1. Diabetic Retinopathy Clinical Research Network
Plasma VEGF Concentrations after Intravitreous Anti-VEGF Therapy for DME During the DRCR.net Protocol-T 1

2. Financial Disclosures
Funding/Support: Cooperative Agreement with NEI and NIDDK of NIH, U.S. Department of Health and Human Services.
Additional Contributions: Regeneron Pharmaceuticals, Inc. provided the aflibercept; Genentech Inc. provided the ranibizumab. Genentech Inc. also provided funding for blood pressure cuffs and collection of plasma and urine that are not part of the main study reported herein
A complete list of all DRCR.net investigator financial disclosures can be found at
Personal Disclosures
None

3. DRCR.net Protocol T

4. Objectives Pre-planned Secondary Analysis
Assess the effect of intravitreous aflibercept, bevacizumab and ranibizumab on systemic free VEGF-A levels and compare the changes in levels across treatment groups

5. Methods
Plasma samples collected at baseline, 4, 52 and 104 week visits prior to any ocular treatment
Concentration of free VEGF* was measured in plasma using a commercial immunoassay
Performed by an independent laboratory at the Fred Hutchinson Cancer Research Center where the samples were stored at -80°C
Human VEGF Quantikine ELISA immunoassay (R&D Systems)
436 (66%) of all 660 Protocol T participants provided separate consent to participating in the plasma ancillary study.
*2 duplicate readings were taken from each sample and the average of these measurements & the coefficient of variation (CV) was calculated. Only samples with CV < 20% (n=16 excluded) were included. A minimum quantifiable VEGF concentration of 7.8 pg/ml was used. VEGF concentration was analyzed using a natural log scale (ln) as recommended by the DRCR.net lead statistician (D. Liu and M. Melia) as the values were positively skewed. Change in VEGF concentration was truncated at 3 standard deviations from the mean pooling all treatments and visits (n=16 truncated). 436 (66%) of all 660 Protocol T participants provided separate consent to participating in the plasma ancillary study.

6. Change in Plasma VEGF Concentrations*
* For the purpose of analysis, only samples with a CV less than 20% were included. VEGF concentrations were truncated at 7.8 pg/ml and were analyzed in the natural log scale (ln) since the values were positively skewed. To minimize the impact of extreme outliers, the mean change from baseline was truncated at 3 standard deviations from the mean pooling all treatments and visits.

7. Mean Plasma VEGF Concentrations
Afl
Bev
Ran
N = 139
N = 130
N = 141
Baseline (pg/ml)
28.50
31.45
37.55
4 weeks (pg/ml)
23.44
24.06
30.43
- The data on this slide only include patients with data at BL and week 4 (both visits).
P-values adjusted for baseline ln(VEGF) concentration and multiple treatment group comparisons.
Only including participants with available data at both baseline and 4-week visit
ln(VEGF) = natural log of VEGF measurement

8. Mean Plasma VEGF Concentrations & Change from Baseline to Week 4
P = 0.89 (A v. B)
P < (A v. R)
P < (R v. B)
Afl
Bev
Ran
N = 139
N = 130
N = 141
Baseline (pg/ml)
28.50
31.45
37.55
4 weeks (pg/ml)
23.44
24.06
30.43
Change ln(VEGF)
-0.30
-0.31
-0.02
- The data on this slide only include patients with data at BL and week 4 (both visits).
P-values adjusted for baseline ln(VEGF) concentration and multiple treatment group comparisons.
Only including participants with available data at both baseline and 4-week visit
ln(VEGF) = natural log of VEGF measurement

9. Plasma VEGF Concentration
Change in Plasma VEGF Concentrations Baseline to Week 52 for All Patients
P = 0.07 (A v. B)
P = 0.07 (A v. R)
P < (B v. R)
Plasma VEGF Concentration
Afl
Bev
Ran
N = 132
N = 115
N = 130
Baseline (pg/ml)
28.10
31.69
33.47
52 weeks (pg/ml)
26.40
27.05
34.89
Change ln(VEGF)
-0.10
-0.24
-0.03
P-values adjusted for baseline ln(VEGF) concentration and multiple treatment group comparisons.
Only including participants with available data at both baseline and 52-week visit
ln(VEGF) = natural log of VEGF measurement

10. No Injection within 1 month Yes Injections within 1 month
Change in Plasma VEGF Concentrations Baseline to Week 52 by Timing of Injection
No Injection within 1 month
(N = 76 / 70 / 82 = 60%)
P = 0.78 (A vs. B)
P = 0.95 (A vs. R)
P = 0.78 (B vs. R)
Yes Injections within 1 month
(N = 56 / 45 / 48)
P = 0.03 (A vs. B)
P = (A vs. R)
P < (B vs. R)
In order to be included in the analysis, the patient must have a 52-week sample, which means he/she must complete the 52-week visit.
52-week cohort N=377 [132/115/130 for A/B/R]: NO injection 1 month N=228 (60%) [76 (58%)/70 (61%)/82 (63%) for A/B/R], YES injection 1 month N=149 (40%) [56 (42%)/45 (39%)/48 (37%) for A/B/R]
Change in ln(VEGF) Concentration
P-values adjusted for baseline ln(VEGF) concentration and multiple treatment group comparisons. Only including participants with available data at both baseline and 52-week visit

11. No Injection within 2 months Yes Injections within 2 months
Change in Plasma VEGF Concentrations Baseline to Week 52 by Timing of Injection
No Injection within 2 months
(N = 43 / 42 / 50 = 36%)
P = 0.43 (A vs. B)
P = 0.79 (A vs. R)
P = 0.44 (B vs. R)
Yes Injections within 2 months
(N = 89 / 73 / 80)
P = 0.15 (A vs. B)
P = 0.02 (A vs. R)
P < (B vs. R)
In order to be included in the analysis, the patient must have a 52-week sample, which means he/she must complete the 52-week visit.
52-week cohort N=377 [132/115/130 for A/B/R]: NO injection 2 months N=135 (36%) [43 (33%)/42 (37%)/50 (38%) for A/B/R], YES injection 2 months N=242 (64%) [89 (67%)/73 (63%)/80 (62%) for A/B/R]
Change in ln(VEGF) Concentration
P-values adjusted for baseline ln(VEGF) concentration and multiple treatment group comparisons. Only including participants with available data at both baseline and 52-week visit

12. Objectives & Methods Pre-planned Secondary Analysis
Evaluate for a correlation between plasma VEGF levels and key systemic factors
Adverse Events
Recorded prospectively
Mean Arterial Pressure (MAP)
Calculated from 3 sets of BP at baseline, 4, 52 & 104 week visits
Urine ACR
Albumin-creatinine ratios calculated from urine samples* at baseline & 52-week visits
Urine samples were collected at baseline and 52-week visits and were frozen at -20°C and shipped within 7 days of collection to the Advanced Research and Diagnostic Laboratory at the University of Minnesota for measurement of urinary albumin and creatinine
Urine samples were frozen at -20°C and shipped within 7 days of collection to the Advanced Research and Diagnostic Laboratory at the University of Minnesota for measurement of urinary albumin and creatinine
*Urine samples were frozen at -20°C and shipped within 7 days of collection to the Advanced Research and Diagnostic Laboratory at the University of Minnesota for measurement of urinary albumin and creatinine

13. Occurrence of APTC Events Through 52 Weeks
Aflibercept
Bevacizumab
Ranibizumab
Total
No. of Participants*
132
115
130
377
Non-fatal MI 2 1 3
Non-fatal Stroke 4
Any APTC Event 7
* Only including participants with available plasma VEGF data at both baseline and 52-week visit

14. VEGF Concentration by APTC Event Through 52 Weeks
* Only including participants with available plasma VEGF data at both baseline and 52-week visit

15. VEGF Concentration & Mean Arterial Pressure At Week 52 & Change from Baseline
We did look at MAP vs. VEGF levels at each of the plasma sample collection visits. There was no significant correlation identified.
The figure above on the left shows 52-week MAP vs. 52-week VEGF, and 52-week MAP change vs. 52-week VEGF log change on the right.

16. VEGF Concentration & Urine Albumin-Creatinine Ratio: At 52 Weeks
Urine ACR
No-albuminuria (N = 133)
UACR < 30 pg/g
Microalbuminuria (N = 112)
30 ≤ UACR ≤ 300 pg/g
Macroalbuminuria (N = 128)
UACR > 300 pg/g
- We did look at the baseline VEGF by baseline UACR subgroups. No significant difference was identified (P=0.83)
P=0.09 from Analysis of Variance, combing all treatment groups

17. Limitations Plasma VEGF levels only measured at 4 points
Blood levels of the anti-VEGF agents not measured
Assay uncertainties
Systemic half lives of the drugs differ with ranibizumab cleared fastest
Uncertainties over whether VEGF levels can be measured reliably in the presence of competing VEGF binding agents
Protocol T not designed or powered to study systemic complications

18. Conclusions
At 4 weeks, the decreases in VEGF levels were greater with aflibercept and bevacizumab compared with ranibizumab. This was still true for bevacizumab at 1 and 2 years, but not for aflibercept.
In the pre-specified subgroups who did not receive an injection within 1 month prior to week 52, no differences in changes in VEGF levels were identified among anti-VEGF agents
In patients who had received an injection within 1 or 2 months prior to the 52 week visit, the decreases in VEGF levels were less with ranibizumab than the other two drugs.

19. Conclusions
There are no data from this analysis to suggest that patients with lower or higher VEGF plasma levels were at an increased risk of complications, but complication rates were low & samples were not necessarily obtained in proximity to the event