|Year : 2016 | Volume
| Issue : 6 | Page : 427-433
Change of retinal pigment epithelial atrophy after anti-vascular endothelial growth factor treatment in exudative age-related macular degeneration
Moosang Kim1, Eung Suk Kim2, Kyung Hoon Seo2, Seung-Young Yu2, Hyung-Woo Kwak2
1 Department of Ophthalmology, School of Medicine, Kangwon National University, Chuncheon, Korea
2 Department of Ophthalmology, Kyung Hee University Hospital, Kyung Hee University, Seoul, Korea
|Date of Submission||06-Jan-2016|
|Date of Acceptance||28-May-2016|
|Date of Web Publication||3-Aug-2016|
Department of Ophthalmology, Kyung Hee University Hospital, Kyung Hee University, Seoul
Source of Support: None, Conflict of Interest: None
Purpose: The study aimed to investigate the quantitative changes of retinal pigment epithelial (RPE) atrophy during a 24-month follow-up period of anti-vascular endothelial growth factor (VEGF) for exudative age-related macular degeneration (AMD). Materials and Methods: This is a retrospective study. Sixty-five eyes of 62 consecutive patients with naοve exudative AMD who had received treatment with anti-VEGF therapy and followed for more 24 months were enrolled. All patients received three initial monthly injections of anti-VEGF (ranibizumab or bevacizumab), followed by pro re nata or treat-and-extend protocol. Color fundus image, optical coherence tomography, and fundus autofluorescence were evaluated for RPE atrophy. Multiple regression analysis was performed to investigate the predictive factors found during univariate analysis to identify an association with increased RPE atrophic areas. Results: The mean number of anti-VEGF treatments was 9.18. RPE atrophic area was 1.293 ± 1.298 mm 2 at baseline and enlarged to 2.394 ± 1.940 mm 2 after 24 months, which differed significantly (P = 0.001). Multiple regression analysis revealed that larger areas of RPE atrophy at month 4 and larger numbers of anti-VEGF treatments were associated with increased RPE atrophic areas. Conclusions: RPE atrophy progresses in eyes with exudative AMD during anti-VEGF treatment. Larger areas of RPE atrophy at month 4 and larger numbers of anti-VEGF injections were associated with an increased risk of progression of RPE atrophy the following treatment. These ﬁndings may be useful to clinicians using intravitreal anti-VEGF for the treatment of exudative AMD, both for selecting an appropriate treatment plan and for predicting the progression of RPE atrophy.
Keywords: Anti-vascular endothelial growth factor, exudative age-related macular degeneration, retinal pigment epithelial atrophy
|How to cite this article:|
Kim M, Kim ES, Seo KH, Yu SY, Kwak HW. Change of retinal pigment epithelial atrophy after anti-vascular endothelial growth factor treatment in exudative age-related macular degeneration. Indian J Ophthalmol 2016;64:427-33
|How to cite this URL:|
Kim M, Kim ES, Seo KH, Yu SY, Kwak HW. Change of retinal pigment epithelial atrophy after anti-vascular endothelial growth factor treatment in exudative age-related macular degeneration. Indian J Ophthalmol [serial online] 2016 [cited 2020 Feb 28];64:427-33. Available from: http://www.ijo.in/text.asp?2016/64/6/427/187659
Moosang Kim∗, Eung Suk Kim∗
∗These authors contributed equally to this work.
Exudative age-related macular degeneration (AMD) is characterized by the development of choroidal neovascularization (CNV), often leading to intra- or sub-retinal exudation and hemorrhage.  Anti-vascular endothelial growth factor (VEGF) treatments have become the standard treatment for patients with exudative AMD. After monthly regimens, excellent visual outcomes can be obtained with an expectation of more than 90% of patients to maintain their vision, and 30-40% to experience visual gain. , Although the introduction of anti-VEGF therapies has substantially reduced early vision loss, ≥7 years of long-term data suggest that retinal pigment epithelial (RPE) atrophy and vision loss eventually ensued in most patients.  The Comparison of Age-related Macular Degeneration Treatments Trials (CATT) reported that patients treated monthly were more likely to develop RPE atrophy than those treated pro re nata (PRN). , A retrospective study by Lois et al. has also shown that the development of RPE atrophy, as measured using fundus autoﬂuorescence (FAF) imaging, was signiﬁcantly associated with the number of intravitreal ranibizumab injections.  However, none of the above had conducted a quantitative analysis of RPE atrophy which enables researchers to provide an objective evaluation of changes in atrophic area. In this study, therefore, we focused, especially on the quantitative changes in RPE atrophy during the 24-month follow-up period of anti-VEGF treatment in eyes with exudative AMD. In addition, we investigated the risk factors for the progression of RPE atrophy.
| Materials and Methods|| |
The current research followed the tenets of the Declaration of Helsinki, and all patients provided informed consent after explanation of the study protocol. The Institutional Review Board at our hospital approved this retrospective study. This retrospective study involved a series of 65 eyes with exudative AMD from March 2009 to December 2012. The following inclusion criteria were used: (1) no previous treatment before diagnosis, (2) treatment with anti-VEGF only, and (3) a minimum follow-up period of 24 months. Exclusion criteria included the following: (1) presence of an RPE tear at baseline or during the follow-up period, (2) significant media opacity, enough to affect the quality of imaging, and (3) subretinal hemorrhages and exudates that obscured evaluation of FAF. All patients underwent a complete ophthalmologic examination, including an assessment of best-corrected visual acuity (BCVA), spectral-domain optical coherence tomography (OCT), fluorescein angiography (FA), indocyanine green angiography (ICGA), and FAF imaging before the first intravitreal anti-VEGF injection. Neovascular lesion subtypes were classified based on FA and ICGA as occult CNV, classic CNV, polypoidal choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP).
All patients initially received three monthly intravitreal bevacizumab (Avastin ® ; Genentech Inc., San Francisco, California, USA) or ranibizumab (Lucentis ® ; Genentech Inc., San Francisco, California, USA) injections (loading phase), followed by PRN or treat-and-extend protocol. Patients who treated with PRN protocol were followed up every 2 or 3 months after month 4, preceding the monthly loading doses. This study was a retrospective study, and as such strict criteria for follow-up and retreatment were not established. Patients were retreated when signs of CNV activity were present.
In this study, FAF imaging was analyzed at four follow-up visits, including baseline, month 4, 12, and 24. FAF imaging was obtained with a Spectralis ® HRA-OCT (HRA2; Heidelberg Engineering, Heidelberg, Germany), which included OCT and confocal scanning laser ophthalmoscopy to visualize FAF. Confocal scanning laser ophthalmoscopy used blue light at a wavelength of 488 nm and a barrier filter at 500 nm to illuminate the fundus and detect fluorescence signals from the retinal and RPE layers. The dramatic decrease of the FAF signal in the area of RPE atrophy compared with the nonatrophic retinal areas is used by the Region Finder (version 2.4; Heidelberg Engineering) software, which can semi-automatically quantify atrophic areas and allows FAF images to be processed directly, for the segmentation of the atrophic areas [Figure 1]. 
|Figure 1: Screenshot of Region Finder software. The fundus autofluorescence reference image is shown in the left panel. The working image is shown on the right panel. The atrophic region is colored in blue. The number of regions with their size is shown at the bottom. This corresponded to the number of times the reader recalibrated the algorithm using a new reference pixel. It did not define the multifocal nature of the lesion. The total atrophic area is also shown at the bottom of the picture|
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Semiautomated atrophy detection and quantification were independently performed by the one reader. RPE atrophy was recognized as well-demarcated black areas (hypofluorescent signals) corresponding to the dead or absent RPE. Fovea and retinal blood vessels can exhibit similar intensities as RPE atrophy. If fovea and retinal blood vessels interfered with atrophic areas on FAF images, those areas were excluded using the constraint tool of the software. After processing every atrophic area measure, screenshots and reports were generated and recorded for analysis. Once atrophic areas and constraints had been defined for the baseline visit, they could be easily copied to any subsequent image that belonged to the same follow-up series. Reports included the name of the patient, total area of atrophy, number of spots, and areas of spots. In addition, the defined lesion areas and any corresponding manually applied constraints are shown in the report [Figure 2]. The reader manually positions a seeding point inside the atrophic area to initiate an automatic identification algorithm that identifies demarcated areas of severely decreased FAF signal. Considering the measurement error of the atrophic area, we used mean value after the second measurement. Intraobserver reliability was evaluated by the intraclass correlation, which was calculated from the measurements of the RPE atrophic areas on different days by one reader.
|Figure 2: Screenshot of the constraint tool in Region Finder software. The atrophic region is colored in blue. The red line is a manual constraint that the reader could use to perform his measurements more precisely|
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We investigated the area of RPE atrophy at baseline, month 4 (after anti-VEGF loading phase), 12, and 24. Change in the area of RPE atrophy was analyzed by the Student's t-test. Some possible factors that may affect changes in the area of RPE atrophy during 24-month anti-VEGF treatment were considered. The data recorded were age, gender, VA at baseline, area of RPE atrophy at baseline, area of RPE atrophy at month 4, presence of RPE detachment (pigment epithelial detachment) at baseline, number of anti-VEGF treatments, presence of intraretinal fluid (IRF) and subretinal fluid (SRF) at baseline, and type of neovascular lesion. To study the association between the change of RPE atrophic area during anti-VEGF treatment and the factors measured on a continuous variable (i.e., age, VA, area of RPE atrophy, and number of anti-VEGF treatments), simple regression analysis was performed. In the categorical variables, the differences in change of RPE atrophic area during anti-VEGF treatment between two mean values of categories or among more than three categories were analyzed using the Student's t-test or one-way analysis of variance test, respectively. To evaluate the predictive factors of an increased area of RPE atrophy during 24-month anti-VEGF treatment, multiple regression analysis was performed using the factors that achieved P < 0.15 in the univariated analysis. The logarithm of the minimum angle of resolution (logMAR) VA converted from the decimal VA was used to analyze the VA. SPSS version 18.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses, and the significance level was set at ≤0.05.
| Results|| |
A total of 65 eyes (30 male eyes and 35 female eyes) with a mean age of 69.1 ± 8.3 years were included in this study; the majority had occult CNV (35/65 eyes, 53.8%). Classic CNV was present in 6 of 65 eyes (9.2%), PCV in 18 of 65 eyes (27.8%), and RAP in 6 of 65 eyes (9.2%). BCVA (logMAR) at baseline was 0.70 ± 0.43. After three monthly anti-VEGF injections, BCVA of month 4 was 0.49 ± 0.37, which significantly improved from that of baseline (0.70 ± 0.43, P = 0.001). BCVA was maintained until month 12 (0.49 ± 0.36, P = 0.001), then aggravated; there was no significant difference between baseline at month 24 (0.57 ± 0.37, P = 0.068). The mean number of intravitreal anti-VEGF injections received during the 24 months was 9.18 ± 5.46. The patients' baseline characteristics are summarized in [Table 1].
[Figure 3] shows a change of RPE atrophy area over time after anti-VEGF treatment. The mean area of RPE atrophy was 1.293 ± 1.298 mm 2 at baseline and slightly decreased to 1.007 ± 1.043 mm 2 (P = 0.101) at month 4, which did not differ significantly. Twelve months after treatment, the atrophic area was 1.657 ± 1.375 mm 2 (P = 0.077) and significantly increased to 2.394 ± 1.940 mm 2 (P = 0.001) after 24 months. [Figure 4] and [Figure 5] show representative imaging examples of patients in this study.
|Figure 3: The change of retinal pigment epithelial atrophic area over time after anti- vascular endothelial growth factor treatment. Area of retinal pigment epithelial atrophy at month 24 was significantly different from that of baseline †P < 0.05, significantly different from baseline|
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|Figure 4: (a) A 74-year-old female with 6 months' history of decreasing vision in the left eye (visual acuity, 20/100). Optical coherence tomography image of the patient at baseline, choroidal neovascularization with intraretinal fluid was seen. Atrophic area was 0.645 mm2. (b) Color fundus, fundus autofluorescence, and optical coherence tomography images of month 4 after three intravitreal ranibizumab injections. Area of the atrophic region was 0.296 mm2, which decreased from baseline. The intraretinal fluid was completely absorbed. (c) Color fundus, fundus autofluorescence, and optical coherence tomography images of month 12. Area of the atrophic region was 1.002 mm2, and intraretinal fluid was detected in optical coherence tomography image. (d) Color fundus, fundus autofluorescence, and optical coherence tomography images of month 24. Area of the atrophic region was 3.164 mm2. The intraretinal fluid was still observed, and size of choroidal neovascularization increased in optical coherence tomography image. Patient received intravitreal ranibizumab injections 9 times during 24-month follow-up|
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|Figure 5: (a) A 70-year-old female with 3 months' history of decreasing vision in the right eye (visual acuity, 20/200). Optical coherence tomography image of the patient at baseline, choroidal neovascularization with subretinal fluid was seen. Area of the atrophic region was 1.221 mm2. (b) Color fundus, fundus autofluorescence, and optical coherence tomography images of month 4 after three intravitreal ranibizumab injections. Area of the atrophic region was 0.913 mm2, which decreased from baseline. Subretinal fluid was completely absorbed. (c) Color fundus, fundus autofluorescence, and optical coherence tomography images of month 12. Area of the atrophic region was 0.939 mm2. Subretinal fluid is not observed in optical coherence tomography image. (d) Color fundus, fundus autofluorescence, and optical coherence tomography images of month 24. Area of the atrophic region increased to 1.456 mm2, though no subretinal fluid is visible on optical coherence tomography image. The patient received intravitreal ranibizumab injections 6 times during 24-month follow-up|
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Univariate analysis showed that larger areas of RPE atrophy at month 4, and the number of anti-VEGF injections were the risk factors for increased RPE atrophic areas during the 24-month follow-up period [Table 2]. The multiple stepwise regression analysis also showed that larger areas of RPE atrophy at month 4 (P = 0.001) and larger numbers of anti-VEGF injections (P = 0.001) were significantly associated with increased RPE atrophy areas during the 24-month follow-up period [Table 3]. The intraobserver agreement was relatively good (intraclass correlation, 0.90; 95% confidence interval, 0.87-0.97) between the two measurement times.
|Table 2: Univariate analysis for the association between each factor and increased retinal pigment epithelial atrophic area during the 24 months|
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|Table 3: Multiple stepwise regression analysis of risk factors for increased areas of retinal pigment epithelial atrophy during 24-month follow-up period|
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| Discussion|| |
RPE degeneration or atrophy is one of the central hallmarks in the pathogenesis of AMD. RPE is a single layer of polygonal cells, uniform in size, which separates the choroid from the sensory retina. This epithelial layer plays a critical role in the normal functioning of the retina. It is responsible for phagocytosis and lysosomal breakdown of pigmented outer segments of photoreceptors, which allows the renewal process necessary to maintain photoreceptor excitability. Over the course of a lifetime, each RPE cell will phagocytose 3 billion outer segments.  With aging, incomplete or partial breakdown of these segments in the postmitotic RPE cells causes the accumulation of lipofuscin. The first visible change of geographic atrophy is drusen formation. Drusen is thought to be accumulations of vast amounts of lipofuscin. The pathogenesis of drusen is not completely understood; however, oxidative stress and/or blue light damage along with an age-related decline in lysosomal enzyme function have been implicated. , RPE cell dysfunction can be seen in pigment irregularities with either hyper- or hypo-pigmentation, which often presents together with drusen. As the disease progresses, drusen might disappear while RPE atrophy becomes more significant. 
In this study, we evaluated the quantitative changes in RPE atrophy in eyes with exudative AMD, and a significant enlargement of RPE atrophy areas was documented after anti-VEGF treatment. Lois et al. reported that advanced AMD is associated with increased RPE atrophy and a larger overall number of injections in exudative AMD.  This suggests that suppression of VEGF could provoke the incidence of RPE atrophy, excluding the natural progression of exudative AMD. Kurihara et al. developed a mouse model to explain why inhibiting VEGF to dry the macula might induce RPE atrophy or similar conditions.  They deleted VEGF-A gene, responsible for VEGF-A production, from adult mouse RPE cells. Deletion of VEGF-A resulted in dramatic and rapid loss of endothelial cells of the choriocapillaris and severe vision loss due to cone cell death. They said this suggests that RPE-derived VEGF plays an essential functional role in supporting the adult subretinal vasculature, including the choriocapillaris, which nurtures the cone photoreceptors, and maintains central vision. Therefore, anti-VEGF treatments could have an effect on the development and progression of RPE atrophy. These results have raised a number of important safety questions among clinicians using intravitreal anti-VEGF treatment in AMD patients.
Our study provides two important pieces of information regarding the risk factors for the development and apparent progression of RPE atrophy in eyes receiving intravitreal anti-VEGF therapy for exudative AMD. Although this study did not directly address any causal relationship, we found that the total number of intravitreal anti-VEGF injections is associated with growth of existing RPE atrophy, which is consistent with the results of Lois et al.  It is becoming apparent that repeated anti-VEGF injections have the ability to promote RPE atrophy over time, thus potentially negatively impacting vision. For now, limiting the number of injections to the minimum amount necessary appears to be warranted. In addition, a larger area of RPE atrophy at month 4 predicted the progression of atrophic areas during anti-VEGF treatment. Previously, Biarnιs et al. reported that both the baseline area of RPE atrophy and the duration of follow-up were the risk factors of RPE atrophy progression in patients with nonexudative AMD.  In this study, however, the progression of RPE atrophy was significantly associated with the atrophy area after the initial loading phase of anti-VEGF treatment, whereas the area of atrophy at baseline showed no association. This result may be explained as follows. To date, there is no convincing evidence supporting the idea that a loading phase is superior to an immediate PRN scheme. The most widely applied treatment scheme is a so-called loading phase with three initial consecutive intravitreal injections of anti-VEGF agents followed by a PRN or treat-and-extend scheme. The baseline SRF or IRF is identified in most of the exudative AMD eyes, which shows partial or complete vanishment following the loading phase of anti-VEGF. According to our results, 26 eyes (40%) obtained dry maculas after initial loading doses. We presume that the decreased SRF and IRF could result in an increase of FAF in month 4. After the loading phase of anti-VEGF, the dark area of FAF decreased in consequence of decreased blocked fluorescence, subsequent to the prominent loss of fluid [Figure 4] and [Figure 5]. The initial response to anti-VEGF therapy was suggested to be indicative of the long-term response in previous studies. , In this respect, decreased RPE atrophic area after the loading phase may be an important predictor for a good long-term visual outcome.
To the best our knowledge, this is the ﬁrst study to quantify the RPE atrophy area using the Region Finder software in patients receiving anti-VEGF for exudative AMD. Semiautomated atrophy detection and quantiﬁcation of GA due to AMD using the Region Finder software was ﬁrst described by Schmitz-Valckenberg et al., reporting that the intraobserver and interobserver agreements were superior to manual outlining methods with reproducible and time-efﬁcient measurements.  However, especially in RPE atrophy associated with exudative AMD, lesion boundaries seem ill-deﬁned, and thus, the Region Finder may be less reliable for the identiﬁcation and quantiﬁcation of atrophic areas. Moreover, hypofluorescent signals on FAF are also found in fresh hemorrhages, lipid deposits, dense hyperpigmentation, and RPE tears. To eliminate such lesions, fundus photos and OCT findings were used as references. We also used the mean value of the atrophic area after the second measurement to minimize the subjective nature of quantification. The intraobserver repeatability of performing the RPE atrophy area calculation was assessed, and the intraclass correlation of area measurement was 0.90 (95% confidence interval, 0.87-0.97), which showed favorable repeatability.
The results of our study should be taken cautiously given methodological limitations, such as its retrospective nature, the relatively small number of patients included, the absence of a control group, the assessment of FAF images by the same observer, and the relatively short follow-up period. Another limitation is that this study did not directly address any causal relationship, such as whether anti-VEGF treatment itself causes acceleration of RPE atrophy or whether the development of RPE atrophy is part of the underlying disease progression of exudative AMD.
| Conclusions|| |
In summary, RPE atrophy progresses in eyes with exudative AMD during anti-VEGF treatment. Larger areas of RPE atrophy at month 4 and larger numbers of anti-VEGF injections were associated with an increased risk of RPE atrophy progression following treatment. These ﬁndings may be useful to clinicians using intravitreal anti-VEGF for the treatment of exudative AMD, both for selecting an appropriate treatment plan and for predicting the progression of RPE atrophy.
Financial support and sponsorship
This study was supported by 2015 Kangwon National University Hospital Grant.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]