Original Paper Received: June 3, 2013 Accepted: July 7, 2013 Published online: October 22, 2013

Ophthalmic Res 2014;51:1–8 DOI: 10.1159/000354328

VEGF Gene Polymorphism and Response to Intravitreal Ranibizumab in Neovascular Age-Related Macular Degeneration Carlos Eduardo dos Reis Veloso a Luciana Negrão Frota de Almeida a Franco Maria Recchia b David Pelayes c Márcio Bittar Nehemy a   

 

 

a

 

 

Department of Ophthalmology, Federal University of Minas Gerais, Belo Horizonte, Brazil; b Vanderbilt Eye Institute, Nashville, Tenn., USA; c Department of Ophthalmology, Buenos Aires University, Buenos Aires, Argentina  

 

 

Abstract Background/Aims: To investigate the association between VEGF gene polymorphism and response to ranibizumab in  neovascular age-related macular degeneration (AMD). Methods: A total of 92 patients were genotyped for the VEGF rs1413711 single nucleotide polymorphism. Patients with neovascular AMD initially received 3 monthly ranibizumab intravitreal injections and were retreated as needed. Visual acuity (VA) and central retinal thickness (CRT) were measured before and 1, 3, 6 and 12 months after treatment. Results: For patients with TT and CT genotypes, paired comparisons of mean VA showed improvement when the data obtained at all visits were compared with baseline values, in contrast to patients with the CC genotype. CRT statistically improved at all visits for all genotypes. Conclusion: Patients with the CC genotype showed poorer long-term functional and anatomical response to anti-VEGF therapy. © 2013 S. Karger AG, Basel

© 2013 S. Karger AG, Basel 0030–3747/14/0511–0001$39.50/0 E-Mail [email protected] www.karger.com/ore

Introduction

Age-related macular degeneration (AMD) is a progressive disorder that affects the central retina with primary involvement of the outer retinal layers [1]. It is considered the leading cause of severe visual acuity (VA) loss in industrialized countries and is responsible for a poor quality of life among the affected population [2, 3]. Neovascular AMD occurs when a choroidal neovascular membrane grows under the retinal pigment epithelium (RPE) or between the RPE and neurosensory retina, leading to subretinal hemorrhage or leakage of fluid and subsequent scar tissue formation. Although the etiology of AMD remains largely unknown, many studies have established age, smoking and genetic predisposition as key factors and cardiovascular risk factors (such as hypertension and hyperlipidemia) as inconsistent contributors to disease manifestation [4–6]. The AMD-associated genes might interact with other genes or nongenetic risk factors to produce the clinical phenotypes. Recent studies have shown that single nucleotide polymorphisms (SNP) in the complement factor H (CFH) and LOC387715 genes confer an increased risk for the development of AMD [7, 8]. The vascular endothelial growth factor (VEGF) gene, which encodes the VEGF protein, a major player in the Carlos Eduardo dos Reis Veloso Rua dos Otoni 881, 13o andar, Santa Efigênia Belo Horizonte, MG 30150-270 (Brazil) E-Mail cerveloso @ hotmail.com

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Key Words Age-related macular degeneration · Genetics · Polymorphism · Ranibizumab · Intravitreal injection

Material and Methods This study is part of a retrospective review of prospectively acquired data on patients with AMD that included the identification of VEGF gene polymorphism and its relationship with the devel-

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Ophthalmic Res 2014;51:1–8 DOI: 10.1159/000354328

opment of the disease and the therapeutic response to anti-VEGF treatment. All subjects were informed about the nature of the study and signed a written consent in accordance with the guidelines of the Declaration of Helsinki. The ethics committees of both the Federal University of Minas Gerais and the Institute of Vision in Belo Horizonte, Brazil, approved the study. All patients with AMD diagnosed between 2008 and 2012 at the Institute of Vision underwent a complete ophthalmological examination, including biomicroscopy, retinography, fluorescein angiography and optical coherence tomography (OCT). When indicated, indocyanine green angiography was performed for better evaluation of neovascular AMD subtypes. All patients had a blood sample taken in order to study the genetic SNP possibly associated with this disease. Inclusion criteria were: (a) age >50 years; (b) diagnosis of neovascular AMD; (c) VA better than 20/400 (if both eyes were affected, the one with worse VA was selected); (d) loading dose with 3 intravitreal injections of ranibizumab (Lucentis; Novartis, Basel, Switzerland, and Genentech Inc., San Francisco, Calif., USA), administered 1 time per month over 3 months; and (e) follow-up period of at least 12 months. Exclusion criteria were: (a) choroidal neovascularization secondary to any cause other than AMD; (b) previous treatment for neovascular AMD; (c) patients with indication for combined treatment; (d) eyes with polypoidal choroidal vasculopathy; (e) eyes previously submitted to posterior vitrectomy; and (f) other diseases that could affect VA. All intravitreal anti-VEGF injections using ranibizumab (0.5 mg/0.05 ml) were performed in the operating room, by aseptic technique, including the prophylactic use of topical iodopovidone 5%. All patients were subjected to a treatment protocol that included a loading dose with 3 intravitreal injections of ranibizumab at 1-month intervals. After the third dose, they followed a pro re nata regimen. Retreatment criteria were: (a) persistence of or increase in intra- or subretinal fluid; (b) increase in RPE detachment; (c) worsening by at least 1 line of VA; and (d) new subretinal hemorrhage. All patients were retreated with ranibizumab. Best-corrected VA (BCVA) and central retinal thickness (CRT), obtained using spectral-domain OCT (Spectralis OCTTM; Heidelberg Engineering, Heidelberg, Germany), were measured at baseline and 1 month after each intravitreal injection. The automated segmentation of retinal boundaries was used and any segmentation error was corrected manually. CRT was determined based on central 1-mm subfield thickness. Snellen VA was recorded in a standardized manner for all patients during all visits and, for statistical analysis purposes, was converted to logarithm of the minimal angle of resolution (logMAR) values. VA measurements, OCT and intravitreal injections were performed by different investigators in a double-masked fashion. Genotyping Genomic DNA was isolated from whole blood based on the high-salt method of Lahiri and Nurnberger [38]. We selected one SNP which was genotyped using TaqMan® SNP Genotyping Assays (Applied Biosystems, Foster City, Calif., USA). The probe used corresponds to the VEGF rs1413711 SNP. The polymorphism was chosen using the HapMap database (www.hapmap.org). Retypes of 10% of the whole sample were performed for quality control. Genotypings were read using PCR-Realtime in the allelic discrimination mode (Strategene Mx3005 with MxPro QPCR software, 2007). PCR protocols followed the instructions for use of the

dos Reis Veloso/Frota de Almeida/ Recchia/Pelayes/Nehemy

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control of angiogenesis, has also been studied. A few papers suggested that the VEGF gene could play a role in the pathogenesis of AMD [9–15]. However, other studies showed no association [16–18]. In a recent report, we observed a prevalence of homozygosity (TT genotype) of 18.1% for the rs1413711 polymorphism among AMD cases compared with 5.8% among controls (p < 0.002) in a Brazilian cohort, suggesting that VEGF TT genotype is associated with AMD among Brazilian patients [15]. Interindividual differences in drug response are partially attributed to genetic variations, which have led several groups to conduct pharmacogenetic studies with a hope of offering personalized treatment for AMD patients. Some authors have studied the effect of genotypes on the response to nutritional supplements, showing a positive association [19]. Other studies evaluated the relationship between genotypes and photodynamic therapy (PDT) with controversial results [20–27]. The association between gene polymorphism and response to anti-VEGF therapy for neovascular AMD has also been studied. Anti-VEGF therapy was the first treatment that consistently improved VA in a large number of patients, representing a remarkable advance in the treatment of neovascular AMD. However, there is a broad range in response rates to anti-VEGF therapy, and genetic variants may be partially responsible [6]. Several reports evaluated CFH genotype association with the response to bevacizumab and ranibizumab intravitreal injections, suggesting a potential pharmacogenetic relationship [28–32]. For the VEGF gene, different studies involving different SNP showed conflicting results [31– 37]. There are only three studies that evaluated the association between VEGF SNP rs1413711 and response to AMD treatment. Kloeckener-Gruissem et al. [34] and Boltz et al. [37] did not show any influence in therapeutic response with ranibizumab. McKibbin et al. [35] found that patients homozygous for the risk allele showed trends toward a more favorable outcome. Since there are only a few controversial published articles, we undertook this study to contribute to a better elucidation of this topic of paramount importance. The purpose of this paper is to investigate the association of VEGF rs1413711 (C674T) SNP with response to ranibizumab therapy in neovascular AMD.

Table 1. Pretreatment findings according to VEGF genotypes

Genotypes

Age1, years Male/female, n VA1, logMAR CMT1, μm

p

CC (n = 38)

CT (n = 38)

TT (n = 16)

73.9±8.8 (75.0) 22/16 0.53±0.3 (0.45) 340±87 (334.5)

74.3±8.7 (76.5) 17/21 0.63±0.4 (0.5) 329±87 (305)

73.1±8.0 (76.5) 5/11 0.64±0.3 (0.75) 357±103 (332)

0.92223 0.17822 0.43293 0.60053

CMT = Central macular thickness. 1 Means ± pattern deviation, with medians in parentheses. 2  2 χ test. 3  Kruskal-Wallis test.

 

 

 

 

 

 

Statistical Analysis Descriptive statistics for all demographic and clinical variables were calculated. When quantitative variables were compared with qualitative variables with three categories, the F (ANOVA) test was employed if the values were sampled from a Gaussian distribution (verified by the Hosmer-Lemeshow test), and otherwise by a Kruskal-Wallis test. Comparisons between proportions of qualitative variables were analyzed by the χ2 test. Paired comparisons were made using the paired t test if the values were sampled from a Gaussian distribution (verified by the Hosmer-Lemeshow test), and otherwise by a Wilcoxon test.

Results

of treatment were compared with baseline values (table 2). For patients with the TT and CT genotypes, paired comparisons of VA also showed statistically significant improvement when the data obtained at all visits were compared with baseline. However, patients with the CC genotype did not show statistically significant improvement when VA obtained at visits 1, 3, 6 and 12 months after the beginning of treatment were compared with the baseline value. For the whole group of patients, paired comparisons of CRT showed statistically significant improvement when the data obtained at visits 1, 3, 6 and 12 months after the beginning of treatment were compared with baseline values (table 3). For patients with the TT and CT genotypes, paired comparisons of CRT also showed statistically significant improvement when the data obtained at all visits were compared with baseline values. However, patients with the CC genotype showed statistically significant improvement only when CRT measurements obtained at 3 and 6 months were compared with the baseline. The mean number of intravitreal injections required between the 3rd and 12th month was 2.6 ± 1.0 (median: 2.0) for patients homozygous for the C allele, 2.3 ± 1.3 (median: 2.0) for patients with the CT genotype, and 2.0 ± 0.8 (median: 2.0) for patients with the TT genotype (p = 0.20; Kruskal-Wallis test).

A total of 585 eyes of 362 AMD patients were evaluated, and 92 that met the inclusion criteria were selected. The mean age of the 92 patients was 73.9 ± 8.8 years (range: 54–91 years) and 48 (52.2%) were female. The mean pretreatment BCVA was 0.59 ± 0.4 logMAR and mean pretreatment CRT was 339 ± 90 μm. Pretreatment BCVA, central macular thickness, gender distribution and age were not statistically different for the three different genotypes (table 1). No serious local and no systemic adverse effect was noted in any case. Sixteen patients (17.4%) were homozygous for the risk allele (TT), 38 (41.3%) had at least one risk allele (CT), and 38 (41.3%) were homozygous for the C allele (CC). For all 92 patients, paired comparisons of VA showed statistically significant improvement when the data obtained at visits 1, 3, 6 and 12 months after the beginning

Genetic factors are known to play a major role in the pathogenesis of AMD and have been suggested to influence the response to different modalities of AMD therapy including oral antioxidants, PDT and anti-VEGF agents

VEGF Polymorphism and Response to Ranibizumab in AMD

Ophthalmic Res 2014;51:1–8 DOI: 10.1159/000354328

Discussion

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TaqMan Genotyping Master Mix (Applied Biosystems), as follows: 3.5 μl of mix, 0.1 μl of probe, 3.4 μl of deionized water and a 1.0-μl DNA concentration 50 ng/μl, to a total volume of 8 μl. PCR conditions were: 1 cycle (10 min at 95 ° C) and 50 cycles (95 ° C for 15 s, 60 ° C for 1 min).

Table 2. Comparison of mean VA, according to VEGF genotypes, between baseline and 1, 3, 6 and 12 months after beginning of treatment

CC genotype (n = 38) VA1, logMAR p CT genotype (n = 38) VA1, logMAR p TT genotype (n = 16) VA1, logMAR p CC, CT and TT genotypes VA1, logMAR p

Baseline

1 month

3 months

6 months

12 months

0.53±0.3 (0.45)

0.58±0.5 (0.5) 0.92182

0.52±0.5 (0.4) 0.23042

0.52±0.5 (0.35) 0.09532

0.56±0.5 (0.4) 0.36062

0.63±0.4 (0.5)

0.53±0.4 (0.4) 0.00092

0.51±0.4 (0.4) 0.00092

0.54±0.4 (0.5) 0.02292

0.51±0.5 (0.4) 0.00462

0.64±0.3 (0.75)

0.46±0.3 (0.5) 0.00102

0.33±0.3 (0.2) 0.00052

0.34±0.3 (0.3) 0.00052

0.37±0.5 (0.3) 0.01102

0.59±0.4 (0.5)

0.54±0.4 (0.5) 0.00102

0.49±0.4 (0.4)

VEGF gene polymorphism and response to intravitreal ranibizumab in neovascular age-related macular degeneration.

To investigate the association between VEGF gene polymorphism and response to ranibizumab in neovascular age-related macular degeneration (AMD)...
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