Conditions: Corneal Neovascularization Corneal Graft Failure
Keywords: Corneal Neovascularization Cornea Blood Vessels Corneal Graft Failure High-Risk Penetrating Keratoplasty Corneal Transplant
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 75
Healthy volunteers: no

Brief summary:
The goal of this study is to investigate whether using bevacizumab (Avastin®) is both safe and effective at decreasing the likelihood of a high-risk corneal graft rejection. Patients who are "high-risk" for rejection have blood vessels growing from the white of the eye into the cornea (clear, front region of the eye). The medication is used at the time of surgery and in the weeks following surgery. Participants have a 50/50 chance at receiving the active study medication or a placebo medication.

Detailed description:
The purpose of this study is to test the effectiveness of a drug, bevacizumab (Avastin), in preventing blood vessels that often occur after a corneal transplantation which are considered at "high-risk" for rejection. In many cases these blood vessels lead to the graft rejection and eventual failure of the corneal transplant. It is hoped that this treatment will increase the chances of corneal graft survival.

The medication used in this study is called bevacizumab or Avastin (Genentech, Inc). It works by inhibiting the action of a molecule called vascular endothelial growth factor (VEGF). VEGF is a substance molecule that binds to certain cells to stimulate new blood vessel formation. When VEGF is bound to the drug, it cannot stimulate the formation and growth of new blood vessels. Growth of blood vessels into the cornea is a complication which can worsen the prognosis of your corneal transplant and put the transplant at a higher risk for rejection.

Arms:
arm 1: Treatment will begin on Day 0, immediately upon the conclusion of the penetrating keratoplasty procedure with an injection of 0.1 mL (2.5 mg) bevacizumab. Starting Day 1 post-transplant surgery, subjects will begin treatment with topical bevacizumab (1% solution). Topical treatment will be self-administered 4 times a day for 4 weeks.

The study treatments are to be given in addition to standard of care treatments. Also, all patients will follow a standard post-operative follow-up visit schedule. arm 2: Treatment will begin on Day 0, immediately upon the conclusion of the penetrating keratoplasty procedure with an injection of 0.1 mL 0.9% NaCl. Starting Day 1 post-transplant surgery, subjects will begin treatment with topical Refresh Liquigel. Topical treatment will be self-administered 4 times a day for 4 weeks.

The study treatments (both topical and subconjunctival injection) are to be given in addition to standard of care treatments. Also, all patients will follow a standard post-operative follow-up visit schedule.

Interventions: intervention 1: Avastin® (bevacizumab) intervention 2: 0.9% NaCl & Refresh Liquigel
Intervention descriptions: intervention 1: One time subconjunctival injection of 0.1 mL (2.5 mg) bevacizumab followed by topical treatment with 1% solution bevacizumab four times a day for four weeks. intervention 2: One-time subconjunctival injection of 0.1mL 0.9% NaCl followed by topical treatment with Refresh Liquigel four times a day for four weeks.

Protocol document:
Prot_SAP_000.pdf: 
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COVER PAGE 
Clinical Protocol Title: A Multi-Center Study of the Safety and Efficacy of 
Bevacizumab in High-Risk Corneal Transplant Survival 
HSC #: 13-113H 
IRBNet ID: 512431 
Version number and date: Version 2.3 (submitted 08September2015) 
Phase of clinical investigation: Phase I/II 
Participating sites:  
Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114 
Bascom Palmer Eye Institute, University of Miami, 900 N.W. 17th Street, Miami, FL 
33136 
Bascom Palmer Eye Institute, University of Miami, 8100 S.W. 10th Street, Crossroads 
Business Park, Building 3, Plantation, FL 33324 
New York-Presbyterian Hospital, Weill Cornell Medical College, 1305 York Avenue- 
12th Floor, New York, NY 10065 
 
IND number: FDA IND #: 106,247 
 
Investigational drug: Bevacizumab (Avastin®) 
Regulatory Sponsor:  
Reza Dana, MD, MPH, MSc 
 Claes Dohlman Professor of Ophthalmology, Harvard Medical School 


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 Vice Chairman, Harvard Department of Ophthalmology, and Associate Chief of 
Ophthalmology for Academic Programs, Massachusetts Eye and Ear Infirmary 
 Senior Scientist & W Clement Stone Scholar, Schepens Eye Research Institute 
 Director, Cornea & Refractive Surgery, Massachusetts Eye and Ear Infirmary 
 Director, Harvard Medical School Department of Ophthalmology Cornea Center 
of Excellence 
 Mass. Eye and Ear, 243 Charles St., Boston, MA 02114 
Funding Sponsor: United States Department of Defense 
Research Monitor: Joseph Rizzo, MD 
Investigators: 
Massachusetts Eye and Ear Infirmary: 
Principal Investigator: 
Reza Dana, MD, MPH, MSc 
Co-Investigators: 
Ula Jurkunas, MD 
Joseph Ciolino, MD  
 
Bascom Palmer Eye Institute: 
Victor Perez, MD (Site PI) 
Sonia Yoo, MD 
 
New York Presbyterian Hospital: 
Jessica Ciralsky, MD (Site PI) 
Kimberly Sippel, MD 
 
Trial Biostatistician: 
Debra Schaumberg, Sc.D., O.D., M.P.H. 
 


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Institutions Providing Clinical Study Services: 
Massachusetts Eye and Ear Infirmary, 9th floor, 243 Charles Street, Boston, MA 02114 
Bascom Palmer Eye Institute, University of Miami, 900 N.W. 17th Street, Miami, FL 
33136 
Bascom Palmer Eye Institute, University of Miami, 8100 S.W. 10th Street, Crossroads 
Business Park, Building 3, Plantation, FL 33324 
New York-Presbyterian Hospital, Weill Cornell Medical College, 1305 York Avenue- 
12th Floor, New York, NY 10065 


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STUDY DESIGN SCHEMATIC 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1. CLINICAL PROTOCOL 
 
1.1 Background 
The Public Health Relevance of Corneal Blindness: A large analysis of the published 
literature related to the prevalence and causes of blindness worldwide [1] has 
concluded that the second leading cause of blindness (after cataract) is corneal 
disease. Childhood keratitis, trauma, and microbial keratitis among adults, can often 
lead to vascularized corneal scars, the most frequent causes of corneal blindness. As 
reported by the Bulletin of the World Health Organization [2] in addition to the many 
millions of corneally blind individuals are 1.5-2.0 million cases of monocular 
blindness per year due to corneal pathology. Many of these cases remain unregistered 
as legal “blindness”, since this term refers to vision in the best eye (suggesting 
bilateral disease). Numerous reports have concluded that in addition to suboptimal 
eye banking and infrastructural support for transplantation in many areas around the 
globe, a universal barrier to alleviating the burden of corneal blindness is the poor 
outcome of grafting in vascularized beds, often requiring repeat procedures, as 
outlined below. Development of more effective strategies to tackle this unmet need is 
therefore a priority in ophthalmology. In addition, importantly, corneal disease is the 
most common type of ocular pathology experienced by deployed military personnel. 
Significant eye injuries have been experienced by 10-12% of the active-duty personnel 
who have been deployed since 2002 [26]. 
 
The Burden of Transplant Rejection: Corneal transplantation has emerged as the 
most common form of solid tissue transplantation. Of the >100,000 cases performed 
annually worldwide, over 38,000 are performed in the US alone [2007 Eye Banking 
Statistical Report]. In uncomplicated first allografts performed in non-vascular host 


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beds (‘normal-risk’ grafts), the 2-year 
survival rate can approach 85-90% 
under cover of topical steroids, 
without systemic immune 
suppression. However, this success 
rate is completely abrogated in hosts 
with inflamed and vascularized host 
beds – in so called ‘high-risk’ 
transplantation where rejection rates 
easily surpass 50% [3]. Many of the 
features of the healthy cornea that 
promote transplant acceptance—the 
avascularity of the tissue, the rarity of 
professional antigen-presenting cells in the graft bed, and a spectrum of locally 
produced immunomodulatory cytokines that suppress immunogenic inflammation 
and complement activation [4]—are lost in high-risk transplants. The frequent 
rejection of high-risk grafts and the less frequent rejection of the larger pool of 
normal-risk grafts continues to cumulatively add to the burden of transplant rejection 
(see Fig. 1) as regrafting a host eye for a rejected transplant is associated with a 
considerably increased risk of rejection. Accordingly, a significant number of host 
eyes are getting grafted for a second, third, or fourth time due to the incrementally 
increased risk of rejection with each surgery. Indeed, repeat grafting due to previous 
failure has become the second leading indication for corneal transplantation as 
reported in the 2007 Eye Banking Statistical Report. In the aggregate, as suggested by 
Fig. 1, close to 30% of corneal grafts (~12,000 cases in US, and ~30,000 cases globally) 
are at risk of rejection annually. 
  
The Role of Angiogenesis in Exacerbating Transplant Rejection Risk: Allograft 
rejection is the leading cause of corneal graft failure and thus a leading indication for 
repeat transplantation [5]. Importantly, stratification of risk factors for immunologic 


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rejection of corneal transplants has identified recipient vascularization as the leading 
proximal cause for earlier and more fulminant rejection episodes [3, 6]. Grafting into 
these vascularized high-risk beds leads to a high rejection rate even with a strict 
regimen of topical and systemic immunosuppressive drugs [3]. Furthermore, it has 
been shown that graft rejection in a previously grafted eye relates more to the 
number of blood vessels in the cornea than to the number of previous grafts [7]. 
Finally, enhanced angiogenesis into the graft bed often heralds an acute rejection as it 
enables amplification of the alloimmune response [8]. In summary, vascularization of 
the cornea is the principal clinically detectable poor prognosticator for corneal graft 
survival. 
The exact reasons why the relative immunological quiescence of the eye, which is a 
central facet of its immune privileged state, is disturbed in a setting of corneal 
neovascularization (NV) is not fully understood [9]. However, experimental evidence 
strongly suggests that molecular factors such as the local immunosuppressive 
cytokine milieu (i.e., transforming growth factor-β, and α-melanocyte stimulating 
hormone) and functional attributes (i.e., anterior chamber-associated immune 
deviation) that play a critical role in maintaining the physiologic quiescence in the 
anterior segment, are subverted in the presence of corneal NV [9]. Thus, treatment of 
corneal NV in the setting of corneal transplantation can potentially limit both the 
afferent (sensitization) and efferent (rejection) arms of alloimmunity, and hence 
reduce the propensity for immuno-inflammatory reactions that can jeopardize graft 
survival [9]. 
The use of corticosteroids in the prophylaxis and treatment of corneal transplant 
rejections has represented the most significant contribution to the prolongation of 
corneal transplant survival over the last half century [10]. However, the local and/or 
systemic use of corticosteroids, or alternatively general immunosuppressants, is 
often associated with significant complications, such as infection, cataracts, 
glaucoma, and corneal thinning. Therefore, it is apparent that development of 
molecular strategies that can specifically target a critical ligand may prove to be an 
effective modality for circumventing the problems inherent in nonspecific treatments. 


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The Role of VEGF in Corneal NV: Vascular endothelial growth factor (VEGF) is 
thought to be a key mechanistic mediator of corneal NV. The prominent role of VEGF 
in the pathophysiology of corneal NV has been demonstrated in experimental models 
of corneal angiogenesis [11]. In humans, pathological studies have confirmed that 
VEGF and its receptors are present in higher concentrations in corneal buttons with 
corneal NV than in normal corneas irrespective of the cause of neovascularization 
[12]. Furthermore, it has been also shown that systemic inhibition of angiogenesis by 
neutralization of VEGF can promote corneal graft survival in animals [13]. VEGF 
inhibitors, including pegaptanib sodium, ranibizumab, and bevacizumab are currently 
used with considerable success for the treatment of neovascular age-related macular 
degeneration (AMD) [14].  Bevacizumab (Avastin) is a full
-length, recombinant humanized monoclonal 
immunoglobulin-G1 that binds to and inhibits the activity of VEGF-A thereby 
inhibiting angiogenesis. It was the first anti-VEGF antibody to be approved by the U.S. 
Food and Drug Administration specifically for the treatment of metastatic colon 
cancer, and recently, for non-small cell lung cancer and metastatic breast cancer. 
Bevacizumab is also used off-label to treat choroidal neovascularization, central 
retinal vein occlusion, proliferative diabetic retinopathy, and iris neovascularization. 
Bevacizumab has now been widely adopted and is arguably part of the standard of 
care for the treatment of neovascular AMD for many participants [14]. Recently, there 
has been a growing interest in using topical as well as subconjunctival bevacizumab 
for the treatment of corneal angiogenic pathologies but the use of anti-VEGF 
strategies in clinical transplantation has not been systematically investigated to date 
[15-17]. The approach proposed in this application may prove both feasible and of 
considerable value for several reasons: 1) bevacizumab has shown significant efficacy 
in treatment of retinopathies and anterior eye diseases (iris neovascularization); 2) it 
has a good safety profile; and 3) the drug has a relatively low cost, especially for the 
limited amounts needed for corneal application.  
 


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Military Relevance: The significant majority of military personnel with ocular injuries 
have experienced trauma to the cornea and anterior segment of the eye, inducing 
scarring, inflammation, and, in severe cases, growth of new blood vessels 
(angiogenesis) into the normally clear and avascular cornea. Corneal disease 
constitutes the most common type of ocular pathology experienced by deployed 
warriors [26]. Importantly, due to changes in munitions use and survival amongst 
trauma victims, the proportion of surviving military personnel afflicted with serious 
anterior segment eye injuries has continuously escalated from WWI to WWII, Korea, 
Vietnam, and now Iraq and Afghanistan.  
The highly variable efficacy and myriad side-effects (cataract, glaucoma, and increase 
risk of infection) with conventional treatments, such as corticosteroids, creates a need 
for more effective treatments for deployed military personnel. This is particularly the 
case since the final recourse for civilians and military personnel afflicted with a  
corneal scar is often a corneal transplant, which when done in a high-risk setting, as 
often occurs following ocular trauma, has a high potential for failure. 
 
1.2 Rationale 
Limited data are available regarding the safety and efficacy of bevacizumab for the 
prevention of corneal allograft rejection administered either topically or 
subconjunctivally. In clinical studies to treat corneal NV, bevacizumab has been used 
topically in different concentration regimens from 0.5% to 2.5% (5 to 25 mg/ml), two 
to four times a day for durations of 3 weeks to 6 months [15, 16, 19, 20]. In our 
completed study of 24 eyes of 24 participants, topical bevacizumab 1% in a dose 
regimen of 4 times a day for 3 weeks has shown significant effect in the treatment of 
clinically stable corneal NV which was evidenced by a nearly 50% reduction in 
neovascular area and vessel caliber [16]. None of these participants treated with the 
same regimen as proposed in this study showed any systemic or local adverse events. 
Systemic blood pressure remained stable at the baseline level, and no serious side 
effects occurred during the follow-up period. Similarly, from an ocular standpoint, 


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topical bevacizumab 1% was tolerated very well in all participants. No local irritation, 
allergic reaction, or surface epitheliopathy was observed. This is in contrast with a 
60% rate of spontaneous loss of epithelial integrity as reported by Kim et al [15]. In 
their study, the investigators used topical bevacizumab at a higher concentration 
(1.25%) and for a much longer period (3 months), and adverse effects generally 
appeared during the second month of treatment. This suggests that the duration of 
treatment may well determine the safety of topical bevacizumab because prolonged 
blockade of VEGF may impair wound healing and the regeneration of corneal nerves 
[21], which may cause a loss of epithelial integrity in cornea. Given our systematic 
prospective analysis of the 24 eyes of 24 participants receiving topical bevacizumab 
in whom we have noted no local or systemic adverse events, we believe that this 
pharmacotherapeutic approach is safe.    
Bevacizumab has also been used subconjunctivally in the treatment of corneal NV in 
doses of 1.25 mg/0.05 ml, 2.5 mg/0.1 ml, and 5.0 mg/0.2 ml [17]. You et al. have 
shown that one time subconjunctival injection of 2.5 mg or greater reduces corneal 
NV significantly without any serious systemic side effects. However, they reported 
minor localized side effects including injection site pain, subconjunctival hemorrhage 
and ocular irritation. In a comparative experimental study to determine the effects of 
topical and subconjunctival bevacizumab in a mouse model of high-risk corneal graft 
survival, our group has shown that both topical and subconjunctival bevacizumab 
treatment can diminish the severity of corneal NV [27]; however, the regression of 
corneal NV is more profound when treated subconjunctivally. Moreover, only 
subconjunctival bevacizumab treatment significantly promotes graft survival in the 
high-risk setting. It is likely that the limited efficacy of topical bevacizumab in corneal 
graft survival is due (at least in part) to a lack of adequate penetration through the 
corneal epithelium. Relatedly, bevacizumab is a humanized monoclonal antibody, and 
thus its potency in murine models would be significantly impaired—suggesting that 
the effect of bevacizumab in the clinical setting could be appreciably more than that 
suggested by murine data referenced from our laboratory. Given the encouraging 
clinical results of topical and subconjunctival bevacizumab in the treatment of 


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corneal NV and our experimental results in high-risk corneal transplantation, it would 
be reasonable to employ a mixed subconjunctival/topical bevacizumab treatment 
regimen as proposed herein. We thus propose that a one-time subconjunctival 
injection of 2.5 mg /0.1 ml bevacizumab at the conclusion of corneal transplantation 
surgery followed by 4 weeks of topical bevacizumab 1%, four times a day starting at 
post-operative day 1 following transplantation surgery would combine safety, efficacy 
and convenience of both routes of drug administration.   
 
Treatments: Participants in the treatment arm will receive a one-time subconjunctival 
injection of 2.5 mg/0.1 ml of bevacizumab (vs. 0.9% saline [NaCl] in the control group) 
in the treatment eye at the conclusion of corneal transplantation surgery. Participants 
will initiate topical bevacizumab (1.0% solution) or vehicle (Refresh Liquigel) four 
times a day on post-op day-1 and will continue its application for 4 weeks. 
 
 
Study Population: Ninety eyes from 90 adults with corneal NV who are candidates for 
corneal transplantation will be recruited at the following study centers. The name of 
the center is followed by the site’s target enrollment. Target enrollment for each site 
is 30 subjects, however, enrollment distribution can increase or decrease by a 
maximum of 10 subjects in order to meet the overall enrollment goal.  
 Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA: 30 
subjects +/- 10 subjects 
 New York Presbyterian Hospital, Weill Cornell Medical College, New York, NY: 
30 subjects +/- 10 subjects 
 Bascom Palmer Eye Institute, University of Miami, Miami, FL: 30 subjects +/- 10 
subjects 
 
 


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1.3 Potential Limitations of Approach 
Similar to any study, this proposal has certain limitations, and we acknowledge these 
and intend to incorporate our understanding of these in our analyses. 
Drug bioavailability and pharmacokinetics. As described above, there is limited 
information on the pharmacokinetics of topical bevacizumab. However, our 
laboratory studies (unpublished data) indicate that a single subconjunctival injection 
of bevacizumab leads to appreciable drug retention by the cornea for at least 3 weeks. 
It is conceivable that from the standpoint of NV regression, a longer period of 
treatment or more frequent applications of topical bevacizumab would be more 
effective than the regimen proposed here. However, (i) our clinical data suggest that 
significant benefit can be seen after a 3-week treatment, and (ii) this has an acceptable 
safety profile [16]. 
Age disparity between donors and recipients. There is no mandatory guideline in 
our protocol for age matching between donors and recipients. We understand that 
there is some concern that tissue from older donors will be more apt to lead to failure 
(due to on average lower endothelial cell counts). We do not believe this will be a 
major issue for this study: (i) We have instituted guidelines (detailed above) that limit 
death to preservation time <12 hours and storage time <5 days to ensure particularly 
high quality tissue; (ii) participating surgeons may each have their own standards for 
tissue age selection but given the randomized and masked design of the study there 
is no reason why this would preferentially bias the results in one direction; (iii) 
interim data from the Cornea Donor Study Investigator Group [25] suggest that high 
donor age alone is not a significant predictor of graft failure. 
Limited Follow-up to 1 year. This study is limited to a minimum follow-up of one 
year. It is conceivable that the intervention will have benefit, but this does not reach 
statistical significance within this time frame. This is possible but unlikely for the 
vast majority of high-risk graft rejections occur in the first year [The Australian 
Corneal Graft Registry 2007 Report]. If we notice that there is a beneficial effect to the 
treatment at 12 months, but this is of borderline statistical significance, the involved 


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sites will report on the subjects’ clinically indicated follow-up visits in the 2nd year 
after surgery (as increasing the number of follow-up visits will give added statistical 
power) and we will report on these data in a follow-up report.  
Effect of Confounders. As in any clinical study, there are a large number of potential 
confounders, including concurrent ocular and systemic morbidities and the 
heterogeneity of subjects (with variable immune reactivity) and transplant 
indications. While all of these factors cannot be controlled for, the randomized and 
masked design of the trial will lower the chances of significant confounding. 
Additionally, variables with known confounding effect on the primary endpoint (1st vs. 
regraft) will be managed through stratification of randomization as detailed above. 
Finally, certain variables may confound results, but these can be accounted for in our 
analyses. For example, an endothelial rejection episode will lead to increasing 
corticosteroid usage (as detailed above), and this could affect the degree of NV and 
other parameters. However, this does not affect the primary endpoint of interest 
(endothelial rejection rate) and will be considered in our analyses of secondary 
endpoints.  
 
1.4 Risk/Benefits Assessment  
Risks: Systemic administration of bevacizumab is reported to have a low incidence of 
adverse effects such as hypertension and thrombosis [23]. However, a report by the 
International Intravitreal Bevacizumab Safety Survey suggested that small doses of 
drug injected intravitreally would not produce serious systemic effects [24]. There has 
not been any pharmacokinetics data on use of topical and subconjunctival 
bevacizumab. It is noteworthy that no drug related serious systemic adverse events, 
definitively linking the drug to the adverse events, have been reported in any of the 
studies using bevacizumab for the treatment of ocular neovascularization, including 
for age-related macular degeneration. This issue remains somewhat controversial 
however and we will ‘err’ on the side of being too conservative—hence all the 
exclusion criteria listed and detailed above. It seems reasonable that a very low dose 


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of bevacizumab instilled over the surface of the eye or injected subconjunctivally 
would be safer overall than higher doses given systemically for the purpose the drug 
was originally approved (colorectal cancer). However, low levels of bevacizumab may 
reach the systemic circulation. The significance of this is not well understood. 
Therefore, the risk of the side effects of bevacizumab, which were described in 
participants taking the drug systemically for the treatment of colon or lung cancer, is 
unknown, but expected to be very low for the regimen proposed in this study.  
The potential ocular surface side effects of bevacizumab, including local irritation, 
visual disturbances, allergic reaction, or surface epitheliopathy and other ocular 
surface toxicity responses, are yet to be fully known. In a completed study at MEEI, 24 
eyes of 24 participants were treated with topical bevacizumab 1% in frequencies of 
two and four times a day for 3 weeks did not show any ocular surface side effects 
[16]. None of these participants experienced any ocular adverse events; however, in a 
study using topical bevacizumab at a higher dose (1.25%) for a much longer period (3 
months) did report did report ocular adverse events (loss of corneal epithelial 
integrity) during the second month of treatment [15]. These data strongly suggest 
that the lower concentration of bevacizumab given for a short duration of three 
weeks will be safe.     
 
Potential Benefits: Considering the encouraging results of clinical studies using 
subconjunctival and topical bevacizumab for the treatment of corneal NV combined 
with the promising results of animal studies of bevacizumab in promoting corneal 
graft survival in high-risk setting, some therapeutic benefits are most likely 
anticipated to the participating individuals. It is hoped that the information from this 
study will help us to bring a new treatment modality in prevention of corneal graft 
rejection in high-risk corneal transplantation. 


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2. STUDY OBJECTIVES 
 
2.1 Primary Objective 
Determine the safety of local (subconjunctival + topical) bevacizumab treatment in 
participants who have undergone high-risk corneal transplantation using the 
following measures: 
 Systemic safety: Incidence and severity of systemic adverse events during 
the study (based on physical examination, subject self-reporting, and 
changes in vital sign). 
 Ocular safety: Incidence and severity of ocular adverse events during the 
study (based on ophthalmic examination and subject self-reporting). 
 
2.2 Secondary Objective(s) 
Determine the efficacy of local (subconjunctival + topical) bevacizumab treatment in 
preventing graft rejection in subjects who have undergone high-risk corneal 
transplantation. This will be tested using the following outcomes measures: 
 Primary Endpoints: 
 Twelve-month endothelial rejection rate 
 Secondary Endpoints:  
 Time from surgery to occurrence of any rejection episode (endothelial, 
epithelial, subepithelial) 
 Time from surgery to overall graft failure (regardless of cause) 
 Incidence of delayed epithelial healing at Day 7  
 Endothelial cell density at Weeks 26 and 52 


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 Central corneal thickness at Weeks 4,16, 26, and 52 
 Frequency of primary graft failure 
 Corneal NV Metrics at the Weeks 4,16, 26, and 52  
o Neovascular Area (NA), measuring the area of the corneal vessels 
themselves 
o Vessel Caliber (VC), measuring the mean diameter of the corneal 
vessels 
o Invasion Area (IA), measuring the fraction of corneal area in which 
vessels are present 
 


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3. STUDY DESIGN 
 
3.1 Study Design Description 
This is a Phase I/II multicenter, randomized, double masked, vehicle-controlled study 
evaluating the safety and efficacy of locally delivered bevacizumab (in addition to 
standard treatment with corticosteroids) in subjects who are candidates for high-risk 
corneal transplantation. Investigators and subjects will be double-masked to 
treatment.  
As detailed in the ‘Statistical Methods’ section below, we aim to enroll 90 subjects 
into this study (45 per arm). Subjects will be followed in an outpatient setting for 
safety and efficacy for 52 weeks (12 months) following the corneal transplant surgery. 
A detailed layout of subject visits and study procedures can be found in the Schedule 
of Events (Appendix 6). 
 
3.2 Allocation to Treatment  
Ninety subjects will be randomized according to a predetermined randomization 
scheme within each of the three participating centers. A stratified, blocked, 
randomization scheme will be determined by our trial statistician, Dr. Debra 
Schaumberg. Randomization will be stratified by center, as well as first graft vs. 
regraft. Thus there will be a total of 6 strata. Stratification will protect against chance 
departures between the two arms of the study. A number of blocks of varying size 
will be determined (e.g. 5 blocks of size 6, 6 blocks of size 4, and 7 blocks of size 3) 
and randomization assignments will be prepared accordingly. Since we have 6 strata, 
we plan to use relatively small block sizes to minimize the chance of large departures 
from the desired allocation ratio that could occur if some of the blocks in individual 
strata remained unfilled by the time of completion of participant recruitment. 


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Although the ability of the clinical staff to predict future treatment assignments is 
increased with smaller block sizes, we do not think that this will be a major issue in 
the present study. 
Description of the Recruitment Process: This study will be conducted in accordance 
to the Department of Defenses (DOD) regulations for research involving human 
subjects. All institutions and research staff will comply with the guidelines described 
in Title 32 Code of Federal Regulations (CFR) Part 219, “Protection of Human 
Subjects”, and title 45 CFR 46 Subparts B, C and D. This study will be using an 
investigational medication and, therefore, will abide by the Food and Drug 
Administration’s (FDA) regulations outlined in 21 CFR Parts 50, 56, 312 and 812. In 
addition, all institutions involved in this study will follow the guidelines set forth in 
the Health Insurance Portability and Accountability Act (HIPAA), which is written in 
45 CFR 160-164. 
All institutions and investigators involved in this research study understand the 
regulations and responsibilities for human research with volunteers from the 
Department of the Army as described in the DoDi directive 3216.02.  
Potential study participants will be identified by the study investigators at the 
designated study sites (Massachusetts Eye and Ear Infirmary, Boston, MA; New York 
Presbyterian Eye Hospital, New York, NY; Bascom Palmer Eye Institute, Miami, FL). 
Participants in each investigator’s clinic will be pre-screened for possible eligibility in 
the study. A research technician, study coordinator, research fellow and/or 
investigator will identify participants who are considering a corneal transplant and 
are considered at high-risk for rejection. Once identified, the study team will evaluate 
whether or not the participant may be able to participate in the study based on their 
ophthalmic medical record and the study eligibility criteria. If no definite reasons to 
exclude the participant are found during the review, the participant will be 
approached by their physician (investigator). A recruitment script can be found in 
Appendix 2. 


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Other physicians within the institutions will be able to refer participants to the 
participating investigators who they anticipate may be eligible for participation. Per 
HIPAA regulations, participants referred to an enrolling site must contact the site 
themselves. Outside physicians may not refer eligible participants to the enrolling site 
by name.  
 All qualified subjects who meet the below inclusion/exclusion criteria will be invited 
to participate in the study. Those participants who volunteer to participate (after 
signing the written informed consent) will be enrolled consecutively during routine 
visits to the study sites. The study eye will be identified at the screening visit. If both 
eyes are eligible for the study, the worse eye will be selected for entry into the study. 
Only one eye of each participant will be enrolled into the study. An enrolled 
participant may re-enroll with the 2nd eye at the end of his/her study participation 
with the 1st eye. Volunteers will not be given a stipend for participation in the 
research study. Study medications and all non-standard assessments will be covered 
by the study funds.  
This study does not exclude, or intentionally recruit, subjects based on race or 
ethnicity, as data does not support any significant differences between these 
subgroups’ susceptibility to the risk of rejection or failure post transplantation [28]. 
The subjects in this study will therefore, most likely reflect the full range of gender 
and minorities seen at the participating hospitals which is quite broad given that they 
are in urban centers. 
A sample recruitment script can be found in Appendix 2. 
3.2.1 Randomization Procedures 
Assignment sheets for randomization will be prepared by the statistician and 
maintained by MEEI study staff. When a participant is enrolled into the study, the 
Coordinator at each site will notify MEEI and obtain a masked randomization 
assignment, which will be communicated to the Mass. Eye and Ear’s Pharmacist, who 
will be in charge of preparing the appropriate medication (active or vehicle) and 
dispensing to all sites. Codes will be used to indicate different treatment assignments 


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so that the Site Coordinator and Investigators will remain masked to the subjects’ 
assignment.  
The statistician will use each randomization assignment in order according to a set of 
predetermined rules. Briefly, allocation of active and vehicle treatments will be 
determined in 4 steps: 1) arrange the initial treatment assignments arbitrarily, 2) 
generate via computer a list of pseudorandom numbers, 3) link the initial treatment 
codes with the pseudorandom numbers, 4) order the pseudorandom numbers (e.g. 
smallest to largest) with associated treatment codes to determine the final order of 
treatment assignments. Codes will be used to indicate different treatment 
assignments so that the participants and study investigators will remain masked to 
the subjects’ assignment. For the first participant enrolled, the statistician will mark 
the first sheet according to the stratum of this participant (center and first vs. 
regraft), and this participant's ID number will be entered on the line next to the first 
treatment assignment. Each consecutive participant who falls into the same stratum 
will be assigned from the next treatment assignment on this sheet until all 
assignments have been used. 
Whenever a participant is 
enrolled who falls into a stratum 
for which no sheets have been 
started or for which all previous 
sheets have been completed, the 
next sheet will be used and 
marked according to the 
appropriate stratum, and so on.  
The examining ophthalmologist 
will not be involved in allocating 
the randomization assignments. 
The Mass. Eye and Ear pharmacist 
will provide the masked 
treatment packages to the site study staff to distribute to the study participant. Thus, 


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it is extremely unlikely that the clinical staff would be able to alter the treatment 
assignments in such a way as to bias the outcome of the study. Subjects will be 
randomized to bevacizumab or vehicle with 1:1 allocation at the baseline visit 
(Screening Visit) (Fig. 2) provided they have satisfied all entry criteria.  
 
3.2.2 Masking Procedures 
Identifiers & Masking: Ninety subjects will be randomized according to a 
predetermined randomization scheme within each of the three participating centers. 
A stratified, blocked, randomization scheme will be determined by trial 
epidemiologist and statistician Dr. Debra Schaumberg. Randomization will be 
stratified by center, as well as first graft vs. regraft. Thus there will be 6 strata. 
Stratification will protect against chance departures between the two arms of the 
study. A number of blocks of varying size will be determined (e.g. 5 blocks of size 6, 6 
blocks of size 4, and 7 blocks of size 3) and randomization assignments will be 
prepared accordingly. Since we have 6 strata, we plan to use relatively small block 
sizes to minimize the chance of large departures from the desired allocation ratio 
that could occur if some of the blocks in individual strata remained unfilled by the 
time of completion of participant recruitment. Although the ability of the clinical 
staff to predict future treatment assignments is increased with smaller block sizes, 
we do not think that this will be a major issue in the present study. 
Assignment sheets for randomization will be prepared by the statistician and 
maintained by MEEI study staff. When a participant is enrolled into the study, the 
Coordinator at each site will notify MEEI and obtain a masked randomization 
assignment, which will be communicated to the Mass. Eye and Ear’s Pharmacist, who 
will be in charge of preparing the appropriate medication (active or vehicle) and 
dispensing to all sites. Codes will be used to indicate different treatment assignments 
so that the Site Coordinator and Investigators will remain masked to the subjects’ 
assignment.  


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Each clinical site will be given a unique study site number. When a participant is 
enrolled, the study site will then assign the participant a study identification number 
(ID) prefaced with the study site number and completed with the subject ID, which is 
assigned sequentially starting from 01.  All sites’ first subject ID will end with 01 (e.g., 
the first subject enrolled at MEEI will have subject ID 101 because MEEI’s study site ID 
is 1).  
If a participant signs a consent form and does not complete surgery within 90 days, 
he/she will be considered lost to follow-up. If the participant reschedules surgery and 
elects to participate, he/she must re-screen and sign a new consent form. The 
participant may use the study ID number and randomization code assigned in the 
first screening visit.  
The statistician will use each randomization assignment in order according to a set of 
predetermined rules. Briefly, allocation of active and vehicle treatments will be 
determined in 4 steps: 1) arrange the initial treatment assignments arbitrarily, 2) 
generate via computer a list of pseudorandom numbers, 3) link the initial treatment 
codes with the pseudorandom numbers, 4) order the pseudorandom numbers (e.g. 
smallest to largest) with associated treatment codes to determine the final order of 
treatment assignments. Codes will be used to indicate different treatment 
assignments so that the participants and study investigators will remain masked to 
the subjects’ assignment. For the first participant enrolled, the statistician will mark 
the first sheet according to the stratum of this participant (center and first vs. 
regraft), and this participant's ID number will be entered on the line next to the first 
treatment assignment. Each consecutive participant who falls into the same stratum 
will be assigned from the next treatment assignment on this sheet until all 
assignments have been used. Whenever a participant is enrolled who falls into a 
stratum for which no sheets have been started or for which all previous sheets have 
been completed, the next sheet will be used and marked according to the appropriate 
stratum, and so on.  
 


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3.2.3 Breaking the Mask 
The study drugs (subconjunctival and drops) will be labeled by the pharmacy with a 
unique identifier provided by the statistician, instructions for storage, the name of 
the study, an expiration date, and a notice that the bottle may contain active or 
vehicle solution for investigational use. The pharmacy will maintain a master list of 
the unique identifier, the coded treatment assignment, and participant ID. The “key” 
for the coded treatment assignments will also be provided to the pharmacy by the 
statistician, but the key will be kept separate from the master list to guard against the 
chance that the participant, coordinator, or other study staff will inadvertently 
become unmasked to the participant’s treatment assignment. However, a participant’s 
medication may be unmasked at any point for a medical emergency or to supplement 
a serious adverse event (SAE) report. 
 
 
 
 
 
 
 
 


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4. SUBJECT SELECTION 
 
4.1 Subject Inclusion Criteria 
The following eligibility criteria are designed to select subjects for whom the protocol 
treatment is considered appropriate. The criteria must be met at the screening visit. 
All relevant medical and non-medical conditions will be taken into consideration 
when deciding whether this protocol is suitable for a particular subject. Subjects who 
have provided informed consent will be screened using the following inclusion and 
exclusion criteria.  
A. Inclusion Criteria: 
 Age > 18 years 
 Participant willing and able to provide written informed consent  
 Willing and able to comply with study assessments for the full duration 
of the study 
 High-risk characteristics for penetrating keratoplasty:  
 Presence of corneal NV in one or more quadrants (≥ 3 clock hours NV ≥ 
2mm from the limbus) OR 
 Extension of corneal NV to graft–host junction in a previous failed graft  
 In generally good stable overall health 
4.2 Subject Exclusion Criteria 
 History of Stevens-Johnson syndrome or ocular pemphigoid  
 Ocular or periocular malignancy  
 Non-healing epithelial defect of at least 0.5x0.5 mm in host corneal bed 
lasting ≥6 weeks preoperatively 


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 Uncontrolled glaucoma 
 Currently on dialysis 
 Has received treatment with anti-VEGF agents (intraocular or systemic) 
within 45 days of study entry 
 Concurrent use of systemic anti-VEGF agents 
 Change in topical corticosteroid regimen within 14 days of 
transplantation 
 Use of systemic immunosuppressive for indication other than corneal 
graft rejection 
 Pregnancy (positive pregnancy test) or lactating  
 Pre-menopausal women not using adequate contraception (Reliable 
intrauterine devices, hormonal contraception or a spermicide in 
combination with a barrier method) 
 Uncontrolled hypertension defined as systolic blood pressure (BP) ≥150 
or diastolic BP ≥90 mmHg 
 History of thromboembolic event within 12 months prior to study entry 
 Participation in another simultaneous medical investigation or trial 
 


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5. STUDY DRUG  
 
5.1 Study Drug Information 
 
 
 
 
 
 
 
 
 
 
 Bevacizumab (Avastin) is a full
-length, recombinant humanized monoclonal 
immunoglobulin-G1 that binds to and inhibits the activity of VEGF-A thereby 
inhibiting angiogenesis. It was the first anti-VEGF antibody to be approved by the U.S. 
Food and Drug Administration specifically for the treatment of metastatic colon 
cancer, and recently, for non-small cell lung cancer and metastatic breast cancer. 
Bevacizumab is also used off-label to treat choroidal neovascularization, central 
retinal vein occlusion, proliferative diabetic retinopathy, and iris neovascularization. 
Bevacizumab has now been widely adopted and is arguably part of the standard of 
care for the treatment of neovascular AMD for many participants [14]. Recently, there 
has been a growing interest in using topical as well as subconjunctival bevacizumab 
for the treatment of corneal angiogenic pathologies but the use of anti-VEGF 
strategies in clinical transplantation has not been systematically investigated to date 
[15-17]. The approach proposed in this application may prove both feasible and of 
Group 1: Active Treatment 
Subconjunctival injection of bevacizumab 2.5mg/0.1ml 
Topical bevacizumab (1.0% solution) prepared with 
Refresh Liquigel 
 
Group 2: Control Treatment 
Subconjunctival injection of 0.9% saline [NaCl] 
Topical Refresh Liquigel 


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considerable value for several reasons: 1) bevacizumab has shown significant efficacy 
in treatment of retinopathies and anterior eye diseases (iris neovascularization); 2) it 
has a good safety profile; and 3) the drug has a relatively low cost, especially for the 
limited amounts needed for corneal application. 
In this study, participants are randomized to Avastin or placebo and receive either 
receive either:  
 AVASTIN 2.5mg/0.1ml Subconjunctival Injection and AVASTIN 1% Study 
Ophthalmic Solution 
 Sodium Chloride 0.1ml Subconjunctival Injection and Refresh Liquigel Study 
Ophthalmic Solution 
 
Injection of Bevacizumab or Placebo occurs once at the conclusion of surgery. 
Administration of Avastin or placebo topical drops is four times per day for four 
weeks.  
5.2 Study Drug/Device Compliance/Adherence  
During participant follow-up visits at Day 7 and Week 4, participants will be queried 
on compliance of study and concomitant medications. The decision to withdraw a 
participant due to non-compliance or adherence to study medications, standard-of-
care medications, or study visits will be made by the principal investigator, Dr. Reza 
Dana. 
If withdrawn, participants will not be replaced. They will be asked to return for the 
follow-up care of the corneal transplantation. 
 
 
 
 
 
 
 


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5.3 Study Drug Supplies 
 Formulation and Packaging 
Subconjunctival bevacizumab will be aseptically prepared from commercially 
available intravenous bevacizumab by the hospital pharmacy. One tenth of 
milliliter containing 2.5 mg of bevacizumab will be transferred into a sterile 
insulin syringe and refrigerated at 2°C - 8°C (36°F - 46°F) in an amber colored 
(light-protected) plastic bag till the time of use. One tenth of 1mL of normal 
saline, aseptically transferred into a sterile insulin syringe, will be used as the 
subconjunctival ‘placebo’ control injection.  
Topical bevacizumab solution will be formulated and aseptically prepared from 
commercially available intravenous bevacizumab (Avastin; Genentech Inc., San 
Francisco, CA), and transferred into a sterile, light-protected dropper container 
by the hospital pharmacy. A formulation of 1.0% (10 mg/ml) concentration of 
bevacizumab with 1% (10 mg/ml) Refresh Liquigel (Allergan, Irvine, CA) will be 
used. The participants will be instructed to only refrigerate the study drugs. 
The participants will be instructed to never freeze the study medication. 
 Preparing and Dispensing 
The recipes for the treatment formulations have been written by the 
Massachusetts Eye and Ear Infirmary Clinical Pharmacy. The Mass. Eye and Ear 
pharmacy will prepare, label and distribute the study medication to all enrolling 
sites.  
All study medications (topical and subconjunctival injection of Avastin or 
placebo) for participants at NY Presbyterian (Cornell) and Bascom Palmer (U. 
Miami) will be shipped to the non-Mass. Eye and Ear sites via overnight Federal 
Express for arrival prior to surgery. The medications will be packaged and sent 
in the appropriate packaging to ensure that the medication is kept cool during 
transportation.  


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 Administration 
Day 0: At the conclusion of surgery, 0.1 mL (2.5 mg) of bevacizumab or vehicle 
(0.9% NaCl) will be injected subconjunctivally at the 12-o'clock position 1 mm 
posterior to the limbus. 
 
Day 1: In addition to standard treatment with corticosteroids, participants will 
receive topical bevacizumab (1% solution) or vehicle (Refresh Liquigel), four 
times a day (every 4 hours while awake) for 4 weeks starting on Day 1 post-
transplant surgery. Subjects will self-administer study treatment topically in the 
study eye. Participants will be instructed to stop using the study treatment by 
the end of week 4, and return all unopened or partially-filled drug bottles to the 
study site. Any empty bottles may be disposed of by the participant. 
 
Participants will be given a study medication instruction sheet that will specify 
which date to start and stop the medication. The sheet will also provide 
instructions on storing the medication.  
 
During treatment period: If a subject contracts infectious conjunctivitis, they 
will be instructed to discard the remainder of the study drug and new drug will 
be sent from the host site pharmacy, Massachusetts Eye and Ear Infirmary. 
 
5.4 Study Drug/Device Storage and Accountability 
 Participants will be asked to protect the study medication from light and will be 
instructed to refrigerate the study drugs. At the conclusion of the 4-week 
treatment period, participants will be asked to return any remaining study 
medication to be destroyed per pharmacy regulations. 
 
 
 


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5.5 Other Medications 
 Corticosteroid 
All participants will receive topical 1% prednisolone acetate drops six times a 
day (every 2-3 hours while awake) for the first 2 weeks after surgery. The 
frequency of administration of topical steroids will be gradually tapered as 
follows: 4x/day for 6 weeks, 3x/day for 4 weeks, 2x/day for 6 months, and then 
once a day indefinitely, unless intervening complications (e.g. acute rejection) 
require that the regimen be altered (see section 7.3, ‘Corneal Graft Rejection’ ). 
In addition to standard treatment with corticosteroids, participants will receive 
topical bevacizumab (1% solution) or vehicle (Refresh Liquigel), four times a day 
(every 4 hours while awake) for 4 weeks starting on Day 1 post-transplant 
surgery.  
 
 Antibiotic 
All participants will receive an antibiotic eye drop four times a day until full 
graft epithelialization, or as necessary with regard to each participant's needs. 
Use of bandage contact lens is allowed if necessary. Selective suture removal 
will be initiated as early as 6 months after the surgery unless the sutures get 
loose or broken earlier. All subjects will be instructed in the signs and 
symptoms of rejection (decrease in vision; redness, pain or irritation persisting 
for more than a few hours; painful sensitivity to strong light) and urged to 
contact their ophthalmologist immediately should they develop such 
symptoms.  
 
 Considerations with Concomitant Medications 
The use of long-acting forms of corticosteroids (e.g., triamcinolone) injected 
subconjunctivally at the end of corneal transplant surgery is prohibited 
(subconjunctival injection of rapid-acting steroids such as dexamethasone at 
the end of surgery is allowed) in this protocol. Use of systemic 


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immunosuppressant medications is prohibited throughout the study, except 
oral steroids which are allowed for treatment of corneal graft rejection 
episodes as stated above. Concomitant medications other than these prohibited 
agents are permitted as necessary. Subjects using topical steroid preparations 
prior to transplantation must be on a stable regimen (i.e., neither intensified 
nor augmented) for 14 days prior to surgery. 
 
Subjects who develop elevated intraocular pressure secondary to steroids 
should be managed, if possible, by initiating anti-hypertensive therapy as per 
standard ‘glaucoma’ care regimens. If this proves inadequate and the 
intraocular pressure management becomes difficult, the dose (frequency and 
potency) of the topical corticosteroid will be reduced, especially if there is a 
history of steroid-responsiveness.  If maximally tolerated medical therapy does 
not sufficiently control intraocular pressure, alternative approaches to 
intraocular pressure control should be considered. Elective surgery post 
transplantation (e.g., for cataract removal) should be deferred if possible until 
after the 12-month follow-up period has expired.  
 
Therapy with steroids can be altered at the discretion of the investigator for 
treatment of graft rejection, as described in section 7.3, ‘Treatment of Corneal 
Graft Rejection’.  
 
 


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6. BIOSPECIMEN COLLECTION  
 
No biospecimens will be collected for this study.  


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7. STUDY PROCEDURES 
 
7.1 Screening Procedures 
Prospective participants, as defined by the inclusion/exclusion criteria, will be 
considered for entry into this study. The study design and treatment regimen will be 
discussed with each participant. Written informed consent will be obtained before any 
study-specific screening evaluations are performed. Screening evaluations will be 
performed within ninety (90) days preceding Day 0 (Surgery Day). The following 
evaluations and procedures will be performed for all subjects during the screening 
period: 
 Written informed consent 
 Record current ocular and systemic medications  
 Record significant medical/surgical history in the past 5 years 
 Record demographic data, including date of birth, sex, and race/ethnicity 
 Review of systems 
 Measurement of systolic and diastolic blood pressures 
 Urine pregnancy test for women of childbearing potential, if appropriate 
 Ocular assessments (OU) 
 Best spectacle-corrected visual acuity (BSCVA) (OU) 
 Slit-lamp examination (OU) 
 Intraocular pressure (OU) 
 Dilated funduscopy (Study eye) 
 Corneal photography (Study eye)  


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 Pachymetry (OU) 
 Classification of depth of blood vessels (superficial/anterior-, mid-, or 
deep-stromal)* (OU) 
(* if at multiple depths will classify at depth where preponderance of neovessels 
reside) 
Once a participant is deemed eligible and enrolled into the study they will be given a 
unique study identification number. The Mass. Eye and Ear Pharmacy will assign the 
treatment code that corresponds with the unique Study ID based on a pre-established 
randomization scheme created by the trial statistician. Each participant will be 
randomized to one of two randomization groups in equal allocations.  
 
7.2 Enrollment/Baseline Procedures 
A list of baseline procedures can be found in the Schedule of Events.  
 
Description of the Informed Consent Process: Subjects are required to sign an 
informed consent and Health Insurance Portability and Accountability Act (HIPAA) 
before participating in the study.  
Given that the Investigators, Research Fellows and Study Coordinators are responsible 
for maintaining a detailed knowledge of the study protocol, safety profile and 
previous work with the medication, these individuals will discuss the protocol with 
participants and obtain informed consent. The study team will review the study 
procedures, visit schedule, risk and benefits, alternative treatments and financial 
responsibilities with all potential subjects. Each participant will be informed of their 
right to withdraw at any time from the study without affecting their care or 
relationship with the treating physician and participating institution. A study member 
will also explain and discuss with the participant their confidentiality rights as 
described in the HIPAA form. Consent will be obtained from each participant prior to 


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any study procedures being performed by the Investigator, Research Fellow or Study 
Coordinator within the clinic.  
A note will be made on the study record that the informed consent was signed by the 
participant. The informed consent will follow the guidelines set forth by the FDA and 
relevant site’s IRB. A copy of the consent form will be given to the participant.  
We do not anticipate a need to obtaining on-going consent or re-assessing capacity 
over the course of the study. No minors will be eligible to participate.  
 
7.3 Study Drug or Device Procedures 
Surgery Day (Day 0): Transplant surgery (penetrating keratoplasty) may occur up to 
ninety days after the screening visit. If available, study staff will document 
endothelial cell count of donor tissue. 
 
Penetrating Keratoplasty Procedure: Participants will undergo penetrating 
keratoplasty under general or retrobulbar/peribulbar anesthesia. Tissue matching for 
HLA or ABO will not be performed in this study. The donor cornea will be trephined 
from the endothelial surface. Trephine size will be selected according to recipient 
corneal size (range 7.0–8.0 mm), and the donor–recipient disparity will be 0.25–0.50 
mm. 10-0 nylon suture material will be used. The technique of suturing will be 
interrupted since the risk of suture-related complications with running sutures is 
higher in vascularized host beds. The donor endothelium will be protected at all 
times by maintaining a formed anterior chamber with viscoelastic substances. At the 
conclusion of surgery, 0.1 mL (2.5 mg) of bevacizumab or vehicle (0.9% NaCl) will be 
injected subconjunctivally at the 12-o'clock position 1 mm posterior to the limbus. 
Steroid and antibiotic will be injected subconjunctivally at the 6-o'clock position. 
Penetrating keratoplasty may be combined with other procedures such as cataract 
extraction, intraocular lens implantation, and vitrectomy procedures as necessary 
with regard to each participant's needs.  


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Postoperative Care: All participants will receive topical 1% prednisolone acetate 
drops six times a day (every 2-3 hours while awake) for the first 2 weeks after 
surgery. The frequency of administration will be gradually tapered as follows: 4x/day 
for 6 weeks, 3x/day for 4 weeks, 2x/day for 6 months, and then once a day 
indefinitely, unless intervening complications (e.g. acute rejection) require that the 
regimen be altered (see below, ‘Corneal Graft Rejection’ section). In addition to 
standard treatment with corticosteroids, participants will receive topical bevacizumab 
(1% solution) or vehicle (Refresh Liquigel), four times a day (every 4 hours while 
awake) for 4 weeks starting on Day 1 post-transplant surgery. Subjects will self-
administer study treatment topically in the study eye. Participants will be instructed 
to stop using the study treatment by the end of week 4, and return all unopened or 
partially-filled drug bottles to the study site. Any empty bottles may be disposed of 
by the participant. All participants will receive an antibiotic eye drop four times a day 
until full graft epithelialization, or as necessary with regard to each participant's 
needs. Use of bandage contact lens is allowed if necessary. Selective suture removal 
will be initiated as early as 6 months after the surgery unless the sutures get loose or 
broken earlier. All subjects will be instructed in the signs and symptoms of rejection 
(decrease in vision; redness, pain or irritation persisting for more than a few hours; 
painful sensitivity to strong light) and urged to contact their ophthalmologist 
immediately should they develop such symptoms.  
 
Corneal Graft Rejection. Corneal allograft rejection will be diagnosed and classified 
according to its clinical manifestations as endothelial rejection (“definite”, “probable”, 
and “possible”), epithelial rejection, subepithelial infiltrates, or any combination 
thereof [18]:  
 The diagnosis of ‘definite’ endothelial rejection will be made when an eye 
that has had a clear graft for at least 2 weeks shows the onset of 
inflammatory reaction in the anterior chamber (anterior chamber cells or 


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keratic precipitates) as well as both stromal edema and an endothelial 
(Khodadoust) rejection line. 
 The diagnosis of ‘probable’ endothelial rejection will be made when an 
eye satisfies the diagnosis of ‘definite’ rejection but does not have an 
endothelial rejection line. ‘Probable’ rejection may alternatively be 
diagnosed by the presence of keratic precipitates on the graft 
endothelium without stromal edema. 
 ‘Possible’ endothelial rejection will be defined as acute graft edema 
without obvious signs of anterior chamber inflammation in a graft that 
had previously been clear for 2 weeks.  
 Epithelial rejection will be diagnosed by the presence of an elevated 
epithelial line that stains with fluorescein.  
 Subepithelial infiltrates will be diagnosed by the appearance in the 
anterior stroma of grey-white deposits, 0.2 to 0.5 mm in diameter, 
randomly distributed throughout the graft.  
Treatments associated with these clinical manifestations are part of a patient’s 
clinical care, not a research procedure. 
Treatment of Corneal Graft Rejection.  
All cases of endothelial rejection will be treated with topical 1% prednisolone acetate 
drops applied hourly when the participant is awake. In the setting of ‘definite’ or 
‘probable’ endothelial rejection, oral corticosteroids will be added if the endothelial 
rejection presents with extensive (involving >one-half the en-face surface area) and 
significant (>20% relative increase in corneal thickness compared to baseline) graft 
edema. These participants will need several additional visits, and corticosteroid 
therapy will be tailored according to the clinical findings at the time. Epithelial 
rejection and subepithelial infiltrates will be treated with topical 1% prednisolone 
acetate drops every 2 hours while participants are awake. These participants will be 


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reexamined within 1 week, and the corticosteroid drops will be tapered over ensuing 
weeks.  
Subjects who experience an endothelial rejection episode have reached the primary 
study endpoint, but will continue to be followed for other (secondary) endpoints 
through Week 52. The assessment of subjects being treated for graft rejection at times 
outside of the prescribed set protocol will be in accord with week 26 endpoints’ 
assessment.  
A flow diagram of Classification and Treatment of Graft Rejection can be found in 
Appendix 4. 
Corneal Photography. Prior to corneal transplant surgery at the screening visit, and 
at Weeks 4, 16, 26, and 52, digital corneal photography will be done using a slit-lamp 
with a digital camera attachment and a flash-through-the-slit illumination system. By 
fine focusing on the area of the cornea where NV predominates, the entire cornea will 
be pictured using diffuse illumination and 10x magnification. If lids are drooping, the 
photographer will attempt to gently remove them from area of focus with a cotton 
swab. If the lids occlude the cornea, a lid speculum may be used to facilitate whole-
cornea photography. Participants with dark irises, which may prohibit a clear image 
of the blood vessels, will be dilated with tropicamide only (without use of an 
adrenergic agent that may induce vascular ‘blanching’) at each visit. Upon dilation, 
photos will be taken with retro illumination of the cornea. Further guidance on 
imaging can be found in the photography protocol (Appendix 5). 
Immediately after a study visit, captured image files will be transmitted in a masked 
fashion (containing only the participant study identification number and visit 
number) by each site to StudyTrax.  Mass. Eye and Ear will serve as the central image 
repository and analyzing site. The assigned Clinical Research Fellow, trained and 
experienced in NV quantification, will perform this task. The investigator who 
performs  


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the quantification will be masked 
to the treatment regimen. 
Quantification of Corneal NV. 
Digital slit-lamp corneal pictures 
will be analyzed using graphics 
editing software (Photoshop) and a 
mathematical program (Matlab 
script). After the total corneal area 
is delineated, the blood vessels are 
isolated using Photoshop software. 
To allow for precise quantification 
of corneal NV, three metrics will be 
computed using a Matlab script: 
Neovascular Area (NA), which measures the area of the corneal vessels themselves; 
Vessel Caliber (VC), which determines an approximate mean diameter of the corneal 
vessels; Invasion Area (IA), which measures the fraction of corneal area in which 
vessels are present [16] (Fig. 3).  
The investigator will assess the depth of blood vessels during the slit lamp 
examination and note whether the vessels are considered predominantly 
superficial/anterior-stromal, mid-stromal or deep-stromal. 
Endothelial Cell Density. Endothelial cell density will be assessed at  week-26 and 
week-52 visits via confocal/specular microscopy at each center based on the 
preferences of the site. All measurements on a given subject and at each site will be 
made utilizing the same technique throughout the study.  
Corneal Thickness. Central corneal Pachymetry will be performed on the study eye at 
Week-4, 16, 26 and 52 visits using an ultrasound pachymeter. Three readings of 
central corneal thickness will be obtained at each of these visits. 
Both Endothelial cell density and corneal thickness will serve as secondary endpoints. 
In general, endothelial loss and corneal graft edema are both significantly associated 


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with graft failure. Tracking these metrics will assist in evaluating the potential graft-
protective function of local VEGF blockade.  
 
7.4 Standard of Care Procedures (procedures regardless of study participation) 
 Record current ocular and systemic medications  
 Record significant medical/surgical history in the past 5 years 
 Record demographic data, including date of birth, sex, and race/ethnicity 
 Review of systems 
 Ocular assessments (OU) 
 Best spectacle-corrected visual acuity (BSCVA) (OU) 
 Slit-lamp examination (OU) 
 Intraocular pressure (OU) 
 Dilated funduscopy (Study eye) 
 Pachymetry (OU) 
 
7.5 Follow-up Procedures  
To determine the safety and efficacy of subconjunctival/topical bevacizumab in 
promoting graft survival, follow-up visits will be scheduled at Day 1 (≤90 days from 
screening) and 7 (±6 days), Week 4 (±10 days), 8 (±2 weeks), 16 (±2 weeks), 26 (±2 
weeks), 39 (±2 weeks), and 52 (±2 weeks). 
 
A list of follow-up procedures can be found in Appendix 1 and 6.  
 
 


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7.6 Unscheduled Visits 
Unscheduled visits have also been built into the study protocol to ensure that 
participant’s care is not limited by the investigational medication or study events. In 
the event of an unscheduled visit, participants can be seen by the investigator, fellow, 
or resident and imaging is not required. 
 
7.7 Early Termination 
Subjects who voluntarily withdraw from the study prior to completion will be asked 
to return for an early termination evaluation in 7 days (±3 days) for monitoring of all 
adverse events. The schedule of assessments for early termination is the same as that 
for the final visit (Week 52). 
 
7.8 Schedule of Activities  
A Schedule of Events can be found in Appendix 6. 
 


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8. SAFETY AND EFFECTIVENESS ASSESSMENTS 
 
8.1 Safety Assessments 
The safety of the study medication will be evaluated at every visit following surgery 
and will be defined by the incidence of related adverse events. Specifically, we will 
evaluate:  
 Systemic safety: Incidence and severity of systemic adverse events during the 
study (based on physical examination, subject self-reporting, and changes in 
vital sign). 
 Ocular safety: Incidence and severity of ocular adverse events during the study 
(based on ophthalmic examination and subject self-reporting). 
All adverse events will be reviewed by the principal investigator Reza Dana within 24 
hours of occurrence and reported on the following schedule:  
 
 Possibly, Probably, or Definitely Related Expected AE – Report to IRB on annual 
basis  
 Possibly, Probably, or Definitely Related Expected Serious AE – Report to IRB 
on annual basis  
 Possibly, Probably, or Definitely Related Unexpected AE – Report to IRB within 
30 days of event  
 Possibly, Probably, or Definitely Related Unanticipated Problem – Report to IRB 
within 7 days of event (24 hours for death of data loss)  
 Possibly, Probably, or Definitely Related Unexpected Serious AE – Report to IRB 
within 7 days of event   
 Specify the frequency of safety observation (a number of times, at defined time 
points, after a certain number of subjects have been recruited or as needed).  


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8.2 
Effectiveness Assessments 
The effectiveness of bevacizumab will be judged using the following parameters at 
specified time points:  
 Primary Endpoints: 
 Twelve-month endothelial rejection rate 
 Secondary Endpoints:  
 Time from surgery to occurrence of any rejection episode (endothelial, 
epithelial, subepithelial) 
 Time from surgery to overall graft failure (regardless of cause) 
 Incidence of delayed epithelial healing at Day 7  
 Endothelial cell density at Weeks 26 and 52 
o As measured by confocal/specular microscopy 
 Central corneal thickness at Screening, Weeks 4,16, 26, and 52 
 Frequency of primary graft failure 
 Corneal NV Metrics at Screening, Weeks 4,16, 26, and 52  
o Neovascular Area (NA), measuring the area of the corneal vessels 
themselves 
o Vessel Caliber (VC), measuring the mean diameter of the corneal 
vessels 
o Invasion Area (IA), measuring the fraction of corneal area in which 
vessels are present 
 


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9. ADVERSE EVENT RECORDING AND REPORTING 
 
9.1 Recording Requirements 
Investigators and study staff will be asked to report unanticipated problems or 
adverse events immediately to Dr. Dana to allow for proper reporting. Expected 
adverse events for the study drug injection during surgery include ocular 
pain/discomfort, bleeding/bruising beneath the injection site (conjunctiva), and globe 
penetration.  Expected adverse events for the study drug treatment period include 
corneal thinning, epithelial defect/surface epitheliopathy, delayed epithelial healing, 
ocular burning upon instillation of medication, hypertension, and symptoms related 
to allergic reaction (e.g, rash, difficulty swallowing).  All expected adverse events 
directly related to the standard of care surgery (e.g., ocular infection, loss of eye, 
photophobia, increased tearing) are listed in the surgical consent form and reviewed 
with the subject by the treating physician. 
All participants are informed of the anticipated risks of the trial and asked to report 
any unusual ocular or systemic problems that occur during their participation in this 
trial to the investigator or cornea research staff. In addition, all participants will be 
queried during their visits for occurrence of side effects from the medications, post-
surgical complications, and overall well-being. Any event mentioned by the 
participant will be documented in the CRF.  
All observed or volunteered adverse events (serious or non-serious) and abnormal test 
findings, regardless of study group or suspected causal relationship to the study 
drug(s) will be recorded in the subjects’ case histories (source data, case report form).  
For all adverse events, sufficient information will be pursued and/or obtained so as to 
permit 1) an adequate determination of the outcome of the event (i.e., whether the 


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event should be classified as a serious adverse event) and; 2)  an assessment of the 
causal relationship between the adverse event and the study drug(s) or device(s).    
Adverse events or abnormal test findings felt to be associated with the study drug(s) 
will be followed until the event (or its sequel) or the abnormal test finding resolves or 
stabilizes at a level acceptable to the Sponsor-Investigator. 
Study staff will alert the treating physician immediately of any participant complaints 
or problems that are expressed to them. Dr. Dana will be made aware of any 
unexpected problems that have occurred within 24 hours if considered to be an 
unexpected adverse event or serious adverse event, regardless of the assumed 
relation to the study medication, Dr. Dana and the study staff will follow the 
reporting guidelines below. 
Recording and Reporting Adverse events to HSC 
The study team at NYPH and BPEI and investigators at MEEI will be given the phone 
number and e-mail address of both Dr. Dana and the Cornea Research staff. If the 
event is serious or requires immediate attention Dr. Dana will be called on his cell 
phone or paged. If Dr. Dana is not available, the fellow or attending on call will be 
paged. All enrolling sites have access to an Emergency Room specializing in 
ophthalmic care that can be used, as well, 24 hours a day, 365 days a year. 
 
Adverse events and unanticipated problems will be reported to the DOD and IRB as 
detailed below.  
 
 
 
 
 
 


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Summary of HSC Reporting Requirements:  
Reporting 
Schedule 
Non-Serious AE 
Serious AE 
Unanticipated 
Problem 
Expected 
Unexpected Expected 
Unexpected 7 Days (24 
hours for 
death or data 
loss) 
Possibly, 
probably 
or 
definitely 
related 
Continuing 
Review 
30 Day 
Continuing 
Review 
7 Days 
 
*Any unexpected and study-related death must be reported to MEEI IRB within 24 
hours of the PI’s knowledge of the event by e-mail or telephone. 
 
Recording and Reporting Adverse Events 
For each subject, AEs and SAEs occurring after informed consent is obtained will be 
recorded until the subject has completed his/her participation in the study. 
 
Adverse Event recording will be done in a concise manner using standard, acceptable 
medical terms that reflects the reason for the procedure or the diagnosis based on 
the abnormal measurement. 
 
Any serious adverse event that is ongoing when a subject completes his/her 
participation in the Study will be followed until any of the following occurs: 
 the event resolves or stabilizes; 
 the event returns to baseline condition or value (if a baseline value is 
available); 


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 the event can be attributed to agents(s) other than the Study Product, or to 
factors unrelated to Study conduct. 
 
Any subsequent AE felt by the Investigator to be causally related to the use of the 
Study Product will be reported.  
 
Abnormal Test Findings 
An abnormal test finding will be classified as an adverse event if one or more of the 
following criteria are met: 
o The test finding is accompanied by clinical symptoms 
o The test finding necessitates additional diagnostic evaluation(s) or 
medical/surgical intervention; including significant additional 
concomitant drug treatment or other therapy  
o Note: simply repeating a test finding, in the absence of any of the 
other listed criteria, does not constitute an adverse event.   
o The test finding leads to a change in study drug dosing or 
discontinuation of subject participation in the clinical research study 
o The test finding is considered an adverse event by the Sponsor-
Investigator of the IND application 
 
 
Causality and Severity Assessment 
The Sponsor-Investigator of the IND application will promptly review documented 
adverse events and abnormal test findings to determine 1) if the abnormal test 
finding should be classified as an adverse event; 2) if there is a reasonable possibility 
that the adverse event was caused by the study drug(s); and 3) if the adverse event 
meets the criteria for a serious adverse event. 
If the Sponsor-Investigator’s final determination of causality is “unknown and of 
questionable relationship to the study drug(s)”, the adverse event will be classified as 


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associated with the use of the study drug(s) for reporting purposes.  If the Sponsor-
Investigator’s final determination of causality is “unknown but not related to the 
study drug(s)”, this determination and the rationale for the determination will be 
documented in the respective subject’s case history (source data or case report form).  
 
9.2 REPORTING PROCEDURES 
Reporting of Adverse Events to FDA 
1. Adverse events will be sent to the FDA per the CFR guidelines:  
 Non-serious adverse events will be submitted at time of the annual report.  
 Serious and unexpected adverse events related to the drug will be reported 
within 15 calendar days.  
 Unexpected fatal or life-threatening adverse events related to the drug will 
be reported within 7 calendar days. 
New animal findings that suggest significant risk in human subjects will be 
reported in 15 calendar days. 
2. Telephoned IND Safety Reports – Fatal or life-threatening suspected adverse 
reactions 
 
Reporting Adverse Events to Other External Entities 
Adverse events will be reported to the DOD on a quarterly basis. 
 
9.3 Withdrawal of Subjects due to Adverse Events 
All potential systemic and ocular adverse events will be assessed carefully at follow-
up visits in the study. The safety of subconjunctival and topical bevacizumab will be 
assessed through the collection and analysis of any systemic or ocular adverse events 
during the study based on subject self-reporting, physical and ophthalmic 
examination, and changes in vital sign (blood pressure). All participants will be asked 


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to return to clinic as standard for participants undergoing a penetrating keratoplasty. 
In addition, all standard of care post-operative medications are being maintained for 
the study. Unscheduled visits have also been built into the study protocol to ensure 
that participant’s care is not limited by the investigational medication or study 
events. In the event of an unscheduled visit, participants can be seen by the 
investigator, fellow, or resident and imaging is not required. 
Stopping criteria: development of non-healing epithelial defect of ≥ 2 x 2 mm in the 
graft lasting ≥2 weeks while on investigational treatment. 
Emergency care is available at all participating institutions in their emergency wards, 
which are staffed by ophthalmologists 24 hours per day, 365 days per year. 
Additionally, cornea specialists are on call at all times and can be reached by cell 
phone or pager to attend to any problem that may arise for these participants. 
Emergency care will be billed to the study, participant or third party payer as 
appropriate.  
Special precautions to be taken by the human subjects prior to and during 
participation in this study include: 45 day washout of anti-VEGF agents (intraocular or 
systemic), no change in topical corticosteroid regimen for 14 days prior to 
transplantation, 12 months or greater of no thromboembolic event, control of 
diabetes, and pregnancy prevention.  
Participants will be dilated at the screening, week 16 and week 52 visits. It is not 
recommended that participants drive while their eyes are dilated and are encouraged 
to find alternate transportation. Following the surgical procedure, participants will 
not be allowed to leave their hospital without being accompanied by another adult. 
All participants who experience an adverse event will be asked to complete all 52 
weeks of the study, regardless of severity or if the participant has been asked to 
discontinue use of the study medication. Subjects will not be replaced if withdrawn.  
The Severity of each adverse event will be defined by Mass. Eye and Ear criteria:  


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Adverse Event (AE): Any untoward or unfavorable medical occurrence in a human 
subject, including any abnormal sign (for example, abnormal physical exam or 
laboratory finding), symptom, or disease, temporally associated with the subject’s 
participation in the research, whether or not considered related to the subject’s 
participation in the research.  
AE Related to Research: There is a reasonable possibility that the adverse event, 
incident, experience or outcome may have been caused by the research procedures, 
e.g. blood draw, interview, survey, etc.  
AE Unrelated to Research: Adverse events related to circumstances independent of 
the research, e.g. relating to the underlying disease, disorder, or condition of the 
subject.  
Expected Adverse Event: Any adverse event that does not meet the definition of 
unexpected adverse event.  
External Adverse Events (Safety Reports): In the context of multi-center studies, 
adverse events experienced by research participants enrolled at study sites that are 
not under MEEI HSC’s jurisdiction.  
Internal Adverse Events: Adverse events experienced by research participants enrolled 
at study sites that are under MEEI HSC’s jurisdiction. In the context of a single-center 
study, all adverse events would be considered internal adverse events. 
Non-Serious Adverse Event: Any adverse event that does not meet the definition of a 
serious adverse event.  
Serious Adverse Event (SAE): Any event temporally associated with the subject’s 
participation in research that meets any of the following criteria:  
 Results in death  
 Is life threatening  
 Requires hospitalization/prolongation of hospitalization  
 Results in congenital anomaly  


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 Results in persistent or significant disability/incapacity  
 Required intervention to prevent permanent impairment/damage 
Unexpected Adverse Event: Any adverse event occurring in one or more subjects 
participating in a research protocol, the nature, severity, or frequency of which is not 
consistent with either: (1) the known or foreseeable risk of adverse events associated 
with the procedures involved in the research that are described in the protocol-
related documents; or (2) the expected natural progression of any underlying disease, 
disorder, or condition of the subject(s) experiencing the adverse event and the 
subject’s predisposing risk factor profile for the adverse event. 
 


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10. STATISTICAL METHODS/DATA ANALYSIS 
10.1 Primary endpoint(s) or outcome measure(s): 
 Systemic safety: Incidence and severity of systemic adverse events during the 
study (based on physical examination, subject self-reporting, and changes in 
vital sign). 
 Ocular safety: Incidence and severity of ocular adverse events during the study 
(based on ophthalmic examination and subject self-reporting). 
10.2 Secondary endpoints or outcome measure(s) 
 Primary Endpoints: 
 Twelve-month endothelial rejection rate 
 Secondary Endpoints:  
 Time from surgery to occurrence of any rejection episode (endothelial, 
epithelial, subepithelial) 
 Time from surgery to overall graft failure (regardless of cause) 
 Incidence of delayed epithelial healing at Day 7  
 Endothelial cell density at Weeks 26 and 52 
 Central corneal thickness at Weeks 4,16, 26, and 52 
 Frequency of primary graft failure 
 Corneal NV Metrics at the Weeks 4,16, 26, and 52  
o Neovascular Area (NA), measuring the area of the corneal vessels 
themselves 
o Vessel Caliber (VC), measuring the mean diameter of the corneal 
vessels 


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o Invasion Area (IA), measuring the fraction of corneal area in which 
vessels are present 
10.3 Sample Size Determination 
We based sample size determination on rate of rejection observed in prior studies of 
high-risk corneal transplantation. In those studies, endothelial rejection episodes tend 
to occur in the range of about 60% of transplants [22]. We used rates ranging from 50 
to 70 percent to compute the sample size (number of participants to enroll) for the 
study using the method described by Lachin and Foulkes (LF), as well as the 
Rubenstein, Gail and Santner approach (RGS). We specified the probability of a type 1 
error equal to 0.05, study power equal to 80%, a follow-up period of 12 months, and 
4% loss to follow-up. For practical purposes, we also took into consideration the time 
available for the study and the number of high-risk procedures done at each study 
site. Given these considerations, we estimated that if the treated group were half as 
likely to experience a rejection episode using the active treatment compared with the 
vehicle treatment (e.g. a rejection rate of 30% in the active group vs. 60% in the vehicle 
group), we wanted to be able to detect this difference. Calculations resulted in sample 
size estimates of between 65 and 124 participants to achieve this result. There were 
minor differences between the LF and RGS approaches (Table 2). Using the midrange 
estimate we aim to enroll 90 participants into this study. 
Table 2. Sample Size Calculation 
Cumulative Rejection Rate among 
Control Group 
Total Sample Size 
 
LF 
RGS 
0.50 
122 
124 
0.60 
90 
90 
0.70 
66 
65 
 


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10.4 Analysis Population  
Population analysis is not applicable in this study. 
 
10.5 Effectiveness Analysis  
The primary efficacy analysis will follow an intent-to-treat analysis strategy including 
all randomized eyes. The major goal of this study is to determine whether treatment 
with bevacizumab significantly reduces the occurrence of transplant endothelial 
rejections compared to a control regimens following corneal transplantation in high-
risk eyes. The randomized design should provide for balanced distributions of 
baseline characteristics between the two treatment groups. Nonetheless, we will 
conduct an initial analysis to determine any chance imbalances that may have 
occurred. These analyses will form part of the routine monitoring of the trial and will 
be regularly reported to the DSMB. For continuous and ordinal variables (e.g. age), 
comparisons will be made using the Mann-Whitney Wilcoxon test based on ranks. For 
categorical variables (e.g. sex, race) comparisons will be made using Chi-square or 
Fisher exact tests as appropriate. In addition to inter-group (arm) analysis of different 
time points, the "area under the curve" (severity vs. time) will be evaluated for 
neovascular area (invasion area), length, and caliber between different areas. These 
analyses are intended for data monitoring and quality control, and not to determine 
which baseline covariates to include in efficacy analysis.  
 
The primary endpoint of the trial is the time to first endothelial rejection. We will use 
a proportional hazards regression framework with robust variance estimation to test 
the effectiveness of bevacizumab in reducing the occurrence of transplant rejection 
over time following high-risk corneal transplantation. This analysis will stratify on 
graft/regraft and center as was done in the randomization scheme. We will obtain an 
estimate of the incidence rate ratio (relative risk) of rejection for eyes assigned to 
bevacizumab versus vehicle. This framework allows for the control of covariates, 
which may be unbalanced by chance despite the randomization of treatment 


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assignment. If Kaplan-Meier plots of event free survival by study time, or related plots 
of log (-log) (survival) indicate violations of the proportional hazards assumption, 
then weighted log-rank tests will be used according to strategies described by Peckova 
and Fleming. The clinical significance of any observed benefit associated with 
bevacizumab therapy will also be quantified by the number needed to treat. This 
measure has the advantage of quantifying the absolute, as compared to the relative 
treatment effect. 
Any deviations from the previously described statistical plan will be described and 
justified in a protocol amendment and/or in the final report submitted to this 
application. 
 
10.6 Safety Analysis  
Interim Analysis for Treatment Monitoring: We have established an independent 
Data and Safety Monitoring Board (DSMB) to monitor progress and make 
recommendations regarding early termination of the study as outlined below. In some 
trials, motivated primarily by a desire to avoid putting participants at unnecessary 
risk or to detect as early as possible whether the effect of the intervention is so great 
that further data collection is unnecessary, the committee is aided by setting a 
statistical criterion or “stopping rule” that, when met by the accumulating data, 
indicates that the trial can or should be stopped early. While the frequency of 
meetings and approach to interim monitoring will be the choice of the DSMB, we will 
recommend to conduct one interim analysis at the midway point of the study. We 
recommend that this analysis should examine the primary study endpoint and, 
following the Peto approach, consider early termination of the study if the p-value at 
the interim analysis is <0.001. Although this is a stringent criterion, it is appropriate 
in low- to moderate-risk trials and balances the risk of overestimating treatment 
effects. In addition, the occurrence of any adverse events will be described and 
compared between treatment arms of the study. There is no explicit pre-specified 
harms-related hypothesis as the treatment has been studied and used systemically 


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and is well tolerated in this topical ocular preparation. If interim analyses show 
unexpected differences in benefits or toxicity, the study investigators and DSMB may 
consult with other independent advisors comprised of experts in the field of 
Ophthalmology and Cornea and External Diseases to consider all of the evidence and 
aid them in the decision process. 
 
Secondary Analyses: In addition to the primary comparisons of bevacizumab versus 
vehicle, we plan to conduct a number of pre-specified analyses of secondary study 
endpoints. Analyses of secondary “time-to-occurrence” type endpoints (e.g. any graft 
failure, any graft rejection) will use the proportional hazards regression framework 
(as used for the primary outcome). Analyses of quantitative endpoints (e.g. 
endothelial cell density, corneal thickness) will be performed using the Mann-Whitney 
Wilcoxon test based on ranks for comparison of active versus vehicle groups.   
 


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11. DATA AND SAFETY MONITORING  
 
11. 1 Site Training and Compliance  
Site expectations for data entry, record keeping and regulatory documents are 
outlined in this protocol and are communicated verbally to each site investigator and 
coordinator either during monthly study coordinator conference calls and quarterly 
investigator calls or at the study annual meeting.  
Additional information on monitoring can be found in Section 12. Clarification on 
training and study management can be found in Appendix 3.  
 
11.2 Data and Safety Monitoring Plan  
Research subject safety is a priority. Therefore, a Data and Safety Monitoring Plan to 
ensure the safety of participants and the validity and integrity of the data collected 
has been established for this project. This plan will be shared with each site’s IRB.  
As detailed previously, at each study visit the investigator will query and examine 
each participant regarding potential side effects and adverse reactions. The efficacy 
outcomes that will be monitored include endothelial rejection rate, occurrence of any 
rejection episode, overall graft failure, delayed epithelial healing, endothelial cell 
density, central corneal thickness, occurrence of primary graft failure and 
quantification of corneal neovascularization. The primary safety variable monitored 
will be the occurrence of adverse events. A Data and Safety Monitoring Plan will be in 
place which includes the following: The severity of each adverse event observed 
(ocular and systemic) will be rated from mild (awareness of sign or symptom, but 
easily tolerated) to severe (incapacitating with inability to work or do usual activity). 
The relationship of the event to the study medication will be assessed by the 
investigator as ‘none’, ‘possible’, ‘probable’, or ‘definite’. Safety variables will be 


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evaluated at baseline and at all study visits. If adverse events occur, the first concern 
will be the safety of the study participants. Any serious adverse event occurring 
during the study period will be reported to all site PIs, all site IRBs, the FDA as 
outlined in the Study Management Plan and the designated Data and Safety 
Monitoring Board (DSMB) (see below).  
At each of the visits throughout the study, the investigator will begin querying for 
adverse events by asking each participant a general, non-directed question such as 
“How have you been feeling since the last visit?” Directed questioning and 
examination will then be done as appropriate. The investigator will ask questions to 
subjects at each visit to determine if they have had any changes to the use of 
concomitant medications since the previous visit.  A comprehensive eye examination 
including best-corrected visual acuity, measuring intraocular pressure, evaluation of 
the condition of the lid/lashes, conjunctiva, cornea, anterior chamber, iris/pupil, lens, 
vitreous, macula and optic nerve will be performed. Any changes in the study eye 
from the baseline visit will be recorded.  
The DOD will be informed of any actions taken by the IRB as a result of its continuing 
review. This study holds an IND from the FDA. Therefore, individual adverse event 
reports will be submitted to the DOD, FDA and IRBs in accordance with regulations. 
The research staff at MEEI, the primary site of the protocol, will serve as the central 
reporting entity. They will be responsible for preparing summary reports of adverse 
events and distributing the information to all participating sites. Detailed information 
on adverse event and unanticipated problem reporting is included in the Study 
Management Plan.  
Data and Safety Monitoring Board 
A Data and Safety Monitoring Board (DSMB) has been formed that will review the data 
and any reported adverse events on a periodic basis. This group is comprised of 
researchers and physicians who are not associated with the trial. The members 
include Joseph Rizzo, MD, who will serve as the DSMB Chair, Lou Pasquale, MD, Debra 


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Schaumberg, OD, ScD, MPH and Reverend Stephen Ayres, PhD, Episcopal Diocese of 
Boston. 
Joseph Rizzo, M.D. serves as Director of the Neuro-Ophthalmology Service at 
Massachusetts Eye and Ear Infirmary.  He is board certified in psychiatry and 
neurology as well as ophthalmology and holds the position of David G. Cogan 
Professor of Ophthalmology in the Field of Neuro-Ophthalmology at Harvard Medical 
School.  Dr. Rizzo has conducted clinical research for over 25 years. 
Louis Pasquale, M.D. serves as Director of the Glaucoma Service as well as the 
Ophthalmology Telemedicine Program at Massachusetts Eye and Ear Infirmary.  He is 
board certified in ophthalmology and holds the position of Associate Professor of 
Ophthalmology at Harvard Medical School.  Dr. Pasquale has been involved in clinical 
research for over 25 years. 
Debra Schaumberg, Sc.D., O.D. is Adjunct Professor of Epidemiology at Harvard 
School of Public Health.  She also holds the position of Associate Professor of 
Ophthalmology at Massachusetts Eye and Ear Infirmary.  Dr. Schaumberg has been 
involved in research for over 20 years. 
Reverend Stephen Ayres is Vicar of Christ (Old North) Church in Boston, 
Massachusetts.  He has served as a community representative on the Schepens Eye 
Research Institute’s IRB since 2003. 
Research Monitor 
The Research Monitor, Joseph Rizzo, MD, is responsible to oversee the safety of the 
research and report observations/findings to the MEEI IRB.  The Research Monitor will 
review all unanticipated problems involving risk to volunteers or others associated 
with the protocol and provide an unbiased written report of the event to the MEEI IRB. 
The Research Monitor may discuss the research protocol with the investigators, 
interview human subjects, and consult with others outside of the study about the 
research.  The Research Monitor shall have authority to stop the research protocol in 
progress, remove individual human subjects from the study, and take whatever steps 
are necessary to protect the safety and well-being of human subjects until the IRB can 


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assess the monitor’s report.  The Research Monitor is responsible for promptly 
reporting their observations and findings to the IRB. 
Since this is a multi-center study participants will be drawn from the surrounding 
areas of each participating site. The DSMB plan will be incorporated in site IRB 
applications. The DSMB will ensure the safety of participants and the validity and 
integrity of the data collected. The Statement of Work highlights the timing of the 
DSMB meetings throughout the course of the study. A quarterly update will also be e-
mailed to all sites, the DOD, trial statistician and DSMB. The update will include an 
overview of adverse events, an enrollment update, change in any personnel, reports 
from the Data Safety Monitoring Board.  
All investigators and key personnel will be invited to attend annually a study meeting, 
which will be held at ARVO. The meeting will allow for all investigators to discuss 
progress of the study, issues that have arisen within the past year, feedback from the 
Data Safety Monitoring Board and adverse events.  
 
 


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12. DATA HANDLING, RECORD-KEEPING AND MONITORING 
 
12.1 Data Recording, Record-Keeping and Monitoring  
A Case Report Form (CRF) will be completed for each subject enrolled into the clinical 
study.  The Sponsor-Investigator will review, approve and sign/date each completed 
CRF; the Sponsor-Investigator’s signature serving as attestation of the Sponsor-
Investigator’s responsibility for ensuring that all clinical and laboratory data entered 
on the CRF are complete, accurate and authentic. In this study, the CRF will be 
digitally represented, in the form of a 21 CFR Part 11-compliant online data capture 
program called StudyTrax.  
Source Data are the clinical findings and observations, laboratory and test data, and 
other information contained in Source Documents. StudyTrax will serve as the Source 
Data in addition to the Case Report Forms. 
Case Report Forms (CRF) will be completed at each participant visit on StudyTrax, a 
software designed to capture study related data. Study Staff at New York Presbyterian 
Hospital (NYPH) and Bascom Palmer Eye Institute (BPEI) will enter all study data from 
participant visits directly into StudyTrax. Treatment randomization codes will be 
documented in StudyTrax for each participant. StudyTrax will be designed with 
certain limits, ensuring that all required information is entered. Limits in specific data 
fields can be set to minimize typographical errors. Were StudyTrax not available, 
paper CRFs will be sent over to MEEI via secure e-mail or via fax, and the data will be 
uploaded to StudyTrax by MEEI study staff.  
The Study Coordinator (SC) at MEEI will review each CRF from MEEI, BPEI and NYPH to 
ensure that all required study assessments have been completed. If data is missing, 
the coordinator will query the site to ensure completeness.  Following this, the MEEI 
Clinical Research Fellow assigned to this study will review the documents for safety. 


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The Clinical Research Fellow has in depth knowledge of the study protocol, 
procedures and investigational medication. If there are any safety concerns or 
uncertainty the Fellow will review the documents with Dr. Dana at the time the event 
was discovered or reported. Dr. Dana, who is responsible for overall monitoring of the 
study, will review all CRFs and reports at the following visits per participant: 
screening, and weeks 4, 26, 52. The efficacy outcomes that will be monitored include 
endothelial rejection rate, occurrence of any rejection episode, overall graft failure, 
delayed epithelial healing, endothelial cell density, central corneal thickness, 
occurrence of primary graft failure and quantification of corneal neovascularization. 
Participants will be monitored continuously for systemic and ocular safety during 
their ophthalmic examinations. If there is an adverse event or safety issue, the 
guidelines above will be followed.  
Each site is responsible for verifying the data collected into StudyTrax. A monitoring 
log will be kept to verify the PI review of the data collected during each study visit.  
Investigators, fellows, study coordinators, and technicians will have access to 
participant data through StudyTrax. Study staff at Bascom Palmer and Cornell will 
only be able to view and edit their own participant data. The PI, fellow, and research 
coordinator at MEEI will have access to participant information and data at Bascom 
Palmer and Cornell as to follow the data safety monitoring plan.  
 
Confidentiality 
Identifying health information will be collected from each participant only with their 
permission (obtained in the Informed Consent and HIPAA).  Solely information 
needed for the research project will be collected.  All health information will be 
protected and kept confidential to preserve the confidentiality of all participants in 
accordance with HIPAA regulations.  All subjects will be given a HIPAA authorization 
to sign acknowledging their understanding of their private health information rights.  
Protected health information with participant identifiers will only be accessible to 
research personnel who require it to ensure safety of the participants and integrity of 


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the data. The source documents with identifiable health information will only be 
shared amongst members of the research team involved in the subject’s care. All 
other research personnel will be given data with the participants study identification 
number only.  
 
All paper study records will be kept locked allowing only research staff access. 
Electronic records will be stored on computers that are password protected and 
equipped with anti-virus software within each institution. The Electronic Data 
Capturing system, StudyTrax, is also password protected and access is controlled by 
one administrator at Mass. Eye and Ear. Each site will only have access to their own 
records, except for the primary enrolling site, Mass. Eye and Ear, whose study 
coordinator and clinical research manager will have access to all site information.  
 
Slit lamp photographs of participants will be shared through StudyTrax, which is 
supervised by Mass. Eye and Ear. The University of Miami and Weill Cornell Medical 
College enrolling sites will be given privileges to upload photographs to be attached 
with corresponding patient visits. 
 
All study records are subject to review by representatives of the USAMRMC.   
The data from this project may be used in publications or manuscripts written by the 
research team. 
 
Data Transmission 
Data will be entered at the time of the study visit by the investigators or study staff 
into StudyTrax. StudyTrax allows research teams to create unique case report forms 
(CRFs), determine levels of user access for increased security, extract data into SAS, 
SPSS or excel spreadsheets, query data, create audit trails, and track adverse events 
and participant’s medications. The system is HIPAA and 21 CFR Part 11 compliant. 
The central enrolling site will be able to access the information from each visit on 
StudyTrax and will check to ensure that all assessments are done as described in the 


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protocol. If any information is noted to be missing, the study coordinator from MEEI 
will contact the site coordinator and gather the missing information. The MEEI will 
check all entries and immediately notify the sites of any errors, so that they can be 
addressed in a timely fashion. 
 
Data Security 
All data will be entered into StudyTrax. StudyTrax is a leading provider of electronic 
data capturing and is designed to meet the needs of academic clinical research 
centers. StudyTrax is a web-based system hosted by Partners HealthCare that can be 
used by all study sites with a secure, unique username and password.  
 
Data Extraction 
All participant information and study data can be extracted from the website at any 
given time for any of the study parameters. This functionality can greatly assist in an 
interim analysis or assessment of study adverse events. Data extraction will occur at a 
minimum of every three months and be sent to the Statistician for independent trial 
monitoring. This will assist in making sure that enrollment targets are being met, 
there are no detected errors or concerns with randomization. 
 
Disposition of Data 
Electronic case report forms will be stored in the StudyTrax system. Following the 
data lock at the end of the study, the information collected in the case report forms 
will be exported to excel spreadsheet. The key to the study participant identifications 
will be held in a separate electronic file. The de-identified excel spreadsheets of 
participant data will be saved for two years from the termination of the study. All 
files will be backed up on a secure server during this time. Following data export, the 
StudyTrax account will be closed for all sites. Hard copies of documents (regulatory 
documents, consent forms and other study documentation) will be maintained at 
each site in a secure, locked space. 
 


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Sharing Study Results 
The decision to share study assessments and results completed while participating in 
the study with the participant and/or their other physicians will be evaluated on a 
case-by-case basis.  The decision will ultimately be made by the investigator and/or 
participant. 
 
Sharing of Corneal Photographs  
Individual sites are required to upload slit lamp photos to StudyTrax from visits 
where those images are required.  
 
Regulatory Documents 
Monitoring of the electronic regulatory documents will be conducted monthly. 
Queries will be sent to each site following the review to address any outstanding 
items. All queries and subsequent resolutions will be documented and stored in the 
MEEI regulatory binder.  
 
Routine Reporting 
The MEEI will provide quarterly reports to all sites, including a Performance Report 
and a Quality Control Report. The Performance Report will include enrollment and 
randomization rates, reasons for randomization ineligibility; number of completed 
follow-up visits, number of participants not taking their study drug, and reasons for 
discontinuation. A Quality Control assessment will also be provided and will detail 
any ID and acrostic errors, inappropriate randomizations, missing data broken down 
by form, reasons for missed visits, number of visits taking place outside of the 
designated time windows, etc. Finally, the Data and Safety Monitoring Committee will 
be provided an analysis of treatment safety and efficacy in addition to the routine 
progress and quality control reports. The routine reporting and ongoing QC checks 
described will help ensure maximal data integrity and timely completion of study as 
forecast. 


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The Principal Investigator will comply with the following Department of Defense 
reporting requirements: 
(1) Substantive modifications to the research protocol and any modifications that 
could potentially increase risk to subjects will be submitted to the HRPO for approval 
prior to implementation. The USAMRMC ORP HRPO defines a substantive 
modification as a change in Principal Investigator, change or addition of an 
institution, elimination or alteration of the consent process, change to the study 
population that has regulatory implications (e.g. adding children, adding active duty 
population, etc), significant change in study design (i.e. would prompt additional 
scientific review) or a change that could potentially increase risks to subjects. 
(2) Any changes of the IRB used to review and approve the research will be promptly 
reported to the USAMRMC ORP HRPO. 
(3) All unanticipated problems involving risk to subjects or others must be promptly 
reported by telephone (301-619-2165), by email (usarmy.detrick.medcom-
usamrmc.other.hrpo@mail.mil), or by facsimile (301-619-7803) to the HRPO. A 
complete written report will follow the initial notification. In addition to the methods 
above, the complete report can be sent to the US Army Medical Research and Materiel 
Command, ATTN: MCMR-RP, 810 Schreider Street, Fort Detrick, Maryland 21702-5000. 
(4) Suspensions, clinical holds (voluntary or involuntary), or terminations of this 
research by the Institutional Review Board (IRB), the institution, the Sponsor, or 
regulatory agencies will be promptly reported to the USAMRMC ORP HRPO. 
(5) A copy of the continuing review approval notification by the IRB of Record will be 
submitted to the HRPO as soon as possible after receipt. A copy of the continuing 
review report approved by the IRB will also be provided. Please note that the HRPO 
also conducts random audits at the time of continuing review. Additional information 
and documentation may be requested at that time. 
(6) The final study report, including any acknowledgement documentation and 
supporting documents, will be submitted to the HRPO when available. 


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(7) The knowledge of any pending compliance inspection/visit by the FDA, DHHS 
Office of Human Research Protections (OHRP), or other government agency 
concerning this research, the issuance of Inspection Reports, FDA Form 483, warning 
letters or actions taken by any regulatory agencies including legal or medical actions 
and any instances of serious or continuing noncompliance with the regulations or 
requirements, must be promptly reported to the HRPO. 
 
The USAMRMC ORP HRPO conducts site visits as part of its responsibility for 
compliance oversight. Accurate and complete study records must be maintained and 
made available to representatives of the USAMRMC as a part of their responsibility to 
protect human subjects in research. Research records must be stored in a confidential 
manner so as to protect the confidentiality of subject information. 


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13. STUDY DISCONTINUATION CRITERIA 
 
13.1 Discontinuation of Individual Research Subjects 
Subjects have a right to withdraw from the study at any time. The subject may be 
withdrawn from the study for any reasons; such as if it is deemed in the best interest 
of the subject, due to concurrent illness, adverse events, or worsening condition. The 
primary investigator may request the withdrawal of a subject because of protocol 
violations, administrative reasons, or any other valid and ethical reasons.  
 
Stopping Criterion: development of non-healing epithelial defect of ≥ 2 x 2 mm in the 
graft lasting ≥2 weeks while on investigational treatment. 
 
Early Termination Assessments: Subjects who voluntarily withdraw from the study 
prior to completion will be asked to return for an early termination evaluation in 7 
days (±3 days) for monitoring of all adverse events. The schedule of assessments for 
early termination is the same as that for the final visit (Week 52). 
 
Withdrawal of subjects due to non-compliance/adherence: The decision to withdraw 
a participant due to non-compliance or adherence to study medications, standard-of-
care medications, or study visits will be made by the principal investigator, Dr. Reza 
Dana.  
 
Withdrawal of subjects due to adverse events: Subjects who experience adverse 
events will not be required to withdraw from the study. Rather, subjects will be 
followed throughout the 52 week period, barring voluntary participant withdrawal. 
Voluntary withdrawal effectively stops the collection of study data for the participant 
and only previous study visits can be analyzed.  


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Replacement of Withdrawn Study Participants: Participants who have voluntarily 
withdrawn or have been withdrawn by the PI will not be replaced.  
13.2 Sponsor-Investigator Discontinuation of the Clinical Research Study 
Trial Suspension and Termination: If Dr. Dana and/or the DSMB determines that the 
trial needs to be terminated or temporarily suspended, the investigators, FDA, DOD, 
and HSC will be notified. Dr. Dana, research staff and the HSC will determine the best 
method to notify participants of study termination or suspension to ensure 
participant safety, confidentiality and data integrity. The investigators will notify all 
participants of the termination or suspension and reasoning. Outstanding data from 
outside MEEI will be collected for analysis and reporting purposes.  
If the trial is terminated, active participants will be asked to return to MEEI for routine 
follow-up, yet data will not be recorded for study purposes.  
 
 


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14. REFERENCES CITED 
 
REFERENCES CITED  
1.   Garg P, Krishna PV, Stratis AK, et al. The value of corneal transplantation in 
reducing blindness. Eye 2005;19:1106-14. 
2.   Whitcher JP, Srinivasan M, Upadhyay MP. Corneal blindness: a global perspective. 
Bull World Health Organ 2001;79:214-21. 
3.   Williams KA, Esterman AJ, Bartlett C, et al. How effective is penetrating corneal 
transplantation? Factors influencing long-term outcome in multivariate analysis. 
Transplantation 2006;81:896-901. 
4.   Streilein JW. Immunological non-responsiveness and acquisition of tolerance in 
relation to immune privilege in the eye. Eye 1995;9 ( Pt 2):236-40. 
5.   Coster DJ, Williams KA. The impact of corneal allograft rejection on the long-term 
outcome of corneal transplantation. Am J Ophthalmol 2005;140:1112-22. 
6.   Maguire MG, Stark WJ, Gottsch JD, et al. Risk factors for corneal graft failure and 
rejection in the collaborative corneal transplantation studies. Collaborative Corneal 
Transplantation Studies Research Group. Ophthalmology 1994;101:1536-47. 
7.   Hill JC. The relative importance of risk factors used to define high-risk 
keratoplasty. Ger J Ophthalmol 1996;5:36-41. 
8.   Dana MR, Qian Y, Hamrah P. Twenty-five-year panorama of corneal immunology: 
emerging concepts in the immunopathogenesis of microbial keratitis, peripheral 
ulcerative keratitis, and corneal transplant rejection. Cornea 2000;19:625-43. 
9.   Dana MR, Streilein JW. Loss and restoration of immune privilege in eyes with 
corneal neovascularization. Invest Ophthalmol Vis Sci 1996;37:2485-94. 
10. Hill JC, Maske R, Watson P. Corticosteroids in corneal graft rejection. Oral versus 
single pulse therapy. Ophthalmology 1991;98:329-33. 


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11. Amano S, Rohan R, Kuroki M, et al. Requirement for vascular endothelial growth 
factor in wound- and inflammation-related corneal neovascularization. Invest 
Ophthalmol Vis Sci 1998;39:18-22. 
12. Cursiefen C, Rummelt C, Kuchle M. Immunohistochemical localization of vascular 
endothelial growth factor, transforming growth factor alpha, and transforming 
growth factor beta1 in human corneas with neovascularization. Cornea 2000;19:526-
33. 
13. Cursiefen C, Cao J, Chen L, et al. Inhibition of hemangiogenesis and 
lymphangiogenesis after normal-risk corneal transplantation by neutralizing VEGF 
promotes graft survival. Invest Ophthalmol Vis Sci 2004;45:2666-73. 
14. Pieramici DJ, Rabena MD. Anti-VEGF therapy: comparison of current and future 
agents. Eye 2008;22:1330-6. 
15. Kim SW, Ha BJ, Kim EK, et al. The effect of topical bevacizumab on corneal 
neovascularization. Ophthalmology 2008;115:e33-8. 
16. Dastjerdi MH, Al-Arfaj KM, Nallasamy N, et al. Topical bevacizumab in the 
treatment of corneal neovascularization: results of a prospective, open-label, 
noncomparative study. Arch Ophthalmol 2009;127:381-9. 
17. You IC, Kang IS, Lee SH, et al. Therapeutic effect of subconjunctival injection of 
bevacizumab in the treatment of corneal neovascularization. Acta Ophthalmol 2008. 
18. Alldredge OC, Krachmer JH. Clinical types of corneal transplant rejection. Their 
manifestations, frequency, preoperative correlates, and treatment. Arch Ophthalmol 
1981;99:599-604. 
19. Uy HS, Chan PS, Ang RE. Topical bevacizumab and ocular surface 
neovascularization in participants with stevens-johnson syndrome. Cornea 
2008;27:70-3. 
20. Bock F, Konig Y, Kruse F, et al. Bevacizumab (Avastin) eye drops inhibit corneal 
neovascularization. Graefes Arch Clin Exp Ophthalmol 2008;246:281-4. 


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21. Yu CQ, Zhang M, Matis KI, et al. Vascular endothelial growth factor mediates 
corneal nerve repair. Invest Ophthalmol Vis Sci 2008;49:3870-8. 
22. Thompson RW, Jr., Price MO, Bowers PJ, et al. Long-term graft survival after 
penetrating keratoplasty. Ophthalmology 2003;110:1396-402. 
23. Fernando NH, Hurwitz HI. Targeted therapy of colorectal cancer: clinical 
experience with bevacizumab. Oncologist 2004;9 Suppl 1:11-8. 
24. Fung AE, Rosenfeld PJ, Reichel E. The International Intravitreal Bevacizumab 
Safety Survey: using the internet to assess drug safety worldwide. Br J Ophthalmol 
2006;90:1344-9. 
25. Lass JH, Gal RL, Dontchev M, et al. Donor age and corneal endothelial cell loss 5 
years after successful corneal transplantation. Specular microscopy ancillary study 
results. Ophthalmology 2008;115:627-32. 
26. Ward, TP. Eye Injuries in modern warfare. Third Biennial Military Vision Research 
Symposium: Boston. 
27. Dastjerdi M, Saban DR, Okanobo A, Nallasamy N, Sadrai Z, Chauhan SK, 
Hajrasouliha A, Dana R. Effects of topical and subconjunctival bevacizumab (Avastin) 
in high-risk corneal transplant survival. Invest Ophthalmol Vis Sci 2010; 51: 2411-
2417. PMC2868492  
28. Maguire MG. Risk factors for corneal graft failure and rejection in the 
Collaborative Corneal Transplantation Studies. CCTS Research Group. Ophthalmology 
1994; 101:1536-47. 


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15. APPENDICES 
 
15.1 Appendix 1: Outline of Study Visits and Procedures  
15.2 Appendix 2: Recruitment Script 
15.3 Appendix 3: Study Management Plan 
15.4 Appendix 4: Classification and Treatment of Graft Rejection Summary  
15.5 Appendix 5: Corneal Photography Protocol 
15.6 Appendix 6: Schedule of Events 
15.7 Appendix 7: Acronyms 
 
 


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APPENDIX 1: OUTLINE OF STUDY VISITS AND PROCEDURES 
 
Outline of Study Visits and Procedures 
STUDY VISIT 
 
STANDARD OF CARE 
PROCEDURES 
NON-STANDARD OF CARE 
PROCEDURES 
Screening Visit (Baseline) 
Record current ocular and 
systemic medications  
Review of eligibility criteria 
Record significant 
medical/surgical history in the 
past 5 years 
Written informed consent 
Review of systems 
 
Measurement of systolic and 
diastolic blood pressures 
Record demographic data, 
including date of birth, sex, and 
race/ethnicity 
Urine pregnancy test for women 
of childbearing potential, if 
appropriate 
Slit-lamp examination (OU) 
Corneal photography (Study eye) 
Best spectacle-corrected visual 
acuity (BSCVA) manifest 
refraction (OU) 
 
Intraocular pressure (OU) 
Dilated funduscopy (OU) 
Pachymetry (OU) 
Day 0 
Penetrating keratoplasty 
Subconjunctival injection of 
bevacizumab or saline 
Post-Op Day 1 
Best spectacle-corrected visual 
acuity (BSCVA) (Study eye) 
Blood Pressure 
 
Slit-lamp examination (Study 
eye) 
Start study eye drop 4 times per 
day for 4 weeks 
Intraocular pressure (Study eye) 
Review of systems and ocular 
and systemic medications 
Start topical prednisolone acetate 
1% 6 times per day, continue for 
2 weeks 
Start topical antibiotic 4 times 
per day until graft 
epithelialization or continue as 
necessary 
Post-Op Day 7 
Best spectacle-corrected visual 
acuity (BSCVA) (Study eye) 
Blood Pressure 
 
Review of systems and ocular 
and systemic medications 
Study medication compliance 
check 
Slit-lamp examination (Study 
eye) 
Adverse event query 


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Intraocular pressure (Study eye) 
Continue prednisolone acetate 
1% 6 times per day for one more 
week. At the beginning of post-
op week 3, reduce to 4 times per 
day. Continue this regimen for 6 
weeks (post-op week 8) 
Post-Op Week 4 
Slit-lamp examination (OU) 
Blood Pressure 
Review of systems and ocular 
and systemic medications 
Study medication compliance 
check 
Pachymetry (OU) 
Adverse event query 
Intraocular pressure (OU) 
Stop study eye drop and return of 
all unused bottles 
Best spectacle-corrected visual 
acuity (BSCVA) (OU) 
Corneal Photography (Study eye) 
Continue prednisolone acetate 4 
times per day 
Post-Op Week 8 
Best spectacle-corrected visual 
acuity (BSCVA) (OU) 
Adverse event query 
Slit-lamp examination (OU) 
Blood Pressure 
Intraocular pressure (OU) 
Review of systems and ocular 
and systemic medications 
Taper prednisolone acetate to 3 
times per day. Continue regimen 
for 4 weeks (post-op week 12). 
At post-op week 13 begin 
prednisolone 2 times per day for 
6 months (post-op month 9). 
Post-Op Week 16 
Best spectacle-corrected visual 
acuity (BSCVA) (OU) 
 
Slit-lamp examination (OU) 
Corneal photography (Study eye) 
Intraocular pressure (OU) 
Adverse event query 
Review of systems and ocular 
and systemic medications 
Dilated Funduscopy (Study eye) 
Pachymetry (OU) 
Continue prednisolone acetate 2 
times per day until post-op 
month 9/week 39 
 
 
 
Post-Op Week 26 
Best spectacle-corrected visual 
Adverse event query 


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acuity (BSCVA) (OU) 
Slit-lamp examination (OU) 
Corneal photography (Study eye) 
Intraocular pressure (OU) 
Confocal/specular microscopy 
(Study eye) 
Review of systems and ocular 
and systemic medications 
Pachymetry (OU) 
Continue prednisolone acetate 2 
times per day until post-op 
month 9/week 39 
Post-Op Week 39 
Best spectacle-corrected visual 
acuity (BSCVA) (OU) 
Adverse event query 
Slit-lamp examination (OU) 
Intraocular pressure (OU) 
Review of systems and ocular 
and systemic medications 
Taper prednisolone acetate to 1 
time per day. Continue 
indefinitely unless instructed 
otherwise by the investigator. 
Post-Op Week 52 
Best spectacle-corrected visual 
acuity (BSCVA) manifest 
refraction (OU) 
Adverse event query 
Slit-lamp examination (OU) 
Confocal/specular microscopy 
(Study eye) 
Intraocular pressure (OU) 
Corneal photography (Study eye) 
Review of systems and ocular 
and systemic medications 
Pachymetry (OU) 
Dilated funduscopy (Study eye) 
 
 


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APPENDIX 2: RECRUITMENT SCRIPT 
 
If no definite reasons to exclude the participant are found during the pre-screen 
review (as described in Section III of the Protocol), the participant will be approached 
by their physician (investigator) in the following manner: 
 
Study Coordinator/Research Fellow (After receiving word from the investigator that a 
participant is interested): “Hello Mr./Mrs./Ms. ______________. I am _____, a study 
coordinator/research fellow that works with Dr.________.  Dr. _______ asked me to 
speak with you about a research study because you are considering/have been 
scheduled for a corneal transplant and are considered to be at high-risk for a graft 
rejection. The study Dr. _______ mentioned is looking at the safety and effectiveness of 
an investigational drug, bevacizumab, also known as Avastin.  This study is looking at 
whether this investigational drug helps reduce the risk of graft rejection after surgery.  
 
If you agree to participate, you will receive one of two study treatments: Avastin, an 
investigational drug or a placebo. A placebo is a treatment with no active ingredient. 
Although Avastin is currently approved by the FDA to treat certain cancers, it is not 
FDA approved for how it will be used in this study.  During your surgery, your surgeon 
would either inject into your conjunctiva a dose of Avastin, if you are assigned to the 
active study treatment group, or an injection of saline, if you are assigned to the 
placebo group. Following surgery you would be given eye drops, either containing 
Avastin, the active study treatment, or Refresh Liquigel, an artificial tear for the 
placebo study treatment, to use four times a day for four weeks.  


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We will follow you closely after your surgery to see if you have any side effects related 
to the study medication. We will do other assessments throughout the study to also see 
if you have any signs of rejection. 
 
Our pharmacy will randomly assign you a study treatment. They will give you the 
study medication in plain eye dropper bottles so neither you, Dr. __________, or I will 
know which eye drop you are taking. You will be assigned to either the active study 
treatment group or the placebo group. Depending on the group assigned you will 
receive either an Avastin injection at surgery and Avastin eye drops or a saline 
injection at surgery and Refresh Liquigel eye drops. In addition to the study 
medication, you would receive all standard treatments and medications during your 
surgery and post-operatively.  
 
Overall, there are ten study visits: a screening appointment prior to your surgery, your 
surgery, and then 8 visits following your surgery. The screening visit is the first visit of 
the study and will only occur if you want to participate in the study.  The other visits 
are considered to be standard of care, examinations that you would have done even if 
you didn’t participate in this study.  The last of these visits will occur about one year 
after your surgery.  During these follow-up visits, three extra assessments will be done 
at some of the visits that are considered non-standard of care (meaning the 
assessments are being performed only for study purposes). The first study procedure is 
blood pressure measurements to ensure that your blood pressure remains stable. In 
people who have taken the study medication for systemic use (that is to treat their 
whole body), an increase of blood pressure has been seen and is considered to be a 
potential side effect of the medication.  Therefore, for this study, we will track your 
blood pressure while you are taking the study medication. We will also ask you to have 
confocal/specular microscopy done. Confocal/specular microscopy is a non-invasive 
test which allows us to look at the different layers of your cornea in more detail than 
we are able to during your regular examination. Finally, we will also take pictures of 


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your eye with a slit-lamp camera to document the amount of blood vessels we can see 
on your cornea. The additional study procedures will take between 10 – 20 minutes. 
This time will be in addition to the normal time of your follow-up visits.  The total time 
of each of the follow-up visits will be between 1 and 2 hours. 
Here is a copy of the study consent form, which we would like you to review before 
participating in this study (as Study Personnel review consent form they mention key 
points in each section and stop to ask questions before moving forward to the next 
section):  
 In the first few pages, you will find that the consent form explains why we are 
doing this study, how we think it might help you, and the procedures conducted 
as part of the study. 
 In the following section, you will see that risks of the study medications and the 
imaging procedures are explained.  The treatments used in this study are FDA-
approved for other uses, and therefore, considered to be investigational for this 
purpose. The confocal/specular microscopy and photography are procedures 
routinely performed by trained professionals at Mass Eye and Ear. 
 Next you will see the potential benefits and alternative treatments are explained 
to you. 
 Then you will see the explanations about participant confidentiality, your right to 
withdraw from the study, right to ask questions, costs and compensations, and 
what happens if there are any injuries while you are in the study. You have the 
right to withdraw at any point or ask any questions. The screening visit, study 
medication, and imaging done for research purposes only are all provided at no 
cost to you. Your insurance will not be billed for study related procedures.  
 In the following pages, you will see the detailed list of study procedures for each 
of your ten visits. If you are not clear about any of these, do not hesitate to ask 
what they are or entail. 
 Lastly, before you enroll in the study, we will have you sign the last page.  


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You may take this consent form with you to read over. It is sometimes helpful to review 
it with family or friends and look up the different medications online. After reviewing 
it, please call or speak with me if you have any other questions. I can also help you 
schedule your study appointments with Dr. __________. Here is my card with my office 
number and e-mail address.” 
 
 
 
 
 


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APPENDIX 3: STUDY MANAGEMENT PLAN 
 
Investigator and Personnel Training 
 
To ensure that all staff and investigators are adequately trained on the protocol 
procedures, assessments and regulatory requirements for this clinical trial the 
following will take place: 
1. The Clinical Research Manager and Principal Investigator at Mass. Eye and Ear 
will review all of the protocol procedures and regulatory requirements with 
each Site Principal Investigator prior to the start of the enrollment phase of the 
study.   
2. Each Site Principal Investigator will outline the roles and responsibilities of each 
personnel involved in the study. Copies of these documents will be held in the 
regulatory binder at MEEI. 
3. The Site Principal Investigator will be responsible for training their site 
personnel. Prior to enrollment opening at each site, the Principal Investigator 
will document review and understanding of the protocol and responsibilities of 
all parties involved. This documentation will be held in the MEEI regulatory 
binder. 
4. New study personnel can be included into the study at any time. Prior to study 
involvement documentation of their roles and responsibilities and 
understanding of the protocol will be documented.  
5. Slit lamp photographs taken within the guidelines outlined in the protocol from 
BPEI and NYPH will be sent to MEEI prior to the start of enrollment. The 
photographs will be analyzed to ensure that equipment is functional and staff 
are adequately trained in the imaging techniques required for this study.  
6. The sample photograph will be uploaded through MyFiles, a data sharing 
website that will be used throughout the study. This will ensure that staff are 


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able to access the program and are comfortable using it prior to the start of the 
study. 
7. In the event that a site does not feel comfortable with the outlined procedures 
and data collection methods, the MEEI Principal Investigator, Dr. Dana, or the 
Clinical Research Manager will perform a site initiation visit.  
8. A quarterly update will also be provided to all sites, trial statistician and DSMB. 
The update will include an overview of adverse events, an enrollment update, 
change in any personnel, reports from the Data Safety Monitoring Board. 
9. All coordinators will participate in a monthly phone call to address any 
questions or concerns from any site, provide additional clarification, training or 
education on the protocol and study-related procedures. 
10. All investigators will participate in a quarterly phone call to address any 
questions or concerns from any site, provide additional clarification, training or 
education on the protocol and study-related procedures.  
11. All investigators and key personnel will be invited to attend annually a study 
meeting, which will be held at ARVO. The meeting will allow for all 
investigators to discuss progress of the study, issues that have arisen within 
the past year, feedback from the Data Safety Monitoring Board and adverse 
events.  
 
Regulatory Documentation 
NYPH and BPEI will be required to submit the following documents to MEEI prior to 
the initiation of the study on their site: a signed FDA form 1572, current CV of all 
investigators and all formal communication between the site IRBs overseeing this 
study at NYPH and BPEI, including stamped consent forms, approved protocol and 
approval notice. These documents will be kept on file with the IND and other 
regulatory study documents, including information and approvals by IRBs. 


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These procedures are set in place to allow Dr. Dana the ability to monitor the study 
and ensure that the risk to participants does not increase throughout the course of 
the study. 


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APPENDIX 4: CLASSIFICATION AND TREATMENT OF GRAFT REJECTION 
 


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APPENDIX 5: CORNEAL PHOTOGRAPHY PROTOCOL 
 
The instructions below serve as a guideline for all sites when performing the corneal 
photography.  Actual photography procedures may vary from site to site. 
1. Adjust the backlight at mid to low level 
2. Slit lamp beam will be on at 6V, fully open, through diffuser 
3. Position the participant’s eye so the cornea is facing completely straight. 
Position the cornea to be in the center of the photo, and ensure that the 
participant’s forehead is always pressed against the plastic band and the chin 
resting on the chinrest.  
4. Adjust the position of the light sources to remove the glare from the areas with 
vessels, while still keeping the cornea as illuminated as possible. This can be 
accomplished by physically moving the light sources, or by loosening and 
adjusting the position of the slit beam.  
- If vessels cover the entire cornea, position the glare in the center of the cornea (if no 
vessels exist centrally). If vessels intrude into the center, position the glare outside of 
the corner by loosening the slit beam and positioning the reflex in the limbus or 
conjunctiva.  
- Always keep the position of the glare constant throughout photos from all visits 
- Print out a screenshot of the baseline photo so that the exposure and glare position 
can be replicated at future visits 
5. Keep the aperture at 1 and exposure at 80, however, exposure can be adjusted 
slightly if photo is especially dark or reflective.  
- Vary exposure as needed to create an ideal lighting for visualizing the vessels.  
- Record exposure used for each set of photos so that this can be replicated in the 
following visits.  


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6. With a dark iris where the vessels cannot be visualized well, use 
retroillumination if all other attempts at illuminating the corneal vessels fail.  
7. Ensure that all blood vessels around the cornea are focused when taking the 
photo. 
8. If imaging is impaired due to lid positioning, use a lid speculum to aid in 
keeping lids open.  
Avastin Photo Labeling Protocol 
 
Within each patient’s appropriate visit in StudyTrax, label each photo with the 
following information: 
 
a. Participant ID number 
b. Eye photographed 
c. visit number – screening (w0), week 4 (w4), week 8 (w8), etc.  
d. photo number – p1, p2, p3…  
 
For example, MEEI012_OD_w0_p1 would be the name of the first photo taken of the 
right eye at the screening visit for participant MEEI012. Photos should be exported as 
jpeg images. Images will then be analyzed in a masked fashion by investigators.  
 
 
 
 
 
 


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APPENDIX 6: SCHEDULE OF EVENTS 
 
Table 1: Schedule of Events and Procedures 
Visit 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Time period 
Screening 
Visit 
Day 0 
 
90 days 
from 
screening 
Day 
1 
±0 
days 
Day 
7 
±6 
days 
Week 
4 
±10 
days 
Week 
8 
±2 
weeks 
Week 
16 
±2 
weeks 
Week 
26 
±2 
weeks 
Week 
39 
±2 
weeks 
Week 
52 
±2 
weeks 
Obtain Informed 
Consent 
X 
SURGERY  DAY ( subconjunctival injection of 2.5 mg/0.1 ml of bevacizumab (vs. 
vehicle) 
 
 
 
 
 
 
 
 
Inclusion/ exclusion 
criteria 
X 
 
 
 
 
 
 
 
 
Medical and 
Ophthalmic History 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Pregnancy test3 
X 
 
 
 
 
 
 
 
 
Blood Pressure 
X 
X 
X 
X 
X 
 
 
 
 
BCVA 
X2 
X1 
X1 
X2 
X2 
X2 
X2 
X2 
X2 
Biomicroscopy 
X2 
X1 
X1 
X2 
X2 
X2 
X2 
X2 
X2 
Intraocular Pressure 
X2 
X1 
X1 
X2 
X2 
X2 
X2 
X2 
X2 
Pachymetry 
X2 
 
 
X2 
 
X2 
X2 
 
X2 
Dilated Funduscopy 
X2 
 
 
 
 
X1 
 
 
X1 
Study Rx 
Start 
Stop 
Compliance Check 
 
 
X 
X 
 
 
 
 
 
Adverse Event Query 
 
X 
X 
X 
X 
X 
X 
X 
X 
Confocal/specular   
microscopy 
 
 
 
 
 
 
X1 
 
X1 
Corneal Photography 
X1 
 
 
X1 
 
X1 
X1 
 
X1 
1 = Only the Study Eye Required  
 
 
2 = Both Eyes Required 
3 = If the pregnancy test performed at the screening visit is greater than 30 days from Day 0, an 
additional pregnancy test will be performed within 30 days of a participant’s surgery date. 
 
Participants will be instructed to strictly follow the study visit schedule. This study will not include 
blood sampling for serum levels of bevacizumab or any pharmacokinetic measures. 


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APPENDIX 7: ACRONYMS 
 
AMD 
Age-related Macular Degeneration 
ARVO 
 The Association for Research in Vision and Ophthalmology 
CA 
 California 
CDMRP 
Congressionally Directed Medical Research Programs 
CFR 
Code of Federal Regulations 
CRF 
Case Report Forms 
BCVA 
Best Corrected Visual Acuity 
BPEI 
Bascom Palmer Eye Institute 
BSCVA 
Best Spectacle-Corrected Visual Acuity 
DSMB 
Data Safety Monitoring Board 
DSMP 
Data Safety Monitoring Plan 
DOD 
Department of Defense 
e.g. 
Exempli gratia 
FB 
Foreign Body  
FDA 
Food and Drug Administration 
FL 
Florida 
GS 
Guanosine Synthetase 
HIPAA 
Health Insurance Portability and Accountability Act 
HLA 
Human Leukocyte Antigen 
Hx 
History 
IA 
Invasion Area 
 
ICF 
Informed Consent Form 
ID 
Identification 
IND 
Investigational New Drug 
IRB 
Institutional Review Board (Human Studies Committee) 
LF 
Lachin and Foulkes 
MA 
Massachusetts 
MD 
Doctor of Medicine 


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MEEI 
Massachusetts Eye and Ear Infirmary  
mg 
Milligrams 
ml 
Milliliters 
mm 
Millimeters 
NaCl 
Sodium Chloride 
NYPH 
New York Presbyterian Hospital 
NIH 
National Institutes of Health 
NA 
Neovascular Area 
NV 
Neovascularization 
NY 
New York 
OD 
Right eye 
OS 
Left eye 
OU 
Both eyes 
PH 
Pinhole 
PhD 
Doctor of Philosophy 
PI 
Principal Investigator 
PK 
Penetrating Keratoplasty 
Post-Op 
Post-operative 
QC 
Quality Control 
RGS 
Rubenstein, Gail and Santner  
Rx 
Prescription 
SAS 
Statistical Analysis System 
SPSS 
originally, Statistical Package for the Social Sciences 
UCVA 
Uncorrected Visual Acuity 
U. Miami 
University of Miami 
US 
United States 
VC 
Vessel Caliber 
Vs 
versus  
VEGF 
Vascular Endothelial Growth Factor 
Wk 
Week 
WWI 
World War I 
WWII 
World War II 


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SYMBOLS 
≥ 
Greater than or equal to 
≤ 
Less than or equal to 
> 
Greater than 
< 
Less than 
± 
Plus or minus 
C 
Degrees is Celsius  
F 
Degrees in Fahrenheit  
@ 
at 
% 
percent 
® 
registered sign