AGE-RELATED MACULAR DEGENERATION (AMD) IS

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1 Progression of Geographic Atrophy and Impact of Fundus Autofluorescence Patterns in Age-related Macular Degeneration FRANK G. HOLZ, MD, ALMUT BINDEWALD-WITTICH, MD, MONIKA FLECKENSTEIN, MD, JENS DREYHAUPT, PHD, HENDRIK P. N. SCHOLL, MD, AND STEFFEN SCHMITZ-VALCKENBERG, MD, IN COLLABORATION WITH THE FAM-STUDY GROUP PURPOSE: To test if fundus autofluorescence (FAF) patterns around geographic atrophy (GA) have an impact on GA progression rates over time in atrophic age-related macular degeneration (AMD). DESIGN: Prospective longitudinal multicenter natural history study. METHODS: Standardized digital FAF images were obtained from 195 eyes of 129 patients with GA using confocal scanning laser ophthalmoscopy (excitation 488 nm, emission >500 nm). Areas of GA were quantified and patterns of abnormal FAF in the junctional zone were classified. Repeated FAF images were obtained over a median follow-up period of 1.80 years (interquartile range [IQR], 1.28 to 3.34). RESULTS: Areas of GA (median, 7.04 mm 2 at baseline; IQR, 3.12 to 10.0) showed a median enlargement of 1.52 mm 2 /year (IQR, 0.81 to 2.33). Progression rates in eyes with the banded (median 1.81 mm 2 /year) and the diffuse FAF pattern (1.77 mm 2 /year) were significantly higher compared to eyes without FAF abnormalities (0.38 mm 2 / year) and focal FAF patterns (0.81 mm 2 /year, P <.0001). Within the group of the diffuse pattern, eyes with a diffuse trickling pattern could be identified that exhibited an even higher spread rate (median 3.02 mm 2 / year) compared to the other diffuse types (1.67 mm 2 / year, P.001). CONCLUSIONS: The results indicate that distinct phenotypic FAF patterns have an impact on disease progression in eyes with atrophic AMD and may therefore serve Supplemental Material available at AJO.com. Accepted for publication Nov 3, From the Department of Ophthalmology, University of Bonn, Bonn, Germany (F.G.H., A.B.-W., M.F., H.P.N.S., S.S.-V.); and the Institute for Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany (J.D). Inquiries to Frank G. Holz, MD, Department of Ophthalmology, University of Bonn, Ernst-Abbe-Str. 2, Bonn, Germany; Frank.Holz@ukb.uni-bonn.de as prognostic determinants. The findings underscore the relevance of FAF imaging and the pathogenetic role of excessive retinal pigment epithelium (RPE) lipofuscin (LF) accumulation in GA. Natural history data and identification of high-risk characteristics will be helpful to design interventional studies aiming at slowing the spread of atrophy. (Am J Ophthalmol 2007;143: by Elsevier Inc. All rights reserved.) AGE-RELATED MACULAR DEGENERATION (AMD) IS now the most common cause for severe vision loss in developed countries. 1 5 In late-stage AMD, a central scotoma may result from choroidal neovascularization (CNV), long-standing pigment epithelial detachment, or geographic atrophy (GA) of the retinal pigment epithelium (RPE). Hereby, GA is thought to be the natural end stage of the AMD process when CNV does not develop. 6,7 Although CNV is the most common cause for severe vision loss, GA is responsible for approximately 20% of legal blindness attributable to AMD and will become even more prevalent with the aging population. 5,8,9 Extensive research has been attributed to the pathomechanisms of CNV formation, whereas relatively little attention has been focused on GA. Until now, there has been no proven therapy for GA patients available to stop or to slow down the disease process. The pathophysiologic mechanisms of RPE cell atrophy with a corresponding atrophy of the choriocapillaris and outer neurosensory retinal layers still remain unclear. Several lines of experimental and clinical evidence indicate that lipofuscin (LF) accumulation in the lysosomal compartment of RPE cells plays a critical role in the disease process Life-long phagocytosis of photoreceptor outer segments by postmitotic RPE is thought to contribute to the accumulation of LF. LF has been shown to contain toxic compounds such as A2-E (N-retinylidene- N-retinylethanol-amine) and modified macromolecules that interfere with regular lysosomal degradation and, /07/$ BY ELSEVIER INC. ALL RIGHTS RESERVED. 463 doi: /j.ajo

2 FIGURE 1. Step-by-step approach for the classification of fundus autofluorescence (FAF) patterns in patients with geographic atrophy attributable to age-related macular degeneration (AMD). Eyes with no increased FAF intensity were graded as NONE. The eyes with increased FAF were divided into two groups depending on the configuration of increased FAF surrounding atrophy. Eyes with increased FAF only at the margin of geographic atrophy (GA) were split into three subtypes (FOCAL, BANDED, and PATCHY) while eyes showing areas with increased FAF directly adjacent to the margin of the atrophic patch and elsewhere were called diffuse. This latter group was subdivided in four different subtypes depending on the appearance of the FAF features: Reticular, Branching, Fine Granular, and Fine Granular with Peripheral Punctate Spots. During the analysis of the current study, a new pattern (Trickling) within the diffuse group was identified, which showed not only a specific morphologic pattern but also a significant faster spread of atrophy over time compared to the other diffuse types of abnormal FAF (see Results section). TABLE 1. Frequency and Atrophy Progression Rates for Patterns of Abnormal Fundus Autofluorescence in the Junctional Zone of Geographic Atrophy in the Study Sample Frequency (n 195) Progression Rate (mm 2 /Year) Patterns of Increased FAF in the Junctional Zone of GA Number (%) Median IQR None to 0.79 Focal to 1.07 Band to 2.69 Patchy Diffuse to 2.58 Reticular (12) Branching (45) Fine granular (36) Fine granular with peripheral punctuated spots (10) Trickling (9) (3.02) (2.89 to 3.20) Remaining* to 2.78 FAF fundus autofluorescence; GA geographic atrophy; IQR interquartile ratio. *Fourteen eyes were not grouped to one of these FAF types, because there was no predominant FAF pattern in the junctional zone of GA. In 11 eyes, a classification was not possible attributable to insufficient image quality. subsequently, normal cell function via various molecular mechanisms Excessive LF accumulation represents a common pathogenetic pathway in various degenerative and monogenetic retinal diseases. 18 Confocal scanning laser ophthalmoscopy (cslo) has been introduced for in vivo visualization of fundus autofluorescence (FAF) Spectrophotometric measurements by Delori and associates demonstrated that the FAF signal originates from RPE LF fluorophores. 23,24 We have recently identified distinct patterns of abnormal increased FAF in the junctional zone of GA To test the hypothesis that these different patterns have an impact on the progression and spread of GA areas, we performed a longitudinal analysis as part of the natural history 464 AMERICAN JOURNAL OF OPHTHALMOLOGY MARCH 2007

3 FIGURE 2. Examples of atrophy enlargement over time in patients with geographic atrophy attributable to age-related macular degeneration (AMD) for the fundus autofluorescence (FAF) patterns, which were evaluated in this study. Each pattern is shown in pairs with baseline FAF image (left) and follow-up FAF image (right). Atrophic areas are outlined in white in each image. Time between image pairs and atrophy progression rates are described in the following: (Upper left) Right eye of a 78-year-old patient with no FAF abnormalities (NONE). Minimal atrophy enlargement is observed (0.02 mm 2 /year, follow-up 12 months). (Middle left) Right eye of a 77-year-old patient with only small areas of focally increased autofluorescence at the margin of the atrophic patch (FOCAL). Slow atrophy progression over time can be detected (0.36 mm 2 /year, follow-up 15 months). (Lower left) Right eye of a 66-year-old patient for the patchy pattern (atrophy progression 1.84 mm 2 /year) (PATCHY). Note that follow-up (25.2 months) between the images is much longer compared to the other examples. (Upper right) Right eye of a 55-year-old patient with diffuse fine granular FAF pattern surrounding atrophy (DIFFUSE). Moderate growth of atrophy is noticed over time (1.71 mm 2 /year, follow-up 12 months). (Middle right) Left eye of a 76-year-old patient with a band of increased autofluorescence surrounding the geographic atrophy (BANDED). High atrophy progression is observed (2.52 mm 2 /year, follow-up 18 months). (Lower right) Left eye of a 64-year-old patient with a diffuse trickling FAF pattern (DIFFUSE, TRICKLING) with very high atrophy enlargement over time (3.78 mm 2 /year, follow-up 18 months). FAM-Study (Fundus Autofluorescence in age-related Macular Degeneration). PATIENTS AND METHODS PATIENTS WITH GA SECONDARY TO AMD WERE INCLUDED from the longitudinal natural history arm of the multicenter FAM-Study (registration NCT ). The study followed the tenets of the Declaration of Helsinki and was approved by the local Institutional Review Boards and the local ethics committees at the study centers. Informed consent was obtained from each patient after explanation of the nature and possible consequences of the study. Patients with uni- or multifocal GA and with clear media to allow FAF imaging were enrolled in the study. Exclusion criteria included any history of retinal surgery, VOL. 143, NO. 3 GEOGRAPHIC ATROPHY AND FUNDUS AUTOFLUORESCENCE 465

4 laser photocoagulation, and radiation therapy or other retinal diseases in the study eye, including diabetic retinopathy or hereditary retinal dystrophies. Fluorescein angiography was only performed if there were funduscopic signs present indicative of neovascular AMD in addition to patches of GA. Such eyes were excluded from the study. Best-corrected visual acuity (BCVA) was determined using Early Treatment Diabetic Retinopathy Study (ETDRS) charts on a quasi-logarithmic ordinal scale (for use in regression models, it was ranked one to 10 and treated as quasi-continuous). Before examination, the pupil of the study eye was dilated with 0.1% tropicamide eye drops. A standardized case report form including ophthalmologic history, possible risk factors, and family history was completed for each patient. FAF was recorded using a cslo (Heidelberg Retina Angiograph, HRA classic and HRA 2, Heidelberg Engineering, Dossenheim, Germany), the optical and technical principles of which have been described previously. 22,28,29 An argon blue laser (HRA classic) or an optically pumped solid-state laser (HRA 2) are used for excitation (both 488 nm), and the emitted light above 500 nm is detected using a barrier filter. Maximal retinal irradiance using the HRA is approximately 2 mw/cm 2 for a 10 degrees 10 degrees image and is therefore well below the limits established by the American National Standards Institute and other international standards. 30 Images were immediately digitized and processed using a flexible frame processor and subsequently displayed on a computer screen. FAF images were recorded in accordance with a standard operation procedure including focusing in the red-free reflection mode ( 514 nm for HRA classic, 488 nm for HRA 2), acquisition of a series of 30 degrees 30 degrees images (488 nm), and calculation of mean images after automated alignment (out of nine single images) in order to amplify the signal-to-noise ratio using image analysis software (Heidelberg Eye Explorer, Heidelberg Engineering, Dossenheim, Germany). 31 The image resolution was pixels for the HRA classic and pixels for the HRA 2, respectively. When measuring areas on images, pixels are automatically converted for both versions into millimeters taking into account the original image resolution and the focusing during image acquisition. For the analyses herein, only patient eyes with at least two examinations (minimum follow-up of six months) and with sufficient image quality to accurately determine the size of atrophy were included, screening the FAM database until the end of November The total size of uni- or multifocal GA was measured by outlining dark atrophic areas using image analysis software (Heidelberg Eye Explorer), and atrophy progression was calculated as previously described. 32 In accordance with the published FAF classification in patients with advanced atrophic AMD, FAF images were evaluated for different FAF patterns at the baseline image by the central FAF Reading Center in TABLE 2. Statistical Analysis of Difference in Atrophy Progression Rates/Year Between the Fundus Autofluorescence Patterns (Wilcoxon Rank-Sum Test) Comparison of Fundus Autofluorescence Pattern P value None vs focal.092 None vs diffuse.001 None vs banded.001 Focal vs diffuse.001 Focal vs banded.001 Diffuse vs banded.510 None focal vs diffuse banded.0001 Diffuse without trickling vs diffuse trickling.001 FIGURE 3. Box plots showing the distribution of atrophy progression per year for the pooled data of groups with no abnormal and focal fundus autofluorescence (FAF) pattern and the pooled data of the banded and the diffuse FAF pattern group in patients with geographic atrophy attributable to age-related macular degeneration (AMD). The median enlargement for each group is given below the box plot. Using the Wilcoxon rank-sum test, the difference between the two groups was significant (P <.0001). Bonn, Germany. 27 This classification is based on FAF features in the junctional zone of atrophy in patients with GA attributable to AMD and uses a step-by-step approach for FAF image evaluation (Figure 1). All baseline images were graded separately by two independent readers (S.S.V. and M.F.). In case of discrepancy, a third reader was asked to arbitrate (F.G.H.). As no patients with early age-related macular changes were included, the recently introduced FAF pattern classification of abnormal FAF in eyes with early nonexudative age-related macular disease was not applied in the ongoing study. 33 Statistical analyses included frequency and descriptive statistics. Nonparametric procedures were used to compare different groups: The Wilcoxon rank-sum test and the Spearman rank correlation coefficient. 34 The comparison was performed using the likelihood ratio test in hierarchi- 466 AMERICAN JOURNAL OF OPHTHALMOLOGY MARCH 2007

5 FIGURE 4. Examples for the diffuse trickling fundus autofluorescence (FAF) pattern as distinctive new FAF pattern in patients with geographic atrophy attributable to age-related macular degeneration (AMD). (Left) Left eye of a 56-year-old patient with extrafoveal geographic atrophy. Multifocal patches of atrophy above the fovea appear more grayish rather than dark black, as atrophy is usually characterized by FAF imaging. FAF intensity directly around atrophy is largely enhanced with a diffuse trickling of the FAF signal toward the periphery. (Right) Composite FAF image of the right eye of a 96-year-old patient with the diffuse trickling type showing very advanced geographic atrophy. The huge continuous atrophy includes large retinal areas within the temporal arcades as well as the optic disk and retinal areas nasally to the optic disk. cal models. The Fisher exact test was used to investigate the hypothesis of independence of FAF pattern classification and the total baseline atrophy classification. All statistical calculations were carried out with the software S-Plus 2000 (Mathsoft, Seattle, Washington, USA) and SAS 9.1 (SAS Institute, Cary, North Carolina, USA). RESULTS A TOTAL OF 195 EYES OF 129 PATIENTS (76 FEMALE, 53 MALE) met the inclusion criteria and were analyzed. Median age at first examination was 73.8 years (interquartile range [IQR], 67.6 to 78.2). Bilateral GA was present in 87 patients; in 66 of those patients, both eyes were included into the study, whereas 42 patients had unilateral atrophies. In three study eyes, a CNV developed during the follow-up period (1.5%). Overall, median follow-up for all eyes was 1.80 years (IQR, 1.28 to 3.34; range, 0.52 to 7.14). Areas of GA (median, 7.04 mm 2 at baseline; IQR, 3.12 to10.0) showed a continuous enlargement over time with a median growth rate of 1.52 mm 2 /year (IQR, 0.81 to 2.33). Median visual acuity (VA) at baseline was 0.32 (IQR, 0.13 to 0.50) and was significantly better in eyes with extrafoveal GA (VA, 0.5; IQR, 0.3 to 0.63) compared to eyes with foveal involvement (VA, 0.16; IQR, 0.1 to 0.2; P.0001). At baseline, the area of GA included the foveola in 102 eyes. In 32 eyes with an initial extrafoveal GA, the atrophic area spread to involve the foveola during the review period. The size of the GA at baseline did not significantly correlate with the VA in eyes with extrafoveal GA (P.063). In contrast, a significant correlation was present between VA and size of the GA at baseline examination in eyes with a central atrophic patch at study entry (P.033). No correlation was found between the rate of progression and other characteristics recorded in standardized case report forms including age 80 years (P.720), hypertension (P.684), diabetes (P.202), hyperlipidemia (P.786), actual or previous history of smoking (P.463), body mass index 30 kg/m 2 (P.099), or family history for AMD (P.941). In accordance with the recently published classification system, five different patterns of abnormal FAF in the junctional zone of GA were distinguished in 170 eyes at the baseline image: none, focal increased, banded, patchy, and diffuse. The diffuse pattern is originally further divided into four subtypes: reticular, branching, fine granular, and fine granular with peripheral punctate spots. 27 In a total of 14 eyes, no predominant FAF pattern could be identified; in 11 eyes, insufficient image quality did not allow to accurately grade the abnormal FAF pattern. Frequency data of the FAF patterns and respective atrophy progression rates is provided in Table 1. Atrophy enlargement was the slowest in eyes with no abnormal FAF pattern (median, 0.38 mm 2 /year), followed by eyes with the focal FAF pattern (median, 0.81 mm 2 / year), then by eyes with the diffuse FAF pattern (median, 1.77 mm 2 /year), and by eyes with the banded FAF pattern (median, 1.81 mm 2 /year). The three eyes with the patchy FAF pattern had progression rates of 1.37, 1.84, and 2.94 mm 2 /year, respectively. Because of their small frequency number, eyes with the patchy FAF pattern were not included in the further statistical analysis. Examples for all different FAF patterns and their atrophy progression rates are illustrated in Figure 2. The Wilcoxon rank-sum test showed statistically significant differences in atrophy enlargement per year for different abnormal FAF patterns except between the groups of no abnormal and focal FAF pattern (P.092) and between the groups of banded and diffuse FAF pattern VOL. 143, NO. 3 GEOGRAPHIC ATROPHY AND FUNDUS AUTOFLUORESCENCE 467

6 FIGURE 5. Box plots showing the distribution of atrophy progression per year for the diffuse subgroups without the diffuse trickling subgroup and the diffuse trickling subgroup only. The median progression rate of atrophy for each subgroup is given below the box plot. Using the Wilcoxon rank-sum test, the difference between the two was groups significant (P.001). (P.510; Table 2). Pooling the data of the none and the focal FAF pattern group and comparing to the pooled data of the diffuse and banded FAF patterns, the difference in atrophy progression was highly significant (P.0001; Figure 3). Eyes were ranked according to the progression rate, and morphologic differences in the FAF distribution were analyzed. Within the group of the diffuse pattern, a total of nine eyes with relatively rapid progression rates showed very high FAF intensities directly around atrophy with a diffuse trickling FAF pattern toward the periphery (Figures 2 and 4). This subgroup has not been identified before and is not implemented in the previous FAF classification system. This diffuse trickling FAF pattern was originally classified as diffuse fine granular or diffuse branching but now showed distinguishable FAF features compared to the other diffuse eyes. Overall, atrophy areas in this group did appear more grayish rather than dark black as usually seen by FAF imaging. In eight of nine eyes, peripapillary atrophy was observed. Interestingly, all eyes showed foveal sparing at baseline, and foveal involvement appeared not until extensive extrafoveal atrophy. In two very advancedstage eyes, atrophy did not include just the area between the temporal arcades but also reached extensively to the outer-macula retina nasally to the disk (Figure 4). Median enlargement of these nine eyes with the diffuse trickling abnormal FAF pattern was 3.02 mm 2 /year (IQR, 2.89 to 3.20), while the other diffuse types alone had a median progression rate of 1.67 mm 2 /year (IQR, 0.92 to 2.29). This difference was statistically significant (P.001; Figure 5). To test the influence of the total GA size at baseline on atrophy enlargement, the eyes of the none, focal, banded, and diffuse FAF pattern group were classified according to FIGURE 6. Box plots showing the distribution of atrophy progression per year and baseline total atrophy for the studied eyes according to the classification of Sunness and associates for patients with geographic atrophy attributable to age-related macular degeneration (AMD). 6 Eyes were classified depending on the total size of atrophy at baseline, while one disk area (DA) is equal to an area of 2.54 mm 2. Median atrophy enlargement per year is given below each box plot. Using the Wilcoxon rank-sum test, only the difference between the <1 DA group and the other groups was statistically significant (P <.0001). TABLE 3. Comparison of the Fundus Autofluorescence Pattern Classification and the Classification of Baseline Total Atrophy for all Eyes With the None, Focal, Banded, and Diffuse Fundus Autofluorescence Pattern* Fundus Autofluorescence Pattern at Baseline 1 DA 1to3 DA Total Baseline Atrophy 3to5 DA 5to10 DA 10 DA None focal Diffuse banded DA disk area. *No significant statistical dependence between both classification systems could be found (Fisher exact test, P.070). Sunness and associates 6 The group with a total GA size of greater than mm 2 ( 10 disk areas [DA]) contained only three eyes (atrophy progression 0.658, 1.173, and 2.21 mm 2 /year) in this sample and was therefore excluded in the following statistical analysis. The group of eyes with less than 2.54 mm 2 ( 1 DA) baseline total atrophy (median, 1.27 mm 2 ; range, 0.13 to 2.47) exhibited a significant difference in atrophy progression compared to the other groups (P.0001). The groups with a total GA size at baseline between 1 to 3 DA (2.54 to 7.61 mm 2 ), 3 to 5 DA (7.62 to mm 2 ), and 5 to 10 DA (12.70 to mm 2 ) had nonsignificant differences in progression 468 AMERICAN JOURNAL OF OPHTHALMOLOGY MARCH 2007

7 rates (Figure 6). In a combined analysis of the FAF pattern classification and the classification of baseline total atrophy, there was a trend of a smaller GA size at baseline within the group with no abnormal and focal FAF pattern, but statistical analysis could not abandon the thesis of the independence between both classification systems (P.070; Table 3). DISCUSSION WITH THE ADVENT OF DIGITAL CONFOCAL SCANNING LAser ophthalmoscopy, it has become possible to visualize LF accumulation at the level of the RPE cell monolayer in vivo ,35 FAF imaging gives additional information above and beyond conventional imaging tools such as fundus photography, fluorescein angiography, or optical coherence tomography. In a cross-sectional analysis of the FAM-Study, we recently identified various distinct patterns of abnormal elevated FAF in the junctional zone of GA. 27 This morphologic classification was based on information that is only detectable by FAF imaging. The current study shows that variable GA progression rates depend on the specific phenotype of abnormal FAF patterns at baseline. Phenotypic features of FAF abnormalities had a much stronger impact on atrophy progression than any other risk factor that has been addressed in previous studies on progression of GA attributable to AMD. Interestingly, we identified nine eyes with extremely rapid atrophy progression, which showed distinct FAF features around atrophy that have not been observed before. We introduce the term diffuse trickling for this pattern that is associated with a significantly faster atrophy enlargement compared to all other diffuse eyes. Large population-based studies have indicated a correlation between smoking, hypertension, diabetes, and AMD. 4,36 39 Another natural history study on patients with GA attributable to AMD did not show a more rapid enlargement of atrophy in patients with hypertension and smoking. 6 In this study, none of these potential risk factors contributed significantly to atrophy progression. It has been shown that areas with increased FAF signal, and therefore, excessive RPE LF load, precede the development of new areas of GA or the enlargement of preexisting atrophic patches. 26 Scholl and associates have demonstrated impaired rod photoreceptor function associated with increased FAF by fine-matrix mapping in patients with AMD including GA. 40 Combining SLO microperimetry and FAF imaging, impaired retinal sensitivity has been observed in areas of abnormal FAF in the junctional zone. 31 These findings as well as the results of this study underscore the importance of abnormal FAF intensities around atrophy and the pathophysiologic role of increased RPE LF accumulation in patients with GA attributable to AMD. Recently, we have shown that the extension of the total area with increased FAF surrounding atrophy at baseline in eyes with the diffuse pattern has a positive correlation with atrophy progression rate over time. 41 In a study of eight GA eyes, Hwang and associates have shown that new atrophy occurs not specifically where previously areas of increased FAF have been observed, suggesting that increased FAF is not a risk factor for development or extension of GA. 42 This study had a very small sample size, used a different image acquisition protocol, and allowed moderate image quality. Furthermore, the quantification of FAF intensities in that study must be regarded as problematic or even not feasible until now because of the fluctuations in the FAF signal attributable to inconstant laser power, optical opacities, and eye movements that only seem to allow to evaluate a qualitative comparison of FAF intensities. 31 However, the large database of the FAM-Study shows that localized FAF is not automatically associated with localized occurrence of new atrophy or enlargement of existing atrophy, but that different FAF patterns around atrophy have an overall impact on atrophy progression. The differences in atrophy enlargement between FAF phenotypes in this study may reflect heterogeneity on the cellular and molecular level in the disease process. As there is a high degree of intraindividual symmetry, genetic determinants rather than nonspecific aging changes may be involved. 27,43 Recent findings on the role of polymorphisms in the genes coding for complement factor H, factor B, and LOC are in accordance with the assumption that genetic factors may play a major role in the pathophysiology of AMD In our patients, we obtained blood samples for future molecular genetic analyses to address potential genotype/phenotype correlations. Progressive enlargement of GA over time has been assessed previously. 6,7,52 54 The first quantitative data on the spread of atrophic macular degeneration was published by Schatz and McDonald in They showed in fundus photographs of 50 eyes an average growth rate of 139 m/year in the horizontal direction. Sunness and associates described in fundus photographs of 123 eyes (mean enlargement of 2.2 DA; median, 1.8) in a two-year follow-up period, which would be 2.79 mm 2 /year (2.29 mm 2 /year) assuming 2.54 mm 2 for one DA. 6 Furthermore, they classified their eyes in five different groups according to the total size of atrophy at baseline and stated that the enlargement of atrophy increased with increasing baseline atrophy for up to five DA of baseline atrophy, leveled off for the 5 to 10 DA group, and slightly decreased for the 10 DA group. The studies identified a great difference and range in atrophy enlargement study (0 to 13.8 mm 2 / per year), which could be explained neither by baseline atrophy nor by any other tested demographic factor. The detection and quantification of atrophy was based on fundus photographs and involved several magnification steps. No FAF imaging was included. In the current study, the mean atrophy progression rate with 1.74 mm 2 /year (median, 1.52 mm 2 /year) analyzed by FAF imaging is VOL. 143, NO. 3 GEOGRAPHIC ATROPHY AND FUNDUS AUTOFLUORESCENCE 469

8 slightly smaller than data from Sunness and associates. Statistical analysis of atrophy progression in FAM-Study data has shown that atrophy enlargement is overall not dependent on baseline atrophy, and eyes most likely have a linear atrophy growth over time suggesting that progression is not strongly correlated with baseline atrophy. 32 Stratifying the data per Sunness and associates, we could, however, confirm that the slowest atrophy progression was found to be in the eyes with a total baseline atrophy of 1 DA, but no statistical significant difference in atrophy enlargement was shown for the other DA groups. This remarkable observation is, in our view, most likely caused by the fact that the groups of no abnormal and focal FAF patterns tend to have smaller baseline atrophies compared to the banded and diffuse FAF pattern groups. Overall, the classification according to the FAF pattern at baseline appears to have a much stronger predictive value on atrophy progression over time than the classification in groups of different baseline GA sizes and could furthermore better explain the great range of atrophy progression rate within patients. Recently, two different substances have been described that could serve as a novel therapeutic approach to halt LF accumulation in degenerative retinal diseases. Radu and associates could show dose-dependent reductions in serumretinol and in retinol binding protein and an arrested accumulation of RPE LF autofluorescence in ABCR -/- mice after administration of N-(4-hydroxyphenyl)retinamide. 55 Maiti and associates demonstrated that RPE65 antagonists limit the visual cycle and prevents LF accumulation in the ABCR -/- mouse model. 56 These molecules may serve as candidates for the treatment of forms of macular degeneration whereby LF/A2E accumulation is an important pathophysiologic factor. The results from our study may be helpful in designing such interventional trials in atrophic AMD. The inclusion of patients with high-risk characteristics based on FAF imaging ( rapid progressors ) would markedly reduce required sample sizes and make the detection of a therapeutic effect within a reasonable study time frame more likely. Using the longitudinal data of the FAM-Study population and stratifying patients according to the FAF pattern in two classes (none focal vs banded diffuse), a total of 135 patients (one study eye per patient) would be required to detect a treatment effect of 0.50 mm 2 /year reduction in atrophy progression (alpha 0.05, power 0.9). Such studies may implement automated procedures for the detection and measurements of atrophic patches in FAF images as recently described. 57 Furthermore, the differentiation of FAF pattern in none focal vs banded diffuse serves as a simplification of the complex FAF classification system and may be helpful in a clinical setting to assess progression rates of geographic atrophy in individual patients. Certain limitations have to be taken into account in this study. Two eyes per patient were allowed, and the review period was limited. The acquisition and classification of FAF images is challenging attributable to difficulties with image quality as well as inhomogeneity concerning protocols and FAF classification systems between different studies. However, the FAM-Study presents the largest data pool of GA patients with FAF imaging and longitudinal review so far. Image acquisition protocol as well as image grading have previously been introduced and well evaluated including testing of the reproducibility. 27 (Schmitz- Valckenberg S, Dreyhaupt J, et al. Interobserver variability of the classification of abnormal fundus autofluoresence paticles in the junctional bone of atrophy in patients with AMD. Joint meeting of the Societas Ophthlamologica Europea (SOE) and German Ophthalmological Society (DOG), 2005, Abstract). In summary, FAF imaging using cslo is a suitable noninvasive tool to visualize LF-related topographic variations in FAF in atrophic AMD in vivo. Different patterns of abnormal FAF in the junctional zone of GA identified by digital FAF imaging have an impact on disease progression. FAF patterns may serve as novel prognostic determinants and appear to represent the strongest predictive factor for atrophy progression known so far. Furthermore, the results implicate a role of excessive LF accumulation in the pathogenesis of atrophic AMD. Natural history data and identification of high-risk characteristics will also help to test novel interventions in future clinical trials to slow down spread of atrophy in order to preserve vision in patients with atrophic AMD. THIS STUDY WAS SUPPORTED BY THE DEUTSCHE FORschungsgemeinschaft (DFG), Bonn, Germany, Ho 1926/1-3, Ho 1926/ 3-1, Ma 1723/1-1, DFG Priority Program Age-related Macular Degeneration (SPP 1088); DFG Heisenberg Fellowship SCHO 734/2-1; EU FP6; and Integrated Project EVI-GENORET (LSHG-CT ). The authors indicate no financial conflict of interest. Involved in design of study (F.G.H., A.B.W., H.P.N.S., J.D., S.S.V.); conduct of study (F.G.H., A.B.W., M.F., H.P.N.S., J.D., S.S.V.); collection of the data (F.G.H., A.B.W., M.F., H.P.N.S., S.S.V.); management of the data (F.G.H., A.B.W., M.F., H.P.N.S., J.D., S.S.V.); analysis and interpretation of the data (F.G.H., A.B.W., M.F., H.P.N.S., J.D., S.S.V.); preparation of the manuscript (F.G.H., A.B.W., S.S.V.); and the review and approval of the manuscript (F.G.H., A.B.W., M.F., H.P.N.S., J.D., S.S.V.). The authors acknowledge the centers and members participating in the Fundus Autofluorescence in Age-Related Macular Degeneration (FAM) Study. They are listed in the Appendix (available at AJO.com). REFERENCES 1. van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT. Epidemiology of age-related maculopathy: a review. Eur J Epidemiol 2003;18: Klein R, Klein BE, Tomany SC, Meuer SM, Huang GH. Ten-year incidence and progression of age-related maculopathy: the Beaver Dam Eye Study. 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11 APPENDIX CENTERS AND MEMBERS PARTICIPATING IN THE FUNDUS Autofluorescence in Age-Related Macular Degeneration (FAM) Study: Department of Ophthalmology, University of Bonn, Germany: Investigators: Almut Bindewald-Wittich, MD, Monika Fleckenstein, MD, Felix Roth, MD, Steffen Schmitz-Valckenberg, MD, Hendrik P. N. Scholl, MD, Johannes N. Witten, MS, Arno Göbel, MS, Frank G. Holz, MD Study nurses: Martina Hofmann, Gabriele Wessling. Department of Ophthalmology, University of Heidelberg, Germany: Investigators: Helena Sieber, MS, Jork J. Jorzik, MD, Daniel W. Miller( ), MD, Ute Wiehler, MD, Friderike Mackensen, MD, Stefan Dithmar, MD, Matthias Becker, MD Study nurse: Maria Herrmann. Institute for Medical Statistics and Biometry, University of Heidelberg, Germany: Andreas Deckert, Jens Dreyhaupt, PhD, Maria Pritsch, PhD, Anja Schuldt Department of Ophthalmology, University of Aachen, Germany Investigator: Anja Niewels, MD, Cordula Jung, MD, Andreas Weinberger, MD, Peter Walter, MD Department of Ophthalmology, University of Leipzig, Germany Investigators: Wilma Einbock, MD, Andreas Moessner, MD, Christian Foja, MD, Ute Schnurrbusch-Wolf, MD, Sebastian Wolf, MD, Peter Wiedemann, MD Department of Ophthalmology, University of Wuerzburg, Germany Investigators: Claudia Keilhauer, MD St. Franziskus Hospital Münster, Germany Investigators: Astrid Meister, MD, Georg Spital, MD, Daniel Pauleikhoff, MD Institute for Biometry and Epidemiology, Ludwig- Maximilians-University, Munich, Germany Investigators: Christine Adrion, Peter Kampe, Ulrich Mansmann, PhD VOL. 143, NO. 3 GEOGRAPHIC ATROPHY AND FUNDUS AUTOFLUORESCENCE 472.e1

12 Biosketch Frank G. Holz, MD, is a Professor and Chairman, Department of Ophthalmology, University of Bonn, Germany. He has a particular interest in the pathogenesis and therapy of age-related macular degeneration. A main focus in retinal imaging is fundus autofluorescence imaging using scanning laser ophthalmoscopy. Dr Holz is a founding member of the German priority research program Age-related Macular Degeneration, Editor-in-Chief of the organ of the German Ophthalmological Society Der Ophthalmologe, and president-elect of that society. 472.e2 AMERICAN JOURNAL OF OPHTHALMOLOGY MARCH 2007