Spectral Domain Optical Coherence Tomography Analysis Of Fibrotic Lesions In Neovascular Age-Related Macular Degeneration

Journal Pre-proof Spectral Domain Optical Coherence Tomography Analysis Of Fibrotic Lesions In Neovascular Age-Related Macular Degeneration Eric H. Souied, MD, PHD, Manar Addou-Regnard, MD, Avi Ohayon, MD, Oudy Semoun, MD, Giuseppe Querques, MD, Rocio Blanco-Garavito, MD, Roxane Bunod, MD, Camille Jung, MD, PHD, Anne Sikorav, MD, Alexandra Miere, MD PII:

S0002-9394(20)30069-6

DOI:

https://doi.org/10.1016/j.ajo.2020.02.016

Reference:

AJOPHT 11239

To appear in:

American Journal of Ophthalmology

Received Date: 28 December 2018 Revised Date:

10 February 2020

Accepted Date: 13 February 2020

Please cite this article as: Souied EH, Addou-Regnard M, Ohayon A, Semoun O, Querques G, BlancoGaravito R, Bunod R, Jung C, Sikorav A, Miere A, Spectral Domain Optical Coherence Tomography Analysis Of Fibrotic Lesions In Neovascular Age-Related Macular Degeneration, American Journal of Ophthalmology (2020), doi: https://doi.org/10.1016/j.ajo.2020.02.016. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

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ABSTRACT Purpose: To describe the spectral-domain optical coherence tomography (SD-OCT) features of fibrotic lesions associated with neovascular age-related macular degeneration (nAMD) and to outline the progression pathways from initial macular neovascular lesions (CNV) to fibrosis. Methods: Neovascular AMD patients were retrospectively included when macular subretinal fibrosis was present. Fibrosis was categorized using SD-OCT with respect to retinal pigment epithelium (RPE) in 836 SD-OCT slices from 44 eyes of 39 patients. Additionally, in 47 distinct eyes, 4181 SD-OCT slices were retrospectively reviewed in order to longitudinally assess progression from the initial lesion to the final fibrosis. Results: Cross-sectional analysis classified fibrosis on SD-OCT slices, as type A if located underneath the RPE, as type B if located above the RPE and as type C if the remaining RPE was undistinguishable. The longitudinal analysis series revealed 3 progression pathways from the original CNV: 1. Progression to type A, followed by RPE erosion and subretinal hyperreflective material (SHRM), then type B and type C fibroglial lesion (FGL, 17/47 eyes); 2. Progression to type B, then type C FGL (17/47 eyes); 3. Persistence of type A with development of a flat, fibroatrophic lesion (FAL, 13/47 eyes). SHRM, macular haemorrhage or RPE tear occurred in 14/47, 13/47 and 10/47 eyes. Conclusion: This SD-OCT analysis identified various patterns of macular fibrosis in eyes with neovascular AMD. Three pathways of progression to fibrosis were described including the wellestablished pathway of type 2 CNV progression to FGL and the progression of type 1 fibrovascular CNV to FGL or FAL.

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SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY ANALYSIS OF FIBROTIC LESIONS IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION ERIC H. SOUIED, MD, PHD1,2 MANAR ADDOU-REGNARD, MD,1 AVI OHAYON, MD,1 OUDY SEMOUN, MD,1 GIUSEPPE QUERQUES, MD,1 ROCIO BLANCO-GARAVITO, MD,1 ROXANE BUNOD, MD,1 CAMILLE JUNG, MD, PHD,2 ANNE SIKORAV, MD,1 ALEXANDRA MIERE, MD1,2

1. Department of Ophthalmology, Centre Hospitalier Intercommunal de Créteil, University Paris Est Créteil, Créteil, France; 2. Clinical Research Center, GRC Macula, and Biological Ressources Center, Centre Hospitalier Intercommunal de Créteil, Créteil, France. Reprint requests : Eric H Souied, Department of Ophthalmology, Centre Hospitalier Intercommunal de Créteil, Université de Paris Est Créteil, 40 Avenue de Verdun, 94000 Créteil, France. Tel: (33)145175222 ; Fax: (33)145175227 ; email: [email protected]

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ABSTRACT Purpose: To describe the spectral-domain optical coherence tomography (SD-OCT) features of fibrotic lesions associated with neovascular age-related macular degeneration (nAMD) and to outline the progression pathways from initial macular neovascular lesions (CNV) to fibrosis. Methods: Neovascular AMD patients were retrospectively included when macular subretinal fibrosis was present. Fibrosis was categorized using SD-OCT with respect to retinal pigment epithelium (RPE) in 836 SD-OCT slices from 44 eyes of 39 patients. Additionally, in 47 distinct eyes, 4181 SD-OCT slices were retrospectively reviewed in order to longitudinally assess progression from the initial lesion to the final fibrosis. Results: Cross-sectional analysis classified fibrosis on SD-OCT slices, as type A if located underneath the RPE, as type B if located above the RPE and as type C if the remaining RPE was undistinguishable. The longitudinal analysis series revealed 3 progression pathways from the original CNV: 1. Progression to type A, followed by RPE erosion and subretinal hyperreflective material (SHRM), then type B and type C fibroglial lesion (FGL, 17/47 eyes); 2. Progression to type B, then type C FGL (17/47 eyes); 3. Persistence of type A with development of a flat, fibroatrophic lesion (FAL, 13/47 eyes). SHRM, macular haemorrhage or RPE tear occurred in 14/47, 13/47 and 10/47 eyes. Conclusion: This SD-OCT analysis identified various patterns of macular fibrosis in eyes with neovascular AMD. Three pathways of progression to fibrosis were described including the wellestablished pathway of type 2 CNV progression to FGL and the progression of type 1 fibrovascular CNV to FGL or FAL.

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INTRODUCTION Age-related macular degeneration (AMD) is a leading cause of visual loss in developed countries.1 With a rising prevalence worldwide, an estimated of 288 million people will be affected with AMD by 2040.2 Originating from the choroid or retina, aberrant angiogenesis, leading to the development of retinal pigment epithelial detachment (PED), subretinal fluid, intraretinal fluid or hemorrhages, plays a central role in neovascular AMD (nAMD).3,4 The advent of intravitreal vascular endothelial growth factor (VEGF) inhibitors has greatly improved the visual prognosis in nAMD. Several effective therapeutic strategies using anti-VEGF treatment have been established during the last decade.5 Nevertheless, common complications of nAMD, including submacular hemorrhage, retinal pigment epithelium (RPE) tears, geographic atrophy and the development of fibrotic scars, cannot be avoided despite anti-VEGF treatment, finally leading to a loss of central vision.6,7 As a result, despite the development of efficacious therapeutics, nAMD remains one of the most common vision-threatening diseases of the elderly population.8,9 Fibrosis represents the wound healing response to choroidal neovascularization in nAMD.10 Typically a later complication of nAMD, fibrotic scars respond minimally to anti-VEGF therapies. Risk factors for the development of fibrosis in treatment-naïve nAMD eyes treated with anti-VEGF therapy, according to the Comparison of Age-Related Macular Degeneration Treatment Trials (CATT), include classic choroidal neovascularization, blocked fluorescence with dye based fluorescein angiography, foveal retinal thickness > 212 microns, subfoveal tissue thickness > 275 microns, foveal subretinal fluid and subretinal hyperreflective material (SHRM).11,12 In fact, the presence of predominantly classic choroidal neovascularization (type 2 CNV) was associated with 4.5-fold risk for scarring compared with occult choroidal neovascularization (type 1 CNV).12 Similarly, Bloch and coworkers observed a 6fold risk of macular fibrosis in eyes with type 2 CNV compared with occult CNV (type 1).13 Progression from a neovascular membrane to a fibrovascular lesion and finally to a fibrotic scar leads to destruction of the photoreceptors or the RPE and choriocapillaris layers, ultimately leading to visual acuity loss.6,11 However, the transition from angiogenesis to fibrosis, referred to as the ‘angiofibrotic switch’, in neovascular AMD remains poorly described. Detailed imaging studies of fibrotic progression, especially of type 1 CNV, using spectral-domain optical coherence tomography (SDOCT), is lacking.10,14,15 Currently the assessment of fibrotic lesions is based on multimodal imaging (MMI), including color fundus photography (CF), spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography (FA) or indocyanine green angiography (ICGA).11,13,16 More recently, OCT Angiography (OCTA) has provided a non-invasive assessment of flow within fibrotic choroidal neovascularization (CNV), but no information was available concerning the surrounding fibrotic tissue, which generated signal voids on the choriocapillaris slab.17 Roberts and coworkers have shown, using polarizationsensitive OCT (PS-OCT), that subretinal fibrosis can be identified as an intrinsically bi-refringent structure, allowing a clear separation of neovascular tissue from the fibrous tissue.18 Even though tissue segmentation based on polarization (PS-OCT) and not signal intensity (SD-OCT) has clear advantages,18 in a real-life clinical setting SD-OCT remains the gold standard imaging technique. Several groups have proposed classification systems of nAMD using OCT including the fibrosis stage, in eyes treated by photodynamic therapy (PDT).19 Nonetheless, these classifications did not use high resolution SD-OCT, thus the morphological details of fibrotic lesions were not thoroughly described. Compared to previous OCT generations, SD-OCT delivers faster imaging, enabling depth resolved visualization of the macular layers and adjacent tissues close to microscopic resolution.20 With a resolution of 5 µm and layer-by-layer assessment of intraretinal microstructures, SD-OCT has elucidated the broad phenotypically heterogeneous nature of AMD and its complex morphology.21,22 In the last years, SD-OCT has become an indispensable tool for the diagnosis and follow up of nAMD patients.23,24 Despite the fast evolution of imaging techniques, the potential of SD-OCT to evaluate lesions within the fibrosis spectrum secondary to AMD remains insufficiently explored. On SD-OCT, fibrotic lesions can harbor various phenotypes, presenting a wide range of morphological patterns, including fibrovascular PED versus sub-retinal and/or sub-RPE fibrotic scars. In this cross-sectional study, we aim to describe the SD-OCT morphologic features of a broad spectrum of fibrotic lesions associated with nAMD. Moreover, a distinct retrospective longitudinal analysis was performed, in a well-defined

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large series of patients, in order to delineate retrospectively the progression pathways from the initial neovascular lesion to the fibrotic scar observed on SD-OCT. Our hypothesis is that SD-OCT may be useful to define different types of fibrosis and that different patterns of progression from CNV to fibrosis exist. This may be useful in future investigations to treat and prevent the development of fibrosis.

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METHODS STUDY DESIGN AND STUDY POPULATION Informed consent was obtained from all subjects from our clinic in agreement with the Declaration of Helsinki for research involving human subjects. This study was Institutional Review Board (IRB) approved and was carried out in compliance with local and national IRB guidelines. This study was a retrospective analysis of patients from the Department of Ophthalmology of the University Paris Est Creteil, who presented between March 2008 and March 2018 with fibrotic lesions secondary to advanced nAMD, as diagnosed on fundus biomicroscopy or color photography. All included patients had previously received long-standing anti-VEGF therapy, using a pro re nata (PRN) regimen. This study consisted of 2 separate analyses on 2 distinct series of eyes. The first analysis consisted of a description of the SD-OCT morphological features observed in the wide spectrum of fibrotic lesions associated with neovascular AMD, but without active exudative features at the time of analysis. The second analysis consisted of a retrospective study, in nAMD eyes, of the sequence of progression from the original CNV lesion to the fibrotic scar. In summary, 2 distinct series of eyes were retrospectively included in the present study: I. Cross-sectional series of nAMD eyes with lesions within the fibrotic spectrum II. Series of eyes with fibrotic scars secondary to nAMD, for which a longitudinal long-term follow up (over 5 years) was available.

INCLUSION AND EXCLUSION CRITERIA Eligibility criteria included a diagnosis of long-standing neovascular AMD, based on SD-OCT and FA and the International AMD classification25, and a history of no antiangiogenic treatment for at least 3 months. Absence of exudative signs on SD-OCT -including subretinal and intraretinal fluid - was also required for both the cross-sectional and longitudinal cohorts. On SD-OCT the lesion was required to fit within the borders of the 30 × 30-degree-field-of-view scan raster. For the longitudinal series, follow up of at least 5 years was mandatory. Diagnosis of fibrosis was based on fundus biomicroscopy and color pictures. Only patients that underwent both FA and SD-OCT at diagnosis of nAMD, as well as SD-OCT during follow up, were included in the cross-sectional and longitudinal analysis. On fundus examination, a fibrotic lesion was defined as a well demarcated, elevated mound of yellowish-white tissue.22 On FA, fibrosis secondary to late AMD displayed staining, with minimal or no leakage in the late phase of the angiographic sequence.11,25,26 On SD-OCT, the lesion was defined as fibrotic if more than 50% of its area was occupied by compact, sheet-like hyperreflective material, situated either above or underneath the RPE. In addition, in the longitudinal cohort only patients without scar at the initial examination were included. Exclusion criteria included cases of fibrosis secondary to causes other than AMD. Eyes with diseases other than AMD were excluded, such as cases of adult onset foveomacular vitelliform dystrophy, myopic CNV or pathologic myopia, and inherited macular dystrophies. Patients with media opacities preventing an adequate fundus view were also excluded.

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AMD eyes that progressed from nAMD to GA with no evidence of fibrosis, as well as eyes with subRPE scar/fibrosis/hyperreflective material that did not have a yellow-whitish aspect on fundus photography were excluded. Additionally, patients lost to follow up were excluded. For the crosssectional series, patients harboring a fibroatrophic flat pattern on SD-OCT (thickness of the fibrotic lesion < 100 microns) were excluded

PROCEDURES All patients in the study underwent a complete ophthalmic examination, consisting of best-corrected visual acuity (BCVA), fundus biomicroscopy, FA and SD-OCT. Color fundus photography (Canon CR-2 Retinal Camera, Canon, Tokyo, Japan) was also performed in a subset of eyes. MultiColor imaging (using three monochromatic laser sources: blue reflectance (wavelength 488 nm), green reflectance (wavelenght 515 nm) and infrared reflectance (wavelenght 820 nm) was also performed in a subset of patients.27 MultiColor, FA and SD-OCT were performed using Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany). Each SD-OCT image consisted of a 19 lines horizonal volume scan, 30 × 30-degree-field-of-view, centered on the fovea, with a minimum averaging of 9 frames. I. Cross-sectional Series For the cross-sectional analysis, lesions at the last follow up were assessed on SD-OCT images of study eyes by two readers (MA, AS). In case of disagreement, a further assessment was performed by a third expert reader (ES). The cross sectional analysis was performed on all SD-OCT slices corresponding to the fibrotic lesion of each study eye. An additional exclusion criterion was applied for this series: lesions with features of fibrosis on color images and features of atrophy on SD-OCT were classified as fibroatrophic lesions (FAL) and excluded. FAL lesions exhibit a maximum height < 100 microns and display homogenous reflectivity with SD-OCT. The presence/absence of the following features were evaluated by SD-OCT: presence of abnormalities within the fibrotic lesion (i.e. hyperreflective lamellae, hyporeflective lamellae, hyporeflective spaces), presence of perilesional atrophy, presence of outer retinal tubulations (ORT) or intraretinal degenerative spaces, and loss of adjacent retinal pigment epithelium (RPE) and ellipsoid zone (EZ) bands. The borders of the lesions of the fibrotic scar were classified as well defined or ill defined and the location of the fibrotic scar with respect to the RPE was also noted (Figure 1). SD-OCT measurements included: greatest fibrotic lesion diameter (GLD), maximal subfoveal lesion height (MSFH) and maximal lesion height (MLH). If MLH <100 microns, the lesion was considered as FAL and thus excluded form this cross-sectional study. These measurements were performed manually using the SDOCT caliper tool. Greatest lesion diameter corresponded to the maximal horizontal diameter of the lesion on an SD-OCT slice. Maximal subfoveal lesion height corresponded to the manual measurement of the fibrotic lesion in the subfoveal area, while maximal lesion height corresponded to the greatest lesion height regardless of its location (sub- or extra-foveal). Central macular thickness (CMT) measurements were calculated using the automated SD OCT tool. To determine the location of the lesion (i.e. sub, juxta or extrafoveal), an ETDRS (Early Treatment Diabetic Retinopathy Study) macular subfield grid was placed manually at the center of the macula. II. Longitudinal Series In addition to the cross sectional description of the SD-OCT features of fibrovascular lesions, the progression of the original lesion to the final fibrovascular phenotype was studied. In order to delineate retrospectively the progression pathway of fibrotic scars observed on SD-OCT, we included a distinct series of patients with fibrotic scars secondary to advanced nAMD with long follow up (at least 5 years). In this series, only patients without fibrosis at first examination in our imaging records were included. For the longitudinal series, the retrospective analysis of SD-OCT examinations was performed by two independent readers (ES, AM). In case of disagreement, a further assessment was performed by a third

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expert reader (GQ). A single SD-OCT slice, passing through the center of the lesion, was analyzed for each study eye, on each visit. The SD-OCT line passing through the center of the lesion within the fibrotic spectrum was analyzed retrospectively at each visit, in order to detect the progression pathway from the baseline type of choroidal neovascularization (CNV) to the fibrotic scar. Baseline type 1 CNV was defined on SD-OCT as a neovascular lesion localized underneath the RPE, associated with a PED.4,28 On FA, type 1 CNV was characterized by an ill-defined hyperfluorescent lesion, with heterogenous or stippled late staining and leakage. Type 2 CNV was defined on SD-OCT as a hyperreflective neovascular lesion situated above the RPE. The diagnostic criteria for well-defined CNV on FA included a well-demaracted lacy area of early hyperfluorescence with progressive leakage of dye in the overlying subsensory retinal space during the late phases of the angiogram. The margins of CNV were well-defined in the early phase.28 Type 3 NV was defined as an intraretinal hyperreflective lesion, situated above a (serous/drusenoid) pigment epithelial detachment. Angiographic features for diagnosis of type 3 NV included a discrete late focal area of hyperfluorescence (focal staining) or a typical “hot spot” (focal leakage).29 Aneurysmal type 1 neovascularization (polypoidal choroidal vasculopathy, PCV) was defined as a peaked PED (corresponding to the aneurysmal lesion) adjacent to a shallow irregular PED (corresponding to type 1 neovascularization).30 On ICGA, aneurysmal type 1 CNV (PCV) showed the typical branching vascular network and hyperfluorescent polypoidal dilations. The presence of the following characteristics was assessed from baseline nAMD diagnosis to the last visit: type of baseline CNV, presence/absence of large macular hemorrhage (>1 disc diameter), presence/absence of SHRM, presence/absence of an RPE tear, intergrity of the RPE band, presence/absence of RPE erosion presence/absence of accompanying perilesional atrophy. Focal RPE erosion was defined as a single limited erosion of RPE visible on a SD-OCT scan, whereas a dashed aspect of the RPE was termed RPE erosions. STATISTICAL ANALYSIS Statistical analysis was performed using STATA software (version 13.0, STATACORP LP, College Station, TX, USA) and included descriptive statistics for main clinical features. BCVA was converted from Snellen to ETDRS letters for statistical analysis. Chi Square and Fisher exact test were used to compare qualitative variables and Mann Whitney test was used to compare quantitative data. P=.05 was considered to be the threshold for significance.

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RESULTS I. CROSS-SECTIONAL ANALYSIS OF LESIONS WITHIN THE FIBROTIC SPECTRUM a. Demographics and clinical characteristics Forty-four eyes of 39 patients (fifteen male, twenty four female) with a mean age of 83 ± 6 years (± SD) were included in the cross-sectional analysis. Neovascular AMD was diagnosed on average 68 ± 31 months before inclusion (range: 5-116 months). Mean BCVA at last follow up was of 42± 27 ETDRS letters (Snellen equivqlent 20/160). All patients underwent anti-VEGF treatment (ranibizumab and/or aflibercept) with a mean of 20 (±13) intravitreal injections from diagnosis of nAMD to study inclusion. Study eyes did not undergo antiangiogenic treatment on average the last 10 months (±17) prior to inclusion. In our cross-sectional series, lesions within the fibrotic spectrum were secondary to Type 1 CNV in 13/44 eyes (30%), Type 2 CNV in 9/44 eyes (20 %), mixed Type 1 and 2 CNV in 16/44 eyes (36%), and Type 3 neovascularization in 3/44 eyes (7 %). Type of initial CNV could not be determined for 3/44 eyes. Fibrotic lesions were located in the subfoveal region in 41/44 eyes (93 %), while in only 3/44 eyes (7 %) the lesions were located in the extrafoveal region. In 5/39 patients from the cross sectional series, both eyes exhibited fibrotic lesions and were therefore included according to our criteria, accounting for 10/44 eyes in the series (23 %). Concerning the fellow eyes of the remaining 34/39 patients, it displayed fibrotic lesions in 10 eyes, which did not meet the inclusion criteria (such as history of no antiangiogenic treatment for at least 3 months, absence of exudative signs on SD-OCT or a lesion area within the borders of the 30 × 30-degree-field-of-view scan raster). In the remaining 24/39 patients, the fellow eye failed to show a fibrotic scar and exhibited type 1 CNV in 8 cases, type 3 NV in 1 case, GA in 9 cases, GA+CNV in 2 cases and intermediate AMD in 4 cases. b. SD-OCT features of lesions within the fibrotic spectrum

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We analyzed a total of 836 SD-OCT slices (44 eyes x 19 slices per eye) through all fibrotic lesions in our cross sectional cohort and the detailed features studied are displayed in Figure 1. Fibrosis was located above the RPE in 8/44 eyes (40/836 slices) and underneath the RPE in 43/44 eyes (430/836 slices). The RPE was not visible on SD-OCT in 6/44 eyes (30/836 slices) and was intact in 38 eyes (470/836 slices). On SD-OCT, the borders of the fibrotic lesion were morphologically well defined or fairly defined in 38/44 eyes (well defined in 154/836 slices, fairly defined in 316/836 or poorly defined in 6/44 eyes (30/836 slices). Abnormalities such as hyperreflective lamellae, hyporeflective lamellae or hyporeflective spaces coexisted in 20/44 eyes within lesions belonging to the fibrotic spectrum (120/836 slices; Figure 2). Moreover, the presence of a hyporeflective/slightly hyperreflective band within the width of the fibrotic scar was detected on 22/44 eyes (100/836 slices). Presence of peri-lesional atrophy, surrounding fibrosis, was detected on 24/44 eyes. Outer retinal tubulations (ORT) were present in 29/44 eyes. Complete loss of RPE surrounding the lesion was present in 6/44 eyes. Loss of the EZ was noted in 19/44 eyes. Mean CMT was 299 ± 72 µm in our cohort. Mean GLD averaged 3128 ± 1051 µm, mean MSFH was 177 ± 127 µm and mean MLH was 277 ± 119 µm. Given the above-mentioned features, multiple types of fibrotic lesions emerged, based on the location with respect to the RPE, RPE integrity (i.e. RPE band intact and identified or not) and the morphological aspect on SD-OCT. Type A lesions within the fibrotic spectrum were defined by the sub-RPE location on SD-OCT. The RPE in this group was mainly intact and preserved. Lesions included in this group were well defined on SD-OCT. Type A lesions were a common feature in this series, present in 43/44 eyes (98 %) and 430/836 slices. Among this group of lesions, we distinguished different patterns, based on the sub-RPE reflectivity of the vascularized PED: • Subtype A1 (20/44 eyes) was characterized by a heterogeneous hyperreflective lesion with the presence of intralesional abnormalities, as follows: hyperreflective lamellae, hyporeflective lamellae and hyporeflective spaces. This subtype was present on average in 6 slices/eye, thus on a mean of 120/836 slices. (Figure 2, upper panel) • Subtype A2 (22/44 eyes) was characterized by the presence of a hyporeflective/slightly reflective band within the entire width of the lesion. This subtype was present in average on 5 slices/eye, thus on a mean of 100/836 slices. (Figure 2, middle panel) • Subtype A3 (42/44 eyes) was characterized by the presence of compact, homogeneous hyperreflective lesion situated beneath the RPE, without intralesional irregularities. This subtype was present in average on 5 slices/eye, thus on a mean of 210/836 slices. (Figure 2, lower panel) Type B lesions within the fibrotic spectrum were defined by the subretinal localization on SD-OCT. The RPE in these cases was located within the lesion, separating it in two parts (subretinal and subRPE). Fibrotic lesions included in this type were consistently hyperreflective and well defined. Type B lesions were present in 8/44 eyes (19 %) and 40/836 slices. (Figure 3) Type C lesions within the fibrotic spectrum were defined by the subretinal localization on SD-OCT, with no discernable RPE and an ill-defined pattern. However, RPE was visible on the edges of the fibrotic lesion. This subtype illustrated a heterogeneous, protuberant lesion. Type C fibrotic lesions were present in 6/44 eyes (14 %) and 30/836 slices. (Figure 4) The characteristics of each type and subtype of lesions within the fibrotic spectrum are summarized in Table 1. Figure 5 summarizes the different types of lesions described above. These morphological features and the respective correlations with BCVA, number of intravitreal injections, presence of degenerative signs, and SD-OCT measurements are summarized in Table 2. II. LONGITUDINAL ANALYSIS OF FIBROTIC LESIONS a. Demographics and clinical characteristics Fifty-four eyes, distinct from the cross-sectional series, adhered to the inclusion criteria. Seven eyes were excluded due to previous laser photocoagulation or photodynamic therapy. Forty-seven eyes of forty-seven patients were thus included in the final longitudinal analysis (seventeen male, thirty female). Neovascular AMD was diagnosed on average 89± 35 months before the last visit in the

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longitudinal analysis cohort. Mean BCVA at last follow up was of 35 ± 20 ETDRS letters (Snellen equivqlent 20/200). All patients underwent anti-VEGF treatment (ranibizumab and/or aflibercept) with a mean of 37 (±16) intravitreal injections from diagnosis of nAMD to study inclusion. All patients received a PRN regimen. As for the fellow eye of the 47 patients included in the longitudinal series, 11 eyes exhibited intermediate AMD at the initial visit, 14 showed type 1 CNV, 1 displayed type 2 CNV, 2 exhibited type 3 NV, 6 showed GA, 5 displayed GA complicated by CNV, and 7 exhibited fibrosis (Table 3). b. SD-OCT features of fibrotic lesions during follow up We retrospectively analyzed a total of 4181 SD-OCT slices passing through the center of the fibrotic lesion at each visit for the forty-seven included eyes. The previously described types of lesions within the fibrotic spectrum were used to define the progression pathway of each patient. At baseline nAMD diagnosis, type 1 CNV was present in 26/47 eyes (55 %), type 2 CNV in 12/47 eyes (26 %), mixed type 1 and 2 CNV in 3/47 eyes (6 %), type 3 neovascularization in 5/47 eyes (11 %) and aneurysmal type 1 in 1/47 eye (2 %). Lesions within the fibrotic spectrum developed after 20 ± 19 months from the baseline diagnosis of CNV. Moreover, macular hemorrhage was observed during follow up in 13/47 eyes (28 %). The presence of macular hemorrhage was not statistically associated with a baseline type of CNV (p=1, Fisher exact test). RPE tear occurred during follow up in 10/47 eyes (21 %). RPE tear was not statistically associated with a particular type of baseline CNV (p=.313, Fisher exact test). During follow up, SHRM was noted in 14/47 eyes (Figures 6, 7). This subretinal hyperreflective material situated above the RPE was statistically associated with type 1 CNV at presentation (p=.007 Fisher exact test) and preceded the transition from Type A to Type B lesions within the fibrotic spectrum. RPE erosions (Figure 6, 7, 8, 9) were detected in 28/47 eyes during follow up and was not associated with a type of initial CNV in particular (p=.815, Fisher’s exact test). Perilesional macular atrophy was detected in 13/47 eyes on SDOCT (Figure 9). c. Progression pathways of fibrotic lesions During follow up, evidence of the angiofibrotic switch (defined previously as the transition from the angiogenic phase to the fibrotic phase)14 was noted after a mean of 20 ± 19 months follow up from the baseline diagnosis of CNV to one of the 3 fibrotic lesions: •

Type A lesions in 30/47 eyes. Among the 30 Type A lesions, 13 remained stationary throughout follow up and progressed to FAL (Figure 9). The remaining 17/30 eyes progressed from type A towards Type B and finally to Type C lesions (Figure 6, 7). As shown in Table 4, focal RPE erosion of the RPE associated with SHRM was observed as an early stage during the progression between type A and type B stages, whereas multiple RPE erosions were observed during the progression between type B and type C stages (fibroglial lesion, FGL).

•

Type B lesions in 10/47 eyes. In these 10 eyes, the original neovascular lesions directly progressed to a type B fibrotic scar without evidence of transition through stage A. All eyes (10/10) following this progression pathway evolved towards type C, with progressive RPE erosions and disappearance in all eyes. (Figure 8).

•

Type C lesions in 7/47 eyes. In these 7 eyes, the original neovascular lesion directly evolved to a type C fibrotic scar without evidence of transition through stages A or B. These eyes presented with a protuberant fibrotic lesion with no remaining visible RPE layer after the initial monthly loading dose (Figure 8).

Thus, 3 progression pathways to fibrotic scars were notable after evaluation of all the SD-OCT scans available from baseline nAMD diagnosis to final follow up, using the afore-mentioned scale (A, B, C) of fibrotic lesions: 1.

The first pathway, for which we coined the term ‘A B C’, was characterized by a progression from Type A to Type B and then Type C. This progression pathway appeared in 17/47 eyes from our longitudinal cohort and is illustrated in Figure 6 and Figure 7. It is characterized by the initial presence of a multilayered vascularized PED (Type A), secondary mostly to type 1 CNV (13/17),

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The appearance of SHRM above the RPE during follow up preceded in these cases the development of subretinal fibrosis (Type B). All “type A fibrotic lesions” included the A3 subtype defined above. A focal erosion of the RPE was observed as an early stage of progression, at the onset of SHRM (Figure 7). The final step in this progression pathway was loss of the RPE band as identified by SD-OCT, which marked the development of the type C fibrotic lesion. Besides the original type 1 CNV lesion, we observed 3/17 type 2 or mixed CNV lesions and 1/17 type 3 NV lesions following the same pattern. The latter 4 cases progressed to type A fibrosis and followed the A B C pathway. 2.

The second pathway, for which we coined the term ‘B C’, was characterized by a direct progression, under anti-VEGF therapy, from the baseline type of CNV to type B and/or C fibrotic lesions. A total of 17/47 eyes from our longitudinal series followed this progression pathway. This pathway is illustrated in Figure 8. It is noteworthy that 7/17 eyes transitioned to type C lesions without any evidence of a type B lesion during the follow up. Direct rapid progression to a type C fibrotic lesion was the result of a large subretinal hemorrhage (4/7) or RPE tear (2/7). We cannot exclude the possibility that a type B fibrotic lesion was transient and missed during this progression. The original CNV lesion following this pathway mainly consisted of baseline type 2 or mixed type 1/type 2 CNV (11/17). Besides type 2 CNV, we also observed 3/17 type 1 CNV, 2/17 type 3 CNV and 1/17 aneurysmal type 1 CNV follow this second fibrotic pathway. Among these 6 eyes, RPE tear occurred before the progression to type B fibrosis in 4/6 eyes (1 type 3 CNV and 2 type 1 CNV) and subretinal hemorrhage occurred in 4/6 eyes before progression to type B fibrosis (1 case of aneurysmal type 1 CNV, 2 cases of type 1 CNV and 1 case of type 3 CNV).

3.

The third pathway, described as “A FAL (fibroatrophic lesion)” was associated with perilesional atrophy at the initial visit. The 13/47 eyes following this pathway showed persistent Type A fibrotic lesions, with the fibrosis confined under the RPE, evidenced by a shadow effect on SDOCT. This pathway is illustrated in Figure 9. No large macular hemorrhage was observed during the follow up of these eyes in this pattern of progression.

The presence of SHRM during follow up was significantly associated with CNV progression from Type A to Type B and then to Type C lesions (p<.001, Fisher’s exact test). SHRM appeared in all cases in the interval between Type A and Type B lesions. Macular hemorrhage was not associated with any of the 3 progression pathways, despite being observed in 13/47 cases (4 A→B→C ; 4 B→C ; 5 C direct) (p=0.058 for the occurrence of macular hemorrhage between stage A B and B C). However, macular hemorrhage was absent during follow up for eyes with FAL (type A). RPE erosions were significantly associated with Type C fibrotic lesions (p<.001, Fisher’s exact test). RPE tear occurred in 8/10 cases in the interval between Type A and B lesions, while in 2/10 cases RPE tear occurred in the interval between Type B and Type C lesions. The presence of macular atrophy was significantly associated with Type A lesions (p<.001).

43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59

DISCUSSION In this retrospective study, we described the cross-sectional SD-OCT morphological features of lesions within the fibrotic spectrum secondary to nAMD. In a second analysis, we performed a retrospective long-term longitudinal cohort study to elucidate the progression from the original neovascular lesion to the fibrotic scar. We analyzed a total of 5017 SD-OCT slices in the cross-sectional and longitudinal analyses (836 and 4181, respectively). The cross-sectional analysis distinguished 3 types of lesions within the fibrotic spectrum. Type A corresponded to well-defined sub-RPE lesions, with or without intralesional abnormalities (43/44, 98% of eyes). Type B corresponded to well-defined hyperreflective lesions situated in the subretinal and subRPE space (8/44, 18 % of eyes) with an intact RPE band. Type C consisted of prominent, elevated fibrotic lesions, with a complex pattern and RPE atrophy and loss of an identifiable RPE band (6/44, 14 % of eyes). Interestingly, these phenotypes often coexisted in the same eye. Roger and coworkers proposed, in 2002, a five-stage classification, based on a highspeed fiberoptic OCT scanner device coupled to a standard slit-lamp biomicroscope, that monitored response in eyes

9

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with nAMD treated by PDT.19 The presence of fibrosis was noted in stages IIIa and IIIb based on the severity of subretinal fibrosis and fluid. In stage V with subretinal fluid resolution, a fibrotic lesion merged with the retinal pigment epithelium (RPE) and was associated with overall thinning of the retina.19 With the advent of anti-VEGF therapy, PDT with verteporfin is no longer used for nAMD, being reserved for cases cases of aneurysmal type 1 neovascularization in combination with antiVEGF. 31In nAMD, as shown in the outcomes of the ANCHOR study32 and later confirmed by follow up studies,33 while in the ranibizumab groups (0.3-mg and 0.5-mg) mean visual acuity increased by 8.5 letters and 11.3 letters respectively, in the verteporfin group there was a decrease of 9.5 letters in mean BCVA (p<.001).32 Therefore, a switch in treatment paradigms has occurred, and nearly all nAMD patients receive currently antiangiogenic therapy. In the current literature, the usual annotation of lesions within the fibrotic spectrum varies without consensus, but the most widely used terminology is that of ‘subretinal fibrosis.11,19,34 “Subretinal fibrosis” typically corresponds to type B or type C lesions observed in our cross-sectional series, based on SD-OCT. However, in our study the incidence of these types were rather low: 8/44 eyes for type B and 6/44 eyes for type C. Thus, the phenotypic variability of lesions within the fibrotic spectrum, as relates to their location or/and the integrity of the RPE is much greater than suggested in previous literature. Furthermore, while a careful analysis of multiple sections was performed for each patient, no correlation between clinical factors, such as BCVA and number of injections, versus type of fibrosis was observed. Additionally, different patterns of fibrosis co-existed in the same eye. One explanation could be the fact that foveal involvement was not taken into account in the A/B/C classification. In addition, even though the greatest linear diameter of the lesions was most commonly associated with the lesion subtype A1 (p=.05), there were no significant correlations between fibrosis type and other quantitative criteria, such as number of intravitreal injections or automatic and manual measurements on SD-OCT. In the cross sectional analysis, we observed 3 subtypes of lesions in the type A fibrotic lesion group, which we labeled as A1, A2 and A3. These subtypes included intralesional abnormalities (A1: 20/44, 46 % of eyes), a hyporeflective band across the lesion (A2: 22/44, 50% of eyes) or a grey hypereflective band under the RPE (A3: 42/44, 96 % of eyes). We hypothesize that the A1 lesion subtype corresponds to a multilayered pigment epithelium detachment and may correspond to a preliminary stage in the development of the classically compact, hyperreflective lesions described in the literature.4,27,35-38 Subtype classification of type A fibrosis establishes a continuum between fibrovascular pigment epithelial detachment and the fibrotic lesion. This is confirmed by our longitudinal analysis, where type A3 indeed precedes B and C forms of fibrosis. An important question was thus to delineate the progression pathways from the initial neovascular lesion to the fibrotic scar observed on SD-OCT, by means of a longitudinal analysis on a well-defined cohort of patients with long-standing nAMD and close SD-OCT follow-up. The results of our longitudinal retrospective analysis suggested that CNV and the ultimate development of fibrosis might be seen as a continuum, progressing from the original CNV through the 2 fibrotic lesions types (subRPE Type A, subretinal/subRPE Type B) and finally reaching, after years of follow up under antiVEGF treatment, Type C fibrotic lesions. In the A B C pathway, the presence of SHRM during follow up was significantly associated with CNV progression from Type A to Type B and then to Type C lesions (p<.001, Fisher’s exact test). SHRM appeared in all cases in the interval between Type A and Type B lesions. Our hypothesis would be that a focal erosion of RPE overlying a Type A fibrotic lesion may subsequently cause the fibrotic component to emerge from the sub-RPE to the subretinal space, manifested as a SHRM lesion, with subsequent conversion to Type B (subretinal) fibrosis, followed by a progressive erosion of the RPE and enlargement of the subretinal fibrotic component in this A B C pathway. In the B C pathway, 11/17 eyes had a type 2 CNV or mixed type 1/type 2 CNV as the baseline type of neovascularization. Concerning the A FAL pathway, baseline lesions in this case were mainly secondary to type 1 CNV (10/13 eyes). Our results are consistent with the CATT study analysis at 2 years follow up, stating that baseline CNV type on FA predicted scar formation, with type 1 CNV being less likely to evolve to atrophic macular scar versus type 2 CNV. 11,12The two final morphologies of the fibrotic process in either pathway were either FAL or FGL. It is notable that hemorrhage, SHRM, or an RPE tear consistently preceded the fibrotic switch from type 1 or type 3 NV to FGL (Type B and/or type C lesions). Figure 10 illustrates the main progression pathways from the original CNV to fibroatrophic or fibroglial scarring.

10

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These pathways of progression within our longitudinal cohort support the cross-sectional analysis and validate a continuum or graduate progression of stages in the final development of fibrosis. In our longitudinal cohort, the angiofibrotic switch occurred 20 months from baseline examination. This is also consistent with current literature, stating that in up to 50% of eyes scars develop after 2 years of anti-VEGF treatment.11 In another recent study, subretinal fibrosis appeared after 60 months of antiVEGF therapy in 68.5% of eyes and was a predictor of worse visual outcome.39 Other studies have focused on the functional outcomes of nAMD eyes undergoing anti-VEGF treatment, but were limited by small sample size and short follow up.40-43 Daniel and coworkers described the baseline risk factors of scars (both fibrotic and nonfibrotic) following anti-VEGF treatment with ranibizumab or bevacizumab from the CATT study.11,12 In their study, fibrotic scars developed at 1 year of anti-VEGF treatment in 32% of eyes and the incidence of fibrosis was 56 % at 5-years follow up. Multivariate analysis also identified baseline characteristics that predicted scar formation, such as classic CNV, subretinal fluid and subretinal hyperreflective material.11 In the CATT study, type 2 CNV was associated with 4.5-fold risk of fibrotic scar formation compared with type 1 CNV. Stevens and coworkers similarly reported that the presence of classic CNV increases the risk of development of fibrosis in late AMD.44 In our cross-sectional study, 25/44 of eyes (57 %) exhibited type 2 CNV or mixed type 1 and type 2 CNV at initial onset of nAMD. In our longitudinal analysis, 15/47 of eyes (32 %) presented as a type 2 CNV or mixed type 1 and type 2 CNV. It is clear that type 2 CNV is an important risk factor for fibrotic scar development but it is noteworthy that type 1 CNV was the original lesion in 10/13 eyes (77%) of the FAL and 16/34 eyes (47%) of the FGL in our study. Histologically, it is expected that type 2 CNV, may evolve into subretinal fibrotic lesions (Type B). However, Dolz-Marco and coworkers and our team have shown that type 2 CNV lesions treated with intravitreal antiangiogenic therapy may progress to a type 1 CNV fibrovascular pattern.45,46 Similarly, in our longitudinal series, we observed two type 2 CNV cases and one mixed CNV case evolving into subtype A3, and then following the “A B C” pathway. In the CATT study, a large submacular hemorrhage was associated with a more than 2-fold risk of fibrotic scarring.12 This has been confirmed by other studies.47,48 However, Hwang and colleagues49 showed that subfoveal fibrosis may be identified in eyes without significant subfoveal hemorrhage after anti-VEGF treatment. This is consistent with our findings, in which macular hemorrhage was noted in 13/47 (28 %) of eyes during follow up. However, macular hemorrhage was not associated with a specific subtype of CNV. It is noteworthy that no hemorrhage was observed in the progression pattern type 1 CNV to FAL. On the other hand, the presence of hemorrhage was found in 4/6 cases of non-type 2 CNV that underwent a B C pathway. In our longitudinal analysis, an RPE tear occurred in 10/47 (21 %) of eyes during the follow up from the CNV lesion to fibrosis. Such an association was not previously described in previous studies on the progression of fibrotic lesions.11–13 Sarraf et. al noted that severe fibrotic scarring may be reduced with continued antiVEGF therapy after RPE tear development.50 One of the risk factors for scarring identified by the CATT study was the presence of SHRM.41,51 In our longitudinal cohort, SHRM appeared in 14/47 (almost 30%) of eyes and was associated in a statistically significant manner with eyes presenting with type 1 CNV at baseline (p=.007). Moreover, in all cases (14/47), SHRM preceded the progression to Type B subretinal fibrotic lesions (p<.001). This is significant in the context of multiple studies showing that SHRM persistence despite anti-VEGF therapy was a risk factor for fibrosis development and had a negative impact on BCVA.51-53 Willoughby and colleagues also found that SHRM was present at week 52 in 69.3% of eyes with scars in the CATT cohort.51 In addition, Casalino et. al showed that well-defined SHRM borders on SD-OCT appear to represent fibrotic tissue or mature neovascular lesions.52,53 Pokroy and coworkers and Casalino and colleagues suggested that SHRM persistence is consistent with subretinal fibrosis development.52,53 SHRM may be attributed to a heterogeneous group of lesions, including grey exudative fluid, hemorrhage, vitelliform material, or type 2 CNV.34,54 This hyperreflective subretinal material, lying above the RPE28,52 may consist of a myriad of elements, from neovascular tissue to fibrin, blood and lipid.52,55 OCTA can distinguish the vascular and avascular components of SHRM, as demonstrated by Dansingani and colleagues56 and Kawashima and colleagues.57 Subretinal fibrosis may be the

11

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consequence of either the natural healing process or different therapies. We propose that an A3 lesion may progress due to RPE erosion, leading to eruption of the fibrotic component into the subretinal space, forming a SHRM aspect, which evolves into type B fibrotic lesion. SHRM development may also take place after anti-VEGF treatment. Continuous treatment over long periods of time may resolve or reduce fluid but can generate an increase in the fibrotic component of SHRM, rendering anti-VEGF therapy less efficacious51,52. Anti-VEGF molecules induce the blockage of pathologic vessel growth through inhibition of the migration and proliferation of endothelial cells. This skews the angiofibrotic switch towards connective tissues mediators leading to connective tissue deposition, antiVEGF resistance and consequent scar formation. This resistance to VEGF neutralizing molecules has long been accounted for in the oncologic literature over the past decade.58 Pericytes, that supply VEGF and other factors to the proliferating endothelial cells of CNV, are involved in this process. Pericyte recruitment, survival and maturation are modulated by the platelet-derived growth factor (PDGF). Pericytes may contribute to the development of fibrosis either directly by producing collagen or indirectly by differentiating into myofibroblasts, α-smooth muscle actin expressing and collagen producing cells, responsible for the expansion of fibrotic tissue.59 Other studies have emphasized the role of (prolonged) anti-VEGF treatment in promoting the transition from angiogenesis to fibrosis.14,15 Connective tissue growth factor (CTGF) is a profibrotic factor whose vitreous levels correlate strongly with the degree of fibrosis in multiple vitreo-retinal disorders.10,14 Moreover, the ratio of CTGF and VEGF seems to be the strongest predictor of fibrosis in eyes with proliferative diabetic retinopathy (PDR).14 Several animal models have been applied to study the molecular complexities of subretinal fibrosis in mice and have confirmed the role of macrophage rich peritoneal exudate cells (PEC) in the myofibrotic changes of RPE cells in vitro.60,61 Platelet-activating factor (PAF) and its receptor’s inhibition (PAF-R) seem to suppress induced subretinal fibrosis in another animal model.62 IL-6 and its receptor were also proven to be involved in the development of subretinal fibrosis.63 All in all, the pathogenic sequence of subretinal fibrosis, even if partially understood, seems to consist of leucocyte induced exudation by a highly permeable neovascular lesion, which initiates the inflammation process, stimulating glial proliferation and ultimately generating subretinal fibrosis.60,61,63,64 Prevention of fibrosis in nAMD patients is still a challenging goal. Anti-PDGF therapies to target pericytes have met with failure despite initial excitement65 and an efficacious treatment to prevent the formation of fibrosis is still lacking. Nevertheless, many research groups work on the identification of angiogenic factors involved in wound healing and fibrosis that could lead to emerging therapies. To date, no interventional study has demonstrated the efficacy of any anti-PDGF drugs. The criteria selection of eyes for such studies is of major importance in order to demonstrate efficacy of these drugs. It is possible that development of fibrosis may be an acceptable process without adverse consequences when limited to the subRPE space, as with a multilayered PED (i.e. Type A lesions). However when fibrosis invades the subretinal space, photoreceptor loss and visual decline may ensue. From our analysis, the occurrence of RPE erosion associated with SHRM may be considered an early stage of the progression to a fibroglial scar. Our hypothesis is that anti fibrotic drugs should be considered particularly at this stage, only when fibrosis begins to invade the subretinal space, based on SD-OCT analysis. Limitations of our study include the relatively small sample size and its heterogeneity, with no differentiation of patients according to the anti-VEGF treatment agent or duration of treatment. Moreover, we excluded large fibrotic scars that extended beyond the field of view. A number of patients were lost of follow up. Boulanger et. al demonstrated the dropout rate of nAMD patients after 5 years of follow up was 57% and that age and BCVA at baseline and distance from home to hospital were independently associated with long-term drop out (115/201 patients).66 Stringent inclusion and exclusion criteria for our longitudinal cohort, including a follow up of >= 5 years and strict standards with regards to image quality, size and localization of the fibrotic lesion within the 30x30 degrees field of view, can explain our population size. Our cross sectional series included only 44 eyes, but a total of 836 (19x44) SD-OCT slices was analyzed. Both eyes were included in 5 patients of the cross sectional analysis, which may over represent a particular individual’s response to nAMD as pertains to fibrosis. Our longitudinal series included only 47 eyes, but our long term follow up analysis included a review of a total of 4181SDOCT slices, which allowed a very detailed morphologic analysis of the process from CNV to final fibrosis. The relatively small numbers of each subtype in the cross-sectional series and the imbalance

12

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of baseline lesion types in the longitudinal series (mostly type 1 CNV) is another limitation of our study. However, the predominance of type 1 CNV leading to fibrotic lesions may be considered an important outcome of this study. We demonstrated that macular fibrosis is not only an outcome of type 2 CNV, but that fibrosis also occurs frequently as a consequence of type 1 CNV. Assessment of the presence or absence of RPE may be limited by SD-OCT technology, especially as fibrosis may have the same reflectivity as the RPE on SD-OCT. Given that our study did not use PSOCT, which may have been able to distinguish the RPE from the fibrotic tissue, we assumed that the lack of visualization of the RPE in Group C fibrotic lesions was due to an altered and/or absent RPE. Last but not least, given the reverse follow up methodology used for the longitudinal series, we cannot state how type 1 or type 2 CNV progress over time (reported in the CATT study by Daniel et. al11,12). Nonetheless, from our reverse follow up analysis, we can determine that FAL mainly originated from type 1 CNV, whereas subretinal FGS originated either from type 1, type 2 CNV or type 3 NV. The strengths of the present study were the long-term follow up of patients with fibrotic scars, allowing insights into the expanded spectrum of fibrotic lesions in nAMD. The progression from CNV to fibrosis is more multifaceted than the simple scheme involving type 2 CNV progressing to a FGL. In this study, besides the well-known progression from type 2 CNV to a FGL, we also demonstrated the steps of progression from type 1 fibrovascular CNV to FGL or FAL (fibroatrophic lesion). This study has shown that any erosion or breach of RPE may lead to an invasion of the fibrotic component under the neurosensory retina, progressing to a FGL lesion. Conversely, some type 1 lesions associated with fibrovascular PED did not invade the subretinal space, and these lesions alternatively progressed to flat FAL. We believe that a description of these different phenotypes and pathways using SD-OCT can provide a better understanding of the multifaceted pathogenic process of scarring, leading to fibrosis and contribute to a future international classification of fibrosis and to the development potential therapies to prevent fibrosis and vision loss and blindness. This SD-OCT analysis identified 3 main pathways to macular fibrosis leading to 2 types of advanced fibrotic lesions, either FAL -absence of proliferation under the subretinal space- or the FGL - fibroglial proliferation in the subretinal space- after RPE erosion. A focal RPE erosion associated with SHRM may represent an early sign of progression from fibrovascular PED to subretinal fibrosis. Our study showed that RPE erosion plus SHRM may constitute a prognostic biomarker for occurrence of fibrosis and, may indicate the need for more aggressive treatment and may represent a specific target for future anti fibrotic drugs. We hope that our description may contribute to a better understanding of the stages of the fibrotic process. Understanding of the angiofibrotic switch and detection of the early stages of fibrosis will be of major importance to potentially target a population at greatest risk of scarring and develop a therapeutic approach for the prevention of fibrotic scars. With new therapies promising a brighter future for the management of nAMD, SD-OCT analysis may help to determine the precise treatment-targets and predict the different therapeutic responses.

13

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46. Coscas F, Querques G, Forte R, Terrada C, Coscas G, Souied EH. Combined fluorescein angiography and spectral-domain optical coherence tomography imaging of classic choroidal neovascularization secondary to age-related macular degeneration before and after intravitreal ranibizumab injections. Retina (Philadelphia, Pa). 2012;32(6):1069-1076.

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48. Avery RL, Fekrat S, Hawkins BS, Bressler NM. Natural history of subfoveal subretinal hemorrhage in age-related macular degeneration. Retina (Philadelphia, Pa). 1996;16(3):183-189.

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50. Sarraf D, Joseph A, Rahimy E. Retinal pigment epithelial tears in the era of intravitreal pharmacotherapy: risk factors, pathogenesis, prognosis and treatment (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc. 2014 Jul;112:142-159.

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54. Ores R, Puche N, Querques G, et al. Gray hyper-reflective subretinal exudative lesions in exudative age-related macular degeneration. Am J Ophthalmol. 2014;158(2):354-361.

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57. Kawashima Y, Hata M, Oishi A, et al. Association of Vascular Versus Avascular Subretinal Hyperreflective Material With Aflibercept Response in Age-related Macular Degeneration. Am J Ophthalmol. 2017;181:61-70.

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66. Boulanger-Scemama E, Querques G, About F, et al. Ranibizumab for exudative age-related macular degeneration: A five year study of adherence to follow-up in a real-life setting. J Fr Ophtalmol. 2015 Sep;38(7):620-627.

4 5 6 7

18

SD-OCT morphological features

Location

Lesions within the fibrotic spectrum

Type A

Hyperreflective lesion

RPE totally/ partially preserved

Intralesional abnormalities (hyporeflective spaces, hyperreflective lamellae)

Hyporeflective band through the lesion

Shape

Subtype A1

Sub-RPE

+

+/-

+

-

Well defined

Subtype A2

Sub-RPE

+

+/-

-

+

Well defined

Subtype A3

Sub-RPE

+

+/-

-

-

Well defined

Type B

Subretinal

+

+/-

+/-

-

Well defined

Type C

Subretinal

+

-

+/-

-

Complex shape

Table 1. Cross sectional series. Description of lesions within the fibrotic spectrum according to SD-OCT morphological features.

Type A

Type B

Type C

Subtype A1

Subtype A2

Subtype A3

20/44

22/44

42/44

8/44

6/44

39.7 p=.30*

47.9 p=.14*

42.6 p=.70*

34.6 p=.32*

34.2 p=.46*

IVT mean, (number) (p)

23 p=.24*

17 p=.04*

21 p=.27*

15 p=.38*

15 p=.47*

Degenerative signs (eyes) (p)

14/20 p=.19**

13/22 p=1**

23 p=.6**

3 p=.31**

3 p=.69**

6/20, p=.79** 4/20, p=.5** 6/20, p=.63** 1/20, p=.87**

8/22, p=1** 2/22, p=.66** 8/22, p=1** 2/22, p=1**

16/22, p=.89** 6/22, p=.63** 14/22, p=.25** 3/22, p=.29**

1/8, p=.41** 1/8, p=.69** 4/8, p=.71** 2/8, p=.16**

1/6, p=.97** 1/6, p=.58** 2/6, p=.97** 0/6, p=.97**

307, p=.17* 4025, p=.05* 226, p=.23* 317, p=.1*

306.9, p=.16* 3566.5, p=.8* 227, p=.2* 286, p=.78*

298.2, p=.53* 3681, p=.08* 195.3, p=.25* 277.2, p=.57*

291.5, p=.87* 3695.3, p=1* 225.5, p=.14* 312, p=.08*

315.5, p=.63* 4039.8, p=.33* 271.2, p=.62* 383.5, p=.08*

Eyes (nb)

BCVA letters), Mean (p)

(ETDRS

CNV (number of eyes) Type 1 (p) Type 2 (p) Mixed 1&2 (p) Type 3 (p)

SD-OCT measurements (µ µm), Mean CMT (p) GLD (p) MSFH (p) MLH (p)

Table 2. Cross-sectional series. Correlations between types of lesions within the fibrosis spectrum and quantitative criteria. BCVA stands for best corrected visual acuity IVT stands for intravitreal injection CNV stands for choroidal neovascularization SD-OCT stands for spectral domain optical coherence tomography CMT stands for central macular thickness GLD stands for greatest lesion diameter MSFH stands for maximal subfoveal lesion height MLH for maximal lesion height * Statistical analysis for quantitative analysis was performed using the Student t-test ** Statistical analysis for qualitative analysis was performed using Chi-square nonparametric test

CNV type Baseline CNV type (N=47)

Time between baseline CNV diagnosis and angiofibrotic switch (mean±SD) Fellow eye status

Angiofibrotic switch (N =47)

Type 1 CNV Type 2 CNV Type 3 CNV Mixed type 1 and 2 CNV Aneurysmal type 1 CNV

Percentage

Number of eyes

55 % 26 % 11 % 6%

26 12 5 3

2%

1

20 ±19 months

Intermediate AMD 11/47 Type 1 CNV 14/47 Type 2 CNV 1/47 Type 3 NV 2/47 GA 6/47 GA+CNV 5/47 Fibrosis 7/47 Baseline type of CNV Type 1 CNV N=26 Type 2 CNV N=12 Type 3 CNV N=5 Mixed type 1 and 2 CNV N=3 Aneurysmal type 1 CNV N=1

First fibrosis type Type A 23

Type B 1

Type C 2

3

7

2

3 1

2 1 1

Table 3. Longitudinal series. Distribution of types of CNV within the longitudinal series at baseline (neovascular AMD diagnosis) and at the time of the angiofibrotic switch, according to the initial lesions within the fibrotic spectrum type

1

Progression pathway

Type 1 CNV

Type 2 CNV

Type 3 CNV

A B C

13

2

1

B C A FAL

3 10

9 1

2 2

Mixed type 1 and 2 CNV 1

Aneurysmal type 1

2

1

SHRM n=14/47

Macular hemorrhage n=13/47

RPE tear n=10/47

RPE erosions n=28/47

Perilesional macular atrophy n=13/47

A B*

A B** B C** B C** -

A B

B C***

-

B C -

B C*** -

A A+GA****

-

Table 4. Longitudinal series. Progression pathways towards fibrotic lesions for each type of baseline CNV GA stands for Geographic Atrophy, FAL stands for fibroatrophic lesion. SHRM stands for subretinal hyperreflective material *p<.001, Fisher’s exact test for the association between A B progression pathway and occurrence of macular hemorrhage **p=.058 Fisher’s exact test for the association between progression pathways and macular hemorrhage *** p<.001, Fisher’s exact test for the association between B C progression pathway and occurrence of RPE crumbling ****p<.001, Fisher’s exact test for the association between A A+GA progression pathway and occurrence of perilesional atrophy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

LEGENDS Figure 1. Multimodal imaging of the right eye of an 84 year-old patient with a lesion within the fibrotic spectrum secondary to late nAMD. Upper left panel. Color fundus photography shows a well-demarcated yellow, elevated mound of tissue in the macular area. Upper middle panel. Early fluorescein angiography (FA) reveals a slight staining of the lesion. Upper right panel. Late staining on FA with no accompanying leakage. Lower panels: Horizontal slices of spectral domain optical coherence tomography (SD-OCT) guided by FA reveal that the location of the lesion within the fibrotic spectrum with respect to the RPE is different: on the lower left panel, the lesions is located underneath a clearly visible RPE, while in the lower right panel the fibrotic lesion is subretinal, with no clear visualization of the RPE. Figure 2. Type A lesions within the fibrotic spectrum, 3 eyes issued from the cross sectional analysis. Upper panels: Subtype A1 lesion within the fibrotic spectrum in the left eye of an 81 year-old patient. Multicolor imaging reveals a yellow-greyish lesion in the macular area. Horizontal slice of spectral domain optical coherence tomography (SD-OCT) guided by infrared imaging (IR) shows a hypereflective, sub-retinal pigment epithelium (sub-RPE) (white arrows point to the RPE) lesion with intralesional abnormalities: hyporeflective lamellae (yellow arrowhead) and hyporeflective spaces (asterisks). Middle panels: Subtype A2 lesion within the fibrotic spectrum in the right eye of an 83 year-old patient. Multicolor imaging reveals a yellowish-green lesion in the macular area. Horizontal slice of spectral domain optical coherence tomography (SD-OCT) guided by infrared imaging show a hypereflective, sub-retinal pigment epithelium (sub-RPE) (white arrows point to the RPE) lesion with a hyporeflective band (red dotted arrows) within the entire width of the lesion. Lower panels: Subgroup A3 lesion within the fibrotic spectrum. Color fundus photography reveals a yellowish lesion in the macular area. Horizontal slice of spectral domain optical coherence tomography (SD-OCT) guided by infrared imaging show a compact, homogeneous hypereflective lesion situated beneath the retinal pigment epithelium (RPE, white arrows), without any intralesional abnormalities. Figure 3. Type B lesion within the fibrotic spectrum, 2 eyes issued from the transversal analysis. Each line represents the multimodal imaging of a different case. Upper left panel corresponds to the colour fundus photography revealing a well-defined yellowish lesion in the macular area. Upper middle panel and upper right panels correspond to infrared imaging (upper middle panel) guiding a horizontal slice of spectral domain optical coherence tomography (SD-OCT). SD-OCT guided by infrared imaging shows a subretinal compact, homogeneous, hypereflective lesion. Note that the RPE (blue arrows) in this case was localized within the lesion, separating it in two parts: subretinal (red dotted arrows) and sub-RPE fibrosis (yellow dotted arrows). Lower panels correspond to a 2nd case. Lower left panel corresponds to multicolor imaging, revealing a well-defined, bright-yellow lesion in the macular area. Lower middle panel and lower right panels correspond to infrared imaging (upper middle panel) guiding a horizontal slice of spectral domain optical coherence tomography (SD-OCT). SDOCT guided by infrared imaging shows a subretinal compact, homogeneous, hypereflective lesion. Note that the RPE (blue arrows) in this case was localized within the lesion, separating it in two parts: subretinal (red dotted arrows) and sub-RPE fibrosis (yellow dotted arrows). Figure 4. A Type C lesion within the fibrotic spectrum. Upper left panel: Multicolor imaging reveals a yellowish-grey lesion in the macular area. Upper right panel: Infrared imaging and corresponding Lower panel: Horizontal slice of spectral domain optical coherence tomography (SD-OCT) reveal a hypereflective, heterogeneous, prominent, ill-defined, subretinal lesion on SD-OCT, with no discernable RPE overlaying the lesions. Note that the RPE was visible on the edges of the lesion (white arrows). Figure 5. Illustrative drawing with SD-OCT examples of types of lesions within the fibrotic spectrum. Subtypes A1, A2 and A3 correspond to sub-retinal pigment epithelium (sub-RPE) lesions with (A1, A2) or without (A3) intralesional abnormalities, while Type B corresponds to subretinal fibrosis. A subretinal protuberant lesion with no visible overlaying RPE, however, characterizes type C. Figure 6. Long-term follow up of the right eye of a 71 years old patient, evolving from type 2 CNV, to type A, type B and type C fibrosis. Upper panels. Multimodal imaging at baseline diagnosis of nAMD. Multicolor imaging, early fluorescein angiography (FA) and early indocyanine green angiography (ICGA) show a well demarcated lesion, hyperfluorescent on early frames, suggestive for type 2 CNV. SD-OCT at baseline reveals a hypereflective lesion above the RPE, consistent with the angiographic findings of type 2 CNV. At one year follow-up, we note that the lesion has regressed under the RPE on SD-OCT. Although subretinal fluid is still present, the content of the PED seems rather hypereflective and heterogeneous. This aspect is suggestive for Type A fibrosis. At three years follow-up, we note that fibrosis is located above the RPE (asterisk), as well as underneath the RPE, with a heterogeneous multilayered aspect. This is suggestive for Type B fibrosis. On the magnification

1

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corresponding to type B fibrosis, note the discontinuous RPE erosions (white arrows). At five years from baseline, SD-OCT reveals a hypereflective, protuberant lesion situated in the subretinal space. RPE is not visible within the lesion (white arrowhead for the sudden interruption of the RPE on one edge of the lesion), which is typical for Type C fibrosis. Multimodal imaging (lower left panel) at last follow up reveals, on Multicolor imaging, a grey-green lesion; FA (lower middle panel) reveals staining with no late leakage from the lesion. On early ICGA (lower right panel) the lesion is slightly and heterogeneously hyperfluorescent. Therefore, in this patient the pathway of progression from the initial type 2 CNV to the late-stage fibrotic lesions could be summarized as A B C. Figure 7. Long-term follow up of the right eye of a 82 years old patient, evolving from type 1 CNV to type A, type B and type C fibrosis. Upper panels. Multimodal imaging at baseline diagnosis of nAMD. Early and late frames of fluorescein angiography (FA, upper left panels) and early and late indocyanine green angiography (ICGA, right panels) show a hyperfluorescent, ill defined lesion on early frames, generating a late hyperfluorescent plaque on ICGA, suggestive for type 1 CNV. SD-OCT at baseline reveals a moderately hypereflective pigment epithelial detachment (PED), accompanied by subretinal fluid, consistent with the angiographic findings of type 1 CNV. At one-year follow up we note that the content of the PED seems multilayered, which is suggestive for Type A fibrosis. At two years follow up from baseline, we note that while Type A fibrosis is still present, a well-defined subretinal hypereflective material (SHRM) is also present (white arrowhead). At four years from baseline fibrosis is located both located above the RPE as a homogenous, hypereflective lesion, as well as underneath the RPE, with a heterogeneous multilayered aspect. This is suggestive for Type B fibrosis. Note the chronic subretinal fluid present. On the magnification corresponding to type B fibrosis, note the discontinuous RPE erosions (white arrows). At seven years from baseline, SD-OCT reveals a hypereflective, prominent lesion situated in the subretinal space. RPE is not visible within the lesion, suggesting progression to type C fibrosis. In this patient the pathway of progression from the initial type 1 CNV to the late-stage fibrotic lesions could be summarized as A B C. Figure 8. Long-term follow up of the right eye of a 83 years old patient, evolving from type 2 CNV to type B and type C fibrosis. Upper panels. Multimodal imaging at baseline diagnosis of nAMD. Early fluorescein angiography (FA, upper left panel) reveals a hyperfluorescent subfoveal lesion, as well as a subretinal hemorrhage generating masking. Late indocyanine green angiography (ICGA, right panel) shows a hyperfluorescent late plaque on ICGA. SD-OCT at baseline reveals a hypereflective lesion above the RPE and a moderately hypereflective pigment epithelial detachment (PED), accompanied by subretinal fluid, consistent with mixed type 1 and 2 (minimally classic) neovascularization presenting with subretinal hemorrhage. After the monthly loading dose, we note a hypereflective, homogenous fibrotic scar is located above the RPE, while the initial small PED has a heterogeneous multilayered aspect. Note the discontinuity of the RPE underneath the fibrotic scar (white arrow). This is suggestive for Type B fibrosis. At two years follow-up, SD-OCT reveals a hypereflective, prominent lesion situated in the subretinal space. Note that RPE is not visible within the lesion, suggesting progression from type B to type C fibrosis. In this patient, the pathway of progression from the initial mixed type 1 and 2 CNV to the late-stage fibrotic lesions could be summarized as B C. Figure 9. Long-term follow up of the left eye of a 90 years old patient evolving from type 1 CNV to a fibroatrophic lesion. Upper panels. Multimodal imaging at baseline diagnosis of nAMD. Early fluorescein angiography (FA, upper left panel) reveals an ill-defined hyperfluorescent lesion. Late FA (upper middle panel) reveals late leakage and hyperfluorescent pin-points. Late indocyanine green angiography (ICGA, upper right panel) shows a hyperfluorescent late plaque. SD-OCT at baseline reveals a moderately hypereflective pigment epithelial detachment (PED), accompanied by abundant subretinal fluid, consistent with type 1 neovascularization. The patient underwent the monthly loading dose and an as needed (PRN) regimen afterwards. At one-year follow up, the initial PED seems larger, containing hypereflective and hyporeflective lamellae, which is consistent with type A fibrosis. Throughout time, the aspect of the PED remained suggestive for Type A fibrosis, with a progressive disappearance of the hyporeflective lamellae and a homogenous hypereflective aspect at 3 years follow up. Note that at 7 years follow up, multicolor imaging (lower left panel) reveals the presence of a yellow-grey macular lesion with concave borders (white arrowhead), as well as of well-defined patches of atrophy. The atrophy is also visible on both infrared imaging (lower middle panel) and FAF imaging (lower right panel). Therefore, in this patient the pathway of progression from the initial type 1 CNV to fibrotic lesions falls into the fibroatrophic pathway (A FAL). In all cases, perilesional atrophy ‘blocked’ fibrosis extension in patients presenting with a degree of perilesional atrophy at baseline. Figure 10. Illustrative drawing of all patterns of progression, from CNV to final fibrotic lesions. On the left side are illustrated the 3 types of neovascularization(NV), types 1 CNV, 2 CNV and 3 NV. On the right side are the final stages of fibrotic lesions: fibroglial lesion (FGL) on the upper part and

2

1 2 3 4 5 6 7 8 9 10

fibroatrophic lesion (FAL) on the lower part. The 3 large blue arrows illustrate the 3 main pathways of progression to final fibrotic lesions, from top to bottom: 1. type 2 CNV evolving into type B (subretinal) fibrosis and then FGL (type C); 2. type 1 CNV evolving into type B (subretinal) fibrosis following RPE erosion and SHRM; 3. type 1 CNV evolving into a fibroatrophic lesion (FAL). Thin arrows illustrate some peculiar cases, such as evolvement of any type of neovascularization directly to type B (subretinal) fibrosis and type C fibrosis (FGL) when RPE tears or hemorrhage occur. The shift form type 2 CNV or type 3 NV to a type A fibrosis is also illustrated by thin arrows. For clarity of the figure, aneurysmal type 1 neovascularization is not shown but in integrated in type 1 CNV.

3

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ERIC H. SOUIED:

Conceptualization, Methodology, Writing- Original draft

preparation. MANAR ADDOU-REGNARD:

Data curation

AVI OHAYON: Visualization, Investigation. OUDY SEMOUN: Visualization, Investigation. GIUSEPPE QUERQUES: Supervision ROCIO BLANCO-GARAVITO: Visualization, Investigation. ROXANE BUNOD: Visualization CAMILLE JUNG: Software, Methodology ANNE SIKORAV: Investigation ALEXANDRA MIERE: Writing- Reviewing and Editing , Project administration

1