The Diabetic Retinopathy Clinical Research Network Randomized Clinical Trial Evaluating Intravitreal Ranibizumab or Intravitreal Saline for Vitreous Hemorrhage from Proliferative Diabetic Retinopathy Supported through a cooperative agreement from the National Eye Institute and the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services EY14231, EY14229, EY

Background Vitreous Hemorrhage and Vitrectomy  In at least 5% of cases, proliferative diabetic retinopathy (PDR) can lead to vitreous hemorrhage even after panretinal photocoagulation (PRP) is initiated which can substantially affect vision and may preclude performing complete PRP.  Current treatment for non-clearing vitreous hemorrhage is vitrectomy Surgical complications Recovery time Some patients may fear surgery 2

Background Use of anti-VEGF for vitreous hemorrhage  Intravitreal anti-vascular endothelial growth factor (anti-VEGF) is a potential alternative to vitrectomy VEGF is shown to be major causative factor in PDR While hemorrhage absorbs, anti-VEGF drug may cause temporary or permanent regression of NVD/NVE, reducing the likelihood of additional hemorrhage until PRP is applied Small case series suggest anti-VEGF causes short-term clearing of vitreous hemorrhage 3

Background Use of Saline for vitreous hemorrhage  Unknown if effects seen with anti-VEGF are from fluid injection alone  Prior study found that saline injected eyes were 5 times more likely to have visual improvement at 2 months (26% vs. 6%) and 3 times more likely at 3 months (36% vs. 11%) compared with eyes under observation alone 4

Study Objectives  To determine if intravitreal injections of ranibizumab decrease the proportion of eyes in which vitrectomy is performed compared with saline injections in eyes presenting with vitreous hemorrhage from PDR. Note: This trial was not a comparison of anti-VEGF with observation or sham injection; rather the trial was a comparison of intravitreal anti-VEGF with intravitreal saline injection  To assess the efficacy and safety of anti-VEGF therapy as treatment for vitreous hemorrhage due to PDR. 5

Study Design, Enrollment, Follow-up  At least one eye that met all of the following criteria:  Vitreous hemorrhage causing vision impairment, presumed to be from PDR, and precluding completion of PRP  Immediate vitrectomy is not required  Visual acuity is light perception or better  No prior anti-VEGF treatment for VH Randomized Multicenter Double-Masked Trial Primary Outcome: Proportion of eyes with vitrectomy by 16 weeks. 6

Study Enrollment 261 Eyes Randomized (61 Sites) 261 Eyes Randomized (61 Sites) 12 Week Visit Completion 95% Overall* (96% Ranibizumab Injection; 95% Saline Injection) 12 Week Visit Completion 95% Overall* (96% Ranibizumab Injection; 95% Saline Injection) Intravitreal Injection of 0.5 ranibizumab N = 125 Intravitreal Injection of 0.5 ranibizumab N = 125 Intravitreal Injection 0.9% sodium chloride N = 136 Intravitreal Injection 0.9% sodium chloride N = 136 * One death occurred prior to the 12 week visit. 7

Follow-up Schedule Phase 1 Phase 2 4 WK VISIT 26 WK Phone Call 8 WK VISIT 12 WK VISIT 52 WK VISIT 8

Follow-Up Treatment  Follow-up injections performed at 4 and 8 weeks unless: Vitreous hemorrhage had cleared enough to complete PRP or Vitrectomy had been performed.  All eyes were to be treated with complete PRP as soon as possible. 9

Guidelines for Vitrectomy Prior to 8 Week Study Visit  Vitrectomy was not to be performed unless one of the following was present: Traction retinal detachment involving or threatening the macula Rhegmatogenous retinal detachment Angle neovascularization Progressive iris neovascularization Neovascular glaucoma or ghost cell glaucoma resulting in increased IOP that could not be controlled medically 10

Guidelines for Vitrectomy 8 to 16 Weeks  Vitrectomy could be performed (but was not required) if there was failure of substantial clearing of vitreous hemorrhage such that the study participant’s need for vitrectomy and its associated benefits outweighed its risks.  Primary outcome time point was at 16 weeks to include cases where decision was made at 12 weeks to perform vitrectomy. 11

PRP Treatment  PRP was to be initiated as soon as possible  “Complete” PRP defined as 500 micron size burns on the retina placed no further than 1 to 2 burn widths apart beginning ~3000 µm from macular center and extending to equator for 12 clock hours.  If vitreous hemorrhage cleared and it was determined that “complete” PRP was already given, further treatment was not required. 12

Recruitment Summary 13 Original goal reached Recruitment Resumed Original Goal Reached Recruitment Resumed

Baseline Subject Characteristics 14 Ranibizumab N=125 Saline N=136 Women52%51% Age (yrs) (Median 25,75 th percentile) 61 (50, 67)58 (49, 64) Race White54%51% African-American16% Hispanic or Latino26%25% Other4%8% Type 1 Diabetes17%23% Type 2 Diabetes81%73% Uncertain2%4%

Baseline Subject Characteristics 15 Subject CharacteristicsRanibizumab N=125 Saline N=136 Duration of Diabetes - yrs (Median 25 th,75 th percentile) 19 (12, 28)21 (16, 27) Hemoglobin A1c - % (Median 25th,75th percentile) 7.7 (6.7, 8.7)8.0 (6.9, 9.3) Pre-existing cardiovascular condition 35%44% Pre-existing hypertension84%86%

Baseline Study Eye Characteristics 16 Ranibizumab N=125 Saline N=136 Prior PRP50%57% Prior Treatment for DME42% Prior treatment with anti- VEGF for DME 8%13% E-ETDRS Visual Acuity Median (75 th, 25 th Percentile); Snellen Equivalent 34 (61, 0) 20/200 (20/63, <20/800) 28 (59, 0) 20/320 (20/63, <20/800) OCT Signal Strength = 060%72% > 040%28%

Baseline Study Eye Characteristics Ranibizumab N=125 Saline N=136 Duration of Vitreous Hemorrhage <1 Month53%55% 1-3 Months33%29% 4-6 Months6%8% >6 Months9%8% IOP - mmHg Median (25 th, 75 th percentile) 15 (12, 17)14 (12,17) 17

Total Number of Intravitreal Injections In Eyes without vitrectomy or “complete PRP” prior to 8 weeks Number of InjectionsRanibizumab Injection Saline Injection N = 91N = †1† † One subject was given masked intravitreal study drug at the 12-week follow-up visit when a study injection was not scheduled per protocol. Since an injection was not scheduled, a drug number according to the randomized treatment was not obtained, and it is possible that the wrong randomized treatment was performed. The investigator did believe that an injection was medically indicated for the study participant. 18

Total Number of Intravitreal Injections In Eyes with vitrectomy or “complete PRP” prior to 8 weeks Number of InjectionsRanibizumab Injection Saline Injection N = ¥1¥ ¥ After consenting and randomizing, 1 subject changed their mind and did not want to receive study drug – the participant remained in the study but was never injected. 19

Non-Protocol Study Eye Treatment for DME Prior to 16 weeks Ranibizumab Injection N = 4 Saline Injection N = 4 Number of Eyes with Non-Protocol Study Eye Treatment for DME 34 Intravitreal Bevacizumab (Avastin)1*1** Intravitreal Triamcinolone Acetonide1*0 Focal/Grid Laser Photocoagulation23 Note: only focal/grid laser was permitted per protocol prior to 16 weeks *Same eye received bevacizumab and triamcinolone for DME after vitreous hemorrhage cleared and PRP was considered “complete” **DME treatment performed 1 week post-vitrectomy 20

Vitrectomy Primary Outcome 21

Cumulative Probability of Vitrectomy by 16 Weeks

Vitrectomy Status Over Time Ranibizumab Injection N = 125 Saline Injection N = 136 Vitrectomy performed up to 8 weeks 2% Vitrectomy performed between 8 weeks and 16 weeks 10%14% Vitrectomy performed by 16 weeks 12%16% 23

Cumulative Probability of Vitrectomy by 20 Weeks 24

“Complete” Panretinal Photocoagulation Secondary Outcome 25

Cumulative Probability of “Complete” PRP (in absence of vitrectomy) by 16 Weeks

Visual Acuity at Follow-up Visits Secondary Outcome 27

Mean Change in Visual Acuity from Baseline † Treatment comparison for the mean change in visual acuity at the 12 week visit was performed using a longitudinal mixed model adjusting for baseline visual acuity. † P =

Visual Acuity Binary Outcomes at 12 Week Visit 12 Week Visit Ranibizumab Injection N = 119 Saline Injection N = 129 P value* VA better than 69 letter score (>20/50) and no vitrectomy 45%33%0.10 Severe VA deficiency ≤38 letter score (<20/200) 20%27%0.30 Very severe VA deficiency ≤4 letter score (<20/800) 11%16%0.25 *P-values were obtained using a logistic regression model adjusted by baseline visual acuity. 29

OCT Signal Strength Exploratory Outcome 30

OCT Signal Strength at Follow Up Ranibizumab Injection Saline InjectionP value Baseline N =124N = = 0 60%72% Among Participants with Baseline OCT = 0 4 Week Visit N =70N = > 0* 36%30% 8 Week Visit N = 69N = > 0* 46%38% 12 Week Visit N = 66N = > 0* 52%50% * Defined as a composite outcome of OCT >0 and no vitrectomy prior to the study visit. 31

Safety Outcomes 32

Ocular Adverse Events of Interest Prior to 16 weeks Ocular Events Ranibizumab Injection N = 125 Saline Injection N = 136 Endophthalmitis01 (< 1%) Angle or iris neovascularization 1 ( 1%)4 (3%) Neovascular glaucoma1 (1%)1 (<1%) Cataract Surgery02 (2%) Recurrent vitreous hemorrhage ¥ 8 (6%)23 (17%) ¥ Treatment comparison for recurrent vitreous hemorrhage was performed using Fisher Exact test (P-value = 0.01) 33

Retinal Detachments Prior to 16 weeks Ranibizumab Injection N = 125 Saline Injection N = 136 Traction retinal detachment only 8 (6%)9 (7%) Rhegmatogenous retinal detachment only 1(<1%)2(2%) Combined retinal detachment 1(<1%)0 Any Retinal Detachment10 (8%)11(8%) 34

Elevated Intraocular Pressure Prior to 16 weeks Ranibizumab Injection N = 125 Saline Injection N = 136 Increase of IOP ≥10 mm Hg from baseline 6%8% IOP ≥30 mmHg3% Currently on IOP-lowering medication, but not at baseline 10% Glaucoma surgery at anytime 00 Elevated IOP/Glaucoma13%14% Percentages represent the number of eyes with at least 1 occurrence.

Systemic Adverse Events Cardiovascular Events Ranibizumab Injection N = 125 Saline Injection N = 136 Non-fatal myocardial infarction04 Non-fatal stroke –ischemic or hemorrhagic 12 Vascular death21 Any APTC Event ¥ 26 ¥ According to Antiplatelet Trialists’ Collaboration 36

Systemic Adverse Events of Interest Ranibizumab Injection N = 125 Saline Injection N = 136 Kidney-related events79 Hypertension-related event 49 Cardiovascular-related event 1214 Summary of Causes of Deaths AgeTreatment GroupCause of DeathDays in Study 53RanibizumabUnknown7 Cell counts are the number of participants with at least one of the given event during the study

Discussion 38

Discussion Rationale for Saline Injections  Controls for potential effects of drug vehicle and any physical fluid dynamic effect of an intravitreal injection on VH  Prior study found that saline injected eyes had higher rate of visual acuity improvement than eyes under observation alone  Allowed complete masking of all study staff, which was essential given the subjective primary outcome measure, i.e., undergoing vitrectomy 39

Rates of Vitrectomy 40 TrialsOutcome Anti-VEGF Injection Saline Injection Observation Group DRCR- Protocol N Vitrectomy by 16 weeks N = % N = % - Vitrase Study Persistence of VH by 12 weeks - N = % - Bevacizumab † Vitrectomy by weeks N = 40 10% - N = 40 45% Pegaptanib ¥ Vitrectomy by 16 weeks N = 15 40% -- † Intravitreal bevacizumab and Panretinal Photocoagulation for PDR associated with vitreous hemorrhage, Huang (2009) ¥ Use of pegaptanib for recurrent and non-clearing vitreous hemorrhage in proliferative diabetic retinopathy, Hornan (2010).

Discussion Rates of Vitrectomy  Rate of vitrectomy following intravitreal ranibizumab or saline in DRCR.net trial appears lower than expected with observation 41

Conclusions Primary Outcome  Rate of vitrectomy by 16 weeks was not reduced by intravitreal ranibizumab compared with intravitreal saline in eyes with vitreous hemorrhage due to PDR. 42

Conclusions Secondary Outcomes  Secondary outcomes such as short-term change in visual acuity, completion of PRP, and frequency of recurrent vitreous hemorrhage suggest faster hemorrhage clearing with intravitreal ranibizumab.  Intravitreal ranibizumab does not appear to increase the risk of retinal detachment 43

Other Considerations  Outcomes of intravitreal ranibizumab or saline compared with observation or sham treatment cannot be determined from this study 44

Acknowledgements  Top 10 Site Enrollers Site 46; Maturi Site 100; Friedman Site 127; Gonzalez Site 19; Kim Site 39; Sharuk Site 152; Marcus Site 212; Jamal Site 14; Bhavsar Site 111; Elman Site 207; Stoltz  Top 10 Site Enrollers Site 46; Maturi Site 100; Friedman Site 127; Gonzalez Site 19; Kim Site 39; Sharuk Site 152; Marcus Site 212; Jamal Site 14; Bhavsar Site 111; Elman Site 207; Stoltz  Top 10 enrollers with 100% visit completion. (4, 8, and 12 week visits) Dr. Friedman Dr. Elman Dr. Marcus Dr. Bhavsar  Top 10 enrollers with 100% visit completion. (4, 8, and 12 week visits) Dr. Friedman Dr. Elman Dr. Marcus Dr. Bhavsar 45

Thank You on Behalf of Diabetic Retinopathy Clinical Research Network (DRCR.net)  61 clinical study sites  Study participants who volunteered to participate in this trial  DRCR.net Data and Safety Monitoring Committee  Genentech provided clinical site funding and the ranibizumab for the study and collaborated in a manner consistent with the Network’s DRCR.net Industry Collaboration Guidelines, the DRCR.net had complete control over the design of the protocol, ownership of the data, and all editorial content of presentations and publications related to the protocol.  DRCR.net investigators and staff 46