Correlation of Vascular Endothelial Growth Factor Levels in Anterior Chamber Fluid to Disease State in Patients With Retinal Vein Occlusion Receiving Standard of Care Treatment
Phase: PHASE4
Conditions: Retinal Vein Occlusion
Sponsor: Wake Forest University Health Sciences

VEGF Levels in RVO
IRB00064405
IRB Approval Date 01FEB23

1 
Study Title: Correlation of Vascular Endothelial Growth 
Factor Levels in Anterior Chamber Fluid to Disease State in 
Patients with Retinal Vein Occlusion Receiving Standard of 
Care Treatment 
Principal Investigator -  Mark H. Nelson, MD MBA, Assistant    
Professor of Ophthalmology, Wake Forest Baptist Health 
Co-Investigator: Rebecca Sappington, PhD, Associate 
Professor of Neurobiology and Anatomy, Vice Chair of 
Research, Department of Ophthalmology, Wake Forest 
Baptist Health. 
Co-investigator: Sally S. Ong, MD Assistant Professor of 
Ophthalmology, Wake Forest Baptist Health 
Co-investigator: Joseph Rigdon, PhD, Department of 
Biostatistics and Data-Science, Wake Forest Baptist Health 
Sponsor or funding source: Wake Forest Baptist Health: 
Surgery Ophthalmology  
Background, Rationale and Context 
     Retinal vein occlusion (RVO) is the second most common retinal vascular 
disease after diabetic retinopathy. It presents as a Branch retinal vein 
occlusion (BRVO) or a Central retinal vein occlusion (CRVO). The BRVO is four 
times more common as a CRVO. The pathophysiology of RVO involves the 
intraluminal thrombus formation, which may be associated with systemic 
conditions such as hypertension, hyperlipidemia, diabetes and abnormal 
thrombophilia. The blockage of venous circulation causes an elevation of 
intraluminal pressure in the capillaries, leading to hemorrhages and leakage 
of fluid within the retina. There is associated reduction of retinal perfusion as 
well, which leads to the secretion of vascular endothelial growth factor 
(VEGF). VEGF therefore has a leading role in RVO and leads to the clinical 
finding of macular edema which limits vision. The current treatment for RVO 

 
2 
 
is anti-VEGF therapy, injected into the vitreous, which has largely replaced 
macular photocoagulation. Randomized controlled trials (VIBRANT for BRVO 
(1) and  GALILEO/COPERNICUS (2, 3) for CRVO) have shown efficacy and safety 
with aflibercept (Eylea) injections. It would be advantageous to be able to 
measure VEGF  since our RVO treatment paradigms depend on initiating 
therapy with anti-VEGF drugs, as well as maintenance therapy which 
continues to suppress its production.  
      
     The purpose of this project is to treat patients with RVO with standard of 
care anti-VEGF drug Eylea (aflibercept)  and to then correlate levels of VEGF 
in the ocular fluids to disease state manifested by retinal, specifically macular,  
thickening, which can be objectively measured by Optical Coherent 
Tomography (OCT). The VEGF levels will be measured by ELISA, a laboratory 
test which will be performed by Dr. Sappington. i.e.In addition, further 
evaluation into the cohort of patients (10%) who have persistent exudation or 
who require monthly Eylea will be studied.  ELISA multiplex will be run 
simultaneously with the VEGF analysis to evaluate other proteins in the ocular 
fluids of patients with RVO in order to determine if other classes of cytokines 
are simultaneously responsible for the pathophysiologic exudative process 
found in RVO.           
 
Study Goal and Objective 
      
     The study hypothesis is that measuring VEGF levels from anterior chamber 
samples from paracentesis prior to Eylea (aflibercept) injections will create a 
improved paradigm for treatment which currently looks at OCT measurements  
of foveal thickness and the number of injections.   
 
Overview of Study Design       
      
     Patients will receive standard of care intravitreal injections of Eylea 
(aflibercept) over the course of 52 weeks while undergoing a pre-treatment 
paracentesis at each study visit. Anterior chamber fluid removed from the eye 
during the paracentesis will be used to measure VEGF levels as well as other 
cytokines.  
           

 
3 
 
     The anterior chamber fluid taken at each visit will be divided for both the 
ELISA VEGF analysis immediately and a portion to be frozen for further 
cytokine analysis in the future. 
      
     All study activities will occur at Wake Forest Baptist Health Highland Oaks, 
an outpatient clinic that is part of the Department of Surgery Ophthalmology,  
expect for the ELISA which will be done in a lab located at the Medical Center 
by Dr. Sappington. 
      
Methods and Study Design 
      
1. Recruitment and Consent      
     Recruitment will occur in an outpatient Ophthalmology clinic as patients 
are receiving eye exams for ocular related issues. Patients that are diagnosed 
with BRVO or CRVO will be asked by the Principal Investigator if they are 
interested in learning more about a research study related to their diagnosis. 
Patients that show interest will be presented with the study consent and the 
study will be reviewed with the patients. During this time patients will be 
provided time to review the consent and discuss the study with their families, 
friends or health care providers. Questions will be answered and any concerns 
will be addressed prior to obtaining consent. Patients will be informed that if 
they decide not to participate they will still receive treatment for any eye 
related issues. Other treatment options will be discussed with the patient by 
the Principal Investigator. 
 
    Patient data will not be collected for study related purposes until informed 
consent is obtained. Any data used to contact potential subjects prior to 
obtaining consent will be maintained in the electronic medical record.      
 
 
2. Screening 
     Informed consent will be administered prior to any study related activities 
being performed.  
      
     During the screening phase medical history will be reviewed to determine 
eligibility. All eligibility criteria must be met before a patient can move forward 
with study treatment and testing of VEGF levels.  

 
4 
 
      
     Patients that do not meet enrollment criteria (screen-failed patients) will 
be offered standard of care treatment. 
      
3.Treatment Period and Visit Schedule 
     Study assessments, procedures and standard of care treatment will be 
started on Day 1 for patients successfully enrolled into the clinical trial and 
continue through week 52.  
      
     If a patient is experiencing an ocular issue they will be instructed to 
contact the study doctor to determine if an additional follow up visit is needed.  
 
     The study will end at week 52. There will a need to continue treatment 
since anti-VEGF drugs are not a cure. This indefinite period where Eylea is 
continued will not be paired with saving  anterior chamber samples.  
      
4. Subject Selection Criteria      
     This is a non-randomized exploratory pilot study that will enroll 
approximately 20 subjects; 10 subjects with BRVO and 10 subjects with 
CRVO.  
      
Inclusion Criteria: 
1. Willingness and ability to provide written informed consent 
2. Age ≥18 years 
3. Diagnosis of Retinal Vein Occlusion with macular edema and central 
foveal thickness of ≥300 microns confirmed by intravenous fluorescein 
angiography and Optical Coherence Tomography 
4. Visual Acuity between 20/25 and 5/200 
      
Exclusion Criteria: 
1. Bilateral Retinal Vein Occlusion 
2. Vision worse than 5/200 in study eye. 
3. History of myocardial infarction, ischemia, or cerebrovascular accident 
within 6 weeks of screening  
4. Concurrent Proliferative Diabetic Retinopathy and/or Maculopathy 
5. Concurrent Exudative Age-related Macular Degeneration 

 
5 
 
6. Concurrent optic neuropathy with the presence of an afferent pupillary 
defect 
7. Previous vitrectomy in the study eye 
8. Currently pregnant or planning to become pregnant during the duration 
of the study. Women currently breastfeeding are also excluded  
9. Previous treatment for retinal vein occlusion in the study eye 
10. 
Any current medical condition which, in the opinion of the 
investigator is considered to be uncontrolled 
11. 
History of allergy or hypersensitivity to study treatment, 
fluorescein, or any study procedure and treatment related ingredients 
(e.g. topical anesthetics, betadine, etc.)  
12. 
If any of these conditions start after the initiation of the study, 
the patient will not be allowed to participate in the study, however, will 
be offered the treatment as described above.  
      
      
5. Study Treatment 
     The recommended dose of aflibercept for the treatment of RVO is 2mg (0.05 
mL). All subjects will be administered an intravitreal injection of 2mg (0.05 mL) 
aflibercept in a single-dose pre-filled syringe. All intravitreal injections will be 
performed by the Principal Investigator. 
      
 
      
6. Study Assessments and Procedures 
     During the screening phase, after consent has been obtained, a complete 
medical history, including clinically significant diseases, chronic and ongoing 
conditions, and all current medications will be collected. 
      
     Blood pressure will be collected prior to the paracentesis and intravitreal 
injection.  
      
    Ocular assessments will be performed on both eyes at each study visit and 
will include: 
1. Best Corrected Visual Acuity (BCVA) will be assessed using Snellen 
charts. If a subject’s visual acuity is so poor that the subject can’t see 

 
6 
 
any letters on the charts, finger counting and/or hand motions will be 
checked.  
2. Intraocular Pressure (IOP) will be measured prior to the paracentesis. 
IOP will be measured in both eyes with the results in mmHg. 
3. Slit lamp biomicroscopy will be performed to examine the eye 
structures for both eyes. The slit lamp exam will be performed prior to 
the paracentesis, and will evaluate lids, lashes, lens, conjunctiva, 
cornea, anterior chamber, pupils, cataract status and vitreous.  
4. Dilated indirect ophthalmoscopy will be performed to examine the 
retina of each eye after the pupils have been sufficiently dilated. The 
dilated indirect exam will occur prior to the paracentesis, and will 
evaluate the retina vessels, macula, fovea, periphery, optic nerve and 
vitreous.  
5. Optical Coherence Tomography will be performed prior to the 
paracentesis on both eyes, and will be assessed to determine the 
central foveal thickness. 
      
     A paracentesis will be performed at each study visit prior to anti-VEGF 
treatment. A topical anesthetic such at lidocaine, will be applied to the eye 
prior to the paracentesis.  A lid speculum will be inserted under the eyelids to 
hold the eye open during the paracentesis and the intravitreal injection. An 
application of betadine solution will be applied to the inferior sclera where a 
30 gauge needle attached to a syringe will be injected into the eye to remove 
0.1mL of anterior chamber fluid. Following the paracentesis, while the 
speculum is still in place, the intravitreal injection of aflibercept will occur. 
Directly following the intravitreal injection, betadine will be applied to the 
injection site to minimize the risk of infection. The samples will be divided in 
the clinic. All anterior chamber fluid samples will be stored until ELISA 
analysis has been completed.            
     Lab personnel will be present in the clinic at the time of collection. Upon 
collection, samples will be immediately placed on dry ice in an appropriate 
transport container. Samples will be immediately transported to 4087 Gray 
Building on WFBMC campus by the on-site lab personnel. Samples will be 
transferred from the storage container to the -80 degree freezer located in 
Room 4087. Samples will be stored in this location until the requisite number 
of samples are obtained for Luminex multi-plex ELISA. 
     All subjects will receive aflibercept starting on Day 1.   

 
7 
 
      
 
      
 
9. Adverse Events and Serious Adverse Events 
      The investigator will be responsible for the monitoring of the data and 
safety of study participants. The investigator will be assisted by other 
members of the study staff.       
 
      Adverse events (AEs) will be collected at each study visit by the study 
doctor from the subject. The study doctor will review all relevant information 
related to the AE to determine causality and severity, and if follow up care is 
needed.  
 
      Severe Adverse Events (SAEs) will be reported to the IRB within 24 hours 
of the study doctor being made aware or identifying the SAE. Adverse events 
will also be reported to government agencies as appropriate.       
      
    All AEs, regardless of severity will be collected following the initiation of 
study procedures and until the subject has completed the study. Each AE will 
be followed until resolution or until the AE is stable.       
 
 
 
  
 
11. Outcomes 
 
Primary 
1. Change in macular edema from baseline through Week 52 measured by 
central retinal thickness using optical coherence tomography. 
2. Change in macular volume from baseline through Week 52 measured by 
central retinal thickness using optical coherence tomography. 
3. Changes in VEGF levels in the anterior chamber from baseline through 
Week 52 measured by ELISA. 
 
Secondary 

 
8 
 
1. Change in best corrected visual acuity using Snellen Visual Acuity Charts 
from baseline through Week 52. 
2. Change in intraocular pressure from baseline through Week 52. 
3. Number of intravitreal injections from baseline through Week 52. 
4. Changes in analytes from baseline through week 52 will be studied at a 
later time. Analytes analysis will include: 
• 
CCL2/JE/MCP-1 
• 
CCL11/Eotaxin 
• 
CXCL1/GRO alpha/KC/CINC-1 
• 
CXCL2/GRO beta/MIP-s/CINC-3 
• 
EGF 
• 
FGF basic/FGF2/bFGF 
• 
IFN-alpha 2/IFNA2 
• 
IFN-beta 
• 
IFN-gamma 
• 
IL-1 alpha/IL-1F1 
• 
IL-1 beta/IL-1F2 
• 
IL-1ra/IL-1F3 
• 
IL-2 
• 
IL-3 
• 
IL-4 
• 
IL-5 
• 
IL-6 
• 
IL-7 
• 
IL-8/CXCL8 
• 
IL-10 
• 
IL-12 p70 
• 
IL-13 
• 
IL-15 
• 
IL-17/IL-17A 
• 
IL-17E/IL-25 
• 
IL-33 
• 
PD-L1/B7-H1 
• 
PDGF-AA 
• 
PDGF-AB/BB 
• 
TGF-alpha 
 
 
 
12.Analytical Plan 
 

 
9 
 
     Changes from baseline to week 52 in main study outcomes (retinal 
thickness and volume) will be evaluated using a paired t-test, supposing 
change outcomes for the n=10 study participants are normally distributed. 
Linear mixed effect models, including fixed effects for time and type of vein 
occlusion (BRVO vs. CRVO) and a random effect for study participant, will be 
employed to study longitudinal changes in primary and secondary outcomes. 
The relationship between VEGF and retinal thickness (or volume) will be 
characterized at each time point using scatterplots and Pearson (or Spearman) 
correlations.  
 
Confidentiality and Privacy      
      
      Confidentiality will be protected by collecting only information needed to 
assess study outcomes, minimizing to the fullest extent possible the 
collection of any information that could directly identify subjects, and 
maintaining all study information in a secure manner.  To help ensure subject 
privacy and confidentiality, only a unique study identifier will appear on the 
data collection form.  Any collected patient identifying information 
corresponding to the unique study identifier will be maintained on a linkage 
file, store separately from the data. The linkage file will be kept secure, with 
access limited to designated study personnel.  Following data collection 
subject identifying information will be destroyed (state the anticipated time 
the data will be destroyed, e.g. three years after closure of the study, and the 
method of destruction), consistent with data validation and study design, 
producing an anonymous analytical data set.  Data access will be limited to 
study staff.  Data and records will be kept locked and secured, with any 
computer data password protected.  No reference to any individual participant 
will appear in reports, presentations, or publications that may arise from the 
study. The data will be stored on an Excel spreadsheet in two places – Dr. 
Sappington’s office and in Dr. Nelson’s office, both protected by Wake security 
procedures.  
           
           
References      
1. Clark, W., et al. “Intravitreal Aflibercept for Macular Edema Following 
Branch Retinal Vein Occlusion”. Ophthalmology 2016;123:330-6 

 
10 
 
2. Ogura, Y., et al. “Intravitreal Aflibercept Injection for Macular Edema 
Secondary to Central Retinal Vein Occlusion: 18 Month Results of the 
Phase 3 GALILEO Study”. American Journal of Ophthalmology 2014;158: 
1032-1038.  
3. Heier, J., et al. “Intravitreal Aflibercept Injection for Macular Edema 
Secondary to Central Retinal Vein Occlusion”. Ophthalmology 2014 
121:1414-1420.  
4. Falavarjani KG, Nguyen QD. Adverse events and complications associated 
with intravitreal injection of anti-VEGF agents: a review of literature. Eye 
(Lond). 2013;27(7):787-794. doi:10.1038/eye.2013.107  
5. Eylea (Package insert). Tarrytown, NY: Regeneron Pharmaceuticals, Inc. 
08/2019.