Clinical trial NCT04004208
Conditions: Retinopathy of Prematurity (ROP)
Sponsor: Bayer
Start date: 2019-09-25; Completion date: 2021-02-12
Enrollment: 113 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to demonstrate how well aflibercept works in babies with ROP, comparing it with laser therapy. The study also has the objective to demonstrate how safe aflibercept is when used in babies, and describe how the drug moves into, through and out of the body.

Detailed description:

Arms: arm 1: Subjects randomized to aflibercept will receive a intravitreal (IVT) injection of Dose A aflibercept per eligible eye at baseline and, if needed, up to a defined number of additional injections in each eye. arm 2: Subjects randomized to laser photocoagulation will receive treatment in each eligible eye at baseline. Retreatments may be administered if needed.
Interventions: intervention 1: Eylea (Aflibercept, BAY86-5321) intervention 2: Laser photocoagulation
Intervention descriptions: intervention 1: Solution in a sterile glass vial, Dose A, IVT injection. intervention 2: Transpupillary conventional laser ablative therapy

Protocol documents:
Prot_000.pdf: 
 
Document Type: 
Clinical Study Protocol 
Official Title: 
Open-label, randomized, two–arm, controlled study to assess the 
efficacy, safety, and tolerability of intravitreal (IVT) aflibercept 
compared to laser photocoagulation in patients with retinopathy of 
prematurity (ROP) 
NCT Number: 
NCT04004208 
Document Date: 
23 JUN 2020 
 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  1 of 59
Title Page
Protocol Title: Open-label, randomized, two–arm, controlled study to assess the efficacy, 
safety, and tolerability of intravitreal (IVT) aflibercept compared to laser photocoagulation in 
patients with retinopathy of prematurity (ROP)
Protocol Number:
20090 
Amendment Number:
01
Compound Number:
BAY 86-5321 / aflibercept 
Study Phase:
3 
Short Title: Aflibercept for ROP – IVT injection versus laser therapy
Acronym:
FIREFLEYE
Sponsor Name:  Non-US: Bayer AG
Legal Registered Address:  Non-US: 51368 Leverkusen, Germany
Regulatory Agency Identifier Number(s):  EudraCT: 2018-002611-99
Protocol Amendment Date: 
23 JUN 2020
Medical Monitor name and contact information can be found in the Trial Master File (TMF).
Name:
Role:
Global Clinical Leader
This is an electronically generated document that does not bear any sponsor signatures. The 
signature of the sponsor’s medically responsible person is filed in the TMF and available on 
request.
Confidential
The information provided in this document is strictly confidential and is intended solely for the performance of 
the clinical investigation.  Reproduction or disclosure of this document, whether in part or in full, to parties not 
associated with the clinical investigation or its use for any other purpose without the prior written consent of the 
sponsor is not permitted. 
Throughout this document, symbols indicating proprietary names (®, TM) may not be displayed. Hence, the 
appearance of product names without these symbols does not imply that these names are not protected.
PPD


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  2 of 59
Protocol Amendment Summary of Changes Table
Amendment 01 (23 JUN 2020)
This amendment is considered to be substantial based on the criteria set forth in Article 10(a) 
of Directive 2001/20/EC of the European Parliament and the Council of the European Union.
Overall Rationale for the Amendment:
The current amendment aims at collecting additional pharmacokinetic (PK) data after Week 4 
over the course of the study to further characterize the PK profile in those premature infants 
who were treated with aflibercept, document the further elimination of free 
(pharmacologically active) aflibercept and bound aflibercept from plasma, and provide 
estimates of the elimination half-life. The amendment is supported by the study’s Steering 
Committee and Data Monitoring Committee.
 
 
 
 
To collect the aforementioned additional PK data, a total of 3 PK samples (0.6 mL each) after 
Week 4 will be added, i.e. at Weeks 8, 12 and 24 (end of the study). The consecutive 
additional blood draw volumes are well in line with the limits set by respective regulatory 
guidance (Recommendations of the expert group on clinical trials for the implementation of 
Regulation [EU] No 536/2014, 2017) and recommendations on blood draw volume limits in 
children (Howie, 2011). The addition of the 3 PK samples introduces new blood draws for PK 
data collection at two timepoints (Weeks 8 and 24). At Week 12, the additional blood draw 
for PK analyses can be taken in the context of the already scheduled blood draw at the Week 
12-visit.
Section # 
and Name
Description of Change
Brief Rationale
Section 1.3 Schedule 
of Activities (SoA)
Added PK sampling at Visits 9, 11 
and 14 (EOS) at Weeks 8, 12 and 
24, respectively.
To further characterize the elimination of free
and bound aflibercept from plasma, and 
provide estimates of the elimination half-life. 
Section 8.5 
Pharmacokinetics
Changed the total blood volume 
collected for PK analysis from 1.8 
mL to 3.6 mL; 
Changed the time period for PK 
evaluations from “after the first 
IVT injection up to 4 weeks 
thereafter” to “after the first IVT 
injection up to 24 weeks
thereafter.”
Additional 3 PK samples of 0.6 mL each will 
be added to the Schedule of Activities. These 
additional PK samples will be taken at Weeks 
8, 12 and 24. Blood volumes associated with 
these additional PK samples are well in line 
with the respective regulatory guidance 
(Recommendations of the expert group on 
clinical trials for the implementation of 
Regulation [EU] No 536/2014, 2017) and 
recommendations on blood draw volume 
limits in children (Howie, 2011). 
Added “Pharmacokinetics in 
patients who receive repeated 
injections will be evaluated up to 
the time point prior to the second 
injection. In these patients, 
Clarified PK evaluation for patients receiving 
repeated injections of aflibercept
CCI


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  3 of 59
Section # 
and Name
Description of Change
Brief Rationale
concentration data for sampling 
times beyond the second injection 
will be evaluated exploratively, 
considering total dose and potential 
accumulation. ”
Section 9.5 Interim 
Analysis
Added how to deal with the final 
PK sample results that come in 
later for the intial database release
To ensure the planned initial database release 
as availability of results for PK samples from 
Week 24 takes longer.
Title Page
GCL Changed from 
 to 
 
Study personnel update
Sponsor Signatory
Sponsor Signatory page removed 
and relevant information was 
stated on Title Page.
To follow the e-signature process
Section 2.2 
Introduction
“Zone I with any ROP stages 
without plus disease, or Zone I 
stage 3 without plus disease, or
…”changed to “Zone I with any 
ROP stages with plus disease, or 
Zone I stage 3 without plus 
disease, or…”
Correction of typo
Section 4.1 Overall 
Design
Specified that pharmacokinetic 
evaluation will be performed in 
subjects treated with aflibercept by 
adding “In subjects treated with 
aflibercept,” to the beginning of  
“Pharmacokinetic evaluations will 
investigate plasma concentrations 
of free and bound aflibercept.”
To better orient investigators that such 
laboratory assessment is done only in 
aflibercept-treated subjects, and to harmonize 
with the respective information given in the 
SoA.
Section 4.1 Overall 
Design
In the figure for Aflibercept arm:
“Worsening of ROP requiring 
treatment
And
Interval since the last AFL 
injection < 27 days” changed to 
“…..Interval since the last AFL 
inject ≤27 days”
Correction of typo
Section 8 Study 
Assessments and 
Procedures
Added details for the blood volume 
taken for study-related safety 
assessments as “for analysis of 
study drug (PK, in blood plasma), 
and ADA (in blood serum) in 
conjunction….”
For clarification
PPD
PPD


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  4 of 59
Table of Contents
Title Page................................................................................................................................... 1
Protocol Amendment Summary of Changes Table............................................................... 2
Table of Contents...................................................................................................................... 4
Table of Tables.......................................................................................................................... 6
1.
Protocol Summary............................................................................................................. 7
1.1
Synopsis ........................................................................................................................... 7
1.2
Schema............................................................................................................................. 9
1.3
Schedule of Activities (SoA) ......................................................................................... 10
2.
Introduction ..................................................................................................................... 15
2.1
Study Rationale.............................................................................................................. 15
2.2
Background.................................................................................................................... 15
2.3
Benefit/Risk Assessment ............................................................................................... 16
3.
Objectives and Endpoints............................................................................................... 19
4.
Study Design .................................................................................................................... 20
4.1
Overall Design ............................................................................................................... 20
4.2
Scientific Rationale for Study Design............................................................................ 23
4.3
Justification for Dose ..................................................................................................... 23
4.4
End of Study Definition................................................................................................. 23
5.
Study Population ............................................................................................................. 23
5.1
Inclusion Criteria ........................................................................................................... 24
5.2
Exclusion Criteria .......................................................................................................... 24
5.3
Lifestyle Considerations ................................................................................................ 25
5.4
Screen Failures............................................................................................................... 25
6.
Study Intervention........................................................................................................... 26
6.1
Study Intervention(s) Administered............................................................................... 26
6.1.1
Medical Devices ......................................................................................................... 26
6.2
Preparation/Handling/Storage/Accountability............................................................... 26
6.3
Measures to Minimize Bias: Randomization and Masking ........................................... 27
6.4
Study Intervention Compliance ..................................................................................... 28
6.5
Prior and Concomitant Therapy..................................................................................... 28
6.5.1
Rescue Medicine......................................................................................................... 28
6.6
Dose Modification ......................................................................................................... 29
6.7
Intervention after the End of the Study.......................................................................... 29
7.
Discontinuation of Study Intervention and Participant 
Discontinuation/Withdrawal.......................................................................................... 29
7.1
Discontinuation of Study Intervention........................................................................... 29
7.2
Participant Discontinuation/Withdrawal from the Study............................................... 29
7.3
Lost to Follow-Up.......................................................................................................... 30
8.
Study Assessments and Procedures............................................................................... 30
8.1
Efficacy Assessments..................................................................................................... 31
8.1.1
Other Ocular Assessments.......................................................................................... 32
8.2
Safety Assessments........................................................................................................ 32


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  5 of 59
8.2.1
Ophthalmic Examinations .......................................................................................... 32
8.2.2
Physical Examinations................................................................................................ 32
8.2.3
Vital Signs .................................................................................................................. 32
8.2.4
Clinical Safety Laboratory Assessments .................................................................... 33
8.2.5
Central Nervous System Imaging............................................................................... 33
8.3
Adverse Events and Serious Adverse Events ................................................................ 33
8.3.1
Time Period and Frequency for Collecting AE and SAE Information....................... 34
8.3.2
Method of Detecting AEs and SAEs .......................................................................... 34
8.3.3
Follow-up of AEs and SAEs....................................................................................... 34
8.3.4
Regulatory Reporting Requirements for SAEs........................................................... 34
8.3.5
Pregnancy ................................................................................................................... 35
8.3.6
Medical Device Incidents (Including Malfunctions).................................................. 35
8.3.6.1
Time Period for Detecting Medical Device Incidents............................................. 35
8.3.6.2
Follow-up of Medical Device Incidents.................................................................. 35
8.3.6.3
Prompt Reporting of Medical Device Incidents to Sponsor.................................... 35
8.3.6.4
Regulatory Reporting Requirements for Medical Device Incidents ....................... 36
8.4
Treatment of Overdose................................................................................................... 36
8.5
Pharmacokinetics ........................................................................................................... 36
8.6
Pharmacodynamics ........................................................................................................ 37
8.7
Genetics.......................................................................................................................... 37
8.8
Biomarkers..................................................................................................................... 37
8.8.1
Other Biomarkers........................................................................................................ 37
8.9
Medical Resource Utilization and Health Economics ................................................... 38
9.
Statistical Considerations ............................................................................................... 38
9.1
Statistical Hypotheses .................................................................................................... 38
9.2
Sample Size Determination............................................................................................ 38
9.3
Populations for Analyses ............................................................................................... 38
9.4
Statistical Analyses ........................................................................................................ 39
9.4.1
Efficacy Analyses ....................................................................................................... 40
9.4.2
Safety Analyses .......................................................................................................... 42
9.4.3
Other Analyses............................................................................................................ 43
9.5
Interim Analyses ............................................................................................................ 43
9.5.1
Data Monitoring Committee (DMC).......................................................................... 43
10. Supporting Documentation and Operational Considerations .................................... 43
10.1
Appendix 1: Regulatory, Ethical, and Study Oversight Considerations........................ 43
10.1.1
Regulatory and Ethical Considerations ...................................................................... 43
10.1.2
Financial Disclosure ................................................................................................... 44
10.1.3
Informed Consent Process.......................................................................................... 44
10.1.4
Data Protection ........................................................................................................... 44
10.1.5
Committees Structure ................................................................................................. 44
10.1.6
Dissemination of Clinical Study Data ........................................................................ 44
10.1.7
Data Quality Assurance.............................................................................................. 45
10.1.8
Source Documents...................................................................................................... 45
10.1.9
Study and Site Closure ............................................................................................... 46
10.2
Appendix 2: Aflibercept IVT Administration................................................................ 46
10.2.1
Use of Topical Antibiotic Agents............................................................................... 47
10.2.2
Postinjection Examinations ........................................................................................ 48
10.3
Appendix 3: Clinical Laboratory Tests.......................................................................... 48


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  6 of 59
10.4
Appendix 4: Adverse Events: Definitions and Procedures for Recording, Evaluating, 
Follow-up, and Reporting .............................................................................................. 49
10.4.1
Definition of AE ......................................................................................................... 49
10.4.2
Definition of SAE....................................................................................................... 51
10.4.3
Recording and Follow-Up of AE and/or SAE............................................................ 51
10.4.4
Reporting of SAEs...................................................................................................... 53
10.5
Appendix 5: Medical Device Incidents: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting........................................................................... 54
10.6
Appendix 6: Abbreviations............................................................................................ 55
10.7
Appendix 7: Protocol Amendment History ................................................................... 57
11. References ........................................................................................................................ 57
Table of Tables
Table 10–1: Protocol-Required Safety Laboratory Assessments............................................. 49


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  7 of 59
1.
Protocol Summary
1.1
Synopsis
Protocol Title:  Open-label, randomized, two–arm, controlled study to assess the efficacy, 
safety, and tolerability of intravitreal (IVT) aflibercept compared to laser photocoagulation in 
patients with retinopathy of prematurity (ROP)
Short Title:  Aflibercept for ROP – IVT injection versus laser therapy
Rationale:
Currently available clinical data indicate potential advantages of the 
use of IVT anti-VEGF agents, including aflibercept, compared to laser photocoagulation for 
treatment of ROP, and caused a gradual but steady increase in off-label use of IVT anti-VEGF 
agents in this condition. However, currently available prospective and longer-term data on the 
use of aflibercept for the treatment of ROP are still insufficient. The efficacy of IVT 
aflibercept in a well-characterized population of patients with ROP still needs to be evidenced 
by data from a prospective clinical trial. Knowledge of the benefit-risk profile for this 
population needs to be improved, and the pharmacokinetic parameters of aflibercept need to 
be more fully characterized taking into account the characteristics and limitations of this 
premature newborn population. These data are planned to be generated in a clinical 
development program including this randomized, phase 3, multicenter, 2-arm, open-label 
clinical study to assess the efficacy, safety, and tolerability of IVT aflibercept in subjects with 
ROP.
Objectives and Endpoints:
Objectives
Endpoints
Primary
To assess the efficacy of 
aflibercept in subjects 
diagnosed with retinopathy of 
prematurity (ROP) in 
comparison to laser
Primary endpoint:

Proportion of patients with absence of active ROP and 
unfavorable structural outcomes at 24 weeks after starting 
study treatment
Secondary endpoints addressing primary objective:

Requirement for intervention with a second treatment modality 
from baseline to Week 24

Recurrence of ROP from baseline to Week 24

To explore new Retinopathy of Prematurity Activity Scale 
proposed by the International Neonatal Consortium
Secondary
To assess the safety and 
tolerability of aflibercept
To assess the treatment 
burden of aflibercept and 
laser 
To describe the systemic 
exposure to aflibercept
Secondary endpoints addressing secondary objectives:

Number of aflibercept administrations from baseline to Week 24

Number of laser treatments from baseline to Week 24

Proportion of participants with ocular TEAEs and SAEs from 
baseline to Week 24 

Proportion of participants with systemic TEAEs and SAEs from 
baseline to Week 24 

Systemic exposure to free aflibercept (at expected maximum 
plasma concentration and during elimination period from 
plasma) determined by sparse sampling

Presence of anti-drug antibodies before and 12 weeks after 
aflibercept injection


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  8 of 59
Overall Design:
Intervention Model:
Parallel
Primary Purpose:
Treatment 
Number of Arms: 2
Masking: No masking 
Number of Participants:
A minimum of 102 preterm infants are planned to be randomized to achieve at least 
102 subjects evaluable  for the primary analysis.
The determination of sample size is described in Section 9.2.
Intervention Groups and Duration:
Subjects will be randomized 2:1 to receive treatment with an IVT injection of aflibercept 
0.4 mg/0.01 mL (approximately 68 subjects) or laser photocoagulation (approximately 
34 subjects). Study duration must be planned for at least 24 weeks in the study protocol.
The study comprises the following study phases:

Screening and initial treatment (may be conducted at the same visit or within 10 days)

Continued observation and retreatment and/or rescue treatment as needed (from Week 
1 to Week 23)

Final observation (Week 24; can occur between Weeks 25 and 27 for subjects treated 
between Weeks 21 and 23)
One or both eyes can be treated according to the investigator’s assessment of the study’s 
eligibility criteria. The second eye of subjects who start the study with only one eligible eye
should be kept under observation according to the local ROP screening guidelines or at every 
study visit, whichever is more frequent. Second eyes that develop ROP requiring treatment 
during the study should receive treatment according to the randomization assignment of the 
first eye. 
Retreatment(s) with the subject’s randomized treatment, or rescue treatment (laser for the 
aflibercept arm; aflibercept for the laser arm) are allowed if the specified criteria are met 
during the 23-week treatment period.  
For the efficacy analysis, only data collected up to Week 24 will be considered.  All data 
collected up to Week 27 will be included in the safety analyses. This applies to subjects who 
receive retreatment or rescue treatment between Weeks 21 and 23, as their last follow-up visit 
will be performed 4 weeks after treatment.
Data Monitoring Committee:
Yes


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
                                                                  Page:  9 of 59
1.2
Schema


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
                                                                  Page:  10 of 59
1.3
Schedule of Activities (SoA)
Visit Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14 
EOS
Retreatment or
rescue treatment 
follow-up visits
Additional visits may be performed depending on the 
investigator’s assessment of the subject’s response to 
treatment, as part of local standard of care. 
Visits 1 and 2 can be on same day or within 10 days of 
each other.
If treatment cannot take place at Visit 2, it can be 
administered within 3 days of Visit 2. For Visits 3 to 14, 
the intervals are based on the date of initial treatment.  
The intervals of the rescue/retreatment follow-up visits 
are based on the date of rescue/retreatment.  A 
retreatment/rescue follow-up visit may be combined 
with a regularly scheduled visit.
If a subject’s second eye qualifies for treatment during 
the study, both eyes can follow the visit schedule of the
first eye after the retreatment/rescue treatment follow-
up visit schedule is completed for the second eye.
Visit Name
SCR BL
D1
W1
W2
W3
W4
W6
W8 W10 W12 W16 W20 W24
TRT 
Visit
D1
1 W 
FUP
4 W 
FUP
Visit Window
--
--
+2D ±3D ±3D ±3D +7D ±7D ±7D ±7D ±7D ±7D ±7D
±7D
--
+2D
±3D
+7D
Informed consent
X
In/exclusion criteria
X
X
Enroll (IVRS/IWRS)
X
Demographic
X
Medical history
X
Maternal and subject medical history will be recorded.
Prior/concomitant 
medication
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Includes recording of oxygen supplementation.  
At screening, include any of the medications listed in 
Section 6.5 given to mother during pregnancy and 
breastfeeding.
AEs
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Body weight
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Head circumference 
and body length
X
X
X
X
CNS imaging
X
Imaging is not required at screening if results from an 
imaging exam performed within the previous 10 days 
are available and there are no new neurological 
sign/symptoms.  
Physical exam
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Includes cardiovascular, respiratory, gastrointestinal, 
and neurological systems according to local general 
practice and aiming to evaluate overall health.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
                                                                  Page:  11 of 59
Visit Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14 
EOS
Retreatment or
rescue treatment 
follow-up visits
Additional visits may be performed depending on the 
investigator’s assessment of the subject’s response to 
treatment, as part of local standard of care. 
Visits 1 and 2 can be on same day or within 10 days of 
each other.
If treatment cannot take place at Visit 2, it can be 
administered within 3 days of Visit 2. For Visits 3 to 14, 
the intervals are based on the date of initial treatment.  
The intervals of the rescue/retreatment follow-up visits 
are based on the date of rescue/retreatment.  A 
retreatment/rescue follow-up visit may be combined 
with a regularly scheduled visit.
If a subject’s second eye qualifies for treatment during 
the study, both eyes can follow the visit schedule of the
first eye after the retreatment/rescue treatment follow-
up visit schedule is completed for the second eye.
Visit Name
SCR BL
D1
W1
W2
W3
W4
W6
W8 W10 W12 W16 W20 W24
TRT 
Visit
D1
1 W 
FUP
4 W 
FUP
Visit Window
--
--
+2D ±3D ±3D ±3D +7D ±7D ±7D ±7D ±7D ±7D ±7D
±7D
--
+2D
±3D
+7D
Vital signs
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Temperature, blood pressure (before study treatment, if 
applicable), respiratory and pulse rate.
Anterior segment 
examination
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Can be done using indirect ophthalmoscopy or portable 
slit lamp.
Binocular indirect 
ophthalmoscopy
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
The pupils must be sufficiently dilated to allow 
examination of all ROP features.
Unfavorable structural outcome is defined as retinal 
detachment, macular dragging, macular fold, or 
retrolental opacity.
Tonometry
X
X
Only in subjects receiving aflibercept:
IOP will be measured in both eyes prior to the injection.
IOP will be measured at least once postinjection (only in 
treated eyes) as described in Section 10.2.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
                                                                  Page:  12 of 59
Visit Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14 
EOS
Retreatment or
rescue treatment 
follow-up visits
Additional visits may be performed depending on the 
investigator’s assessment of the subject’s response to 
treatment, as part of local standard of care. 
Visits 1 and 2 can be on same day or within 10 days of 
each other.
If treatment cannot take place at Visit 2, it can be 
administered within 3 days of Visit 2. For Visits 3 to 14, 
the intervals are based on the date of initial treatment.  
The intervals of the rescue/retreatment follow-up visits 
are based on the date of rescue/retreatment.  A 
retreatment/rescue follow-up visit may be combined 
with a regularly scheduled visit.
If a subject’s second eye qualifies for treatment during 
the study, both eyes can follow the visit schedule of the
first eye after the retreatment/rescue treatment follow-
up visit schedule is completed for the second eye.
Visit Name
SCR BL
D1
W1
W2
W3
W4
W6
W8 W10 W12 W16 W20 W24
TRT 
Visit
D1
1 W 
FUP
4 W 
FUP
Visit Window
--
--
+2D ±3D ±3D ±3D +7D ±7D ±7D ±7D ±7D ±7D ±7D
±7D
--
+2D
±3D
+7D
Digital wide-field 
retinal imaging 
X
X
X
X
X
X
X
X
Baseline imaging will be done prior to study treatment
on the treatment day or up to 2 days prior to treatment.
W24 imaging may not be required if the following 
conditions are met: the reading center assessment of
morphological outcomes on the W12 image is in 
agreement with indirect ophthalmoscopy performed by 
the investigator, and the subject shows poor tolerance
for the imaging procedure, preventing acquisition of 
images with adequate quality.
Cases of rescue/retreatment require additional imaging 
before any treatment (on the treatment day or up to 2 
days prior to treatment) as well as 1 week and 4 weeks 
following rescue/retreatment.
Blood sample for ADA 
(aflibercept arm only)
X
X
One 0.6-mL blood sample will be taken at baseline prior 
to dosing.  At W12 one 1.1-mL blood sample will be 
taken to detect anti-drug antibodies (ADAs) and, if 
applicable, the occurrence of potential neutralizing 
antibodies (NAB) (if collection is not possible, the W12 
sample can be taken at W16).


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
                                                                  Page:  13 of 59
Visit Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14 
EOS
Retreatment or
rescue treatment 
follow-up visits
Additional visits may be performed depending on the 
investigator’s assessment of the subject’s response to 
treatment, as part of local standard of care. 
Visits 1 and 2 can be on same day or within 10 days of 
each other.
If treatment cannot take place at Visit 2, it can be 
administered within 3 days of Visit 2. For Visits 3 to 14, 
the intervals are based on the date of initial treatment.  
The intervals of the rescue/retreatment follow-up visits 
are based on the date of rescue/retreatment.  A 
retreatment/rescue follow-up visit may be combined 
with a regularly scheduled visit.
If a subject’s second eye qualifies for treatment during 
the study, both eyes can follow the visit schedule of the
first eye after the retreatment/rescue treatment follow-
up visit schedule is completed for the second eye.
Visit Name
SCR BL
D1
W1
W2
W3
W4
W6
W8 W10 W12 W16 W20 W24
TRT 
Visit
D1
1 W 
FUP
4 W 
FUP
Visit Window
--
--
+2D ±3D ±3D ±3D +7D ±7D ±7D ±7D ±7D ±7D ±7D
±7D
--
+2D
±3D
+7D
Blood sample for PK 
(aflibercept arm only)
X
X
X
X1
X1
X1
Only applicable for subjects treated with aflibercept at 
baseline.
One blood sample (0.6 mL each) for plasma 
concentrations of study drug will be taken on Day 1 
(~24 hours after dosing), Day 14, Day 28, and Weeks 8,
12 and 24. Blood pressure must be measured before 
the PK sample is taken. 
If a subject’s second eye is deemed eligible for
treatment and is treated with aflibercept at Visits 3, 5,
7, 9, 11 and 14 (EOS), the PK sample is taken before 
treatment.  
1 PK samples at Weeks 8, 12 and 24 are optional for 
patients with consent signed prior to the effectiveness 
date of the current amendment (Amendment 01).
Hematology, clinical 
chemistry, urinalysis 
X
A sample is not needed if results from laboratory tests 
within 8 days prior to screening are available and there 
was no change in the clinical situation from the time of 
the sample to the screening visit. 
Urine protein test
X
X
X
X
X
The baseline sample can be taken up to 2 days prior to 
baseline. The Day 1 sample will be ~24 hours after 
study treatment. 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
                                                                  Page:  14 of 59
Visit Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14 
EOS
Retreatment or
rescue treatment 
follow-up visits
Additional visits may be performed depending on the 
investigator’s assessment of the subject’s response to 
treatment, as part of local standard of care. 
Visits 1 and 2 can be on same day or within 10 days of 
each other.
If treatment cannot take place at Visit 2, it can be 
administered within 3 days of Visit 2. For Visits 3 to 14, 
the intervals are based on the date of initial treatment.  
The intervals of the rescue/retreatment follow-up visits 
are based on the date of rescue/retreatment.  A 
retreatment/rescue follow-up visit may be combined 
with a regularly scheduled visit.
If a subject’s second eye qualifies for treatment during 
the study, both eyes can follow the visit schedule of the
first eye after the retreatment/rescue treatment follow-
up visit schedule is completed for the second eye.
Visit Name
SCR BL
D1
W1
W2
W3
W4
W6
W8 W10 W12 W16 W20 W24
TRT 
Visit
D1
1 W 
FUP
4 W 
FUP
Visit Window
--
--
+2D ±3D ±3D ±3D +7D ±7D ±7D ±7D ±7D ±7D ±7D
±7D
--
+2D
±3D
+7D
Aflibercept injection 
(for subjects 
randomized to active)
X
X
The injection should be performed in both eyes on the 
same day, if applicable. After initial treatment, each 
study eye may receive up to 2 additional treatments 
(with a minimum interval of 28 days between injections) 
only if retreatment criteria are met (see Section 4.1).  
After retreatment, the retreatment/rescue treatment 
follow-up visits are also required. Blood pressure must 
be measured before the injection. Details of each 
injection will be recorded in the eCRF.
Laser treatment (for 
subjects randomized 
to control)
X
X
Multiple sessions performed within 1 week after
baseline to complete the procedure will be counted as a 
single treatment. Each study eye may receive 
retreatment only if the retreatment criteria are met (see 
Section 4.1). After retreatment, the retreatment/rescue 
treatment follow-up visits are also required. Blood 
pressure must be measured before laser.
Visual function
X
Refraction
X
Ocular extrinsic 
motility
X


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  15 of 59
2.
Introduction
2.1
Study Rationale
Currently available clinical data indicate potential advantages of the use of IVT anti-VEGF 
agents, including aflibercept, compared to laser photocoagulation for treatment of ROP, and 
caused a gradual but steady increase in off-label use of IVT anti-VEGF agents in this 
condition. However, currently available prospective and longer-term data on the use of 
aflibercept for the treatment of ROP are still insufficient. The efficacy of IVT aflibercept in a 
well-characterized population of patients with ROP still needs to be evidenced by data from a 
prospective clinical trial. Knowledge of the benefit-risk profile for this population needs to be 
improved, and the pharmacokinetic parameters of aflibercept need to be more fully 
characterized taking into account the characteristics and limitations of this premature newborn 
population. These data are planned to be generated in a clinical development program 
including this randomized, phase 3, multicenter, 2-arm, open-label clinical study to assess the 
efficacy, safety, and tolerability of IVT aflibercept in subjects with ROP.
2.2
Background
Retinopathy of prematurity is a proliferative vascular retinopathy caused by an abnormal 
development of the vascularization of the peripheral retina in premature infants (Mutlu and 
Sarici, 2013; Pertl et al, 2015; Salman and Said, 2015). It affects mainly newborns with a 
preterm gestational age (≤32 weeks) and very low birth weight (≤1500 g). ROP remains a 
major cause of childhood blindness globally. The prevalence of childhood blindness caused 
by ROP in developed nations ranges between 6% and 18%, while in developing nations the 
estimate is above 20% (Hartnett, 2014; Gilbert et al, 1997). Approximately 10% to 11% of all 
newborn infants are born preterm (Blencowe et al, 2013; Hellström et al, 2013). Among 
premature infants, the incidence of ROP ranges from 20% to 30%, increasing for younger 
gestational ages. Epidemiological studies have also identified increased risks for ROP due to
genetic variants and environmental factors, such as oxygen exposure (Hartnett et al, 2014). 
The first wave of ROP cases, in the 1940s and 1950s, were identified in preterm infants who 
were exposed to high levels of supplemental oxygen in closed incubators, causing abnormal 
development of the vascular network in the premature retina. Stricter control of supplemental 
oxygen led to a decreased incidence of this condition (Hellström et al, 2013). An English 
study showed that the incidence of ROP later increased from 12.8 per 1000 low birth weight 
infants in 1990 to 125.5 per 1000 low birth weight infants in 2011 (Painter et al, 2015). This 
second wave of ROP cases may be attributed to improved survival of the most extremely
premature infants, introducing a new population of vulnerable newborns more susceptible to 
ROP.
Vascular endothelial growth factor (VEGF) is up-regulated under ischemic conditions. As 
ROP is characterized by incomplete vascularization of the retina in premature infants, it has 
also been associated with increased levels of VEGF. VEGF-A is a member of the VEGF 
family of angiogenic factors and acts through activation of receptor tyrosine kinases VEGFR1 
and VEGFR2 to increase mitosis, chemotaxis and vascular permeability in endothelial cells. 
Upregulation of VEGF-A due to ischemia in the avascular retina may induce pathologic 
neovascularization and subsequently lead to retinal detachment and blindness, as seen in late 
stage ROP (Stone et al,1996; Young et al, 1997). Aflibercept inhibits these effects of VEGF 
by acting as a soluble decoy receptor that binds VEGF-A with higher affinity than the natural 
receptors and thereby inhibiting the binding and activation of these cognate VEGF receptors.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  16 of 59
Aflibercept also inhibits the effects of placental growth factor, which binds only to VEGFR1. 
Placental growth factor can synergize with VEGF-A, enhancing these processes of pathologic 
neovascularization and is also known to promote leukocyte infiltration and vascular 
inflammation. Based on the drug’s mode of action, aflibercept has a high potential to become 
an effective treatment option for the treatment of ROP, and several reports of off-label clinical 
use have shown positive efficacy outcomes without indicating safety concerns (Sukgen and 
Kocluk, 2018; Salman and Said, 2015; Huang et al, 2018). In addition to the evidence 
currently available for aflibercept, the efficacy and safety of anti-VEGF agents for the 
treatment of ROP have been compared to laser photocoagulation in several case series and 2
large randomized controlled trials (BEAT-ROP and RAINBOW). The totality of the clinical 
evidence available thus far indicates a trend towards superior prevention of unfavorable 
anatomical outcomes for anti-VEGF agents compared to laser photocoagulation for ROP in 
central retinal locations (Zone I with any ROP stages with plus disease, or Zone I stage 3 
without plus disease, or Zone II with stages 2 plus or 3 plus disease) and aggressive posterior 
ROP (AP-ROP) and established the proof of concept for this indication (Mintz-Hittner et al, 
2011; Harder et al, 2013; Hwang et al, 2015).
Aflibercept has been previously studied for several adult indications characterized by ocular 
neovascularization or increased permeability of the retinal vascular network, and is approved 
in multiple countries for treatment of indications such as neovascular age-related macular 
degeneration, diabetic macular edema, macular edema secondary to retinal venous occlusion 
and choroidal neovascularization secondary to pathological myopia. Considering the 
consistently positive outcomes provided by aflibercept across these adult VEGF-mediated 
indications, it is deemed appropriate to conduct a study for a pediatric VEGF-mediated 
indication for the treatment of ROP.
A detailed description of the chemistry, pharmacology, efficacy, and safety of aflibercept is 
provided in the Investigator’s Brochure.
2.3
Benefit/Risk Assessment
Aflibercept is marketed for the treatment of adult patients with several retinal diseases that are
characterized by ischemia-induced upregulation of VEGF, are related to pathological 
neovascularization and/or vascular leakage, and can result in retinal thickening and edema, 
which is thought to contribute to vision loss. The efficacy and safety of IVT aflibercept used
in adult patients with retinal diseases are well established; and its benefit-risk profile is 
considered favorable. Inhibition of VEGF activity by aflibercept can be expected to result in 
therapeutic benefit in premature infants with ROP, since ROP is also characterized by the 
pathological development of the retinal vasculature and has been associated with VEGF 
upregulation. Aflibercept has been shown to have demonstrable efficacy in animal models of 
pathological ocular neovascularization after systemic and IVT administration. No differences 
in the mechanism of action of aflibercept in the treatment of neovascular ocular diseases in 
children (eg, ROP) and adults are expected. There is a high and unmet medical need for an 
effective, safe and tolerable approved treatment for preterm infants with vision-threatening 
ROP, a disease that is a leading cause of blindness in developing and developed countries. 
Laser photocoagulation (for peripheral early stage of ROP) is currently considered a standard 
of care, but fails to achieve a normal retinal structure in up to 25% of ROP subjects with 
central retinal disease or AP-ROP. Additionally, laser photocoagulation is associated with 
potential risks of substantial patient impact, including irreversible loss of visual field and high 
myopia. Vitreoretinal surgery is reserved for advanced disease stages with retinal detachment
(Stages 4 and 5). Increasing clinical experience from off-label use of IVT aflibercept for 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  17 of 59
central ROP suggests positive efficacy outcomes, usually after a single treatment session, with 
no major safety concerns as currently reported. Re-administration is rare. Thus, aflibercept has 
the potential to offer improved outcomes in this high risk, vulnerable premature newborn
population with ROP. 
Known and expected benefits
Aflibercept is expected to provide demonstrable anatomical benefits through regression of 
ROP features while allowing the vascularization to resume its usual development towards the 
retinal periphery, thus preventing unfavorable structural outcomes in approximately 90% of 
treated subjects. Visual function is also expected to improve (preservation of peripheral 
visual field, prevention of loss of best-corrected visual acuity, and lower proportion of patients 
developing high myopia). Moreover, improvements of treatment convenience in ROP 
patients are expected, such as the advantage of being less time consuming than laser (an IVT 
injection is often less than 30 minutes, whereas laser treatment may take 2 or more hours), 
potentially less risky by avoiding general anesthesia/sedation, and is not dependent on the 
ocular anatomy to be performed (on the contrary, laser treatment would be difficult in patients 
without adequate visualization of the retina, such as subjects with hazy corneas, iris 
neovascularization, or small pupils). Aflibercept treatment offers a single-injection treatment 
option and can be administered immediately after diagnosis and informed consent by the 
parent(s)/legally authorized representative(s).  Bilateral cases can be treated in a single 
session.  Upon recurrence of vision-threatening ROP, re-treatment with the same dose may be 
given to each treatment-requiring eye, after injection-free intervals of at least 28 days. For 
laser, once avascular retina ablation is complete, re-treatment may not offer additional 
benefits. In addition to all this, laser treatment is more susceptible to operator-dependent 
differences with larger areas of untreated retina, increasing the risk of failure. Achieving 
adequate laser treatment may depend on the experience of an individual practitioner.
Potential risks and mitigation measures
Based on the cumulative safety experience with aflibercept for the treatment of adult patients 
with retinal diseases, the potential risks for the study subjects include:
Ocular risks (intraocular inflammation including endophthalmitis, retinal tear/detachment, 
transient intraocular pressure increase, traumatic cataract). Specific guidance on the 
appropriate use and administration of aflibercept, in order to optimize the technique of IVT
injection in the premature eye and reduce injection procedure-related complications is given 
in Section 10.2.
Subjects with relevant ongoing or significant previous use of immunosuppressive exposure 
levels of systemic steroids are excluded as well, to mitigate any potential increased risk of 
local or systemic infections, and to address any potential risk of worsening clinical conditions 
that have been historically reported in the context of steroid use in preterm infants such as 
more frequent or greater incidence rates of necrotizing enterocolitis, cerebral palsy, and 
gastrointestinal ulcer.
Hypersensitivity/immunogenicity (inherent to all therapeutic proteins) is addressed by 
exclusion of relevant patients (see exclusion criteria in Section 5.2).
Acute systemic effects: A minimal weight at the time of study treatment (see Section 5.1) is 
requested. Subjects will be monitored for any evidence of arterial hypertension, proteinuria, 
worsening of concomitant conditions typical for preterm infants, such as worsening of 
intraventricular hemorrhage I or II, other non-ocular hemorrhages, or arterial thromboembolic 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  18 of 59
events.  Pharmacokinetic samples for the measurement of systemic aflibercept concentrations
(both free aflibercept [the pharmacologically active form] and aflibercept bound to VEGF [the 
inactive form]) will be taken. The study population is vulnerable due to their underlying 
prematurity, increased risk for multi-organ comorbidities (such as bronchopulmonary 
dysplasia, intraventricular hemorrhage, necrotizing enterocolitis, septic conditions), and 
developmental delays compared to term infants.
Given the high unmet medical need, the potential to more effectively treat ROP with a single 
or few injections, and the risks associated with laser treatments, the potential lifelong benefits 
to children treated with aflibercept outweigh the potential risks, and the expected benefit-risk 
profile is favorable.
For details of study procedures, dose, and study design justification see Section 4.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  19 of 59
3.
Objectives and Endpoints
Objectives
Endpoints
Primary
To assess the efficacy of 
aflibercept in subjects 
diagnosed with ROP in 
comparison to laser
Primary endpoint:

Proportion of patients with absence of active ROP and 
unfavorable structural outcomes at 24 weeks after starting study 
treatment *
Secondary endpoints addressing primary objective:

Requirement for intervention with a second treatment modality 
from baseline to Week 24

Recurrence of ROP from baseline to Week 24 *

To explore new Retinopathy of Prematurity Activity Scale 
proposed by the International Neonatal Consortium
Secondary
To assess the safety and 
tolerability of aflibercept
To assess the treatment 
burden of aflibercept and 
laser 
To describe the systemic 
exposure to aflibercept
Secondary endpoints addressing secondary objectives:

Number of aflibercept administrations from baseline to Week 24

Number of laser treatments from baseline to Week 24

Proportion of participants with ocular TEAEs and SAEs from 
baseline to Week 24 

Proportion of participants with systemic TEAEs and SAEs from 
baseline to Week 24

Systemic exposure to free aflibercept (at expected maximum 
plasma concentration and during elimination period from plasma) 
determined by sparse sampling 

Presence of anti-drug antibodies before and 12 weeks after 
aflibercept injection
Other pre-specified
To characterize further 
aspects of the effect of 
aflibercept in the treatment 
of ROP
To further investigate the 
study intervention and 
similar drugs (ie, mode-of-
action-related effects and/or 
safety) and to further 
investigate 
pathomechanisms deemed 
relevant to diseases of the 
eye and associated health 
problems

Evaluation of visual function at Week 24

Time required to perform treatment

Requirement for sedation or general anesthesia

Requirement for treatment with more than one aflibercept 
injection

Time to intervention with a second treatment modality for ROP or 
development of unfavorable structural outcomes *

Time to recurrence of ROP *

Regression of plus disease, regression of pre-retinal 
vascularized ridge and progression of retinal vascularization 
beyond the ridge from baseline to Week 24 *

Progression to Stage 4 or 5 ROP from baseline to Week 24*

Completion of vascularization of the peripheral retina to within 
one disc diameter of the ora serrata at Week 24 *

Time to completion of vascularization *

Number of visits required up to Week 24

Systemic exposure to total aflibercept determined by sparse 
sampling

Various biomarkers (eg, diagnostic, safety, pharmacodynamics, 
monitoring, or potentially predictive biomarkers)
* The primary analysis for these endpoints will be based on the investigators’ assessments.
Note:  Unfavorable structural outcome is defined as retinal detachment, macular dragging, macular 
fold, or retrolental opacity.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  20 of 59
4.
Study Design
4.1
Overall Design
This is a phase 3, multicenter, randomized, 2-arm, open-label clinical study to assess the 
efficacy, safety, and tolerability of IVT aflibercept versus laser photocoagulation in subjects 
with ROP. The study consists of screening/baseline (1 or 2 visits), a 23-week treatment period
(including retreatment and rescue treatment), and a final visit at Week 24 (up to Week 27 for 
subjects treated after Week 21).
Successful screening requires the presence of treatment-naïve ROP classified by the 
investigator according to the International Classification for Retinopathy of Prematurity  in at 
least one eye with one of the following retinal findings:

Zone I Stage 1 plus, 2 plus, 3 non-plus or 3 plus, or

Zone II Stage 2 plus or 3 plus, or 

AP-ROP 
One or both eyes can be treated according to the investigator’s assessment of the study’s 
eligibility criteria. The second eye of subjects who start the study with only one eligible eye 
should be kept under observation according to the local ROP screening guidelines or at every 
study visit, whichever is more frequent. Second eyes that develop ROP requiring treatment 
during the study should receive treatment according to the randomization assignment of the 
first eye. 
Color fundus photography with digital wide-field retinal images will be taken before any 
treatment is applied. These images will be submitted to a central reading center (RC) as soon 
as possible for confirmation of ROP staging, and if deemed eligible, treatment should be 
administered. However, due to the urgency of treatment for this medical condition, in 
situations where the investigator considers that awaiting a response from the RC and thus 
delaying treatment may be detrimental to the outcome, treatment is allowed to be 
administered immediately after the images are acquired, before availability of the RC 
confirmation of ROP staging. Subjects who received treatment and are not confirmed by the 
RC to meet the inclusion criteria will continue being followed in the study and their efficacy 
and safety outcomes will be collected, however, their data will not be in the modified full 
analysis set (mFAS) or the per protocol set (PPS).  
Subjects will be randomized 2:1 to treatment with either aflibercept injection or laser 
photocoagulation, respectively.
Aflibercept arm
Subjects randomized to aflibercept will receive a single IVT injection of aflibercept 
0.4 mg/0.01 mL per eligible eye at baseline.  Thereafter, if required, up to 2 additional IVT
injections of aflibercept 0.4 mg/0.01 mL may be administered in each eye in case retreatment 
criteria are met: 

Presence of ROP requiring treatment AND

The interval since the last aflibercept IVT injection is 28 or more days


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  21 of 59
Rescue treatment with laser may be performed if one of the following conditions is met:

Worsening of ROP compared to the examination before the previous injection during 
the 27 days following that IVT aflibercept injection

Presence of ROP requiring treatment after the subject already received a total of 3 
aflibercept injections and the interval since the last IVT injection is 28 or more days
Laser photocoagulation arm
Subjects randomized to laser photocoagulation will undergo treatment in each eligible eye at 
baseline.  Laser ablation should be as complete as possible as judged by the investigator.  In
case multiple sessions are necessary within 1 week from baseline, they will be counted as a 
single treatment. Treatment will be applied to the entire avascular peripheral retina. Treatment 
should be kept well away from the fovea.
Supplementary laser treatments are allowed during the study. Retreatment with laser is 
allowed if both of the following criteria are met:

Presence of ROP requiring treatment 

Fundus examination reveals laser treatment is incomplete as judged by the investigator
Rescue treatment with aflibercept 0.4 mg/0.01 mL is allowed if the fundus examination 
reveals laser treatment is complete as judged by the investigator and if one of the following 
conditions is met:

Worsening of ROP compared to the prelaser examination

Persistence of ROP requiring treatment 28 or more days after laser treatment
Subjects who initiate aflibercept rescue treatment will thereafter be managed according to the 
aflibercept arm treatment regimen.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  22 of 59
Once rescue treatment is applied to an eye, treatment in that eye with the subject’s 
randomized treatment cannot be reinitiated. However, the fellow eye can still receive the 
subject’s randomized study intervention, if retreatment criteria are met.
Subjects will have mandatory evaluations at regular intervals during the study at the time 
points specified in the SoA. Subjects receiving retreatment and/or rescue treatment will have 
mandatory evaluations 1 day, 1 week, and 4 weeks after the retreatment/rescue treatment to a 
given eye. 
Acquisition and submission of digital wide-field retinal images (eg, RetCam) to the RC is 
required at baseline and at Weeks 1, 4, 12, and 24, and before each retreatment and rescue 
treatment, and after 1 and 4 weeks of retreatment/rescue treatment. Safety will be assessed 
through the evaluation of AEs, ophthalmic and physical examinations (including assessment 
of acute tolerability post-dosing), vital signs, and laboratory tests. In subjects treated with 
aflibercept, pharmacokinetic evaluations will investigate plasma concentrations of free and 
bound aflibercept. The potential emergence of antidrug antibodies (ADAs) will also be 
evaluated by serum sampling at baseline and Week 12.
Additional optional evaluation visits may be performed in accordance with the usual standard 
of care, as medically needed. 
A Phase 3b study (Study 20275; extension study) is planned to assess the long-term outcomes 
of patients who received study intervention in this study. All treated patients must be offered 
participation in a follow-up Study 20275 until they are 5 years of age to assess ocular effects, 
clinical and neurodevelopmental outcomes. 
 
See Section 6.7 for 
details.
CCI


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  23 of 59
4.2
Scientific Rationale for Study Design
Laser photocoagulation of the peripheral avascular retina is the current standard of care 
treatment for ROP. Considering aflibercept’s mode of action, and the current clinical evidence 
describing positive outcomes of ROP with off-label use of anti-VEGF in central retinal 
disease or AP-ROP, a randomized clinical trial to compare the ability of these treatments to 
manage active ROP and prevent the development of ocular complications is necessary.
A fully masked study of IVT aflibercept compared to laser photocoagulation is technically not 
feasible since laser treatment produces burns that are readily detectable to the grader who 
assesses treatment outcomes. Additionally, the substantial amount of already existing data 
describing outcomes of laser photocoagulation in ROP is sufficient to provide an accurate 
understanding of the potential benefits and limitations of this treatment modality. The purpose 
of the current study is primarily to collect the missing data on the outcomes of aflibercept in 
the treatment of ROP, and for this reason, a 2:1 randomization design was defined.
 
The inclusion and exclusion criteria allow the selection of an appropriate subject population 
and increase the likelihood of producing reliable and reproducible results, while guarding 
against exploitation of vulnerable persons. The proposed criteria are based on existing clinical 
knowledge and feedback from key opinion leaders involved in treatment of preterm infants 
with ROP.
4.3
Justification for Dose
Currently available clinical data (Sukgen and Kocluk, 2018; Salman and Said, 2015; 
Sidorenko, 2018) consistently shows promising efficacy, with no identification of major 
safety concerns, when using aflibercept doses ranging from 0.4 mg to 1.0 mg per eye (ie, 1/5 
to ½ of the 2 mg dose for indications in adult patients). In order to limit drug exposure, the 
lowest dose for which positive efficacy was reported (0.4 mg/0.01 mL) was selected for this 
study. 
4.4
End of Study Definition
A subject is considered to have completed the study if he/she has completed all phases of the 
study including the last visit at Week 24 (or up to Week 27 in the event of follow-up visits).
The end of the study as a whole is defined as the date of the last visit of the last subject in the 
study in all centers in all participating countries (EU and non-EU).
Primary completion
The primary completion is defined as the date of the last visit of the last subject for the 
primary outcome.
Subjects who are treated in this study must be offered participation in a follow-up Study
20275, which will include evaluation of visual function and overall neurological development.
5.
Study Population
Prospective approval of protocol deviations to recruitment and enrollment criteria, also known 
as protocol waivers or exemptions, is not permitted.
CCI


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  24 of 59
The study population will consist of male and female preterm infants with treatment-naïve 
ROP Zone I Stage 1 plus, 2 plus, 3 non-plus or plus, or Zone II Stage 2 plus or 3 plus or 
aggressive posterior retinopathy of prematurity (AP-ROP) according to the International 
Classification for ROP.
5.1
Inclusion Criteria
Subjects are eligible to be included in the study only if all of the following criteria are met:
Age
1. Gestational age at birth ≤32 weeks or birth weight ≤1500 g
Type of Participant and Disease Characteristics
2. Subjects with treatment-naïve ROP classified according to the International 
Classification for ROP in at least one eye as:
Zone I Stage 1 plus, or 2 plus, or 3 non-plus or 3 plus, or
Zone II Stage 2 plus or 3 plus, or
AP-ROP
Weight
3. Weight at baseline (day of treatment) ≥ 800 g
Sex
4. Male or female
Informed Consent
5. Signed informed consent from parent(s)/legally authorized representative(s) as 
described in Section 10.1.3, which includes compliance with the requirements and 
restrictions listed in the informed consent form (ICF) and in this protocol.
5.2
Exclusion Criteria 
Subjects are excluded from the study if any of the following “per subject” criteria are met.  A 
potential study eye is excluded from the study if any of the “per eye” criteria are met:
Medical Conditions – per subject
1. Known or suspected chromosomal abnormality, genetic disorder or syndrome
2. Previous exposure to any IVT or systemic anti-VEGF agent, including maternal 
exposure during pregnancy and/or during breastfeeding
3. Clinically significant neurological disease (eg, intraventricular hemorrhage grade 3 or 
higher, periventricular leukomalacia, congenital brain lesions significantly impairing 
optic nerve function, severe hydrocephalus with significantly increased intracranial 
pressure)
4. Pediatric conditions rendering the infant ineligible for study intervention at baseline or 
for repeated blood draws as evaluated by a NICU specialist and a study 
ophthalmologist
5. Presence of active ocular infection within 5 days of the first treatment


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  25 of 59
Medical Conditions – per eye
6. Advanced stages of ROP with partial or complete retinal detachment (ROP Stages 4 
and 5)
7. ROP involving only Zone III
8. Ocular abnormalities that may interfere with the administration of study intervention
or assessment of the study primary endpoint
Prior/Concomitant Therapy – per subject
9. Postnatal treatment with oral or intravenous corticosteroids at an equivalent dose of 
prednisone ≥1 mg/kg/day for > 2 weeks within 14 days of the first study intervention
Prior/Concomitant Therapy – per eye
10. Previous surgical or nonsurgical treatment for ROP (IVT anti-VEGF injection, 
ablative laser therapy, cryotherapy, and vitrectomy)
Prior/Concurrent Clinical Study Experience
11. Participation of the subject or the mother in other clinical trials requiring 
administration of investigational treatments (other than vitamins and minerals) at the 
time of screening, or within 30 days or 5 half-lives of administration of the previous 
study drug, whichever is longer
5.3
Lifestyle Considerations
No restrictions during the study are required.
5.4
Screen Failures
Screen failures are subjects for whom informed consent has been obtained but who do not 
subsequently enter the study.  A minimal set of screen failure information is required to 
ensure transparent reporting of screen failure subjects to meet the Consolidated Standards of 
Reporting Trials (CONSORT) publishing requirements and to respond to queries from 
regulatory authorities. Minimal information includes demography, screen failure details, 
eligibility criteria, and any serious pretreatment events. 
In general, re-starting the defined set of screening procedures to enable the “screen failure” 
subject’s participation at a later time point is not allowed. Thus, in general, participation of an 
initial “screen failure” subject at a later time point is not acceptable, except if the screening 
failure was triggered by missing inclusion criteria 2 and/or 3, or by meeting exclusion criteria 
4, 5, 8, 9 and/or 11, or if the in-/exclusion criteria preventing the subject’s initial attempt to 
participate have been changed via protocol amendment.
Under any of the above exceptions, a subject may only be re-screened once and only within 
10 days of the start of the first screening period. 
To be eligible, rescreened subjects must meet all selection criteria at the re-screening visit. In 
any case, the investigator has to ensure that the repeated screening procedures do not expose 
the subject to an unjustifiable health risk. Also, for re-screening, the subject’s parent(s)/legally 
authorized representative(s) has to sign a new informed consent form, even if it was not 
changed after the subject’s previous screening.
Rescreened subjects should be assigned a new subject number.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  26 of 59
6.
Study Intervention
Study intervention is defined as any investigational intervention(s), marketed product(s), 
placebo, or medical device(s) intended to be administered to a study subject according to the 
study protocol.
6.1
Study Intervention(s) Administered
Intervention Name
Aflibercept 
Laser photocoagulation
Type
Drug
Procedure/surgery
Use
Experimental
Active comparator
Dose formulation
solution in a sterile glass vial
Not applicable
Unit dose strengths
mg (mL) 
Not applicable
Dosage Level(s)
0.4 mg (0.01 mL) 
Not applicable
Route of Administration
IVT injection  
Transpupillary conventional 
laser ablative therapy 
Packaging and Labeling
Study intervention will be 
provided in vials.  Each vial will 
be labeled as required per 
country requirement.
Not applicable
Current/Former Name(s) or 
Alias(es)
BAY 86-5321
Not applicable
6.1.1
Medical Devices
The medical device (not manufactured by or for Bayer) provided for use in this study is the
18-gauge filter needle.  Ancillary components required (30-gauge injection needle and Luer-
lock 1-mL syringe) may also be provided. The ancillary medical devices provided for use in 
this study are CE marked or FDA cleared according to the regulatory requirements specific 
for the country where the study site is located.
Medical device incidents, including those resulting from malfunctions of the device, must be 
detected, documented, and reported by the investigator throughout the study (see Section 
8.3.6).
6.2
Preparation/Handling/Storage/Accountability
Only subjects enrolled in the study may receive study intervention and only authorized site 
staff may supply or administer study intervention. 
The investigator is responsible for study intervention accountability, reconciliation, and record 
maintenance (ie, receipt, reconciliation, and final disposition records).
Further guidance and information for the final disposition/destruction of unused study drugs 
are provided in the Investigator Site File. 
Aflibercept 
The study drug will be supplied in kits that include the following:

Sterile study drug in sealed glass vials (2 mL) with a withdrawable volume of 0.1 mL
(ie, 10-fold the intended injection volume)

Filter needle (18-gauge)
Other ancillary components required for the administration of aflibercept (ie, 30-gauge 
injection needle and a 1-mL Luer-lock syringe) may also be supplied to the study sites.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  27 of 59
Aflibercept must be stored in a secure and monitored (manual or automated) area with access 
limited to the investigator and authorized site staff.  Aflibercept vials are to stored in the 
refrigerator (2°C to 8°C) and must not be frozen. The vial must be kept in the outer carton to 
protect from light. The investigator or designee must confirm that the appropriate temperature 
conditions have been maintained during transit and any discrepancies are reported and 
resolved before use.
When aflibercept vials are removed from the refrigerator, the solution should be inspected 
visually and it should have no evidence of turbidity. If particulates, cloudiness, or 
discoloration are visible, the vial must not be used. Prior to use, the unopened vial may be 
stored at room temperature (below 25°C) for up to 24 hours.
Details on the administration of aflibercept IVT injection and postinjection procedures can be 
found in Section 10.2.
Laser photocoagulation
Transpupillary conventional laser photocoagulation will be given following topical anesthesia,
sedation or general anesthesia, with appropriate respiratory support as required (eg, laryngeal 
mask, endotracheal intubation or similar), and administered according to standard local 
procedures. The pupil of the eye to be treated must be previously dilated (mydriasis) with 2 or 
3 drops of mydriatic agent(s) applied topically to the eye, according to local practice.
Laser power settings should be defined according to the investigator’s practice and applicable 
medical standards at the site. The treatment should cover the entire avascular retina, using a 
confluent laser pattern and extending to the ora serrata. 
The location of study intervention should follow medical standards at each site (eg, bedside in 
the NICU or in a separate operating room following local practice).
6.3
Measures to Minimize Bias: Randomization and Masking
This is an open-label study where the study sites, the study team and the subject and subject’s 
parent(s)/legally authorized representative(s) are unmasked. Potential bias will be reduced by 
central randomization and objective endpoints. Any aggregated treatment-specific interim 
data will be kept strictly confidential and will not be communicated to investigators.
Eligible subjects will be randomized 2:1 to receive either treatment with aflibercept or laser 
photocoagulation, respectively, stratified by Japanese and Non-Japanese sites as well as by 
ROP classification in Zone I, Zone II, or AP-ROP according to investigator assessment. If
both eyes meet the inclusion criteria of the study after screening, the eye with the more severe 
disease will be considered for stratification.
Treatment allocation will be done according to a computer-generated randomization list 
specified by the sponsor's responsible statistician and provided by the sponsor's randomization 
management group. For preventing imbalances, the randomization result of subjects who do 
not complete baseline treatment will be assigned to a subsequent subject once it is known that 
the baseline treatment was not completed.
Subjects will be centrally assigned to randomized study intervention using an Interactive 
Voice/Web Response System (IVRS/IWRS). Before the study is initiated, the telephone 
number and call-in directions for the IVRS and/or the log in information and directions for the 
IWRS will be provided to each site. The investigator will provide the IVRS/IWRS with study 
center identification, the subject´s date of birth (DOB), gender and weight at birth.  The 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  28 of 59
complete DOB (day, month and year) will be entered if allowed by local regulation because it 
is needed in order to correctly determine the paticipant´s chronological and corrected age 
during the study. Because of the particular population in this study, absence of an exact DOB 
will cause a 30-day deviation causing misinterpretation of results and the impossibility to 
understand the outcomes as this is usually compared with controls matched for the same 
corrected gestational age. Additional details are documented in the IVRS/IWRS instruction 
manuals. Study intervention will be dispensed at the study visits summarized in the SoA. 
6.4
Study Intervention Compliance
Study intervention will be administered by a qualified ophthalmologist.  Details of aflibercept 
injection and of the laser procedure will be recorded in the eCRF (eg, time required, type of 
anesthesia, presence or absence of endotracheal intubation, treatment site).
6.5
Prior and Concomitant Therapy
All medications the subject received prior to Visit 1 and any medication or vaccine (including 
any sedation, anesthesia, eye drops used for the study procedures, blood-derived products, 
prescription or over-the-counter medicines, probiotics, vitamins, and herbal supplements) that 
the subject is receiving at the time of enrollment or receives during the study should be 
recorded along with:

The reason for use

Dates of administration including start and end dates

Dosage information including dose and frequency
The use of phototherapy lamp for treating jaundice is allowed.  Appropriate eye protection 
must be provided to the subject. The use of phototherapy for jaundice will be documented in 
the eCRF including the use of eye protection.
The use of oxygen supplementation will be recorded in the corresponding eCRF section with 
special consideration of the method, concentration, duration of administration and the 
subject´s oxygen saturation by pulse oximetry once the supplementation is stable. In the last 
visit of the trial, it will be recorded whether oxygen supplementation is required.
Exposure to any IVT or systemic anti-VEGF agent is not allowed.
The use of medications, recreational drugs or similar substances during pregnancy will be 
recorded on the Maternal Prior Medication section on the eCRF at screening (eg, alcohol,
tobacco, antibiotics, corticosteroids, vaccination, supplements). For breastfeeding mothers, all 
maternal medication or similar products taken after the umbilical cord clamping will be 
recorded in the Maternal Concomitant Medication section of the eCRF only if the transfer of 
these products into human milk is feasible according to the investigator’s judgment. The 
Medical Monitor should be contacted if there are any questions regarding concomitant or prior 
therapy. 
Anti-VEGF agents are not allowed in mothers who are breastfeeding. 
6.5.1
Rescue Medicine
For subjects randomized to aflibercept, rescue treatment with laser may be performed.  For 
subjects randomized to laser, rescue treatment with aflibercept may be performed.  The 
criteria for the allowance of rescue treatment is specified in Section 4.1. Use of rescue 
treatment in either eye must be recorded on the respective eCRF page.  


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  29 of 59
All subjects requiring rescue treatment will be counted as missing the primary endpoint. 
Nevertheless, the subjects should be followed to assess the efficacy and safety outcomes after 
rescue treatment. 
6.6
Dose Modification
Not applicable. Only the 0.4 mg/0.01mL dose of aflibercept will be used for all treatments.
6.7
Intervention after the End of the Study
A Phase 3b study (Study 20275; extension study) is planned to assess the long-term outcomes 
of subjects who received study intervention in this study and accept participation in the 
extension study.  No study treatment will be required in this study, or provided by the 
sponsor.  In case any treatment for ROP is required during Study 20275, the site’s standard of 
care should be administered. Subjects will undergo safety and efficacy assessments until 5 
years of age.
The baseline visit of Study 20275 can be conducted concomitantly with the Week 24 visit or 
the last follow-up visit, whichever is later, or at any later point between this date and 
≤1 month after the date when the subject is 1 year of chronological age.
7.
Discontinuation of Study Intervention and Participant 
Discontinuation/Withdrawal 
7.1
Discontinuation of Study Intervention 
Study intervention discontinuation can be triggered by the subject’s parent(s)/legally 
authorized representative(s) or by the treating physician.
Subjects in which retreatment or rescue treatment are planned at any time during the study but 
not administrated will be considered a temporarily discontinuation of study intervention. If 
study intervention is temporarily discontinued, it can be restarted at any time during the study. 
In case there is a decision against retreatment or rescue treatment, subjects will be considered 
a permanent discontinuation of study intervention. If study intervention is permanently 
discontinued, further treatment is at the investigator’s discretion. 
Study intervention discontinuation must not lead to study discontinuation of a subject. 
If study intervention is permanently discontinued, the subject should continue in the study on 
an observational basis (eg, for study examinations only) for safety evaluation. 
7.2
Participant Discontinuation/Withdrawal from the Study
A subject may be withdrawn from the study at any time at the request of the parent(s)/legally 
authorized representative(s), or at the discretion of the investigator for safety, behavioral, 
compliance, or administrative reasons. This is expected to be uncommon.
Any surgical treatment for ROP other than study interventions is considered a prohibited
treatment, and subject must be withdrawn from the study.
At the time of discontinuing from the study, if possible, an end of study (EOS) visit should be 
conducted, as shown in the SoA. See the SoA for data to be collected at the time of study 
discontinuation and follow-up and for any further evaluations that need to be completed.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  30 of 59
The participant will be permanently discontinued from the study intervention and from the 
study at that time.
If the parent(s)/legally authorized representative(s) withdraw consent for disclosure of future 
information, the sponsor may retain and continue to use any data collected before such a 
withdrawal of consent.
7.3
Lost to Follow-Up
A subject will be considered lost to follow-up if he or she repeatedly fails to return for 
scheduled visits and the study site is unable to contact the subject’s parent(s)/legally 
authorized representative(s). 
The following actions must be taken if a subject fails to return for a required study visit:

The site must attempt to contact the subject’s parent(s)/legally authorized
representative(s) and reschedule the missed visit as soon as possible and counsel the 
subject’s parent(s)/legally authorized representative(s) on the importance of 
maintaining the assigned visit schedule and ascertain whether or not the subject’s 
parent(s)/legally authorized representative(s) wishes to have the subject continue in the 
study.

Before a subject is deemed lost to follow-up, the investigator or designee must make 
every effort to regain contact with the subject’s parent(s)/legally authorized
representative(s) (where possible, 3 telephone calls and, if necessary, a certified letter 
to the subject’s parent[s]/legally authorized representative[s] last known mailing 
address or local equivalent methods). These contact attempts should be documented in 
the subject’s medical record.

Should the subject’s parent(s)/legally authorized representative(s) continue to be 
unreachable, the subject will be considered lost to follow-up.
8.
Study Assessments and Procedures
Study procedures and their timing are summarized in the SoA. Protocol waivers or 
exemptions are not allowed.
Subjects should be seen for all visits on the designated day, with an allowed “visit window” as 
indicated in the SoA.
Safety concerns should be discussed with the sponsor immediately upon occurrence or 
awareness to determine if the subject should continue or discontinue study intervention.
Adherence to the study design requirements, including those specified in the SoA, is essential 
and required for study conduct.
All screening evaluations must be completed and reviewed to confirm that potential subjects 
meet all eligibility criteria. The investigator will maintain a screening log to record details of 
all subjects screened and to confirm eligibility or record reasons for screening failure, as 
applicable. 
The blood volume taken for study-related safety assessments, for analysis of study drug (PK, 
in blood plasma), and ADA (in blood serum) in conjunction with non-study, safety-related 
assessments should be kept to a minimum, at the discretion of the investigator and applying 
local regulations.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  31 of 59
For subjects randomized to aflibercept: in order to monitor subjects for the potential 
development of ADA, one 0.6-mL blood sample for determination of ADA serum titers will 
be taken at baseline prior to aflibercept dosing (Visit 2), and one 1.1-mL blood sample at 
12 weeks after dosing (Visit 11), which can also be used to detect the occurrence of potential 
neutralizing antibodies (NAB). If it is not possible to obtain an ADA sample at Visit 11, it can 
be collected at Visit 12.  A total of approximately 1.7 mL of blood will be collected for ADA 
measurement in serum during the first 12 weeks after treatment.  
For the collection of PK data, one blood sample (0.6 mL each) will be taken at 6 time points 
after dosing: at Day 1 (Visit 3), Week 2 (Visit 5), Week 4 (Visit 7), Week 8 (Visit 9), Week 
12 (Visit 11) and Week 24 (Visit 14 / EOS)1. 
The time and date of samplings must be documented. Details are provided in the sample 
handling manual.  
For PK and ADA, the total sample volume was specified to a minimum of 3.6 mL (Section 
8.5) and 1.7 mL respectively.  No capillary blood is allowed for PK and ADA samples.
Repeat or unscheduled samples may be taken for safety reasons or for technical issues with 
the samples. Study blood samples should be performed in combination with regular blood 
draws to avoid an additional puncture if possible. 
Unscheduled visits for observation follow the list of assessments used for Visit 8 (Week 6).  
In case treatment is required, the assessments listed in the re-treatment visit will be performed.
All results will be recorded in the eCRF.
8.1
Efficacy Assessments 
All ophthalmic evaluations will be conducted according to the schedule detailed in SoA.
Posterior segment assessment to confirm ROP staging or resolution will be evaluated by the 
investigator by binocular indirect ophthalmoscopy or wide-field digital retinal photography 
(eg, RetCam, Phoenix ICON Camera). 
Indirect ophthalmoscopy will be performed according to local medical practice and applicable 
medical standards at the site (eg, usually using a head mounted light source and a 20- or 28-
diopter lens). For this examination, the pupil of the eye must be dilated (mydriasis) with 
topical application of mydriatic eye drops to the eye.
Clinically significant abnormal findings will be reported as AEs in the eCRF.
Color fundus photographs using wide-field digital retinal photography have to be taken at the 
time points specified in the SoA. 
All images taken by wide-field digital retinal imaging photography during the study will be 
submitted to a central reading center to confirm the ROP staging or resolution, and analysis of 
efficacy data. All images are part of the source data for the study and must be retained by the 
investigator site.  A detailed protocol for color fundus photographs image acquisition and 
transmission can be found in the respective manual.
                                                
1 PK samples at Weeks 8, 12 and 24 are optional for patients with consent signed prior to the effectiveness date of the current 
amendment (Amendment 01).


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  32 of 59
8.1.1
Other Ocular Assessments
With all ophthalmological examinations, abnormalities of the retina or optic nerve as well as 
unfavorable ocular structural outcomes in each eye will be assessed. All ocular assessments 
have to be performed bilaterally.
Visual function will be evaluated using a methodology appropriate for the age and 
development status of the child, including evaluation of fixation (eg, central, steady and 
maintained) and fixing and following a 5-cm toy. 
Monocular and binocular evaluation of visual function has to be performed. 
If the subject is not able to cooperate with the above methods, an other suitable method (eg, 
Visual evoked potentials) can be used to evaluate visual function.
Cycloplegic refraction will be measured with retinoscopy in each eye and reported separately 
for each eye.
Ocular motility tests of both eyes (binocular testing) will be assessed to investigate the 
integrity of the extrinsic ocular muscles and their nerves (eg, Cover test or Hirschberg test).
8.2
Safety Assessments 
Planned time points for all safety assessments are provided in the SoA.
Clinically significant abnormal findings will be reported as AEs in the eCRF.
Care should be taken to use the same equipment for all examinations of a certain subject 
whenever possible.
8.2.1
Ophthalmic Examinations
Assessments of ocular safety will include ophthalmologic assessment of the anterior and 
posterior segment (eg, by indirect ophthalmoscopy), and measurement of intraocular pressure 
(IOP; only in subjects receiving aflibercept).
All ophthalmic examinations are to be conducted in both eyes, unless indicated otherwise.
IOP will be measured only in subjects receiving aflibercept using a portable tonometer (eg, 
Tono-Pen, Perkin’s Tonometer, or other locally approved device). The same method of IOP 
measurement should be used in each subject throughout the study.  IOP will be measured in 
both eyes prior to the injection and postinjection only in the study eye(s) according to the 
directions in Section 10.2.  A local anesthetic may be topically applied to the eye being tested 
(eg, 1 drop of oxybuprocain).
8.2.2
Physical Examinations
A routine physical examination will assess cardiovascular, respiratory, gastrointestinal, and 
neurological systems and will follow the standard practice of the site. The assessment will be 
based on the clinical judgment of the physician and aim to evaluate the overall health of the 
baby. Weight, body length, and head circumference will be measure as specified in the SoA.
8.2.3
Vital Signs
Temperature, pulse rate, respiratory rate, and blood pressure will be measured according to 
the local medical practice and regulations.  Study staff trained in the assessment of infants will 
perform these assessments. Blood pressure and pulse measurements should be assessed with a 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  33 of 59
completely automated device, appropriate for use in infants. Blood pressure must be 
measured before any study treatment is administered and before PK sampling, if applicable.
Clinically significant abnormal findings will be reported as AEs in the eCRF.
8.2.4
Clinical Safety Laboratory Assessments
Section 10.3 lists the clinical laboratory tests to be performed and the SoA specifies the 
timing. 
The investigator must review the laboratory report, document this review, and record any 
clinically relevant changes occurring during the study in the AE section of the eCRF. The 
laboratory reports must be filed with the source documents. Clinically significant abnormal 
laboratory findings are those that are not associated with the underlying disease, unless judged 
by the investigator to be more severe than expected for the subject's condition.
All laboratory tests with values considered clinically significantly abnormal during 
participation in the study or within 4 weeks after the last dose of study intervention should be 
repeated until the values return to normal or baseline or are no longer considered clinically 
significant by the investigator.

If such values do not return to normal/baseline within a period of time judged 
reasonable by the investigator, the etiology should be identified and the sponsor 
notified.

If laboratory values from non-protocol specified laboratory assessments performed at 
the institution’s local laboratory require a change in subject management or are 
considered clinically significant by the investigator (eg, SAE or AE or dose 
modification), then the results must be recorded in the eCRF. 
Laboratory (hematology, chemistry, and urinalysis) analyses will be performed and reviewed 
at screening (Visit 1). A sample is not needed if results from laboratory tests within 8 days 
prior to screening are available and there was no change in the clinical situation from the time 
of the sample to the screening visit.
In order to avoid discomfort and reduce the probability of urinary tract infections, collection 
bags will be used to collect urine samples if possible.
Additional samples, including ADA/NAB samples, may be collected at any time during the 
study as determined necessary by the investigator or required by local regulations. This might 
be true for subjects receiving rescue treatment with aflibercept.
8.2.5
Central Nervous System Imaging
Central nervous system (CNS) ultrasound examinations will be performed by a well-
experienced examiner. The minimum source documentation will include electronic or paper 
documentation (medical report). Other modalities of imaging (eg, magnetic resonance 
imaging [MRI]) can be used instead of ultrasound if the alternative test was planned 
independently of the study.
8.3
Adverse Events and Serious Adverse Events
The definitions of an AE or SAE can be found in Section 10.4.
AEs will be reported by the subject’s treating physician, caregiver, surrogate, or the subject's 
legally authorized representative(s) or health care professional not involved in the study.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  34 of 59
The investigator and any qualified designees are responsible for detecting, documenting, and 
recording events that meet the definition of an AE or SAE. They remain responsible for 
following up SAEs, or AEs considered related to the study intervention or study procedures, 
or those that caused the subject to discontinue the study intervention or the study.
8.3.1
Time Period and Frequency for Collecting AE and SAE Information 
All AEs/SAEs will be collected from the start of study intervention at the time points 
specified in the SoA (Section 1.3).
Medical occurrences that begin before the start of study intervention but after obtaining 
informed consent will be recorded on the Medical History/Current Medical Conditions section 
of the eCRF not the AE section. Any relevant maternal medical occurrences during pregnancy 
until the delivery of the newborn that may have impact on the subject´s health will be 
recorded on the Maternal Medical History section of the eCRF (eg, urinary tract infections, 
TORCH infections, high blood pressure, preeclampsia).
All SAEs will be recorded and reported to the sponsor or designee immediately and under no 
circumstances should this exceed 24 hours, as indicated in Section 10.4. The investigator will 
submit any updated SAE data to the sponsor within 24 hours of it being available.
Investigators are not obligated to actively seek AEs or SAEs after the conclusion of study 
participation at Week 24 or at the follow-up, whichever is later. However, if the investigator 
learns of any SAE, including a death, at any time after a subject has been discharged from the 
study, and he/she considers the event to be reasonably related to the study intervention or 
study participation, the investigator must promptly notify the sponsor. 
8.3.2
Method of Detecting AEs and SAEs
The methods of recording, evaluating, and assessing causality of AEs and SAEs and the 
procedures for completing and transmitting SAE reports are provided in Section 10.4.
Care will be taken not to introduce bias when detecting AEs and/or SAEs.  Open-ended and 
non-leading verbal questioning of the subject’s parent(s)/legally authorized representative(s)
is the preferred method to inquire about AE occurrences. 
8.3.3
Follow-up of AEs and SAEs
After the initial AE/SAE report, the investigator is required to proactively follow each subject
at subsequent visits/contacts. All SAEs will be followed until resolution, stabilization, the 
event is otherwise explained, or the subject is lost to follow-up (as defined in Section 7.3). 
Further information on follow-up procedures is given in Section 10.4. 
8.3.4
Regulatory Reporting Requirements for SAEs
Prompt notification by the investigator to the sponsor of an SAE is essential so that legal 
obligations and ethical responsibilities towards the safety of subjects and the safety of a study 
intervention under clinical investigation are met. 
The sponsor has a legal responsibility to notify both the local regulatory authority and other 
regulatory agencies about the safety of a study intervention under clinical investigation. The 
sponsor will comply with country-specific regulatory requirements relating to safety reporting 
to the regulatory authority, Institutional Review Boards (IRBs)/Independent Ethics 
Committees (IECs), and investigators.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  35 of 59
Investigator safety reports must be prepared for suspected unexpected serious adverse 
reactions according to local regulatory requirements and sponsor policy and forwarded to 
investigators as necessary.
An investigator who receives an investigator safety report describing an SAE or other specific 
safety information (eg, summary or listing of SAEs) from the sponsor will review and then 
file it along with the Investigator’s Brochure and will notify the IRB/IEC, if appropriate 
according to local requirements.
8.3.5
Pregnancy
Not applicable.
8.3.6
Medical Device Incidents (Including Malfunctions)
A filter needle is provided for use in this study. In order to fulfill regulatory reporting 
obligations worldwide, the investigator is responsible for the detection and documentation of 
events meeting the definitions of incident or malfunction that occur during the study with such 
devices. 
Definitions of a medical device incident/AE/SAE and device deficiency can be found in 
Section 10.5.
NOTE: Incidents fulfilling the definition of an AE/SAE will also follow the processes 
outlined in Section 8.3.3 and Section 10.4.
8.3.6.1
Time Period for Detecting Medical Device Incidents
Medical device incidents or malfunctions of the device identified, documented, and reported 
during all periods of the study in which the medical device is used. 
If the investigator learns of any incident at any time after a subject has been discharged from 
the study, and such incident is considered reasonably related to a medical device provided for 
the study, the investigator will promptly notify the sponsor.
The method of documenting medical device incidents is provided in Section 10.5.
8.3.6.2
Follow-up of Medical Device Incidents
All medical device incidents involving an AE will be followed and reported in the same 
manner as other AEs (see Section 8.3.3). This applies to all subjects, including those who 
discontinue study intervention.
The investigator is responsible for ensuring that follow-up includes any supplemental 
investigations as indicated to elucidate the nature and/or causality of the incident.
New or updated information will be recorded on the originally completed form with all 
changes signed and dated by the investigator. 
8.3.6.3
Prompt Reporting of Medical Device Incidents to Sponsor
Medical device incidents will be reported to the sponsor within 24 hours after the investigator 
determines that the event meets the protocol definition of a medical device incident.
The Medical Device Incident Report Form will be sent to the sponsor by facsimile 
transmission. If unavailable, then telephone, overnight mail or courier service should be 
utilized.
The same individual will be the contact for the receipt of medical device reports and SAE.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  36 of 59
8.3.6.4
Regulatory Reporting Requirements for Medical Device Incidents
The investigator will promptly report all incidents occurring with any medical device 
provided for use in the study in order for the sponsor to fulfill the legal responsibility to notify 
appropriate regulatory authorities and other entities about certain safety information relating 
to medical devices being used in clinical studies.
The investigator, or responsible person according to local requirements (eg, the head of the 
medical institution), will comply with the applicable local regulatory requirements relating to 
the reporting of incidents to the IRB/IEC.
8.4
Treatment of Overdose
For this study, any instance where the investigator assumes that a single IVT dose of more 
than 0.4 mg (0.01 mL) was administered will be considered an overdose. Overdosing with 
increased injection volume may increase IOP. In these cases, evaluation of IOP and central 
retinal artery perfusion should be performed immediately after the injection and monitored 
until normalized. If there is severe elevation of IOP causing disruption of central retinal artery 
perfusion, immediate performance of an anterior segment paracentesis should be considered.
Additionally, after ensuring that IOP and central retinal artery perfusion are in a safe range, 
the investigator should: 
1. Contact the Medical Monitor immediately.
2. Closely monitor the subject for any further AE/SAE and laboratory abnormalities.
3. Obtain a plasma sample for PK analysis if requested by the Medical Monitor 
(determined on a case-by-case basis).
4. Document the quantity of the excess administered overdose in the eCRF.
Decisions regarding dose interruptions or modifications will be made by the investigator in 
consultation with the Medical Monitor based on the clinical evaluation of the subject.
8.5
Pharmacokinetics
Since aflibercept is systemically demonstrable after IVT administration, systemic 
concentrations of study drug will be collected and described. The PK evaluations will support 
the safety evaluation.  The objective of the PK evaluations are to:

Describe the individual concentrations of free and bound aflibercept at several time
points after the first IVT injection up to 24 weeks thereafter2

Explore the potential influence of demographic and other factors on systemic 
aflibercept concentrations

Explore the relationship of systemic exposure and blood pressure 
PK sampling schedule (only applicable for subjects treated with aflibercept at baseline)
One 0.6-mL blood sample for plasma concentrations of study drug will be taken according to 
the time points in SoA after BP is measured. A total of 3.6 mL of blood will be collected for 
PK analysis during the study. See the SoA in Section 1.3 for details.
                                                
2 PK samples at Weeks 8, 12 and 24 are optional for patients with consent signed prior to the effectiveness date of the current 
amendment (Amendment 01).


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  37 of 59
If the second eye needs to be treated with aflibercept on a day with PK sample collection, the 
PK sample should be collected first.  
To ensure accuracy of the PK analyses, it is critical to accurately record the exact date and 
time (24-hour clock) of all blood samples taken on the eCRF, as well as the exact time of the 
study intervention administration.
Details about the collection, processing, storage and shipment of samples will be provided 
separately in the laboratory manual.  No capillary blood is allowed for PK samples.
Additional samples may be collected at any time during the study as determined necessary by 
the investigator or required by local regulations. 
PK evaluation and reporting
Concentrations of free and bound aflibercept will be listed by sample time point, and 
descriptive statistics will be applied. No formal PK parameters will be evaluated. 
Decline of aflibercept concentrations after the first injection will be described. Drug 
concentration listings will be further grouped by potentially influencing factors (ie, renal 
impairment as determined by serum creatinine values, concomitant medications, body weight, 
ethnicity) and evaluated by means of descriptive statistics.
Pharmacokinetics in patients who receive repeated injections will be evaluated up to the time
point prior to the second injection. In these patients, concentration data for sampling times
beyond the second injection will be evaluated exploratively, considering total dose and 
potential accumulation.
Evaluation of the data will be reported separately and attached to the final clinical study report 
as an appendix. Fundamental results and conclusions of the PK evaluation will also be 
included in the main text of the final clinical study report.
8.6
Pharmacodynamics
Pharmacodynamic parameters are not evaluated in this study.
8.7
Genetics
Pharmacogenetic investigations are not planned.
8.8
Biomarkers
Biomarkers for efficacy will not be evaluated. 
Blood pressure (BP) as a safety biomarker will be measured using an age-appropriate monitor 
at the time points shown in the SoA.  When applicable, measurements will be taken before 
study treatment, and before PK sampling.  
Urine protein will also be measured.  The date and time of urine sampling must be 
documented.
8.8.1
Other Biomarkers
Only a very limited amount of blood is collected; leftovers (if any) may be used for additional 
research on study drug and/or disease.  Blood volume will not be increased for these research 
purposes.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  38 of 59
In addition to the biomarkers described above, further biomarkers related to the mode of 
action or the safety of aflibercept and similar drugs may be examined.  The same applies to 
further biomarkers deemed relevant to diseases of the eye and associated health problems.  
These investigations may include eg, diagnostic, safety, pharmacodynamics, monitoring, or 
potentially predictive biomarkers.
The additional biomarker results may be reported separately.
8.9
Medical Resource Utilization and Health Economics 
Health Economics/Medical Resource Utilization and Health Economics parameters are not 
evaluated in this study. 
9.
Statistical Considerations 
9.1
Statistical Hypotheses
The study will provide estimates of efficacy and success will be concluded based on the 
posterior probability from the Bayesian analysis using the following success criterion: 
Success criterion defined as “response probability for aflibercept is greater than the one for 
laser minus 5 percentage points with at least 95% probability.”
This can be interpreted as non-inferiority hypothesis with a non-inferiority margin of 5 
percentage points.
9.2
Sample Size Determination 
The sample size rationale is based on the primary efficacy endpoint “absence of active ROP 
and unfavorable structural outcomes at 24 weeks after starting study treatment” and the 
success criterion that the response probability for aflibercept is greater than the one for laser 
minus 5 percentage points with at least 95% probability.
With at least 102 subjects evaluable for the primary analysis in the full analysis set (FAS; see 
Section 9.3 for definition) and a randomization ratio of 2:1 (active:control) to receive either 
treatment with aflibercept or laser photocoagulation (corresponds to 68 and 34 subjects, 
respectively), the defined success criterion will be achieved with a power of 81% under
following assumptions: 

The laser response probability for the study is similar to historic data: A Bayesian 
meta-analytical prediction based on BEAT-ROP and RAINBOW resulted in a 
posterior predictive distribution described by a beta(34.7, 13.8) distribution. For the 
power simulations, the true response probability for laser photocoagulation in this trial 
was drawn from this distribution for each simulation run.   

And the response probability for aflibercept is 15 percentage points higher than for 
laser, but not higher than 95%.
Respective power simulations were performed using package rjags (Plummer, 2016) in the 
statistical software R (R Core Team, 2016).
9.3
Populations for Analyses 
For purposes of analysis, the following populations are defined:


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  39 of 59
Population
Description
Full analysis set (FAS)
All subjects who received any type of study treatment, and had a 
baseline and at least one post-baseline assessment of efficacy
Modified full analysis set 
(mFAS)
All subjects with central reading center positive confirmed disease 
stages meeting the inclusion criteria who completed baseline 
treatment, had a baseline and at least one post-baseline central 
reading center assessment of efficacy.
Per protocol set (PPS)
All subjects in the mFAS who have no validity findings or important 
deviations that could affect the primary efficacy variable. 
The status of each subject, with regard to protocol deviations, will be 
determined by the sponsor and documented before database release.
Safety analysis set (SAF)
All subjects who received any type of study treatment
The primary efficacy variable will be analyzed using the FAS, mFAS, and the PPS, where the 
FAS analysis is considered to be the primary one.  The secondary and explorative efficacy 
variables will be analyzed using the FAS and mFAS, where in all cases the analyses with the 
mFAS and PPS are considered as supportive. Safety variables will be analyzed based on the 
SAF.
9.4
Statistical Analyses
The statistical analysis plan (SAP) will be developed and finalized before database release and 
will describe the subject populations to be included in the analyses, and procedures for
accounting for missing, unused, and spurious data. This section is a summary of the planned 
statistical analyses of the primary and secondary endpoints.
Data of the second eye (of subjects who have started the study with only one eligible eye) will
be included in the efficacy and safety analyses if treated before or on Visit 9 (≤8 weeks from 
baseline), whereas second eyes treated after Visit 9 (>8 weeks from baseline) will be included 
in the safety analysis only.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  40 of 59
9.4.1
Efficacy Analyses
Endpoint
Statistical Analysis Methods
Primary: 
Proportion of patients with absence of active 
ROP and unfavorable structural outcomes at 24 
weeks after starting study treatment.
Primary analysis: The data to be analyzed by a 
Bayesian statistical model. (detail below)
Secondary: 

Requirement for intervention with a second 
treatment modality from baseline to Week 24 

Recurrence of ROP from baseline to Week 24

Number of aflibercept administrations from 
baseline to Week 24 

Number of laser treatments from baseline to 
Week 24

To explore new Retinopathy of Prematurity 
Activity Scale proposed by the International 
Neonatal Consortium *
The first two secondary endpoints will be 
analyzed using Bayesian statistical modelling 
as described below.
The number of aflibercept administrations and 
laser treatments as well as the ROP activity 
scale will be analyzed descriptively.
No hypothesis testing will be conducted for the 
secondary endpoints.
Other pre-specified:

Evaluation of visual function at Week 24

Time required to perform treatment

Requirement for sedation or general 
anesthesia

Requirement for treatment with more than one 
aflibercept injection

Time to intervention with a second treatment 
modality for ROP or development of 
unfavorable structural outcomes

Time to recurrence of ROP 

Regression of plus disease, regression of pre-
retinal vascularized ridge and progression of 
retinal vascularization beyond the ridge from 
baseline to Week 24 

Progression to Stage 4 or 5 ROP from 
baseline to Week 24

Completion of vascularization of the peripheral 
retina to within one disc diameter of the ora 
serrata at Week 24 

Time to completion of vascularization

Number of visits required up to Week 24
The analysis of all other prespecified endpoints
will be described in the SAP finalized before 
database release.
*Smith et al, 2018
The primary endpoint is the proportion of patients with absence of active ROP and 
unfavorable structural outcomes at 24 weeks after starting study treatment.
For this endpoint, active ROP is defined as ROP requiring treatment (according to the 
inclusion criteria). Unfavorable structural outcome is defined as retinal detachment, macular 
dragging, macular fold, or retrolental opacity.  
Furthermore, eyes are considered non-responders if rescue treatment was given.
The primary analysis for this endpoint will be based on the investigator assessment of ROP; 
sensitivity analyses based on the central reading center data will be conducted.
The primary endpoint will be analyzed using a Bayesian statistical model with a non-
informative prior probability distribution for the response probability for a single eye (p). One 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  41 of 59
eye or both eyes of a patient to be included into the analysis to determine the primary endpoint 
on a patient level, if treated and meeting the inclusion criteria.
As it is assumed that in most subjects both eyes will be treated, this will be accounted for by 
using a bivariate model including a correlation coefficient ρ with an informative prior 
distribution allowing positive values only. The model is based on following distribution 
assumptions:
li ~ Bernoulli (p): response of subject i in left eye
            ri ~ Bernoulli (p): response of subject i in right eye
            li and ri are correlated with correlation coefficient ρ
            p ~ beta(1,1)     – non-informative prior for the response probability in one eye
          ρ ~ beta(1,1)   – prior for the correlation between the two eyes of one subject
(allowing only a positive correlation)
Bivariate probability distribution:
left eye / right eye
Response
No response
Response
p2+ ρ p(1-p)
p(1-p)(1- ρ)
p
No response
p(1-p)(1- ρ)
(1-p)(1-p(1- ρ))
1-p
p
1-p
Based on this model the primary endpoint “Proportion of patients with absence of active ROP 
and unfavorable structural outcomes at 24 weeks after starting study treatment,” (eg, response 
on a subject level, i.e. p2+ ρ p(1-p)) will be derived for each of the 2 treatment groups.
The efficacy of aflibercept and laser photocoagulation will be characterized by the resulting 
posterior distribution for the probability of response.  90% credible intervals to be provided 
for the probability of response.
In addition mean, median, and mode of the posterior distributions for the response 
probabilities of the 2 treatments as well as for the difference in response probabilities between 
the treatments will be provided together with 90% two-sided credible intervals.
Success of this study will be concluded, if the response probability for aflibercept is greater 
than the one for laser minus 5 percentage points with at least 95% probability.
This is the case if the lower limit of the one-sided 95% credible interval for the treatment 
difference (aflibercept – laser photocoagulation) is greater than -5%.
As non-informative prior distributions are used for the analysis this success criterion 
corresponds to a frequentist  non-inferiority test with a non-inferiority margin of 5 percentage 
points and a significance test of 5% (one-sided test).
This success criterion is reasonable since in historical studies, differentiation between laser 
and anti-VEGF in unfavorable structural outcomes has not been consistently demonstrated 
when treating ROP in Zone II. In addition, laser photocoagulation requires burning and 
destroying retina where vessels have not yet developed, whereas anti-VEGF agents allow 
further development of retinal vascularization and potential future functional benefits such as 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  42 of 59
improved visual fields. Furthermore, laser photocoagulation is associated with development of 
high myopia.  The proposed non-inferiority (NI) margin of 5% is smaller than the smallest 
difference between laser and ranibizumab in RAINBOW, and not greater than the difference 
between the two ranibizumab doses. With the proposed sample size, numerically better 
response (~10 percentage points) needs to be observed for aflibercept compared to laser to 
meet the success criterion and the NI test can be seen as a “relaxed“ superiority test.
If the success criterion is met, in a second step superiority of aflibercept over laser 
photocoagulation is evaluated by comparing the lower limit of the 95% one-sided credible 
interval with 0.
Handling of missing data for the primary endpoint:
The handling of subjects with missing data for the primary endpoint is outlined in the 
following. Further details will be provided in the SAP.
The following rules apply only for eyes not having had an unfavorable structural outcome or 
rescue treatment before dropping out, as in these cases the eye would always be considered as 
non-responding.
If at the last visit before dropping out the subject had no active ROP and Zone II was 
completely vascularized (aflibercept subjects) or laser treatment was completed (laser 
subjects) the respective eye will be considered as responding.
Otherwise, if the subject dropped out at or after week 16, the last ROP staging before 
dropping out will be carried forward and used for determining the response in this eye. If the 
subject dropped out before week 16, the missing information will be imputed as follows:
If there was a clear documentation that the subject dropped out due to lack of efficacy, the 
respective eye will be considered as non-responding; otherwise, a multiple imputation 
approach will be used based on the subject’s treatment group and the initial staging (Zone I 
versus II versus AP-ROP).
For the primary and secondary efficacy endpoints, subgroup analyses by zones (Zone I/II/AP-
ROP) will be performed.
 
9.4.2
Safety Analyses
All safety analyses will be performed on the SAF.  All safety variables including ocular and 
systemic TEAEs and SAEs from baseline to Week 24 and the presence of ADAs before and 
12 weeks after the baseline aflibercept injection will be analyzed descriptively using summary 
statistics or frequency tables as appropriate. Thereby, TEAE is defined as AE that is observed 
or reported after the first and not later than 30 days after the last administration of study 
treatment.
All AEs will be coded using Medical Dictionary for Regulatory Activities (MedDRA). The 
latest available version at the time of coding will be used. The results will be summarized, at 
a minimum, on the level of system organ class and preferred term. Data will also be 
summarized according to intensity and investigator’s causality assessment.
CCI


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  43 of 59
9.4.3
Other Analyses 
PK and biomarker exploratory analyses (see Section 8.5 and 8.8) will be described in the 
SAP. Results of further bioanalytical analyses will be presented separately from the main 
CSR (see Section 8.5).
9.5
Interim Analyses 
No interim analysis is planned. However, safety assessments will be continuously performed.
Database release will occur after all subjects have completed the Week 24 visit or have 
dropped out of the study. In case of the scenario where a subject has a 30-day follow-up visit 
after Week 24 or the final PK sample results come in later and these data cannot be cleaned in 
time for the initial data release, they will be included in a planned re-release of the database.
9.5.1
Data Monitoring Committee (DMC)
The aim of the safety assessments are to determine if the study shows unacceptable risks for 
the subjects, and to discontinue the study if necessary. The assessments will be performed by 
an independent DMC. Detailed information regarding the DMC procedures will be explained 
in a separate DMC charter.
10.
Supporting Documentation and Operational Considerations
10.1
Appendix 1: Regulatory, Ethical, and Study Oversight Considerations
10.1.1
Regulatory and Ethical Considerations

This study will be conducted in accordance with the protocol and with the following:

Consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical 
Sciences (CIOMS) International Ethical Guidelines

Applicable ICH Good Clinical Practice (GCP) Guidelines

Applicable laws and regulations

The protocol, protocol amendments, ICF, Investigator Brochure, and other relevant 
documents (eg, advertisements) must be submitted to an IRB/IEC by the investigator 
and reviewed and approved by the IRB/IEC before the study is initiated. 

Any amendments to the protocol will require IRB/IEC approval before 
implementation of changes made to the study design, except for changes necessary to 
eliminate an immediate hazard to study subjects. 

The investigator will be responsible for the following:

Providing written summaries of the status of the study to the IRB/IEC annually 
or more frequently in accordance with the requirements, policies, and procedures 
established by the IRB/IEC

Notifying the IRB/IEC of SAEs or other significant safety findings as required 
by IRB/IEC procedures

Providing oversight of the study conduct at the site and adherence to all 
applicable requirements (21 CFR, ICH guidelines, the IRB/IEC, European 
regulation 536/2014 for clinical studies, and local regulations)


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  44 of 59
10.1.2
Financial Disclosure
Investigators and sub-investigators will provide the sponsor with sufficient, accurate financial 
information as requested to allow the sponsor to submit complete and accurate financial 
certification or disclosure statements to the appropriate regulatory authorities. Investigators 
are responsible for providing information on financial interests during the course of the study 
and for 1 year after completion of the study.
10.1.3
Informed Consent Process
The investigator or his/her qualified representative will explain the nature of the study to the 
subject’s parent(s)/legally authorized representative(s), including that participation is 
voluntary, and answer all questions regarding the study. 
The subjects’ parent(s)/legally authorized representative(s) will be required to sign a statement 
of informed consent that meets the requirements of all applicable regulations (eg, 21 CFR 50, 
local regulations, ICH guidelines, Health Insurance Portability and Accountability Act 
[HIPAA]) and the IRB/IEC or study center. 
The medical record must include a statement that written informed consent was obtained 
before the subject was enrolled in the study and the date the written consent was obtained. The 
authorized person obtaining the informed consent must also sign the ICF.
If the ICF is revised during the study, re-consent must be obtained, if applicable. 
A copy of the ICF(s) must be provided to the parent(s)/legally authorized representative(s). 
A new ICF is required if a subject is rescreened.
10.1.4
Data Protection
Subjects will be assigned a unique identifier by the sponsor. Any subject records or datasets 
that are transferred to the sponsor will contain the identifier only; subject names or any 
information that would make the subject identifiable will not be transferred. 
The subject’s parent(s)/legally authorized representative(s) must be informed that the 
subject’s personal study-related data will be used by the sponsor in accordance with local data 
protection law. The level of disclosure must also be explained. 
The subject’s parent(s)/legally authorized representative(s) must be informed that the 
subject’s medical records may be examined by Clinical Quality Assurance auditors or other 
authorized personnel appointed by the sponsor, by appropriate IRB/IEC members, and by 
inspectors from regulatory authorities.
10.1.5
Committees Structure
An independent central reading center (RC) will evaluate the digital wide-field retinal 
images.
The sponsor may decide to institute a Steering Committee to guide the trial in all aspects of 
safety and efficacy and must ensure that all relevant information is provided by investigators. 
The composition of the committee, the functional roles, and responsibilities will be specified 
in its charter.
10.1.6
Dissemination of Clinical Study Data
Result summaries of Bayer's sponsored clinical trials in drug development Phases 2, 3, and 4 
and Phase 1 studies in subjects are provided in the Bayer Trial Finder application after 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  45 of 59
marketing authorization approval in line with the position of the global pharmaceutical 
industry associations laid down in the "Joint Position on the Disclosure of Clinical Trial 
Information via Clinical Trial Registries and Databases."  In addition, results of clinical drug 
trials will be provided on the publicly funded website www.ClinicalTrials.gov and EU 
Clinical Trials Register in line with the applicable regulations.
Bayer commits to sharing upon request from qualified scientific and medical researchers 
subject-level clinical trial data, study-level clinical trial data, and protocols from clinical trials 
in subjects for medicines and indications approved in the United States (US) and European 
Union (EU) on or after 01 JAN 2014 as necessary for conducting legitimate research. 
All Bayer-sponsored clinical trials are considered for publication in the scientific literature 
irrespective of whether the results of the clinical trials are positive or negative.
10.1.7
Data Quality Assurance

All subject data relating to the study will be recorded in an eCRF unless transmitted to 
the sponsor or designee electronically (eg, laboratory data). The investigator is 
responsible for verifying that data entries are accurate and correct by electronically 
signing the eCRF. 

The investigator must maintain accurate documentation (source data) that supports the 
information entered in the eCRF. 

The investigator must permit study-related monitoring, audits, IRB/IEC review, and 
regulatory agency inspections and provide direct access to source data documents. 

The sponsor or designee is responsible for the data management of this study including 
quality checking of the data. 

The sponsor assumes accountability for actions delegated to other individuals (eg, 
Contract Research Organizations).

Study monitors will perform ongoing source data verification to confirm that data 
entered into the eCRF by authorized site personnel are accurate, complete, and 
verifiable from source documents; that the safety and rights of subjects are being 
protected; and that the study is being conducted in accordance with the currently 
approved protocol and any other study agreements, ICH GCP, and all applicable 
regulatory requirements. 

Records and documents, including signed ICFs, pertaining to the conduct of this study 
must be retained by the investigator for 25 years after study completion unless local 
regulations or institutional policies require a longer retention period. No records may 
be destroyed during the retention period without the written approval of the sponsor. 
No records may be transferred to another location or party without written notification 
to the sponsor. 
10.1.8
Source Documents

Source documents provide evidence for the existence of the subject and substantiate 
the integrity of the data collected. Source documents are filed at the investigator’s site.

The site must implement processes to ensure availability of all required source 
documentation. A source document checklist (not part of this protocol) will be used at 
the site to identify the source data for key data points collected and the monitor will 
work with the site to complete this.  It is the expectation of the sponsor that all data 
have source documentation available at the site.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  46 of 59

Data entered in the eCRF that are transcribed from source documents must be 
consistent with the source documents or the discrepancies must be explained. The 
investigator may need to request previous medical records or transfer records, 
depending on the study. Also, current medical records must be available.

Definition of what constitutes source data can be found in ICH-GCP guidelines 
E6(R2) § 1.51, 1.52.
10.1.9
Study and Site Closure
The sponsor designee reserves the right to close the study site or terminate the study at any 
time for any reason at the sole discretion of the sponsor. Study sites will be closed upon study 
completion. A study site is considered closed when all required documents and study supplies 
have been collected and a study-site closure visit has been performed.
The investigator may initiate study-site closure at any time, provided there is reasonable cause 
and sufficient notice is given in advance of the intended termination.
Reasons for the early closure of a study site by the sponsor or investigator may include but are 
not limited to:

Failure of the investigator to comply with the protocol, the requirements of the 
IRB/IEC or local health authorities, the sponsor's procedures, or GCP guidelines

Inadequate recruitment of subjects by the investigator

Discontinuation of further study intervention development
10.2
Appendix 2: Aflibercept IVT Administration
IVT injections must be carried out according to medical standards and applicable guidelines 
by a qualified ophthalmologist experienced in administering IVT injections. After opening the 
vial, all preparation steps have to take place under aseptic conditions.
The supplied kit includes a vial of aflibercept 40 mg/mL and an 18-gauge filter needle. The
procedure also requires ancillary components including a 30-gauge injection needle with up to 
13 mm length and a 1-mL Luer-lock syringe.
Aflibercept is formulated as a sterile liquid to a final concentration of 40 mg/mL. The volume 
of injection is 10 L (0.01 mL) for the 0.4 mg dose of aflibercept.  The study drug will be 
withdrawn using aseptic technique through the filter needle attached to the syringe.  The filter
needle will be discarded after withdrawal of the vial contents and is not used for IVT 
injection.  The injection needle is attached to the syringe and the contents should be expelled 
until the plunger is aligned with the line that marks 0.01 mL on the syringe. Special care 
should be taken to ensure accurate alignment of the plunger with the 0.01 mL dose mark, and 
use of magnification equipment such as surgical microscope or an indirect ophthalmoscopy 
lens is recommended. The injection of aflibercept must be completed within 2 hours of the 
start of dose preparation.
The IVT injection will be given following topical anesthesia, sedation or general anesthesia, 
with appropriate respiratory support as required (eg, laryngeal mask, endotracheal intubation 
or similar), and administered according to standard procedures for IVT injection adapted for 
ROP infants. Administration is recommended to be carried out in an inpatient surgical room, 
or in a clean room (as defined by local infection control regulations).  In case both eyes 
require treatment, the procedures should be kept as independent as possible. Each vial should 
only be used for the treatment of a single eye. Each eligible eye will receive an IVT injection 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  47 of 59
of 0.4 mg (0.01 mL) of aflibercept.  This drug administration protocol is based on 
recommendations from the Euretina Expert Consensus (Grzybowski et al, 2018). Detailed 
recommendations are provided below. 
1. Apply topical anesthetic.
2. Apply povidone-iodine ophthalmic solution to eyelid margins, eyelashes, and 
conjunctival surface.
Povidone iodine is the agent of choice for subjects without an iodine allergy.  Of note, 
subjects with an iodine allergy must receive an agent other than povidone iodine. A 
disinfecting agent must be used; however, this can be povidone iodine or another agent, 
according to local clinical standard and the investigator’s decision.
3. Place 1 or 2 drops of povidone-iodine (or alternative agent for subjects with iodine 
allergy) ophthalmic solution on the ocular surface at the intended injection site.
4. Drape.
5. Apply an additional drop of povidone-iodine (or alternative agent for subjects with 
iodine allergy) ophthalmic solution to the site of injection.
6. Sterilized forceps and calipers (speculum) should be used to stabilize the globe and 
measure the injection site; the entry site of the needle for the IVT injection should be 
1.0-2.0 mm from the limbus, usually in the inferotemporal quadrant. Other quadrants 
can be used depending on easy of access.
7. Insert needle at marked injection point. The relative size of the lens in premature 
infants is larger than in adults, therefore the needle should be directed to the posterior 
pole, to avoid damaging the lens with the needle. The needle should only be 
introduced to the depth require to administer the injection (eg, half the length of a 
13 mm needle). It should not be completely introduced into the eye to avoid potential 
trauma to the posterior pole.
8. Gently inject study drug.
9. As the needle is withdrawn, a sterile cotton tip applicator should be rolled over the 
entry site to minimize the risk of drug reflux.  This should be held in place for 
10 seconds.
After the injection, any unused product must be discarded.  
10.2.1
Use of Topical Antibiotic Agents
The use of topical antibiotics as prophylaxis in IVT injections, both in the preparation and 
postinjection varies considerably between different practices. There is no consensus on the use 
of topical antibiotics, the agent to be used and the dose to be administered, especially in 
pediatric settings.  Additionally, treatment should performed be as soon as possible, and the 
injection date should not be postponed to allow antibiotic prophylaxis. If time allows, it is 
recommended that a broad spectrum topical antibiotic be used as part of the preparation for 
the IVT injection, as prophylaxis, in the days immediately preceding and following the 
injection.
Suggested use:
The injection date should be as early as possible, and not be postponed to allow any 
prophylaxis. Cases of infection (eg, conjunctivitis) must be adequately treated before the 
injection is performed. In case the injection date allows, treatment with 1 to 2 drops of 
antibiotic to both eyes 3 times a day can be initiated up to 3 days before the injection day.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  48 of 59
On the injection day, as part of the preparation for injection, instill 1 to 2 drops to the eye 
1 hour before the injection, and another drop 15 minutes before the injection. 
After the injection, administer 1 to 2 drops of antibiotic to the treated eye(s). Subsequently,
administer 1 to 2 drops of the topical antibiotic to the injected eye(s), 3 times a day, for an 
additional 3 days.
10.2.2
Postinjection Examinations
No further special precautions are required for a subject who has had an uneventful recovery 
from IVT injection. In the days following the IVT injection, physicians and nursing staff 
should be instructed to report any symptoms suggestive of endophthalmitis (eg, redness, 
irritation of the eye, ocular discharge, lid swelling, photophobia) without delay.
Cases of postinjection AEs requiring additional monitoring should remain under observation 
and be treated according to the investigator’s medical judgment.
Optic nerve and retina
Visualize the optic nerve to verify reperfusion of the central retinal artery in the immediate 
postinjection period.  Verify that the retina is attached and that there is no new intraocular 
hemorrhage.
Intraocular pressure 
Immediately following the IVT injection, subjects should be monitored for elevation in IOP.  
Appropriate monitoring may consist of tonometry and/or a check for perfusion at the optic 
nerve head.  Sterile equipment for anterior chamber paracentesis should be available, to allow 
immediate performance if required (refer to further guidance below).  If a tonopen is used to 
check pressure, a clean tonopen condom should be placed on the tip before taking each 
measurement.  Similarly, if a rebound tonometry device is used, a new, clean tip should be 
used.  If applanation tonometry is used, the applanator tip should be swabbed with alcohol and 
let to dry before using it to measure IOP.  If required, IOP may be lowered by pharmaceutical 
or surgical intervention.  Treatment should be initiated whenever IOP is increased to the 
extent that the central retinal artery remains closed for more than 1 to 2 minutes.
Paracentesis should be used only in extreme circumstances when the degree of pressure 
elevation poses an imminent and irreversible threat to vision.  In the rare situation when a 
paracentesis is warranted, IOP should be recorded before and after the procedure.  A 0.1-mL 
paracentesis may be performed using a 30-gauge needle if judged necessary by the 
investigator.
IOP must be measured in injected eyes approximately 30 minutes following the injection if 
not done immediately postinjection or when the measurement was abnormal immediately 
postinjection. An abnornal value will be monitored closely until it returns to normal.  If IOP 
was measured immediately after the injection and the value was normal, it does not have to be 
repeated.
Record all IOP measurements and related treatments in the source documents and the 
appropriate pages of the eCRF.
10.3
Appendix 3: Clinical Laboratory Tests
The tests detailed in Table 10–1will be performed and reported by the local laboratory.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  49 of 59
Additional tests may be performed at any time during the study as determined necessary by 
the investigator or required by local regulations. 
Positive results of any prenatal congenital infectious disease screening test (eg, toxoplasmosis, 
syphilis, varicella-zoster, parvovirus B19, rubella, cytomegalovirus [CMV], and herpes 
infections), if available will be recorded as medical history. The results of any serological test 
performed in order to confirm these diseases will be also recorded.
Table 10–1: Protocol-Required Safety Laboratory Assessments
Laboratory 
Assessments
Parameters
Hematology
Platelet count
White blood cell count
Differential (neutrophils, lymphocytes, 
monocytes, eosinophils, basophils) if possible
Red blood cell count
Hemoglobin
Hematocrit
Clinical 
Chemistry
Blood urea 
nitrogen 
Potassium
Aspartate 
aminotransferase 
(AST)/serum glutamic-
oxaloacetic transaminase 
(SGOT)
Total bilirubin
Creatinine
Sodium
Alanine aminotransferase 
(ALT)/serum glutamic-
pyruvic transaminase 
(SGPT)
Total protein
albumin
Glucose 
(nonfasting)
Routine 
Urinalysis
Specific gravity
pH, glucose, protein, blood, ketones, bilirubin, urobilinogen, nitrite, leukocyte 
esterase
Microscopic examination (if blood or protein is abnormal)
Other Screening 
Tests
Any congenital infectious disease test performed during pregnancy that may 
lead to a subject infection should be recorded as medical history.
Investigators must document their review of each laboratory safety report.
10.4
Appendix 4: Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting
10.4.1
Definition of AE 
AE Definition

An AE is any untoward medical occurrence in a subject or clinical study subject, 
associated with the use of study intervention, whether or not considered related to 
the study intervention.

NOTE: An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease (new or exacerbated) associated 
with the use of study intervention.
Events Meeting the AE Definition 

Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) 
or other safety assessments (eg, ECG, radiological scans, vital signs measurements), 
including those that worsen from baseline, considered clinically significant in the 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  50 of 59
medical and scientific judgment of the investigator.

Exacerbation of a chronic or intermittent pre-existing condition including either an 
increase in frequency and/or intensity of the condition.

New conditions detected or diagnosed after study intervention administration even 
though it may have been present before the start of the study.

Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction.

Signs, symptoms, or the clinical sequelae of a suspected overdose of either study 
intervention or a concomitant medication. 

“Lack of efficacy” or “failure of expected pharmacological action” per se will not be 
reported as an AE or SAE. Such instances will be captured in the efficacy 
assessments. However, the signs, symptoms, and/or clinical sequelae resulting from 
lack of efficacy will be reported as AE or SAE if they fulfil the definition of an AE 
or SAE. 
Events NOT Meeting the AE Definition 

Any clinically significant abnormal laboratory findings or other abnormal safety 
assessments that are associated with the underlying disease, unless judged by the 
investigator to be more severe than expected for the subject’s condition.

The disease/disorder being studied or expected progression, signs, or symptoms of 
the disease/disorder being studied, unless more severe than expected for the 
subject’s condition.

Medical or surgical procedure (eg, endoscopy, appendectomy): the condition that 
leads to the procedure is the AE.

Situations in which an untoward medical occurrence did not occur (social and/or 
convenience admission to a hospital).

Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present 
or detected at the start of the study that do not worsen.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  51 of 59
10.4.2
Definition of SAE
An SAE is defined as any untoward medical occurrence that, at any dose:
a.
Results in death
b.
Is life-threatening

The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
subject was at risk of death at the time of the event. It does not refer to an event, 
which hypothetically might have caused death, if it were more severe.
c.
Requires inpatient hospitalization or prolongation of existing hospitalization

In general, hospitalization signifies that the subject has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for 
observation and/or treatment that would not have been appropriate in the physician’s 
office or outpatient setting. Complications that occur during hospitalization are AEs. 
If a complication prolongs hospitalization or fulfills any other serious criteria, the 
event is serious. When in doubt as to whether “hospitalization” occurred or was 
necessary, the AE should be considered serious.

Hospitalization for elective treatment of a pre-existing condition that did not worsen 
from baseline is not considered an AE.
d.
Results in persistent disability/incapacity

The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions.

This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 
and accidental trauma (eg, sprained ankle) that may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption.
e.
Is a congenital anomaly/birth defect
f.
Other situations:

Medical or scientific judgment should be exercised in deciding whether SAE
reporting is appropriate in other situations such as important medical events that 
may not be immediately life-threatening or result in death or hospitalization but may 
jeopardize the subject or may require medical or surgical intervention to prevent one 
of the other outcomes listed in the above definition. These events should usually be 
considered serious.

Examples of such events include invasive or malignant cancers, intensive treatment 
in an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency 
or drug abuse.
10.4.3
Recording and Follow-Up of AE and/or SAE 
AE and SAE Recording


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  52 of 59

When an AE/SAE occurs, it is the responsibility of the investigator to review all 
documentation (eg, hospital progress notes, laboratory reports, diagnostics reports) 
related to the event.

The investigator will record all relevant AE/SAE information in the eCRF.

It is not acceptable for the investigator to send photocopies of the subject’s medical 
records to the sponsor in lieu of completion of the AE/SAE eCRF pages.

There may be instances when copies of medical records for certain cases are 
requested by the sponsor. In this case, all subject identifiers, with the exception of 
the subject number, will be redacted on the copies of the medical records before 
submission.

The investigator will attempt to establish a diagnosis of the event based on signs, 
symptoms, and/or other clinical information. Whenever possible, the diagnosis (not 
the individual signs/symptoms) will be documented as the AE/SAE.
Assessment of Intensity

The investigator will make an assessment of intensity for each AE and SAE reported 
during the study and assign it to 1 of the following categories: 

Mild: An event that is easily tolerated by the subject, causing minimal 
discomfort and not interfering with everyday activities.

Moderate: An event that causes sufficient discomfort and interferes with normal 
everyday activities.

Severe: An event that prevents normal everyday activities. An AE that is 
assessed as severe should not be confused with an SAE. Severe is a category 
utilized for rating the intensity of an event; and both AEs and SAEs can be 
assessed as severe.

An event is defined as ‘serious’ when it meets at least 1 of the predefined outcomes 
as described in the definition of an SAE, NOT when it is rated as severe.
Assessment of Causality

The investigator is obligated to assess the relationship between study intervention 
and each occurrence of each AE/SAE.

A “reasonable possibility” of a relationship conveys that there are facts, evidence, 
and/or arguments to suggest a causal relationship, rather than a relationship cannot 
be ruled out.

The investigator will use clinical judgment to determine the relationship.

Alternative causes, such as underlying disease(s), concomitant therapy, and other 
risk factors, as well as the temporal relationship of the event to study intervention 
administration will be considered and investigated.

The investigator will also consult the Investigator’s Brochure (IB) and/or Product 
Information, for marketed products, in his/her assessment.

For each AE/SAE, the investigator must document in the medical notes that he/she 
has reviewed the AE/SAE and has provided an assessment of causality.

There may be situations in which an SAE has occurred and the investigator has 
minimal information to include in the initial report to the sponsor. However, it is 


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  53 of 59
very important that the investigator always make an assessment of causality for 
every event before the initial transmission of the SAE data.

The investigator may change his/her opinion of causality in light of follow-up 
information and send an SAE follow-up report with the updated causality 
assessment.

The causality assessment is one of the criteria used when determining regulatory 
reporting requirements.
Follow-up of AEs and SAEs

The investigator is obligated to perform or arrange for the conduct of supplemental 
measurements and/or evaluations as medically indicated or as requested by the 
sponsor to elucidate the nature and/or causality of the AE or SAE as fully as 
possible. This may include additional laboratory tests or investigations, 
histopathological examinations, or consultation with other health care professionals.

If a subject dies during participation in the study or during a recognized follow-up 
period, the investigator will provide the sponsor with a copy of any post mortem 
findings including histopathology.

New or updated information will be recorded in the originally completed eCRF.

The investigator will submit any updated SAE data to the sponsor within 24 hours of 
receipt of the information.
10.4.4
Reporting of SAEs
SAE Reporting to the Sponsor via an Electronic Data Collection Tool

The primary mechanism for reporting an SAE to the sponsor will be the electronic 
data collection tool.

If the electronic system is unavailable, then the site will use the paper SAE data 
collection tool (see next section) in order to report the event within 24 hours.

The site will enter the SAE data into the electronic system as soon as it becomes 
available.

After the study is completed at a given site, the electronic data collection tool will be 
taken off-line to prevent the entry of new data or changes to existing data.

If a site receives a report of a new SAE from a study subject or receives updated data 
on a previously reported SAE after the electronic data collection tool has been taken 
off-line, then the site can report this information on a paper SAE form (see next 
section).

Contacts for SAE reporting can be found in the investigator site file.
SAE Reporting to the Sponsor via Paper CRF

Facsimile transmission of the SAE paper CRF is the preferred method to transmit 
this information to the sponsor.

In rare circumstances and in the absence of facsimile equipment, notification by 
telephone is acceptable with a copy of the SAE data collection tool sent by overnight 
mail or courier service.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  54 of 59

Initial notification via telephone does not replace the need for the investigator to 
complete and sign the SAE CRF pages within the designated reporting time frames.

Contacts for SAE reporting can be found in the investigator site file.
10.5
Appendix 5: Medical Device Incidents: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting 
The detection and documentation procedures described apply to the sponsor medical devices
provided for use in the study (see Section 6.1.1).
Medical Device Incident/SAE/AE Definitions

An incident is any malfunction or deterioration in the characteristics or performance 
of a device made available on the market, including use-error due to ergonomic 
features as well as any inadequacy in the information supplied by the manufacturer 
and any undesirable side effect.

An AE is any untoward medical occurrence, unintended disease or injury or any 
untoward clinical signs, including an abnormal laboratory finding, in subjects, users 
or other persons, in the context of a clinical investigation, whether or not related to 
the investigational device.

An SAE is any AE that leads to any of the following:
(a) death
(b) serious deterioration in the health of the subject, that resulted in any of the 
following:
(i) life-threatening illness or injury
(ii) permanent impairment of a body structure or a body function
(iii) hospitalization or prolongation of subject hospitalization
(iv) medical or surgical intervention to prevent life-threatening illness or injury 
or permanent impairment to a body structure or a body function
(v) chronic disease
(c) fetal distress, fetal death or a congenital physical or mental impairment or birth 
defect

A device deficiency is any inadequacy in the identity, quality, durability, reliability, 
safety or performance of an investigational device, including malfunction, use errors 
or inadequacy in information supplied by the manufacturer.

Not all incidents lead to death or serious deterioration in health. The nonoccurrence 
of such a result might have been due to other fortunate circumstances or to the 
intervention of health care personnel.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  55 of 59
Examples of Incidents

A subject, user, caregiver, or healthcare professional is injured as a result of a 
medical device failure or its misuse.

A subject’s study intervention is interrupted or compromised by a medical device 
failure.

A misdiagnosis due to medical device failure leads to inappropriate treatment.

A subject’s health deteriorates due to medical device failure.
Medical Device Incident Documenting

Any medical device incident/device deficiency occurring during the study will be 
documented in the subject’s medical records, in accordance with the investigator’s 
normal clinical practice, and on the Device Event Form of the eCRF and on the 
Product Technical Complaint form for any technical issues identified with the 
device.

For incidents fulfilling the definition of an AE or an SAE, the appropriate AE/SAE 
CRF page will be completed as described in Section 10.4.  The CRF will be 
completed as thoroughly as possible and signed by the investigator before 
transmittal to the sponsor or designee.

It is very important that the investigator provides his/her assessment of causality 
(relationship to the medical device provided by the sponsor) at the time of the initial 
AE or SAE report and describes any corrective or remedial actions taken to prevent 
recurrence of the incident.

A remedial action is any action other than routine maintenance or servicing of a 
medical device where such action is necessary to prevent recurrence of an incident. 
This includes any amendment to the device design to prevent recurrence.
10.6
Appendix 6: Abbreviations
ADA
anti-drug antibodies
AE
adverse event
AFL
aflibercept 
AG
Aktiengesellschaft
ALT
alanine aminotransferase
AP-ROP
aggressive posterior retinopathy of prematurity
AST
aspartate aminotransferase
ATE
arterial thrombotic event
BEAT-ROP
Bevacizumab Eliminates the Angiogenic Threat of Retinopathy of 
Prematurity trial 
BL
Baseline
BP
blood pressure
BW
body weight
CE
Conformité Européenne
CFR
Code of Federal Regulations
CIOMS
Council for International Organizations of Medical Sciences
CMV
Cytomegalovirus


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  56 of 59
CNS
central nervous system
CNV
choroidal neovascularization
CONSORT
Consolidated Standards of Reporting Trials
CRF
case report form
CSR
clinical study report
D
day(s)
DMC
Data Monitoring Committee
DOB
date of birth
ECG
Electrocardiogram
eCRF
electronic case report form
EOS
end of study
EU
European Union
EudraCT
European Clinical Trials Database
FAS
full analysis set
FDA
Food and Drug Administration
FUP
follow-up
GCP
Good Clinical Practice
HIPPA
Health Insurance Portability and Accountability Act
IB
Investigator’s Brochure
ICF
informed consent form
ICH
International Council for Harmonisation
IEC
Independent Ethics Committee
IOP
intraocular pressure
IRB
Institutional Review Board
IVRS
Interactive Voice Response System 
IVT
Intravitreal
IWRS
Interactive Web Response System
MedDRA
Medical Dictionary for Regulatory Activities
mFAS
modified full analysis set
MRI
magnetic resonance imaging
NAB
neutralizing antibodies
NI
non-inferiority
NICU
neonatal intensive care unit
PK
pharmacokinetic(s)
PPS
per protocol set
RAINBOW 
Study
RAnibizumab Compared With Laser Therapy for the Treatment of 
INfants BOrn Prematurely With Retinopathy of Prematurity
RC
reading center
ROP
retinopathy of prematurity
SAE
serious adverse event
SAF
safety analysis set
SAP
statistical analysis plan
SCR
Screening
SGOT
serum glutamic-oxaloacetic transaminase
SGPT
serum glutamic-pyruvic transaminase (SGPT)
SoA
schedule of activities
TEAE
treatment-emergent adverse event
TORCH
toxoplasma gondii, other viruses, rubella, cytomegalovirus, and 
herpes simplex


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  57 of 59
TRT
Treatment
US
United States
VEGF
vascular endothelial growth factor
VEGF-A
vascular endothelial growth factor A
VEGFR1
vascular endothelial growth factor receptor 1
VEGFR2
vascular endothelial growth factor receptor 2
w/wk
Week
10.7
Appendix 7: Protocol Amendment History
The Protocol Amendment Summary of Changes Table for the current amendment is located 
directly before the Table of Contents (TOC).
11.
References
Antithrombotic Trialists’ Collaboration. Collaborative meta-analysis of randomised 
trials of antiplatelet therapy for prevention of death, myocardial infarction, 
and stroke in high risk patients. Br Med J. 2002;324:71-86.
Blencowe H, Lee AC, Cousens S, Bahalim A, Narwal R, Zhong N, Chou D, Say L, 
Modi N, Katz J, Vos T, Marlow N, Lawn JE. Preterm birth-associated 
neurodevelopmental impairment estimates at regional and global levels for 
2010. Pediatr Res. 2013;74 Suppl 1:17-34.
Gilbert C, Rahi J, Eckstein M, O'Sullivan J, Foster A. Retinopathy of prematurity in 
middle-income countries. The Lancet. 1997;350(9070):12-14.
Grzybowski A, Told R, Sacu S, Bandello F, Moisseiev E, Loewenstein A, Schmidt-
Erfurth U, on behalf of the Euretina Board.  2018 Update on intravitreal 
injections:  Euretina expert consensus recommendations.  Ophthalmologica.
2018;239:181-193.
Harder BC, Schlichtenbrede FC, Von Baltz S, Jendritza W, Jendritza B, Jonas JB.  
Intravitreal bevacizumab for retinopathy of prematurity: refractive error 
results.  Am J Ophthalmol. 2013 Jun;155(6):1119-1124.
Hartnett ME. Vascular endothelial growth factor antagonist therapy for retinopathy 
of prematurity. Clin Perinatol. 2014;41:925-943.
Hartnett ME, Morrison MA, Smith S, Yanovitch TL, Young TL, Colaizy T, 
Momany A,  Dagle J, Carlo WA, Clark EAS, Page G, Murray J, DeAngelis 
MM, Cotton CM for the Genomics Subcommittee.  Genetic variants 
associated with severe retinopathy of prematurity in extremely low birth 
weight infants.  Invest Ophthalmol Vis Sci. 2014 Oct;55(10):6194-6203.
Hellström A, Smith LEH,  Dammann O. Retinopathy of prematurity. Lancet. 
2013;382(9902):1445-1457.
Howie, Stephen RC. Blood sample volumes in child health research: review of safe 
limits. Bulletin of the World Health Organization, 2011;89 (1), 46 - 53.
Huang CY, Lien R, Wang NK, Chao AN, Chen KJ, Chen TL, Hwang YS, Lai CC, 
Wu WC. Changes in systemic vascular endothelial growth factor levels after 
intravitreal injection of aflibercept in infants with retinopathy of prematurity. 
Graefes Arch Clin Exp Ophthalmol. 2018;256, 479-487.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  58 of 59
Hwang CK, Baker Hubbard G, Hutchinson AK, Lambert SR.  Outcomes after 
intravitreal bevacizumab versus laser photocoagulation for retinopathy of 
prematurity: a 5-year retrospective analysis.  Ophthalmology. 2015 
May;122(5):1008-1015.
Mintz-Hittner HA, Kennedy KA, Chuang AZ. Efficacy of intravitreal bevacizumab 
for stage 3+ retinopathy of prematurity. N Engl J Med. 2011;364(7):603-15. 
Mutlu FM, Sarici SU. Treatment of retinopathy of prematurity: a review of 
conventional and promising new therapeutic options. Int J Ophthalmol. 
2013;6:228-236.
Painter SL, Wilkinson AR, Desai P, Goldacre MJ, Patel CK. Incidence and 
treatment of retinopathy of prematurity in England between 1990 and 2011:
database study. British Journal of Ophthalmology. 2015;99(6):807.
Pertl L, Steinwender G, Mayer C, Hausberger S, Poschl EM, Wackernagel W, El-
Shabrawi Y, Haas A. A Systematic Review and Meta-Analysis on the Safety 
of Vascular Endothelial Growth Factor (VEGF) Inhibitors for the Treatment 
of Retinopathy of Prematurity. PLoS One. 2015;10:e0129383.
Plummer M. rjags: Bayesian Graphical Models using MCMC. R package version 4-
6. 2016. Available from:  https://CRAN.R-project.org/package=rjags.
Recommendations of the expert group on clinical trials for the implementation of 
Regulation (EU) No 536/2014 on clinical trials on medicinal products for 
human use. Revision 1. 18 September 2017. Ethical considerations for 
clinical trials on medicinal products conducted with minors. Available from: 
https://ec.europa.eu/health/sites/health/files/files/eudralex/vol-
10/2017_09_18_ethical_consid_ct_with_minors.pdf (Accessed 08-June 
2020).
R Core Team. R: A language and environment for statistical computing. R 
Foundation for  Statistical Computing, Vienna, Austria. 2016.  Available 
from: https://www.R-project.org/.
Salman AG, Said AM. Structural, visual and refractive outcomes of intravitreal 
aflibercept injection in high-risk prethreshold type 1 retinopathy of 
prematurity. Ophthalmic Res. 2015;53:15-20.
Sidorenko EE.  Treatment of retinopathy of prematurity with aflibercept in Russia.  
Presented at: 17th International Conference on Clinical and Experimental 
Ophthalmology.  2018 Oct 01-03. Moscow, Russia. Available from:  
https://ophthalmology.conferenceseries.com/abstract/2018/treatment-of-
retinopathy-of-prematurity-with-aflibercept-in-russia 
Smith LEH, Hellström A, Stahl A, Fielder A, Chambers W, Moseley J, Toth C, 
Wallace D, Darlow B, Aranda J, Hallberg B, Davis JM, for the Retinopathy 
of Prematurity Workgroup of the International Neonatal Consortium.  
Development of a retinopathy of prematurity activity scale and clinical 
outcome measures for use in clinical trials. JAMA Ophthalmol.  
2019;137:305-11.


CONFIDENTIAL
Integrated Clinical Study Protocol
BAY 86-5321 / 20090
23 JUN 2020
Version 2.0
Page:  59 of 59
Stone J, Chan-Ling T, Pe'er J, Itin A, Gnessin H, Keshet E.  Roles of Vascular 
Endothelial Growth Factor and Astrocyte Degeneration in the Genesis of 
Retinopathy of Prematurity. Invest Ophthalmol Vis Sci. 1996;37:290-99.
Sukgen EA, Kocluk Y. Comparison of clinical outcomes of intravitreal ranibizumab 
and aflibercept treatment for retinopathy of prematurity. Graefe’s Archive for 
Clinical and Experimental Ophthalmology.  Revised 2018 Apr 16.  Available 
from: https://doi.org/10.1007/s00417-018-4168-5.
The Royal College of Ophthalmologists.  Intravitreal injection therapy. Revised 
August 2018. Available from: https://www.rcophth.ac.uk/wp.../Intravitreal-
Injection-Therapy-August-2018-2.pdf.
Young TL, Anthony DC, Pierce E, Foley E, Smith LE.  Histopathology and vascular 
endothelial growth factor in untreated and diode laser-treated retinopathy of 
prematurity.  J AAPOS. 1997;1:105-10.

SAP_001.pdf: 
 
Document Type: 
Statistical Analysis Plan 
Official Title: 
Open-label, randomized, two-arm, controlled study to assess the 
efficacy, safety, and tolerability of intravitreal (IVT) aflibercept 
compared to laser photocoagulation in patients with retinopathy of 
prematurity (ROP) 
NCT Number: 
NCT04004208 
Document Date: 
10 MAY 2021 
 


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 1 of 30
Title page
Open-label, randomized, two-arm, controlled study to assess the efficacy, 
safety, and tolerability of intravitreal (IVT) aflibercept compared to laser 
photocoagulation in patients with retinopathy of prematurity (ROP)
Aflibercept for ROP – IVT injection versus laser therapy (FIREFLEYE)
Bayer study drug
BAY 86-5321 / Aflibercept 
Study purpose:
Primarily to collect data on the efficacy and safety outcomes in 
patients after treatment of ROP with aflibercept versus laser surgery  
Clinical study 
phase:
3
Date:
10 MAY 2021
Study No.:
20090
Version:
4.0
Author:
Confidential
The information provided in this document is strictly confidential and is intended 
solely for the guidance of the clinical investigation.  Reproduction or disclosure of this 
document, whether in part or in full, to parties not associated with the clinical 
investigation or its use for any other purpose without the prior written consent of the 
sponsor is not permitted.
Throughout this document, symbols indicating proprietary names (®, TM) are not displayed.  Hence, 
the appearance of product names without these symbols does not imply that these names are not 
protected.
This Statistical Analysis Plan is produced on a word-processing system and bears no signatures.
The approval of the Statistical Analysis Plan is documented in a separate Signature Document.
PPD
PPD


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 2 of 30
Table of Contents
Title page................................................................................................................................... 1
Abbreviations............................................................................................................................ 3
1.
Introduction....................................................................................................................... 3
2.
Study Objectives................................................................................................................ 4
3.
Study Design ...................................................................................................................... 4
4.
General Statistical Considerations .................................................................................. 5
4.1
General Principles............................................................................................................ 5
4.2
Handling of Dropouts ...................................................................................................... 5
4.3
Handling of Missing Data................................................................................................ 6
4.4
Interim Analyses and Data Monitoring ........................................................................... 7
4.5
Data Rules........................................................................................................................ 7
4.6
Blind/Masked Review...................................................................................................... 8
5.
Analysis Sets ...................................................................................................................... 8
5.1
Assignment of Analysis Sets ........................................................................................... 8
6.
Statistical Methodology .................................................................................................... 9
6.1
Population Characteristics ............................................................................................... 9
6.1.1
Sample Sizes, Subject Validity Status and Subject Disposition................................... 9
6.1.2
Demography, Baseline Characteristics and Medical History..................................... 10
6.1.3
Exposure and Compliance to Study Treatment .......................................................... 11
6.1.4
Prior/Concomitant Medication ................................................................................... 11
6.1.5
COVID-19 pandemic-related assessments................................................................. 12
6.2
Efficacy.......................................................................................................................... 12
6.2.1
Primary Efficacy Endpoint ......................................................................................... 12
6.2.2
Secondary Efficacy Endpoints.................................................................................... 18
6.2.3
Other Pre-Specified Efficacy Endpoints..................................................................... 20
6.2.4
Subgroup Analyses..................................................................................................... 23
6.2.5
Other exploratory analyses ......................................................................................... 23
6.3
Pharmacokinetics/Pharmacodynamics........................................................................... 24
6.4
Safety ............................................................................................................................. 26
6.4.1
Adverse Events, Serious Adverse Events, and Device Events................................... 26
6.4.2
Surgical Procedures after Study Start......................................................................... 27
6.4.3
Ophthalmic Examinations .......................................................................................... 27
6.4.4
Physical Examinations and Vital Signs...................................................................... 27
6.4.5
Laboratory Safety Variables....................................................................................... 27
6.4.6
Central Nervous System Imaging............................................................................... 27
6.5
Development of Anti-drug Antibodies .......................................................................... 28
7.
Document History and Changes in the Planned Statistical Analysis ......................... 29
8.
References ........................................................................................................................ 29
9.
Appendix.......................................................................................................................... 30
9.1
Multiple imputation approach for missing data in primary efficacy analysis ............... 30


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 3 of 30
Abbreviations
ADAs
Anti-drug antibodies
AE
Adverse event
AP-ROP
aggressive posterior ROP
CRF
Case Report Form
CSP
Clinical Study Protocol
DMC
Data Monitoring Committee
FAS
Full Analysis Set
ICROP
International Classification of Retinopathy of Prematurity
IOP
intraocular pressure
IVT
intravitreal
LLOQ
Lower Limit Of Quantification
LOCF
Last Observation Carried Forward
MedDRA
Medical Dictionary for Regulatory Activities
mFAS
Modified Full Analysis Set
PKS
Pharmacokinetic Analysis Set
PMDA
Pharmaceuticals and Medical Devices Agency
PPS
Per-protocol set
PT
Preferred term
PTP
Planned Timepoint
RC
Reading Center
ROP
Retinopathy of Prematurity
SAE
Serious adverse event
SAF
Safety Analysis Set
SAP
Statistical Analysis Plan
SD
Standard Deviation
SOC
System organ class
TEAE
treatment-emergent adverse event
TESAE
treatment-emergent serious adverse event
TLFs
Tables, listings, figures
WHO
World Health Organization 
1.
Introduction
The study is designed for providing evidence for efficacy, safety, and tolerability of IVT
aflibercept versus laser photocoagulation in subjects diagnosed with ROP.
ROP is a proliferative vascular retinopathy caused by an abnormal development of the 
vascularization of the peripheral retina in prematurely born infants. It affects mainly newborns 
with a preterm gestational age (≤ 32 weeks) and very low birth weight (≤1500 g).  ROP 
remains a major cause of childhood blindness globally.  
The present Statistical Analysis Plan (SAP) reflects the final statistical analyses to be 
performed for study 20090 and is based on the Integrated Clinical Study Protocol (CSP), 
Version 2.0, dated 23 JUN 2020 and the local protocol amendment for Japan (amendment 
number JPN-1), dated 09 MAY 2019. The aforementioned integrated CSP version 2.0 (23 
JUN 2020) was amended from version 1.0 (22 MAR 2019) for non-COVID 19-pandemic 
related reasons, i.e. in order to allow for additional collection of PK data beyond 4 weeks after 
initial aflibercept injection.


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 4 of 30
2.
Study Objectives
The primary objective of this study is to assess the efficacy of aflibercept in subjects 
diagnosed with ROP in comparison to laser.
The secondary objectives of this study are: 

To assess the safety and tolerability of aflibercept

To assess the treatment burden of aflibercept and laser 

To describe the systemic exposure to aflibercept
Further objectives are:

To characterize further aspects of the effect of aflibercept in the treatment of ROP

To further investigate the study intervention and similar drugs (ie, mode-of-action-
related effects and/or safety) and to further investigate pathomechanisms deemed 
relevant to diseases of the eye and associated health problems
3.
Study Design
This study is a prospective, multicenter, parallel-group, randomized, unmasked for study
treatment Phase 3 clinical study with two treatment arms comparing IVT administered 
aflibercept versus laser photocoagulation.
After one or two screening/baseline visit/s, eligible subjects will be randomized with a ratio of 
2:1 to treatment with either aflibercept injection or laser photocoagulation, respectively. After 
the initial treatment with a single injection of aflibercept per eligible eye for subjects 
randomized to the aflibercept arm or the initial treatment with laser photocoagulation for 
subjects randomized to the control arm (multiple sessions within 1 week to complete the 
procedure are allowed), a 23-week treatment period (including possible retreatment and 
rescue treatment), and a final visit at Week 24 (up to Week 27 for subjects treated after Week 
21) follow.
One or both eyes can be treated according to the investigator’s assessment of the study’s 
eligibility criteria. The second eye of subjects who start the study with only one eligible 
eyeshould be kept under observation. Second eyes which develop ROP requiring treatment 
during the study should receive treatment according to the randomization assignment of the 
first eye. Eyes treated as randomized before or on study visit 9 (≤8 weeks from baseline) will 
be included in the efficacy and safety analyses as appropriate, whereas those treated after visit 
9 (>8weeks from baseline) will be included in the safety analysis only.
Subjects will be stratified for randomization according to Japanese and Non-Japanese study 
sites as well as by ROP classification in Zone I, Zone II, or AP-ROP according to investigator 
assessment. If both eyes are treated at baseline, the eye with the more severe disease will be 
considered for stratification. To prevent imbalances, the randomization result of subjects who 
do not complete baseline treatment will be assigned to a subsequent subject once it is known 
that the baseline treatment was not completed.
If required, each treated eye can receive re-treatment (up to two times in the aflibercept arm at 
least 28 days after the previous injection). For subjects randomized to aflibercept, rescue 
treatment with laser may be performed.  For subjects randomized to laser, rescue treatment 
with aflibercept may be performed.  The criteria for the allowance of retreatment and rescue 
treatment are specified in the CSP.  


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 5 of 30
Database release will occur when all subjects have completed the Week 24 visit or have 
dropped out of the study.  In case of the scenario where a subject has a 30-day safety follow-
up visit after Week 24 or the final PK sample results come in later and these data can not be 
cleaned in time for the initial data release, these additional safety data will be included in a 
planned re-release of the database.
Definition of treatment completion: Subject had assessments of re-treatment / rescue criteria 
up to and including Week 23 and received all intended treatments (retreatment and rescue 
treatment) as prescribed in the study protocol. A subject is considered a treatment completer if 
treatment was completed for at least one eye.
Definition of study completion: Subject completed Week 24 or Week 27 if indicated. A 
subject is considered also as a completer of the study if the study intervention is discontinued 
and the subject continues only on observational basis for safety evaluations.
4.
General Statistical Considerations
4.1
General Principles
The statistical evaluation will be performed by using the software package SAS release 9.2 or 
higher (SAS Institute Inc., Cary, NC, USA). All variables will be analyzed by descriptive 
statistical methods. The number of data available and missing data, mean, standard deviation
(SD), minimum, quartiles, median, and maximum will be calculated for metric data. 
Frequency tables will be generated for categorical data.
Descriptive analyses will be shown where applicable

for all subjects 
and separately 

for subjects treated in both eyes (bilateral cases)

for subjects treated in one eye only (unilateral cases)
within the respective analysis set.
For time to event analysis, the start date will be the date of randomization (i.e. the baseline 
visit).
4.2
Handling of Dropouts
In case a subject qualifies for retreatment or rescue treatment and there is a decision against 
retreatment or rescue treatment and subjects permanently discontinue study interventions (ie, 
does not meet the definition of treatment completion, see Section 3), the subject should 
continue in the study on an observational basis for safety evaluation.
Subjects who prematurely discontinue the study (participant discontinuation/ withdrawal from 
study or in the case of lost to follow-up) or permanently discontinue the study treatment for 
any reason will not be replaced. 


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 6 of 30
The number of subjects who prematurely discontinue the study or permanently discontinue 
the study treatment for any reason will be reported and the corresponding reasons will be 
shown. Potential differences between the treatment groups in the proportion of patient 
withdrawals or in the timing of withdrawals will be evaluated using Kaplan-Meier plots for 
“Time to end of study” and “Time to permanently discontinuation of study treatment”. 
Potential dropout patterns with respect to 

ROP classification (Zone I vs. Zone II vs. AP-ROP)

Gestational age at birth (“<=26” vs “>26” weeks) and 

Number of aflibercept administrations / laser treatments or requirement for rescue 
treatment from baseline to Week 24 

For subjects affected by the COVID-19 pandemic (based on reported important  
protocol deviations) vs subjects not affected by the COVID-19 pandemic. 
will be described.
The details for the handling of missing data due to dropouts are described in Section 4.3.
4.3
Handling of Missing Data
All missing or partial data will be presented in the subject data listing as they are recorded on 
the Case Report Form (CRF). 
Additional descriptive analyses in the presence of missing data
The number, timing, and pattern for missing values of the primary endpoint will be displayed 
by means of descriptive statistics and visually displayed if applicable. Data exploration will 
include investigation of 

potential missing data imbalance between the two treatment groups and ROP 
classification (Zone I vs. Zone II vs. AP-ROP)

baseline characteristics of subjects with and without missing values

the direction of change over time with time courses of subjects with and without 
missing data. 
General rules
When appropriate, the following rules will be implemented so as not to exclude subjects from 
statistical analyses due to missing or incomplete dates:

Safety Variables                                                                                                                         
In general, missing data will not be imputed for the safety analysis. If dates of adverse 
events are missing or partially missing so that the determination of whether or not the 
event is treatment-emergent is questionable, the event will be presumed to be 
treatment-emergent.

Prior/concomitant medication data
For the tabulation of prior and concomitant medication, missing or partially missing 
start dates of the medication will be imputed by the earliest possible time point, 
missing or partially) missing stop dates will be imputed by the latest possible time 
point.


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 7 of 30

Date of discontinuation of study                                                                         
Partially missing dates will be imputed by the minimum approach, i.e., the earliest 
possible date.
Details for the handling of missing data for the efficacy analysis are described in Section 6.2.
4.4
Interim Analyses and Data Monitoring
No interim analysis is planned.  However, safety assessments will be continuously performed.
During the study, an independent Data Monitoring Committee (DMC) will perform regular 
safety assessments to determine if the study shows unacceptable risks for the subjects, and 
issue recommendations to proceed or terminate the study. Detailed information regarding the 
DMC procedures are explained in a separate DMC charter.
4.5
Data Rules
Definition of Screen Failure: A screen failure is any subject for whom informed consent was 
signed but did not complete the screening period and was therefore not randomized.
Definition of Baseline: Baseline assessment is defined as the latest, valid assessment 
available before first administration of study treatment (including unscheduled assessments).
Unscheduled assessments: Extra assessments (laboratory data or vital signs associated with 
non-protocol visits or obtained in the course of investigating or managing adverse events 
(AEs)) will be included in listings, but not in summaries. If more than one value is available
for an unscheduled assessment, all observations will be presented in the listings. 
Handling of repeated measurements at the same visit: If measurements were repeated at 
the same visit, the value actually used for statistical summaries and analyses will be the 

Last non-missing repeated measurement, if respective visit is before start of treatment, and

First non-missing repeated measurement, if respective visit is after start of treatment.
Handling of Week 24/ Early Termination Visit:
The study eCRF collects the final assessment per subject in a single visit folder. In case a 
subject completed the study, this information refers to Week 24, otherwise the information 
refers to the early discontinuation of a subject and will be assigned to an early termination
visit. The information is included in VISITNUM=700000. For the analysis visit (AVISITN), 
the information will be split into AVISITN=168 if the subject completed and into 
AVISITN=950 for subjects who discontinued early. 
Correction of AP-ROP definition based on central reading center (RC): The RC graded 
AP-ROP as:

YES, AGGRESSIVE NV WITHOUT RIDGE AND WITH PLUS

YES, ZONE I/II WITH STAGE II/III AND PLUS,
which can be identified in the data with PARAMCD=”ROPAGGR” and OEEVALN=27 in 
ADOE. 


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 8 of 30
For the RC grading of “AGGRESSIVE NV WITHOUT RIDGE AND WITH PLUS”, the RC 
actually allowed less than 4 quadrants to be affected by plus disease, causing a discrepancy to 
the agreed criteria in the RC charter.
To correctly apply the AP-ROP definition based on the RC data, AP-ROP should only be 
assigned in case 
1) The RC assessed AP-ROP as “YES, ZONE I/II WITH STAGE II/III AND PLUS”
2) The RC assessed AP-ROP as “YES, AGGRESSIVE NV WITHOUT RIDGE AND 
WITH PLUS” and reported that 4 quadrants have been affected with plus disease. 
The number of quadrants with plus disease is documented in ADOE with PARAMCD=”
NUMQUAAF” and OEEVALN=27. AVAL=5 reflects 4 quadrants with plus disease.
4.6
Blind/Masked Review
In this document, the analysis of an open-label study is described. Validity reviews will be 
also unmasked. The results of the final data assessment will be documented in the final list of 
important deviations, validity findings and assignment to analysis set(s).
5.
Analysis Sets
5.1
Assignment of Analysis Sets
Final decisions regarding the assignment of subjects/eyes to analysis sets will be made during 
the review of study data and documented in the final list of important deviations, validity 
findings and assignment to analysis set(s).
In general, a subject is assigned to an analysis set if at least one eye meets the respective 
criteria. Eye level analyses include only eyes that meet the respective criteria, i.e. single eyes 
could be excluded from an analysis while the subject itself is valid for the analysis. 
The primary and secondary efficacy variables will be analyzed using the FAS, mFAS, and the 
PPS.  The explorative efficacy variables will be analyzed using the FAS and mFAS. In all 
cases, the analysis with the FAS is considered as primary and mFAS and PPS are considered 
as supportive. Safety variables will be analyzed based on the SAF. Pharmacokinetic related 
analyses will be performed based on the PKS. 
Full Analysis Set (FAS)
All subjects who received any type of study treatment, and had a baseline and at least one 
post-baseline assessment of efficacy.
The analysis on the FAS will be performed according to the treatment assigned at baseline (as 
randomized).
Modified Full Analysis Set (mFAS)
All subjects with central reading center positive confirmed disease stages meeting the 
inclusion criteria who completed baseline treatment, had a baseline and at least one post-
baseline central reading center assessment of efficacy.


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 9 of 30
The analysis on the mFAS will be performed according to the treatment assigned at baseline 
(as randomized).
Per Protocol Set (PPS)
All subjects in the mFAS who have no validity findings or important deviations that could 
affect the primary efficacy endpoint.
The analysis of the PPS will be performed according to the treatment the subject actually 
received (as treated; determined by the first study treatment that the subject actually received
during the study).
The status of each subject, with regard to protocol deviations, will be determined by the 
sponsor and documented before database release.
Safety Analysis Set (SAF)
All subjects who received any type of study treatment.
The analysis of the SAF will be performed according to the treatment the subject actually 
received (as treated; determined by the first study treatment that the subject actually received
during the study).
Pharmacokinetic Analysis Set (PKS)
All subjects who received aflibercept treatment at the baseline visit and who had at least one 
non-missing PK assessment following the first dose of study drug. 
Immunogenicity analysis sets
Anti-drug antibody (ADA) data will be analyzed using the ADA Analysis Set (AAS) and 
neutralizing antibody (NAb) data will be analyzed using the NAb Analysis Set (NAS).
The AAS will include all participants who received study intervention with aflibercept and 
had at least 1 non-missing result in the ADA assay following the first study dose.
The NAS will include all participants who received any study intervention with aflibercept 
and with at least 1 non-missing result in the NAb assay. Participants who are negative in the 
ADA assay and those who are positive in the ADA assay but are not treatment-emergent will 
be set to negative in the NAb analysis set. Analysis of both immunogenicity analysis sets will 
be performed according to the treatment the participant actually received (as treated).
6.
Statistical Methodology
6.1
Population Characteristics
6.1.1
Sample Sizes, Subject Validity Status and Subject Disposition 
The following will be summarized descriptively: 

Study sample size by country and site

Protocol deviations, screen failures and premature discontinuations

The number of subjects with important protocol deviations

The number of screened subjects and primary reasons for screening failures


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 10 of 30

The total number and percentage of randomized subjects (ie, subjects who received a 
randomization number)

The number and percentage of subjects who qualified as FAS, mFAS, PPS, PKS, and 
SAF (defined in Section 5.1) and primary reasons for exclusions

The total number and percentage of subjects who permanently discontinued the study 
treatment with the reasons for discontinuation (ie, does not meet the definition of 
treatment completion, see Section 3). This description will be on a subject level 
considering a subject on treatment, as long as at least one eye is on treatment.

The total number and percentage of subjects who discontinued the study with the 
reasons for discontinuation
6.1.2
Demography, Baseline Characteristics and Medical History 
Demographic and baseline assessments to be summarized will include:

Chronological age at randomization (date of randomization - date of birth + 1)

Gestational age at birth (in weeks and days)

Post-menstrual age at randomization (Gestational age at birth + Chronological age at 
randomization)

Gender

Race

Body weight, head circumference and body length at birth and baseline

APGAR score at 1, 5, 10 and 15 minutes after birth  categorized as 0-3, 4-7,8-10
Baseline disease characteristics to be summarized will include:

ROP classification (Zone I, Zone II, plus status and AP-ROP)

ROP classification according to International Classification of Retinopathy of 
Prematurity stages (Table 6–1)

O2 supplementation at baseline (yes, no)

History of sepsis (yes, no)

History of necrotizing enterocolitis (yes, no)

History of intraventricular hemorrhage (yes, no)
Demographic data and baseline characteristics variables will be summarized using descriptive 
statistics for all four analysis sets (ie, FAS, mFAS, PPS, and SAF).
Maternal and subject medical history will be recorded and will be coded according to latest 
available version of Medical Dictionary for Regulatory Activities (MedDRA). Medical 
history data will be evaluated by frequency tables, showing number of subjects with medical 
history findings by primary system organ class (SOC) and preferred term (PT).


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 11 of 30
6.1.3
Exposure and Compliance to Study Treatment
Study intervention will be administered by a qualified ophthalmologist. Recorded details of 
aflibercept injections and of laser procedures will be described descriptively and include:

Number of aflibercept administrations from baseline to Week 24 (see secondary 
efficacy endpoint described in Section 6.2.2.3) – per eye and per subject

Number of laser treatments from baseline to Week 24 (see secondary efficacy 
endpoint described in Section 6.2.2.3) – per eye and per subject

Time required to perform treatment (analysed as “Other Pre-Specified Efficacy 
Endpoints”, see further description in Section 6.2.3.2)  - per eye and subject

Requirement for sedation or general anesthesia (analysed as “Other Pre-Specified 
Efficacy Endpoints”, see further description in Section 6.2.3.3) - per eye and subject:
o
type of anesthesia (general, sedation, local or other)
o
need for additional respiratory assistance and type (endotracheal intubation, 
non-invasive or other)
o
treatment site

Injection volume (for aflibercept treatment) - per eye

Number of burns, wave length, spot sizes, power and duration of laser pulse (for laser 
treatment) - per eye

Time to first rescue treatment – per subject
Usually a single (and mostly bilateral) aflibercept treatment session is planned with up to 2 
retreatments per eye after baseline if needed, compliance with study medication will not be 
calculated. 
6.1.4
Prior/Concomitant Medication
Prior and concomitant medication will be analyzed based on the SAF.
All medications the subject received prior to the Screening Visit or during the study as well as
maternal prior medications taken during pregnancy or for breastfeeding mothers after the 
umbilical cord clamping (if transfer of these products into human milk is feasible) will be 
tabulated by substance according to the World Health Organization (WHO) Drug Global.
Prior medication is defined as any medication that was taken before start of study treatment 
and that has a stop date before the treatment start date.
Concomitant medication is defined as any medication that was taken after first study 
intervention, i.e., any medication with a stop date after the start of study intervention or which 
is ongoing after the end of study.
Whenever applicable in displays, a distinction is made between:

maternal prior medications taken during pregnancy (i.e. medication taken by the 
mother with start day prior to date of birth of child but after conception)

prior medication taken by the breastfeeding mother (i.e. medication taken by the 
mother with start date after date of birth but prior to the start of study drug and end 
date prior to the start of study drug)

concomitant medication taken by the breastfeeding mother 

prior medication taken by the subject 


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 12 of 30

concomitant medication taken by the subject.
The number of subjects with O2 supplementation at each visit, as well as at the end of the 
study of each subject, will be presented descriptively. 
6.1.5
COVID-19 pandemic-related assessments
The study started patient enrollment in September 2019 already prior to the worldwide spread 
of the novel Coronavirus disease (COVID- 19) caused by the severe acute respiratory 
syndrome Coronavirus 2 (SARS-CoV2). The WHO declared this Coronavirus disease in 
March 2020 a pandemic. The study was never paused since then , and is ongoing. 
Apart from protocol deviations for non-pandemic related reasons, any deviations from the 
protocol for pandemic-related reasons were collected on an ongoing basis. The following 
statstical analyses are planned to assess any potential impact on the efficacy and safety 
evaluation and interpretation of the collected study data and the overall conduct of the study:

A listing of all subjects affected by COVID-19 pandemic, whether due to the specific 
protocol deviations, an early discontinuation of the study due to COVID-19 pandemic
or whether a subject reported COVID-19 as an adverse event by country and 
investigator. 

The number and percentage of subjects affected by COVID-19 pandemic per analysis 
population

A summary for the number of subjects who did not complete a study period 
(Treatment/Follow-up) for COVID-19 pandemic-related reasons.

Presentation of COVID-19 pandemic-related protocol deviations, overall and per 
subject, as well as by treatment arm, region and country.

Present primary efficacy analysis for subjects with COVID-19 pandemic-related 
protocol deviations and for subjects without any reported COVID-19 pandemic-
related protocol deviation. 
6.2
Efficacy
6.2.1
Primary Efficacy Endpoint
The primary efficacy endpoint is “Proportion of patients with absence of active ROP and 
unfavorable structural outcomes at 24 weeks after starting study treatment”, where 

Active ROP is defined as ROP requiring treatment (according to the inclusion 
criterion).

Unfavorable structural outcome is defined as 
o
retinal detachment, 
o
macular dragging, 
o
macular fold, or 
o
retrolental opacity.  


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 13 of 30
One eye or both eyes of a subject will be included into the analysis to determine the primary 
endpoint on a subject level, if treated and meeting the inclusion criteria. Data of a second eye
of subjects who started the study with only one eligible eye and that develops ROP during 
study will be included in the efficacy analyses only if treated before or on Visit 9 
(approximately 8 weeks from baseline).
All subjects requiring rescue treatment will be counted as missing the primary endpoint.
A subject for whom both eyes have been included in the analysis is defined as responder if 
both eyes show the defined absence. If only one eye has been treated this fact is accounted for 
by the statistical model (see below).
The number and percentage of eyes with active ROP or any of the unfavorable structural 
outcomes (overall and by type of unfavorable structural outcome) defined for the assessment 
of this endpoint at Week 24 will be displayed separately by treatment group using descriptive 
statistics.
Primary Analysis
The primary analysis for this endpoint will be based on the investigators’ assessments of ROP
and analyzed for FAS and repeated for mFAS and PPS
As it is assumed that in most subjects both eyes will be treated, this will be accounted for by 
using the following  bivariate binomial model:
li ~ Bernoulli (p): response of subject i in left eye
ri ~ Bernoulli (p): response of subject i in right eye
li and ri are correlated with correlation coefficient ρ
Bivariate probability distribution:
left eye / right eye
Response (ri=1)
No response (ri=0)
Response (li=1)
p2+ ρ p(1-p)
p(1-p)(1- ρ)
p
No response (li=0)
p(1-p)(1- ρ)
(1-p)(1-p(1- ρ))
1-p
p
1-p
Based on this model the probability for a patient to be a responder is
π = p2+ ρ p(1-p)).
The primary efficacy endpoint will be analyzed using a Bayesian statistical model with a non-
informative prior probability distribution for the response probability for a single eye (p). For 
the correlation coefficient ρ, an informative prior distribution allowing positive values only
will be assumed. The model is based on following distribution assumptions:
p ~ beta(1,1):  non-informative prior for the response probability in one eye
ρ ~ beta(1,1):  prior for the correlation between the two eyes of one subject    
(allowing only a positive correlation)
Based on this model, the primary endpoint “Proportion of subjects with absence of active 
ROP and unfavorable structural outcomes at 24 weeks after starting study treatment,” (e.g. 


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 14 of 30
response on a subject level, ie π = p2+ ρ p(1-p)) will be analyzed for each of the 2 treatment 
groups.
This will be implemented by SAS using the following code (variable and dataset names are 
for illustration only and may be subject to change):
proc mcmc data=oerespm_ seed=1 nbi=5000 nmc=50000 outpost=regOut monitor=(prespa prespl diff) 
stats(alpha=0.1) = all plots=all;
  *peyea, peyel: priors for eye level reponse probability for aflibercept and laser, 
             rho: correlation;
    parms peyea peyel rho;  *eye level reponse probability for aflibercept and laser;
    prior peyea ~ beta(1, 1);
    prior peyel ~ beta(1, 1);
    prior rho ~ beta(1, 1);
    peye = dtreat*peyea + (1-dtreat)*peyel;
    prespa = peyea**2 + rho*peyea*(1-peyea);
    prespl = peyel**2 + rho*peyel*(1-peyel);
    diff = prespa - prespl;
    model first
~ binary(peye);
    model second ~ binary(first*(peye + rho*(1-peye)) + (1-first)*((1-rho)*peye));
RUN;
The efficacy of aflibercept and laser photocoagulation will be characterized by the resulting 
posterior distribution for the probability of response.  Mean, median, and mode of the 
posterior distributions for the response probabilities of the two treatments as well as for the 
difference in response probabilities between the treatments will be provided in addition to 
90% equal tail credible intervals. For the mode, the “half sample mode” (HSM) will be 
calculated based on all simulated posterior outcomes. The half sample mode will be 
calculated as described by Robertson T. and Cryer J.D. (1974).   
Success of this study will be mainly concluded, if the posterior probability:                                              
P(response probability for aflibercept > (response probability for laser -5%) ) ≥ 95%.
This is the case if the lower limit of the one-sided 95% credible interval for the treatment 
difference (aflibercept – laser photocoagulation) is greater than -5%. As non-informative prior 
distributions are used for the analysis this success criterion corresponds to a frequentist  non-
inferiority test with a non-inferiority margin of 5 percentage points and a significance test of 
5% (one-sided test).
The posterior distribution of both response probabilities, as well as the difference in response 
probabilities will be displayed using normal kernel densities. 
If the success criterion is met, in a second step superiority of aflibercept over laser 
photocoagulation is evaluated by comparing the lower limit of the 95% one-sided credible 
interval with 0. As a supportive analysis, the Japanese success criterion will be analysed to 
demonstrate superiority in response proportion over 66% in the aflibercept group.
Handling of missing data for primary analysis
All subjects are required to have efficacy assessments 24 weeks after starting study 
intervention (with an allowed visit window of +/- 7 days). Missing Week 24 data for a treated 
eye will be imputed as described in the following as long as respective eyes not having had an 
unfavorable structural outcome or rescue treatment before dropping out, as in these cases the 
eye would always be considered as non-responding:


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 15 of 30
1. If at the last visit before dropping out the subject had no active ROP and Zone II was 
completely vascularized (aflibercept subjects) or laser treatment was completed (laser 
subjects) the respective eye will be considered as responding.
2. Otherwise, if the subject dropped out at or after Week 16, the last non-missing post-
baseline ROP staging before dropping out will be carried forward and used for 
determining the response in this eye (last observation carried forward (LOCF) 
approach).
3. If the subject dropped out before Week 16, the missing information will be imputed as 
follows: 
a.
If there was a clear documentation that the subject dropped out due to lack of 
efficacy, the respective eye will be considered as non-responding; 
b. otherwise, a multiple imputation approach will be used giving the same 
probability of success as subject’s having the same treatment group and initial 
staging (Zone I versus II versus AP-ROP). Further details are provided in 
Appendix 9.1.
Sensitivity Analyses
A sensitivity analysis will be performed where the RC data will be used instead of the 
investigator assessments of ROP at week 24 for FAS, mFAS and PPS.
After release of the final database, one of the diagnostic criteria for AP-ROP applied by the 
RC was identified to be incorrect, and in deviation from the international classification of 
ROP (ICROP, 2005) and the agreed RC charter. As per RC charter the presence of plus 
disease in all 4 quadrants of the posterior retina is one prerequisite for the diagnosis of AP-
ROP. Section 4.5 decribes the data rules to correctly apply the AP-ROP definition according 
to the charter and based on the available RC data. 
A full re-run of the tables, listings and figures (TLF) documents is needed after implementing 
the updated AP-ROP definition.
Furthermore sensititivity analysis to evaluate the impact of missing data will be conducted:

considering all drop-outs as non-responders unless completely vascularized
(aflibercept subjects) or laser treatment was completed (laser subjects) for FAS, mFAS 
and PPS (worst case imputation)
As a further sensitivity analysis, the “Proportion of patients with absence of active ROP and 
unfavorable structural outcomes at 24 weeks after starting study treatment” according to the 
investigator assessment will be analyzed by the corresponding frequentist approach for FAS.
Reading center results will be analysed for mFAS and PPS.
Asymptotic 90% confidence intervals for the difference in response rates will be calculated 
based on a 2x2 contingency table for response (Yes, No) vs. Treatment (Aflibercept injection, 
Laser photocoagulation). In case that both eyes of a subject are treated, the subject will only 
be considered as a responder if both eyes responded, if only one eye is treated, then the 
subject will be considered as a responder if this eye responded.  Aflibercept will be 
considered to be non-inferior to laser with regards to the sensitivity analysis if the 90% 
confidence interval of the difference lies entirely above -5%. Asymptotic 90% confidence 
intervals will be presented in addition for for the Mantel-Haenszel weighted treatment 
difference calculated using normal approximation and adjusting for each individual  
stratification factor (baseline ROP classification and Japan vs Non-Japan). The following 
methodology (Koch et al, 1990) is used:


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 16 of 30










h
h
h
hc
ht
h
w
p
p
w
d
/
)
ˆ
ˆ
(
, where 
)
/(
hc
ht
hc
ht
h
n
n
n
n
w


.
Then 


2
2
/
)1
/(
)
ˆ
1(
ˆ
)1
/(
)
ˆ
1(
ˆ
)ˆ
r(
aˆ
v




















h
h
ht
ht
ht
hc
hc
hc
h
h
w
n
p
p
n
p
p
w
d
.
With this, the 90% confidence interval (CI) can be given as: 
)ˆ
r(
aˆ
v
ˆ
2
/
d
z
d


(
2
/
z
being the lower 
2
/

quantile of the standard normal distribution).
In the formulae, 

h : number of strata

ht
p : proportion of subjects with absence of active ROP and unfavorable structural 
outcomes at 24 weeks after starting study treatment in the aaflibercept arm, in 
stratum h ,

hc
p : proportion of subjects with absence of active ROP and unfavorable structural 
outcomes at 24 weeks after starting study treatment in the laser arm, in stratum h ,

ht
n : number of subjects in the aflibercept arm in stratum h,

hc
n : number of subjects in the laser arm in stratum h .
 
 
 
 
 
 
CCI


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 17 of 30
 
 
 
 
CCI


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 18 of 30
6.2.2
Secondary Efficacy Endpoints
6.2.2.1
Requirement for Intervention with a Second Treatment Modality from 
Baseline to Week 24 
A second treatment modality for ROP in this study can be either rescue treatment as defined 
per protocol or treatment with any other surgical or nonsurgical treatment for ROP (e.g. IVT 
anti-VEGF injection, ablative laser therapy, cryotherapy, or vitrectomy captured as 
concomitant medication or surgeries after study start).
Second treatment modalities other than rescue treatment will be identified by:

Any concomitant medication starting after start of study treatment with standardized 
medication term in ("BEVACIZUMAB" "PEGAPTANIB" "RANIBIZUMAB" 
"AFLIBERCEPT" "BROLUCIZUMAB")

Any ocular surgery after start of study treatment assessed by the medical experts as 
second treatment modality. 
The same Bayesian statistical model as described for the primary analysis of the primary 
endpoint will be used to estimate probabilities for a requirement of intervention with a second 
treatment modality within the time between baseline and Week 24. As for the primary 
analysis, a subject might contribute with a single eye or with both eyes. Analyses will be 
presented for FAS, mFAS and PPS. 
Handling of missing data 
Missing data regarding requirements for intervention with a second treatment modality will be 
imputed analogously to the approach used for the primary endpoint and as described in the 
following as long as respective eyes did not have had a second treatment modality before 
dropping out:
1. If at the last visit before dropping out the subject had no active ROP and Zone II was 
completely vascularized (aflibercept subjects) or laser treatment was completed (laser 
subjects) the respective eye will be considered as not requiring intervention with a 
second treatment modality.
2. Otherwise, if the subject dropped out at or after Week 16, the respective eye will be 
considered as not requiring for intervention with a second treatment modality.
3. If the subject dropped out before Week 16, the missing information will be imputed as 
follows: 
a.
If there was a clear documentation that the subject dropped out due to lack of 
efficacy, the respective eye will be considered as requiring intervention with a 
second treatment modality; 
b. otherwise, a multiple imputation approach will be used giving the same 
probability of success as subjects having the same treatment group and initial 
staging (Zone I versus II versus AP-ROP).


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 19 of 30
6.2.2.2
Recurrence of ROP from Baseline to Week 24
Recurrence of ROP until Week 24 aims to monitor the disease activity during the study and is 
defined for this study as a need for retreatment or rescue treatment in cases where the question 
“presence of active ROP requiring treatment” had been previously answered with “No” .
This binary endpoint will be analysed using the same Bayesian statistical model as described 
for the primary analysis of the primary endpoint. Analyses will be presented for FAS, mFAS 
and PPS. 
Handling of missing data 
Missing data regarding recurrence of ROP will be imputed analogously to the approach used 
for the primary endpoint and as described in the following as long as respective eyes did not 
have had a recurrence before dropping out:
1. If at the last visit before dropping out the subject had no active ROP and Zone II was 
completely vascularized (aflibercept subjects) or laser treatment was completed (laser
subjects) the respective eye will be considered as not having a recurrence.
2. Otherwise, if the subject dropped out at or after Week 16, the respective eye will be 
considered as not having a recurrence.
3. If the subject dropped out before Week 16, the missing information will be imputed as 
follows: 
a.
If there was a clear documentation that the subject dropped out due to lack of 
efficacy, the respective eye will be considered as having a recurrence; 
b. otherwise, a multiple imputation approach will be used giving the same 
probability of success as subjects having the same treatment group and initial 
staging (Zone I versus II versus AP-ROP).
6.2.2.3
Number of Aflibercept Administrations/ Laser Treatments from 
Baseline to Week 24 
The number of aflibercept treatments and the number of laser treatments from baseline to 
Week 24 will be described descriptively per treatment arm in frequency tables.
In case multiple sessions of laser treatment are necessary within 1 week from baseline, they 
will be counted as a single treatment. The same approach will be applied for laser treatment as 
re-treatment or rescue treatment.
Accounting for eventual reduced follow-up times due to dropping-out of subjects, the number 
of aflibercept treatments and the number of laser treatments from baseline to Week 24 will be 
also displayed descriptively only for treatment completers (see Section 3 for definition).
6.2.2.4
To explore new Retinopathy of Prematurity Activity Scale proposed by 
the International Neonatal Consortium
Based on the International Classification of Retinopathy of Prematurity (ICROP) 
classification, an activity scale and severity classification into three subcategories of ROP 


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 20 of 30
(mild, moderate, severe) was derived (Smith et al, 2018). Classifications are shown in Table 
6–1.
Table 6–1: ROP activity scale and ROP subcategories derived from the ICROP 
classification
Zone
Stage
ROP Activity Scale
Category
none
none
0
mild
III
1
1
III
2
2
II
1
3
III
1+
4
III
3
5
III
2+
6
II
2
7
II
3
8
moderate
III
3+
9
I
1
10
II
1+
11
I
2
12
II
2+
13
severe
II
3+
14
I
1+
15
I
3
16
I
2+
17
I
3+
18
Any zone
AP-ROP
19
Any zone
4a
20
Any zone
4b
21
Any zone
5
22
The distribution and changes from baseline over time of the ROP Activity Scale and the three 
subcategories as assessed by the central reading center will be described descriptively per eye 
by summary statistics (inclunding quartiles), frequency and shift tables. The number and 
percentage of subjects with at least a two step decrease by visit will be presented. Analyses 
will be presented for FAS, mFAS and PPS. 
6.2.3
Other Pre-Specified Efficacy Endpoints
6.2.3.1
Evaluation of Visual Function at Week 24
Visual function per eye will be presented by descriptive statistics for the following variables:

abnormalities of the retina or optic nerve as well as unfavorable ocular structural 
outcomes 

fixation (central, steady and maintained)

fixing and following a 5-cm toy


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 21 of 30

in case visual function cannot be assessed with fixation, visual evoked potentials can 
be used for instance to evaluate if the visual function is normal or abnormal

cycloplegic refraction (sphere, cylinder, axis, pseudophakia or intraocular vision 
correction)

ocular motility tests (if motility test done: assessment of ocular palsy with affected 
nerves, assessment of strabismus, and nystagmus).
6.2.3.2
Time Required to Perform Treatment
The start and stop times for any aflibercept administrations or laser procedures (irrespective 
of initial treatment, retreatment or rescue treatment) will be captured. The required time to 
perform aflibercept administrations or laser treatments will be calculated by “stop time - start 
time” per eye and subject, respectively and summarized with descriptive statistics by 
treatment arm.
The total time of administrations for each study intervention will be analyzed per eye and per 
subject, i.e. the sum over all initial and retreatment and rescue treatment administrations. 
6.2.3.3
Requirements During Study Intervention
Frequencies of the following during all aflibercept administrations or laser treatments 
(including also retreatment or rescue treatments) will be described descriptively per eye and 
per subject:
o
type of anesthesia (general, sedation, local or other)
o
need for additional respiratory assistance and type (endotracheal intubation, 
non-invasive or other)
o
treatment site
6.2.3.4
Requirement for Treatment with more than one Aflibercept Injection
The number of eyes, as well as the number of subjects, with more than one aflibercept 
injection will be described descriptively by frequency tables.
6.2.3.5
Time to Intervention with a second Treatment Modality for ROP or 
Development of Unfavorable Structural Outcome 
The time to either

the first intervention with a second treatment modality for ROP (see definition of 
secondary endpoint “requirement for intervention with a second treatment modality 
from baseline to Week 24”) for at least one eye per subject
Or

the development of unfavorable structural outcome defined as retinal detachment, 
macular dragging, macular fold, or retrolental opacity based on the investigator’s
assessment of ROP for at least one eye per subject
will be displayed descriptively by treatment group on subject level (for all subjects and 
separately for bilateral cases and for unilateral cases) and descriptive statistics using Kaplan-
Meier estimates. Descriptive statistics include 

number of subjects with event

number of subjects censored


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 22 of 30

median, 25th and 75th percentiles

as well as rates of subjects without any event at Week 6, 12 and 24 (42, 84 and 168 
days) with corresponding 95% confidence intervalls).
Partially missing event dates will be imputed as the minimum possible date on or after start of 
study intervention. 
Handling of censored data:
Subjects without an event will be censored at the date of last visit if they prematurely 
discontinued from the study. All censored data will be treated as censored completely at 
random. 
6.2.3.6
Time to Recurrence of ROP 
Recurrence of ROP until Week 24 on subject level is considered as an event for this endpoint 
and is defined as follows:
For at least one eye of a subject a need for retreatment or rescue treatment  in cases where the 
question “presence of active ROP requiring treatment” had been previously answered with 
“No” for the same eye.
The time to the first event will be analysed on subject level analougously to the approach 
described in Section 6.2.3.5.
6.2.3.7
Regression of Plus Disease, Regression of Pre-Retinal Vascularized 
Ridge and Progression of Retinal Vascularization beyond the Ridge 
from Baseline to Week 24 
Plus disease describes an increase in severity of ROP where blood vessels are particularly 
enlarged and twisted. The frequency of regression of plus disease, i.e. that plus disease is 
present at baseline, but not at Week 24, will be described descriptively per eye.
There are 6 stages (stages 1, 2, 3, 4a, 4b, 5) indicating the severity of the disease. For the 
regression of the pre-retinal vascularized ridge from baseline to Week 24, the 
decrease/improvement of the stage is analysed. A reduction of at least one stage as well as the 
amount of change will be described descriptively.
There are three zones indicating the extent to which blood vessel growth (vascularization) has 
been completed. A progression of retinal vascularization beyond the ridge from baseline to 
Week 24, i.e. if the normal vascularization has grown towards the periphery is indicated by a 
higher zone classification (improvement of the disease). An increase of at least one zone as 
well as the amount of change will be described descriptively.
The primary analysis for this endpoint will be based on the investigators’ assessments while 
the reading center assessment will be presented in addition.
6.2.3.8
Progression to Stage 4 or 5 ROP from Baseline to Week 24 
The frequency of a progression to stage 4 or 5 at any time until Week 24 based on the 
investigators’ assessments will be described per eye by visit and treatment group.
6.2.3.9
Completion of Vascularization of the Peripheral Retina to within one 
Disc Diameter of the Ora Serrata at Week 24 
Whether the vascularization is complete or not at Week 24 based on the investigators’ 
assessments will be described per eye by frequency tables.


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 23 of 30
6.2.3.10
Time to Completion of Vascularization
The time to the investigator’s first assessment where the vascularization is complete will be 
analysed analougously to the approach described in Section 6.2.3.5. This will be analysed on 
subject level for all subjects, all bilateral cases as well as all unilateral cases.
6.2.3.11
Number of Visits required up to Week 24
The number of visits up to Week 24 including mandatory vists according to the protocol as 
well as additional optional visits will be described.
Accounting for eventual reduced follow-up times due to dropping-out of subjects, the number 
of visits up to Week 24 will be also displayed descriptively only for treatment completers (see 
Section 3 for definition).
6.2.4
Subgroup Analyses
For the primary and secondary efficacy endpoints, subgroup analyses by zones (Zone I/II/AP-
ROP) will be performed. The subgroup will be defined twice, once based on investigator 
assessment and once for reading center assessment. 
6.2.5
Other exploratory analyses
6.2.5.1
Recurrence following complete regression of ROP from Baseline to 
Week 24
Recurrence following complete regression of ROP until Week 24 is defined for this study as
the presence of ROP at a visit after ROP has been documented as “absent” for at least one 
previous visit.
This binary endpoint will be analysed using the same Bayesian statistical model as described 
for the primary analysis of the primary endpoint.
Handling of missing data 
Missing data regarding recurrence of ROP will be imputed analogously to the approach used 
for the primary endpoint and as described in the following as long as respective eyes did not 
have a recurrence before dropping out:
1. If at the last visit before dropping out the subject had no active ROP and Zone II was 
completely vascularized (aflibercept subjects) or laser treatment was completed (laser 
subjects) the respective eye will be considered as not having a recurrence.
2. Otherwise, if the subject dropped out at or after Week 16, the respective eye will be 
considered as not having a recurrence.


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 24 of 30
3. If the subject dropped out before Week 16, the missing information will be imputed as 
follows: 
a.
If there was a clear documentation that the subject dropped out due to lack of 
efficacy, the respective eye will be considered as not having a recurrence;
b. otherwise, a multiple imputation approach will be used giving the same 
probability of success as subjects having the same treatment group and initial 
staging (Zone I versus II versus AP-ROP).
6.3
Pharmacokinetics/Pharmacodynamics
Biomarkers for efficacy are not evaluated in this study. 
Pharmacokinetic (PK) evaluations will describe plasma concentrations of free, adjusted bound 
and total aflibercept. Adjusted bound will be calculated from ‘bound aflibercept’ by 
multiplication with a factor of 0.717. Total aflibercept will be calculated as the sum of free 
and adjusted bound aflibercept. 
The results of the bioanalytical analyses will be reported separately and attached to the final 
clinical study report as an appendix.
The objectives of the PK evaluations include:

Describe the individual concentrations of free,adjusted bound and total aflibercept at 
several time points after the first IVT injection up to 24 weeks thereafter

Explore the potential influence of demographic factors such as age and weight on 
systemic aflibercept concentrations

Explore the relationship of systemic exposure and blood pressure (as a 
pharmacodynamic safety marker) and urine protein
PK evaluation
Concentrations of free,adjusted bound and total aflibercept will be listed by sample time point 
throughout the treatment period, and summarized by descriptive statistics overall and by 
baseline weight (<1000 gr, 1000 gr - < 1500 gr, 1500 gr - < 2000 gr, 2000 gr - < 2500 gr, ≥ 
2500 gr), gender and race subgroups including the subgroup of Japanese patients. 
Mean concentration time curves (plus/minus one SD) will be presented. 
PK concentrations will only be considered for the analysis, if the actual measurement took 
place at the scheduled time point within the allowed time window according to the protocol.
I.e., PK samples will only be analysed if measured:

D1 post treatment: For a maximum of 3 days after aflibercept injection

Week 2: If measured 14 ± 3 days after aflibercept injection

Week 4: If measured 28 ± 7 days after aflibercept injection

Week 8: If measured 56 ± 7 days after aflibercept injection

Week 12: If measured 84 ± 7 days after aflibercept injection

Week 24: If measured 168 ± 30 days after aflibercept injection.


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 25 of 30
To describe the deviations from the planned PK assessment time point, descriptive summary 
statistics will be presented by time point for:

Time deviation = (Actual PK sample date – aflibercept injection date) – planned 
timepoint (PTP) (with the PTP = 1 for Day 1, PTP = 14 for Week 2 and PTP = 28 for 
Week 4 as well as PTP = 56  for Week 8, PTP = 84 for Week 12 and PTP = 168 for 
Week 24). 
PK concentrations from samples taken outside the defined time window will be listed together 
with the actual sampling date.
Relationship of systemic exposure and clinical outcome  
The relationship of exposure to specific safety parameters (blood pressure, urine protein, 
treatment emergent serious AEs) will be explored graphically. For each sampling time point 
scatterplots for individual aflibercept concentrations (adjusted bound and free) vs. each 
pharmacodynamics safety marker (systolic, diastolic blood pressure, change from baseline in 
systolic blood pressure (SBP) and diastolic blood pressure (DBP)) will be presented.
Aflibercept concentrations measured as below lower limit of quantification (<LLOQ) will be 
presented with a value ½ LLOQ. Subjects with unilateral and bilateral treatment will be 
presented differently, as well as subjects with treatment emergent serious AEs after the initial 
aflibercept administration (within 30 days after first injection) and subjects without. 
Similar scatter plots will be created for the absolute values and change in SBP/DBP at Week 4 
vs gestational age presenting subjects for the aflibercept group differently to subjects from the 
laser group. Similarly, additional scatter plots for absolute values and change in SBP/DBP at 
Week 4 vs baseline weight will be presented.
Optional Population Pharmacokinetic Analysis 
Optionally, a population PK analysis may be considered as part of a meta-analysis of PK data 
obtained with aflibercept. In this case, aflibercept concentrations collected during the study 
will be analyzed using nonlinear mixed effects models. Mixed effects models, or population-
type PK models, describe the relationship between dose and time and variables such as drug 
plasma concentrations. Both structural and random effects are involved in this relationship. A 
population PK compartmental model may be developed using the concentration of aflibercept 
as the dependent variable.
Results obtained by the optional population PK modeling may then be further specified and 
presented in a separate report that may also include PK data from other studies with Eylea and 
aflibercept.
 
 
CCI


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 26 of 30
 
 
 
 
6.4
Safety
All safety variables will be analyzed for the safety analysis set. 
6.4.1
Adverse Events, Serious Adverse Events, and Device Events
All AEs will be coded using Medical Dictionary for Regulatory Activities (MedDRA). The
latest available version at the time of coding will be used. 
Adverse events will be summarized, at a minimum, on the level of system organ class (SOC) 
and preferred term (PT) and summaries will be provided displaying AEs within each system 
organ class (SOC) in decreasing order of total frequency according to the numbers of subjects 
reporting the AE (not number of events). Data will also be summarized according to intensity 
and investigator’s causality assessment.
Ocular and systemic TEAEs and SAEs from baseline to Week 24 will be analyzed 
descriptively using summary statistics or frequency tables as appropriate. Thereby, TEAE is 
defined as AE that is observed or reported after the first and not later than 30 days after the 
last administration of study treatment. This definition includes that the length of the time 
window for potential treatement emergence varies from subject to subject.
Overview tables will be presented for both AEs and TE(S)AEs for ocular events and systemic 
events as well as for all adverse events (ocular and systemic). Ocular events will be presented 
overall, and for bilaterally and unilaterally treated subjects separately.
Ocular events on treated eyes will be presented by SOC and PT for:

AE

TEAE

Serious adverse event (SAE)

TESAE

Aflibercept related TEAE

Injection procedure-related TEAE

Photocoagulation related TEAE

Aflibercept related TESAE

Injection procedure-related TESAE

Photocoagulation related TESAE

Non serious AE

TEAE resulting in discontinuation from aflibercept

TEAE resulting in discontinuation from photocoagulation

AE by intensity
CCI


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 27 of 30

TEAE by intensity

TESAE by intensity

Aflibercept related TEAE by intensity

Injection procedure-related TEAE by intensity

Photocoagulation related TEAE by intensity
Ocular events for non treated eyes will be reported with a reduced number of tables:

AE

SAE

Non serious AE

AE by intensity
The same analyses as for ocular events for treated eyes will be repeated for systemic adverse 
events. 
Device events will be listed only.
A listing including all COVID-19 adverse events will be presented. 
6.4.2
Surgical Procedures after Study Start
Any surgical procedures after study start and the type of any anesthesia used, will be 
displayed using number and percentage of subjects.
6.4.3
Ophthalmic Examinations
Any clinically significant abnormal findings:

before treatment and changes from baseline over time in the anterior segment

post treatment in the posterior segment (other than ROP) 
will be displayed using descriptive statistics.
For subjects in the aflibercept arm, the intraocular pressure (IOP) will be measured prior to 
the injection and at least once post-injection. Summary statistics and changes from pre- to 
post-injection will be displayed. In case of multiple post injection IOP assessments per eye, 
the first post injection assessment will be analyzed. Analyze first and possible second and 
third injection per subject separately. 
6.4.4
Physical Examinations and Vital Signs
Measurements from physical examinations (weight, body length, head circumference) and 
vital signs (body temperature, heart rate, respiratory rate, and blood pressure) will be 
displayed using descriptive statistics. Mean (plus/minus one SD) blood pressure time curves 
will be presented and repeated for the aflibercept arm for subjects with free or adjusted bound 
aflibcercept concentrations >2000 ng/mL at any time point. 
6.4.5
Laboratory Safety Variables
Results of laboratory analyses (hematology, chemistry, urinalysis, proteinuria) will be listed.
Proteinuria results over time will be displayed with frequency counts. 
6.4.6
Central Nervous System Imaging
Procedure findings will be listed.


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 28 of 30
6.5
Development of Anti-drug Antibodies
Antibodies to aflibercept will be evaluated in serum samples collected from all participants at 
baseline and at Week 12.
Immunogenicity will be characterized by ADA responses and titers observed in patients in the 
ADA analysis set. ADA response categories and titer categories are defined as follows:. 

ADA Negative: all time points negative and those that exhibit a pre-existing response, 
regardless of any missing samples
o
 
 

ADA positive: defined as those that exhibit a treatment emergent or treatment boosted 
ADA response, regardless of any missing sample
o
 
o
 
ADA titer will additionally be summarized with number and percentage of sujects for 
categories:



NAb status will be evaluated for the samples that are positive in the ADA assay and have 
sufficient volume to analyse. The number and proportion of participants positive in the NAb
assay will be summarized for the NAS.
If applicable, further analyses for ADA positive or NAb positive subjects  will include:

Plots of drug concentrations will be examined and the influence of ADAs and NAbs 
on individual PK profiles evaluated. 

Primary efficacy endpoint

Ocular TEAE for study eye and fellow eye

non-ocular TEAE
Potential association between immunogenicity variables and safety may be explored with a 
primary focus on the following safety events during the TEAE period: 

Injection site reaction (serious or severe and lasting 24 hours or longer)

Infusion reactions 
CCI
CCI
CCI
CCI
CCI
CCI


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 29 of 30

Hypersensitivity (SMQ: Hypersensitivity [Narrow])

Anaphylaxis (SMQ: Anaphylaxis [Narrow])
7.
Document History and Changes in the Planned Statistical Analysis

Version 1.0 of the SAP dated 10 JUL 2019
Summary of changes:

Addition of COVID-19 pandemic related analyses

Addition of frequentist analysis approach for primary efficacy variable

Addition of further exploratory endpoint: “Recurrence following complete regression 
of ROP from Baseline to Week 24”

Further details on analyses of PK and immunogenicity data

Version 2.0 of SAP dated 09 FEB 2021
Summary of changes:

Definition of screen failure added

Version 3.0 of SAP data 24 FEB 2021
Summary of changes:

Addition of data rules to correct the AP-ROP assessment of the RC to follow the RC 
charter

Editorial changes to the PK figures
8.
References
Smith LEH, Hellström A, Stahl A, Fielder A, Chambers W, Moseley J, Toth C, 
Wallace D, Darlow B, Aranda J, Hallberg B, Davis JM, for the Retinopathy of 
Prematurity Workgroup of the International Neonatal Consortium.  Development of 
a retinopathy of prematurity activity scale and clinical outcome measures for use in 
clinical trials. JAMA Ophthalmol.  2018 Dec 13 (corrected 10 Jan 2019). Available 
from: doi:10.1001/jamaophthalmol.2018.5984.
Robertson T. and Cryer J.D. (1974). An iterative procedure for estimating the mode. 
J. Amer. Statist. Assoc., 69(348):1012-1016.
Koch GG, Carr GJ, Amara IA, Stokes ME, Uryniak TJ (1990). Categorical Data 
Analysis in Statistical methodology in the pharmaceutical sciences
International Committee for the Classification of Retinopathy of Prematurity. The 
International Classification of Retinopathy of Prematurity revisited. Arch 
Ophthalmol. 2005 Jul;123(7):991-9.


Statistical Analysis Plan
BAY 86-5321 / Aflibercept
Page: 30 of 30
9.
Appendix
9.1
Multiple imputation approach for missing data in primary efficacy 
analysis
For the primary efficacy analysis, a multiple imputation approach is planned in case that the 
efficacy data for Week 24 is not available and the otherwise provided data is not sufficient to 
assess the endpoint otherwise, i.e. if the subject was followed up only shortly and there is no 
clear information that the subject is a non responder (rescue treatment provided or 
documentation of ocular unfavorouble structural outcome). 
For  the multiple imputation approach only a single eye will be imputed (regardless whether 
the subject was uni- or bilaterally treated) and the same probability of success as subject’s 
having the same treatment group and initial staging (Zone I versus II versus AP-ROP) will be 
assumed. For subjects where one eye is assessable and the second eye would need imputation, 
no further imputation will be considered.
For each subject with missing data, the imputation step will be repeated 20 times and the 
resulting posterior distributions for the probability of response will be combined from all 
imputation steps. Summaries of the resulting posterior distribution (mean, median and mode) 
will be derived from the combined posterior distribution.