorin intervention 2: Leucovorin
Intervention descriptions: intervention 1: Bevacizumab 5 mg/kg intravenous infusion; Oxaliplatin 85 mg/m2 intravenous infusion; 5-FU 400 mg/m2 intravenous bolus; Arfolitixorin 60 mg/m2 intravenous bolus; 5-FU 2400 mg/m2 intravenous infusion; Arfolitixorin 60 mg/m2 intravenous bolus intervention 2: Bevacizumab 5 mg/kg intravenous infusion; Oxaliplatin 85 mg/m2 intravenous infusion; Leucovorin 400 mg/m2 intravenous infusion; 5-FU 400 mg/m2 intravenous infusion; 5-FU 2400 mg/m2 intravenous infusion

Protocol document:
Prot_000.pdf: 
   
 
 
 
A randomized, multicenter, parallel-group, Phase III study to compare the 
efficacy of arfolitixorin versus leucovorin in combination with 5-fluorouracil, 
oxaliplatin, and bevacizumab in patients with advanced colorectal cancer 
 
Protocol Number: IS0-CC-007 
Drug Product: arfolitixorin 
 
Global Coordinating Investigator: Josep Tabernero, MD, PhD, Prof 
US Coordinating Investigator: Heinz-Josef Lenz, MD, Prof 
IND/IDE number: 127894 
EudraCT number: 2017-004154-41 
 
Sponsor: Isofol Medical AB (publ), Gothenburg, Sweden 
 
Version Number: 9.0 USA 
22 April 2022 
 
Summary of Changes from Previous Version: 
Version no. 
Date 
Type of change (Amendment or Admin) 
1.0 
21 September 2018 
Initial version 
2.0 
13 November 2018 
Administrative change 
3.0 USA 
25 January 2019 
Local amendment 
4.0 USA 
29 August 2019 
Global amendment 
5.0 USA 
07 October 2019 
Administrative change 
6.0 USA 
20 February 2020 
Global amendment 
7.0 USA 
20 August 2021 
Global amendment 


NIH-FDA Phase 2 and 3 IND/IDE Clinical Trial Protocol Template   
 
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Global Amendment. Never submitted, changes included in 
v 9.0 
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Global Amendment 
For more detailed information regarding the changes, see section 10.3 in this study protocol. 


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Table of Contents 
STATEMENT OF COMPLIANCE ...................................................................................................... 1 
CONFIDENTIALITY STATEMENT .................................................................................................... 1 
SIGNATURE PAGE Protocol approval ............................................................................................. 2 
SIGNATURE PAGE Study Coordinators .......................................................................................... 3 
SIGNATURE PAGE Investigator ....................................................................................................... 4 
1 
PROTOCOL SUMMARY ......................................................................................................... 5 
1.1 
Synopsis ........................................................................................................................... 5 
1.2 
Schema ........................................................................................................................... 14 
1.3 
Schedule of Activities (SoA) ........................................................................................ 15 
2 
INTRODUCTION .................................................................................................................... 17 
2.1 
Study Rationale ............................................................................................................. 17 
2.1.1 
Advanced Colorectal Cancer ..................................................................... 17 
2.1.2 
Current standard of care ............................................................................ 17 
2.1.3 
Mechanism of action of Arfolitixorin.......................................................... 18 
2.2 
Background .................................................................................................................... 19 
2.2.1 
Non-clinical Background ............................................................................ 19 
2.2.2 
Clinical Background .................................................................................... 19 
2.3 
Risk/Benefit Assessment ............................................................................................. 21 
2.3.1 
Known Potential Risks ................................................................................ 21 
2.3.2 
Known Potential Benefits ........................................................................... 21 
2.3.3 
Assessment of Potential Risks and Benefits .......................................... 21 
3 
OBJECTIVES AND ENDPOINTS ........................................................................................ 24 
4 
STUDY DESIGN ..................................................................................................................... 25 
4.1 
Overall Design ............................................................................................................... 25 
4.2 
Scientific Rationale for Study Design ......................................................................... 26 
4.3 
Justification for Dose .................................................................................................... 27 
4.4 
End of Study Definition ................................................................................................. 29 
5 
STUDY POPULATION ........................................................................................................... 29 
5.1 
Inclusion Criteria............................................................................................................ 29 
5.2 
Exclusion Criteria .......................................................................................................... 30 
5.3 
Lifestyle Considerations ............................................................................................... 31 
5.4 
Screen Failures ............................................................................................................. 31 
5.5 
Strategies for Recruitment and Retention ................................................................. 31 
6 
STUDY INTERVENTION ....................................................................................................... 32 
6.1 
Study Intervention(s) Administration .......................................................................... 32 
6.1.1 
Study Intervention Description .................................................................. 32 
6.1.2 
Dosing and Administration ......................................................................... 32 
6.2 
Preparation/Handling/Storage/Accountability ........................................................... 33 
6.2.1 
Acquisition and accountability ................................................................... 33 
6.2.2 
Formulation, Appearance, Packaging, and Labelling ............................ 34 
6.2.3 
Product Storage and Stability .................................................................... 35 
6.2.4 
Preparation ................................................................................................... 35 
6.3 
Other Medications ......................................................................................................... 36 
6.3.1 
5-FU 
36 
6.3.2 
Oxaliplatin ..................................................................................................... 36 


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6.3.3 
Bevacizumab ............................................................................................... 37 
6.3.4 
Dose adjustements ..................................................................................... 37 
6.4 
Measures to Minimize Bias: Randomization and Blinding ...................................... 38 
6.4.1 
Randomization ............................................................................................. 38 
6.4.2 
Blinding 38 
6.5 
Study Intervention Compliance ................................................................................... 38 
6.6 
Concomitant Therapy ................................................................................................... 39 
6.6.1 
Prohibited Therapies................................................................................... 39 
6.6.2 
Authorized Medication ................................................................................ 39 
6.6.3 
Rescue Medicine ......................................................................................... 39 
7 
IMP DISCONTINUATION AND PATIENT DISCONTINUATION .................................... 39 
7.1 
Discontinuation of Study Intervention ........................................................................ 39 
7.2 
Participant Discontinuation/Withdrawal from the Study .......................................... 40 
7.3 
Lost to Follow-Up .......................................................................................................... 40 
8 
STUDY ASSESSMENTS AND PROCEDURES ................................................................ 41 
8.1 
Efficacy Assessments .................................................................................................. 41 
8.1.1 
CT scan / MRI .............................................................................................. 41 
8.1.2 
Pharmacokinetics ........................................................................................ 43 
8.1.3 
ECOG performance status ........................................................................ 45 
8.1.4 
EQ-5D Quality of life ................................................................................... 45 
8.1.5 
Survival Status ............................................................................................. 45 
8.2 
Safety and Other Assessments .................................................................................. 46 
8.2.1 
Laboratory safety assessments ................................................................ 46 
8.2.2 
Vital signs, ECG and physical findings related to safety ....................... 46 
8.2.3 
PRO CTCAE ................................................................................................ 47 
8.2.4 
Pharmacogenetics ...................................................................................... 47 
8.2.5 
RESECTION/REMOVAL OF TUMOR LESION(S) ................................ 48 
8.3 
Study Schedule ............................................................................................................. 48 
8.3.1 
Screening and Enrollment ......................................................................... 48 
8.3.2 
Baseline (Cycle 1) ....................................................................................... 49 
8.3.3 
Treatment Period......................................................................................... 49 
8.3.4 
End of treatment visit (EOT) ...................................................................... 50 
8.3.5 
Post-Treatment visits .................................................................................. 50 
8.4 
Adverse Events and Serious Adverse Events .......................................................... 51 
8.4.1 
Definition of Adverse Events (AE) ............................................................ 51 
8.4.2 
Definition of Serious Adverse Events (SAE) ........................................... 51 
8.4.3 
Classification of an Adverse Event ........................................................... 52 
8.4.4 
Time Period and Frequency for Event Assessment and Follow-Up ... 54 
8.4.5 
Adverse Event Reporting ........................................................................... 54 
8.4.6 
Adverse Event of Special Interest ............................................................ 54 
8.4.7 
Serious Adverse Event Reporting ............................................................ 54 
8.4.8 
Reporting Events to Participants .............................................................. 55 
8.4.9 
Reporting of Pregnancy ............................................................................. 55 
9 
STATISTICAL CONSIDERATIONS ..................................................................................... 56 
9.1 
Statistical Hypotheses .................................................................................................. 56 
9.2 
Sample Size Determination ......................................................................................... 56 


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9.2.1 
Primary efficacy endpoint: ORR ............................................................... 56 
9.2.2 
Secondary efficacy endpoint: PFS ........................................................... 57 
9.2.3 
Hierarchical testing approach .................................................................... 57 
9.2.4 
Overall survival ............................................................................................ 58 
9.2.5 
Accrual rate .................................................................................................. 58 
9.3 
Populations for Analyses ............................................................................................. 58 
9.4 
Statistical Analyses ....................................................................................................... 59 
9.4.1 
General Approach ....................................................................................... 59 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) .......................................... 59 
9.4.3 
Analysis of the Secondary Endpoint(s) .................................................... 59 
9.4.4 
Safety Analyses ........................................................................................... 60 
9.4.5 
Baseline Descriptive Statistics .................................................................. 60 
9.4.6 
Planned Interim and final Analyses .......................................................... 60 
9.4.7 
Sub-Group Analyses................................................................................... 63 
9.4.8 
Tabulation of Individual participant Data ................................................. 63 
9.4.9 
Exploratory Analyses .................................................................................. 63 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS ........ 64 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ...................................... 64 
10.1.1 
Informed Consent Process ........................................................................ 64 
10.1.2 
Study Discontinuation and Closure .......................................................... 65 
10.1.3 
Confidentiality and Privacy ........................................................................ 65 
10.1.4 
Future Use of Stored Specimens and SAMPLES .................................. 65 
10.1.5 
Key Roles and Study Governance ........................................................... 65 
10.1.6 
Safety Oversight .......................................................................................... 66 
10.1.7 
Clinical Monitoring ....................................................................................... 66 
10.1.8 
Quality Assurance ....................................................................................... 67 
10.1.9 
Data Handling and Record Keeping ......................................................... 68 
10.1.10 
Protocol Deviations ..................................................................................... 69 
10.1.11 
Publication and Data Sharing Policy ........................................................ 70 
10.1.12 
Insurance ...................................................................................................... 70 
10.1.13 
Conflict of Interest Policy ........................................................................... 70 
10.2 
Abbreviations ................................................................................................................. 72 
10.3 
Protocol Amendment History ...................................................................................... 74 
11 
REFERENCES ........................................................................................................................ 79 
 
 
 
 
 
 
 


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Tables 
Table 1: Schedule of Activities (SOA) ............................................................................................. 15 
Table 2: Components of arfolitixorin................................................................................................ 34 
Table 3: Laboratory tests .................................................................................................................. 46 
Table 4: Hazard ratio for significance in function of the number of PFS events ...................... 57 
Table 5: Guidelines for sample size increase at interim analysis ............................................... 62 
 
 
Appendices 
Appendix 1: ECOG performance status scale 
Appendix 2: NCI PRO CTCAE 
Appendix 3: EuroQol EQ-5D


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STATEMENT OF COMPLIANCE 
 
The trial will be conducted in accordance with International Conference on Harmonization Good 
Clinical Practice (ICH GCP), the provisions of the Helsinki Declaration, and the relevant legislation in 
force. For US based clinical sites, the study will be conducted under an IND. For sites based outside of 
the US (Non-IND sites), the study will be conducted according to GCP and other applicable local laws 
and regulation. The Principal Investigator will assure that no deviation from, or changes to the protocol 
will take place without written approval by an adequately constituted Institutional Review 
Board/Ethics Committee and Regulatory Authorities, except where necessary to eliminate an 
immediate hazard(s) to the study patients. All personnel involved in the conduct of this study have 
completed ICH GCP Training and have appropriate knowledge about other applicable regulation, 
including Human Subject Protection and GDPR. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the Ethics Committee and/or Regulatory Authorities for review and approval. Approval 
of both the protocol and the consent form must be obtained before any participant is enrolled. Any 
amendment to the protocol will require review and approval by the Ethics Committee and/or 
Regulatory Authorities before the changes are implemented to the study.  All changes to the consent 
form will be Ethics Committee and/or Regulatory Authorities approved; a determination will be made 
regarding whether a new consent needs to be obtained from participants who provided consent, using 
a previously approved consent form. 
 
CONFIDENTIALITY STATEMENT 
 
This Clinical Study Protocol (CSP) contains information that is confidential and proprietary to Isofol 
Medical AB (publ) (Sponsor). This information is being provided to you for the purpose of conducting 
a clinical study sponsored by Isofol. You may disclose the contents of this CSP to study personnel under 
your supervision who need to know the contents for this purpose, as well as to your Independent 
Ethics Committee/Institutional Review Board (IEC/IRB). The contents of this CSP may not be disclosed 
to any other person or entity without the prior written permission of Isofol and may not be used for 
any other purpose than the conduct of this study. The foregoing shall not apply to disclosure required 
by governmental regulations or laws; however, you will give prompt notice to Isofol of any such 
disclosure. 
 
Any supplemental information that may be added to this document is also confidential and proprietary 
to Isofol and must be kept in confidence in the same manner as the contents of this CSP. 
 
Any person who receives this CSP without due authorization from Isofol is requested to return it to 
Isofol or to promptly destroy it. 
 
 
 


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SIGNATURE PAGE PROTOCOL APPROVAL 
 
The signatures below constitute the approval of this protocol and the appendices, and provides the 
necessary assurances that this protocol has been designed and the study will be conducted according 
to all stipulations of GCP and according to all applicable legal and regulatory requirements. 
 
 
Sponsor’s Representative: 
 
 
 
 
Roger Tell, MD, PhD 
Date: 
Chief Medical Officer 
 
Isofol Medical AB (publ) 
 
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SIGNATURE PAGE STUDY COORDINATORS 
 
I have read and understood this study protocol, and I agree to conduct the study according to the 
study protocol, GCP and applicable local regulation. 
 
Biostatistician: 
 
 
 
 
Koenraad D`Hollander, MSc  
Date:  
IDDI 
Louvain-la-Neuve, Belgium 
 
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Global Coordinating Investigator: 
 
 
 
 
Josep Tabernero, MD, PhD, MSc  
Date:  
Vall D’Hebron Institute of Oncology 
Barcelona, Spain 
 
 
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US Coordinating Investigator: 
 
 
 
 
Heinz-Josef Lenz, MD, FACP 
Date: 
University of Southern California  
Norris Comprehensive Cancer Center 
Los Angeles, USA 
 
 
 
 


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SIGNATURE PAGE INVESTIGATOR 
 
I have read and understood this study protocol, and I agree to conduct the study according to the 
study protocol, GCP and applicable local regulation. 
 
Site investigator signature 
 
Principal Investigator (Sign): 
 
 
Name (Block letters): 
Date: 
 
Site: 
 
 
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1 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS  
 
 
 
Study title 
A randomized, multi-center, parallel-group, Phase III study to compare the efficacy 
of arfolitixorin versus leucovorin in combination with 5-fluorouracil, oxaliplatin, 
and bevacizumab in patients with advanced colorectal cancer. 
 
Global 
Coordinating 
Investigator 
Josep Tabernero, MD, PhD, MSc 
Vall D’Hebron Institute of Oncology 
Barcelona, Spain 
 
US Coordinating 
Investigator 
Heinz-Josef Lenz, MD, PhD 
Division of Medical Oncology, University of Southern California 
Norris Comprehensive Cancer Center 
Los Angeles, CA, USA 
 
Study Duration 
Recruitment period: 
Q4 2018 – Q2 2021 
Study period: 
 
Q4 2018 – Q1 2023 
Participant 
duration 
All patients will be treated until progressive disease (PD), or toxicity, and followed 
for survival until end of study. 
 
Short Summary 
This is a multicenter, randomized, parallel-group, Phase III study in at least 440 
patients with advanced colorectal cancer to compare the efficacy of treatment 
with arfolitixorin versus Leucovorin in combination with 5-fluorouracil, oxaliplatin, 
and bevacizumab according to modified FOLFOX-6 until PD according to RECIST 
1.1 criteria.  
Primary 
Endpoint 
Overall Response Rate (ORR) 
Background 
5-fluorouracil (5-FU) in combination with the folate Leucovorin (LV) has been the 
cornerstone in the treatment of colorectal cancer (CRC) for decades.  All folates 
currently approved for use in the clinical setting need to be metabolically activated 
to [6R] 5,10-methylenetetrahydrofolic acid ([6R]-MTHF), which is the active 
thymidylate synthase co-substrate that potentiates the effect of 5-FU.  
Arfolitixorin does not need multi step metabolic activation like currently available 
folates. It is therefore hypothesized that the administration of arfolitixorin will 
result in higher, and more consistent, intracellular concentrations of the active 
thymidylate synthase co-substrate [6R]-MTHF in all patients compared to LV 
administration.   
Indication 
First line advanced CRC patients eligible for treatment with 5-FU, oxaliplatin, and 
bevacizumab. 
 
 


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Objectives and 
endpoints 
 
Objective: 
Endpoint/Outcome Measure: 
Primary 
Overall response rate (ORR) 
Best ORR, defined as the best response 
recorded from the start of the study 
treatment until the end of treatment. All 
responses will be confirmed 8 weeks 
after onset of response. All assessments 
including confirmation of response will 
be performed by BICR.     
Key Secondary 
Progression-free survival (PFS) 
PFS, defined as the time from 
randomization to first occurrence of 
tumor progression assessed by BICR 
(RECIST 1.1) based on CT-scans/MRIs 
conducted every 8 weeks after start of 
treatment, or death from any cause.  
Duration of Response (DOR) 
The duration of overall response (DOR) 
is measured from the first time point at 
which criteria are met for complete 
response (CR) or partial response (PR) 
through the last time point when overall 
response has been objectively 
documented.  
  
Other Secondary (efficacy) 
Overall survival (OS) 
OS, defined as time from randomization 
to death from any cause 
Quality of Life 
Quality of Life assessed using the EQ-5D 
patient reported outcome questionnaire 
(PRO). 
Other Secondary (safety) 
Safety and tolerability 
Number and severity of adverse events 
(AEs), including clinically significant 
abnormal laboratory findings, regardless 
of causal relationship to arfolitixorin or 
LV. Specific AEs will be followed using 
PRO (NCI PRO-CTCAE). 
Patients 
undergoing 
curative 
metastasis resection. 
Defined as the number of patients 
qualifying for curative metastasis 
resection after treatment with study 
drug. 
Exploratory 
Daily living abilities 
Daily living abilities as assessed by the 
Eastern Cooperative Oncology Group 
(ECOG) Performance Status Scale 


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To determine the pharmacokinetic 
(PK) characteristics of arfolitixorin and 
LV in patients with advanced CRC 
The plasma concentration of methylene 
tetrahydrofolate ([6R]-MTHF), methyl-
tetrahydrofolate (methyl-THF), and 
tetrahydrofolate (THF) will be 
determined in a limited number of 
patients in both treatment arms. 
To investigate folate metabolism- and 
transportation-related gene 
expression levels in patients with 
advanced CRC 
Gene expression levels of folate 
metabolism- and transportation-related 
genes, analyzed by sub-group low/high 
gene expression levels. 
 
Recurrence Free Survival (RFS) for 
patients undergoing metastatic 
resection. 
Defined as the time between the first 
surgery with complete removal of the 
metastasis and recurrence of the 
disease or death from any cause. 


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1  
Female patients must be post-menopausal for more than one year or must provide a negative urine 
pregnancy test and use an efficient method of contraception (i.e., a method with less than 1% failure rate [e.g., 
sterilization, hormone implants, hormone injections, some intrauterine devices, or vasectomized partner]) 
during the study and for 1 month (or more if requested by non-IMP labels) after the end of the study (last dose 
of IMP). 
2  
Unless the partner of a male patient is post-menopausal or using an efficient method of contraception 
as described above, male patients must agree to use condoms during the study treatment and for 1 month 
after the end of the study treatment. 
 
Main Selection 
Criteria 
 
 
Inclusion criteria  
For randomization in the study, patients must fulfill all the following criteria: 
1. Colorectal adenocarcinoma verified by biopsy. 
2. Availability of biopsy material, from the primary tumor or metastasis, allowing 
for analysis of tumor gene expression. 
3. Non-resectable metastatic CRC planned for first line therapy with 5-FU, 
Leucovorin, oxaliplatin, and bevacizumab.   
4. Evaluable disease with at least one measurable lesion of metastatic disease 
(≥10 mm in longest diameter on axial image on CT-scan or alternatively MRI 
with <5 mm reconstruction interval) or lymph node (≥ 15 mm in shortest axis 
when assessed by CT) obtained within 28 days of randomization. 
5. Life expectancy of more than 4 months. 
6. ECOG performance status 0 or 1. 
7. Hemoglobin (Hb) > 80 g/L, Absolute neutrophil count (ANC) > 1.5x109/L. 
Thrombocytes > 100x109/L.  
8. Creatinine clearance > 50 mL/min, Total bilirubin < 1.5 x ULN, AST and ALT < 3 
x ULN (and < 5 x ULN in case of liver metastases). 
9. Male or female ≥18 years of age. 
10. Female patients of childbearing potential must have a negative urine 
pregnancy test and use adequate contraceptive measures1.  Male patients 
must use adequate contraceptive measures2. 
11. Voluntarily signed informed consent before performance of any study related 
procedure not part of normal medical care, with the understanding that 
consent may be withdrawn by the patient at any time without prejudice to 
future medical care. 
 
Exclusion Criteria 
Patients meeting one or more of the following criteria are ineligible to 
participate in the study:  
1. Malignant tumors other than colorectal adenocarcinomas (current or within 
the previous five years), with the exception for curatively treated non-
melanoma skin cancer or in situ carcinoma of the cervix. 


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2. Less than 6 months between randomization and completion of the last anti-
cancer treatment (chemotherapy/radiotherapy/immunotherapy, etc.). (NB: 
Rectal cancer treatment shorter than 8 weeks of chemo/radiation therapy is 
allowed.) 
3. Confirmation of progressive disease within 6 months after completion of prior 
adjuvant anti-cancer treatment.  
4. Indication for any metastatic Colo-rectal Cancer (mCRC) surgery or anti-cancer 
treatment other than study treatment. 
5. Prior treatment with arfolitixorin. 
6. Indication for treatment with a 5-FU analogue, or 5-FU for a condition other 
than mCRC. 
7. Known Dihydropyrimidine Dehydrogenase Deficiency (DPD) deficiency. 
8. Known or suspected central nervous system (CNS) metastases. 
9. Unresolved bowel obstruction, uncontrolled Crohn’s disease, or ulcerative 
colitis. 
10. History of cardiac disease with a New York Heart Association Class II or greater, 
congestive heart failure, myocardial infarction, or unstable angina at any time 
during the 6 months prior to randomization, or serious arrhythmias requiring 
medication for treatment. 
11. Current CTCAE ≥ grade 3 diarrhea. 
12. Current chronic infection or uncontrolled serious illness causing 
immunodeficiency. 
13. Known or suspected hypersensitivity or intolerance to arfolitixorin, LV, 5-FU, 
oxaliplatin, or bevacizumab. 
14. Breastfeeding patients. 
15. Patient who received investigational drugs in other clinical trials within 28 
days, or 5 half-lives of the investigational drug, prior to randomization. 
16. Patient with serious medical or psychiatric illness likely to interfere with 
participation in this clinical study. 
17. Ongoing drug or alcohol abuse, as deemed by the Investigator. 
18. Any condition that, in the opinion of the Investigator, could compromise the 
patient's safety or adherence to the study protocol. 
19. Involvement, or related to people involved in the planning or conduct of the 
study (applies to both Isofol Medical AB (publ) staff and staff at the study site) 
20. Surgery (excluding previous diagnostic biopsy) in the 28-day period before 
randomization 


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Interventions 
Investigational Medicinal Product (IMP) 
• 
Study drug: Arfolitixorin ([6R] 5,10-methylenetetrahydrofolic acid) is 
formulated as a lyophilized white to light yellowish or light beige powder 
containing 100 mg arfolitixorin per vial.  Each vial of arfolitixorin must be 
reconstituted with 10 mL water for injection before administration as 
rapid IV bolus injections. 
• 
Comparator: Leucovorin (LV; folinic acid) 400 mg/m2 IV infusion.   
Non-investigational medicinal products 
• 
5-FU, oxaliplatin, and bevacizumab. 
Treatment regimens 
Eligible and consenting patients will be randomized 1:1 to one of two treatment 
groups (A and B), administered according to the treatment regimens described 
below. Each new treatment cycle should start on day 14 (+7 days) after the 
previous treatment cycle (unless delayed due to toxicity): 
Group A (ARFOX + bevacizumab): 
• 
Bevacizumab 5 mg/kg, IV infusion in accordance with the label   
↓ followed by 
• 
Oxaliplatin 85 mg/m2 IV infusion in accordance with the label   
↓ followed by 
• 
5-FU 400 mg/m2 IV bolus (2–4 minutes) 
↓ followed by (30 minutes (± 5 minutes) after 5-FU bolus) 
• 
Arfolitixorin 60 mg/m2 rapid IV bolus (less than 3 minutes) 
↓ followed by 
• 
5-FU 2400 mg/m2 continuous IV infusion over 46 hours 
↓ followed by (30-60 minutes after first arfolitixorin bolus) 
• 
Arfolitixorin 60 mg/m2 rapid IV bolus (less than 3 minutes) 
 
Group B (modified FOLFOX-6 + bevacizumab): 
• 
Bevacizumab 5 mg/kg, IV infusion in accordance with the label   
↓ followed by 
• 
Oxaliplatin 85 mg/m2 IV infusion in accordance with the label 
↓ followed by* 
• 
LV 400 mg/m2 IV infusion  
↓ followed by 
• 
5-FU 400 mg/m2 IV bolus (2–4 minutes) 
↓ followed by  
• 
5-FU 2400 mg/m2 continuous IV infusion over 46 hours 
*Oxaliplatin and LV may be administered at the same time through two different 
injection ports. NB: Oxaliplatin and LV must not be mixed. 
The doses of arfolitixorin and LV should not be adjusted. 5-FU and LV should be 
administered separately to avoid the formation of a precipitate. Following a 
treatment-related adverse event the doses of 5-FU, oxaliplatin, and bevacizumab 


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may be adjusted or delayed in accordance with the label and section 6.3.4 in the 
protocol. Oxaliplatin cannot be substituted but caution should be taken regarding 
toxicity and in case of toxicity, dose modifications (decrease in infusion rate and/or 
dose reductions) according to label should be performed. For patients with 
platinum 
hypersensitivity, 
pre-medication 
with 
IV 
dexamethasone, 
chlorphenamine, and ranitidine can be given at the Investigator’s discretion. 
Prohibited concomitant therapies 
Treatment with nucleoside analogues, known DPD inhibitors, additional anti-
cancer treatment, additional experimental medications, and radiation therapy are 
prohibited during the screening period or in conjunction with the IMP. 
Study visits 
Study visits include a screening visit, a randomization/baseline visit, regular 
treatment visits every 2 weeks, and CT/MRI assessments every 8 weeks.  
Treatment visits will continue until progressive disease (PD) as assessed by the 
Investigator. The assessment visits will continue until centrally confirmed PD 
according to RECIST 1.1 criteria. An end of treatment visit will occur following PD 
or premature discontinuation of IMP. Follow-up (visit or phone call) for OS occurs 
every 12 weeks thereafter until end of study.     
Study design 
This is a randomized, multicenter, parallel-group, Phase III study to compare the 
efficacy of arfolitixorin versus LV in patients with advanced CRC treated by 5-FU, 
oxaliplatin, and bevacizumab. 
Patients will be randomized in a 1:1 ratio to either the investigational arm 
(arfolitixorin) or the comparator arm (Leucovorin), following the completion of all 
screening assessments and after confirmation of their eligibility, using a stratified 
permuted block randomization. Randomization will be stratified for the following 
baseline factors:  
• 
Geographic region (Europe versus North America versus Australia versus 
Japan) 
• 
Primary tumor location (left colon versus right colon versus rectal cancer) 
• 
Previous neo-adjuvant/adjuvant CRC treatment (yes versus no) 
 
Tumor evaluations, based on CT/MRI, will be performed at baseline and every 8 
weeks until PD by a blinded independent central review committee (according to 
RECIST 1.1 definitions).  All patients will be treated according to the study protocol 
until PD as determined by the Investigator, unless prematurely discontinued from 
treatment for any reason. A BICR review will be performed to assess all tumor 
responses and progression in accordance with RECIST 1.1 definitions. BICR 
committee members will be blinded to all treatment data and perform their 
reviews in closed-meeting sessions. All activities and processes related to the BICR 
will be outlined in the BICR Charter. 
Final analysis of ORR and PFS will be done when at least 235 PFS events are 
reached. An adaptive design allows for a re-estimation of the sample size based on 
interim results. If the sample size is increased to 660 patients, the final analysis of 
ORR and PFS will be done when approximately 450 PFS events have been observed. 
All patients will be followed for OS after end of treatment (regardless whether due 
to discontinuation/withdrawal or PD) until 60% OS events have been reached.    
 


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Premature 
discontinuation 
of treatment 
Patients may withdraw consent to participation in the study or voluntarily 
discontinue treatment with IMP at any time without providing a reason and 
without any prejudice to future treatment.  The Investigator may also, at his/her 
discretion, discontinue IMP at any time if it is in the best interest of the patients’ 
well-being.  The Sponsor can instruct the Investigator to discontinue IMP (e.g. due 
to severe non-compliance with the protocol). Patients discontinuing treatment 
will be asked to continue with follow-up assessments, including ongoing CT/MRI 
assessments until centrally confirmed PD or end of study. 
Data and Safety 
Monitoring 
Board (DSMB) 
An independent DSMB will be established and regularly monitor the safety profile 
and study progress to ensure that the study is being conducted with the highest 
safety, scientific and ethical standards.  The DSMB Charter specifies the DSMB 
procedures and constitution, statistically derived interim analysis decision 
guidelines, as well as time points for DSMB evaluations. Recommendations from 
the DSMB to the Sponsor will comprise, in addition, increasing sample size, as well 
as early termination of the study or any other relevant recommendations. 
An interim analysis will be performed when 16-week BICR evaluation has been 
performed for the 330th patient. The objective of the interim analysis is to 
calculate the conditional power for both ORR and PFS to guide continuing the 
study as planned or increasing the sample size.  The guidelines for sample size 
increase decisions are defined in the DSMB charter and protocol section 9.4.6.  
There is no intention to claim efficacy for ORR or PFS at this stage. The interim 
results will only serve to guide the decision rules regarding sample size re-
estimation. Results of this analysis will remain blinded to everyone, except the 
DSMB. Regardless of the efficacy results of the interim analysis, the trial will 
continue to accrue patients, until the total planned sample size is reached. 
Number of 
planned 
patients & 
replacement 
Approximately 490 patients will be randomized 1:1 to arfolitixorin or LV at 
approximately 80-100 study sites in Europe, Canada, the United States, Australia 
and Japan. Patients randomized but prematurely discontinuing before receiving 
any study treatment will be replaced. 
The sample size calculation is based on the following assumptions: 
• 
The ORR is 60% in Group A 
• 
The ORR is 45% in Group B 
For a two-sided test with 𝛼𝛼=0.05 and a power of 80%, 440 patients need to be 
treated to detect this difference, and also allow for an interim analysis on 330 
patients. Adjustment of the significance level in the interim analysis and final 
analysis has been performed, using a Rho spending function, with ρ=5 for the 
primary endpoint (ORR) at the interim analysis, so that the overall 2-sided 
significance level does not exceed the desired 𝛼𝛼 of 0.05. Although significance will 
not be claimed based on the interim results, this small alpha spending is foreseen 
to take into account the interim look at the results for ORR and PFS. 
Initially, approximately 440 were planned to be randomized. However, Japanese 
authorities requested that approximately 12.7% of the total study population are 
enrolled from Japan. For this reason, additional patients from Japan were 
randomized. In the end a total of 490 patients were randomized. This decision to 
randomize extra Japanese patients was made before the interim analysis. The 
decision to include the Japanese patients in the ITT population (see Section 9.2.2) 
for the primary efficacy analysis was made after the interim analysis. The addition 
of extra Japanese patients leads to a statistical power greater than 80% for ORR. 


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Statistical 
Methods 
 
A statistical analysis plan will be in place before including patients in the study.  
The analysis will be stratified for the following factors: 
• 
Geographical region (Europe versus North America versus Australia versus 
Japan) 
• 
Primary tumor location (left colon versus right colon versus rectal cancer) 
• 
Previous neo-adjuvant/adjuvant CRC treatment (yes versus no) 
All efficacy analyses will be performed on both the ITT and the per-protocol 
analysis sets. The ITT analysis will be the primary basis for interpreting study 
results. 
ORR will be analyzed using a Cochran-Mantel-Haenszel test (CMH), stratified for 
the stratification factors used for randomization (geographic region, primary 
tumor location and previous neo-adjuvant/adjuvant CRC treatment). 
PFS, DOR and OS will be analyzed using Kaplan-Meier curves, the logrank test and 
a stratified Cox proportional hazards model, using the same stratification factors 
as for randomization (geographic region, primary tumor location and previous 
neo-adjuvant/adjuvant CRC treatment). The assumption of proportional hazards 
will be tested. For AEs absolute and relative frequencies will be presented in total 
and by system organ class/preferred term, intensity, and relatedness. 


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1.2 
SCHEMA 
 
 
 
1:1 Randomization 
  
Group A 
arfolitixorin, 5-FU, 
oxaliplatin + 
bevacizumab 
 
Group B 
Leucovorin, 5-FU, 
oxaliplatin + 
bevacizumab 
 
Interim analysis 
when 330 patients 
are evaluable for 
response after at 
least 16 weeks of 
treatment, 
analyzing both ORR 
and PFS. 
  
Depending on the 
conditional powers, 
the trial will either 
continue as 
planned, or the 
sample size will be 
increased for a 
total of 660 
patients. 
Final analysis of 
ORR and PFS to 
be performed 
when at least 
235 PFS events 
have been 
observed.  
 
If the sample size 
is increased to 
660 patients the 
analysis will be 
performed when 
450 PFS events 
have been 
observed.  
Follow-up for survival 
every 12 weeks until 
60% of patients have 
reached OS. 
Imaging 
evaluations 
done at baseline 
and every 8 
weeks 
throughout the 
study. 
 
Confirmatory 
scans performed 
8 weeks after 
response. 


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1.3 
SCHEDULE OF ACTIVITIES (SOA) 
 
Table 1: Schedule of Activities (SOA) 
 
 
PRE-TREATMENT  
TREATMENT PERIOD  
END OF TREATMENT 
FOLLOW-UP 
Screening 
 
Administer IMP 
Assessment 
visit 
Administer IMP 
Assessment 
visit 
End of treatment 
visit 
Follow-up visits 
until centrally 
confirmed PD 
Survival follow-up 
 
Cycle 1 
 
Cycles 2-4 
 
Cycles 5 - PD or 
discontinuation 
 
 
Only patients 
prematurely 
discontinuing IMP 
prior to centrally 
confirmed PD 
All patients 
 
Timing 
 
Day 1 
Baseline 
14 days 
after 
previous 
admin 
visit 
Week 8 from 
day 1 
14 days after 
previous admin 
visit 
Weeks 16, 
24, etc. from 
day 1 
Within 30 days after 
last dose of IMP and 
before start of new 
anti-cancer 
treatment 
Every 8 weeks 
from end of 
treatment visit 
until centrally 
confirmed PD 
Every 12 weeks 
after centrally 
confirmed PD 
until death or end 
of study 
CSP 
Section 
Visit window 
Day -28 to  
Day -1 
-3 daysa,f 
+7 days 
±7 days  
+7 days 
±7 days  
 
±7 days  
±7 days  
Screening procedures 
10.1.1 
Informed consent 
X 
 
 
 
 
 
 
 
 
8.3 
Patient Study ID 
X 
 
 
 
 
 
 
 
 
5.1/5.2 
Eligibility criteria 
X 
X 
 
 
 
 
 
 
 
8.3 
Demographics/medical history 
X 
 
 
 
 
 
 
 
 
6.4 
Randomization 
 
Xa 
 
 
 
 
 
 
 
General procedures 
6.6 
Concomitant therapy 
X 
X 
X 
X 
X 
X 
X 
 
 
8.2 
Physical examination 
X 
X 
 
X 
 
X 
X 
 
 
8.2 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
 
 
8.2 
Height  
X 
 
 
 
 
 
 
 
 
8.2 
Weighte 
X 
X 
X 
X 
X 
X 
X 
 
 
8.2 
Biopsy available for Pharmacogenetics 
X 
 
 
 
 
 
 
 
 
6.1/6.2 
Administer and record study treatment 
 
X 
X 
 
X 
 
 
 
 
6.6 
Record other anti-cancer treatment 
 
 
 
 
 
 
X 
X 
X 
8.1 
ECOG 
X 
X 
 
X 
 
X 
X 
X 
 


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PRE-TREATMENT  
TREATMENT PERIOD  
END OF TREATMENT 
FOLLOW-UP 
Screening 
 
Administer IMP 
Assessment 
visit 
Administer IMP 
Assessment 
visit 
End of treatment 
visit 
Follow-up visits 
until centrally 
confirmed PD 
Survival follow-up 
 
Cycle 1 
 
Cycles 2-4 
 
Cycles 5 - PD or 
discontinuation 
 
 
Only patients 
prematurely 
discontinuing IMP 
prior to centrally 
confirmed PD 
All patients 
 
Timing 
 
Day 1 
Baseline 
14 days 
after 
previous 
admin 
visit 
Week 8 from 
day 1 
14 days after 
previous admin 
visit 
Weeks 16, 
24, etc. from 
day 1 
Within 30 days after 
last dose of IMP and 
before start of new 
anti-cancer 
treatment 
Every 8 weeks 
from end of 
treatment visit 
until centrally 
confirmed PD 
Every 12 weeks 
after centrally 
confirmed PD 
until death or end 
of study 
CSP 
Section 
Visit window 
Day -28 to  
Day -1 
-3 daysa,f 
+7 days 
±7 days  
+7 days 
±7 days  
 
±7 days  
±7 days  
Efficacy assessments 
8.1 
Plasma PK sample 
 
Xb 
Xb 
 
 
 
 
 
 
8.1 
CT-scan/MRIc 
X 
 
 
X 
 
X 
X 
X 
 
8.1 
PRO EQ-5D 
 
X 
 
X 
 
X 
X 
 
 
8.1 
Survival Status 
 
 
 
 
 
 
 
 
Xg 
Safety assessments 
8.4 
AEs  
 
X 
X 
X  
X 
X 
Xd 
Xd 
 
8.2 
PRO CTCAE 
 
X 
X 
X 
 
X 
X 
 
 
8.2 
Hematology and clinical chemistrye 
Xf 
 
X 
 
X 
 
X 
 
 
8.2 
Urinalysise 
Xf 
 
X 
 
X 
 
X 
 
 
8.2 
ECGh 
 
X 
X 
 
 
 
 
 
 
8.2 
Pregnancy test 
Xf 
 
 
 
 
 
X 
 
 
a 
Randomisation can occur on the same day as study treatment initiation, or up to 3 days before study treatment initiation.  Study treatment must be initiated within 28 days from start of screening. 
b 
Plasma PK samples will be collected during Cycle 1 and Cycle 2. 
c 
CT-scans/MRIs must be performed on thorax, abdomen and pelvis. Post-randomisation CT-scans/MRIs should be performed every 8 weeks (±7 days) from Day 1 to PD confirmed by BICR. End of Treatment visit scans will be taken if the 
previous scan is >14 days. 
d 
AEs are to be recorded until the day of the End of Treatment Visit. After the End of Treatment Visit, only SAEs related to study specific procedures or considered to be at least possibly related to study drug should be recorded.  
e 
Laboratory, urine samples and weight are to be collected according to local practice (7 days before study treatment).  The laboratory and urinalysis reports must be obtained and reviewed prior to administration of chemotherapy for each 
cycle.   
f 
Haematology, clinical chemistry, urinalysis, and pregnancy testing must be performed within 7 days prior to study treatment initiation and must be assessed before randomization 
g 
Survival follow-up visit could be conducted either by phone. ordinary visits, hospital records or other means found suitable. 
h 
12-lead ECG should be performed during administration visits 1-4.  Directly prior to and 10 min (±5 min) after each arfolitixorin dose, and directly prior to and 10 min (±5 min) after initiation of Leucovorin infusion, during administration 
visits 1-4.  


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2 
 
INTRODUCTION 
 
2.1 
STUDY RATIONALE  
 
2.1.1 ADVANCED COLORECTAL CANCER 
Colorectal cancer (CRC) is the third most common cancer in men (10% of the total) and the second in 
women (9.2%), with over 1.3 million cases (746 000 men and 614 000 women) reported worldwide 
during 2012. The geographic incidence of CRC varies widely across the world, and the geographical 
patterns are very similar in men and women. Incidence rates vary ten-fold in both sexes worldwide, 
the highest estimated rates being in Australia/New Zealand (the age standardized rate (ASR) 44.8 and 
32.2 per 100 000 in men and women respectively), and the lowest in Western Africa (4.5 and 3.8 per 
100 000). The incidence increases with age and is highest amongst the elderly, i.e., 60-64 years: 67.4; 
65-69 years: 95.1; 70-74 years: 127.8; and ≥ 75 years: 196.2 per 100 000 [1]. 
Approximately 40-50% of the affected patients develop metastatic disease and more than half a 
million deaths are reported annually as a consequence of CRC [2]. CRC accounted for 694 000 deaths 
worldwide solely during 2012 (8.5% of total cancer deaths) [1]. 
 
2.1.2 CURRENT STANDARD OF CARE 
CRC patients are usually treated surgically and, in most circumstances, with curative intent. Surgery, 
in fact, remains the primary modality of treatment for malignancies of the lower gastrointestinal tract, 
and standard resection is the only therapy required for early-stage cancer [3].  
As the stage of the tumor advances, in terms of depth of penetration and lymph node involvement, 
the chance of cure with surgery alone diminishes and the rate of local recurrence increase. In such 
cases, surgery may either be combined with adjuvant treatment or be performed for palliative control 
of symptoms only. 
Standard first-line systemic therapy for metastatic colorectal cancer (mCRC), in accordance with 
current European Society for Medical Oncology (ESMO) guidelines, is chemotherapy with 5-
fluorouracil (5-FU) + Leucovorin (LV) + oxaliplatin/irinotecan ± bevacizumab [4].  
2.1.2.1 CYTOTOXIC AGENTS 
The backbone of ﬁrst-line palliative chemotherapy alone, as well in combination with targeted agents, 
consists of a ﬂuoropyrimidine (FP; intravenous [IV] 5-FU or the oral FP capecitabine) in various 
combinations and schedules [5, 6].  
LV is a part of a standard treatment regimen for advanced CRC in combination with the anti-metabolite 
5-fluorouracil (5-FU) as it greatly improves the efficacy of 5-FU [7, 8]. Arfolitixorin is an endogenous 
folate-based compound designed to replace LV  
The oral FP capecitabine is an alternative to IV 5-FU/LV [7, 8]. Combination chemotherapy with 5-
FU/LV/oxaliplatin (FOLFOX) or 5-FU/LV/irinotecan (FOLFIRI) provides higher response rates (RRs), 
longer PFS and better survival than 5FU/LV alone [5, 6, 9, 10]. FOLFOX and FOLFIRI as chemotherapy 
alone have similar activity and are both partners for biologics but have a different toxicity proﬁle: 


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more alopecia and, in most trials, more severe diarrhea for irinotecan and more polyneuropathy for 
oxaliplatin [5, 11]. They also have potentially different interactions with biologicals. 
2.1.2.2 BIOLOGIC AGENTS 
Bevacizumab, an antibody that binds circulating VEGF-A, increases the activity of any active cytotoxic 
regimen. Bevacizumab has been shown to improve RR, PFS and/or OS when used as first-line 
treatment in combination with 5-FU/LV/irinotecan and in combination with 5-FU/LV or capecitabine 
alone [12, 13, 14, 15]. Bevacizumab has also been shown to improve the PFS but not RR or OS in 
combination with an FP plus oxaliplatin in the ﬁrst-line treatment of mCRC [16, 17]. Bevacizumab is 
usually continued in combination with a cytotoxic agent/combination until PD or toxicity. 
2.1.3 MECHANISM OF ACTION OF ARFOLITIXORIN 
The Active ingredient of the Drug substance is arfolitixorin or [6R]-5,10-methylene-tetrahydrofolic acid 
([6R]-5,10-MTHF). It is present as such in the reconstituted solution from the Drug product 
lyophilizate. The principal mechanism of action is ultimately the same for arfolitixorin as for LV or L-
LV [18], used together with 5-FU as [6R]-5,10-MTHF is the key endogenous active metabolite of these 
drugs, and is a necessary co-substrate in the synthesis of deoxy-thymidine monophosphate (dTMP) 
from deoxy-uridine monophosphate (dUMP), a reaction catalyzed by the enzyme thymidylate 
synthase (TS). dTMP is in turn a necessary substrate for DNA synthesis and repair and is thus essential 
for cell division and life [19]. Indeed, the antitumor agent 5-FU is known to exert its cytotoxic activity, 
to a major extent, through inhibition of the TS enzyme [18]. 
For either synthesis of dTMP from dUMP via TS catalysis, or inhibition of TS by 5-FdUMP (a metabolite 
of 5-FU), a ternary complex is formed between TS, [6R]-5,10-MTHF, and the respective third 
component, either dUMP or 5-FdUMP.  In the (normal) synthesis situation dUMP picks up the 
methylene (the 1-carbon unit) from [6R]-5,10-MTHF while bound in the ternary complex to form 
dTMP. dTMP is then released during complete decomposition of the ternary complex. The 
transformation of dUMP into dTMP occurs simultaneously with a 5-H abstraction from the dUMP 
molecule. If instead 5-FdUMP has become bound into the ternary complex, the 5-F atom cannot be 
abstracted. The ternary complex instead remains stable and the TS enzyme is inhibited. This is because 
the enzyme TS cannot be released from the 5F-containing ternary complex for a renewed catalytic 
reaction [18].  
The relatively stable FdUMP- containing “inhibitory” ternary complex can, however, release its [6R]-
5,10-MTHF moiety and initiate complete decomposition of this ternary complex. Since the release 
step is an equilibrium reaction, increased concentration of [6R]-5,10-MTHF will stabilize the 
(“inhibitory”) ternary complex and thereby strengthen the TS inhibition. This means that the higher 
the concentration of [6R]-5,10-MTHF, the higher the stability of the FdUMP-containing “inhibitory” 
ternary complex.   
In vivo tetrahydrofolic acid (THF) and 5-methyl-THF are rapidly formed after administration of both 
arfolitixorin and LV/L-LV and these two metabolites together constitute a “metabolite pool”, which 
can re-generate [6R]-5,10-MTHF [18]. Plasma concentration data from a PK/PD study, showing very 
high (transient) plasma levels of [6R]-5,10-MTHF immediately after the administration of arfolitixorin 
and hardly any after L-LV [20].  These much higher levels during the first 30 minutes after arfolitixorin 
may be particularly relevant for efficacy. Our selected dose regime for arfolitixorin, involving two bolus 
doses separated by 30 minutes, is thought to be supported by the PK/PD situation. 
Arfolitixorin, [6R]-5,10-MTHF, circumvents the genetically influenced multistep metabolic activation 
necessary for biological activity of LV/L-LV and consequently, is thought to be efficacious in a larger 


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proportion of patients and with significantly less inter- and possibly intra-individual variability [18]. 
This together with all the direct PK/PD advantages is thought to serve to make arfolitixorin a better 
candidate for improved outcome of 5-FU-based chemotherapy regimens in advanced CRC provided 
that an acceptable benefit/risk ratio can be maintained.  
 
 
 
 
2.2 
BACKGROUND  
 
2.2.1 NON-CLINICAL BACKGROUND 
The nonclinical data are described in the current version of arfolitixorin Investigator’s brochure. 
Arfolitixorin has been evaluated in preclinical GLP (Good Laboratory Practice) rat and dog studies. In 
summary, twice daily IV administration of arfolitixorin for up to 28 days is well tolerated in rats at 
doses up to 100 mg/kg/day and in beagle dogs at doses up to 50 mg/kg/day. 
The Active Pharmaceutical Ingredient (API) of arfolitixorin has also been evaluated in various non-GLP 
mouse models in combination with different anti-metabolites such as pemetrexed and 5-FU. No 
indications of safety issues related to arfolitixorin have been observed in these studies. 
A 13-week (3 months) toxicology program in rats and beagle dogs concluded that the twice daily 
intravenous administrations of arfolitixorin to Sprague-Dawley rats at doses of up to 300 mg/kg (150 
mg/kg b.i.d) for 13 weeks was well tolerated, causing non-adverse histopathological changes at the 
site of administration and the spleen (extramedullary hematopoiesis) The majority of findings showed 
clear evidence of recovery during the two-week off-dose period, and those without recovery were all 
considered non-adverse. Therefore, the highest dose of 300 mg/kg (150 mg/kg b.i.d) was considered 
to be the no-observed-adverse-effect level (NOAEL).   
In Beagle dogs for a 13 weeks administration in cycles of 5 out of 7 days during 3 weeks on and 1 week 
off was well tolerated and resulted in findings in the kidneys of females, and a non-toxicologically 
relevant higher ALT and AST activity and an increase in urea and creatinine level, at 375 mg/kg/day. 
All renal changes had resolved after a 2-week recovery period. Therefore, the dose of 100 mg/kg/day 
arfolitixorin was considered the NOAEL (No Observed Adverse Effect Level). 
2.2.2 CLINICAL BACKGROUND 
The complete clinical data are described in the current version of the arfolitixorin Investigator’s 
brochure. 
One phase I/II clinical trial with arfolitixorin in patients diagnosed with operable CRC was performed 
with the aim of comparing PK/PD profiles in plasma, tumors, and tumor adjacent mucosa. In this 
clinical trial (ISO-CC-002), 60 or 200 mg/m2 of arfolitixorin or L-LV was administered as single doses 
prior to surgery. Thirty-two patients were included in this trial, of whom 16 were exposed to 
arfolitixorin. The report from Clinical Trial ISO-CC-002 has been finalized and the results have been 
published [21]. The study explored four folate metabolites: [6R]-MTHF, [6S]-5-methyl-
tetrahydrofolate (5-methyl-THF), and [6S]-5-THF (THF). The distribution/PK results from the final 
report show that arfolitixorin administration resulted in higher levels of [6R]-MTHF and THF in tumors 
and the surrounding mucosa than did L-LV. Arfolitixorin to L-LV ratios in the tissues were 2.5-4.0 for 


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[6R]-MTHF and 2.4-5.6 for THF. Arfolitixorin and L-LV administration resulted in [6S]-5-methyl-THF 
levels of the same magnitude in tumors and the surrounding mucosa. Arfolitixorin and L-LV gave 
similar concentrations of [6S]-5-methyl-THF in plasma.  
In another phase I/II clinical trial in 24 patients with operable rectal cancer (ISO-MC-091) arfolitixorin 
was administered at a dose of 10, 50, 100, or 500 mg/m2 once every week for 10 weeks in combination 
with Pemetrexed in a neo-adjuvant setting. The primary objective was to evaluate the feasibility of 
arfolitixorin and Pemetrexed as neo-adjuvant treatment and to identify the optimal dose of 
arfolitixorin. Secondary aims were to evaluate pathological complete response rate, sphincter-saving 
surgery, and levels of arfolitixorin and its metabolites in tumors, normal mucosa and plasma. The study 
was terminated prematurely after four of the five intended doses of arfolitixorin had been 
administered (10, 50, 100, and 500 mg/m2). The results obtained by then did not indicate any 
improvement in Pemetrexed efficacy with any dose level of arfolitixorin and it was concluded that no 
further information would be gained by administering the 200 mg/m2 dose. The findings on safety and 
adverse events (AEs) have been published [22]. There were no AEs in the study that could specifically 
be ascribed to arfolitixorin.  
Another clinical trial, ISO-MTX-003, with arfolitixorin as a rescue agent after high dose methotrexate 
(HDMTX) administration in osteosarcoma patients has been completed. ISO-MTX-003 was a multi-
center study designed to (i) investigate the safety of arfolitixorin rescue therapy, (ii) identify a 
recommended dose for further study, (iii) and evaluate the feasibility of arfolitixorin as rescue therapy 
for HDMTX treatment. Eight (8) patients in two dose cohorts, 15 mg/m2 and 7.5 mg/m2 were exposed 
to arfolitixorin. The study findings suggest that the arfolitixorin dose of 15 mg/m2 works as rescue 
treatment at least as well as the standard of care (with calcium folinate) of 15 mg/m2 while the lower 
arfolitixorin dose of 7.5 mg/m2 seems to involve a slightly lower recue ability in relation to standard 
of care. The dose of 15 mg/m2 was selected for further clinical development. 
Yet another trial in colorectal cancer has been initiated, the ISO-CC-005. The trial investigates the 
tolerability of arfolitixorin in patients with stage IV CRC simultaneously treated with 5-FU alone or 
together with oxaliplatin (85 mg/m2) or irinotecan (180 mg/m2) and also to define a dose for 
subsequent trials. Toxicity will be evaluated for four dose levels (30, 60, 120 and 240 mg/m2). The 
selected dose from the arfolitixorin, 5-FU and oxaliplatin treatment arm will then be evaluated for 
safety together with bevacizumab prior to study start. 105 patients in this trial have been exposed to 
arfolitixorin. 
According to IB version 14, dated 1 Oct 2021, seven hundred and thirteen (713) patients have been 
enrolled in Isofol Medical AB’s clinical development program and four hundred and twenty (420) have 
received at least one administration of arfolitixorin in doses of 7.5 to 500 mg/m2 alone or in 
combination with different cytostatic agents. The 420 patients include 388 patients with rectal/colon 
cancer, 8 patients with osteosarcoma and 24 adult healthy male subjects. A total of 226 serious 
adverse events (SAEs) have been reported for patients exposed to arfolitixorin, 25 of which were 
assessed as related to arfolitixorin.   
Consistent with the outcomes reported from previous studies, in particular ISO-CC-005, a review of 
reported events in study ISO-CC-007 to date indicates that fatigue and gastrointestinal adverse events 
(AEs), such as nausea and diarrhea, were the most commonly reported AEs among those patients 
exposed to arfolitixorin.   
The collected knowledge on arfolitixorin indicates that the drug is safe and well tolerable at all doses 
tested (7.5 to 500 mg/m2). Treatment emergent AEs are often attributable to the co-administered 
chemotherapy agents rather than to arfolitixorin itself, in fair agreement with the known safety 


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profiles for other folates. The results show that treatment with the chosen Selected phase 2 dose 
(SP2D) 120 mg/m2 arfolitixorin is safe and feasible in dosing settings similar to the well-established 
mod-FOLFOX-6 and FOLFIRI.  
Based upon existing non-clinical and clinical data to date, there are no safety concerns identified. 
2.3 
RISK/BENEFIT ASSESSMENT   
 
2.3.1 KNOWN POTENTIAL RISKS  
No toxicity directly related to arfolitixorin was observed in toxicity studies performed in rat and dog.  
In a healthy volunteer study (ISO-FF-001) to investigate QT effects, 33 subjects were randomized to 
placebo (n=9) and three doses of arfolitixorin, given as a 2-minute bolus injection in the following 
doses: 200 mg/m2 (n=8); 350 mg/m2 (n=8); or 500 mg/m2 (n=8). The study data indicate that in relation 
to baseline, arfolitixorin causes a dose-dependent increase in QTcF that is transient. For the lowest 
dose level 200 mg/m2 the effect is small and not considered to add risks or warrant increased 
surveillance for patients. 
Safety data from two initial investigations in CRC patients show no evidence of any safety concerns 
for patients after either single high-doses or multiple doses of arfolitixorin.  These clinical data indicate 
that arfolitixorin, in doses of up to 500 mg/m2are well tolerated as bolus IV administration in CRC 
patients.   
Arfolitixorin, in doses of 30, 60, 120, or 240 mg/m2, were administered to CRC patients in combination 
with 5-FU every 2 weeks for at least 8 weeks in a Phase I/II dose-finding and safety study (ISO-CC-005).  
Interim data from this study indicate that all doses are well tolerated with no indications of safety 
concerns.  
Further details from the non-clinical and clinical studies can be found in the Investigator’s Brochure 
(IB). 
2.3.2 KNOWN POTENTIAL BENEFITS  
Arfolitixorin is the active metabolite of LV and in contrast to LV does not require genetically regulated 
enzymatic activation. There is a statistically significant correlation between the level of expression of 
certain key genes and the rate of metabolic activation, as measured by tissue concentrations of [6R]-
MTHF [20]. The level of expression of folate-relevant genes correlates with outcome and survival in 
advanced CRC patients treated with 5-FU and folates. Approximately two thirds of advanced CRC 
patients fall into the category of low gene expression having a worse prognosis [23]. 
It has been demonstrated that the administration of arfolitixorin will result in greater bioavailability 
and higher intracellular concentrations of the active metabolite than does LV administration in all 
patients, as several metabolic steps are bypassed by arfolitixorin administration and varying genetic 
profiles are rendered unimportant [23]. Further it is anticipated that arfolitixorin may reduce potential 
interpatient variability and as a consequence allow for safe and more adaptable and predictable 
treatments. The hypothesis is therefore that administration of arfolitixorin compared to LV in 
combination with 5-FU will result in better efficacy. 
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  


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Arfolitixorin is expected to be more effective than LV in potentiating the effect of 5-FU, particularly in 
patients with low folate-relevant gene expression. An exploratory endpoint of this study is to compare 
the impact of certain folate transportation and metabolism related genes on treatment outcome for 
arfolitixorin and LV, to establish the magnitude of the hypothesized improvement.  
To minimize the number of patients exposed to the investigational drug, the design of the study is 
adaptive, and contains interim analysis and a final analysis (see section 9.4.6). The interim analysis 
includes an opportunity to increase the sample size of the trial adaptively, based on the difference in 
the primary endpoint results. 
A DSMB consisting of at least three experienced members including at least an oncologist and one 
biostatistician will be appointed to review, on a regular basis, accumulating safety data as well as 
efficacy data according to the interim analysis plan. The DSMB will review unblinded data that will not 
be shared with anyone else (Sponsor or Investigators). At each of their meetings, the DSMB will 
recommend a course of action: stop the trial, amend the trial protocol, or continue the trial 
unchanged. The DSMB recommendation will be based on safety considerations and, when 
appropriate, on efficacy considerations as outlined in the interim analysis plan. 
The sponsor (or designee) will be responsible to safeguard the ethical and safety interests of the 
patients, Investigators and the sponsor; to assess the safety of the study’s interventions; to monitor 
the overall conduct of the study and to protect its validity and credibility. The sponsor (or designee) 
will meet at predefined times (or also ad hoc, if deemed necessary), to assess at intervals the progress 
of the clinical study and the critical efficacy endpoints (e.g. ORR and PFS) and will decide whether to 
continue, modify or stop the study for safety.  
The study will be reviewed and not initiated before approval from relevant IRB/IEC have been 
obtained. The study will also be monitored, according to GCP and local requirements, securing the 
trial patients’ well-being and the integrity of data collected. 
The patients in this study will be closely monitored at the participating study sites following the visit 
schedule (see section 8.3). 
Arfolitixorin is the active metabolite of the commercially available prodrug LV and is intended as an 
improvement of established care. The efficacy is presumed to be as good as or better than that of LV. 
A comparative study is necessary to assess the magnitude of efficacy improvement. The study design, 
the close monitoring of the study patient from the Investigators, the DSMB and the study monitors 
contributes to preserving the well-being of the trial patients minimizes the risk of study participation. 
2.3.3.1 ADVERSE EVENTS OF SPECIAL INTEREST  
The hypothesis is that arfolitixorin in combination with 5-FU will increase tumor cell killing by 
increasing the active metabolites MTHF and THF within the tumor. However, the increases in these 
metabolites may also occur in the normal tissue. The two tissues that may have increased toxicity with 
arfolitixorin in combination with 5-FU are bone marrow and the mucous membranes. Therefore, these 
will be monitored and assessed as Adverse Events of Special Interest (AESI) during the trial and by the 
DSMB. 
Seventy patients have been treated with arfolitixorin in study ISO-CC-005 and are evaluable after a 
30-day cut off from their first dose of therapy across the arfolitixorin dosage administered which 
ranged from 7 patients treated at 240 mg/m2, 13 subjects treated at 30 mg/m2, 20 patients treated at 
60 mg/m2 and 35 patients treated at 120 mg/m2. The incidence of mucositis in the combination of 


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arfolitixorin + 5-FU with or without other chemotherapeutic agent’s ranges from 1-5% of all reported 
AEs. Similarly, bone marrow adverse events are also between 1-5% of all reported AEs in in patients 
treated with arfolitixorin + 5-FU combinations. The incidence of either mucositis or bone marrow 
suppression (neutropenia and thrombocytopenia) appear to be similar to what is reported in clinical 
practice for patients treated with FOLFOX + Bevacizumab. 
 


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3 
OBJECTIVES AND ENDPOINTS 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
Overall response rate (ORR) 
Best ORR, defined as the best 
response recorded from the 
start of the study treatment 
until the end of treatment. All 
responses will be confirmed 8 
weeks after onset of response. 
All assessments including 
confirmation of response will 
be performed by BICR.     
The antitumor effect 
attributable directly to the 
drug, not the natural history of 
the disease. 
Key Secondary 
 
 
Progression-free survival (PFS) 
PFS, defined as time from 
randomization to first 
occurrence of tumor 
progression (RECIST 1.1) 
assessed by BICR based on CT-
scans/MRIs conducted every 8 
weeks after treatment, or 
death from any cause. 
PFS is a relevant endpoint to 
measure clinical effect as it 
also includes stable disease. 
Duration of Response (DOR) 
The duration of overall 
response (DOR) is measured 
from the first time point at 
which criteria are met for 
complete response (CR) or 
partial response (PR) through 
the last time point when 
overall response has been 
objectively documented. 
DOR measures the duration of 
confirmed CR and PR, which is 
an important aspect of the 
clinical significance of DOR. 
Secondary 
 
 
Overall survival (OS) 
OS, defined as time from 
randomization to death from 
any cause.  
OS measures potential 
detrimental effects of therapy, 
and possibly a direct benefit. 
Quality of Life 
Quality of Life assessed using 
the EQ-5D patient reported 
outcome questionnaire. 
PRO EQ-5D measures patients 
self-experienced quality of life. 
Safety and tolerability 
Number and severity of 
adverse events (AEs), including 
clinically significant abnormal 
laboratory findings, regardless 
of causal relationship to 
arfolitixorin or LV. Specific AEs 
will be followed using patient 
reported outcome 
questionnaire (NCI PRO 
CTCAE). 
Relevant endpoint to measure 
safety and tolerability. 
Patients undergoing curative 
metastasis resection. 
Defined as the number of 
patients qualifying for curative 
To understand study 
treatment effects on 


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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
metastasis resection after 
treatment with study drug. 
metastasis become curatively 
resectable. 
Exploratory  
 
 
Daily living abilities 
Daily living abilities as assessed 
by the Eastern Cooperative 
Oncology Group (ECOG) 
Performance Status Scale 
Physicians professional 
estimation of the patient’s 
status. 
To determine the 
pharmacokinetic (PK) 
characteristics of arfolitixorin 
and LV in patients with 
advanced CRC 
The plasma concentration of 
[6R]-MTHF, methyl-THF, THF, 
will be determined in a limited 
number of patients in both 
treatment arms. 
To increase the understanding 
of the study drug, the study 
treatment and also in relation 
to the comparator arm. 
To investigate folate 
metabolism- and 
transportation-related gene 
expression levels in patients 
with advanced CRC 
Gene expression levels of 
folate metabolism- and 
transportation-related genes, 
analyzed by sub-group 
low/high gene expression 
levels.  
To increase the understanding 
of the study drug. 
Recurrence Free Survival (RFS) 
for patients undergoing 
metastatic resection. 
Defined as the time between 
the first surgery with complete 
removal of the metastasis and 
recurrence of the disease or 
death from any cause. 
To increase the understanding 
of disease recurrence after 
metastasis resection. 
 
 
4 
STUDY DESIGN  
 
4.1 
OVERALL DESIGN 
This is a randomized, multicenter, parallel-group, Phase III study to compare the efficacy of 
arfolitixorin versus LV in patients with advanced CRC treated by 5-FU, oxaliplatin, and bevacizumab. 
To be eligible for the study, patients must have advanced CRC, without indication for resection, with 
at least one measurable lesion of metastatic disease according to RECIST 1.1 criteria [24] within 28 
days of randomization. 
Approximately 490 patients will be randomized 1:1 to receive either arfolitixorin or LV as a component 
of standard first line therapy for advanced CRC. The study will consist of a screening visit, a 
randomization visit during which patients will receive their first course of IMP, regular treatment visits 
every 2 weeks thereafter, and CT/MRI assessments every 8 weeks from baseline. Treatment visits are 
to continue until PD, as identified by the Investigator based on locally-performed assessment of CT-
scans/MRIs. In the event of PD, Investigators are to discontinue IMP and manage the patient according 
to local routine. Patients will be followed thereafter until death.   
Randomization minimizes potential bias. Multiple sites across geographic regions allows for widely 
applicable results. Baseline factors considered in the randomization will include:  
• 
Geographical region (Europe versus North America versus Australia versus Japan) 
• 
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• 
Previous neo-adjuvant/adjuvant CRC treatment (yes versus no) 
Patient and Investigator blinding is not possible as there is a difference in the color of the injection 
fluids and the method of administration. However, as a measure to minimize any bias arising either 
from the inability to blind patients and Investigators or from potential inconsistencies in local tumor 
response assessments across study sites, analyses of the tumor response endpoints will be based on 
blinded, retrospective, centrally-adjudicated assessment of CT-scans/MRIs according to a pre-
specified Image Review Charter. 
An interim analysis will be performed when 16-week BICR evaluation has been performed for the 
330th patient. The objective of the interim analysis is to calculate the conditional power for ORR and 
PFS to continue the study as planned or increasing the sample size. The guidelines for sample size 
increase decisions is clearly defined in the DSMB charter and in section 9.4.6. Regardless of the efficacy 
results of the interim analysis, the trial will continue to accrue patients, until the total planned sample 
size is reached. 
Initially, approximately 440 were planned to be randomized. However, Japanese authorities requested 
that approximately 12.7% of the total study population are enrolled from Japan. For this reason, 
additional patients from Japan were randomized. In the end a total of 490 patients were randomized. 
This decision to randomize extra Japanese patients was made before the interim analysis. The decision 
to include the Japanese patients in the ITT population (see Section 9.2.2) for the primary efficacy 
analysis was made after the interim analysis. 
The decision-making process to terminate a trial early due to evidence of harm or benefit is complex 
and is defined in section 9.4.6. 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
Standard first-line systemic therapy for mCRC, is chemotherapy with 5-FU + LV + oxaliplatin/irinotecan 
± bevacizumab. LV is a prodrug of the active substance MTHF, which has a central role in the 
potentiation of the 5-FU-mediated inhibition of TS and subsequently halting DNA synthesis and cell 
proliferation. 
Arfolitixorin is the active metabolite of LV and in contrast to LV does not require genetically influenced 
multistep metabolic activation. Distribution results have shown that arfolitixorin versus LV 
administration of equimolar doses resulted in higher levels of [6R]-MTHF and THF in tumors and the 
surrounding mucosa for arfolitixorin [20].  
Arfolitixorin is therefore expected to be more effective than LV, particularly in patients with low folate-
relevant gene expression. Further it is suggested that arfolitixorin may reduce potential interpatient 
variability and as a consequence allow for safe and more adaptable and predictable treatments. The 
purpose of this study is to compare arfolitixorin to LV to establish the magnitude of the hypothesized 
improvement.  
The study is designed as a superiority study. To minimize the number of patients exposed to the 
investigational drug, the design of the study is adaptive, and contains interim analysis and a final 
analysis (see section 9.4.6 for details). The interim analysis includes an opportunity to increase the 
sample size of the trial adaptively, based on the conditional power for ORR and PFS. 
Patient and Investigator blinding is not possible as there is a difference in color of the injection fluids 
and the method of administration (intravenous bolus injection versus infusion). However, as a 


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measure to minimize any bias arising either from the inability to blind patients and Investigators or 
from potential inconsistencies in local tumor response assessments across study sites, analyses of the 
tumor response endpoints will be based on blinded, retrospective, centrally-adjudicated assessment 
of CT-scans/MRIs according to a pre-specified Image Review Charter. 
 
 
4.3 
JUSTIFICATION FOR DOSE 
 
The mechanism of action and the rationale for combining folate derivatives with 5-FU is universally 
accepted and applied [18, 19]. In treatment of colorectal cancer with cytotoxic combinations 
comprising 5-FU together with folate derivatives, the principal role of the folate component is to 
potentiate and prolong the inhibitory effect of 5-FdUMP (a 5-FU metabolite) on TS and thereby reduce 
the supply of thymidine for DNA synthesis and cell replication whilst maintaining an acceptable clinical 
benefit-risk ratio. Despite universal use of this combination for decades, the literature is scarce 
regarding systematic studies aiming to define optimal doses of each drug in the combination. Instead 
the focus is on the nature of the disease and the intrinsic statistical variance with ensuing large sample 
sizes on one hand and the demand for therapeutic progress on the other necessitated a different route 
of advancing. The folate predominantly used in this context has been LV, which requires multistep 
enzymatic conversion to the active metabolite [6R]-5,10-methylenetetrahydrofolate. In this pivotal 
study, ISO-CC-007, the logical reference therapy is LV and the reference treatment regimen is modified 
FOLFOX-6 plus bevacizumab, which is a frequently used standard regimen in the treatment of 
advanced CRC patients. For the experimental arm, the LV infusion of the modified FOLFOX-6 therapy 
is exchanged to arfolitixorin. Arfolitixorin is however not suitable for infusion due to a short half-life. 
Arfolitixorin is therefore administered as bolus injection. 
 
The Sponsor has considered the following aspects when selecting the dose for future development of 
arfolitixorin: 
 
1. The Sponsor has taken previous efforts by Adventrx Inc in developing the dual 
diastereoisomers [6R,S]-5,10-methylenetetrahydrofolate into account. The development 
was unsuccessful for various reasons, particularly due to the infusion administration of 
[6R,S]-5,10-methylenetetrahydrofolate, which is highly unstable in aqueous solution over 
2 hours.  
 
2. Comparison of dose levels with marketed reduced folates (prodrugs of arfolitixorin) i.e., LV 
and L-LV and failed attempts to develop [6R]-MTHF. LV is and has been used in doses from 
15 mg/m2 every six hours IV to 1500 mg/m2 every six hours IV as rescue after high dose 
methotrexate treatment [25, 26]. Furthermore, in combination with 5-FU in colorectal 
cancer, doses from 20 mg/m2 IV to 500 mg/m2 IV over 2 hours every second week have been 
used [4, 20]. 
 
3. PK and tissue concentration data of arfolitixorin and LV collected from previous studies, i.e. 
the ISO-CC-002 and ISO-CC-005 studies. The results obtained demonstrate that arfolitixorin 
administration leads to much greater exposure and levels of MTHF and THF compared to 
administration of an equimolar L-LV dose. The identified differences in PK/PD with respect to 
[6R]-MTHF and THF levels in tumor, surrounding mucosa, and plasma after administration of 
arfolitixorin and L-LV suggest that it might be possible to modify the outcome of present well-
established treatments within different cancer therapeutic areas that are based on 
chemotherapy regimens including folates. 
 


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4. The amalgamated pre-clinical safety experience for arfolitixorin shows no toxicity or adverse 
effect directly related to arfolitixorin in either the preliminary or 28-day toxicity studies 
performed in rats and dogs. At that time, the highest dose tested was considered to be the 
maximum feasible dose. Roughly determined, maximum exposure in rats and dogs was more 
than 50-fold compared to estimated exposure at 120 mg/m2. The initiated 3-month toxicity 
studies in the same species are expected to further confirm the safety of arfolitixorin and will 
also investigate higher concentrations with the aim of reaching the maximum tolerated dose 
(MTD). 
 
5. The arfolitixorin Investigators Brochure was last updated in 9 July 2018. Up until 20 August 
2018, arfolitixorin has been administered to over 145 patients and administered more than 
1000 times in dosages varying from 7.5 to 500 mg/m2 alone or in combination with different 
cytostatic agents. Results from the ongoing ISO-CC-005 study, designed to investigate the 
tolerability of various doses, has evaluated dose levels of 30, 60, 120 and 240 mg/m2 of 
arfolitixorin in combination with 5-FU and oxaliplatin with monitoring of clinical and 
laboratory safety data. Preliminary results show that arfolitixorin in a dose range of 30-240 
mg/m2 is not linked to worsened tolerability compared to historical experience. 
 
6. The main mechanism of action is a folate-enhanced and prolonged inhibition of TS. Plasma 
deoxyuridine levels (pdUrd) is a marker for the global (all cells and organs in the organism) 
inhibition of TS. The influence on pdUrd of Leucovorin, an equimolar and several incremental 
doses of arfolitixorin have now been established. 
 
Arfolitixorin gives enhanced levels of pdUrd compared to an equimolar standard dose of Leucovorin 
and a dose dependent increase of pdUrd with incremental doses of arfolitixorin which levels off 
between the doses 60 and 240 mg/m2 BSA. Doses above 120 mg are therefore unlikely to result in 
much higher efficacy and the doses of arfolitixorin of both 60 and 120 m/m2 are deemed adequate 
for efficacy under the above hypothesis. 
The decision to use two separate injections is supported by the following data based on both safety 
and efficacy considerations: 
ISO-CC-005 study data demonstrates no dose-related increase in toxicity when 28 patients were 
dosed with 120 mg/m2 administered with two separate injections of 60 mg/m2 each. 
PK and metabolism data from the ISO-CC-002 study suggest attaining a high Cmax on two occasions 
achieved with the two injections of 60 mg/m2 may move more arfolitixorin into the cells. In the 
ISO-CC-002 study, a bolus dose of 60 mg/m2 arfolitixorin provides a pronounced transient increase of 
the [6R]-5,10-MTHF levels in plasma but disappears within 30 min.  
Furthermore, 5-FU given as a bolus is known to be rapidly (in 10 to 20 minutes) converted into 
5-FdUMP, which binds highly efficiently to the TS enzyme in tumors under the formation of a binary 
complex. The binary complex can be further strengthened by supplying arfolitixorin at the early 
stage after 5-FU administration [18]. For this reason, the high transient plasma level of 
[6R]-5,10-MTHF should co-exist with the high concentration of 5-FdUMP in the tumor. An overall 
optimal treatment should therefore be obtained by dividing the 120 mg/m2 dose of arfolitixorin into 
two bolus doses, and to achieve a high [6R]-5,10-MTHF intracellular concentrations over a longer 
period of time the bolus doses should be separated by 30-60 minutes. 
LV 400 mg/m2 IV infusion has been chosen as the comparator as this is the agent recommended in 
advanced CRC treatment guidelines [4].   


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Arfolitixorin or LV will be administered in combination with 5-FU, oxaliplatin, and bevacizumab.  This 
is standard first-line adjuvant therapy according to current ESMO and NCCN guidance. All patients in 
this study will receive bevacizumab as it appears to improve PFS when used in combination with 5-
FU/LV/oxaliplatin [4, 27]. The doses of 5-FU, oxaliplatin, and bevacizumab specified in this protocol 
are those commonly used in clinical practice and according to their labels. 
4.4 
END OF STUDY DEFINITION 
A patient is considered to have completed the study if he or she has completed all phases of the study 
including the last scheduled procedure shown in the Schedule of Activities (SoA), Section 1.3, i.e. the 
last contact (phone, hospital records or other means found suitable) to collect the death date. 
The final analysis of ORR and PFS will be performed when at least 235 PFS events have been observed. 
If the sample size is increased to 660 patients, the final analysis of ORR and PFS will be performed 
when approximately 450 PFS events have been observed. All study patients will be followed for OS a 
until 60% of OS events are achieved for the study population, i.e. 294 OS events in case the sample 
size is not increased, and 396 OS events in case the sample size is increased to 660 patients. 
At the time of final analysis of ORR and PFS, patients still on IMP treatment will continue IMP 
treatment until locally assessed progressive disease or discontinuation of IMP for other reasons. After 
completed End of Treatment visit these patients have reached the End of Study definition and will 
thereafter move into survival follow-up until 60% of OS events are achieved for the study population.  
Patients already in follow-up phase at the time of final analysis of ORR and PFS have reached the End 
of Study definition and will move into survival follow-up until 60% of OS events are achieved for the 
study population. 
 
5 
STUDY POPULATION 
 
5.1 
INCLUSION CRITERIA 
For randomization in the study, patients must fulfill all of the following criteria: 
1. Colorectal adenocarcinoma verified by biopsy. 
2. Availability of biopsy material, from the primary tumor or metastasis, allowing for analysis of 
tumor gene expression. 
3. Non-resectable metastatic CRC planned for first line therapy with 5-FU, Leucovorin, 
oxaliplatin, and bevacizumab.   
4. Evaluable disease with at least one measurable lesion of metastatic disease (≥10 mm in 
longest diameter on axial image on CT-scan or alternatively MRI with <5 mm reconstruction 
interval) or lymph node (≥15 mm in shortest axis when assessed by CT) within 28 days of 
randomization. 
5. Life expectancy of more than 4 months. 
6. ECOG performance status 0 or 1. 


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7. Hemoglobin (Hb) > 80 g/L, Absolute neutrophil count (ANC) > 1.5x109/L. Thrombocytes > 
100x109/L.  
8. Creatinine clearance > 50 mL/min, Total bilirubin < 1.5 x ULN,  
AST and ALT < 3 x ULN (and < 5 x ULN in case of liver metastases). 
9. Male or female ≥18 years of age. 
10. Female patients of childbearing potential must have a negative urine pregnancy test and use 
adequate contraceptive measures1. Male patients must use adequate contraceptive 
measures2. 
11. Voluntarily signed informed consent before performance of any study related procedure not 
part of normal medical care, with the understanding that consent may be withdrawn by the 
patient at any time without prejudice to future medical care. 
1 Female patients must be post-menopausal for more than one year or must provide a negative pregnancy test and use an efficient 
method of contraception (i.e., a method with less than 1% failure rate [e.g., sterilization, hormone implants, hormone injections, some 
intrauterine devices, or vasectomized partner]) during the study and for 1 month (or more if requested by non-IMP labels) after the end of 
the study (last dose of IMP). 
2 Unless the partner of a male patient is post-menopausal or using an efficient method of contraception as described above, male patients 
must agree to use condoms during the study treatment and for 1 month after the end of the study treatment. 
5.2 
EXCLUSION CRITERIA 
Patients meeting one or more of the following criteria are ineligible to participate in the study:  
1. Malignant tumors other than colorectal adenocarcinomas (current or within the previous five 
years), with the exception for curatively treated non-melanoma skin cancer or in situ 
carcinoma of the cervix. 
2. Less than 6 months between randomization and completion of the last anti-cancer 
treatment (chemotherapy/ radiotherapy/immunotherapy, etc.). (NB: Rectal cancer 
treatment shorter than 8 weeks of chemo/radiation therapy is allowed.) 
3. Confirmation of progressive disease within 6 months after completion of prior adjuvant anti-
cancer treatment.  
4. Indication for any mCRC surgery or anti-cancer treatment other than study treatment.  
5. Prior treatment with arfolitixorin. 
6. Indication for treatment with a 5-FU analogue, or 5-FU for a condition other than mCRC. 
7. Known DPD deficiency. 
8. Known or suspected central nervous system metastases. 
9. Unresolved bowel obstruction, uncontrolled Crohn’s disease, or ulcerative colitis. 
10. History of cardiac disease with a New York Heart Association Class II or greater, congestive 
heart failure, myocardial infarction or unstable angina at any time during the 6 months prior 
to randomization, or serious arrhythmias requiring medication for treatment. 


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11. Current CTCAE ≥ grade 3 diarrhea. 
12. Current chronic infection or uncontrolled serious illness causing immunodeficiency. 
13. Known or suspected hypersensitivity or intolerance to arfolitixorin, LV, 5-FU, oxaliplatin, or 
bevacizumab. 
14. Breastfeeding patients. 
15. Patient who received investigational drugs in other clinical trials within 28 days, or 5 half-lives 
of the investigational drug, prior to randomization. 
16. Patient with serious medical or psychiatric illness likely to interfere with participation in this 
clinical study. 
17. Ongoing drug or alcohol abuse, as deemed by the Investigator. 
18. Any condition that, in the opinion of the Investigator, could compromise the patient's safety 
or adherence to the study protocol. 
19. Involvement, or related to people involved in the planning or conduct of the study (applies 
to both Isofol Medical staff and staff at the study site). 
20. Surgery (excluding previous diagnostic biopsy) in the 28-day period before randomization 
 
5.3 
LIFESTYLE CONSIDERATIONS 
Not applicable 
5.4 
SCREEN FAILURES 
Screening failures are patients who consent to participate in the clinical trial but are not subsequently 
assigned to the study intervention or entered in the study due to a failure to meet inclusion/exclusion 
criteria. These patients should have the reason for study withdrawal recorded as ‘Eligibility Criteria 
Not Fulfilled’. Screening failure patients will not be allowed to be rescreened into the study. 
If a patient does not meet all the eligibility criteria but is randomized in error, or incorrectly started on 
treatment, the Investigator should inform the Sponsor immediately, before a decision is made to 
continue or discontinue the patient from treatment. 
All AEs that occur after the consent form is signed and before treatment allocation/randomization 
must be reported by the Investigator if the AE causes the patient to be excluded from the study, or 
are the result of a protocol-specified intervention.    
5.5 
STRATEGIES FOR RECRUITMENT AND RETENTION 
Approximatively 80-100 sites across Europe, Canada, the US, Australia and Japan will participate in the 
study. The recruitment will be competitive between the study sites. With an estimated recruitment 
rate of 0.36 patients per site and month, 440 patients should be recruited in approximately 19 months 
including 6 months ramp up period until all sites are activated. The patients will be recruited from the 
sites ordinary pool of patients. No study site should include more than 10 % of the total number of 
patients in the study (i.e. not exceeding 44 patients, or 66 patients if the sample size is increased). 


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The recruitment will continue during the interim analysis. Depending on the conditional powers, the 
trial will either continue as planned, or the sample size of the trial will be increased for a total of 660 
randomized patients. 
 
Initially, approximately 440 were planned to be randomized. However, Japanese authorities 
requested that approximately 12.7% of the total study population are enrolled from Japan. For this 
reason, additional patients from Japan were randomized. In the end a total of 490 patients were 
randomized. This decision to randomize extra Japanese patients was made before the interim 
analysis. The decision to include the Japanese patients in the ITT population (see Section 9.2.2) for 
the primary efficacy analysis was made after the interim analysis. 
 
Study sites should keep up to date contact information for all study patients in their records to 
ensure patient retention throughout the study, including the survival follow up phase. 
 
6 
STUDY INTERVENTION 
 
6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
Eligible and consenting patients will be randomized 1:1 to one of two treatment groups (A and B) and 
administered one of the following treatment regimens starting within 3 days of randomization. 
Following treatment cycles should be started 14 (+ 7) days after the previous administration. 
• 
Group A “Experimental”:  ARFOX (Arfolitixorin + 5-FU + Oxaliplatin) + Bevacizumab  
• 
Group B “Comparator”:    mFOLFOX-6 (Leucovorin + 5-FU + Oxaliplatin) + Bevacizumab 
6.1.2 DOSING AND ADMINISTRATION 
Group A (ARFOX + bevacizumab): 
• 
Bevacizumab 5 mg/kg, IV infusion in accordance with the label   
↓ followed by 
• 
Oxaliplatin 85 mg/m2 IV infusion in accordance with the label   
 
↓ followed by 
• 
5-FU 400 mg/m2 IV bolus (2–4 minutes) 
 
↓ followed by (30 minutes (± 5 minutes) after 5-FU bolus) 
• 
Arfolitixorin 60 mg/m2 rapid IV bolus (less than 3 minutes) 
 
↓ followed by 
• 
5-FU 2400 mg/m2 continuous IV infusion over 46 hours  
↓ followed by (30-60 minutes after the first arfolitixorin bolus) 


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• 
Arfolitixorin 60 mg/m2 rapid IV bolus (less than 3 minutes) 
 
Group B (modified FOLFOX-6 + bevacizumab): 
• 
Bevacizumab 5 mg/kg, IV infusion in accordance with the label   
↓ followed by 
• 
Oxaliplatin 85 mg/m2 IV infusion in accordance with the label   
↓ followed by* 
• 
LV 400 mg/m2 IV infusion  
 
↓ followed by 
• 
5-FU 400 mg/m2 IV bolus (2–4 minutes) 
 
↓ followed by  
• 
5-FU 2400 mg/m2 continuous IV infusion over 46 hours 
*Oxaliplatin and LV may be administered at the same time through two different injection ports. NB: 
Oxaliplatin and LV must not be mixed. 
The doses of arfolitixorin and LV should not be adjusted.  
5-Fluorouracil and LV should be administered separately to avoid the formation of a precipitate. 
Following a treatment-related adverse event the doses of 5-FU, oxaliplatin, and bevacizumab may be 
adjusted or delayed in accordance with the label and section 6.3.4 in the protocol. Oxaliplatin cannot 
be substituted but caution should be taken regarding toxicity and in case of toxicity, dose 
modifications (decrease in infusion rate and/or dose reductions) according to label should be 
performed [4, 28]. 
For patients with platinum hypersensitivity, pre-medication with IV dexamethasone, chlorphenamine, 
and ranitidine can be given at the Investigator’s discretion. 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
The IMP (arfolitixorin and LV) will be sent from Creapharm, with depots in France and in the US, direct 
to the study sites. All shipments of IMP to site will be managed through an Interactive Web Response 
System (IWRS).  
At all times, the number of supplied, used and remaining treatment doses should match. The 
Investigator (or designee) will be responsible for IMP accountability, reconciliation, and record 
maintenance. In accordance with all applicable regulatory requirements, the Investigator (or 
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• 
Receipt of IMP boxes at the study site; 
• 
IMP administered to each patient (including vial numbers); 
• 
Inventory of IMP at study site; 
• 
Log of any discarded/unused/expired IMP; 
• 
Monitoring of the storage conditions. 
These records will be monitored and verified by the local monitor regularly, as part of the routine 
monitoring procedure.  
Any unused or expired IMP should be destroyed at site if possible only after Sponsor approval. If 
returned to the supplier locally approved safety procedures and documentation should be used. 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELLING 
6.2.2.1 STUDY DRUG: ARFOLITIXORIN 
The active pharmaceutical ingredient (API) of arfolitixorin is the hemisulfate salt of [6R]5,10-
methylene-tetrahydrofolic acid ([6R]-MTHF-HS). The API is manufactured in accordance with Good 
Manufacturing Practice (GMP) by Merck & Cie (Schaffhausen, Switzerland). 
The drug product, arfolitixorin powder for injection 100 mg, is a lyophilized white to light yellowish or 
light beige powder (100 mg arfolitixorin per vial, calculated as free acid) for administration after 
reconstitution with 10 mL of water for injection. The drug product is manufactured in accordance with 
GMP by Recipharm (Wasserburg, Germany) 
Table 2: Components of arfolitixorin 
Component 
Amount/vial 
Function  
Sodium hydroxide 
0.031 g 
For pH adjustment 
Trisodium citrate dihydrate 
0.2 g  
Buffer (to guarantee a defined stable pH after 
reconstitution in water) 
Arfolitixorin 
0.1 g  
Active Ingredient 
 
1) 
The listed amount of Sodium hydroxide (2 N in WFI) can vary slightly since the final amounts are dependent on 
the pH-adjustment. During the lyophilization process, water is removed.  
Reconstitution is performed prior to administration (see section 6.2.4.1). The resulting solution for 
injection is isotonic and has a pH of 8.5. The final API concentration is 10 mg/mL calculated as the free 
acid.  
The primary packaging is 10 mL type 1 glass vials with lyophilization stopper (20 mm, siliconized, 
Helvoet FM157/1, light grey) and flip-off cap (20 mm with white synthetic disc). 
Arfolitixorin vials are labeled by Creapharm in accordance with GMP and local regulatory guidelines.  
The labels fulfil GMP Annex 13 requirements for labelling. Label text will be translated into local 
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6.2.2.2 COMPARATOR DRUG: LEUCOVORIN 
 
The comparator, Leucovorin (calcium folinate) 10 mg/mL is a commercialized specialty which will be 
provided to the sites by Creapharm. 
Each vial of 20 mL solution contains 10 mg/mL of folinic acid provided as calcium folinate. 
List of excipients 
• 
Sodium Chloride 
• 
Water for Injection 
Calcium folinate vials are labeled by Creapharm in accordance with GMP and local regulatory 
guidelines. The labels fulfil GMP Annex 13 requirements for labelling.  Label text is translated into local 
language.  
6.2.3 PRODUCT STORAGE AND STABILITY 
6.2.3.1 STUDY DRUG: ARFOLITIXORIN 
Stability tests according to ICH (International Conference on Harmonization) guidelines are ongoing. 
The study drug product should be stored at 2 – 8°C. Reconstituted powder (for details see 6.2.4.1) can 
be handled at room temperature and should be administrated within 2 hours. 
6.2.3.2 COMPARATOR DRUG: LEUCOVORIN 
The comparator is commercialized Leucovorin (calcium folinate) and should be stored at 2 – 8°C, 
protected from light. 
6.2.4 PREPARATION 
 
The study drug is allocated using the IWRS. The doses are based on the patient’s body surface area 
and is calculated using the DuBois formula: 
BSA = 0.007184 x Height0.725 x Weight0.425 
6.2.4.1 STUDY DRUG: ARFOLITIXORIN 
 
1. Arfolitixorin solution for injection should be prepared by trained personnel, for example at 
the hospital pharmacy or by a registered nurse. 
 
2. Use aseptic technique.  
 
3. Reconstitute each vial (100 mg) with 10 mL water for injection and note the time point for 
reconstitution. Gently swirl each vial until the powder is completely dissolved. The solution 


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should be clear and range in colour from colourless to yellow. The solution contains 10 
mg/mL arfolitixorin. 
 
4. Inspect the solution visually for particles. If particles are present, discard the solution. 
 
5. The solution can be kept in the vial, alternatively, directly after the visual inspection 
transferred to a syringe according to standard procedure. 
 
6. Storage conditions for the solution: 
 
a. If the solution is stored at room temperature it must be administered within 2 hours 
from the time of reconstitution. Document the expiry time. 
 
b. If the solution is stored in the refrigerator (2-8 °C) it must be administered within 6 
hours from the time of reconstitution. Document the expiry time. 
 
c. If the solution is not administered before it expires, it must be discarded. 
6.2.4.2 COMPARATOR DRUG: LEUCOVORIN (LV) 
The preparation should be performed according to the product preparation instructions.  
For intravenous infusion, LV may be diluted with 0.9% sodium chloride solution or 5% glucose solution 
before use. Refer also to section 6.3. 
5-Fluorouracil and LV should be administered separately to avoid the formation of a precipitate. 
As the LV used in this study is Bendafolin which contains the excipient trometamol, LV may not be 
mixed with oxaliplatin during the administration of the study drugs.  
6.3 
OTHER MEDICATIONS 
6.3.1 5-FU 
5-FU is formulated as injection solution. 
5-FU Dosing: 
Per label, 5-FU will be administered as a 400 mg/m2 IV bolus (2–4 minutes) on Day 1 followed by 
continuous 2400 mg/m2 IV infusion over 46 hours in each treatment cycle. The treatment will be 
repeated every two weeks until PD or premature discontinuation of IMP. 
An administered 5-FU IV bolus dose should not surpass the maximum recommended daily dose of 
1000 mg, regardless of the body surface area.  
6.3.2 OXALIPLATIN 
Oxaliplatin is formulated as a concentrated infusion solution.  
Oxaliplatin Dosing: 


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Per label, Oxaliplatin will be administered as 85 mg/m2 IV infusion over 15 – 120 minutes on Day 1 in 
each treatment cycle and repeated every two weeks until PD or premature discontinuation of IMP. 
Caution will be taken regarding toxicity and in case of toxicity, dose modifications (decrease in infusion 
rate and/or dose reductions) according to label should be performed. 
6.3.3 BEVACIZUMAB 
Bevacizumab is formulated as a concentrated infusion solution. 
US licensed bevacizumab biosimilars are allowed for treatment. A biosimilar is a biological product 
that is highly similar to and has no clinically meaningful differences from an existing biologic therapy. 
Several biosimilars are available on the U.S. market, including bevacizumab-awwb and -bvzr that have 
been FDA approved for the treatment of mCRC. The biosimilars are however not interchangeable and 
no patient may therefore change between bevacizumab and any biosimilar treatment or between 
different biosimilars during the course of the study. 
Bevacizumab Dosing: 
Per label, Bevacizumab administered as an intravenous infusion as 5 mg/kg of body weight given once 
every 2 weeks. It will be administered on Day 1 of every 2-week cycle and repeated every two weeks 
until PD, premature discontinuation of IMP or unacceptable toxicity. The initial dose should be 
delivered over 90 minutes as an intravenous infusion. If the first infusion is well tolerated, the second 
infusion may be administered over 60 minutes. If the 60-minute infusion is well tolerated, all 
subsequent infusions may be administered over 30 minutes. It should not be administered as an 
intravenous push or bolus.  
6.3.4 DOSE ADJUSTEMENTS 
On the planned day of treatment, chemotherapy may be administered only if: 
• 
ANC is ≥ 1.0 × 103/µL (1.0 × 109/L)† 
• 
Platelet count is ≥ 75 × 103/µL (75 × 109/L) 
• 
Chemotherapy-related gastrointestinal toxicity is ≤ Grade 1 
† In situations where ANC is < 1.5 × 103/µL (1.5 × 109/L) but ≥ 1.0 × 103/µL (1.0 × 109/L) and institutional 
guidelines (or Investigator’s discretion) recommend not administering chemotherapy, then 
chemotherapy may be delayed. 
General dose-modification considerations are as follows: 
• 
All study patients should receive standard approaches to dosing and dose modification for 
adverse reactions that are consistent with approved product labeling for the individual drugs 
within the combination chemotherapy regimen.   
• 
If 5-FU administration is delayed, oxaliplatin, bevacizumab, arfolitixorin or LV must also be 
postponed 
• 
If 5-FU treatment is discontinued the patient will be withdrawn from the study treatment and 
followed for OS. 
• 
5-FU dose reductions are allowed in case of toxicity, but the 5-FU bolus may not be removed 
if the 5-FU treatment continues. Instead both the 5-FU bolus and 5-FU infusion dose should 
be reduced according to the rates described in the 5-FU summary of product characteristics 
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• 
If oxaliplatin or bevacizumab administration is delayed or discontinued, 5-FU/arfolitixorin or 
LV may be continued. 
• 
Dose reductions for arfolitixorin or LV are not allowed; however, arfolitixorin or LV will always 
be withheld when 5-FU is withheld. 
All dose modifications and treatment discontinuations will be captured in the eCRF. 
 
 
6.4 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
6.4.1 RANDOMIZATION 
Patients will be randomized in a 1:1 ratio to either the experimental arm (arfolitixorin) or the 
comparator arm (Leucovorin), following the completion of all screening assessments and after 
confirmation of their eligibility, using a stratified permuted block randomization. Randomization will 
be stratified for the following baseline factors:  
• 
Geographical region (Europe versus North America versus Australia versus Japan) 
• 
Primary tumor location (left colon versus right colon versus rectal cancer) 
• 
Previous neo-adjuvant/adjuvant CRC treatment (yes versus no) 
Randomization will be performed via an interactive web response system (IWRS). The system enables 
time and date stamped entries that in turn allow real time compliance monitoring.  
The system is compliant with the FDA 21 CFR Part 11 standards ensuring the system’s secured access 
and integrity of data collected. In this respect, any person involved with the IWRS will be identified by 
a personal username and will need a secret code to access the system.  
6.4.2 BLINDING 
Patient and Investigator blinding of the study treatment is not possible as there is a difference in the 
color of the injection fluids and the method of administration. To minimize any bias arising either from 
the inability to blind patients and Investigators or from potential inconsistencies in local tumor 
response assessments across study sites, analyses of the tumor response endpoints will be based on 
BICR, i.e. blinded, retrospective, centrally-adjudicated assessment of CT-scans/MRIs according to a 
pre-specified Image Review Charter. 
6.5 
STUDY INTERVENTION COMPLIANCE 
Date and procedures of each study visit will be entered in the electronic case report form (eCRF) to 
document patient compliance to the study schedule as well as to the study procedures (for instance 
complies with the prohibited concomitant therapies). 
In addition, during monitoring visit done at the Investigator’s site, the monitor will check that: 
• 
All IMPs and procedures for the trial are to be used exclusively within the limits defined by the 
protocol; 
• 
Inventory records of study products are in order and that there are sufficient supplies; 
• 
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• 
The storage conditions for study products are adequate;  
• 
Procedures and records of returned and/or unused study products are complete. 
 
6.6 
CONCOMITANT THERAPY 
6.6.1 PROHIBITED THERAPIES 
The following treatments are not permitted: 
• 
nucleoside analogues (e.g., sorivudine) or known DPD inhibitors (e.g., brivudin) 
• 
additional anti-cancer treatment (e.g., chemotherapy, biologics, etc.) except what is described 
in Section 6.3 
• 
experimental medications other than arfolitixorin 
• 
therapeutic radiation therapy. Palliative radiation can be approved by sponsor on a case by 
case basis. 
If any of the foregoing treatments are considered essential during the study the patient is to 
discontinue IMP as per section 7.1.  
6.6.2 AUTHORIZED MEDICATION 
Throughout the study, patients will receive medication required for their health condition at the 
discretion of the Investigator or treating physician, if not included in the list of prohibited medication 
(section 6.6.1). All concomitant treatment administered at any time, from the screening visit until the 
End of Treatment visit, will be recorded in the eCRF on a medication form (including the generic name 
of the medication, the medical indication/AE, the daily dose, the route of administration and the start 
and end dates). After IMP discontinuation, further anti-cancer therapies will be recorded in the eCRF, 
until death or end of study. After the last administration of study treatment, the patients will be 
treated according to local clinical practice. 
Also, the use of vitamins and dietary supplements will be recorded in the eCRF. 
6.6.3 RESCUE MEDICINE 
Not applicable 
7 
IMP DISCONTINUATION AND PATIENT DISCONTINUATION 
 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
The IMP treatment can be discontinued based on the decision of the patient, the Investigator, or the 
Sponsor, as follows: 
• 
Patients may voluntarily discontinue the IMP at any time without providing a reason and 
without any prejudice to future treatment.  


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• 
The Investigator may, at their discretion, discontinue the IMP at any time if deemed to be in 
the best interest of the patient (including AEs, complete response, or symptomatic 
deterioration), or due to incorrect randomization, or pregnancy.   
• 
The Sponsor may also instruct the Investigator to discontinue treatment with IMP in the event 
of severe non-compliance with the protocol (e.g., refusal to adhere to the treatment schedule 
or other protocol related assessments), or a protocol deviation that could affect the safety or 
well-being of the patient, or if new safety information showing unfavorable data becomes 
available.   
Patients prematurely discontinuing the IMP are to be treated according to local routine. Patients will 
be asked to complete an end of treatment visit within 30 days of last dose of IMP and before start of 
new anti-cancer treatment. 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Patients are free to withdraw from the study at any time, without necessarily providing a reason and 
without prejudice to further treatment. 
A patient who withdraws consent will always be asked the reason(s) for the withdrawal and the 
presence of any AEs. Patients will also be asked the following: 
• 
To complete an End of Treatment visit. 
• 
To permit the Investigator to follow-up (including by telephone) and provide data to the 
Sponsor regarding OS and anti-cancer therapy. 
• 
If the patient does not have PD at the time of withdrawal, to permit the Investigator to 
continue to provide data to the Sponsor regarding PD (either or both the Investigator’s 
determination of PD or any future CT-scans/MRIs) and anti-cancer therapy. 
Every effort will be made to minimize the number of patients withdrawing from the trial or lost to 
follow-up. A patient may withdraw from study treatment, however still continue to be followed until 
PD and/or death. Only patients who explicitly withdraw their consent or who are completely lost to 
follow-up will be considered “dropouts”. Data for these patients will be kept in the data base, unless 
the patient request that all his/her data to be deleted from the data base. 
Randomized patients who have received IMP and withdraws from participation in the study will not 
be replaced. 
7.3 
LOST TO FOLLOW-UP 
To minimize lost to follow-up in the study, each site should ensure contact details (including relative) 
are updated when the patient is entering the study. A study patient will be considered lost to follow-
up if he or she fails to return for scheduled visits and is unable to be contacted by the study site staff 
for six months after last study visit. Should the participant continue to be unreachable, he or she will 
be considered to have withdrawn from the study with a primary reason of lost to follow-up. 
Before declaring a patient to be “Lost to follow-up”, reasonable effort should be spent in following 
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• 
The site will attempt to contact the participant and reschedule the missed visit and counsel 
the participant on the importance of maintaining the assigned visit schedule and ascertain if 
the participant wishes to and/or should continue in the study. 
• 
Before a participant is deemed lost to follow-up, the Investigator or designee will make every 
effort to regain contact with the participant (where possible, 3 telephone calls and, if 
necessary, a certified letter to the participant’s last known mailing address or local equivalent 
methods). These contact attempts will be documented in the participant’s medical record or 
study file.  
8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 
EFFICACY ASSESSMENTS  
8.1.1 CT SCAN / MRI  
CT-scans or MRIs will be performed of the thorax, abdomen, and pelvis at screening and every 8 weeks 
after randomization until centrally confirmed PD. A radiologist at each study site/hospital (preferably 
the same radiologist for all patients at that hospital) is to evaluate each scan for target lesions. The 
following information is to be collected: 
• 
Patient ID 
• 
Date of procedure 
• 
Type of procedure 
• 
Progressive disease (Yes/No) 
It is preferred that the scans for a patient is taken with the same technique (CT or MRI) throughout 
the study. On occasions where it is not possible, for whatever reason, to use the same technique as 
for the screening scan the other technique should be used. The technique used for each scan will be 
documented in the eCRF. 
To minimize bias and have an objective assessment of the tumor response, a central imaging vendor 
(Banook Group) will perform independent and blinded response evaluations retrospectively. 
The central reading process will be done from screening until centrally confirmed PD by two different 
Central Readers (double review with adjudication). All the images will be sent to the Central Readers 
in a continues chronological sequence as outlined in the Site Procedure Manual. In order to remove 
the bias of interpolation and ensure the reader concentrates on the latest time-point images and has 
no idea that there are more to follow, each time-point images will be displayed, scored and locked in 
the database before the assessment of the following time point. 
The methodology applied at baseline regarding the identification of the target and non-target lesions 
is based on a common lesions selection process and an identical usage of those selected lesions by 
both Central Readers.  
The rationale for this lesion selection process at baseline is based on the fact that readers can 
potentially identify different target and non-target lesions that each considers representative of the 
patient's overall extent of disease. There is also the potential for the Central Readers to identify the 


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same lesions but classify them differently between target and non-target lesions. Discordance in lesion 
selection can reach 40% or even more. In addition, since lesions do not respond or progress at the 
same rate, differences between readers with regard to response or progression of lesions can be 
observed in the subsequent time-points. 
To minimize such important risk of discordance, the following process for the initial CT-scan or MRI 
will be applied. 
One of the two primary Central Readers is randomly selected to perform lesions selection on initial 
images: 
• 
Determines the number of target lesions and non-target lesions based on RECIST 1.1, 
• 
Communicates the target and not target lesions identification to the second primary Central 
Reader, via the central imaging vendor to preserve the independence of the two Central 
Readers. 
• 
Tracks lesions locations and labelling for up to 5 (2 per organ) target lesions and 10 non-target 
lesions on the subsequent time points. 
 
After having defined the type of targets and others lesions to be selected, the two primary Central 
Readers will carry out a separate and blinded measurement of the target lesions at Baseline:  
• 
If there is an agreement between Central Readers (Δ ∑ <20% and Δ <15% for each lesion), 
Central Reader will keep his own ∑ for the next time points, 
In case of discrepancies between Central Readers (Δ ∑ >20% or Δ >15% for any lesion), the Central 
Chair/adjudicator) will adjudicate the result of the two previous Central Readers and will provide the 
reference values for the baseline (for each target lesion). This value will be considered as reference by 
both Central Readers for the next time point analyses. 
8.1.1.1 INITIAL CT-SCAN OR MRI 
 
Tumor imaging at screening must be performed within 28 days prior to the date of randomization. 
Scans performed as part of routine clinical management are acceptable for use as screening tumor 
imaging if they are of diagnostic quality and performed within 28 days prior to the date of 
randomization and fulfill the requirements stipulated by the central imaging vendor.  
 
Overall review of the TAP (thorax, abdomen, pelvis) CT-scan or MRI for response assessment will only 
be performed on patients delivering an evaluable baseline time point and at least one evaluable 
follow-up time point. 
 
8.1.1.2 SUBSEQUENT TIME POINTS 
The two Central Readers will carry out a continuous reading at each complete patient in a blind way 
one versus the other. Initial images are intended to act as a lesion reference map for lesion 
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• 
The Central Readers perform lesion(s) measurements at each subsequent time point as 
described in the Central Review Charter,  
• 
The Central Readers assess the outcome of non-target lesions, 
• 
The Central Readers determine, if any, the number and location of new lesion(s), 
• 
The Central Readers provide an overall response assessment.  
8.1.1.3 RECIST CRITERIA 
Tumor response will be evaluated according to the following definitions, as per RECIST 1.1 criteria. 
(NB: Investigators are to rely on the locally-performed assessment for treatment guidance). The 
centrally-performed assessment will be the basis for evaluation of the study endpoints: 
Complete Response (CR) 
Disappearance of all target lesions. Any pathological lymph 
nodes (whether target or non-target) must have reduction 
in short axis to <10 mm. 
 
Partial Response (PR) 
At least a 30% decrease in the sum of the diameters of target 
lesions, taking as reference the baseline sum diameters. 
Progressive Disease (PD) 
At least a 20% increase in the sum of diameters of target 
lesions, taking as reference the smallest sum on study (this 
includes the baseline sum if that is the smallest on study). 
In addition to the relative increase of 20%, the sum must 
also demonstrate an absolute increase of at least 5 mm. 
(Note: the appearance of one or more new lesions is also 
considered progression). 
 
Stable Disease (SD) 
Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest 
sum diameters while on study. 
8.1.2 PHARMACOKINETICS  
The plasma concentration of [6R]-MTHF, methyl-THF, THF will be determined in approximately 20 
patients per treatment group. The sample size is not based on pop-PK modelling, but on feasibility as 
the sampling requires access to laboratory facilities at the site. Blood samples for determination of 
folate PK characteristics will be collected from those patients randomized at clinical sites qualified to 
ensure correct handling of samples in accordance with study-specific instructions.  
PK sampling procedures 
Plasma sampling will occur during Cycle 1 (Week 1) and Cycle 2 (Week 3). 
PK sampling for the experimental arm (Timings of PK samplings are approximate and actual sampling 
times will be captured in the PK sample log): 
1. Directly prior to the first arfolitixorin administration  
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3. 1 hour after the first arfolitixorin administration (NB: This sample will be taken after the 
second arfolitixorin dose)  
4. Directly prior to the second arfolitixorin administration 
5. 10 minutes after the second arfolitixorin administration  
6. 1 hour after the second arfolitixorin administration 
PK sampling schedule for the comparator arm (Timings of PK samplings are approximate and actual 
sampling times will be captured in the PK sample log): 
1. Directly prior to the initiation of the Leucovorin infusion  
2. 10 minutes after completion of the Leucovorin infusion  
3. 10 min after the 5-FU Bolus 
4. 1 h after initiation of 5-FU infusion 
In total 12/8 PK samples will be collected from each patient in the arfolitixorin/Leucovorin group 
participating in the PK part of the study yielding approximately 60/40 mL blood collected for this 
purpose. 
The study site should strive to take the blood samples as close to the suggested time-points as 
possible.  The exact sampling time will be recorded in the eCRF and will be used for the PK calculations. 
Plasma samples will be collected, handled, stored, and sent to Charles River Laboratories, (UK and 
USA) for analysis as described in study-specific laboratory instructions.  All samples collected for each 
patient will be analyzed at the same time. 
Bioanalytical method 
A validated bioanalytical method for the determination of levels of [6R]-MTHF, methyl-THF and THF is 
described in a separate bioanalytical method protocol. 
8.1.2.1 POPULATION PHARMACOKINETIC ANALYSIS 
The pharmacokinetics of [6R]-MTHF will be analyzed using a population PK approach. The specific 
objectives of the analysis are to: 
• 
Characterize the single and multiple dose pharmacokinetics, including associated variability, of 
[6R]-MTHF.  
• 
Evaluate the impact of potential covariates (body size, age, sex, race, ethnicity and renal 
function) on the pharmacokinetics of [6R]-MTHF. 
In addition to the concentration data collected in study ISO-CC-007 (see section 8.1.2 for the 
sampling schedule and anticipated number of patients), data from three Phase I/II studies in patients 
(ISO-MC-091, ISO-CC-002 and ISO-CC-005) and from one Phase I study in healthy volunteers (ISO-FF-
001) will be included in the population PK analysis. 


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The population PK analysis will be performed in a stepwise manner as outlined below.  
1. Exploratory graphical analysis of plasma concentrations versus time data and covariate 
distributions, for identification of potential outliers and to inform the further analysis. 
2. Develop a population PK model for [6R]-MTHF: 
a. Evaluate alternative structural and stochastic models to describe the typical [6R]-
MTHF PK profile. 
b. Investigate and characterize covariates that influence the volume of distribution, 
and clearance for [6R]-MTHF. 
c. Evaluate, and if necessary refine, the candidate final model. 
The population PK analysis will be described in a population PK report. The report will contain a 
thorough description of the data used in the analysis, including any data exclusions and handling of 
missing data; the key components of the model development, including the base model, covariate 
analysis and model evaluation; and full description and discussion of the final model.  
8.1.2.2 EXPOSURE-RESPONSE ANALYSIS 
The aim of the exposure-response analysis is to evaluate the suitability of the dose of 120 mg/m2. 
The individual [6R]-MTHF exposure (AUCaverage and Cmax), as established from the population PK 
analysis described above, will be used in the exposure-response analysis (see section 8.1.2 for 
anticipated number of patients). The following efficacy and safety parameters will be evaluated for a 
correlation with exposure:  
• 
ORR 
• 
Dose limiting toxicities 
• 
Adverse events  
 
In addition to the exposure-response data collected in this study (ISO-CC-007), data from study ISO-
CC-005 will be included in the exposure-response analysis. The analysis will be described in a 
separate report. The exposure-response report will contain a thorough description of the data used 
in the analysis, including any data exclusions and handling of missing data.  
8.1.3 ECOG PERFORMANCE STATUS  
The ECOG performance status scale will be used to quantify the extent to which the disease affects 
the daily living abilities of the patient on a scale from 0 to 4 (Appendix 1). The assessment will be 
performed at screening, baseline (cycle 1), each assessment visit, at the end of treatment visit, and at 
CT/MRI Follow-up visits until centrally confirmed PD. The scored values will be recorded in the eCRF.  
8.1.4 EQ-5D QUALITY OF LIFE 
EQ-5D (Appendix 3) patient reported outcome questionnaire will be used to assess the disease effects 
on the patient’s quality of life. The EQ-5D questionnaire will be completed by the patient prior to any 
study related activity at the visit for first administration of IMP, the assessment visits until PD and at 
the End of Treatment visit. The EQ-5D questionnaire will be completed by the patient on paper and 
then transferred to the eCRF by the study site staff. 
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Patients discontinuing study treatment and/or with centrally confirmed PD will move into the survival 
follow-up phase. Survival status will be assessed by telephone, ordinary visits, hospital records or 
other means found suitable every 12 weeks until death or the end of the study, whichever occurs first. 
Information regarding post-study anti-cancer therapies will be collected if new treatment is initiated. 
8.2 
SAFETY AND OTHER ASSESSMENTS 
8.2.1 LABORATORY SAFETY ASSESSMENTS  
The laboratory safety samples (hematology, clinical chemistry, and urinalysis), including pregnancy 
test, will be collected according to the protocol Schedule of Activities and in accordance with local 
practice and analyzed by local laboratories.  
The observed values will be recorded and assessed as “normal” or “abnormal” in the eCRF.  Abnormal 
values will be assessed by the Investigator as “clinically significant” or “not clinically significant”.  
Clinically significant values will be captured as Medical history or AEs, as applicable. 
The laboratory tests are to be performed within 7 days before next treatment and the results must be 
reviewed by the Investigator or qualified designee and found to be acceptable prior to the 
administration of study treatment. 
All required laboratory tests are specified in Table 3 . 
Table 3: Laboratory tests 
HEMATOLOGY 
CLINICAL CHEMISTRY 
URINALYSIS 
Hemoglobin 
 
Sodium 
Protein 
Hematocrit 
Potassium 
Glucose 
WBC 
Calcium 
Blood 
Neutrophils 
Glucose  
 
Lymphocytes 
BUN or urea  
Monocytes 
Creatinine  
Eosinophils 
AST  
Basophils 
ALT  
 
Platelet count 
Alkaline phosphatase  
 
 
Total bilirubin  
 
 
Total protein  
 
 
Albumin  
 
 
LDH  
 
 
gGT 
 
8.2.2 VITAL SIGNS, ECG AND PHYSICAL FINDINGS RELATED TO SAFETY  
Vital signs collected will include resting heart rate (beats per minute), systolic and diastolic blood 
pressure (mmHg; to be measured after at least five minutes of supine rest), weight (within 7 days prior 
to each study drug administration), and height (screening only).  
The physical examination is to be performed according to local clinical practice, and include 
neurological status, skin, and mucous membranes. 
A 12-lead ECG will be performed at treatment cycles 1 to 4. Information about the ECG examination 
must be present in the source documentation at the study site. Single 12-lead safety ECGs will be 


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recorded in supine or in reclining position (should be the same position for all measurements during 
the same day) using the site's ECG machine. 
• 
Group A (Arfolitixorin): ECG should be assessed directly prior to each arfolitixorin 
administration, and 10 min (± 5 min) after the completion of each administration (i.e. 4 ECG 
per treatment cycle). 
• 
Group B (Leucovorin): ECG should be assessed directly prior to Leucovorin infusion, and 10 
min (± 5 min) after start of Leucovorin infusion (i.e. 2 ECG per treatment cycle). 
Safety ECGs will be reviewed and interpreted on-site by the investigator. Significant findings present 
prior to the start of study treatment administration must be recorded in the Medical history section 
of the eCRF. Clinically significant findings made after the start of study treatment administration, and 
which meet the definition of an AE, must be recorded in the eCRF. QTc abnormalities should be 
reassessed after 10 minutes to ensure normalization. If a patient develops a change-from-baseline in 
QTcF > 60 ms or a QTcF interval of > 500 ms, ECG should be monitored until normalized, and the 
patient should be discontinued from IMP treatment. 
8.2.3 PRO CTCAE 
The PRO CTCAE (Appendix 2) adverse event self-assessment questionnaire will be completed by the 
patient immediately after completion of the EQ-5D questionnaire during the first 4 IMP administration 
visit, during the assessment visits until PD and at the End of Treatment visit. The PRO CTCAE will be 
completed by the patient on paper, and then transferred to the eCRF by the site staff. The PRO CTCAE 
questionnaire will only be used in countries where there is a validated translation available at the time 
of study submission to IRB/IEC. 
The Investigator must always conduct an ordinary AE assessment during all study visits independently 
of the information provided by the patient in the PRO CTCAE. 
AEs to be assessed by PRO CTCAE is: 
• 
Mucositis 
• 
Nausea 
• 
Vomiting 
• 
Diarrhea 
• 
Pain in the abdomen 
• 
Neuropathy 
• 
Fatigue 
The above AEs are the most frequently reported AEs from the previously conducted and ongoing 
Phase I/II studies with arfolitixorin in combination with 5-FU, and also including the most commonly 
reported AEs for the anti-cancer treatment combination used in this protocol (modified FOLFOX-6 + 
bevacizumab) [14, 29]. 
8.2.4 PHARMACOGENETICS 
Level of gene expression will be analyzed at a central laboratory (TATAA Biocenter, Gothenburg, 
Sweden) to determine drug metabolism- and transportation-related gene expression levels in all 
patients.  The specifications and logistics for the gene analysis (preparation and shipment of FFPE 
tissue slices from tumor biopsies to the central laboratory) are described in a separate study-specific 


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laboratory instruction provided to the sites.  The relative gene expression levels will be analyzed for 
the following genes: ABCC1, ABCC3, ABCC5, CES2, DPYD, ERCC1, FGF7, MTHFD2, SHMT1, SHMT2, 
SLC46A1, SLC19A1, SLC24A32, TGFB1, TYMS, ACTB and PPIA. The gene expression analysis will be 
performed retrospectively. 
8.2.5 RESECTION/REMOVAL OF TUMOR LESION(S)  
For patients assessed by the investigator as eligible for metastasis and/or primary tumor resection 
during the IMP treatment phase, treatment with the study regimen may be continued at the 
investigator discretion. The therapy will be registered in the eCRF including if continued study 
treatment is intended as well as planned delay until continued treatment. The EoT visit will be 
conducted when the IMP treatment has been completed. If the patient discontinues IMP treatment 
in conjunction with the resection, the EoT visit will be conducted within 30 days after last dose of 
IMP. Patients undergoing metastasis and/or primary tumor resection will be followed for disease 
recurrence by central imaging and Overall Survival until death or the end of the study. 
 
8.3 
STUDY SCHEDULE 
8.3.1 SCREENING AND ENROLLMENT 
Investigators must keep a record of all patients enrolled in to the study. 
During the screening period, Investigators will: 
• 
Obtain signed informed consent from the patient before any study specific procedures are 
performed 
• 
Assign the patient a unique Patient Study Identifier (ID) 
• 
Determine patient eligibility 
• 
Record patient demographic data (date of birth, gender and race) and gene mutations (BRAF, 
KRAS, NRAS and other) where this information is available in the patient´s medical records. 
• 
Record medical history 
• 
Record all concomitant therapies 
• 
Conduct physical examination and record vital signs 
• 
Assess ECOG status 
• 
Record weight and height 
• 
Collection of blood sample for hematology (within 7 days before IMP administration) 
• 
Collection of blood sample for clinical chemistry (within 7 days before IMP administration) 
• 
Collection of urine sample for urinalysis (within 7 days before IMP administration) 
• 
Conduct pregnancy test for all women of childbearing potential (within 7 days before IMP 
administration) 
• 
Conduct CT scan / MRI 
• 
Ensure availability of tumor tissue biopsy 
If a patient withdraws from participation in the study, then his/her ID code must not be reused. 
Investigators must file Patient Identification Lists of all patients enrolled that include sufficient 
information to link eCRF data with clinical records.  This list should be available at the study site for 
possible future inspections/audits. Sponsor staff or representatives should only have access to the list 
for monitoring or auditing purposes. 


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If a CT-scan/MRI was performed before enrolment a new scan is not required, provided that the scan 
occurred no more than 28 days before start of treatment and can be assessed by the central imaging 
vendor. 
Study treatment should be initiated within 28 days after enrolment. 
8.3.2 BASELINE (CYCLE 1) 
During the first treatment visit the following will occur: 
• 
Collect PRO EQ-5D (prior to any other study specific activity) 
• 
Collect PRO CTCAE (immediately after EQ-5D) 
• 
Assessment of the inclusion/exclusion criteria 
• 
Randomization (should be done within three days before study treatment initiation) 
• 
Record adverse event assessment between visits 
• 
Record all concomitant therapies added and/or changed 
• 
Conduct physical examination and record vital signs 
• 
Record weight  
• 
Conduct ECG  
• 
Assess ECOG status 
• 
Administer and record study treatments (i.e. 1st cycle) 
• 
Collection of blood sample for PK (only at pre-selected sites)  
8.3.3 TREATMENT PERIOD 
8.3.3.1 TREATMENT VISITS 
Treatment visits will occur every 14 days (+7 days) until PD.  
During all following treatment visits, the following will occur: 
• 
Collect PRO CTCAE, treatment visits 2-4 (prior to any other study specific activity) 
• 
Record adverse event/toxicity assessment between visits and during IMP administration 
• 
Record all concomitant therapies added and/or changed 
• 
Record vital signs 
• 
Record weight 
• 
Conduct ECG, treatment visits 2-4 
• 
Collection of blood sample for hematology (results evaluated prior to IMP administration) 
• 
Collection of blood sample for clinical chemistry (results evaluated prior to IMP 
administration) 
• 
Collection of urine sample for urinalysis (results evaluated prior to IMP administration) 
• 
Administer and record study treatments 
• 
Collection of blood sample for PK at cycle 2 (only at pre-selected sites)  
At each treatment visit, the collection of blood and urine samples and the recording of vital signs must 
occur before start of treatment. Following a treatment-related adverse event the doses of 5-FU, 
oxaliplatin, and bevacizumab can be adjusted or delayed in accordance with the label and protocol 
section 6.3.4. Oxaliplatin cannot be substituted but caution should be taken regarding toxicity and in 


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case of toxicity, dose modifications (decrease in infusion rate and/or dose reductions) according to 
label should be performed. In case the 5-FU is delayed the IMP will be delayed. 
8.3.3.2 ASSESSMENT VISITS (EVERY 8 WEEKS UNTIL PD) 
Assessment visits are to occur every 8 weeks (±7 days) after the baseline visit (1st IMP administration 
day). During these visits, the following will occur: 
• 
Collect PRO EQ-5D (prior to any other study specific activity) 
• 
Collect PRO CTCAE (immediately after EQ-5D) 
• 
Record adverse event/toxicity assessment between visits 
• 
Record all concomitant therapies added and/or changed 
• 
Conduct physical examination and record vital signs 
• 
Record weight 
• 
Assess ECOG status 
• 
Conduct CT scan / MRI 
• 
Evaluate disease status 
Assessment visits should continue until PD or premature discontinuation of IMP.  
8.3.4 END OF TREATMENT VISIT (EOT) 
All patients discontinuing IMP should complete an EOT visit within 30 days of discontinuation (or as 
soon as possible thereafter). The EOT visit should preferably be conducted before initiation of new 
anti-cancer therapy. During this visit, the following will occur: 
• 
Collect PRO EQ-5D (prior to any other study specific activity) 
• 
Collect PRO CTCAE (immediately after EQ-5D) 
• 
Record adverse event/toxicity assessment prior this visit.  
• 
Record all concomitant therapies added and/or changed 
• 
Conduct physical examination and record vital signs 
• 
Record weight 
• 
Collection of blood sample for hematology 
• 
Collection of blood sample for clinical chemistry 
• 
Conduct pregnancy test for all women of childbearing potential 
• 
Collection of urine sample for urinalysis 
• 
Assess ECOG status 
• 
Conduct CT scan / MRI (if previous scan is >14 days) 
8.3.5 POST-TREATMENT VISITS 
At the time of final analysis of ORR and PFS, patients still on IMP treatment will continue IMP 
treatment until locally assessed progressive disease or discontinuation of IMP for other reasons. After 
completed End of Treatment visit these patients have reached the End of Study definition and will 
thereafter move into survival follow-up until 60% of OS events are achieved for the study population.  
Patients already in follow-up phase at the time of final analysis of ORR and PFS have reached the End 
of Study definition and will move into survival follow-up until 60% of OS events are achieved for the 
study population. 


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8.3.5.1 FOLLOW-UP VISITS UNTIL PROGRESSIVE DISEASE 
Patients who discontinue IMP for reasons other than centrally confirmed PD will move into the Follow-
up phase and will be continuing to be assessed every 8 weeks. During this visit, the following will occur: 
• 
Conduct CT scan / MRI 
• 
Assess ECOG status 
• 
Post-study anti-cancer therapies will be collected if new treatment is initiated 
• 
Collect SAEs related to study procedures  
• 
Collect SAEs considered to be at least possibly related to IMP 
Every effort should be made to collect information regarding disease status until PD, death, or the end 
of the study. 
8.3.5.2 SURVIVAL FOLLOW-UP 
Patients discontinuing study treatment and/or with centrally confirmed PD will move into the survival 
follow-up phase. Patient survival status will be assessed every 12 weeks until death or the end of the 
study, whichever occurs first. Patient survival data can be collected by phone, visit, medical records, 
contact with relatives etc. Date of death, or the Investigators best estimate of the date of death, 
should be entered in the eCRF. 
Information regarding post-study anti-cancer therapies will be collected if new treatment is initiated. 
8.4 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.4.1 DEFINITION OF ADVERSE EVENTS (AE) 
 
Any untoward medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. An adverse event can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding, for example), symptom or disease temporally associated with the use 
of a medicinal product, whether or not considered related to the medicinal product. 
Progression of the cancer under study is not considered an adverse event since the development of 
the cancer will be followed and reported separately. 
 
8.4.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
A serious adverse event is any AE occurring at any dose that results in any of the following outcomes: 
• 
Death 
• 
Is life-threatening  
- 
A life-threatening adverse drug experience is any AE that places the patient, in the 
view of the Investigator, at immediate risk of death from the reaction as it occurred. 
This does not include a reaction that, had it occurred in a more severe form, might 
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• 
A persistent or significant disability/incapacity 
• 
Inpatient hospitalization or prolongation of existing hospitalization, except that planned 
hospitalization during days in clinic and surgery or hospitalization for the social reasons or the 
convenience of the patient or physician shall not be considered an SAE  
• 
A congenital anomaly/birth defect 
• 
Important medical event 
- 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be serious when, based upon appropriate medical judgment, the 
event may jeopardize the patient and require medical or surgical intervention to 
prevent one of the outcomes listed above. 
Examples of such medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. 
The following events are not considered to be SAE:  
• 
progression of the cancer under study since the development of the cancer will be followed 
and reported separately. 
• hospitalizations for the routine treatment or monitoring of the studied indication not 
associated with any deterioration in condition.  
• 
hospitalizations for the treatment, which was elective or pre-planned, for a pre-existing 
condition that is unrelated to the indication under trial and did not worsen 
• 
hospitalizations for the admission to a hospital or other institution for general care, not 
associated with any deterioration in condition 
• 
hospitalizations for the treatment on an emergency, outpatient basis for an event not 
fulfilling any of the definitions of serious given above and not resulting in hospital admission.  
8.4.3 CLASSIFICATION OF AN ADVERSE EVENT 
8.4.3.1 SEVERITY OF EVENT 
The severity (intensity) of AE in physical signs or symptoms will be graded by the investigator 
according to the NCI-CTCAE Version 5.0 [30]. For AEs not listed in NCI-CTCAE, the severity of AE in 
physical signs or symptoms will be classified as follows: 
• 
Grade 1: Mild asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated.  
• 
Grade 2: Moderate; minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental activities of daily living*.   
• 
Grade 3: Severe or medically significant but not immediately life-threatening; hospitalization 
or prolongation of hospitalization indicated; disabling; limiting self-care activities of daily 
living**.  
• 
Grade 4: Life-threatening consequences; urgent intervention indicated.  
• 
Grade 5: Death related to AE. 


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*Instrumental activities of daily living refer to preparing meals, shopping, using the telephone, 
managing money, etc.  
**Self-care activities of daily living refer to: bathing, dressing and undressing, eating, using the toilet, 
taking medications, and ability to get out of the bed. 
 
 
8.4.3.2 
RELATIONSHIP TO STUDY INTERVENTION 
Causality regarding relationship to administration of any of the medications in the study treatment 
regimen, including both IMP (arfolitixorin/Leucovorin) and non-IMP (5-FU, oxaliplatin, and 
bevacizumab) will be assessed by the investigator as: 
Related 
A clinical event, including laboratory test abnormality, with a reasonable time 
sequence to administration of the treatment, unlikely to be attributed to 
concurrent disease or other drugs or chemicals, which follows a clinically 
reasonable response on withdrawal (de-challenge), and which is definitive 
pharmacologically or phenomenologically (i.e., an objective and specific medical 
disorder or a recognised pharmacological phenomenon).  Re-challenge 
information may be required to fulfil this definition. 
Probable 
A clinical event, including laboratory test abnormality, with a reasonable time 
sequence to administration of the treatment, unlikely to be attributed to 
concurrent disease or other drugs or chemicals, and which follows a clinically 
reasonable response on withdrawal (de-challenge).  Re-challenge information is 
not required to fulfil this definition. 
Possible 
A clinical event, including laboratory test abnormality, with a reasonable time 
sequence to treatment, but which could also be explained by concurrent disease 
or other drugs or chemicals.  Information on IMP withdrawal may be lacking or 
unclear. 
Unlikely 
A clinical event, including laboratory test abnormality, with a temporal 
relationship to treatment which makes a causal relationship improbable, and in 
which other drugs, chemicals, or underlying disease provide plausible 
explanations. 
Not Applicable 
Expected only when the drug has not been administered. 
For SUSAR reporting, the three following criteria, Related, Probable and Possible will correspond to 
the criteria “Reasonable possibility of a causal relationship”. The Unlikely criteria will correspond to 
the criteria “No reasonable possibility of a causal relationship”. 
 
8.4.3.3 EXPECTEDNESS  
 
Expected adverse reactions are AEs that are known to occur for the study intervention being studied.  
Expectedness is assessed based on the awareness of AEs previously observed, not on the basis of 
what might be anticipated from the properties of the study intervention.  


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An adverse reaction is considered as unexpected if the nature or severity of this is not consistent 
with the applicable product information presented in the Investigator's Brochure or in a summary of 
product characteristics (SPC) for an authorized product. 
The IB presents a review of the safety data from clinical trials which identified fatigue and nausea as 
the most common ARs. However, they were no more frequent than could be expected for the same 
cytotoxic agents in the presence of other folates. 
The Sponsor will be responsible for determining whether an SAE is expected or unexpected.  An SAE 
will be considered unexpected if the nature, severity or frequency of the event is not consistent with 
the risk information previously described. 
 
8.4.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
AEs and SAEs will be reported in the eCRF from the time of signing informed consent form until End 
of Treatment visit, whether or not considered to be drug related.  
• 
From the time of signing informed consent form until randomization, only AEs causing the 
patient to drop out of the study or being related to study procedure will be reported in the 
eCRF.  
• 
After the End of Treatment visit, only SAEs related to study specific procedures or considered 
to be at least possibly related to study drug should be recorded 
All SAEs and AESIs will be entered in the eCRF and followed until satisfactory resolution or until the 
site Investigator deems the event to be chronic or the participant is stable. Other supporting 
documentation of the event may be requested by the study sponsor and should be provided as soon 
as possible. 
Before database closure, reconciliation between the SAEs entered in the safety database and the 
study database will be performed. 
8.4.5 ADVERSE EVENT REPORTING  
AEs reported by the patient or observed by the Investigator will be individually listed in the eCRF. 
Including diagnosis, signs and symptoms (if a diagnosis is not available), date of onset, duration, action 
taken, severity, outcome to date, relationship to any of the treatment regimen drugs or study 
procedure and seriousness will be recorded. The severity of AEs will be graded according to the 
National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE Version 5.0) 
toxicity criteria. 
At each visit, the Investigator will be prompted to report causality of AEs as “Related”, “Probable,” 
“Possible” or “Unlikely” related to the IMP, any anti-cancer treatment regimen or study procedure. 
8.4.6 ADVERSE EVENT OF SPECIAL INTEREST  
AEs related to mucositis and bone marrow toxicity will be followed as AESIs in this study, to ensure 
that there are no significant safety issues when arfolitixorin, instead of Leucovorin, is combined with 
5-FU in the mFOLFOX-6 + Bevacizumab regimen. AESI will be followed as ordinary AEs but reported 
separately in the study report as described in the SAP. 
8.4.7 SERIOUS ADVERSE EVENT REPORTING  


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The reporting of SAEs will be done through the eCRF. Only in the event of the eCRF not being available 
SAEs should be reported by fax or e-mail on the paper SAE report form found in the Study File.  
Fax: +33 (0) 467 107 253 
or 
Email: isofol-safety@vigipharm.fr 
The Investigator is responsible for ensuring that all SAEs are reported to the Sponsor immediately, but 
in any event no later than 24 hours of any site staff becoming aware of the event.  Initial reports should 
be followed as soon as possible by detailed reports.  The initial and follow-up reports should identify 
patients by unique study code (Study ID). The patients’ names, personal identification numbers, and 
addresses must not be included in the report. The following information is mandatory for the initial 
report: 
• 
Patient Study ID 
• 
Study treatment  
• 
Start date (time, if relevant) of the study treatment 
• 
Brief description of the event (diagnosis, or signs/symptoms if diagnosis is not available) 
• 
Start date (time, if relevant) of the event 
• 
Seriousness criteria 
• 
Causality assessment 
Detailed instructions describing the procedure of reporting SAEs including SUSARs will be distributed 
to the site and all involved parties. 
For reported deaths, the Investigator should supply the Sponsor and the IRB/IEC (if applicable) with 
any additional requested information (e.g., autopsy reports and terminal medical reports). 
The investigator is responsible to report the events to the Sponsor according to the protocol to allow 
the Sponsor to meet their SUSAR reporting obligations. The Sponsor is responsible for reporting 
SUSARs to the competent authorities. The IRB/IEC will be informed according to local requirements 
by either the Sponsor or the PI.   
8.4.8 REPORTING EVENTS TO PARTICIPANTS  
Sponsor will inform Investigators of any SUSAR, discovered in this or in any other study during the 
study period. It is the investigators responsibility to inform the patients if deemed necessary. 
8.4.9 REPORTING OF PREGNANCY  
Female patients will be instructed to notify the Investigator immediately if they become pregnant 
during the study.  Male patients will be instructed to notify the Investigator immediately if their 
partner becomes pregnant.  Pregnant patients will be withdrawn from further study treatment.  The 
patients will also be instructed to report pregnancies discovered after the last visit if they believe that 
conception occurred during their participation in the study.   
A pregnancy is not an AE, unless there is a possibility that the IMP has interfered with the efficiency 
of any contraceptive measures.  However, the Investigator should report pregnancies according to the 
procedures and timelines described for reporting of SAEs. The pregnancy report form should be used 
instead of the SAE form. 


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Pregnant patients or partners will be followed until the end of the pregnancy.  Any complication during 
the pregnancy will be reported as an AE.  The outcome of the pregnancy must be reported on the 
pregnancy report form.  Any spontaneous abortion, stillbirth, birth defect/congenital anomaly, death, 
or other serious infant condition must be reported and followed up as an SAE. 
 
 
 
9 
STATISTICAL CONSIDERATIONS  
 
9.1 
STATISTICAL HYPOTHESES 
The primary end-point is ORR. Null and alternative hypotheses are expressed in terms of ∆ORR, the 
difference in ORR between the randomized treatment arms:  
• 
H0: ∆ORR=0 
• 
H1: ∆ORR≥15% 
 
 
9.2 
SAMPLE SIZE DETERMINATION 
 
9.2.1 PRIMARY EFFICACY ENDPOINT: ORR  
 
For this adaptive design, the initial sample size calculation is based on an improvement by 15% in 
ORR; specifically, ORR is assumed to be 45% in the control arm vs. 60% in the experimental arm. It is 
assumed that up to 10% of patients will be unevaluable for response in both treatment arms. These 
patients will be considered non-responders and will not contribute to the difference between 
experimental and control (hence the expected difference is 13.5% rather than 15%). For a two-sided 
test with 𝛼𝛼=0.05 and a power of 80% to detect this difference, at least 440 patients need to be 
randomized. At a sample size of 330 (75% of the information fraction for ORR), interim results will be 
used for adaptive sample size re-estimation, based on the conditional power for both ORR and PFS, 
without a formal interim analysis of efficacy or futility. This is detailed in the interim analysis plan. 
The statistical design allows for a 50% sample size increase (to 660 patients), should interim results 
be promising enough to warrant such a sample size increase. In case the sample size is increased, 
this provides 94% power to detect the same difference, i.e. 13.5%, for ORR. 
 
Initially, approximately 440 were planned to be randomized. However, Japanese authorities 
requested that approximately 12.7% of the total study population are enrolled from Japan. For this 
reason, additional patients from Japan were randomized. In the end a total of 490 patients were 
randomized. This decision to randomize extra Japanese patients was made before the interim 
analysis. The decision to include the Japanese patients in the ITT population (see Section 9.2.2) for 
the primary efficacy analysis was made after the interim analysis. The addition of extra Japanese 
patients leads to a statistical power greater than 80%. 
 
Blinded Independent Central Review (BICR) is the process by which radiographic exams obtained as 
part of a clinical study are submitted to a central location for review by independent physicians who 
are not involved in the clinical study. Regulatory authorities have recommended BICR of all patients 
(or a cohort of patients) for oncology registration studies when the primary study endpoint is based 
on tumor measurements, such as PFS, DOR or ORR [31]. 


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As the ISO-CC-007 study is an open-label study, it is essential to keep the assessors for the primary 
endpoint blinded to treatment allocation. 
 
The comparator arm in the ISO-CC-007 study consist of Leucovorin, administered together with 5-
FU, oxaliplatin (the mFOLFOX-6 regimen) + bevacizumab. The efficacy for this treatment 
combination has been assessed by blinded independent central review only in a few clinical Phase III 
studies [14, 27]. The ORR in these trials range from 38 to 48 %. For the ISO-CC-007 study, we have 
therefore assumed an ORR of 45% for the comparator arm.   
 
9.2.2 SECONDARY EFFICACY ENDPOINT: PFS 
 
The study is also powered to detect a hazard ratio of 0.725, which corresponds to a clinically 
meaningful difference in PFS (median PFS equal to 10 months in the control arm vs. 13.8 months in 
the experimental arm).  For a power of 80%, 300 PFS events need to be observed using a 1-sided 
𝛼𝛼= 0.025. The statistical design allows for a 50% sample size increase (to 660 patients), should 
interim results be promising enough to warrant such a sample size increase. In case the sample size 
is increased, the target number of events will be increased to 450 PFS events, which provides 80% 
power to detect a hazard ratio of 0.77 (median PFS equal to 10 months in the control arm vs. 13 
months in the experimental arm). 
 
At the time of writing of this protocol amendment, the interim analysis with the possibility of an 
adaptive sample size increase, already took place. The DSMB did not advice to increase the sample 
size. Furthermore, as the number of patients censored because of new anti-cancer therapies, was 
higher than expected, it became clear during the study that the 300 PFS events could not be 
reached. Therefore it was decided to include all Japanese patients in the ITT population, increasing 
the total sample size from the planned 440 to 490 patients in total, and to decrease the number of 
PFS events at the final analysis from 300 to at least 235.  
Table 4 shows, for 300 and 235 PFS events, the hazard ratio for which the trial would have 80% 
power, the hazard ratio for which the trial would just reach statistical significance, and the 
corresponding median PFS in the treatment arm, assuming a 10-month median PFS in the control 
arm. 
 
Table 4: Hazard ratio for significance in function of the number of PFS events  
 
Design 
Final analysis 
Number of 
PFS events 
Median PFS  
Control arm 
Hazard 
ratio 
Median PFS  
Treatment arm 
Hazard ratio for 
significance 
Median PFS  
Treatment arm 
300 
10 months 
0.725 
13.8 months 
0.80 
12.5 months 
235 
10 months 
0.692 
14.5 months 
0.77 
12.9 months 
 
A hazard ratio of 0.77 was considered a clinically relevant threshold for statistical significance.  The 
final PFS analysis will therefore be performed when at least 235 PFS events will be reached. 
 
PFS analysis will be based on the randomized treatment. The censoring rules and details of the 
sensitivity analyses are specified in the study specific statistical analysis plan.   
9.2.3 HIERARCHICAL TESTING APPROACH 
 


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A fixed sequence hierarchical test procedure will be applied, with a pre-defined order in which the 
hypotheses are to be tested. Each test will be performed without a multiplicity adjustment for 
multiple endpoints, i.e. at the nominal 𝛼𝛼-level foreseen by the group sequential design. The 
secondary endpoint PFS can only be tested formally after the first hypothesis, i.e. the null hypothesis 
for ORR, is rejected at the final analysis. 
 
9.2.4 OVERALL SURVIVAL 
 
An analysis of OS at the same time as the final PFS analysis has a 30% power to demonstrate a 
hazard ratio of 0.8 (corresponding to a median OS equal to 25 months in the control arm vs. 31.25 
months in the experimental arm), when approximately 180 events are observed, using a 1-sided 
α=0.025. If the sample size is increased to 660 patients, analyzed at the same time as the final PFS 
analysis, this power is increased to 45%, when approximately 270 events are observed. The addition 
of the extra Japanese patients (see Section 9.2.2) to the 440 initial patients, resulting in a total 
number of 490 randomized patients, leads to a statistical power slightly greater than 30%. No 
interim efficacy analysis is foreseen for OS. Note that this trial is underpowered for OS, and that 
there is no intention to claim efficacy for OS.  
 
Given the key relevance of this endpoint in an overall assessment of benefit/risk, it is important to 
power the OS analysis to exclude a significant detrimental effect of arfolitixorin as compared to 
leucovorin in terms of OS. The OS analysis is based on the following considerations: 
 
1) Collection of vital status will continue in all randomized patients after the final PFS analysis, until 
the OS analysis. Of note, the aim for this continued follow-up is to monitor the patients’ safety, 
rather than showing superior efficacy of arfolitixorin in terms of OS.  
2) Arfolitixorin is not expected to cause serious toxicity. It is therefore reasonable to assume that if 
superior efficacy of arfolitixorin is demonstrated, OS is unlikely to be inferior in the arfolitixorin arm 
compared to the leucovorin arm. The magnitude of the OS difference is hard to predict, for example 
due to the multiple lines of active therapies currently available for the treatment of patients with 
advanced colorectal cancer. Assuming similar post-progression survival in both trial arms, a 
reasonable assumption is that the difference in median PFS between arms will “carry over” to the 
difference in median OS.  
3) A “significant detrimental effect on OS” will be taken equal to the benefit of leucovorin estimated 
in the meta-analysis of all trials comparing 5-fluroruracil alone with 5-fluroruracil + leucovorin [33]. 
The overall OS HR of leucovorin was equal to 0.9 in these trials; hence, an OS hazard ratio equal to 
1/0.9 = 1.11 in the current trial may be considered a significant detriment, to the extent that it will 
outweigh the benefit observed historically with leucovorin.  
4) Collection of vital status will continue after the ORR and PFS analysis, until 60% of the patients 
have had a death event. If the trial accrues 490 patients, 294 deaths, or 60% of 490, will be 
observed. 
 
9.2.5 ACCRUAL RATE 
 
The accrual rate will be assumed to ramp up as follows: 2, 3, 7, 11, 14, 22 and 25 patients 
respectively in the 1st to 7th months after study start, and 28.8 patients monthly thereafter. 
 
9.3 
POPULATIONS FOR ANALYSES 
 
All enrolled patients who were randomized will be included in the analyses. The following  
analysis datasets will be considered: 
 


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• Intention-to-Treat (ITT) analysis set consists of all randomized participants. Analyses will 
be based on the randomized treatment. 
• Intention-to-Treat Excluding Additional Japanese Patients (ITTE) analysis set consists of 
all randomized participants excluding additional Japanese patients. Analyses will be based 
on the randomized treatment. 
• Per-Protocol (PP) analysis set defines a subset of the participants in the ITT 
analysis set without major protocol deviations. The definition of major protocol 
deviations will be agreed upon, and all cases of such major deviations adjudicated prior to 
database lock. Analyses will be based on the randomized treatment. 
• Safety analysis set defines the subset of participants for whom safety analyses will be 
conducted. It includes all patients who received at least one dose of study medication. 
Analysis will be based on the actual treatment. 
• PK analysis set defines a subset of the participants in the Safety analysis set for 
whom PK samples were analyzed. Analyses will be based on the actual treatment. 
 
9.4 
STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
 
Categorical data will be summarized in contingency tables presenting frequencies and percentages. 
Continuous data will be summarized using number of non-missing values (n), mean, standard 
deviation, median, minimum and maximum values. Significance tests will be performed at the α = 
0.05 significance level for two-tailed tests and at α = 0.025 for one-tailed tests. Methods to calculate 
confidence intervals will be specified in Statistical Analysis Plan.  
 
9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) 
 
The primary end-point is ORR based on BICR assessment of CT/MRI scans, and will be analyzed both 
for the ITT and PP population.  
ORR will be analyzed using a Cochran-Mantel-Haenszel test (CMH), stratified for the stratification 
factors used for randomization (geographic region, primary tumor location and previous neo-
adjuvant/adjuvant CRC treatment).   
 
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
 
Secondary efficacy endpoints are PFS, DOR, OS, quality of life (EQ-5D questionnaire), curative 
metastasis resection, and safety and tolerability (including PRO CTCAE).   
 
PFS and DOR will be based on BICR assessment of the patients CT/MRI scans. PFS, DOR and OS will be 
analyzed using Kaplan-Meier curves, the logrank test and a stratified Cox proportional hazards model, 
using the same stratification factors as for randomization (geographic region, primary tumor location 
and previous neo-adjuvant/adjuvant CRC treatment). The assumption of proportional hazards will be 
tested. In addition to the p-value, based on a stratified logrank test, and the Hazard Ratio (with 95%CI), 
descriptive statistics will also be presented for time to event endpoints (Number of patients used for 
analysis, number of events, median months to event and 95%CI).  
 
The EQ-5D questionnaire results will be analyzed in a descriptive way, e.g. by means of graphical 
representation. Number of patients undergoing curative metastasis resection will be compared 


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between the treatment groups. Further evaluation of the results will be specified in the statistical 
analysis plan which will be in place before including the first patient in the study.  
 
 
9.4.4 SAFETY ANALYSES 
 
Safety variables include the reported AEs, SAEs, laboratory tests, vital signs and physical examination. 
AEs will be coded and evaluated for severity using NCI-CTCAE Version 5.0 and will be summarized by 
MedDRA system organ class and preferred term. Separate summaries and listings will include the 
following: 
• 
All adverse events 
• 
Adverse events of CTC grade ≥3 
• 
Related adverse events 
• 
Fatal adverse events 
• 
Discontinuations due to adverse events or deaths 
• 
Serious adverse events  
• 
Deaths on IMP treatment or within 30 days of study drug discontinuation 
• 
Patient reported outcome on specific AEs on PRO CTCAE (See Appendix 2) for countries where 
valid translations are available (See Appendix 2) 
 
The NCI PRO-CTCAE questionnaire results will be analyzed in a descriptive way, e.g. by means of 
graphical representation. Further evaluation of the results will be specified in the statistical analysis 
plan.  
 
Laboratory tests will be summarized at each timepoint, including change from baseline. Summaries 
will be presented separately for hematology, blood chemistry and urinalysis parameters. Other safety 
data (vital signs, physical examination) will also be tabulated and listed.  
 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
 
The study treatment groups will be compared on baseline characteristics, including demographics 
and laboratory measurements, using descriptive statistics. No inferential statistics will be used. 
 
9.4.6 PLANNED INTERIM AND FINAL ANALYSES  
 
9.4.6.1  ANALYSIS PLAN 
The trial design will include the following analyses (and adaptations if indicated): 
• 
Based on the interim results of 330 patients (75% information fraction for ORR), who have a 
follow-up of at least 16 weeks, the DSMB may recommend a 50% sample size increase (from 
440 to 660 patients) according to the guidelines in Table 5.  
• 
if the sample size is not increased: 
o the final analysis of ORR and PFS will take place after approximately 34 months with 
300 PFS events  
• 
if the sample size is increased: 
o the final analysis of ORR and PFS will take place after approximately 38 months with 
450 PFS events  
 


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At the time of writing of this protocol amendment, the interim analysis with the possibility of an 
adaptive sample size increase, already took place. The DSMB did not advice to increase the sample 
size. Furthermore, as the number of patients censored because of new anti-cancer therapies, was 
higher than expected, it became clear during the study that the 300 PFS events could not be 
reached. Therefore it was decided to include all Japanese patients in the ITT population, increasing 
the total sample size from the planned 440 to 490 patients in total, and to decrease the number of 
PFS events at the final analysis from 300 to at least 235 (see also Section 9.2.2). 
 
9.4.6.2 INTERIM ANALYSIS 
 
An interim analysis will be performed when 16-week BICR evaluation has been performed for the 
330th patient. For the interim analysis of ORR, the significance level will be determined using a Rho 
spending function, with 𝜌𝜌= 5, resulting in a very conservative boundary.  Assuming an information 
fraction of 75% for ORR at this interim analysis, the 1-sided significance level for efficacy will be 
0.006, and it will be reached if the difference in ORR is larger than 13.8%. This small alpha spending 
is foreseen to take into account the interim look at the efficacy primary endpoint, i.e. ORR. However, 
there is no intention to claim efficacy for ORR at this stage. The interim results, i.e. the conditional 
power for both ORR and PFS, will only serve to guide the decision rules regarding sample size re-
estimation. Results of this analysis will remain blinded to everyone, except the DSMB. Regardless of 
the efficacy results of the interim analysis, the trial will continue to accrue patients, until the total 
planned sample size is reached. 
 
As for ORR, there is no intention to claim efficacy for PFS at this early stage either. A small alpha 
spending is foreseen to take into account the interim look. For this, a very conservative Rho 
spending function boundary, with 𝜌𝜌= 5, is used. Assuming an information fraction of 37% for PFS at 
this interim analysis, the 1-sided significance level for efficacy will be 0.000173, and it will be 
reached if the hazard ratio is less than 0.51.  
 
9.4.6.3 SAMPLE SIZE ADAPTATION 
 
The conditional power for both ORR and PFS will be calculated at the interim analysis, assuming  
that the estimated treatment difference at interim analysis is the true effect. Depending on the 
conditional powers, the trial will either continue as planned to accrue 440 patients, or the sample 
size of the trial will be increased by 50%, for a total sample size of 660 patients. The guidelines 
provided in Table 5 will be used by the DSMB to make a recommendation to the Sponsor. Two 
possible decisions can be communicated to the Sponsor by the DSMB: “sample size increase” or “no 
sample size increase”. 
 


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Table 5: Guidelines for sample size increase at interim analysis  
ORR = overall response rate; PFS = progression-free survival S=Success, F=Failure, CP=Conditional 
Power assuming that the estimated treatment difference at interim analysis is the true effect, 
CP440= conditional power at N=440, CP660= conditional power at N=660.  m=months, NA=not 
applicable 
* Including type I error rate adjustment, using the Cui, Hung and Wang method [32] 
 
The conditional power for ORR would be equal to approximately 80% if the observed difference in 
ORR, at the time of the interim analysis (75% information fraction) were equal to 11.5%. The 
conditional power for PFS would be equal to 25% if the observed hazard ratio, at the time of the 
interim analysis (37% information fraction) were equal to 0.85.  Note that a HR=0.85 corresponds 
with a Median survival time Control/Experiment of 10/11.75, which is considered clinically 
meaningful. The conditional power for PFS would be equal to 50% if the observed hazard ratio, at 
the time of the interim analysis (37% information fraction) were equal to 0.80. The conditional 
power for PFS would be equal to 75% if the observed hazard ratio, at the time of the interim analysis 
(37% information fraction) were equal to 0.75. 
 
 
 
ORR 
PFS 
Recommendation 
CP660 for  
CP440 
Observed 
ORR 
difference 
CP440 
Observed HR 
ORR 
PFS 
≥ 80% 
≥11.5% 
≥ 75% 
≤0.75 
(≥3.3 m) 
Continue trial unchanged 
NA 
NA 
50-75% 
>0.75; ≤0.8  
(≥2.5<3.3m) 
Increase sample size 
≥ 90% 
70-90% 
25-50% 
>0.8; ≤0.85 
(≥1.8;<2.5m) 
Increase sample size* 
≥ 90% 
>40-<70% 
<25% 
>0.85 
(<1.8m) 
Continue trial unchanged 
NA 
NA 
60-80% 
≥ 10%;< 
11.5% 
≥ 75% 
≤0.75 
(≥3.3 m) 
Increase sample size 
80-90% 
≥90% 
50-75% 
>0.75; ≤0.8  
(≥2.5<3.3m) 
Increase sample size 
80-90% 
70-90% 
25-50% 
>0.8; ≤0.85 
(≥1.8;<2.5m) 
Increase sample size* 
80-90% 
>40-<70% 
<25% 
>0.85 
(<1.8m) 
Continue trial unchanged 
NA 
NA 
< 60% 
<10% 
≥ 75% 
≤0.75 
(≥3.3 m) 
Continue trial unchanged 
NA 
NA 
50-75% 
>0.75; ≤0.8  
(≥2.5<3.3m) 
Continue trial unchanged 
NA 
NA 
25-50% 
>0.8; ≤0.85 
(≥1.8;<2.5m) 
Continue trial unchanged 
NA 
NA 
<25% 
>0.85 
(<1.8m) 
Continue trial unchanged 
NA 
NA 


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9.4.6.4 FINAL ANALYSIS OF ORR WITHOUT SAMPLE SIZE INCREASE 
 
The final analysis of ORR will be performed when at least 235 PFS events have been observed. The 1-
sided significance level for efficacy will be 0.024. 
 
9.4.6.5 FINAL ANALYSIS OF ORR WITH SAMPLE SIZE INCREASE 
 
If the sample size is increased to 660 patients, the final analysis of ORR will be performed when 450 
PFS events have been observed. The 1-sided significance level for efficacy will be 0.025.  
 
9.4.6.6 FINAL ANALYSIS OF PFS WITHOUT SAMPLE SIZE INCREASE 
 
The final analysis of PFS will be performed when at least 235 PFS events have been observed. The 1-
sided significance level for efficacy will be 0.025.  
 
9.4.6.7 FINAL ANALYSIS OF PFS WITH SAMPLE SIZE INCREASE 
 
If the sample size is increased to 660 patients, the final analysis of PFS will be performed when 450 
PFS events have been observed. The 1-sided significance level for efficacy will be 0.025. 
 
9.4.7 SUB-GROUP ANALYSES 
 
Subgroup analyses will be performed for ORR and PFS with respect to the trial’s stratification factors 
(geographic region, primary tumor location and previous adjuvant CRC treatment) and other 
prognostic factors, which will be specified in the SAP.  
 
The consistency of the treatment effect across prognostic subgroups will be assessed visually using 
forest plots. 
 
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
 
Individual participant data will be listed by measure and timepoint 
 
9.4.9 EXPLORATORY ANALYSES 
 
Exploratory analyses are:  
• 
Daily living abilities: analyses will be descriptive.  
• 
Pharmacokinetic characteristics of arfolitixorin 
• 
To determine folate metabolism- and transportation-related gene expression levels in 
patients with advanced CRC  
• 
Evaluation of Recurrence Free Survival for patients undergoing complete metastatic 
resection. 
 
 
 
 
 
 
 
 


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10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 
 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO 
PARTICIPANTS 
Consent forms, in local language and in layman’s terms, describing in detail the study interventions, 
study procedures, and risks and possible benefits are given to the patient and written documentation 
of informed consent is required prior to any study specific procedures are performed. 
The protocol, consent form(s), recruitment materials and all patient material (and any changes to 
these documents) will be submitted to the IRB/IEC for review and approval. If the Patient 
Information/Informed Consent Form is amended, a determination will be made regarding whether 
previously consented patients need to re-consent. 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
Informed consent is a process that is initiated prior to the patient’s agreeing to participate in the study 
and continues throughout the individual’s study participation. Consent forms will be IRB/IEC-
approved, and the patient will be asked to read and review the document. Furthermore, the consent 
forms will include any requirements from the ethics committee. The Investigator will explain the 
research study to the patient, be the primary contact person and answer any questions that may arise. 
A verbal explanation will be provided in terms suited to the patient’s comprehension of the purposes, 
procedures, and potential risks of the study and of their rights as research patients.  Patients will have 
the opportunity to carefully review the written consent form and ask questions prior to signing. The 
patients should have the opportunity to discuss the study with their family or surrogates or think about 
it prior to agreeing to participate. 
Patients must be informed that participation is voluntary and that they may withdraw from the study 
at any time, without prejudice. 
The patients (or their legally acceptable representative or witness, as applicable) will sign and 
personally date the informed consent document prior to any study specific procedure. A copy of the 
informed consent document will be given to the patients. Another copy of the signed consent form 
will be saved in the Investigator’s file. The informed consent process will be conducted and 
documented in the source document (including the date), and the form signed, before the patient 
undergoes any study specific procedures. The rights and welfare of the patients will be protected by 
emphasizing to them that the quality of their medical care will not be adversely affected if they decline 
to participate in this study. 
The informed consent includes information that data will be recorded, collected, processed and may 
be transferred to countries outside the patient’s country of residence. The informed consent shall 
comply, in all respects, all applicable laws and regulations concerning personal data protection in the 
country in which the study takes place. 
The informed consent includes information about the intended purposes of the processing of personal 
data, the right to obtain access to personal data, the right to correct incorrect personal data, the right 


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to withdraw their consent of the processing of personal data at any time and the right to delete 
personal data already collected in the study. 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause. Written notification, documenting the reason for study suspension or termination, will be 
provided by the suspending or terminating party to patients, Investigator, and regulatory authorities. 
If the study is prematurely terminated or suspended, the PI will promptly inform, the IRB/IEC and, 
patients will be contacted, if applicable. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping    
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, 
and satisfy the Sponsor, IRB/IEC and/or Regulatory Authorities. 
10.1.3 CONFIDENTIALITY AND PRIVACY  
Patient confidentiality and privacy is strictly held in trust by the participating Investigators, their staff, 
and the Sponsor. This confidentiality is extended to cover testing of biological samples and genetic 
tests in addition to the clinical information relating to participants. Therefore, the study protocol, 
documentation, data, and all other information generated will be held in strict confidence. No 
information concerning the study, or the data will be released to any unauthorized third party without 
prior written approval of the Sponsor.  
The study monitor, other authorized representatives of the Sponsor, representatives of the IRB/IEC, 
regulatory authorities may inspect all documents and records required to be maintained by the 
Investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy 
records for the participants in this study. The clinical study site will permit access to such records. 
The patient’s contact information will be securely stored at each clinical site for internal use during 
the study. At the end of the study, all records will continue to be kept in a secure location for as long 
a period as dictated by the reviewing IRB/IEC, Institutional policies, or Sponsor requirements. 
10.1.4 FUTURE USE OF STORED SPECIMENS AND SAMPLES  
No specimens and samples will be used or kept after the end of the study. 
10.1.5 KEY ROLES AND STUDY GOVERNANCE 
 
Sponsor 
Sponsor’s Medical Expert 
Isofol Medical AB (publ) 
Arvid Wallgrens Backe 20 
S-413 46 Gothenburg, Sweden 
Roger Tell, MD, PhD 
Phone: +46 (0) 708 63 06 95 
E-mail: roger.tell@isofolmedical.com 


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Co-ordinating Investigator 
Josep Tabernero, MD, PhD 
Spain 
Phone: +34 (0)93 489 4301 
E-mail: jtabernero@vhio.net 
 
 
 
 
Chairman of DSMB 
Alberto Sobrero, MD, PhD 
Italy 
Phone: +33 (0) 52 567 40 
E-mail: alberto.sobrero@hsanmartino.it 
 
 
 
10.1.6 SAFETY OVERSIGHT 
An independent Data and Safety Monitoring Board (DSMB) will be constituted. The DSMB will protect 
the safety and well-being of the patients participating in the study and ensuring the ethical conduct 
of this clinical trial, through a regular review of the accumulated unblinded safety data reported by 
the independent DSMB statistician. The DSMB will make recommendations to the Sponsor or designee 
after each meeting, regarding the continuation of the trial, modification of the protocol or any other 
changes they will judge appropriate. Closure of the study may be recommended if the DSMB deems 
there is an unacceptable risk / benefit ratio of the participants.  
In addition, for Japan the DSMB will review PK data, safety and tolerability after completion of two 
treatment cycles for the first ten randomized Japanese patients, including patients from both 
treatment arms. In case of safety concerns or substantial differences in PK profile compared to the 
other study patients, the DSMB may halt the inclusion of study patients in Japan to gather more 
information. After review of the data, the DSMB will provide a recommendation to continue, or stop, 
inclusion of Japanese study patients. 
The DSMB will be composed of at least three members with the appropriate expertise, including at 
least one oncologist and one biostatistician. Members of the DSMB are independent from the study 
conduct and free of conflicts of interests.  A DSMB Charter, finalized before the start of the study, 
specifies the DSMB procedures, constitution and statistically derived guidelines for sample size 
increase. The safety monitoring analyses by the DSMB will be conducted every six months or more 
often if needed, commencing with completion of the first three months of study accrual. 
Recommendations from the DSMB to Sponsor will comprise, in addition, considerations on sample 
size increase at interim analysis, as well as early termination of the study or any other relevant 
recommendations. 
10.1.7 CLINICAL MONITORING 
In accordance with applicable regulations including GCP, study monitors will contact the site prior to 
the start of the study to review with the site staff the protocol, study requirements and facilities, and 
their responsibilities to satisfy regulatory, ethical, and Sponsor requirements. 
Study monitors will be delegated responsibility to monitor the study and site activity to verify that the: 
• 
Data are authentic, accurate, and complete; 


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• 
Safety and rights of patients are being protected; and 
• 
Study is conducted in accordance with the currently approved protocol and any other study 
agreements, GCP, and all applicable regulatory requirements. 
• 
Data collection is accurate and reliable which will be assured by verification and cross-check 
of selected data in the eCRFs against the Investigator’s records by the study monitor (source 
document verification). 
• 
Location of source documents are clearly identified. 
The monitor will contact and visit the Investigator regularly and must be permitted access to all source 
documents needed to verify the entries on the eCRF and other protocol-related documents, provided 
that patient confidentiality is maintained in accordance with local regulations. It will be the monitor’s 
responsibility to inspect the eCRFs at regular intervals throughout the study, to verify adherence to 
the protocol and the completeness, consistency and accuracy of the data being entered on the eCRFs. 
Sponsor or CRO monitoring standards require full verification that informed consent has been 
provided, and verification of adherence to the inclusion/exclusion criteria, documentation of SAEs, 
and the recording of the main efficacy, safety and tolerability endpoints. Additional checks of the 
consistency of the source data with the eCRFs will be performed according to the Clinical Monitoring 
Plan (CMP). Any data reported in the eCRF must be verifiable in the site source documents, if not 
clearly specified that the eCRF can be the source for a specific data point.  
Details of clinical site monitoring are documented in a CMP. The CMP describes in detail who will 
conduct the monitoring, at what frequency monitoring will be done, at what level of risk-based 
monitoring and SDV will be performed, and the distribution of monitoring reports 
The Investigator must ensure that patient anonymity is maintained. On CT-scans, MRIs, lab reports or 
other documents submitted to Sponsor or to other parties outside the study site, patients must be 
identified by assigned study ID number only, and never by name or other identifiable information. The 
Investigator must keep a patient identification code list showing the patient’s name, date of birth, or 
any other locally accepted identifiers. Documents identifying the patients (e.g., signed informed 
consent forms) should not be sent to Sponsor, and must be kept in strict confidence by the 
Investigator. 
The Investigator and co-Investigators agree to cooperate with the monitor(s) to ensure that any issues 
detected in the course of these monitoring visits are resolved. If the patient is hospitalized or dies in 
a hospital other than the study site, the Investigator is responsible for contacting that hospital in order 
to document the SAE. 
The Investigator must on request supply the Sponsor with any required background data from the 
study documentation or clinical records. This is particularly important when errors in data 
transcription are suspected. In the case of special problems and/or governmental queries, it is also 
necessary to have access to the complete study records, provided that patient confidentiality is 
protected. 
10.1.8 QUALITY ASSURANCE 
This study will be conducted in compliance with the protocol, the ICH Guideline for GCP, and any 
applicable local regulations (for US sites, the study will be conducted under an IND). Sponsor is 


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responsible for independent quality assurance (QA) audits of the clinical study processes, if deemed 
appropriate. Audit of the study sites may be conducted to assess and help assure compliance with 
GCP and applicable regulatory requirements. The study sites may be subject to a QA audit by Sponsor 
or its representatives, may be reviewed by an independent QA department, or may be inspected by 
regulatory authorities. Auditors/inspectors will have the right to inspect the study sites at any time 
during and/or after completion of the study and will have access to source documents, including 
patients’ medical records. By participating in this study, the Investigator agrees to this requirement. 
10.1.9 DATA HANDLING AND RECORD KEEPING  
 
10.1.9.1 PERSONAL DATA PROTECTION 
 
Patient personal data will be collected and processed in this study to answer the scientific questions 
investigated. The legal basis for the processing of personal data is the informed consent collected 
from all patients. Patients are informed about their rights connected to the processing of their 
personal data in the informed consent process.   
The Sponsor is the controller of all personal data recorded, collected and processed in this study. The 
Sponsor follows applicable laws and regulations concerning personal data protection in the countries 
where the study is being conducted. All personal data processed by the Sponsor is coded, and the 
Sponsor does not know the identity of the individual patients (this information will only be available 
at the study site of the respective patient). 
The sites are responsible for handling of their patients’ personal data collected as part of the patient’s 
treatment. The Sponsor is responsible for the processing of the coded data received from the sites. 
Both sites and the Sponsor will take the necessary measures in order to comply with all applicable 
Data Protection laws.  
Coded patient data for statistical analysis and scientific reporting, will be transmitted to various 
recipients (Sponsor, and sponsor representatives, Health Authorities, IRB/IEC, or other participating 
clinical study sites). The transferred data will not include the participant’s contact or identifying 
information. Individual participants and their research data will be identified by a unique identification 
number. The Sponsor is not able to un-identify the code, as the key to the code is available to 
designated study staff at each clinical site only. The study data entry and study management systems 
used by clinical sites and by the Sponsor will be secured and protected with a personal password. 
Isofol Medical has set up appropriate restrictions and controls to ensure that patient data remains 
protected. If study data is transferred to the US, it will only be transferred to companies who 
participate in and comply with the so-called Privacy Shield, which is an agreement between EU and 
USA for the protection of personal data in the US. 
The Sponsor has an integrity policy for clinical studies. This policy includes details of the legal basis for 
processing of personal data and the Data Protection Officer appointed at the Sponsor (contact 
information can be found in this protocol and on the Sponsor’s website). 
10.1.9.2 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
An eCRF will be used to record all data required by the protocol, 
Prior to the start of the study, the Investigator will complete a "Delegation of significant study-related 
duties" form, defining any person who is authorized to make or change entries in the eCRF and any 
person authorized to sign the eCRF.  


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Role-specific training sessions will be completed before a person will be granted access to the eCRF 
(e.g. Investigators and site staff, Sponsor staff and contract research organization staff, including 
project managers, clinical research associates and data managers). 
Support is available in the form of general eCRF user manuals (monitor and site manuals) and study 
specific Data Entry Instructions. 
The eCRF used for this study is validated and fulfils the ICH GCP requirements, and European, US FDA 
(21 CFR Part 11), Australian and Japanese regulations. The data capture system includes password 
protection and internal quality checks, such as automatic range checks, to identify data that appear 
inconsistent, incomplete, or inaccurate. Clinical data will be entered directly, or from defined source 
documents by an authorized person. 
All source documents should be completed in a neat, legible manner to ensure accurate interpretation 
of data. Hardcopies of the study visit worksheets may be provided for use as source document 
worksheets for recording data for each patient enrolled in the study. Data recorded in the eCRF 
derived from source documents should be consistent with the data recorded on the source 
documents. The Investigator is responsible for the management and accuracy of the information in 
the eCRF. At each monitoring visit, the eCRF should be at the monitor’s disposal for review. 
Changes to the data in the eCRF must be made at the site by an authorized person. The eCRF will have 
an audit trail with appropriate functionality for data capture, tracking, and documentation of any 
queries or changes.  Electronic signatures will be used to lock the data and identify the person entering 
or changing the data. 
Before database closure, reconciliation between the SAEs entered in the safety database and the study 
database will be performed. 
10.1.9.3 STUDY RECORDS RETENTION  
Following closure of the study, the Investigator or the head of the medical institution (where 
applicable) must maintain all site study records, except for those required by local regulations to be 
maintained by someone else, in a safe and secure location. The records must be maintained to allow 
easy and timely retrieval, when needed (e.g., audit or inspection), and, whenever feasible, to allow 
any subsequent review of data in conjunction with assessment of the facility, supporting systems, and 
staff. 
Sponsor will inform the Investigator/institution of the required time period for retaining these records 
in order to be compliant with all applicable regulatory requirements. The minimum retention time will 
meet the strictest standard applicable to that study site, as dictated by ICH GCP E6 Section 4.9, any 
institutional requirements or local laws and regulations, or Sponsor standards/procedures. 
The Investigator must notify Sponsor of any changes in the archival arrangements, including, but not 
limited to, the following: archival at an off-site facility, transfer of ownership of the records in the 
event the Investigator leaves the site. In addition, the Investigator should seek the written approval 
of the Sponsor prior to disposing any of the archived records. 
10.1.10 
PROTOCOL DEVIATIONS  


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A protocol deviation is any noncompliance with the clinical trial protocol or ICH GCP. The 
noncompliance may be either on the part of the patient, the Investigator, or the study site staff. As a 
result of deviations, corrective actions are to be developed by the site and implemented promptly.  
These practices are consistent with ICH GCP:  
• 
4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
• 
5.1 Quality Assurance and Quality Control, section 5.1.1  
• 
5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
It is the responsibility of the site Investigator to use continuous vigilance to identify and report 
deviations within 7 working days of identification of the protocol deviation. All deviations must be 
documented and reported to Sponsor. If relevant, protocol deviations must be sent to the reviewing 
IRB/IEC per their policies. The site Investigator is responsible for knowing and adhering to such IRB/IEC 
requirements.  
10.1.11 
PUBLICATION AND DATA SHARING POLICY 
Upon finalization of the ORR and PFS analysis, an integrated clinical and statistical study report will be 
written by the Sponsor in consultation with the Coordinating Investigators. This report will be based 
on the items detailed in this study protocol. When the clinical study report is completed, Sponsor will 
provide the Investigators with a full summary of the study results. The Investigators are encouraged 
to share the summary results with the patients, as appropriate. 
An update of the study report will be done when the follow-up is completed including overall survival 
results. 
The first resulting publication will be a full publication of all data from all participating sites, 
coordinated by Sponsor. Any secondary publications by the Investigators (abstracts in journals, oral 
presentations etc.) will reference the original publication and will require pre-submission review by 
the Sponsor. Note that the Sponsor is entitled to delay any proposed secondary publication, in order 
to obtain patent protection, if required. 
Authorship for Investigators will be assigned on the basis of their recruitment contribution, as well as 
intellectual and administrative input. Ranking will be according to the number of patients randomized 
as well as contribution to the study conduct and preparation of final manuscript.  
10.1.12 
INSURANCE 
Patients will be covered under Sponsor’s liability insurance policy throughout the course of the study. 
The certificate of insurance will be available in the Investigator Study File at each study site and may 
be provided upon request. The participating patients are also protected in accordance with national 
regulations as applicable. Essential information about the insurance coverage will be provided to the 
participating patients through the Informed Consent process. 
10.1.13 
CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the design, 
conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, 
persons who have a perceived conflict of interest will be required to have such conflicts managed in a 
way that is appropriate to their participation in the design and conduct of this trial. The study 


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leadership has established policies and procedures for all study group members to disclose all conflicts 
of interest and will establish a mechanism for the management of all reported dualities of interest. 
Certification and disclosure of any applicable financial interests of all clinical investigators shall be 
done per the requirements of 21 CFR 54 (Financial Disclosure by Clinical Investigators). 
 
 


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10.2 ABBREVIATIONS 
 
5-FU 
5-fluoroucacil 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest 
ALT 
Alanine Transaminase 
ANC 
Absolute Neutrophil Count 
API 
Active Pharmaceutical Ingredient 
AST 
Aspartate Transaminase 
BICR 
Blinded Independent Central Review  
BOR 
Best Overall Response 
CFR 
Code of Federal Regulations 
CR 
Complete Response 
CRA 
Clinical Research Associate 
CRF 
Case Report Form 
CSP 
Clinical Study Protocol 
CTCAE 
Common Terminology Criteria for Adverse Events 
DOR 
Duration of Response 
DPD 
Dihydropyrimidine Dehydrogenase 
DSMB 
Data and Safety Monitoring Board 
EC 
Ethics Committee 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic Case Report Forms 
EGFR 
Epidermal Growth Factor Receptor 
EMA 
European Medicines Agency 
EORTC 
European Organization for Research and Treatment of Cancer 
EOT 
End of Treatment Visit 
ESMO 
European Society for Medical Oncology 
FDA 
Food and Drug Administration 
FFPE 
Formalin-Fixed Paraffin-Embedded 
FP 
Fluoropyrimidine 
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
HDMTX 
High Dose Methotrexate 
IB 
Investigator’s Brochure 
ICH 
International Council for Harmonization of Technical Requirements for 
Pharmaceuticals for Human Use 
IMP 
Investigational Medicinal Product 
IRB 
Institutional Review Board 
IRC 
Independent Review Committee  
ITT 
Intention-To-Treat 
IWRS 
Interactive Web Response System 
LV 
Leucovorin 
mCRC 
Metastatic Colorectal Cancer 
MedDRA Medical Dictionary for Regulatory Activities 
MRI 
Magnetic resonance imaging 


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MTHF 
Methylene Tetrahydrofolate 
ORR 
Overall Response Rate 
OS 
Overall Survival 
PD 
Progressive Disease 
PFS 
Progression-Free Survival 
PI 
Principal Investigator 
PK 
Pharmacokinetic 
PR 
Partial Response 
QA 
Quality Assurance 
PRO 
Patient Reported Outcome 
RECIST 
Response Evaluation Criteria In Solid Tumours 
RR 
Response Rate 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SD 
Stable Disease 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
SPC 
Summary of Product Characteristics 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TAP 
Thorax Abdomen Pelvis 
THF 
Tetrahydrofolate 
TS 
Thymidylate Synthase 
ULN 
Upper Limit of Normal 
UP 
Unanticipated Problem 
VEGF 
Vascular Endothelial Growth Factor 
WHO 
World Health Organization 
 
 
 


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10.3 PROTOCOL AMENDMENT HISTORY 
 
Version 
Date 
Description of Change  
Brief Rationale 
1.0 
21Sep2018 
Initial version 
Initial version 
2.0 
21Sep2018 
In case of Oxaliplatin caused toxicity, 
dose modifications according to label 
should be performed. 
 
Update of OS analysis to exclude a 
significant detrimental effect of 
arfolitixorin as compared to leucovorin 
in terms of OS. 
FDA SPA request 
3.0 USA 
13Nov2018 
Inclusion criteria 3: Addition of 
leucovorin. 
 
Inclusion criteria 7: Change Hgb level 
from 100 g/L to 80 g/L 
 
 
Add collection of baseline mutations 
in the eCRF (KRAS, BRAF, NRAS) at 
screening if available. 
 
Update of interim analysis conditions. 
 
 
 
Clarifying CT/MRI follow-up after 
metastasis resection. 
To clarify the components of 
standard FOLFOX treatment. 
 
To align protocol inclusion 
criteria regarding Hgb levels with 
similar phase III mCRC studies.  
 
To enable sub-group analysis.  
 
 
 
To avoid increasing sample size in 
case of poor interim study 
results. 
 
Protocol clarification.  
4.0 USA 
29Aug2019 
Exclusion criteria 2 and 20: Change 
from >6 months to >28 days from 
surgery to randomization 
 
Exclusion criteria 3: adding “adjuvant” 
anti-cancer treatment 
 
Oxaliplatin and Leucovorin should be 
administered in sequence or in two 
different injection ports. 
To align protocol exclusion 
criteria regarding pre-study 
surgery with clinical praxis. 
 
Clarifying the intention of the 
exclusion criteria. 
 
To ensure study patients get full 
benefit from their oxaliplatin 
treatment as described in the 
SPC. 
5.0 USA 
07Oct2019 
Section 8.2.2.  
Clarifying ECG assessments according 
to the previously updated Oxaliplatin 
and Leucovorin administration. 
To correct the information 
according to the Summary of 
Changes document dated 29 Aug 
2019. 
6.0 USA 
20Feb2020 
2.2.1 Addition of non-clinical data. 
 
2.2.2 Addition of clinical data. 
 
3. Update of the outcome measure for 
Duration of Response objective. 
 
 
3. New exploratory objective 
investigating relevant tumor 
biomarkers levels.   
Additional data available. 
 
Additional data available. 
 
To align wording with 
recommendations from 
competent authorities.  
 
Increase understanding of the 
relation between biomarker 


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3. New exploratory objective 
investigating Recurrence Free Survival 
for patients undergoing metastatic 
resection. 
 
4.1 Clarifying geographical 
stratification. 
 
5.1 Inclusion criteria 4: Addition of 
lymph nodes. 
 
5.5 Update of number of sites and 
regions 
 
6.1.2 and 6.3.1 Defining 5-FU bolus 
duration. 
 
6.2.4 Body Surface Area calculated 
according to DuBois formula. 
 
6.2.4.2 Leucovorin administration 
 
 
6.3.3 Allowing use of bevacizumab 
biosimilars. 
 
6.3.4 5-FU dose reduction 
 
6.4.1 Geographical region update 
 
8.1.2 PK sampling timing.  
 
8.2.2 Clarifying patients position 
during blood pressure and ECG 
measurements. 
 
8.2.5 Clarifying how to manage 
patients becoming eligible for 
metastasis resection. 
 
8.3.4 and 8.4 Update of safety section 
 
9.2.4 Overall Survival analysis 
updated. 
 
9.4.9 Addition of recurrence free 
survival. 
 
10.1.5 Update of contact details 
 
10.1.9.2 Addition of study specific 
Data Entry Instructions and update reg 
eCRF validation. 
 
levels and the treatment 
outcome. 
 
To compare Recurrence Free 
Survival data between the 
treatment groups. 
 
 
Addition on Australia and Japan. 
 
 
Protocol clarification in 
accordance with RECIST 1.1 
 
Protocol clarification. 
 
 
Protocol clarification. 
 
 
Protocol clarification. 
 
 
The supplied Bendafolin contains 
trometamol. 
 
Protocol clarification. 
 
 
Protocol clarification 
 
Protocol clarification 
 
Protocol clarification 
 
Protocol clarification 
 
 
 
Protocol clarification 
 
 
 
Protocol clarification 
 
Clarifying according to FDA 
request. 
 
Protocol clarification 
 
 
Protocol clarification 
 
Protocol clarification 
 
 
 


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10.1.6 Adding details to the DSMB 
review. 
Japan added to the study. 
7.0 USA 
20Aug2021 
1.2 Schema Updated 
 
 
 
2.1.3 Minor clarifications 
 
 
 
4.4 Follow-up process for patients 
post primary endpoint analysis 
 
8.1.2 Update of patients included in 
PK analysis 
 
8.1.2.1 Update of analyte in 
population PK analysis and studies 
included in the analysis 
 
 
 
8.2.2 Patients experiencing a QTc 
prolongation should be taken of study 
drug 
 
 
8.2.4 Section updated and clarified 
 
 
8.2.5 Section updated and clarified 
 
 
8.3.5 Follow-up process for patients 
post primary endpoint analysis 
 
9.2.2 Section updated and clarified 
 
 
9.2.4 Section updated  
 
 
 
9.3 Section updated and clarified 
 
 
9.4.6.1 Section updated and clarified 
 
 
9.4.6.3 Corrections in Table 5 of errors 
 
 
9.4.6.4 Section updated 
 
 
 
 
Schema updated as ORR and PFS 
analysis are performed at the 
same timepoint 
 
Minor clarifications done to 
improve understanding of 
mechanism of action 
 
Process clarified as time point for 
ORR and PFS analysis is updated  
 
Number of patients needed in PK 
evaluation is adjusted 
 
Population PK analysis will only 
be done on the active compound 
and not on the metabolites. One 
previous mentioned study will 
not be included in the analysis 
 
Previous version stated that 
patient should be excluded from 
study but patient should only be 
taken off study drug 
 
Genes included in the analysis 
are updated 
 
Updated as not only metastases 
are resected during study 
 
Process clarified as time point for 
ORR and PFS analysis is updated  
 
Correction to ensure wording is 
not contradicting the SAP 
 
ORR included as the analysis of 
ORR and PFS will be done at the 
same timepoint 
 
Correction to align wording with 
SAP 
 
Correction to align wording with 
SAP 
 
To be aligned with SAP and DSMB 
charter 
 
ORR included as the analysis of 
ORR and PFS will be done at the 
same timepoint 


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9.4.6.5 Section updated 
 
 
 
9.4.7 Section updated and clarified 
 
 
10.1.11 Section clarified related to 
Clinical Study Report 
 
 
 
 
Minor editorial changes 
ORR included as the analysis of 
ORR and PFS will be done at the 
same timepoint 
 
To be aligned with SAP and DSMB 
charter 
 
CSR will be written when the 
analysis for the primary 
endpoints are available. An 
update of the CSR will done when 
OS data are available 
 
 
8.0 USA 
(Never 
submitted, 
changes 
included in 
version 9.0) 
22Feb2022 
Number of patients included in the 
study updated in multiple sections 
 
 
Number of PFS events updated in 
multiple sections 
 
1.1 Synopsis 
 
 
 
2.2.2 Section updated  
 
 
 
4.1 and 5.5 Sections updated 
 
 
 
 
9.2.1 Section updated 
 
 
 
 
9.2.2 Section updated 
 
 
 
 
9.2.4 Section updated 
 
 
 
 
 
 
 
9.3 Section updated 
 
 
 
 
Change from 440 patients to 490 
patients as all Japanese patients 
are included 
 
Change from 300 PFS events to 
230 PFS events 
 
Study recruitment period 
updated to reflect the actual 
recruitment period 
 
Updated with latest safety 
information from IB V14.0 
1Oct2021 
 
Information added clarifying that 
the addition of patients to the ITT 
population was taken after the 
interim analysis 
 
Addition of information on how 
statistical analysis of ORR is 
affected after study population is 
changed to 490 
 
Addition of information on how 
statistical analysis of PFS is 
affected after change in number 
of PFS events 
 
Approximate timelines removed. 
Considerations for OS analysis is 
updated, and wording is deleted 
as PFS hazard ratio has been 
updated but timepoint of OS 
analysis at 60% remains 
unchanged 
 
“Main study” removed as all 490 
patients are included in the 
analysis 
 


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9.4.6 Section updated 
 
 
 
9.4.7 Section updated 
 
 
 
10.1.5 CMO phone number updated 
 
Minor editorial changes 
Text updated and added as this 
protocol update is done after the 
Interim analysis  
 
Information updated in 
accordance with current version 
of the SAP 
 
Phone number changed 
9.0 USA 
22Apr2022 
Number of PFS events updated in 
multiple sections 
 
 
 
9.3 Addition of ITTE analysis set 
 
Change from 300 PFS events to at 
least 235 PFS events, to align 
with statistical analysis plan and 
regulatory authority feedback. 
 
Intention-to-Treat Excluding 
Additional Japanese Patients 
(ITTE) analysis set added, to be in 
line with current SAP. 
 
 
 


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Jackman AL. Patterns of elevation of plasma 2'-deoxyuridine, a surrogate marker of thymidylate 
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specific TS inhibitors raltitrexed (Tomudex) and ZD9331. Clin Cancer Res. 2002 Jan;8(1):103-9. 
[22] Gustavsson B, Carlsson G, Swartling T, Kurlberg G, Derwinger K, Björkqvist H, Odin E, Gibson F 
Phase 1 dose de-escalation trial of the endogenous folate [6R]-5,10-methylene tetrahydrofolate in 
combination with fixed-dose pemetrexed as neoadjuvant therapy in patients with resectable rectal 
cancer. Invest New Drugs. 2015 Oct;33(5):1078-85 
[23] Glimelius B, Lahn M.  Window-of-opportunity trials to evaluate clinical activity of new molecular 
entities in oncology.  Ann Oncol.  2011 Aug;22(8):1717-25. 
[24] Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al.  New response 
evaluation criteria in solid tumours: revised RECIST guideline (version 1.1).  Eur J Cancer.  2009 
Jan;45(2):228-47. 
[25] Gorin A. MD, Strother D. MD, Poplack D. MD, Letvak L. MD, George M. MD, Link M. MD. Safety 
and Efficacy of l-Leucovorin Rescue Following High-Dose methotrexate for Osteosarcoma. Med 
Pediatric Oncol. 1995 Jun;24(6):362-7. 


ISO-CC-007 
Version 9.0 USA 
ISOFOL Medical AB (publ) 
22 April 2022 
 
81 
[26] Jaffe N, Jorgensen K, Robertson R, George M, Letvak L, Barrett G. Substitution of l-leucovorin for 
d,l-leucovorin in the rescue from high-dose methotrexate treatment in patients with osteosarcoma. 
Anticancer Drugs. 1993 Oct;4(5):559-64. 
[27] Hecht J R et al. A randomized phase IIIB trial of chemotherapy, bevacizumab and panitumab 
compared with chemotherapy and bevacizumab alone for metastatic colorectal cancer J Clin Oncol; 
2009; 27(5): 672-680 
[28] National Comprehensive Cancer Network (NCCN). NCCN Clinical Practice Guidelines in Oncology. 
Colon Cancer Version 3.2018. 08/07/18  
[29] Van Cutsem E et al. Safety and efficacy of first-line bevacizumab with FOLFOX, XELOX, FOLFIRI 
and fluoropyrimidines in metastatic colorectal cancer: the BEAT study. Annals of Oncology; Volume 
20, Issue 11, 1 November 2009: 1842-1847 
[30] https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_50 
[31] United States Food and Drug Administration Guidance for Industry: Clinical Trial Endpoints for 
the Approval of Cancer Drugs and Biologics. Rockville, MD: US Department of Health and Human 
Services; 2007 
[32] Cui L, Hung HM, Wang SJ. Modification of sample size in group sequential clinical trials. 
Biometrics—1999—; 55:853–7 
[33] Meta-Analysis Group In Cancer (2004). Modulation of fluorouracil by leucovorin in patients with 
advanced colorectal cancer: an updated meta-analysis. J Clin Oncol 22: 3766-3775. 
[34] Per-Anders Larsson, Göran Carlsson, Bengt Gustavsson, Wilhelm Graf & Bengt Glimelius (1996) 
Different Intravenous Administration Techniques for 5-Fluorouracil Pharmacokinetics and 
Pharmacodynamic Effects, Acta Oncologica, 35:2, 207-212 
 
 
 
 
 

SAP_001.pdf: 
Signature Page for ISO_CC_007_SAP
Study ISO-CC-007 v8.0
Signature Page for ISO_CC_007_SAP
Study ISO-CC-007 v8.0
eSignature for Approval
Karin Gedda
Sponsor
21-Apr-2022 15:52:06 GMT+0000
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TPL N°: eTPL SA-02-01 
TPL Version: 02 
Of SOP SA-02.08 
TPL Effective Date: 31 Mar 2016 
 
 
 
Statistical Analysis Plan 
 
 
 
 
Sponsor:  
ISOFOL 
Protocol Title:  
A randomized, multicenter, parallel-group, Phase III 
study to compare the efficacy of arfolitixorin versus 
leucovorin in combination with 5-fluorouracil, oxaliplatin, 
and bevacizumab in patients with advanced colorectal 
cancer 
Study Code: 
ISO-CC-007 
Protocol Version 
V4.0 20Aug2021 
 
Of note, this version of the SAP was written after the 
interim analysis. 
 
 
 
For IDDI: 
Author: Nicolas Dubois, Biostatistician 
Reviewers:  
- Perrine Bamps, Senior Biostatistician  
- Koenraad D’Hollander, Senior Biostatistician 
 
For Sponsor: 
Reviewer: Karin Gedda, VP Clinical Operations 
 
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TPL N°: eTPL SA-02-01 
TPL Version: 02 
Of SOP SA-02.08 
TPL Effective Date: 31 Mar 2016 
 
Table of Contents 
 
1. 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ............................... 4 
2. 
INTRODUCTION ........................................................................................... 7 
3. 
STUDY DESIGN AND OBJECTIVES ................................................................ 7 
3.1 
Study Objectives...................................................................................... 7 
3.1.1 
Primary Objective .............................................................................. 7 
3.1.2 
Secondary Objectives ........................................................................ 7 
3.1.3 
Exploratory Objectives ....................................................................... 8 
3.2 
Study Design ........................................................................................... 8 
3.2.1 
Study intervention description ............................................................ 8 
3.2.2 
Measures to minimize bias: randomization and blinding ....................... 10 
a) 
Randomization ................................................................................... 10 
b) 
Blinding ............................................................................................. 10 
3.2.3 
Visits ............................................................................................. 10 
a) 
Treatment period ................................................................................ 10 
b) 
Assessment visits (every eight weeks until PD) ...................................... 10 
c) 
End of treatment visit (EOT) ................................................................ 10 
d) 
Post treatment visits: Follow-up visits until centrally confirmed progressive 
disease .................................................................................................... 11 
e) 
Post treatment visits: Survival follow-up ............................................... 11 
3.3 
Sample Size Justification ......................................................................... 15 
3.3.1 
Hierarchical testing approach ............................................................ 15 
3.3.2 
Accrual rate .................................................................................... 15 
3.3.3 
Primary endpoint - Overall Response Rate (ORR) ................................ 15 
3.3.4 
Key secondary endpoint - Progression‐free survival (PFS) ..................... 15 
3.3.5 
Other secondary endpoints ............................................................... 17 
3.3.6 
Japanese study ............................................................................... 17 
4. 
GENERAL ANALYSIS DEFINITIONS ............................................................ 18 
4.1 
Study Period and Visit Window Definitions ................................................. 18 
4.2 
Planned analyses ................................................................................... 18 
4.2.1 
Interim analysis4 ............................................................................. 18 
a) 
Interim analysis of Overall Response Rate (ORR) .................................... 18 
b) 
Interim analysis of Progression Free Survival (PFS) ................................ 19 
c) 
Sample size adaptation ....................................................................... 19 
4.2.2 
Final analysis of ORR and PFS ........................................................... 21 
a) 
Overall Response Rate (ORR) ............................................................... 21 
b) 
Progression Free Survival (PFS) ............................................................ 21 
c) 
Overall Survival (OS) .......................................................................... 21 
d) 
Other efficacy endpoints ...................................................................... 21 
e) 
Safety analysis ................................................................................... 21 
4.2.3 
Final analysis of Overall Survival (OS) ............................................... 21 
4.2.4 
Analysis of Japanese patients ........................................................... 22 
4.3 
Definition of Populations ......................................................................... 22 
4.4 
Subgroup Definitions .............................................................................. 22 
4.5 
Estimand definitions ............................................................................... 26 
4.5.1 
Estimand definitions for ORR ............................................................ 26 
4.5.2 
Estimand definitions for PFS ............................................................. 26 
4.6 
Calculated Variables ............................................................................... 27 
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4.7 
Partial Dates ......................................................................................... 30 
4.8 
Methods To Be Used For Handling Missing Data ......................................... 31 
5. 
STUDY PATIENTS ....................................................................................... 31 
5.1 
Disposition of Patients ............................................................................ 31 
5.2 
Protocol Deviations ................................................................................ 31 
5.3 
In- and Exclusion Criteria ........................................................................ 31 
6. 
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS ........................ 31 
7. 
MEDICAL HISTORY .................................................................................... 32 
8. 
PRIOR AND CONCOMITANT TREATMENT AND PROCEDURES ...................... 32 
9. 
EFFICACY EVALUATION ............................................................................. 33 
9.1 
Response assessment ............................................................................. 33 
9.1.1 
Overall Response Rate (ORR)............................................................ 33 
9.1.2 
Progression free Survival (PFS), Duration of Response (DOR) and Overall 
Survival (OS) ............................................................................................... 34 
9.1.3 
Recurrence Free Survival (RFS) ......................................................... 34 
9.2 
PRO EQ‐5D questionnaire ........................................................................ 34 
9.3 
Patients undergoing metastasis resection .................................................. 35 
10. 
SAFETY EVALUATION ............................................................................. 35 
10.1 
Extent of Exposure ................................................................................. 35 
10.2 
Adverse Events ...................................................................................... 35 
10.3 
Deaths and Serious Adverse Events ......................................................... 36 
10.4 
NCI PRO‐CTCAE questionnaire ................................................................. 36 
10.5 
Clinical Laboratory Determination ............................................................ 36 
10.6 
Vital Signs, Physical Findings and Other Observations Related to Safety ....... 37 
11. 
PHARMACOKINETICS EVALUATIONS ...................................................... 38 
12. 
REFERENCES .......................................................................................... 38 
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TPL N°: eTPL SA-02-01 
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TPL Effective Date: 31 Mar 2016 
 
1. 
List of Abbreviations and Definition of Terms 
Abbreviation 
Term 
 
5-FU  
5-fluorouracil  
 
AE  
Adverse Event 
 
AESI 
Adverse Event of Special Interest 
 
ALT  
Alanine Transaminase  
 
AST  
Aspartate Transaminase  
 
ATC 
Anatomical Therapeutic Chemical 
 
AUC 
Area Under the Curve (=AUClast) 
 
BICR 
Blinded Independent Central Review 
 
BOR 
Best Overall Response 
 
BUN 
Blood Urea Nitrogen 
 
Cmax 
Maximum (or peak) plasma concentration 
 
CMH 
Cochran-Mantel-Haenszel test 
 
CP 
Conditional Power 
 
CR  
Complete Response  
 
CRC 
Colorectal Cancer 
 
CRF  
Case Report Form  
 
CSP  
Clinical Study Protocol  
 
CT 
Computed Tomography 
 
CTCAE  
Common Terminology Criteria for Adverse Events  
 
DOR  
Duration of Response  
 
DSMB  
Data Safety Monitoring Board  
 
ECG  
Electrocardiogram  
 
ECOG  
Eastern Cooperative Oncology Group  
 
eCRF  
Electronic Case Report Forms  
 
EOT  
End of Treatment Visit  
 
gGT 
Gamma-Glutamyl Transferase 
 
HR 
Hazard Ratio 
 
ICH  
International Council for Harmonisation   
 
IMP  
Investigational Medicinal Product  
 
ITT  
Intention-To-Treat analysis set 
 
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Abbreviation 
Term 
 
ITTE 
Intention-to-Treat Excluding Additional Japanese Patients 
analysis set 
 
ITTJ 
Intention-To-Treat Japanese analysis set 
 
IWRS 
Interactive Web Response System 
 
LDH 
Lactodehydrogenase 
 
LLOQ 
Lower Limit of Quantification 
 
LV  
Leucovorin  
 
MedDRA  
Medical Dictionary for Regulatory Activities  
 
MRI  
Magnetic Resonance Imaging  
 
Methyl THF  
Methyl Tetrahydrofolate, metabolite 
 
MTHF 
Methylene Tetrahydrofolate, parent drug 
 
NCI 
National Cancer Institute 
 
NE 
Not Evaluable 
 
ORR  
Overall Response Rate  
 
OS  
Overall Survival  
 
PD  
Progressive Disease  
 
PFS  
Progression Free Survival  
 
PK  
Pharmacokinetic  
 
PP 
Per-Protocol analysis set 
 
PPJ 
Per-Protocol Japanese analysis set 
 
PR  
Partial Response  
 
PRO  
Patient Reported Outcome  
 
RECIST  
Response Evaluation Criteria In Solid Tumours  
 
RFS 
Recurrence Free Survival 
 
RR  
Response Rate  
 
SAE  
Serious Adverse Event  
 
SAP  
Statistical Analysis Plan  
 
SD  
Stable Disease  
 
SOC  
System Organ Class  
 
SOP  
Standard Operating Procedure  
 
THF  
Tetrahydrofolate, metabolite  
 
Tlast 
Time at which last plasma concentration is observed 
 
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TPL N°: eTPL SA-02-01 
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TPL Effective Date: 31 Mar 2016 
 
Abbreviation 
Term 
 
Tmax 
Time at which maximum plasma concentration is observed 
 
VAS 
Visual Analogue Scale 
 
WBC 
White Blood Cell 
 
WHO  
World Health Organization  
 
 
 
 
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TPL N°: eTPL SA-02-01 
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2. 
Introduction 
This Statistical Analysis Plan was written for the clinical trial IS0‐CC‐007 conducted 
in the EU, Canada, USA, Australia and Japan. The ICH guideline E3 “Structure and 
Content of Clinical Study Reports” was used as a guide to the writing of the plan. 
This Statistical Analysis Plan is based on the current version of the clinical study 
protocol. This current version of the SAP was written after the interim analysis. 
3. 
Study Design and Objectives 
3.1 Study Objectives 
3.1.1 Primary Objective 
The primary objective is to compare the effect of treatment with arfolitixorin versus 
Leucovorin (LV), in combination with 5‐FU, oxaliplatin, and bevacizumab in patients 
with advanced Colorectal cancer (CRC), on the Overall Response Rate (ORR). 
This is defined as the best confirmed overall response recorded from the start of the 
study treatment until the end of treatment or the last available assessment at 
database cutoff. All responses will be confirmed 8 weeks after onset of response. All 
assessments including confirmation of response will be performed by BICR.     
3.1.2 Secondary Objectives 
The key secondary objectives are 
• 
To estimate the Progression‐free survival (PFS), defined as the time from 
randomization to first occurrence of tumor progression assessed by BICR 
(RECIST 1.1) based on CT-scans/MRIs conducted every 8 weeks after start 
of treatment, or death from any cause. Patients undergoing metastasis 
resection will not be censored for PFS. Recurrence of disease after surgery 
will be counted as a progression event. 
• 
To estimate the Duration of Response (DOR). The duration of overall 
response (DOR) is measured from the first time point at which criteria are 
met for complete response (CR) or partial response (PR) through the last time 
point when overall response has been objectively documented.  
The other secondary objectives are 
• 
To estimate the Overall survival (OS), defined as time from randomization 
to death from any cause. 
• 
To assess the Quality of Life, assessed using the EQ-5D patient reported 
outcome questionnaire (PRO). 
• 
To assess the Safety and tolerability by counting number and severity of 
adverse events (AEs), including clinically significant abnormal laboratory 
findings, regardless of causal relationship to arfolitixorin or LV. Specific AEs 
will be followed using PRO (NCI PRO-CTCAE). 
• 
To assess Patients undergoing curative metastasis resection after 
treatment with study drug, defined as the number of patients qualifying for 
curative metastasis resection after treatment with study drug. 
 
 
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3.1.3 Exploratory Objectives 
The exploratory objectives*1 are 
• 
To assess the Daily living abilities, as assessed by the Eastern Cooperative 
Oncology Group (ECOG) Performance Status Scale. 
• 
To determine the pharmacokinetic (PK) characteristics of arfolitixorin and 
LV in patients with advanced CRC. The plasma concentration of methylene 
tetrahydrofolate ([6R]-MTHF), methyl-tetrahydrofolate (methyl-THF), and 
tetrahydrofolate (THF) will be determined in a limited number of patients in 
both treatment arms. 
• 
Recurrence Free Survival (RFS) for patients undergoing metastatic 
resection. Defined as the time between the first surgery with complete 
removal of the metastasis and recurrence of the disease or death from any 
cause. 
• 
To investigate folate metabolism- and transportation-related gene 
expression levels in patients with advanced CRC. Gene expression levels 
of folate metabolism- and transportation-related genes, analyzed by sub-
group low/high gene expression levels. 
3.2 Study Design 
This is a randomized, multicenter, parallel-group, Phase III study to compare the 
efficacy of arfolitixorin versus LV in patients with advanced CRC treated by 5-FU, 
oxaliplatin, and bevacizumab. To be eligible for the study, patients must have 
advanced CRC, without indication for resection at study inclusion, with at least one 
measurable lesion of metastatic disease according to RECIST 1.1 criteria within 28 
days of randomization. 
3.2.1 Study intervention description 
490 eligible and consenting patients were randomized 1:1 to one of two treatment 
groups (A and B) and administered one of the following treatment regimens starting 
within 3 days of randomization. Following treatment cycles should be started 14 (+ 
7) days after the previous administration. 
• 
Group A “Experimental”: ARFOX (Arfolitixorin + 5‐FU + Oxaliplatin) + 
Bevacizumab   
• 
Group B “Comparator”: mFOLFOX‐6 (Leucovorin + 5‐FU + Oxaliplatin) + 
Bevacizumab  
Initially, approximately 440 were planned to be randomized. However, Japanese 
authorities requested that approximately 12.7% of the total study population are 
enrolled from Japan. For this reason, additional patients from Japan were 
randomized. In the end a total of 490 patients were randomized (cf. section 3.3.6). 
This decision to randomize extra Japanese patients was taken before the moment of 
the interim analysis, and without looking at the data. To prevent a potential bias, 
the statistician of the final analysis used a dummy randomization file at the time of 
the interim analysis. Only an independent statistician and the DSMB members had 
access to the unblinded interim analysis report. 
                                           
1 * This SAP does not apply to gene expression level exploratory objectives. 
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TPL Effective Date: 31 Mar 2016 
 
 
 
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3.2.2 Measures to minimize bias: randomization and blinding 
a) Randomization 
Patients are randomized in a 1:1 ratio to either the experimental arm (arfolitixorin) 
or the comparator arm (Leucovorin), following the completion of all screening 
assessments and after confirmation of their eligibility, using a stratified permuted 
block randomization. Randomization is stratified for the following baseline factors: 
• 
Geographical region (Europe / North America / Australia / Japan), 
• 
Primary tumor location (left colon/right colon/rectal cancer), 
• 
Previous neo-adjuvant/adjuvant CRC treatment (yes/no) 
Randomization is performed via an interactive web response system (IWRS). The 
system enables time and date stamped entries that in turn allow real time compliance 
monitoring. 
b) Blinding 
Patient and Investigator blinding of the study treatment is not possible as there is a 
difference in the color of the injection fluids and the method of administration. To 
minimize any bias arising either from the inability to blind patients and Investigators 
or from potential inconsistencies in local tumor response assessments across study 
sites, analyses of the tumor response endpoints is based on blinded, retrospective, 
centrally‐adjudicated assessment of CT‐scans/MRIs according to a pre-specified 
Imaging Review Charter (Banook 2021).  
3.2.3 Visits 
The study consists of a screening visit, a randomization visit during which patients 
receive their first course of IMP, regular treatment visits every 2 weeks thereafter, 
and CT/MRI assessments every 8 weeks from baseline. Treatment visits are to 
continue until PD, as identified by the Investigator based on locally‐performed 
assessment of CT-scans/MRIs. In the event of PD, Investigators are to discontinue 
IMP and manage the patient according to local routine. Patients are followed 
thereafter until death. 
a) Treatment period 
Treatment visits occur every 14 days (+7 days) until PD. 
b) Assessment visits (every eight weeks until PD) 
Assessment visits are to occur every 8 weeks (+/‐7 days) after the baseline visit (1st 
IMP administration day). Assessment visits should continue until PD or premature 
discontinuation of IMP. 
c) End of treatment visit (EOT) 
Patients prematurely discontinuing IMP and those reaching PD should complete an 
end of treatment visit within 30 days after last dose of IMP. The EOT visit should be 
conducted before initiation of new anti‐cancer therapy. 
 
 
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d) Post treatment visits: Follow-up visits until centrally confirmed progressive 
disease 
Patients who discontinue IMP for reasons other than centrally confirmed PD move 
into the Follow‐up phase and are assessed every 8 weeks. 
Every effort should be made to collect information regarding disease status until 
disease progression, death, or the end of the study. 
e) Post treatment visits: Survival follow-up 
Patients discontinuing study treatment and/or with centrally confirmed PD move into 
the survival follow-up phase. Patient survival status is assessed every 12 weeks until 
death or the end of the study, whichever occurs first. Patient survival data can be 
collected by phone, visit, medical records, contact with relatives etc. Date of death, 
or the Investigators best estimate of the date of death, should be entered in the 
eCRF.  
Information regarding post‐study anti‐cancer therapies is collected if new treatment 
is initiated. 
 
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The schedule of activities is shown in the table below. 
Table 1: Schedule of activities 
 
 
PRE-TREATMENT  
TREATMENT PERIOD  
END OF TREATMENT 
FOLLOW-UP 
Screening 
 
Administer IMP 
Assessment 
visit 
Administer IMP 
Assessment 
visit 
End of treatment 
visit 
Follow-up visits 
until centrally 
confirmed PD 
Survival follow-up 
 
Cycle 1 
 
Cycles 2-4 
 
Cycles 5 - PD or 
discontinuation 
 
 
Only patients 
prematurely 
discontinuing IMP 
prior to centrally 
confirmed PD 
All patients 
 
Timing 
 
Day 1 
Baseline 
14 days 
after 
previous 
admin 
visit 
Week 8 from 
day 1 
14 days after 
previous admin 
visit 
Weeks 16, 
24, etc. from 
day 1 
Within 30 days after 
last dose of IMP and 
before start of new 
anti-cancer 
treatment 
Every 8 weeks 
from end of 
treatment visit 
until centrally 
confirmed PD 
Every 12 weeks 
after centrally 
confirmed PD 
until death or end 
of study 
CSP 
Section 
Visit window 
Day -28 to  
Day -1 
-3 daysa,f 
+7 days 
±7 days  
+7 days 
±7 days  
 
±7 days  
±7 days  
Screening procedures 
10.1.1 
Informed consent 
X 
 
 
 
 
 
 
 
 
8.3 
Patient Study ID 
X 
 
 
 
 
 
 
 
 
5.1/5.
2 
Eligibility criteria 
X 
X 
 
 
 
 
 
 
 
8.3 
Demographics/medical history 
X 
 
 
 
 
 
 
 
 
6.4 
Randomization 
 
Xa 
 
 
 
 
 
 
 
General procedures 
6.6 
Concomitant therapy 
X 
X 
X 
X 
X 
X 
X 
 
 
8.2 
Physical examination 
X 
X 
 
X 
 
X 
X 
 
 
8.2 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
 
 
8.2 
Height  
X 
 
 
 
 
 
 
 
 
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PRE-TREATMENT  
TREATMENT PERIOD  
END OF TREATMENT 
FOLLOW-UP 
Screening 
 
Administer IMP 
Assessment 
visit 
Administer IMP 
Assessment 
visit 
End of treatment 
visit 
Follow-up visits 
until centrally 
confirmed PD 
Survival follow-up 
 
Cycle 1 
 
Cycles 2-4 
 
Cycles 5 - PD or 
discontinuation 
 
 
Only patients 
prematurely 
discontinuing IMP 
prior to centrally 
confirmed PD 
All patients 
 
Timing 
 
Day 1 
Baseline 
14 days 
after 
previous 
admin 
visit 
Week 8 from 
day 1 
14 days after 
previous admin 
visit 
Weeks 16, 
24, etc. from 
day 1 
Within 30 days after 
last dose of IMP and 
before start of new 
anti-cancer 
treatment 
Every 8 weeks 
from end of 
treatment visit 
until centrally 
confirmed PD 
Every 12 weeks 
after centrally 
confirmed PD 
until death or end 
of study 
CSP 
Section 
Visit window 
Day -28 to  
Day -1 
-3 daysa,f 
+7 days 
±7 days  
+7 days 
±7 days  
 
±7 days  
±7 days  
8.2 
Weighte 
X 
X 
X 
X 
X 
X 
X 
 
 
8.2 
Biopsy available for Pharmacogenetics 
X 
 
 
 
 
 
 
 
 
6.1/6.
2 
Administer and record study treatment 
 
X 
X 
 
X 
 
 
 
 
6.6 
Record other anti-cancer treatment 
 
 
 
 
 
 
X 
X 
X 
8.1 
ECOG 
X 
X 
 
X 
 
X 
X 
X 
 
Efficacy assessments 
8.1 
Plasma PK samples 
 
Xb 
Xb 
 
 
 
 
 
 
8.1 
CT-scan/MRIc 
X 
 
 
X 
 
X 
X 
X 
 
8.1 
PRO EQ-5D 
 
X 
 
X 
 
X 
X 
 
 
8.1 
Survival Status 
 
 
 
 
 
 
 
 
Xg 
Safety assessments 
8.4 
AEs  
 
X 
X 
X  
X 
X 
Xd 
Xd 
 
8.2 
PRO CTCAE 
 
X 
X 
X 
 
X 
X 
 
 
8.2 
Hematology and clinical chemistrye 
Xf 
 
X 
 
X 
 
X 
 
 
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PRE-TREATMENT  
TREATMENT PERIOD  
END OF TREATMENT 
FOLLOW-UP 
Screening 
 
Administer IMP 
Assessment 
visit 
Administer IMP 
Assessment 
visit 
End of treatment 
visit 
Follow-up visits 
until centrally 
confirmed PD 
Survival follow-up 
 
Cycle 1 
 
Cycles 2-4 
 
Cycles 5 - PD or 
discontinuation 
 
 
Only patients 
prematurely 
discontinuing IMP 
prior to centrally 
confirmed PD 
All patients 
 
Timing 
 
Day 1 
Baseline 
14 days 
after 
previous 
admin 
visit 
Week 8 from 
day 1 
14 days after 
previous admin 
visit 
Weeks 16, 
24, etc. from 
day 1 
Within 30 days after 
last dose of IMP and 
before start of new 
anti-cancer 
treatment 
Every 8 weeks 
from end of 
treatment visit 
until centrally 
confirmed PD 
Every 12 weeks 
after centrally 
confirmed PD 
until death or end 
of study 
CSP 
Section 
Visit window 
Day -28 to  
Day -1 
-3 daysa,f 
+7 days 
±7 days  
+7 days 
±7 days  
 
±7 days  
±7 days  
8.2 
Urinalysise 
Xf 
 
X 
 
X 
 
X 
 
 
8.2 
ECGh 
 
X 
X 
 
 
 
 
 
 
8.2 
Pregnancy test 
Xf 
 
 
 
 
 
X 
 
 
a 
Randomisation can occur on the same day as study treatment initiation, or up to 3 days before study treatment initiation.  Study treatment must be initiated within 28 days from 
start of screening. 
b 
Plasma PK samples will be collected during Cycle 1 and Cycle 2. 
c 
CT-scans/MRIs must be performed on thorax, abdomen and pelvis. Post-randomisation CT-scans/MRIs should be performed every 8 weeks (±7 days) from Day 1 to PD confirmed by 
BICR. End of Treatment visit scans will be taken if the previous scan is >14 days. 
d 
AEs are to be recorded until the day of the End of Treatment visit. After the End of Treatment visit, only SAEs related to study specific procedures or considered to be at least possibly 
related to study drug should be recorded.  
e 
Laboratory and urine samples and weight are to be collected according to local practice (7 days before study treatment).  The laboratory and urinalysis reports must be obtained and 
reviewed prior to administration of chemotherapy for each cycle.   
f 
Haematology, clinical chemistry, urinalysis, and pregnancy testing must be performed within 7 days prior to study treatment initiation and must be assessed before randomization 
g 
Survival follow-up visit could be conducted either by phone. ordinary visits, hospital records or other means found suitable. 
h 
12-lead ECG should be performed during administration visits 1-4.  Directly prior to and 10 min (±5 min) after each arfolitixorin dose, and directly prior to and 10 min (±5 
min) after initiation of Leucovorin infusion, during administration visits 1-4. 
 
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3.3 Sample Size Justification 
3.3.1 Hierarchical testing approach 
A fixed sequence hierarchical test procedure will be applied, with a pre-defined order in 
which the hypotheses are to be tested. Each test will be performed without a multiplicity 
adjustment for multiple endpoints, i.e. at the nominal 𝛼-level foreseen by the group 
sequential design. The secondary endpoint PFS can only be tested formally after the first 
hypothesis, i.e. the null hypothesis for ORR, is rejected at the final analysis. 
3.3.2 Accrual rate 
The accrual rate was assumed to ramp up as follows: 2, 3, 7, 11, 14, 22 and 25 patients 
respectively in the 1st to 7th months after study start, and 28.8 patients monthly 
thereafter. 
3.3.3 Primary endpoint - Overall Response Rate (ORR) 
The primary endpoint is the Overall Response Rate (ORR). Null and alternative hypotheses 
are expressed in terms of ΔORR, the difference in ORR between the randomized treatment 
arms: 
• 
H0: ΔORR=0 
• 
H1: ΔORR≥15% 
For this adaptive design, the initial sample size calculation is based on an improvement by 
15% in ORR; specifically, ORR is assumed to be 45% in the control arm vs. 60% in the 
experimental arm. 
It was assumed that up to 10% of patients would be non-evaluable for response in both 
treatment arms. These patients are to be considered non‐responders and do not contribute 
to the difference between experimental and control (hence the expected difference is 
13.5% rather than 15%). 
For a two‐sided test with α=0.05 and a power of 80% to detect this difference, at least 
440 patients were needed to be randomized. Of note, the addition of extra Japanese 
patients leads to a statistical power greater than 80%. An interim calculation of the 
conditional power for ORR as well as PFS took place when 16-week BICR evaluation had 
been performed for the 330th patient. At this analysis, the DSMB did not recommend a 
50% sample size increase (from 4402 to 660 patients). Statistical considerations regarding 
the interim analysis are explained in Section 4.2.1. 
3.3.4 Key secondary endpoint - Progression‐free survival (PFS) 
The study was initially powered to detect a hazard ratio of 0.725, which corresponds to a 
clinically meaningful difference in PFS (median PFS equal to 10 months in the control arm 
vs. 13.8 months in the experimental arm). 
For a power of 80%, 300 PFS events had to be observed using a 1-sided alpha=0.025. 
This number allowed performing one interim analysis. The statistical design allowed for a 
50% sample size increase (to 660 patients), should interim results be promising enough 
to warrant such a sample size increase2. In case the sample size had had to be increased, 
the target number of events would have been increased to 450 PFS events, which provides 
                                           
2 The interim analysis was performed before the decision to add the 50 extra Japanese 
patients to the efficacy and safety analyses. 
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80% power to detect a hazard ratio of 0.77 (median PFS equal to 10 months in the control 
arm vs. 13 months in the experimental arm). 
Due to a number of patients censored because of new anti-cancer therapies higher than 
expected, it became clear during the study that the 300 PFS events could not be reached. 
The following table shows, for 300 and 235 PFS events, the hazard ratio for which the trial 
would have 80% power, the hazard ratio for which the trial would just reach statistical 
significance, and the corresponding median PFS in the treatment arm, assuming a 10-month 
median PFS in the control arm. 
Table 2: Hazard ratio for significance in function of the number of PFS events 
 
Design 
Final analysis 
Number 
of PFS 
events 
Median 
PFS  
Control 
arm 
Hazard ratio 
Median PFS  
Treatment  
arm 
Hazard ratio 
for 
significance 
Median PFS  
Treatment  
arm 
3003 
10 months 
0.725 
13.8 months 
0.80 
12.5 months 
235 
10 months 
0.692 
14.5 months 
0.77 
12.9 months 
A hazard ratio of 0.77 was considered a clinically relevant maximal threshold for statistical 
significance. The 235 number of PFS events is expected to have occurred on 10 April 2022. 
The final PFS analysis will therefore be performed when at least 235 PFS events will be 
reached. Adjustment of the significance level in the interim analysis and final analysis has 
been performed using a Rho family (𝜌= 5) boundary. 
 
                                           
3 Initial version of the protocol 
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3.3.5 
Other secondary endpoints 
An analysis of OS at the same time as the final PFS analysis, with 440 patients, had a 30% 
power to demonstrate a hazard ratio of 0.8 (corresponding to a median OS equal to 25 
months in the control arm vs. 31.25 months in the experimental arm), when approximately 
180 events are observed, using a 1-sided α=0.025. If the sample size had been increased 
to 660 patients, analyzed at the same time as the final PFS analysis , this power would 
have increased to 45%, when approximately 270 events are observed. The addition of the 
extra Japanese patients to the 440 initial patients, leads therefore to a statistical power 
slightly greater than 30%. No interim efficacy analysis is foreseen for OS. Note that this 
trial is underpowered for OS, and that there is no intention to claim efficacy for OS. 
Given the key relevance of this endpoint in an overall assessment of benefit/risk, it is 
important to power the OS analysis to exclude a significant detrimental effect of 
arfolitixorin as compared to leucovorin in terms of OS. The OS analysis is based on the 
following considerations:  
1) Collection of vital status will continue in all randomized patients after the final PFS 
analysis, until the OS analysis. Of note, the aim for this continued follow-up is to monitor 
the patients’ safety, rather than showing superior efficacy of arfolitixorin in terms of OS.  
2) Arfolitixorin is not expected to cause serious toxicity. It is therefore reasonable to 
assume that if superior efficacy of arfolitixorin is demonstrated, OS is unlikely to be inferior 
in the arfolitixorin arm compared to the leucovorin arm. The magnitude of the OS 
difference is hard to predict, for example because of the multiple lines of active therapies 
currently available for the treatment of patients with advanced colorectal cancer. Assuming 
similar post-progression survival in both trial arms, a reasonable assumption is that the 
difference in median PFS between arms will “carry over” to the difference in median OS.  
3) A “significant detrimental effect on OS” will be taken equal to the benefit of leucovorin 
estimated in the meta-analysis of all trials comparing 5-fluorouracil alone with 5-
fluorouracil + leucovorin (Meta-Analysis Group In Cancer 2004). The overall OS HR of 
leucovorin was equal to 0.9 in these trials; hence, an OS hazard ratio equal to 1/0.9 = 
1.11 in the current trial may be considered a significant detriment, to the extent that it 
will outweigh the benefit observed historically with leucovorin.  
4) Collection of vital status will continue after the PFS analysis, until 60% of the patients 
have had a death event. In function of the results of the interim analysis 264 deaths (60% 
of 440 patients) or 396 deaths (60% of 660 patients) would have been required. However, 
with the addition of the extra patients 294 deaths (60% of 490 patients) will be required 
instead of 264 (60% of 440 patients). 
3.3.6 
Japanese study 
Japanese enrollment continued beyond completion of the global enrollment. Approximately 
12.7% of the total study population had to be enrolled from Japan to obtain required 
probability of at least 70% that the ratio (effect in Japanese patients)/(effect in all 
patients) is at least 0.5 i.e. 
𝐷𝐽𝑎𝑝𝑎𝑛
𝐷𝐴𝑙𝑙
> 0.5, with 𝐷𝐽𝑎𝑝𝑎𝑛 the difference in ORR between 
randomized treatment arms, exclusively in Japanese patients, and 𝐷𝐴𝑙𝑙 the difference in 
ORR between randomized treatment arms (Ministry of Health 2007). The proportion of 
Japanese patients needed was calculated according to the methods described in (Ikeda 
and Bretz, 2010). 
 
 
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4. 
General Analysis Definitions 
Data will be analyzed using SAS (Version 9.4 or higher). Graphs will be produced in SAS 
or R. 
Descriptive statistics will be tabulated as follows:  
• 
Categorical data will be summarized in contingency tables presenting frequencies 
and percentages. By default, those percentages will be calculated on the number 
of patients in the analysis population. If this is not the case, it will specifically be 
mentioned in the footer of the table. 
• 
Continuous data will be summarized using number of non-missing values (n), 
mean, standard deviation, median, minimum and maximum values. 
There will be 3 columns (i.e. overall population, arfolitixorin arm and Leucovorin arm). 
Listings with individual values will be provided for all data presented in the tables. 
Significance tests will be performed at the α = 0.05 significance level for two-tailed tests 
and at α = 0.025 for one-tailed tests. When a confidence interval is to be calculated, Wald 
confidence intervals will be used, unless otherwise specified. 
Adjustment of the significance level in the interim analysis and final analysis has been 
done according to group sequential methods, as explained in Section 3.3.1.  
4.1 Study Period and Visit Window Definitions 
Visit windows will not be used, tables will assume that observations are from the recorded 
visit irrespective of the date specified. 
4.2 Planned analyses 
The trial design will include the following analyses (and adaptations if indicated): 
• 
An interim calculation of the conditional power for ORR and PFS took place 
when 16-week BICR evaluation had been performed for the 330th patient. At 
this analysis, the DSMB could have recommend a 50% sample size increase 
(from 440 to 660 patients), according to the guidelines in Table 34. 
• 
In the current case where the sample size is not increased, the final analysis 
of ORR and PFS of the main study will take place when at least 235 PFS events 
are observed. 
• 
The final analysis of OS of the main study will be performed when 60% of the 
patients have had a death event. 
4.2.1 Interim analysis4 
a) Interim analysis of Overall Response Rate (ORR) 
An interim analysis took place when 16-week BICR evaluation had been performed for the 
330th patient. For the interim analysis of ORR, the significance level was determined using 
a Rho spending function, with 𝜌= 5, resulting in a very conservative boundary. Assuming 
an information fraction of 75% for ORR at this interim analysis, the 1-sided significance 
level for efficacy was 0.006, and it would have been reached if the difference in ORR had 
been larger than 13.8%. This small alpha spending was foreseen to take into account the 
interim look at the efficacy primary endpoint, i.e. ORR. However, there was no intention 
                                           
4 The interim analysis was performed before the decision to add the extra Japanese 
patients to the efficacy and safety analyses. 
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to claim efficacy for ORR at this stage. The interim results, i.e. the conditional power for 
both ORR and PFS, only served to guide the decision rules regarding sample size re-
estimation. Results of this analysis remained blinded to everyone, except the DSMB. 
Regardless of the efficacy results of the interim analysis, the trial continued to accrue 
patients, until the total planned sample size is reached. 
 
The interim ORR analysis was performed on a subset of the Intent-to-treat analysis set 
(cf. section 4.3) for subjects who were evaluable for the 16-week BICR assessment or who 
discontinued the study before any evaluation by the BICR. Patients who discontinued the 
study before the 16-week assessment were considered non-responder. They were counted 
in the denominator of the ORR but not in the numerator. All scans evaluated by the BICR 
until the end of treatment available at the time of the interim analysis were used.  
b) Interim analysis of Progression Free Survival (PFS) 
As for ORR, there was no intention to claim efficacy for PFS at this early stage either. A 
small alpha spending was foreseen to take into account the interim look. For this, a very 
conservative Rho spending function boundary, with 𝜌= 5, was used. Assuming an 
information fraction of 37% for PFS at this interim analysis, the 1-sided significance level 
for efficacy was 0.000173, and it would have been reached if the hazard ratio had been 
less than 0.51. 
 
The interim analysis of the PFS was performed on the ITT analysis set (cf. section 4.3) and 
used all scans evaluated by the BICR and available at the time of the interim analysis. 
c) Sample size adaptation 
The conditional power for both ORR and PFS were calculated at the interim analysis, 
assuming that the estimated treatment difference at interim analysis was the true effect. 
Depending on the conditional powers, the trial could have either continue as planned to 
accrue 440 patients, or the sample size of the trial would have been increased by 50%, 
for a total sample size of 660 patients. The guidelines provided in Table 3 were used by 
the DSMB to make a recommendation to the Sponsor. Two possible decisions could be 
communicated to the Sponsor by the DSMB: “sample size increase” or “no sample size 
increase”. 
 
The conditional power for ORR would be equal to approximately 80% if the observed 
difference in ORR, at the time of the interim analysis (75% information fraction) was equal 
to 11.5%. The conditional power for PFS would be equal to 25% if the observed hazard 
ratio, at the time of the interim analysis (37% information fraction) was equal to 0.85. 
Note that a HR=0.85 corresponds with a Median survival time Control/Experiment of 
10/11.75, which is considered clinically meaningful. The conditional power for PFS would 
be equal to 50% if the observed hazard ratio, at the time of the interim analysis (37% 
information fraction) was equal to 0.80. The conditional power for PFS would be equal to 
75% if the observed hazard ratio, at the time of the interim analysis (37% information 
fraction) was equal to 0.75. 
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Table 3: Guidelines for sample size increase at interim analysis 
ORR = overall response rate; PFS = progression-free survival S=Success, F=Failure, CP=Conditional Power 
assuming that the estimated treatment difference at interim analysis is the true effect, CP440= conditional 
power at N=440, CP660= conditional power at N=660.  m=months, NA=not applicable 
* Including type I error rate adjustment, using the Cui, Hung and Wang method (Cui et al., 1999) 
 
 
ORR 
PFS 
Recommendation 
CP660 for  
CP440 
Observed 
ORR 
difference 
CP440 
Observed HR 
ORR 
PFS 
 80% 
11.5% 
 75% 
≤0.75 
(≥3.3 m) 
Continue trial unchanged 
NA 
NA 
50-75% 
>0.75; ≤0.8 
(≥2.5<3.3m) 
Increase sample size 
 90% 
70-90% 
25-50% 
>0.8; ≤0.85 
(≥1.8;<2.5m) 
Increase sample size* 
 90% 
>40 - <70% 
<25% 
>0.85 
(<1.8m) 
Continue trial unchanged 
NA 
NA 
60-80% 
 10%;< 
11.5% 
 75% 
≤0.75 
(≥3.3 m) 
Increase sample size 
80-90% 
90% 
50-75% 
>0.75; ≤0.8 
(≥2.5<3.3m) 
Increase sample size 
80-90% 
70-90% 
25-50% 
>0.8; ≤0.85 
(≥1.8;<2.5m) 
Increase sample size* 
80-90% 
>40 - <70% 
<25% 
>0.85 
(<1.8m) 
Continue trial unchanged 
NA 
NA 
< 60% 
<10% 
 75% 
≤0.75 
(≥3.3 m) 
Continue trial unchanged 
NA 
NA 
50-75% 
>0.75; ≤0.8 
(≥2.5<3.3m) 
Continue trial unchanged 
NA 
NA 
25-50% 
>0.8; ≤0.85 
(≥1.8;<2.5m) 
Continue trial unchanged 
NA 
NA 
<25% 
>0.85 
(<1.8m) 
Continue trial unchanged 
NA 
NA 
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4.2.2 Final analysis of ORR and PFS  
Initially, the final analysis of ORR and PFS should have taken place when 300 events are 
observed, if the sample size was not increased. While if the sample size was increased, 
then the final analysis of ORR and PFS should have taken place when 450 PFS events are 
observed. 
Given the decision of the DSMB at the interim analysis to continue the study unchanged, 
and given the updated timing of the final analysis, the final analysis of ORR and PFS will 
take place when 235 PFS events are observed (see also Sections 3.2.1 & 3.3.4). 
a) Overall Response Rate (ORR) 
The 1-sided significance level for efficacy will be 0.024. If the sample size would have been 
increased to 660 patients, the 1-sided significance level for efficacy would have been be 
0.025. More details about the calculation of the ORR are provided in section 4.5. 
All scans evaluated by the BICR at the time of the final analysis of the ORR and PFS will 
be used. 
b) Progression Free Survival (PFS) 
The 1-sided significance level for efficacy will be 0.025. 
If the sample size would have been increased to 660 patients, the 1-sided significance 
level for efficacy would have been 0.025. 
c) Overall Survival (OS) 
The Overall Survival analysis will be presented at the time of the final analysis of ORR and 
PFS as supportive analysis. 
d) Other efficacy endpoints 
All other efficacy endpoints will be presented at the time of the final analysis of ORR and 
PFS. 
e) Safety analysis 
The safety analyses will be performed on the Safety analysis set (cf. section 4.3). 
4.2.3 Final analysis of Overall Survival (OS)  
The final analysis of OS will be performed when 60% of the patients have had a death 
event. 
The overall survival analysis will be presented. The other efficacy endpoints and the safety 
assessment will be re-evaluated based on the data available at the time of the overall 
survival analysis. The efficacy endpoints will be assessed both on the ITT and the PP 
analysis sets. The safety analyses will be performed on the Safety analysis set (cf. section 
4.3). 
 
 
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4.2.4 Analysis of Japanese patients  
The analysis of the results for the Japanese patients will be performed at the time of the 
final ORR and PFS analysis. 
For this analysis, all analyses planned in this SAP will be repeated on the corresponding 
Japanese analysis subsets (cf. section 4.3) except if specified otherwise. 
4.3 Definition of Populations 
All enrolled patients who were randomized will be included in the analyses. The following 
analysis sets will be considered: 
• 
Intention-to-Treat (ITT) analysis set consists of all randomized participants. 
Analyses will be based on the randomized treatment. 
• 
Intention-to-Treat Japanese (ITTJ) analysis set consists of all randomized 
Japanese participants. Analyses will be based on the randomized treatment. 
• 
Intention-to-Treat Excluding Additonal Japanese Patients (ITTE) analysis 
set consists of randomized participants excluding additional Japanese patients. 
Analyses will be based on the randomized treatment. 
• 
Per-Protocol (PP) analysis set defines a subset of the participants in the ITT 
analysis set without major protocol deviations. The definition of major protocol 
deviations will be agreed upon, and all cases of such major deviations adjudicated 
prior to database lock. Analyses will be based on the randomized treatment. 
• 
Per-Protocol Japanese (PPJ) analysis set defines a subset of the participants 
in the ITTJ analysis set without major protocol deviations. The definition of major 
protocol deviations will be agreed upon, and all cases of such major deviations 
adjudicated prior to database lock. Analyses will be based on the randomized 
treatment. 
• 
Safety analysis set defines the subset of participants for whom safety analyses 
will be conducted. It includes all patients who received at least one dose of study 
medication. Analysis will be based on the actual treatment. 
• 
Safety Japanese analysis set defines the subset of Japanese participants for 
whom safety analyses will be conducted. It includes all Japanese patients who 
received at least one dose of study medication. Analysis will be based on the actual 
treatment. 
• 
PK analysis set defines a subset of the participants in the Safety analysis set for 
whom PK samples were analysed. Analyses will be based on the actual treatment. 
• 
PK Japanese analysis set defines a subset of the participants in the Safety 
Japanese analysis set for whom PK samples were analysed. Analyses will be 
based on the actual treatment. 
4.4 Subgroup Definitions 
Subgroup analyses for the efficacy endpoints: ORR and PFS (all estimands) with 
respect to the trial’s stratification factors (Geographic region, 
Primary 
tumor 
location and previous adjuvant CRC treatment) and the prognostic factors described 
hereunder will be performed. Of note, all factors involved in the subgroup definition 
will be based on baseline information. The confidence intervals and the p-values will 
be displayed only for exploratory purposes with no intention to claim efficacy. 
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The consistency of the treatment effect across prognostic subgroups will be assessed 
visually using forest plots analyses based on observed results. 
 
Table 4: Subgroup definitions 
Factors 
Subgroup levels 
Primary tumor location 
Left colon,  
Rectal cancer, 
Right colon 
Previous adjuvant CRC treatment 
Yes, 
No 
Primary tumor resected 
Yes, 
No 
ECOG performance status 
0, 
1 
Tumor mutation status BRAF 
Wild-type, 
Mutant 
Tumor mutation status KRAS 
Wild-type, 
Mutant 
Tumor mutation status NRAS 
Wild-type, 
Mutant 
Liver metastasis 
Yes, 
No 
Lung metastasis 
Yes, 
No 
Peritoneal metastasis 
Yes, 
No 
Other metastasis 
Yes, 
No 
Measurable lesion burden 
< 25th percentile 
[25th perc. – 75th perc.] 
> 75th percentile 
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Factors 
Subgroup levels 
Geographic region 
North America,  
Europe,  
Australia,  
Japan 
Sex 
Male, 
Female 
Age 
<65 years 
≥65 years 
>75 years 
Body Mass Index (BMI) 
<25 
25+  
Albumin 
Low 
Normal 
High 
Alanine Aminotransferase 
 
Low 
Normal 
High 
Aspartate Aminotransferase 
 
Low 
Normal 
High 
Gamma Glutamyl Transferase 
 
Low 
Normal 
High 
Leukocytes 
Low 
Normal 
High 
Hemoglobin 
Low 
Normal 
High 
Platelets 
Low 
Normal 
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Factors 
Subgroup levels 
High 
Neutrophils 
Low 
Normal 
High 
Neutrophils/Leukocytes ratio 
 
Low 
Normal 
High 
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4.5 Estimand definitions 
According to the FDA guidance document E9(R1) Statistical Principles for Clinical Trials: 
Addendum: Estimands and Sensitivity Analysis in Clinical Trials (May 2021), and the EMA 
guideline document ICH E9 (R1) addendum on estimands and sensitivity analysis in clinical 
trials to the guideline on statistical principles for clinical trials (30 July 2020), the primary 
endpoint ORR and the secondary endpoint PFS are formulated using the estimand 
framework, taking into account relevant intercurrent events.  
4.5.1 Estimand definitions for ORR 
The primary estimand for ORR is defined by:  
• 
Treatments: experimental vs comparator (see Section 3.2.1) 
• 
Population: ITT analysis set (see Section 4.3) 
• 
Variable: proportions of patients with ORR as BOR (See Section 4.6 for definitions) 
• 
Intercurrent events and strategies to address those:  
o 
Early death, metastasis resection, new anticancer treatment and treatment 
discontinuation causing an non-evaluable or missing result for ORR: a 
composite strategy will be used, by imputing any missing or unevaluable 
response data as failure (non-responder imputation).  
• 
Population-level summary: The common difference in ORR between the two arms 
and its associated 95% confidence interval will be estimated  using the Mantel-
Haenszel estimate of the common risk difference by using Mantel-Haenszel stratum 
weights (Mantel and Haenszel 1959) and the Sato variance estimator of this 
difference (Sato 1989). (see Section 9.1.1). 
No supplemental estimands are defined for ORR.  
4.5.2 Estimand definitions for PFS 
The primary estimand for PFS is defined by:  
• 
Treatments: experimental vs comparator (see Section 3.2.1) 
• 
Population:  
o 
Population defined through the inclusion/exclusion criteria  
o 
Analysis set: ITT analysis set (see Section 4.3) 
• 
Variable: time from randomization to event time (see Section 4.6 PFS definition) 
• 
Intercurrent events and strategies to address those:  
o 
Start of a new anticancer treatment (except metastasis resection) before 
any documented progressive disease or death: a hypothetical strategy will 
be used, by censoring patients with this ICE at the last tumor assessment 
with documented non-progression prior to this ICE (see Section 4.6, Table 
5). 
o 
Metastasis resection:  patients with metastasis resection continue to be 
scanned until disease progression, defined as new lesion appearance or 
significant increase of residual lesions (See Section 4.6 under BOR). For this 
ICE a treatment policy is followed, i.e. the ICE will be ignored. 
• 
Population-level summary: hazard ratio, estimated by a stratified Cox proportional 
hazards model (see Section 9.1.2). 
The first supplemental estimand for PFS is defined by:  
• 
Treatments: experimental vs comparator (see Section 3.2.1) 
• 
Population:  
o 
Population defined through the inclusion/exclusion criteria  
o 
Analysis set: ITT analysis set (see Section 4.3) 
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• 
Variable: time from randomization to event time (see Section 4.6 PFS definition) 
• 
Intercurrent events and strategies to address those:  
o 
Start of a new anticancer treatment (except metastasis resection) before 
any documented progressive disease or death: a treatment policy strategy 
will be followed, by ignoring this ICE (see Section 4.6, Table 6). 
o 
Metastasis resection:  patients with metastasis resection continue to be 
scanned until disease progression, defined as new lesion appearance or 
significant increase of residual lesions (See Section 4.6 under BOR). For this 
ICE a treatment policy is followed, i.e. the ICE will be ignored. 
• 
Population-level summary: hazard ratio, estimated by a stratified Cox proportional 
hazards model (see Section 9.1.2). 
The second supplemental estimand for PFS is defined by:  
• 
Treatments: experimental vs comparator (see Section 3.2.1) 
• 
Population:  
o 
Population defined through the inclusion/exclusion criteria  
o 
Analysis set: ITT analysis set (see Section 4.3) 
• 
Variable: time from randomization to event time (see Section 4.6 PFS definition) 
• 
Intercurrent events and strategies to address those:  
o 
Start of a new anticancer treatment (except metastasis resection) before 
any documented progressive disease or death: a composite strategy will be 
used: patients with this ICE will be considered to have a progression event 
on the date of start of the ICE (see Section 4.6, Table 7). 
o 
Metastasis resection:  patients with metastasis resection continue to be 
scanned until disease progression, defined as new lesion appearance or 
significant increase of residual lesions (See Section 4.6 under BOR). For this 
ICE a treatment policy is followed, i.e. the ICE will be ignored. 
• 
Population-level summary: hazard ratio, estimated by a stratified Cox proportional 
hazards model (see Section 9.1.2). 
4.6 Calculated Variables 
• 
Additional Japanese patients are defined as the additional patients enrolled 
to address the Japanese authorities’ request that approximately 12.7% of the 
total study population are enrolled from Japan. A flag variable in a SDTM dataset 
will be used to distinguish these additional Japanese participants. 
• 
Baseline is defined as the last non-missing value measured or collected before 
the first dose of any study treatment. If time not available, assessments done on 
the date of study treatment administration are assumed to take place before the 
administration, unless specified otherwise. 
• 
Best confirmed Overall Response (BOR) 
The estimand related to this outcome is specified in Section 4.5.1.  
 
The evaluations of the Overall Response and the Best confirmed Overall Response 
(BOR) are performed by the BICR using RECIST 1.1 (Eisenhauer et al., 2009). 
 
When there is a discordance between assessments of the two radiologists then the 
adjudicated assessment will be used. More details about the evaluation of the 
confirmed response is provided in the BICR Imaging Review Charter (Banook 2021). 
 
BOR will not be derived by IDDI, instead the BICR BOR results, based on RECIST 1.1, 
will be used. 
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Patients with metastatic resection are continued to be scanned until disease 
progression. The BICR assessments of the overall response of patients with a 
metastasis resection will be as follows:  
- 
Patients having complete metastasis surgery will have the overall response 
as NE until new lesion appearance leading to PD (Progressive Disease);  
- 
Patients having partial metastasis surgery will have the overall response as 
NE too until new lesion appearance or significant increase of the residual 
lesion(s) leading to PD (Progressive Disease) as per RECIST 1.1 criteria.  
The process of the BICR evaluation of the CT-scans/MRIs is described in detail in the 
BICR Imaging Review Charter (Banook 2021). 
 
• 
Overall response rate (ORR) RECIST 1.1 
The overall response rate (ORR) is defined as the proportion of subjects for whom the 
best confirmed overall response (BOR), as per BICR, is complete response (CR) or 
partial response (PR) as determined by the BICR using RECIST 1.1. 
 
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑠 𝑤𝑖𝑡ℎ 𝑎 𝑐𝑜𝑛𝑓𝑖𝑟𝑚𝑒𝑑 𝐵𝑂𝑅 𝑜𝑓 𝐶𝑅 𝑜𝑟 𝑃𝑅
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑃𝑎𝑡𝑖𝑒𝑛𝑡𝑠 𝑖𝑛𝑐𝑙𝑢𝑑𝑒𝑑 𝑖𝑛 𝑡ℎ𝑒 𝑂𝑅𝑅 𝑎𝑛𝑎𝑙𝑦𝑠𝑖𝑠
 
 
• 
Progression-free survival (PFS) 
= (date of death due to any cause or date of PD, where PD is defined as the first 
occurrence of tumor progression based on CT‐scans/MRIs, whichever comes first, as 
per BICR) – randomization date + 1. The PFS will be expressed in months. 
 
In addition, some anticipated special cases or intercurrent events will be taken into 
account. Three estimands concerning PFS are specified in Section 4.5.1: the primary 
estimand, for which Table 5 is to be used, the first supplemental estimand, for which 
Table 6 is to be used, and the second supplemental estimand, for which Table 7is to 
be used. 
 
Table 5: Censoring rules for the primary PFS estimand  
Situation / Intercurrent Event 
Handling 
No documented progression and no death (no 
observed event) 
Censored at last tumor assessment with 
documented non-progression 
Missing (or incomplete) baseline tumor 
assessments 
Censored at randomization  
Progression or death documented immediately 
after 2 or more consecutive missed 
assessment visits  
Censored at last tumor assessment with 
documented non-progression (before missing 
visits) 
Death occurring before any documented 
progressive disease (and not immediately 
following 2 consecutive missed assessment 
visits) 
Considered as an event at the date of death 
New anticancer treatment (except metastasis 
resection) started before any documented 
progression or death 
Censored at last tumor assessment with 
documented non-progression (before start of 
new treatment) 
 
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Situation / Intercurrent Event 
Handling 
Metastasis resection, started before any 
documented progression or death 
These patients will continue to be followed up 
until death or PD, as documented by BICR. 
Considered as an event at date of progression 
or death. 
 
Table 6: Censoring rules for the first supplemental estimand for PFS 
Situation / Intercurrent Event 
Handling 
No documented progression and no death (no 
observed event) 
Censored at last tumor assessment with 
documented non-progression 
Missing (or incomplete) baseline tumor 
assessments 
Censored at randomization  
Progression or death documented immediately 
after 2 or more consecutive missed 
assessment visits  
Censored at last tumor assessment with 
documented non-progression (before missing 
visits) 
Death occurring before any documented 
progressive disease (and not immediately 
following 2 consecutive missed assessment 
visits) 
Considered as an event at the date of death 
New anticancer treatment (except metastasis 
resection) started before any documented 
progression or death 
These patients will continue to be followed up 
until death or PD, as documented by BICR. 
 
Metastasis resection, started before any 
documented progression or death 
These patients will continue to be followed up 
until death or PD, as documented by BICR. 
Considered as an event at date of progression 
or death. 
 
Table 7: Censoring rules for the second supplementary estimand for PFS 
Situation / Intercurrent Event 
Handling 
No documented progression and no death (no 
observed event) 
Censored at last tumor assessment with 
documented non-progression 
Missing (or incomplete) baseline tumor 
assessments 
Censored at randomization  
Progression or death documented immediately 
after 2 or more consecutive missed 
assessment visits  
Censored at last tumor assessment with 
documented non-progression (before missing 
visits) 
Death occurring before any documented 
progressive disease (and not immediately 
following 2 consecutive missed assessment 
visits) 
Considered as an event at the date of death 
New anticancer treatment (except metastatic 
resection) started before any documented 
progression or death 
Considered as a progression event at the date 
of start of new anticancer treatment. 
Metastasis resection, started before any 
documented progression or death 
These patients will continue to be followed up 
until death or PD, as documented by BICR. 
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Situation / Intercurrent Event 
Handling 
Considered as an event at date of progression 
or death. 
 
• 
Overall Survival (OS) 
= date of death due to any cause - randomization date + 1. 
The OS will be expressed in months. 
 
In addition, some anticipated special cases or intercurrent events will be taken into 
account as described in the Table 8. 
 
Table 8: Censoring rules for OS 
Situation / Intercurrent Event 
Handling 
No documented death (no observed event) 
Censored at the last contact date or the 
randomization date (whichever comes later) 
Lost to follow-up 
Censored at the last contact date or the 
randomization date (whichever comes later) 
 
• 
Duration of Response (DOR) 
= (date of death due to any cause or date of PD, where PD is defined as the first 
occurrence of tumor progression based on CT‐scans/MRIs as per BICR, whichever 
comes first) – date of first confirmed response of CR or PR (the start date of response, 
not the date when response was confirmed) + 1. 
The DOR will be expressed in months. 
 
Patients with no response of CR or PR or in whom CR or PR is never confirmed will not 
be included for this endpoint. 
 
The anticipated special cases or intercurrent events described in Table 5: Censoring 
rules for the primary PFS estimand also apply for DOR. Note: patients with metastatic 
resection are continued to be scanned until disease progression. 
 
• 
Recurrence Free Survival (RFS) for patients undergoing metastatic resection 
= (date of death due to any cause or date of PD, where PD is defined as the first 
occurrence of tumor progression based on CT‐scans/MRIs as per BICR, whichever 
comes first ) – date of first surgery with removal of the metastasis + 1. The RFS will 
be expressed in months. 
 
The anticipated special cases or intercurrent events described in Table 5: Censoring 
rules for the primary PFS estimand also apply for RFS. Note: patients with metastatic 
resection are continued to be scanned until disease progression. 
4.7 Partial Dates 
Partial adverse event (AE) start date will not be imputed.  
If a medication date is missing or partially missing, in a way that it cannot be 
determined if it was taken prior or concomitantly (using the date and the eCRF 
variable that indicates if the medication was taken prior to the date of first dose of 
study treatment), it will be considered as concomitant medication. 
 
 
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4.8 Methods To Be Used For Handling Missing Data 
As general rules, missing data will not be imputed. 
Per definition of ORR in section 4.5, any subjects with missing response information 
to treatment will be counted as non-responders for ORR. 
For PK data, the values under the LLOQ will be imputed to ½ times the concentration 
value of the LLOQ. 
 
5. 
Study Patients 
5.1 Disposition of Patients 
The study disposition of all enrolled patients will be summarized with descriptive 
statistics. It will present the number of patients: 
• 
Screened, 
• 
Screen failure, 
• 
ITT analysis set (see definitions in Section 4.3), 
• 
Safety analysis set (see definitions in Section 4.3), 
• 
Per-Protocol analysis set (see definitions in Section 4.3). 
Note: for the analysis of Japanese patients the number of patients in the ITTJ, the 
PPJ and the Safety Japanese analysis sets will be presented. 
For treatment discontinuation, the following categorical outcomes will be presented:  
• 
Patients with ongoing treatment, 
• 
Patients who discontinued the treatment, and the reason for discontinuation. 
The frequency and primary reason for terminating the study will also be summarized, 
as will be death and cause of death. 
5.2 Protocol Deviations 
Major protocol deviations will be summarized for the ITT analysis set. Deviations to 
the protocol will be classified as major or minor by Isofol in a list sent to IDDI before 
database lock. 
5.3 In- and Exclusion Criteria 
Listing of all in- and exclusion criteria not met will be provided for all screened 
patients. 
6. 
Demographic and other Baseline Characteristics 
Descriptive statistics with respect to patient characteristics at baseline will be 
displayed for the ITT analysis set. Intervention groups (Overall, Arfolitixorin and 
Leucovorin) will be compared on baseline characteristics, including demographics 
and laboratory measurements. No inferential statistics will be used. 
The variables to be summarized are: 
• 
Gender, Age (continuous), 
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• 
Race, ethnicity (If race and ethnicity are collected), 
• 
Primary tumor location (left colon/right colon/rectal cancer) from IWRS, 
• 
Previous neo-adjuvant/adjuvant CRC treatment (yes/no) from IWRS, 
• 
Geographic region (Europe / North America / Japan / Australia) from IWRS, 
• 
Pregnancy test. 
7. 
Medical history 
Colorectal Cancer History will summarize by treatment arms: 
• 
The primary tumor resected (Yes, No missing), 
• 
The primary tumor still in place (Yes, No, missing), 
• 
The location of metastatic cancer (Liver, Lung, Other), 
• 
The time since original diagnosis (in months), 
• 
The time since metastatic diagnosis (in months). 
 
Other medical history will be tabulated by system organ class and preferred term (per 
MedDRA), with three separate tables:  
o 
Overall, 
o 
‘ongoing’ medical history, 
o 
‘not ongoing’ medical history. 
8. 
Prior and Concomitant Treatment and procedures 
Prior and Concomitant medications will be classified according to World Health 
Organization Drug Dictionary, WHODrug Global C3. The number and percentage of 
participants receiving a prior or concomitant medication will be displayed by Anatomical 
Therapeutic Chemical (ATC) level 1 and 4 for the ITT analysis set. Medications will be 
reported as prior when they start before the first day of study treatment and if they end 
before the first day of study treatment. Medications will be reported as concomitant when 
they start before, on or after first day of study treatment and continue afterwards. 
There will be two separate tables, one for prior medications and one for concomitant 
medications.  
Prior and Concomitant medication summaries will be sorted alphabetically by ATC level 1 
and 4. A listing of all medications recorded on the prior and concomitant medications CRF 
page will provide details including indication, dose, route, frequency, and start and stop 
dates. 
Concomitant procedures and surgeries will be classified using the Medical Dictionary for 
Regulatory Activities (MedDRA) and will be tabulated by system organ class and preferred 
term for the ITT analysis set. 
Two tables will report the other anti-cancer therapies, one for the metastasis resection 
and a second one for the anti-cancer therapies excluding metastasis resection. The 
metastasis resection will be displayed by MedDRA System Organ Classes and preferred 
terms. The number and percentage of participants receiving other anti-cancer therapies 
will be displayed by WHODrug ATC level 1 and 4 for ITT analysis set as the other 
medication tables. The other anti-cancer therapies and the metastatic resection are 
entered by investigator in a distinct CRF form than the other concomitant treatments and 
procedures. Variables in the SDTM datasets will allow identifying other anti-cancer 
therapies and metastasis resections. 
 
 
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9. 
Efficacy Evaluation 
All efficacy analyses will be performed on both the ITT and the per‐protocol analysis sets. 
The analyses performed on the ITT analysis set are considered as primary. 
9.1 Response assessment 
9.1.1 Overall Response Rate (ORR) 
ORR will be analyzed using a Cochran-Mantel-Haenszel test (CMH), stratified for the 
stratification factors used for randomization (geographic region, primary tumor location 
and previous neo-adjuvant/adjuvant CRC treatment). 
The ORR will be estimated along with 95% confidence limits for proportion. 
The table for ORR will show the following results 
• 
The best confirmed overall response (CR/PR/SD/PD/NE), 
• 
Overall response rate with 95% confidence limits and p-value from a stratified 
Cochran-Mantel-Haenszel test. 
• 
The common difference in ORR between the two arms and its associated 95% 
confidence interval will be estimated  using the Mantel-Haenszel estimate of 
the common risk difference by using Mantel-Haenszel stratum weights 
(Mantel and Haenszel 1959) and the Sato variance estimator of this 
difference (Sato 1989).   
If the ORR response is coded 0 for failure and 1 for response then the option 
“column=2” must be used and the treatment variable must be coded “1”= 
experimental treatment and “2”=control treatment, so that p1-p2 represents the 
desired difference. 
This test will be performed using the SAS PROC FREQ with statements 
PROC FREQ data=dataset; 
 
  
TABLES strata*trt*orr  / COMMONRISKDIFF(CL=MH TEST=MH column=2); 
RUN; 
The 1-sided significance level for the final analysis of ORR will be 0.024 if no sample 
size increase and 0.025 in case of sample size increase. 
This ORR analysis will be repeated on the Intention-to-Treat Excluding additional 
Japanese patients (ITTE) analysis set in order to account for potential bias due to 
non-concurrent enrollment of the additional Japanese patients. 
A listing will be provided for 
• 
Time point response information (sum of target lesions, target lesion 
response, non-target lesion response, overall response), as entered in the 
CRF at each tumor assessment, including baseline (only for sum of target 
lesions) and unscheduled assessments. 
Note: For the analysis of the Japanese patients: 
- In addition to the analyses planned above, the ratio (effect in Japanese patients)/(effect 
in all patients) will be compared to 0.5 i.e. 
𝐷𝐽𝑎𝑝𝑎𝑛
𝐷𝐴𝑙𝑙
> 0.5 as mentioned in section 3.3.6.  
- The tests mentioned above will be performed without use of stratification factors. 
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9.1.2 Progression free Survival (PFS), Duration of Response (DOR) and Overall 
Survival (OS) 
PFS, DOR and OS will be analyzed using the same methodology: 
• 
The stratified logrank test will be produced with the SAS procedure PROC 
LIFETEST using the stratification factors applied for randomization. 
Descriptive statistics will also be presented. Kaplan-Meier curves will be 
created with SAS PROC LIFETEST or with R. 
• 
A stratified Cox proportional hazards model (using the same stratification 
factors as for randomization and the option “ties=discrete”) will be performed 
with the SAS procedure PROC PHREG. The assumption of proportional hazards 
will be assessed. 
• 
The 1-sided significance level for the final analysis of PFS, DOR and OS will 
be 0.025. 
The following results will be presented: 
• 
Kaplan-Meier Graphical display (including numbers at risk), 
• 
Number of patients, 
• 
Number of events, 
• 
Median months to event + 95% confidence interval (Brookmeyer and Crowley 
confidence interval), 
• 
Stratified logrank test statistic and its p-value and 
• 
Hazard Ratio (with 95%CI) from the Cox regression. 
Note: For the analysis of the Japanese patients, the tests mentioned above will be 
performed without use of stratification factors. 
Sensitivity analyses (not for the analysis of Japanese patients) 
In addition to the primary analysis of PFS, two sensitivity analyses will be conducted, 
following the censoring rules as displayed in Table 6 and Table 7 of section 4.5. The results 
of those sensitivity analyses will be presented similarly to the primary analysis of PFS. 
Analyses on the ITTE analysis set 
The Progression Free Survival (PFS), the Duration of Response (DOR) and the Overall 
Survival (OS) analyses will be repeated on the Intention-to-Treat Excluding additional 
Japanese patients (ITTE) analysis set. 
9.1.3 Recurrence Free Survival (RFS) 
For patients having metastasis resection done during the study, treatment with the study 
regimen may be continued at the investigator discretion. 
The exploratory endpoint Recurrence Free Survival (RFS) will be analyzed using the same 
methodology as PFS, DOR and OS (cf. section 9.1.2). 
The exploratory endpoint Recurrence Free Survival (RFS) will not be done for the analysis 
of Japanese patients. 
9.2 PRO EQ‐5D questionnaire 
The PRO EQ‐5D questionnaire results will be analyzed using descriptive statistics and 
graphical representation. 
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PRO EQ‐5D has five dimensions: mobility, self-care, usual activities, pain/discomfort and 
anxiety/depression. Each dimension has 5 levels: no problems, slight problems, moderate 
problems, severe problems and extreme problems (Van Reenen et al., 2015). The number 
and the proportion of patients will be presented by treatment arm in a table for each level, 
for each dimension and for each visit. 
The EQ VAS records the respondent’s self-rated health on a vertical, visual analogue scale 
where the endpoints are labelled ‘Best imaginable health state’ and ‘Worst imaginable 
health state’. EQ VAS data will be described by the mean, the median, the standard 
deviation, the first quartile and the second quartile as recommended by EQ-5D-5L user 
guide (Van Reenen et al., 2015). 
9.3 Patients undergoing metastasis resection 
Defined as the number of patients qualifying for curative metastasis resection after 
treatment with study drug will be compared between the treatment groups. This will 
be summarized by treatment groups using descriptive statistics. 
10. Safety Evaluation 
All safety analyses will be done on the safety analysis set. 
10.1 Extent of Exposure 
The extent of exposure will be summarized descriptively for each arm. 
10.2 Adverse Events 
Adverse events (AEs) will be coded using the Medical Dictionary for Regulatory 
Activities (MedDRA 22.1) and will be graded according to the National Center 
Institute Common Terminology Criteria for AEs (NCI-CTCAE criteria [v5.0]). Uncoded 
terms will be presented under “SOC uncoded”, with their reported term. 
Adverse events will be analyzed in terms of their type, incidence, severity and 
relationship to the study treatment. Related AEs are defined as events with a 
relationship to study treatment equal to ‘Possible’, ‘Probable’, or ‘Related’ or with 
missing relationship. 
A summary table will present by treatment arm the number and percentage of 
patients with at least one: 
• 
AE 
• 
Serious AE 
• 
Adverse event of CTC grade ≥3 
• 
Related AE 
• 
AE leading to permanent discontinuation of the study treatment 
• 
Fatal AE 
• 
AE of special interest (according to current version of List of AESI PT) 
Of note, the list of preferred terms to be considered as AESI will be provided by 
sponsor to IDDI for programming purposes. 
In addition, tabulations of the number of patients who experienced AEs as well as 
severity of the events will be presented by system organ class and preferred term. 
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Patients will only be counted once for each preferred term. In case a patient 
experienced the same event more than once, the worst severity will be presented. 
The following tabulations will be presented: 
• 
All AEs 
• 
AEs leading to permanent discontinuation of the study treatment 
• 
AEs related to the study treatment 
• 
AE of special interest (according to current version of List of AESI PT) 
Listing of all adverse events by treatment arm will be provided, flagging the ones 
that are AESI, including the patient identifier, age, race, sex, verbatim, preferred 
term, duration of the event, severity, action taken, outcome, causality, and date of 
onset. 
In addition, a listing of AESIs will be provided similarly to the listing of all AEs. 
10.3 Deaths and Serious Adverse Events  
Serious adverse events (SAEs), fatal AEs and NCI/CTC grade ≥3 AEs will be 
summarized grouped by system organ class and preferred term. In addition, listings 
of SAEs, fatal AEs and grade ≥3 AEs will be provided, similarly to the listing of all 
AEs. 
The number of deaths will be tabulated together with the primary cause of death. 
The details of the ‘other cause’ will be included in the listing. 
The number of deaths on IMP treatment or within 30 days of study drug 
discontinuation will be displayed by treatment arm. 
The number of deaths occurring in the follow-up after 30 days of study drug 
discontinuation will be displayed by treatment arm. 
10.4  NCI PRO‐CTCAE questionnaire 
The NCI PRO‐CTCAE questionnaire results will be analyzed using descriptive statistics 
and graphical representations. 
The number and the proportion of patients will be presented by treatment arm in a 
table for each categorical response level of each question and for each visit. 
10.5 Clinical Laboratory Determination 
The laboratory safety samples (hematology, clinical chemistry, and urinalysis) will 
be collected according to the protocol schedule of activities and in accordance with 
local practice and analyzed by local laboratories. 
The observed values will be recorded and assessed as “normal” or “abnormal” in the 
eCRF. Abnormal values will be assessed by the Investigator as “clinically significant” 
or “not clinically significant”. 
All required laboratory tests are specified in Table 9. 
Table 9: Laboratory tests 
HEMATOLOGY  
CLINICAL CHEMESTRY 
URINALYSIS  
Hemoglobin  
  
Sodium  
Protein 
Hematocrit  
Potassium  
Glucose 
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HEMATOLOGY  
CLINICAL CHEMESTRY 
URINALYSIS  
WBC 
Calcium  
Blood 
Neutrophils  
Glucose   
 
Lymphocytes 
Monocytes  
BUN or urea  
Creatinine   
 
Eosinophils  
AST   
 
Basophils  
ALT   
  
Platelet count 
 
Alkaline phosphatase   
Total bilirubin   
Total protein   
Albumin 
LDH 
gGT  
  
      
Laboratory tests will be summarized using descriptive statistics for actual values and 
change from baseline for continuous laboratory test. For categorical laboratory test, 
frequencies and percentages of patients in each category will be used. Summaries 
will be presented separately for hematology, blood chemistry and urinalysis 
parameters. 
Grading of laboratory values will be assigned programmatically as per the NCI-CTCAE 
criteria [v5.0]. The calculations of CTCAE grades will be based on the observed 
laboratory values only, clinical assessments will not be taken into account. CTCAE 
grade 0 will be assigned for all non-missing values not graded as 1 or higher. For 
laboratory tests where grades are not defined by the CTCAE, results will be 
categorized as normal/abnormal based on laboratory normal ranges. 
For all laboratory tests where grades are defined by the CTCAE grading, shift tables 
from the baseline CTCAE grade to the worst CTCAE grade during study will be 
produced.  
For the calculation of the worst CTCAE grade, both scheduled and unscheduled values 
available post baseline will be used. 
Shift tables from baseline abnormality grade (Normal/Abnormal) to worst 
abnormality grade during study will be displayed for all laboratory tests where grades 
are not defined by the CTCAE grading. 
10.6 Vital Signs, Physical Findings and Other Observations Related to 
Safety 
Descriptive statistics of systolic and diastolic blood pressure, heart rate and body 
weight will be presented by visit. Both absolute values and change from baseline will 
be presented. In case of multiple measurements, the highest for post-baseline blood 
pressure and heart rate data and the lowest for post-baseline weight will be kept for 
the calculations. 
Descriptive statistics of ECGs results (normal, abnormal not clinically significant, 
abnormal clinically significant) will be presented for the worst outcome (normal< 
abnormal not clinically significant< abnormal clinically significant). The ECG results 
will also be presented as a shift table comparing baseline vs worst. 
Data from physical examination and neurological examination will only be listed. 
Descriptive statistics of ECOG results will be presented by a shift table, comparing 
the worst value over the study versus baseline.  
Isofol - ISO-CC-007                                                                   Version: 8.0
Approved in Vault: 21 Apr 2022


 
 
 
Confidential 
 
 
 
Page 38 of 38 
 
TPL N°: eTPL SA-02-01 
TPL Version: 02 
Of SOP SA-02.08 
TPL Effective Date: 31 Mar 2016 
 
11. Pharmacokinetics evaluations 
All individual plasma concentrations of MTHF, methyl-THF and THF will be listed by 
treatment group. Summary statistics will include geometric mean, median, 
arithmetic mean, standard deviation, coefficient of variation, minimum, maximum 
and the number of measurements under the LLOQ. 
The values under the LLOQ will be imputed to ½ times the concentration value of 
the LLOQ. 
The concentration/time curves will be presented in linear/linear or log/linear scale 
individual (spaghetti plots) and also summarized by treatment group including error 
bars. 
The following PK parameters will be listed as individual data divided in treatment 
groups as well as summary statistics by treatment group, for MTHF (parent drug) 
and each metabolite (methyl-THF and THF): 
• 
AUC (=AUClast) 
• 
Cmax 
• 
tmax 
• 
tlast 
The pharmacokinetics evaluation will be performed on the PK analysis set. 
12. References 
Banook, Image Review Charter ISO_CC-007, (version 9.0), 24Mars2021  
Cui L, Hung HM, Wang SJ. Modification of sample size in group sequential clinical trials. 
Biometrics—1999—; 55:853–7 
Eisenhauer EA, Therasse P, Bogaerts J et al. New response evaluation criteria in solid 
tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 2009;45:S228-47 
Ikeda K, Bretz F. Sample size and proportion of Japanese patients in multi-regional trials. 
Pharmaceut. Statist. 2010;9:207-216 
Mantel, N., and Haenszel, W. (1959). Statistical Aspects of Analysis of Data from 
Retrospective Studies of Disease. Journal of the National Cancer Institute 22:719–748, 
1959 
Meta-Analysis Group In Cancer (2004). Modulation of fluorouracil by leucovorin in patients 
with advanced colorectal cancer: an updated meta-analysis. J Clin Oncol 22: 3766-3775 
Ministry of Health, Labour and Welfare. Basic Principles on Global Clinical Trials, 2007. 
Sato, 
T. 
(1989). 
On 
the 
Variance 
Estimator 
of 
the 
Mantel-Haenszel 
Risk 
Difference. Biometrics 45:1323–1324. Letter to the editor 
Van Reenen M., Janssen B. EQ-5D-5L User Guide (version 1.2), EuroQol Research 
Foundation, 2015 
    
 
 
Isofol - ISO-CC-007                                                                   Version: 8.0
Approved in Vault: 21 Apr 2022