Hyperreflective foci (HFs) have been frequently observed

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1 Multidisciplinary Ophthalmic Imaging Behavior of SD-OCT Detected Hyperreflective Foci in the Retina of Anti-VEGF Treated Patients with Diabetic Macular Edema Carsten Framme, Paul Schweizer, Manfred Imesch, Sebastian Wolf, and Ute Wolf-Schnurrbusch PURPOSE. Hyperreflective foci (HFs) are observable within the neurosensory retina in diabetic macular edema (DME) using spectral domain optical coherence tomography (SD-OCT). HFs have also been seen in wet age-related macular degeneration (AMD), although the origin is still unknown; however, they reduced significantly during anti-vegf (vascular endothelial growth factor) therapy, and their baseline amount seemed to correlate with treatment success. In this study the behavior of HFs was evaluated during anti-vegf therapy for DME. METHODS. Fifty-one patients (mean age: 67 years) underwent SD-OCT before and one month after one anti-vegf injection (ranibizumab: n ¼ 30; bevacizumab: n ¼ 21). The HFs were semiquantitatively counted, assigned to three groups (group A: HFs n ¼ 1 10; group B: n ¼ 11 20; group C: n > 20), and correlated to the course of visual acuity and foveal thickness (paired t-test). Additionally the baseline HbA1c was categorized and correlated to baseline HFs (Spearman Rho). RESULTS. In all eyes, HFs of various amounts were detected in the foveal and parafoveal area. The mean number of HFs reduced significantly from to in all patients (P ¼ 0.000), whereas foveal thickness reduced from to lm (P ¼ 0.000) and visual acuity increased from 62.0 to 66.0 ETDRS letters (P ¼ 0.003). Regarding the three HF groups, a reduction of the level stages was observed in 43.1% (stable: 54.9%; more: 2.0%). This reflects a HF distribution change from 31.4% to 62.7% (group A), from 45.1% to 31.4% (group B), and from 23.5% to 5.9% (group C). The HbA1c correlated significantly to the overall HF amount at baseline (0.880; P ¼ 0.000); however, no distinct overall correlation was found between the HF reduction and the course of visual acuity or retinal thickness. Only in cases of complete edema resolution (25%) did HFs reduce significantly (P ¼ 0.008). CONCLUSIONS. As in wet AMD, HFs are frequently found in DME and behave similarly under anti-vegf therapy. Thus, a HF reduction was observable mainly in cases of complete edema resolution; however, no distinct correlation with visual acuity was noticed, presumably mainly due to the enhanced inhomogeneity in the disease progress of DME. Interestingly, the baseline HF amount seems to correlate positively with From the University Eye Hospital Bern, Inselspital, Bern, Switzerland. Submitted for publication March 30, 2012; revised June 25, July 5, and July 10, 2012; accepted July 18, Disclosure: C. Framme, Novartis(R);P. Schweizer, None;M. Imesch, None; S. Wolf, Novartis (R); U. Wolf-Schnurrbusch, Novartis (R) Corresponding author: Carsten Framme, University Eye Hospital Bern, Freiburgstrasse 10, CH-3010, Bern, Switzerland; carsten.framme@insel.ch. HbA1c values indicating the severity of disease. (Invest Ophthalmol Vis Sci. 2012;53: ) DOI: / iovs Hyperreflective foci (HFs) have been frequently observed within the central neurosensory retinal structures in patients with neovascular age-related macular degeneration (AMD) with use of enhanced spectral-domain optical coherence tomography technique (SD-OCT). 1 SD-OCT provides an image of the retina and retinal pigment epithelium (RPE) based on the reflective properties of the various cellular layers. The varying intensities seen on the OCT scan correlate well with the different layers of the retina and the RPE. 2 The origin of the HFs in AMD has remained unclear and was initially suggested to be leukocytes or RPE cells representing accompanying retinal inflammation. 1 Other authors attributed hyperreflective intraretinal foci in diabetic maculopathy (DME), 3 5 where they occur more frequently than they do in AMD, to subtreshold hard exudates. In our previous publication on AMD, we used the term small dense particles to refer to these foci. 1 In this publication on DME, we adopt the term hyperreflective foci used by the other authors, because dense implies a physical property that may or may not be assessable by OCT and small is an imprecise descriptor of size. Interestingly, in AMD the HF amount significantly reduced after intravitreal ranibizumab (Lucentis; Novartis, Basel, Switzerland) upload therapy with three injections, and this reduction was positively correlated to the best corrected visual acuity (BCVA). 1 Moreover, an initial larger number of HFs revealed better outcomes of ranibizumab therapy regarding edema resolution and BCVA. 1 Thus, the amount of baseline HFs was suggested to be a predictive factor for the therapy outcome. Ranibizumab is a recombinant monoclonal antibody fragment neutralizing all active forms of vascular endothelial growth factor (VEGF) A, and had been shown in large clinical trials to improve the course of neovascular AMD significantly. 6 8 Since October 2011, intravitreal ranibizumab is also approved as a therapy for DME in Switzerland. Large prospective clinical multicenter studies as RESOLVE and RESTORE indicated substantial benefit regarding improved BCVA in DME. 9,10 For both entities, SD-OCT is usually performed to monitor the postoperative status of the macula; however, due to different posology for AMD and DME treatment in Switzerland, OCT guidance seems to be more important in AMD than in DME to prevent recurrent vision loss Before approval of ranibizumab, bevacizumab (Avastin; Roche, Basel, Switzerland) might be used in an off-label approach in DME patients suffering from foveal edema. In contrast to ranibizumab, bevacizumab represents a full antibody including the Fc fragment. From a clinical standpoint, the efficacy of the two anti-vegf drugs is regarded as similar. Investigative Ophthalmology & Visual Science, August 2012, Vol. 53, No Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.

2 IOVS, August 2012, Vol. 53, No. 9 Anti-VEGF Effects on Retinal Hyperreflective Foci in DME 5815 It was the aim of this study to evaluate the behavior of the SD-OCT detected HFs in DME patients undergoing anti-vegf therapy (intravitreal therapy, IVT) and to correlate them to retinal edema and the BCVA. Moreover, we were also interested to see whether there might be a correlation between the baseline amount of HFs and the level of diabetic pathology represented by HbA1c. MATERIALS AND METHODS This clinical study was retrospective and was approved by the cantonal ethical committee of Bern (KEK 044/07; Insel No. 1339). Images of 51 consecutive patients (mean age: 67 years) suffering from DME and visual loss due to foveal involvement, and receiving first-time intravitreal injection with anti-vegf using either ranibizumab (n ¼ 30) after approval or bevacizumab (n ¼ 21), were included. SD-OCT As in the AMD study setup, 1 OCT images were acquired with a combined SD-OCT and scanning laser ophthalmoscope (SLO) imaging system (Spectralis HRAþOCT; Heidelberg Engineering, Heidelberg, Germany). The system is able to acquire en face SLO images in angiographic, autofluorescence (AF), and reflectance imaging modes as well as cross-sectional SD-OCT images. An 870 nm superluminescent diode (SLD) is used for OCT imaging. In SD-OCT mode, the retina is scanned at 40,000 A-scans per second, presenting highly detailed images of the retinal structure. The OCT depth resolution (FWHM) is 7 lm. The images of the SLO and OCT modes can be overlaid and automatically spatially coregistered. Images can also be coregistered over time for different visits of a patient. Using this setup throughout the study made it possible to achieve similar OCT sections before and after therapy. Image Acquisition All consecutive patients, who were first-time diagnosed for anti-vegf treatment for DME and assigned to standard intravitreal anti-vegf therapy, 15 underwent pre- and posttreatment SD-OCT examinations in a regular clinical setting. The posttreatment examination took place usually four weeks after the first injection. OCTs were performed using volume scans with 49 single sections. Same sections were performed during the follow-up to ensure matching sections for evaluation. Images were displayed using the Heidelberg Engineering Eye Explorer software. For HF evaluation, similar sections from the two images (preand posttreatment examination) were evaluated in the foveal area of approximately 1 mm in length. As in the AMD study, 1 the number of HFs which are hyperreflective foci of round or oval shape and of different sizes within the parafoveal area was subjectively determined in all patients by grading them into three stages (A ¼ few, representing 2 10 HFs; B ¼ moderate, representing HFs; C ¼ many, representing 21 or more HFs). Figure 1 illustrates all three categories with arrows indicating the specific HFs. As displayed in Figure 1a, less than 10 HFs were counted within the parafoveal area of this central section, which was thus graded to category A. Figures 1b and 1c show the number of HFs in category B (between 11 and 20 foci) and category C (more than 20 foci), respectively, according to the baseline used and postoperative HF grading. Grading was performed by two different graders (CF, PS). Staging into the three groups matched in 90 of 102 cases (88.2%) for baseline and postoperative OCT sections; for the remaining cases, a third evaluation step was performed by the two graders together. Additional measurements included the BCVA before and after treatment, the central retinal thickness (CRT) using standard protocols of the Heidelberg software, and the qualitative posttreatment judgment of retinal pathology regarding the status of the edema (1 ¼ stable, no change in edema; 2 ¼ improved, but edema still present; 3 ¼ dry FIGURE 1. Examples of baseline HF distribution in the central and paracentral retinal neurosensory layers as detected by SD-OCT. The HF amount was subjectively graded as category A (few HFs) (a), category B (moderate HFs) (b), and category C (many HFs) (c). Arrows indicate HF accumulation. retina). For baseline correlations also the HbA1c value was collected for all patients and graded into five categories (HbA1c <7% [1]; <8% [2]; <9% [3]; <10% [4]; >10% [5]). Statistical analysis for correlations was performed using Spearman s Rho and for paired samples the t-test (SPSS for Windows 17.0; SPSS Inc., Chicago, IL). Standard deviation is abbreviated SD. RESULTS In all eyes, HFs of various amounts were detected in the foveal and parafoveal area. The mean number of HFs reduced significantly from (SD þ8.09) to (SD þ8.46) in all patients (P ¼ 0.000), whereas foveal thickness reduced from lm (SD þ106.13) to lm (SD þ93.96) (P ¼ 0.000) and visual acuity increased from 62.0 (SD þ11.8) to 66.0 (SD þ12.0) ETDRS letters (P ¼ 0.003). Overall, a reduction of HFs was observed in 43.1% of all patients; a stable HF amount was seen in 54.9%, and in only 2% was an increase detected. Regarding the three groups of HF distribution, this reflects a proportion change from 31.4% to 62.7% (group A), from 45.1% to 31.4% (group B), and from 23.5% to 5.9% (group C). Thus, a clear decay of HFs could be noticed after IVT. The HbA1c correlated significantly to the overall HF amount at baseline (0.880; P ¼ 0.000); this means that a high HbA1c value was associated with a larger amount of HFs within the central neurosensory retinal layers. In the overall group, no distinct correlation was found between the HF reduction and the course of visual acuity or the decrease of retinal thickness. However, depending on the

3 5816 Framme et al. IOVS, August 2012, Vol. 53, No. 9 postoperative status of edema, reduction of HF amount and retinal thickness was observable. Four weeks after one injection, there was complete resolution of edema in 25.5% (grade 3), less edema in 31.4% (grade 2), and no reduction in 43.1% (grade 1) of all cases. Thus, if edema improved, retinal thickness obviously also reduced (correlation coefficient: 0.448; P ¼ 0.001), and moreover, the HF amount reduced significantly (correlation coefficient: 0.369; P ¼ 0.008). However, visual acuity did not improve significantly in dependence of edema resolution (correlation coefficient: 0.225; P ¼ 0.113). The figures display examples of the course of HF behavior and the morphologic retinal change in SD-OCT for four cases (Figs. 2 5). Herein, most of these examples showed a reduced expression of the HF amount after intravitreal anti-vegf therapy. In Figure 2, HFs reduced from baseline category C (many HFs) to category B (moderate number of HFs) while central foveal thickness (CFT) reduced by 198 lm and BCVA increased by 10 ETDRS letters. The cystic appearance of the fovea dramatically regressed (Figs. 2a, 2b). Significant retinal thickness reduction was also observed in the second and third cases, correlating in part with huge increases of BCVA, while HF reduction from category B to A accompanied the morphologic retinal improvement (Figs. 3, 4). In contrast to these nascent HFs, Figure 5 shows ophthalmoscopically visible hard exudates that are definitely larger in the OCT sections, which obviously accumulated and further expanded in this case four weeks after one anti-vegf treatment. In this case, HFs were categorized as stage C before and also after injection (Fig. 5). DISCUSSION FIGURE 3. Left eye of a 55-year-old female patient. SD-OCT revealed significant macular edema (CFT: 412 lm; BCVA: 37 ETDRS letters) and multiple HFs throughout the retinal layers judged as category B (a). At one month after anti-vegf treatment, retina was dry (CFT: 282 lm; BCVA: 75 ETDRS letters) and HF expression significantly reduced (category A) (b). Arrows indicate a broad area of HF reduction. HFs as a nascent form of hard exudates are frequently seen in SD-OCT sections of DME patients and seem to reduce under anti-vegf therapy. In cases of postoperative complete resolution of retinal edema, as seen in approximately one-quarter of our patients after one injection, HF reduction was significantly better than in cases of no or less resolution. Thus, the HF amount seems to demarcate the severity status of the disease. Fewer HFs might reflect better tissue integrity whereas the presence of many HFs reflects tissue disintegrity representing more severe DME conditions. Interestingly, in this context the HF amount correlated positively with HbA1c values also indicating the general severity of disease. FIGURE 2. Left eye of a 60-year-old male patient. SD-OCT revealed significant macular edema (CFT: 517 lm; BCVA: 50 ETDRS letters) and multiple HFs throughout the retinal layers but predominantly seen in the apical layers, more enhanced temporal to the foveal cystic formation and judged as category C (a). At one month after anti-vegf treatment, retinal edema was reduced but retina was not dry (CFT: 319 lm; BCVA: 60 ETDRS letters); HF amount was obviously reduced and was judged as category B (b). Circle indicates an area of significant HF reduction. FIGURE 4. Right eye of a 79-year-old male patient. SD-OCT revealed significant macular edema (CFT: 630 lm; BCVA: 50 ETDRS letters) and multiple HFs throughout the retinal layers, judged as category B (a). At one month after anti-vegf treatment, retina was dry (CFT: 283 lm; BCVA: 65 ETDRS letters) and HF amount significantly reduced (category A) (b). Arrows indicate area of HF reduction.

4 IOVS, August 2012, Vol. 53, No. 9 Anti-VEGF Effects on Retinal Hyperreflective Foci in DME 5817 FIGURE 5. Right eye of a 55-year-old male patient. CFT was 603 lm and BCVA was 65 ETDRS letters, and multiple enhanced HFs were judged as category C (a). At one month after anti-vegf treatment, retina was nearly unchanged but visual acuity increased (CFT: 565 lm; BCVA: 73 ETDRS letters). HF amount did not change significantly, but expression was enhanced (category C) (b). The fundus photograph revealed already significant hard exudates (c). As also observed in AMD, 1 there was an overall significant reduction in HFs after anti-vegf treatment for DME. However, HFs decreased more in eyes showing complete edema resolution or significant edema reduction in contrast to eyes without edema reduction. The morphological improvement did not significantly correlate with visual acuity improvement as it did in AMD. This reflects the typical inhomogeneity in the DME disease progress and therapeutic success and might be comparable to what has been seen in vitrectomy studies on DME treated with epiretinal membrane peeling. 16 In the Diabetic Retinopathy Clinical Research Network study, central foveal thickness reduced significantly six months after treatment ( 160 lm), but visual acuity stayed stable at This clearly suggests that in nonischemic DME, retinal thickness is not the only factor predicting visual acuity and that multifactorial intraretinal changes contribute to the maximal possible visual acuity. As discussed in the previous paper, the origin of HFs in AMD has been unclear. 1 It was presumed that these particles might reflect leukocytes, which are able to invade the extracellular spaces in inflammatory regions. An inflammatory component in neovascular AMD has been discussed during recent years. 17 On the other hand, these particles could also reflect RPE migrating cells because there is similar expression as for the RPE layer providing the most highly reflective surface, and usually appears as a hyperreflective (or colorcoded bright orange-red) layer on the OCT scan. 1 Discussions on this paper finally led to the suggestion that HFs might predominantly represent subtreshold hard exudates. Ophthalmoscopically visible hard exudates are typically uncommon in wet AMD; however, within a small series of patients we were able to correlate high-resolution fundus photographs in 16 treatment-naïve wet AMD patients with the SD-OCT images and observed definite subtreshold hard exudates in approximately 50% of the cases that were undetectable in regular funduscopy. These findings led us to the impression that HFs in fact reflect hard exudates even in AMD. The hypothesis HFs ¼ hard exudates for DME seems to be obvious and has already been underlined by earlier publications from various authors. Therefore, it might be concluded that HFs in the neurosensory layers of SD-OCT sections are always reflecting the same material independently of the underlying macular pathology. In the studies on DME, SD-OCT techniques were used to detect and describe distinct hyperreflective foci distributed throughout all retinal layers. 3 5 Bolz et al. first described such hyperreflective dots in 12 patients with DME using different OCT techniques. 3 These dots were interpreted as the morphologic sign of lipid extravasation obviously forming hard exudates, and they were not observable in classic examinations such as those using ophthalmoscopy or fundus photographs. 3 As also in our study, such HFs could be demonstrated in both forms of DME, the focal and the diffuse. The authors concluded that the SD-OCT derived visualization of these deposits could be the missing link in retinal imaging between the breakdown of the inner blood retina barrier secondary to diabetes mellitus detectable on fluorescein angiography and the osmotic swelling of retinal layers visible on biomicroscopy, fluorescein angiography, and OCT. 3 Thus, the presence of HFs in nonedematous retina might be the early sign of barrier breakdown. Moreover, the amount seems to correlate with the HbA1c value also representing the severity of the disease. Regarding such precursors of hard exudates, Ota et al. presented a retrospective chart review in 28 eyes of 19 patients that described hyperreflective dots accompanying serous retinal detachment in DME. 4 A clear correlation of these SD-OCT based dots to biomicroscopically observable hard exudates was possible in approximately 50%. 4 Since the correlation obviously cannot be 100%, in fact the presence of HFs seems to reflect subtreshold tissue pathology. A recent paper by Uji et al. revealed an association of hyperreflective foci predominantly in the outer retina with disrupted external limiting membrane (ELM) and the junction of the inner and outer segment line of photoreceptors (IS/OS). 5 This condition was correlated with decreased visual acuity in DME. 5 Also herein the paper by Uji et al. it is suggested that the localized presence of such HFs might be an indicator maybe also an early one of disease severity. Since damaged ELM and/ or IS/OS is not necessarily accompanied by macular edema in DME, this might be a good explanation for why visual acuity does not always increase after anti-vegf therapy if macular edema is successfully treated and even if HFs are decreasing. In our study, the amount and sizes of HFs, as well as the exact location within the neurosensory retinal layers, appeared to be highly variable. This is similar to what is seen in AMD. 1

5 5818 Framme et al. IOVS, August 2012, Vol. 53, No. 9 However, in contrast, the HF reduction was heterogeneous and in the overall group less than that found in the AMD study. 1 Since the upload therapy in the DME study was not as concise as in the wet AMD study, which used a standardized three-time loading phase, and since postoperative observation in the present study took place four weeks after only one injection, no exact comparison is possible. However, as with AMD, in most DME patients a positive treatment effect is already observable after the first injection. It can be speculated that visual acuity might have been better in the overall group if examination had been performed after three injections (and this can be seen as a drawback of the study), but even in those 25% of patients showing completely dry retina after one treatment, no visual improvement was measured. Since in DME, anti-vegf treats edema resolution but not predominantly vessel growth as in AMD, one might expect maximally possible treatment success even after one injection in dry retinal conditions; however, this was not observed. To summarize the results of the present study, HFs are frequently observable in DME, and they react to anti-vegf treatment. Earlier publications suggested that HFs represent small subtreshold hard exudates. The distribution is throughout all retinal layers, and they are observable in AMD as well as in DME. In the latter, HFs seem to reflect the breakdown of the blood retina barrier, leading to extravasation of fluid and finally to hard exudates and macular edema. The amount correlates with the HbA1c value and the integrity of the neurosensory central layer. After anti-vegf treatment, HF reduction is observable, representing enhanced tissue integrity. In contrast to what occurred with the AMD anti-vegf treatment, no better treatment success was observed if the initial number of HFs was larger. Thus it is questionable whether the baseline HF amount in DME might be a predictor of treatment success in this disease evidencing very heterogeneous courses with and without treatment. As in the AMD study, 1 the subjective judgment and grading of OCT-derived appearances of edema, and especially HFs, is a definite drawback of this study. Due to HF accumulation, nonregular retinal structures from macular edema, and possible heterogeneous image qualities in older patients, a very exact counting of HFs is not always possible. Therefore, evaluation was performed by two independent graders, and HF amount was staged into the three broader groups to achieve more readily understandable results. A specifically software-based demarcation and calculation of HFs might be an interesting tool for future examination of intraretinal pathologies in an SD-OCT derived setting. Then a better and more objective discrimination of HFs in different macular diseases would be possible and more precise information about the appearance and impact of HFs could be achieved. References 1. Framme C, Wolf S, Wolf-Schnurrbusch U. Small dense particles within the neurosensory retinal layers observable by spectral domain optical coherence tomography in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2010;51: Wolf S, Wolf-Schnurrbusch U. Spectral-domain optical coherence tomography use in macular diseases: a review. Ophthalmologica. 2010;224: Bolz M, Schmidt-Erfurth U, Deak G, Mylonas G, Kriechbaum K, Scholda C; Diabetic Retinopathy Research Group Vienna. Optical coherence tomographic hyperreflective foci: a morphologic sign of extravasation in diabetic macular edema. 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