Identifying Ocular and Systemic Biomarkers for Response to Aflibercept in Asian Patients With Centre Involving Diabetic Macular Edema: A Prospective Clinical Trial
Phase: PHASE4
Conditions: Diabetic Macular Edema
Sponsor: Singapore National Eye Centre

DME Protocol Version 4 Dated 26 Feb 2019 
Page 1 of 21 
 
 
 
 
STUDY PROTOCOL 
 
PROTOCOL TITLE: 
Identifying  ocular and systemic biomarkers for response to aflibercept in Asian patients with centre 
involving diabetic macular edema: A prospective clinical trial  
 
PROTOCOL NUMBER: R1436/19/2017 
 
 
PROTOCOL VERSION:  
4.0  
PROTOCOL DATE:  
26 Feb 2019 
STUDY PERIOD: 
16 months from the initiation date of the contract with Santen  
 
 
PRINCIPAL INVESTIGATOR:  
Dr Gavin Tan Siew Wei 
Consultant,  
Singapore Eye Research Institute 
Singapore National Eye Centre  
11 Third Hospital Avenue, Singapore 168751 
Contact No: 6322 7433 
 
REPRESENTATIVE OF SANTEN PHARMACEUTICAL 
Masanao Toshimori 
Santen Pharmaceutical Co., Ltd 
4-20 Ofukacho, Kita-ku, Osaka, 
Japan 530-8552 
 
Santen’s PRINCIPAL INVESTIGATOR:  
Takahiro Imanaka 
Santen Pharmaceutical Co., Ltd 
4-20 Ofukacho, Kita-ku, Osaka, 
Japan 530-8552 
 

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CO-INVESTIGATORS: 
A/Prof Ian Yeo, Senior Consultant, Singapore National Eye Centre 
A/Prof Gemmy Cheung, Senior Consultant, Singapore Eye Research Institute, Singapore National Eye Centre 
Prof Wong Tien Yin, Professor, Singapore Eye Research Institute, Singapore National Eye Centre, Duke-NUS 
Graduate Medical School 
Dr Anna Tan, Consultant, Singapore Eye Research Institute, Singapore National Eye Centre 
Dr Kelvin Teo, Associate Consultant, Singapore National Eye Centre 
Dr Daniel Ting, Associate Consultant, Singapore National Eye Centre 
Dr Wong Chee Wai, Associate Consultant, Singapore National eye Centre 
Dr Cheung Ning (Danny), Senior Fellow, Singapore National Eye Centre  
Dr Yasuo Yanagi, Clinician Scientist, Singapore National Eye Centre 
COLLABORATORS: 
Dr Chan Choi Mun, Senior Consultant, Singapore Eye Research Institute, Singapore National Eye Centre 
Dr Ranjana Mathur, Senior Consultant, Singapore Eye Research Institute, Singapore National Eye Centre 
Dr Laurence Lim, Consultant, Singapore National Eye Centre 
A/Prof Lee Shu Yen, Senior Consultant, Singapore National Eye Centre 
A/Prof Edmund Wong, Senior Consultant, Singapore National Eye Centre 
 
Santen’s CO-INVESTIGATORS: 
Kohei Honda, Santen Pharmaceutical Co., Ltd 
 
RESPONSIBILITY: 
Singapore Eye Research 
Institute, Singapore National Eye 
Centre 
Research planning and management 
Implementation of clinical treatment and sample/information acquisition 
Sample and data analysis 
Sample and data storage for future study 
Santen Pharmaceutical Co., Ltd 
Research planning and management 
Sample and data analysis for proteomic vitreous biomarkers 
 
 
 

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 Table of Contents 
1. 
BACKGROUND AND RATIONALE ................................................................................................. 5 
2. 
HYPOTHESIS AND OBJECTIVES .................................................................................................. 6 
3. 
EXPECTED RIKS AND BENEFITS ................................................................................................. 7 
4. 
STUDY POPULATION ................................................................................................................... 7 
4.1. 
LIST THE NUMBER AND NATURE OF SUBJECTS TO BE ENROLLED. ................................................................................. 7 
4.2. 
CRITERIA FOR RECRUITMENT AND RECRUITMENT PROCESS ....................................................................................... 8 
4.3. 
INCLUSION CRITERIA ................................................................................................................................................ 8 
4.4. 
EXCLUSION CRITERIA ............................................................................................................................................... 8 
5. 
STUDY DESIGN AND PROCEDURES/METHODOLOGY ............................................................... 10 
6. 
SAFETY MEASUREMENTS .......................................................................................................... 16 
6.1. 
DEFINITIONS .......................................................................................................................................................... 16 
6.2. 
COLLECTING, RECORDING AND REPORTING OF ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS TO CIRB ............ 17 
6.3. 
SAFETY MONITORING PLAN .................................................................................................................................... 18 
6.4. 
COMPLAINT HANDLING ........................................................................................................................................... 18 
7. 
DATA ANALYSIS ........................................................................................................................ 18 
7.1. 
DATA QUALITY ASSURANCE .................................................................................................................................... 18 
7.2. 
DATA ENTRY AND STORAGE .................................................................................................................................... 18 
8. 
SAMPLE SIZE AND STATISTICAL METHODS ............................................................................. 18 
8.1. 
DETERMINATION OF SAMPLE SIZE ........................................................................................................................... 18 
8.2. 
STATISTICAL AND ANALYTICAL PLANS ..................................................................................................................... 19 
9. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS ..................................................................... 19 
10. QUALITY CONTROL AND QUALITY ASSURANCE ....................................................................... 19 
11. ETHICAL CONSIDERATIONS ..................................................................................................... 19 
11.1. 
INFORMED CONSENT .............................................................................................................................................. 20 
11.2. 
CONFIDENTIALITY OF DATA AND PATIENT RECORDS ............................................................................................... 20 
12. PUBLICATIONS ......................................................................................................................... 20 
13. RETENTION OF STUDY DOCUMENTS ........................................................................................ 20 
14. FUNDING AND INSURANCE ...................................................................................................... 20 
 
 
 
 
 
 
 
 

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PROTOCOL SIGNATURE PAGE 
 
 
Protocol Title: Identifying ocular and systemic biomarkers for response to aflibercept in Asian patients 
with center involving diabetic macular edema: A prospective interventional trial  
 
 
Protocol Number: R1436/19/2017 
 
Protocol Version/ Date: Version 4.0/ 26 Feb 2019 
 
Sponsor Name: IAF_ICP (Singapore) and Santen Pharmaceutical Co., Ltd. 
 
 
 
Declaration of Investigator 
 
I confirm that I have read the above-mentioned protocol and its attachments. I agree to conduct the described 
study in compliance with all stipulations of the protocol, regulations and ICH E6 Guideline for Good Clinical 
Practice (GCP).  
 
  
 
 
SERI Principal Investigator Name:  Dr Gavin Tan Siew Wei 
                                                            
 
Principal Investigator Signature:  
 
                                                            
 
Date:  05/03/2019 
 
Santen Principal Investigator Name: Takahiro Imanaka 
 
 
Santen Principal Investigator Signature: 
 
 
 
Date: 05/03/2019 
                                                            
 
 

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1. 
BACKGROUND AND RATIONALE 
Diabetic Macular Edema is a major Public Health Problem in Singapore and Worldwide. 
Diabetes is chronic disease with significant morbidity from multi systemic complications. It is 
estimated that by 2030, more than 400 million persons worldwide will be affected by this disease. 
Diabetic retinopathy (DR) is a specific microvascular complication of diabetes and a major public 
health problem in Singapore and globally.1-4 We have led a global effort to show that there are now 
nearly 100 million people with DR, of whom nearly 30 million have vision-threatening DR, including 
diabetic macular edema (DME), now considered the major cause of vision loss in diabetes.4,5 Our 
group has made substantial discovery of novel risk factors for DR from population-based and clinic-
based studies in Singapore and around the world.1,3,4,6-20 
 
DME is a major cause of visual impairment in persons with diabetes. DME is an advanced, vision-
limiting manifestation of DR in which swelling of the central retina causes loss of central vision. The 
Wisconsin Epidemiologic Study found that the prevalence of DME increases from 0–3% in recently 
diagnosed individuals to 28–29% in those living with type 1 or 2 diabetes for at least 20 years.21-
25 Without treatment, 33% of eyes in the Early Treatment Diabetic Retinopathy study (ETDRS) with 
center involved DME experience moderate visual loss (15 or more letter decrease in visual acuity) 
over a three year period. Historically, laser photocoagulation was the gold standard treatment for 
DME based on the results of the ETDRS. However, less than 3% of treated eyes in the ETDRS 
demonstrated an improvement in visual acuity of 15 or more letters.26-32 
 
The current therapeutic approach is limited to Anti-Vascular Endothelial Growth Factor (VEGF) 
agents.  
Anti- VEGF agents such as ranibizumab, aflibercept and bevacizumab have emerged as the 
treatment of choice for center involving diabetic macular edema. The RISE and RIDE trial, 
randomized trials assessing ranibizumab for diabetic macular edema found that a significantly 
greater proportion of patients receiving ranibizumab (33.6 to 45.7%) gained 15 letter or more in best 
corrected visual acuity compared with sham injection (12.3 to 18.1%) at 2 years.33 The Diabetic 
Retinopathy Clinical Research Network (DRCRnet) demonstrated that patients treated with 
ranibizumab plus laser had significantly better visual outcomes (9±12 letter gain) compared with 
those treated with sham injections plus prompt laser (3±13 letter gain).34,35 A Prospective 
Randomized Trial of Intravitreal Bevacizumab or Laser Therapy in the Management of Diabetic 
Macular Edema (BOLT study) randomized 80 eyes from 80 study participants to intravitreal 
bevacizumab (given every six weeks with a minimum of three injections in the first 12 months) or 
macular laser treatment and found that whereas the bevacizumab group gained a median of eight 
letters in visual acuity over 12 months, the laser group lost a median of 0.5 letters over the same 
time period (P = 0.0002).36 In the Da Vinci study, subjects with DME gained up to 13.1 letters at 
one year in the 2mg aflibercept every 4 weekly group.37  
 
However, there is a significant group of DME patients who are non-responders to Anti-VEGF agents, 
and do not improve vision. In the Rise and Ride trial, 20-25% of subjects had no change (<4 letter 
change) or worsening of visual acuity.33 In the DRCRnet DME study, 28% had no significant 
improvement in vision (<4 letter improvement) at 3 years. In the DRCRnet protocol T comparing the 
efficacy of intravitreal aflibercept, bevacizumab, and ranibizumab in the treatment of diabetic 
macular edema, only 50-60% of subject benefited from a significant visual improvement of at least 
10 ETDRS letters.38 
 
There is a need to identify poor and non-responders to antiVEGF and discover novel 
biochemical/genetic biomarkers for DME.  
Accumulating evidence suggests that several overlapping pathophysiological mechanisms play an 
important role in the development of DR, including inflammation, microvascular dysfunction, 
increased oxidative stress, endothelial dysfunction and genetic factors.1 Clinical, systemic and 

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imaging features in DME patients may be able to predict the response to anti-VEGF agents. 
Mediators other than VEGF may be involved in the pathogenesis of DME, which may account for 
the poor response to anti-VEGF in these patients. Ocular and blood biomarkers of poor response 
may be potential therapeutic targets. 
 
Significance 
This study will contribute to the unmet needs of the 40 million persons worldwide with diabetic 
macular edema. Clinicians treating patients with DR are looking accessible biomarkers that will help 
them predict disease outcomes and response to therapy. They also need alternatives to anti-VEGF 
treatment for these diseases. This is the case not only for the roughly 30-40% of patients who have 
poor or suboptimal response to current therapy, but also for the patients that must undergo repeated 
intravitreal injections for control of DME. In addition to alternatives to anti-VEGF treatment, add-on 
therapeutics that can potentially enhance the effects of current front line therapies and/or prolong 
their effectiveness (thus reducing the numbers of injections) are also of considerable potential value 
to clinicians, their patients, as well as the pharmaceutical industry.  
 
 
2. 
HYPOTHESIS AND OBJECTIVES 
Aim 1: To investigate whether ocular and systemic biomarkers predict treatment response to 
intravitreal aflibercept in a cohort of patients with DME.  
Hypothesis: That novel ocular and systemic biochemical markers can predict response to intravitreal 
aflibercept in subjects with DME. 
 
3. 
EXPECTED RISKS AND BENEFITS 
The benefits to this patient will be that by studying the blood and biomarker study we will be able to 
better predict prognosis and treatment response in future patients. The recruited patient will also 
have a direct benefit in receiving free intravitreal aflibercept treatment during the course of the study. 
Risks to the patient would be the adverse events from the intravitreal injections like endophthalmitis, 
cerebral or cardiac complications, which are very rare; the risk of infection from blood which is rare; 
and the risk of minor trauma from tear collection which is uncommon. 
 
4. 
STUDY POPULATION 
4.1. 
List the number and nature of subjects to be enrolled.  
Up to 36 study participants with DME in at least 1 eye are expected to be enrolled. Study participants 
can only have one study eye. If both eyes are eligible for the study, the eye without previous 
intravitreal anti-VEGF treatment will be selected. If both eyes are treatment naïve, the eye with 
worse VA should be selected as the study eye, or the eye with worse central retinal thickness will 
be chosen as the study eye. If both the VA and CRT are equal then the right eye will be chosen. 
The non-study eye will continue to receive treatment as per current clinical standard of care. They 
can receive treatment at the same time as the study eye to ensure there is no delay of treatment in 
the non-study eye. 
 
 

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4.2. 
Criteria for Recruitment and Recruitment Process 
Potential eligibility will be assessed as part of a routine-care examination. Prior to completing any 
procedures or collecting any data that are not part of usual care, written informed consent will be 
obtained. For potential study participants who are considered potentially eligible for the study based 
on a routine-care exam, the study protocol will be discussed with the potential study participant by 
a study investigator and clinic coordinator. The potential study participant will be given the Informed 
Consent Form to read. Potential study participants will be encouraged to discuss the study with 
family members and their personal physician(s) before deciding whether to participate in the study.  
 
4.3. 
Inclusion Criteria 
      Participant 
1. Age >=21 years  
2. Diagnosis of Diabetes Mellitus (Type 1 or type 2)  
a. Current regular use of insulin or oral hypoglycemic agents for treatment of diabetes 
b. Documented diabetes by ADA and/or WHO criteria. 
3. Able and willing to provide informed consent. 
 
Study Eye 
1. Best corrected ETDRS visual acuity score <= 78 (i.e. 20/32 or worse) 
2. On clinical examination, definite retinal thickening due to diabetic macular edema involving 
the center of the macula. 
3. Diabetic macular edema present on optical coherence tomography (OCT) (central subfield 
thickness on OCT >=340um with Spectralis (Heidelberg)  
4. Media clarity, pupillary dilation and individual cooperation sufficient for study procedure 
including fundus photography. 
 
4.4. 
Exclusion Criteria 
Participant 
1. End stage renal failure requiring hemodialysis or peritoneal dialysis. 
2. Medical condition that, in the opinion of the investigator, would preclude participation in the 
study (e.g., unstable medical status including blood pressure, cardiovascular disease, and 
glycemic control).  
3. Participation in an investigational trial within 30 days of enrolment which involves treatment 
with unapproved investigational drug 
4. Known allergy to any component of aflibercept or any other medication to be used in the 
study.  
5. Blood pressure > 180/110 (systolic above 180 OR diastolic above 110 on repeated 
measurements). If blood pressure is brought below 180/110 by anti-hypertensive treatment, 
individual can become eligible.  
6. Myocardial infarction, other acute cardiac event requiring hospitalization, stroke, transient 
ischemic attack, or treatment for acute congestive heart failure within 6 months prior to 
participation to the study. 
7. Systemic anti-VEGF or pro-VEGF treatment within 6 months prior to randomization or 
anticipated use during the study.  
8. For women of child-bearing potential: pregnant or lactating or intending to become pregnant 
within the next 24 months. Women who are potential study participants should be questioned 
about the potential for pregnancy.  

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9. Patient with non-study eye VA: counting finger or worse (i.e. only one seeing eye) will be 
excluded. 
   
Study Eye 
1.  Macular edema is considered to be due to a cause other than diabetic macular edema. An 
eye should not be considered eligible if: (1) the macular edema is considered to be related 
to ocular surgery such as cataract extraction or (2) clinical exam and/or OCT suggest that 
vitreoretinal interface abnormalities (e.g., a taut posterior hyaloid or epiretinal membrane) 
are the primary cause of the macular edema.  
2. An ocular condition is present such that, in the opinion of the investigator, visual acuity loss 
would not improve from resolution of macular edema (e.g., foveal atrophy, pigment 
abnormalities, dense subfoveal hard exudates, nonretinal condition). 
3. An ocular condition is present (other than diabetes) that, in the opinion of the investigator, 
might affect macular edema or alter visual acuity during the course of the study (e.g., vein 
occlusion, uveitis or other ocular inflammatory disease, neovascular glaucoma, etc.) 
4. Substantial cataract that, in the opinion of the investigator, is likely to be decreasing visual 
acuity by more than three lines (i.e., cataract would be reducing acuity to worse than 20/40 
if eye was otherwise normal). 
5. History of an anti-VEGF treatment for DME in the past 6 months or history of any other 
treatment for DME at any time in the past 6 months (such as focal/grid macular 
photocoagulation, intravitreal or peribulbar corticosteroids). 
6. History of pan-retinal photocoagulation in the past 6 months or anticipated need for 
immediate pan-retinal photocoagulation. (e.g. Proliferative diabetic retinopathy. Cases with 
severe non-proliferative diabetic retinopathy will still be eligible) 
7. History of ocular anti-VEGF treatment for a disease other than DME in the past 6 months 
8. History of major ocular surgery (including vitrectomy, cataract extraction, scleral buckle, any 
intraocular surgery, etc.) within prior 6 months or anticipated during the study. 
9. History of YAG capsulotomy performed within two months prior to recruitment. 
10. Aphakia. 
11. Exam evidence of external ocular infection, including conjunctivitis, chalazion, or significant 
blepharitis. 
12. History of intravitreal steroids within the last 6 months. 
 
 
5. 
STUDY DESIGN AND PROCEDURES/METHODOLOGY 
This is a prospective interventional non-comparative clinical trial. Treatment received will be 
based on study design.  All study eyes will receive intravitreal aflibercept with the initial 
injection given on within 2 weeks of the screening visit and x 4 monthly injections including 
at month 3.   
 
 
 
Screening Evaluation and Baseline Testing 
 
Historical Information & Study Questionnaires & The Brief Impact of Vision Impairment Profile 
(B_IVI)  
A history will be elicited from the potential study participant and extracted from available medical 
records. Socio-demographic and medical history details via a face-to-face interview in a study 
questionnaire.  Data to be collected will include: age, gender, ethnicity and race, diabetes history 
and current management, other medical conditions, medications being used, as well as ocular 

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diseases, surgeries, and treatment.  Visual impact quality of life will also be assessed via the brief 
impact of vision impairment profile (B IVI) questionnaire.   
Baseline Testing Procedures  
 
The following procedures are needed to assess eligibility and/or to serve as baseline measures for 
the study.  
• If a procedure has been performed (using the study technique and by study certified personnel) 
as part of usual care, it does not need to be repeated specifically for the study if it was performed 
within the defined time windows specified below or within 8 days of the baseline injection otherwise 
they need to be repeated. 
 
1. 
Best-corrected Visual Acuity: 'best corrected visual acuity (BCVA) will be measured using 
the ETDRS VA protocol following manifest refraction. 
2. 
 Optical Coherence Tomography: OCT and OCT-angiography (OCTA) on both eyes will be 
performed. Both standard and enhanced depth imaging scans will be performed. 
3. 
Baseline ocular examination on each eye including slit lamp, measurement of intraocular 
pressure, lens assessment, and dilated ophthalmoscopy.  
4. 
Fundus Photography & ultra-wide field photography. 
5.         Anthropometric and blood pressure measurements 
6. 
Blood Sample: 30mls of blood sample will be obtained for HbA1c measurement as well as 
assessment of biomarkers and genetics. 
7.     Vitreous sample: 0.1-0.2 ml of vitreous humour will be obtained (detailed procedure outlined 
below) together with the first intravitreal injection at baseline. 
8.    Tears sample  
 
 
Follow-up visits and re-treatment 
 
Visit schedule 
Follow-up visits occur every 1 month (within +/- 7 days; and a minimum of 21 days between visits) 
during the first 4 months.   
 
Testing procedures 
The following procedure will be performed at each protocol visit as per the protocol schedule in table  
1. 
Best corrected visual acuity / ETDRS visual acuity 
2. 
OCT and OCT A on both eyes 
3. 
Ocular examination on both eyes including slit lamp examination, lens assessment, 
measurement of intraocular pressure and dilated ophthalmoscopy.  
4. 
Fundus photography & ultra-wide field photography 
6.         Anthropometric and blood pressure measurements  
7.     Vitreous sample: 0.1-0.2 ml of vitreous humour will be obtained (detailed procedure outlined 
below) at the month 3 visit together with the 4th intravitreal injection. 
8.     Tears sample 
    
 
 
Treatment during follow- up 
All subjects will receive an intravitreal anti-VEGF injection monthly for a total of 4 mandatory 
intravitreal treatments.  

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Response to aflibercept will be assess based on CRT and VA at 4 months 
 
Considerations in follow-up 
 
Endophthalmitis  
Diagnosis of endophthalmitis is based on investigator’s judgment. In the case of suspected 
endophthalmitis, the patient will be immediately referred to the retina and uveitis team for 
management. This will include obtaining cultures of vitreous and/or aqueous fluid, expedient 
intravitreal antibiotic treatment for presumed endophthalmitis, and vitrectomy where clinically 
indicated. The cost of treatment for endophthalmitis will be covered by the study institution. 
 
Surgery for Vitreous Hemorrhage and Other Complications of Diabetic Retinopathy  
A study eye could develop a vitreous hemorrhage and/or other complications of diabetic retinopathy 
that may cause visual impairment. The timing of vitrectomy for the complications of proliferative 
diabetic retinopathy such as vitreous hemorrhage is left to investigator discretion. 
 
Panretinal Photocoagulation  
Panretinal photocoagulation (PRP) can be given if it is indicated in the judgment of the investigator. 
In general, PRP should not be given if the study participant has less than severe non-proliferative 
diabetic retinopathy. In general, PRP should be given promptly for previously untreated eyes 
exhibiting proliferative diabetic retinopathy with high-risk characteristics and can be considered for 
persons with non high-risk proliferative diabetic. 
 
Study Participant Withdrawal and Losses to Follow-up  
A study participant has the right to withdraw from the study at any time. If a study participant is 
considering withdrawal from the study, the principal investigator should personally speak to the 
individual about the reasons, and every effort should be made to accommodate him or her.  
 
Study participants who withdraw will be asked to have a final closeout visit at which the testing 
described for the protocol visits will be performed. Study participants who have an adverse effect 
attributable to a study treatment or procedure will be asked to continue in follow-up until the adverse 
event has resolved or stabilized.  
 
   
Treatment details 
 
Intravitreal Injections 
 
Anti-VEGF agent 
Study eyes will receive a dose of 2.0mg in 0.05 ml of Aflibercept (Eylea)  
 
Intravitreal Injection Technique  
Antibiotics in the pre-, peri-, or post-injection period are not necessary and will not be prescribed in 
this study. 
 
Prior to the injection, the study eye will be anaesthetized with topical anesthetic, followed by a 
povidone iodine prep of the conjunctiva. (Instill 5% povidone iodide on to the ocular surface and 
allow adequate time prior to injection)  
 

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The injection will be performed using sterile technique. Investigator will use a surgical hand 
disinfection technique and wear sterile gloves. Periocular skin and eyelid margins and eye lashes 
will be cleaned with 5-10% povidone iodine. 
Skin will be dried and drape applied. Investigator will insert eyelid speculum, ensuring that it is well 
positioned underneath the eyelids to direct the eyelashes away from the field. Callipers should be 
used to mark the injection site. The entry site of the needle should be 3.0-3.5 mm from the limbus 
in pseudophakic patients, and 3.5-4.0 mm in phakic patients.  
 
The conjunctiva may be displaced anteriorly using either forceps or cotton tipped applicator so that 
no direct route between vitreous and ocular surface remains. The needle is inserted perpendicular 
through sclera with the tip aimed towards the centre of the globe (to avoid any contact with the 
posterior lens). A vitreous tap will be performed at baseline and before the month 3 injection where 
0.1-0.2 ml of the vitreous will be drawn out before injection of the intravitreal medication.  
 
IOP measurement post-injection is not mandatory. While small volume injections (0.05ml) are 
unlikely to cause IOP rise, it should be considered inpatients with ocular hypertension or glaucoma, 
and in all cases where patients are symptomatic for pain or reduced vision immediately following 
injection. Should a high intraocular pressure resulting in non-perfusion of the central retinal artery 
occur, indicated by no perception of light (NPL) in the treated eye, an anterior chamber paracentesis 
is indicated. Such decompression needs to be achieved within 3-5 minutes. Patients should be 
instructed to report any symptoms regarding eye pain or discomfort, increased redness of the eye, 
or additional blurring of vision (which may indicate endophthalmitis) to the treating ophthalmologist 
without delay. 
 
 
Vitreous and tears collection procedures    
Vitreous Tap 
Done at baseline injection and repeated with month 3 injection. Subconjunctival lignocaine  
anaesthesia. Sterile caliper was used to mark 4.0 mm and 3.5 mm posterior to the corneoscleral 
limbus for phakic and pseudophakic patients, respectively. A 27 or 25-gauge, 5/8-inch needle will 
be introduced through the marked site into the mid vitreous cavity. 0.1-0.2 mL of vitreous fluid will 
be gently aspirated into a 1-mL or 3-mL syringe. Procedure will be abandoned if it is a dry tap 
(<0.05ml of vitreous aspirated).  
 
Vitreous Humour samples should be collected into Protein Lo bind Eppendorf tubes. The sample 
will be placed on ice and centrifuge at 5000 rpm, 4°C for 10 minutes (Only for samples with 
debris/blood). The supernatant will be transferred into a new Lo bind Eppendorf tube labelled 
correctly* (Sample volume will be measured). The sample in the tube will be snap-freeze sample in 
dry ice / liquid nitrogen, after which it will be stored at -80°C promptly. 
 
 
Tears Collection  
Done at baseline injection and repeated with month 4. Tears will be collected using a 5x35mm strip 
of filter paper will be placed in the lower lid with your eyes closed, and kept in place for 5 minutes. 
Special care will be taken not to touch the lid margin or ocular surface. 
Storage and Disposal 

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All samples of blood, vitreous fluid and tear will be collected into sterile tubes, placed on ice, 
centrifuged to remove cells and debris, and stored at –80°C for up to 15 years until analysis at SERI. 
Unused samples will be stored indefinitely for future research as permitted by the IRB. Waste 
samples will undergo proper disposal as per SERI biohazard waste requirements. 
 
Laboratory analysis  
Samples will undergo proteomic, metabolomic and cytokine discovery assessment using, mass 
spectrometry, gas and liquid chromatography as well as ELISA based platforms.  
In the analyses, proteomic analysis of vitreous will be conducted as collaborative research between 
SERI and Santen for the purpose of biomarker investigation. The protein analysis will be conducted 
by service provider outside of Singapore under the contract research agreement with SERI and 
Santen. Preliminary test will be conducted to evaluate detectability of proteins by technology of the 
service provider. Vitreous fluid which was obtained and stored in SERI with the protocol of 
R1256/62/2015 (title: Ocular Biomarker Study for Retinal Angiogenic Diseases) will be used for the 
preliminary test. 
 
Delay in Giving Injections  
If a scheduled injection is not given by the end of the visit window, it can still be given up to one 
week prior to the next visit window opening. If it is not given by that time, it will be considered missed. 
If an injection is given late, the next scheduled injection should occur no sooner than three weeks 
after the previous injection. 
 
Non-Study Eye Injections  
The non-study eye will continue to receive treatment as per current clinical standard of care. They 
can receive treatment at the same time as the study eye to ensure there is no delay of treatment in 
the non-study eye. If the non-study eye is going to be treated for any condition which requires 
treatment with an anti-VEGF agent, where possible, the non-study eye should be treated with the 
same antiVEGF as the study eye.  
 
 
 
Focal/Grid Photocoagulation  
If focal/grid photocoagulation will be deferred until after completion of the month 4 visit.  
 
 
Discontinuation of Study  
The study may be discontinued by the Data and Safety Monitoring Committee [DSMC] prior to the 
preplanned completion of follow-up for all study participants. 
 
Study Participant Reimbursement  
The study will be providing the study participant with $50 per completed protocol visit to cover travel 
and other visit-related expenses.  
 
 

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6. 
SAFETY MEASUREMENTS 
6.1. 
Definitions 
An adverse event (AE) is any untoward medical occurrence in a patient or clinical investigation 
subject administered a pharmaceutical product and which does not necessarily have a causal 
relationship with this treatment. 
 
Recording of Adverse Events  
Throughout the course of the study, all efforts will be made to remain alert to possible adverse 
events or untoward findings. The first concern will be the safety of the study participant, and 
appropriate medical intervention will be made.  
All adverse events whether volunteered by the subject, discovered by study personnel during 
questioning, or detected through physical examination, laboratory test, or other means will be 
reported to the study committee. 
The study investigator will assess the relationship of any adverse event to be related or unrelated 
by determining if there is a reasonable possibility that the adverse event may have been caused by 
the treatment (including treatment of the non-study eye with study treatment). 
Adverse events that continue after the study participant’s discontinuation or completion of the study 
will be followed until their medical outcome is determined or until no further change in the condition 
is expected. 
Reporting Serious or Unexpected Adverse Events  
A serious adverse event (SAE) or reaction is any untoward medical occurrence that : 
• 
results in death 
• 
is life-threatening; (a non life-threatening event which, had it been more severe, might 
have become life-threatening, is not necessarily considered a serious adverse 
event). 
• 
requires inpatient hospitalisation or prolongation of existing hospitalisation 
• 
results in persistent or significant disability/incapacity or 
• 
is a congenital anomaly/birth defect 
• 
is a medical event that may jeopardize the patient and may require medical or 
surgical intervention to prevent one of the outcomes listed above. 
 
Unexpected adverse events are those that are not identified in nature, severity, or frequency in the 
current study protocol 
 
 
6.2. 
Collecting, Recording and Reporting of Adverse Events and Serious Adverse Events 
to CIRB 
 
Reporting of adverse events involves the PI submitting to the approving CIRB the completed SAE 
Reporting Form within the stipulated timeframe. PI is responsible for informing the institution 
representative (local SAE resulting in death), sponsor or regulatory bodies as required and 
appropriate. 
 

DME Protocol Version 4 Dated 26 Feb 2019 
Page 14 of 21 
Reporting timeline to CIRB:  
 
• 
SAE that result in death, regardless of causality, should be reported immediately - 
within 24 hours of the PI becoming aware of the event.  
• 
Local life-threatening (unexpected/ expected) SAE should be reported no later than 
7 calendar days after the Investigator is aware of the event, followed by a complete 
report within 8 additional calendar days. 
• 
Local unexpected SAE that are related events, but not life-threatening, should be 
reported no later than 15 calendar days after the investigator is aware of the event. 
• 
An increase in the rate of occurrence of local expected SAE, which is judged to be 
clinically important, should be reported within 15 calendar days after the PI is aware 
of the event.  
• 
Local expected SAE should be reported annually (together with Study Status Report 
for annual review). 
• 
Local unexpected and unlikely related SAE that are not life-threatening should also 
be reported annually (together with Study Status Report for annual review). 
• 
Local unexpected AE that are related events should be reported at least annually 
(together with Study Status Report for annual review). 
• 
Non-local 
unexpected 
SAE 
that 
are 
fatal 
or 
life 
threatening 
and 
definitely/probably/possibly related should be reported not later than 30 calendar 
days after the PI is aware of the event. 
 
6.3. 
Safety Monitoring Plan 
A Data and Safety Monitoring Committee (DSMC) will approve the protocol, template 
informed consent form, and substantive amendments and provide independent monitoring 
of adverse events. Cumulative adverse event data are semi-annually tabulated for review 
by the DSMC. Following each DSMC data review, a summary will be made available for 
submission to Institutional Review Board. A list of specific adverse events to be reported to 
the DSMC expeditiously will be compiled and included as part of the DSMC Standard 
Operating Procedures. 
 
6.4. 
Complaint Handling  
Complaints will be handled by the research co-ordinators and if required the Quality 
Assurance team at the Singapore National Eye Centre  
 
7. 
DATA ANALYSIS 
7.1. 
Data Quality Assurance 
Quality control and quality assurance will be conducted in this study. The Investigator(s)/ 
Singapore National Eye Centre will permit study-related monitoring audits, Medical Clinical 
Research Committee (MCRC) and or Ethics Committee (EC) review and regulatory 
inspection(s), providing direct access to source data/ document. 
 
7.2. 
Data Entry and Storage 
Research data should be stored in a password-protected database on the SERI shared 
drive, accessed via a password-protected delegated staff. As for the hard copies, it will be 
stored in academia, in a locked cabinet within SERI. 

DME Protocol Version 4 Dated 26 Feb 2019 
Page 15 of 21 
All investigators, coordinators as well as the Database Management team declared in this 
CIRB application will have access to the research data. The research coordinator will grant 
access to the research data, and the requestor's information will first be noted in hard copy 
and filed. 
 
8. 
SAMPLE SIZE AND STATISTICAL METHODS 
8.1. 
Determination of Sample Size  
Based on an outcome of 33% non-responders (<20% reduction of CRT on OCT), a sample 
of 30 DME patients will 10 non responders which will enable as to identify biomarkers for 
further validation studies. Up to 36 patients will be enrolled based on expectation of 20% of 
patients will have a dry vitreous tap. 
 
8.2. 
Main Statistical and Analytical Plans 
The proportion of study patients achieving positive response in primary outcome will be 
estimated with 95% confidence interval. Summary statistics will be tabulated to describe 
patient demographics and characteristics. Univariate logistic regression will be performed to 
assess the effects of individual biomarkers, and multiple logistic regression analyses to 
assess associations between biomarkers and treatment outcome while adjusting for 
potential confounders. Some of exploratory analysis will be conducted to evaluate 
biomarkers. Incidence rates of adverse events will be computed as the number of events 
divided by the number of person-years at risk, and the events described in detail in the paper. 
 
9. 
DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
The SERI’s investigator(s)/institution(s) will permit study-related monitoring, audits and/or 
IRB review and regulatory inspection(s), providing direct access to source data/document. 
10. 
QUALITY CONTROL AND QUALITY ASSURANCE 
Quality control and quality assurance will be conducted in this study. The Investigator(s)/ 
Singapore National Eye Centre will permit study-related monitoring audits, MCRC and or EC 
review and regulatory inspection(s), providing direct access to source data/ document. 
 
11. 
ETHICAL CONSIDERATIONS 
This study will be conducted in accordance with the ethical principles that have their origin 
in the Declaration of Helsinki and that are consistent with the Good Clinical Practice, 
Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects 
and the applicable regulatory requirements. 
 
This final study protocol, including the final version of the Patient Information and Informed 
Consent Form, must be approved in writing by the Centralised Institutional Review Board 
(CIRB), and Ethical Review Committee in Japan prior to enrolment of any patient into the 
study. 
 
The principle investigator is responsible for informing the CIRB of any amendments to the 
protocol or other study-related documents, as per local requirement. 
 
Conflicts of interest (COI) 
This study is funded by IAF_ICP (Singapore) and Santen Pharmaceutical Co., Ltd. 
COIs of SERI researchers are deliberated by IRB in Singapore.  
Employees of Santen pharmaceutical Co., Ltd. are involved in this research. 

DME Protocol Version 4 Dated 26 Feb 2019 
Page 16 of 21 
11.1. Informed Consent 
Potential eligibility will be assessed as part of a routine-care examination. Prior to completing 
any procedures or collecting any data that are not part of usual care, written informed 
consent will be obtained. For potential study participants who are considered potentially 
eligible for the study based on a routine-care exam, the study protocol will be discussed with 
the potential study participant by a study investigator and clinic coordinator. The potential 
study participant will be given the Informed Consent Form including the following items to 
read. Potential study participants will be encouraged to discuss the study with family 
members and their personal physician(s) before deciding whether to participate in the study. 
(1) Title of the research and the fact that approval of the chief executive of the research 
implementing entity has been given concerning its implementation; 
(2) Names of the research implementing entity and the principal investigator (including 
names of the collaborative research implementing entity(s) and principal investigators 
of such collaborative research implementing entity(s), when the research is conducted 
collaboratively with other research implementing entity(s)); 
(3)  Purpose of the research study 
(4) Study procedure and visit schedule: Method and time period of the research (including 
purpose of the utilization of specimens or information acquired from the research 
subject; administration of study drug and description of study procedures) 
(5) Patient’s responsibilities in this study 
(6) What is not standard care or experimental in this study; 
(7) Possible risks, discomforts and inconveniences  
 (8) Potential benefits: when the research involves any financial expenditure on or 
remuneration for the research subject, etc., a statement to that effect and details of 
such; 
(9) Important information for women participant  
(10) Alternatives: When the research involves any medical technique beyond usual 
medical practice, description of alternative procedure(s) or course(s) of treatment; 
(11) Costs of participation  
(12) Participant’s rights: The fact that research subjects, etc. may withdraw their consent 
at any time even after they have given consent with regard that the research is 
commenced or continued (when it can be difficult to take measures that follow the 
withdrawal made by the research subject, etc., a statement to that effect and the reason 
for the difficulty); 
 (13) Withdrawal from study : The fact that the refusal or withdrawal of consent by a 
research subject, etc. with regard that the research is to be commenced or continued 
does not cause any disadvantage to such research subject, etc. 
(14) Research related injury and compensation: When the research involves any 
invasiveness, whether or not compensation will be offered for research-related injury 
and details of such compensation; 
(15) Confidentiality of study and medical records  
(16) Who to contact if you have questions regarding the study  
(17) Who has reviewed the study 
 
 
 
11.2. Confidentiality of Data and Patient Records 
All personal information and biological materials will be handled in a manner that is 
consistent with the Singapore Personal Data Protection Act (PDPA) and the Singapore 
Human Biomedical Research Act. Only personnel directly involved in the study will have 
access to study information. All Data collected will be anonymised by the SERI Data 

DME Protocol Version 4 Dated 26 Feb 2019 
Page 17 of 21 
Management group with the identify key kept separate on a password protected computer 
at SERI which cannot be accessed without the SERI’s PI consent.  
Anonymised clinical information and laboratory analysis results will be transferred to Santen 
only for the purpose of vitreous biomarker investigation. Santen and their affiliate subsidiary 
will only have access to the anonymised data. The following documents will be substituted 
for data transfer records. 
Data collected from each site will be anonymized before sending to the SERI internal data 
management team for data entry into electronic system. All records will be accessible for 
inspection and copying by authorized authorities. The research data will be stored for at least 
15 years and then deleted/destroyed. 
l 
Documents stored at SERI 
Ø 
Study protocol and project agreement as records of items provided to Santen. List 
of the items will be prepared as additional document when the items are transferred, 
and then stored at SERI. 
Ø 
Informed consent form as records of patient’s information and his/her consent 
l 
Documents stored at Santen 
Ø 
Study protocol and project agreement as records of items provided from SERI and 
procedure of data acquisition. List of the items will be prepared as additional 
document when the items are transferred, and then stored at Santen. 
The study related documents at Santen will be stored for five years after the final report of 
this study or three years of the final publication, whichever is later, and then destroyed or 
deleted. 
Data collected from each site will be anonymized before sending to the SERI internal data 
management team for data entry into electronic system. All records will be accessible for 
inspection and copying by authorized authorities. The research data will be stored for at least 
15 years and then deleted/destroyed. 
 
 
12. 
PUBLICATIONS 
This study will adhere to the Singapore Eye Research Institute and Santen Master Research 
Collaboration Agreement (DATED the 10th day of October 2014) publication policy. 
 
Report 
When research is finished (including the case of discontinuance), the principal investigator 
of the research shall report to the chief executive of the research institute.  
 
Clinical Research Database 
This 
study 
will 
be 
registered 
on 
JAPIC 
iyakuSearch 
database 
(http://database.japic.or.jp/is/top/index.jsp) 
in 
Japan, 
ClinicalTrial.gov 
(https://clinicaltrials.gov/), 
and 
Health 
Sciences 
Authority  
(http://www.hsa.gov.sg/content/hsa/en.html). 
 
 
13. 
RETENTION OF STUDY DOCUMENTS 
The research data will be stored for at least 15 years and then destroyed/deleted. 
 

DME Protocol Version 4 Dated 26 Feb 2019 
Page 18 of 21 
14. 
FUNDING and INSURANCE 
The study will be funded by IAF_ICP (Singapore) and Santen Pharmaceutical Co., Ltd.  
This is an investigator initiated trial which will be covered by the institutional clinical trial 
insurance. 
 
 
                                      
List of Attachments: 
 
 
 
 
 
 
 
  
Month 
0 
Month 1 
Month 2 
Month 3 
Month 4 
Clinic consultation 
(Follow-up visit)  
X 
X 
X 
X 
X 
Refraction 
X 
X 
X 
X 
X 
Study 
Questionnaires  
X 
 
 
 
 
B_IVI 
X 
 
 
 
 
OCT & OCTA 
X 
X 
X 
X 
X 
Fundus 
Photography (For 
DR Grading) 
X 
 
 
 
X 
HbA1c 
X 
 
 
 
 
Blood Pressure 
X 
X 
X 
X 
X 
Blood specimen 
X 
 
 
 
 
Tears collection 
X 
 
 
 
X 
Vitreous tap 
X 
 
 
X 
 
Intravitreal 
Aflibercept 
X 
X 
X 
X 
 

DME Protocol Version 4 Dated 26 Feb 2019 
Page 19 of 21 
 
References 
1. 
Cheung, N., Mitchell, P. & Wong, T.Y. Diabetic retinopathy. Lancet 376, 124-136 (2010). 
2. 
Lamoureux, E.L. & Wong, T.Y. Diabetic retinopathy in 2011: further insights from new epidemiological studies 
and clinical trials. Diabetes care 34, 1066-1067 (2011). 
3. 
Mohamed, Q., Gillies, M.C. & Wong, T.Y. Management of diabetic retinopathy: a systematic review. JAMA 298, 
902-916 (2007). 
4. 
Yau, J.W., et al. Global prevalence and major risk factors of diabetic retinopathy. Diabetes care 35, 556-564 
(2012). 
5. 
Ding, J. & Wong, T.Y. Current epidemiology of diabetic retinopathy and diabetic macular edema. Current 
diabetes reports 12, 346-354 (2012). 
6. 
Cugati, S., et al. Five-year incidence and progression of vascular retinopathy in persons without diabetes: the 
Blue Mountains Eye Study. Eye (London, England) 20, 1239-1245 (2006). 
7. 
Klein, R., Klein, B.E., Moss, S.E. & Wong, T.Y. The relationship of retinopathy in persons without diabetes to 
the 15-year incidence of diabetes and hypertension: Beaver Dam Eye Study. Transactions of the American 
Ophthalmological Society 104, 98-107 (2006). 
8. 
Lim, L.S., et al. C-reactive protein, body mass index, and diabetic retinopathy. Investigative ophthalmology & 
visual science 51, 4458-4463 (2010). 
9. 
Nguyen, T.T., et al. Inflammatory, hemostatic, and other novel biomarkers for diabetic retinopathy: the multi-
ethnic study of atherosclerosis. Diabetes Care 32, 1704-1709 (2009). 
10. 
Sasongko, M.B., et al. Serum apolipoprotein AI and B are stronger biomarkers of diabetic retinopathy than 
traditional lipids. Diabetes Care 34, 474-479 (2011). 
11. 
Sasongko, M.B., et al. Serum apolipoproteins are associated with systemic and retinal microvascular function in 
people with diabetes. Diabetes 61, 1785-1792 (2012). 
12. 
Sasongko, M.B., et al. Novel versus traditional risk markers for diabetic retinopathy. Diabetologia 55, 666-670 
(2012). 
13. 
See, J.L., Wong, T.Y. & Yeo, K.T. Trends in the pattern of blindness and major ocular diseases in Singapore 
and Asia. Ann Acad Med Singapore 27, 540-546 (1998). 
14. 
Tapp, R.J., et al. Longitudinal association of glucose metabolism with retinopathy: results from the Australian 
Diabetes Obesity and Lifestyle (AusDiab) study. Diabetes care 31, 1349-1354 (2008). 
15. 
Wong, T.Y., et al. Prevalence and risk factors for diabetic retinopathy: the Singapore Malay Eye Study. 
Ophthalmology 115, 1869-1875 (2008). 
16. 
Wong, T.Y., et al. Three-year incidence and cumulative prevalence of retinopathy: the atherosclerosis risk in 
communities study. American journal of ophthalmology 143, 970-976 (2007). 
17. 
Wong, T.Y., et al. Relation between fasting glucose and retinopathy for diagnosis of diabetes: three population-
based cross-sectional studies. Lancet 371, 736-743 (2008). 

DME Protocol Version 4 Dated 26 Feb 2019 
Page 20 of 21 
18. 
Wong, T.Y., Mohamed, Q., Klein, R. & Couper, D.J. Do retinopathy signs in non-diabetic individuals predict the 
subsequent risk of diabetes? Br J Ophthalmol 90, 301-303 (2006). 
19. 
Wong, T.Y., et al. Rates of progression in diabetic retinopathy during different time periods: a systematic review 
and meta-analysis. Diabetes care 32, 2307-2313 (2009). 
20. 
Zheng, Y., et al. Prevalence and risk factors of diabetic retinopathy in migrant Indians in an urbanized society in 
Asia: the Singapore Indian eye study. Ophthalmology 119, 2119-2124 (2012). 
21. 
Klein, R., Klein, B.E., Moss, S.E. & Cruickshanks, K.J. The Wisconsin Epidemiologic Study of Diabetic 
Retinopathy. XV. The long-term incidence of macular edema. Ophthalmology 102, 7-16 (1995). 
22. 
Klein, R., Klein, B.E., Moss, S.E., Davis, M.D. & DeMets, D.L. The Wisconsin epidemiologic study of diabetic 
retinopathy. IV. Diabetic macular edema. Ophthalmology 91, 1464-1474 (1984). 
23. 
Klein, R., Klein, B.E., Moss, S.E., Davis, M.D. & DeMets, D.L. The Wisconsin epidemiologic study of diabetic 
retinopathy. II. Prevalence and risk of diabetic retinopathy when age at diagnosis is less than 30 years. Arch 
Ophthalmol 102, 520-526 (1984). 
24. 
Klein, R., Lee, K.E., Gangnon, R.E. & Klein, B.E. The 25-year incidence of visual impairment in type 1 diabetes 
mellitus the wisconsin epidemiologic study of diabetic retinopathy. Ophthalmology 117, 63-70 (2010). 
25. 
Klein, R., Lee, K.E., Knudtson, M.D., Gangnon, R.E. & Klein, B.E. Changes in visual impairment prevalence by 
period of diagnosis of diabetes: the Wisconsin Epidemiologic Study of Diabetic Retinopathy. Ophthalmology 
116, 1937-1942 (2009). 
26. 
Early Treatment Diabetic Retinopathy Study design and baseline patient characteristics. ETDRS report number 
7. Ophthalmology 98, 741-756 (1991). 
27. 
Early photocoagulation for diabetic retinopathy. ETDRS report number 9. Early Treatment Diabetic Retinopathy 
Study Research Group. Ophthalmology 98, 766-785 (1991). 
28. 
Photocoagulation for diabetic macular edema. Early Treatment Diabetic Retinopathy Study report number 1. 
Early Treatment Diabetic Retinopathy Study research group. Arch Ophthalmol 103, 1796-1806 (1985). 
29. 
Treatment techniques and clinical guidelines for photocoagulation of diabetic macular edema. Early Treatment 
Diabetic Retinopathy Study Report Number 2. Early Treatment Diabetic Retinopathy Study Research Group. 
Ophthalmology 94, 761-774 (1987). 
30. 
Grading diabetic retinopathy from stereoscopic color fundus photographs--an extension of the modified Airlie 
House classification. ETDRS report number 10. Early Treatment Diabetic Retinopathy Study Research Group. 
Ophthalmology 98, 786-806 (1991). 
31. 
Focal photocoagulation treatment of diabetic macular edema. Relationship of treatment effect to fluorescein 
angiographic and other retinal characteristics at baseline: ETDRS report no. 19. Early Treatment Diabetic 
Retinopathy Study Research Group. Arch Ophthalmol 113, 1144-1155 (1995). 
32. 
Fundus photographic risk factors for progression of diabetic retinopathy. ETDRS report number 12. Early 
Treatment Diabetic Retinopathy Study Research Group. Ophthalmology 98, 823-833 (1991). 
33. 
Nguyen, Q.D., et al. Ranibizumab for diabetic macular edema: results from 2 phase III randomized trials: RISE 
and RIDE. Ophthalmology 119, 789-801 (2012). 

DME Protocol Version 4 Dated 26 Feb 2019 
Page 21 of 21 
34. 
Diabetic Retinopathy Clinical Research, N., et al. Randomized trial evaluating ranibizumab plus prompt or 
deferred laser or triamcinolone plus prompt laser for diabetic macular edema. Ophthalmology 117, 1064-1077 
e1035 (2010). 
35. 
Diabetic Retinopathy Clinical Research, N., et al. Intravitreal ranibizumab for diabetic macular edema with 
prompt versus deferred laser treatment: three-year randomized trial results. Ophthalmology 119, 2312-2318 
(2012). 
36. 
Rajendram, R., et al. A 2-year prospective randomized controlled trial of intravitreal bevacizumab or laser 
therapy (BOLT) in the management of diabetic macular edema: 24-month data: report 3. Arch Ophthalmol 130, 
972-979 (2012). 
37. 
Do, D.V., et al. One-year outcomes of the DA VINCI Study of VEGF Trap-Eye in eyes with diabetic macular 
edema. Ophthalmology 119, 1658-1665 (2012). 
38. 
Diabetic Retinopathy Clinical Research, N., et al. Aflibercept, bevacizumab, or ranibizumab for diabetic macular 
edema. The New England journal of medicine 372, 1193-1203 (2015).