Acta Ophthalmologica 2013

PhD Thesis Implementation studies of ranibizumab for neovascular age-related macular degeneration Sara Brandi Bloch Department of Ophthalmology, Glostrup Hospital, Glostrup, Denmark Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Acta Ophthalmol. 2013: 91, thesis 7: 1–22 ª 2013 The Author Acta Ophthalmologica ª 2013 Acta Ophthalmologica Scandinavica Foundation

doi: 10.1111/aos.12272

Introduction Age-related macular degeneration (AMD) has for decades been the most common cause of irreversible blindness in the elderly population in Denmark and in other developed countries (Vinding 1995; Bressler 2004; Buch et al. 2004). The vast majority of vision loss resulting from the disease occurs in individuals with the neovascular form of AMD, primarily during the first 2 years after onset of choroidal neovascularization (CNV; Klein et al. 1995; Vinding 1995; Attebo et al. 1996; Bressler et al. 1988; Macular Photocoagulation Study Group 1991a; Treatment of Age-Related Macular Degeneration with Photodynamic Therapy (TAP) Study Group 1999; Wong et al. 2008). In advanced neovascular AMD, vision impairment is caused by severe morphological damage to the retina following growth and involution of subfoveal CNV (Chandra et al. 1974; Green & Enger 1993). The introduction of ranibizumab, a selective inhibitor of all isoforms of VEGF-A, markedly improves the visual prognosis in neovascular AMD, if given repetitively at fixed 4-week intervals, compared with the natural history of the disease and other known categories of pharmacotherapy (Macular Photocoagulation Study Group 1991a; TAP Study Group 1999; Gragoudas et al. 2004; Brown et al. 2006; Rosenfeld et al. 2006). Treatment with ranibizumab for neovascular AMD was introduced on a

national scale in Denmark in 2006. A fixed monthly treatment regimen with ranibizumab for neovascular AMD has only rarely been used in clinical practice, where various approaches have been taken to reduce the need for retreatment guided by a variety of retreatment criteria (Fung et al. 2007; Lalwani et al. 2009; Rothenbuehler et al. 2009; Gupta et al. 2010, 2011; Kaiser et al. 2012; Larsen et al. 2012). Part of the rationale for a reduced injection frequency is that in preclinical experiments, VEGF has been shown to have the characteristics of a neuroprotector. Thus, iatrogenic suppression of VEGF in the eye is thought to promote neurodegeneration (Rosenfeld et al. 2011). In Denmark, ranibizumab therapy for neovascular AMD was implemented under guidelines describing an individualized dosing regimen after three fixed, monthly loading doses. Retreatment was guided by CNV activity criteria. One aim of the studies described in this thesis was to describe outcomes in a routine clinical practice setting under this regimen. The risks of ocular and systemic complications of ranibizumab injections reported from clinical trials with up to 2 years of follow-up are low and dominated by a risk of endophthalmitis that is generally below 65 medium-sized hard drusen or extrafoveal geographical atrophy (GA)] and advanced AMD (Bressler et al. 2006b). The advanced forms of AMD are divided into a nonneovascular atrophic type with a sharply outlined ‘geographical’ disappearance of the RPE and a neovascular type characterized by the development of new vessels derived from the choroid that grow into the subpigment epithelial and/or subretinal space (Bressler et al. 2006a,b). Occasionally, neovascularization can arise from the retinal vessels (retinal angiomatous proliferation). In advanced atrophic AMD, the affected areas have no visual function because the gradual local disappearance of the RPE in GA is intimately coupled with the disappearance of rod function. In the absence of CNV, GA becomes the end stage of AMD, accounting for approximately 25% of cases with severe central vision loss (Klein et al. 1997; Bressler et al. 2006b). Advanced neovascular AMD in its end stage is associated with some degree of persistent subretinal neovas-

The pathogenesis of AMD is incompletely understood due to its complexity. We know that the pathogenesis of AMD is modulated by genetic and environmental risk factors, the latter consisting primarily of increasing age, family history of AMD, smoking, obesity, diet and hypertension (Vinding 1995; Vingerling et al. 1995b; Klaver et al. 1998; Cho et al. 2001; Klein et al. 2001, 2003; Seddon et al. 2001, 2003; Smith et al. 2001; van Leeuwen et al. 2003). Several gene polymorphisms coding for complement proteins associated with the progression of AMD have been identified, with the most significant genetic variants found within the alternative complement pathway in complement factor H (CFH; Haines et al. 2005; Klein et al. 2005), complement factor B/C2 (CFB/ C2), component 3 (C3) and complement factor I (CFI). Variants in the latter three appear to have a substantially weaker association with AMD susceptibility compared with variations in the CFH gene (Swaroop et al. 2009; Priya et al. 2012). Outside the complement pathway, an AMD risk variant has been found within two tightly linked genes of an unknown pathway [age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement A1 (HTRA1); Fig. 2; Rivera et al. 2005; Dewan et al. 2006; Yang et al. 2006; Fritsche et al. 2008]. Genetic variants may interact with behavioural and environmental risk

factors such as smoking to enhance the probability of developing AMD (Despriet et al. 2006; Seddon et al. 2006). A large oxygen gradient towards the inner sensory retina together with abundant photosensitizers and light exposure supports a highly oxidative milieu in the retina and RPE cells (Fig. 1). The RPE cell is in a delicate balance between physiological oxidative stress mediated by reactive oxidative species and pathological oxidative stress with excessive reactive oxidative species derived mainly from intracellular sources (the mitochondria and photosensitizers) or exogenous influences (smoking, diet, etc.; Jarrett & Boulton 2012). Protection against oxidative damage of the RPE is mediated through antioxidant systems (e.g. vitamins C and E, catalase, superoxide dismutase, glutathione peroxidase and carotenoids) and efficient repair systems. The carotenoids, lutein and zeaxanthin are abundantly distributed in the retina and act as potent scavengers of a variety of oxidative species. Unfortunately, as we age, the oxidative damage of the RPE increases, the repair systems are impaired, and the antioxidant capacity decreases (Handa 2012; Jarrett & Boulton 2012). With age, accumulation of A2E, a bisretinoid that is a constituent of ocular lipofuscin and a photoinducible generator of reactive oxidative species, occurs within the RPE cell. Accumulation of lipofuscin/ A2E may induce oxidative damage; reduce phagocytic capacity; and cause membrane disruption, lysosomal dysfunction and finally RPE cell death (Kennedy et al. 1995; Sundelin et al. 1998; Boulton & Dayhaw-Barker 2001;

Fig. 2. Different stages of age-related macular degeneration versus physiological age changes in the retinal pigment epithelium (RPE), Bruch’s membrane and choroid. ECM, extracellular matrix; CNV, choroidal neovascularization (Zarbin 2004).

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Boulton et al. 2001). It is hypothesized that microglia are recruited to the subretinal space in response to a failing and ageing RPE layer, to facilitate the removal of retinal debris and apoptotic RPE cells (Xu et al. 2008). Senescent subretinal microglia, migrated from the inner to the outer retina, accumulate lipofuscin/A2E, which at sublethal levels may induce significant alterations in complement activation (Combadiere et al. 2007; Ma et al. 2013). The sources of lipofuscin/A2E in subretinal microglia are most likely derived by phagocytosis of photoreceptor outer segments and/or RPE cells. Activated subretinal microglia loaded with lipofuscin/A2E favour complement activation and reduce the ability to rescue photoreceptors undergoing oxidative stress (Xu et al. 2008; Ma et al. 2013). Furthermore, significant age changes in Bruch’s membrane are seen with thickening, impermeability and accumulation of heterogeneous debris (Fig. 2). An age-related decline in the density and lumen diameter of the choriocapillaris contributes to a decrease in clearance of debris from the Bruch’s membrane, which leads to further membrane thickening (Guymer et al. 1999). Altogether, these agerelated changes can lead to chronic pathological oxidative stress of the RPE and microglia, which induces cell dysfunction and damage. AMD involves changes in the outer retina observed with ageing and immune dysfunction. As previously described, susceptibility to AMD is dependent on genetic and environmental factors being involved in the pathological sequence of events. In AMD, poorly degradable RPE debris, Bruch’s membrane components and oxidative injury of the choriocapillaris and RPE cells may trigger a chronic inflammatory response that mediates the accumulation of abnormal extracellular material (Zarbin 2004). When the accumulation of sub-RPE deposits is ophthalmoscopically visible, it is termed ‘drusen’. Histologically, the abnormal extracellular material is located between the plasmalemma and the RPE basement membrane (basal laminar deposits) and outside the RPE basement membrane within the inner collagenous zone of Bruch’s membrane (basal linear deposits; Green & Enger 1993; Zarbin 2004). The presence of subretinal microglia also leads to subretinal drusenoid deposits (Raoul et al.

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2010). Aberrations in the function of CFH proteins, among others, which are present in sub-RPE drusen and subretinal drusenoid deposits, may interfere with the control of the complement pathway and cause excessive inflammation (Gorin 2007). Furthermore, upregulation of drusen-related transcript proteins mediates apoptosis of the RPE through the production of cytotoxic proteins, ultimately leading to atrophy of the RPE, choriocapillaris and photoreceptors. The pattern of the up-regulated expression of proteins is heterogeneous, with some RPE cells expressing more than others, which could explain the heterogeneous macular distribution of drusen and RPE atrophy (Rabin et al. 2013). Changes in Bruch’s membrane composition with abnormal extracellular material together with oxidative damages of the RPE and the choriocapillaris create a hypoxic environment with altered diffusion of waste products and nutrients between the RPE and choriocapillaris, which may lead to further injury of the RPE and the photoreceptors (Zarbin 2004). In response to such metabolic distress, the RPE cells produce a protein, VEGF, which acts on endothelial cells to promote angiogenesis. It is suggested that this is what drives neovascular AMD (Zarbin 2004; Rabin et al. 2013). During the initial stage of CNV, macrophages are recruited to the choroidal side of the Bruch’s membrane and interact with the retinal pigment epithelial cells (Grossniklaus et al. 2002). Vascular endothelial growth factor (VEGF) is expressed in ischaemic retina and allows leakage of fluids from proliferating blood vessels with the absence of endothelial tight junctions. The retinal pigment epithelial cells are believed to be the major sources of the VEGF that stimulates CNV and act together with various inflammatory reactions in the RPE–choroid microenvironment. But macrophages, photoreceptor cells and M€ uller cells also express VEGF and other cytokines in association with CNV. It has been proposed that the transcripts of VEGF and complement activation are mutually interactive to promote a consistent up-regulation of VEGF expression (Grossniklaus et al. 2002; Nozaki et al. 2006; Zhao et al. 2010; Kunchithapautham & Rohrer 2011). Constitutive secretion of low levels of VEGF’s is critical for the maintenance of healthy

blood vessels in the retina and choroid, but excessive VEGF expression results in fluid leakage and angiogenesis, as seen in neovascular AMD, diabetic retinopathy, ocular ischaemia, some tumours and many other conditions. Recent studies suggest that common pathogenic mechanisms are active in Alzheimer’s disease and AMD. The molecular components of drusen in AMD are very similar to the composition of senile plaques in Alzheimer’s disease, with accumulation of amyloid b that triggers inflammatory and angiogenic responses (Ohno-Matsui 2011). Finally, there is increasing evidence that AMD is related to increased systemic complement activation and elevated cytokine levels (Reynolds et al. 2009; Charbel et al. 2011; Singh et al. 2013). Epidemiology

The prevalence and incidence of AMD in Denmark (Vinding 1995; Buch et al. 2005) are largely comparable to what have been found in Caucasian populations in the USA (Klein et al. 1992, 2002), Australia (Mitchell et al. 1995, 2002) and Northern Europe (Vingerling et al. 1995a; Klaver et al. 2001). A Danish study from 2005 estimated that the annual incidence rate of early AMD in individuals aged 60 years and older was 3.4%, while that of advanced AMD was 1.3% (Buch et al. 2005). The current prevalence of advanced AMD in at least one eye in Denmark in persons aged 65 years and older is estimated to be 5.1%, corresponding to 47 000 citizens (Lindekleiv & Erke 2013). The prevalence of AMD is expected to increase due to a growing elderly population (Rein et al. 2009; Lindekleiv & Erke 2013). Nonetheless, a population-based study from 2011 found that the prevalence of AMD in the USA had decreased over the recent decades (Klein et al. 2011). This may reflect changes in lifestyle, general health and modifiable conditions such as arterial hypertension and poor dietary habits. Age-related macular degeneration is the most common cause of legal blindness in people 65 years of age and older in Denmark (Buch et al. 2004). The prevalence and incidence of legal blindness from AMD in Denmark were most recently assessed in 2002, after the introduction of PDT, but before antioxidant prevention therapy, and

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intravitreal inhibitors of VEGF (Buch et al. 2005). Symptoms and clinical examinations

Persons with onset of neovascular AMD have a wide spectrum of symptoms such as accelerated blurred vision, central scotomas, metamorphopsia, macropsia/micropsia and colour desaturation. In advanced cases with severe visual loss, complex visual hallucinations can arise (Charles Bonnet syndrome). The onset of symptoms can vary considerably from hours to days to months. To verify the diagnosis of neovascular AMD, several modes of clinical examinations can be utilized. Best-corrected visual acuity

Best-corrected visual acuity (BCVA) is measured over time to evaluate the degree of vision loss. In clinical practice, visual acuity is often scored by the use of a Snellen chart at 6 m distance, which is efficient and a convenient design for refraction. Nonetheless, it is inferior to the ETDRS chart in accuracy across all ranges of vision, and it is particularly unsuited to low levels of visual acuity, such as seen in AMD (Falkenstein et al. 2008; Kaiser 2009). BCVA measured as the number of letters read on an ETDRS chart at a starting distance of 2 m has become the method of choice in monitoring BCVA in individuals with AMD. Indirect biomicroscopy and colour fundus photography

The biomicroscopic examination or colour photographs of the retina will often reveal bleeding or oedema with retinal elevation in an eye with drusen or abnormalities of the pigment epithelium in AMD.

Fig. 3. An optical coherence tomographic image of the neuroretina, retinal pigment epithelium, Bruch’s membrane and choroid in a healthy person. Reprinted with minimal modifications from Keane et al. (2012), with permission from Elsevier.

the new vessels causes exudation of fluid and blood, which can be seen on the OCT as hyporeflective areas (Grossniklaus & Green 2004; Keane et al. 2008a, 2012). Tomographic images of the retina have been widely adopted for the evaluation of disease activity in neovascular AMD, as it may illustrate increasing fluid or oedema in the macula even before visual deterioration is recognized. OCT is therefore a critical imaging modality in monitoring the morphological response to anti-VEGF treatment (Brown & Regillo 2007; Drexler & Fujimoto 2008; Keane et al. 2009). Activity in neovascular AMD is most often detected on OCT by an increase in central retinal thickness or accumulation of subretinal or intraretinal fluid (Fig. 4). Fluorescein angiography

Fluorescein angiography (FA) is a crucial imaging modality to identify the presence, location and size of the neovascular complex. For clinical practice, a non-stereoscopic digital FA is used to produce instant images that can be altered by computer software. In

most clinical trials, stereoscopic viewing of the images is preferable because it enables the appreciation of thickening and attenuation of the structures of the posterior pole (Chamberlin et al. 1989; Macular Photocoagulation Study Group 1991b; Barbazetto et al. 2003; Brown et al. 2006; Rosenfeld et al. 2006). Before injection of fluorescein dye into the bloodstream, a red-free photograph is taken to highlight the retinal vessels. After injection of fluorescein, fundus photographs are taken during the early transit phase (typically from 15 to 45 seconds), the mid-phase (60– 90 seconds and 2–3 min) and the late phase (5 and 10–15 min after injection). The evaluation procedure may describe the angiographic subtypes and measure the total CNV area, the total lesion area, the leakage area and abnormalities not included in the neovascular lesion complex. Characterization of the CNV may be made by observing how the CNV behaves in the different phases of the fluorescein angiogram. Classic CNV is defined by a well-demarcated, sometimes ‘lacy’,

Optical coherence tomography

Optical coherence tomography (OCT), first described by Huang et al. (1991), is based on the selective imaging of directly reflected light from within transparent or semitransparent tissues such as the retina. In a non-invasive manner, OCT allows high-resolution cross-sectional images of the neurosensory retina and deeper structures to be obtained (Fig. 3; Huang et al. 1991). As the CNV proliferates through breaks in the Bruch’s membrane into the sub-RPE or subretinal space, the structural immaturity of

Fig. 4. Optical coherence tomographic image showing subretinal and intraretinal fluid as well as drusenoid pigment epithelial detachments in an eye with neovascular age-related macular degeneration. Bruch’s membrane (black arrow) is visible underneath the retinal pigment epithelium (yellow arrow).

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Fig. 5. Fluorescein angiographic images showing a well-demarcated lacy pattern with a central hypofluorescence in the early phase (left) with intense progressive leakage of fluorescein in the late phase of the angiogram (right), which is consistent with a classic choroidal neovascularization in age-related macular degeneration.

area of hyperfluorescence observed in the early phase of the angiogram with progressive leakage of dye later in the angiogram (Fig. 5). Occult CNV varies in appearance and can be difficult to identify. Occult CNV has two characteristic patterns. The first pattern stains less than a classic CNV and does not expand with time. It is associated with elevation of the RPE, and it is often stippled with hyperfluorescent dots in the early and mid-phase of the fluorescein angiogram. In the late-phase frames, the hyperfluorescent areas intensify, but may or may not show leakage beyond the boundaries of fluorescence observed in the early- and mid-phase frames, depending upon whether there is an associated detachment of the neurosensory retina to leak under. The second pattern of occult CNV has poorly demarcated areas of leakage in the late-phase frames of the angio-

gram without early hyperfluorescence (Fig. 6). The total CNV area is defined by the leakage area from the image where CNV first appears. The total lesion area is the sum of the total CNV area and the area of each of the non-CNV lesion components such as blocked fluorescence, thick blood, serous PED and subretinal fibrous tissue considered likely to obscure the boundaries of the CNV. Predominantly, classic lesions are defined as having 50% or more of the total lesion area comprised of classic CNV, whereas an occult lesion is composed of occult CNV only (Chamberlin et al. 1989; Macular Photocoagulation Study Group 1991b; Barbazetto et al. 2003). Between the two is the class of minimally classic CNV. The structural background is that classic CNV is characterized by CNV that grows between the RPE and the sensory retina, often with a smaller

Fig. 6. Fluorescein angiographic images illustrating an occult choroidal neovascularization in agerelated macular degeneration with stippled hyperfluorescence in the early phase to mid-phase (left) and an intensified hyperfluorescence in the late-phase frames of the angiogram (right).

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fibrovascular component under the RPE (type 2 growth pattern), whereas occult CNV is located between the RPE and Bruch’s membrane (type 1 growth pattern; Gass 1997; Grossniklaus & Gass 1998; Lafaut et al. 2000; Grossniklaus & Green 2004; Submacular Surgery Trials Research Group 2006). The more prominent fluorescein leakage that accompanies classic CNV corresponds to a predominantly subretinal growth pattern that can more readily produce a cleavage plane in the subretinal space between the photoreceptors and the RPE, whereas the RPE does not detach so easily (Submacular Surgery Trials Research Group 2006). Subretinal fibrosis and atrophy of the retina

Recurrent or persistent epithelial injury is an essential element in the pathogenesis of fibrosis in chronic diseases of the lungs, liver, heart and kidneys (Friedman et al. 2013). In the eye, development of subretinal fibrous tissue is mediated by multiple responses to cellular injury that are involved in the pathogenesis of neovascular AMD. Subretinal fibrous tissue appears to form as a consequence of RPE cell death, injury-triggered inducers of fibrosis, inflammatory responses and hypoxia-/inflammation-driven neovascularization. Injured pigment epithelial cells express transforming growth factor-beta (TGF-b), connective tissue growth factor (CTGF) and plateletderived growth factor (PDGF), among other proangiogenic and profibrotic cytokines that activate adjacent fibroblasts and trigger fibrosis in the neighbouring RPE cells (Kent & Sheridan 2003; Friedlander 2007; Friedman et al. 2013). Fibrous tissue thus formed under the RPE, with the gradual decomposition of the latter, proliferates into the subretinal space with or without accompanying new vessels (Bressler et al. 2006a). The fibrous tissue that accompanies CNV contains apoptotic stromal RPE, endothelial cells and macrophages (Kent & Sheridan 2003; Friedlander 2007). Subretinal fibrous tissue usually accompanies CNV, but it is not always apparent at the first ophthalmoscopic examination. When fibrous tissue in type 2 CNV or atrophic type 1 lesions becomes readily visible on ophthalmoscopy, it is in the form of subretinal mounds of whitish

Acta Ophthalmologica 2013

Fig. 7. Colour fundus photograph of a disciform scar in age-related macular degeneration. The photograph shows a whitish material under the retina and atrophy of the retinal pigment epithelium.

or yellowish material that obscures the RPE and choroid (Fig. 7; Bressler et al. 2006a). In contrast to fibrin and dehemoglobinized blood, fibrous tissue has a stranded appearance with tension lines, curved edges, occasional pigment infiltration and an outline that is more irregular than that produced by expanding fluid. In the late phase of subretinal fibrosis, the term ‘disciform fibrovascular scar’ is often used to describe the condition. Subretinal fibrin is a differential diagnosis of fibrosis and often a precursor of fibrosis. Frequently, vascular anastomoses are observed between the retina and the fibrovascular complex. Fibrovascular scars may on FA be defined by blocked fluorescence and/or staining depending on the extent of RPE within the scar (Fig. 8; Chamberlin et al. 1989; Macular Photocoagulation Study Group 1991b; Barbazetto et al. 2003).

The disciform scar formation appears on OCT as a well-demarcated highly hyper-reflective lesion sometimes with a loss of the overlying photoreceptor layer (Fig. 10; Keane et al. 2012). Severe central vision loss below 20/ 200 is often a direct consequence of fibrovascular scarring in AMD because the fibrous tissue often involves the fovea. Persistent subretinal fibrosis after arrest of new vessel growth under the fovea in eyes with occult or minimally classic lesions has been observed in eyes treated with VEGF inhibitor treatment of neovascular AMD in 12 months without any clear relation to visual loss (Kaiser et al. 2007a). Failure to recover normal visual acuity following VEGF inhibitor treatment in CNV secondary to AMD has in part been associated with atrophic scars. Angiographic images of atrophic scars are defined by angiographic hyperfluorescence that does not begin early in the angiogram, as a transmission defect would do, and does not fade in the late phase of the angiogram. Atrophic scars may as such be accompanied by a ‘thin’ layer of RPE or fibrous tissue (Rosenfeld et al. 2011). An accurate non-invasive imaging method to detect and quantify GA in advanced non-neovascular AMD is fundus autofluorescence (SchmitzValskenberg et al. 2007). Treatment for neovascular AMD: a short update of history

Two decades ago, thermal laser therapy was the only treatment option for neovascular AMD. Argon laser photo-

Fig. 8. Fluorescein angiographic images of a subretinal disciform scar showing a well-demarcated staining in the late phase of the angiogram (right) that has no progressive leakage compared with the early phase (left).

coagulation for CNV in AMD was validated on a large scale in 1982 (first of three trials in the Macular Photocoagulation Study), but the frequency of cases with lesions suitable for photocoagulation treatment was low, and adoption of this treatment modality was limited (Argon Laser Photocoagulation for Senile Macular Degeneration 1982). PDT for subretinal neovascularization using verteporfin (Visudyne; QLT Pharmaceuticals, Vancouver, British Columbia, Canada) was first given in Denmark on 16 September 1999 (TAP Study Group 1999). This timing is relevant for epidemiological data that will be presented below. Implementation followed gradually and was not subject to centralized planning. Treatment with PDT was aimed at individuals with specific lesion characteristics, and while it limited vision loss after 24 months compared with the natural course of the disease, PDT did not improve visual acuity in the average patient with neovascular AMD (Bressler 2001; Bressler et al. 2002). The AREDS showed in 2001 that high doses of vitamins C end E, betacarotene, copper and zinc reduced the risk of progression from severe intermediate AMD (extensive intermediate drusen, large drusen or non-central GA in at least one eye or advanced AMD in one eye or vision loss due to non-advanced AMD in one eye) to advanced AMD with 34% over 5 years (AREDS Research Group 2001a). The use of non-prescription dietary supplements in patients with AMD has been common in Denmark since 2003. The first intravitreal injection of a VEGF inhibitor (bevacizumab, Avastin; Genentech Inc., South Francisco, CA, USA) in Denmark was given on 4 November 2005. Intravitreal ranibizumab (Lucentis; Genentech Inc.) therapy was officially defined as the treatment of choice for neovascular AMD in the public healthcare system in Denmark as of January 2007 after European Medicines Agency (EMA) approval. Intravitreal treatment with ranibizumab given at a fixed 4-week interval led to an increase in visual acuity after 24 months of treatment in the average number of patients in the pivotal preregistration studies, which is remarkably better than natural history (Rosenfeld et al. 2006; Brown

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et al. 2009). Bevacizumab was used as off-label therapy for neovascular AMD in Denmark since 2005. Pegaptanib (Macugen; OSI Eye Tech, New York, NY, USA) was officially approved for the treatment of neovascular AMD in Denmark at about the same time, but because the treatment is less potent than bevacizumab, it was never used on a large scale before the approval and introduction of ranibizumab (Gragoudas et al. 2004). New anti-VEGF agents blocking different points in the VEGF signalling pathway are in the pipeline, whereas VEGF Trap-Eye (Eylea; Bayer HealthCare, Berlin, Germany) was approved by the EMA for treatment of neovascular AMD in December 2012 (Heier et al. 2012). Growth factor inhibitors

Vascular endothelial growth factor-A (VEGF-A) is a major mediator of angiogenesis and has been implicated in the pathogenesis of neovascular AMD (Aiello et al. 1994; Kvanta et al. 1996; Kliffen et al. 1997; Boyd et al. 2002). The observation that VEGF-A may play a crucial part in the angiogenesis leading to neovascular AMD has made it a target of continuous investigation. Four inhibitors of VEGF-A are currently available for clinical use (pegaptanib, bevacizumab, ranibizumab and aflibercept), and some are emerging (sirna-027, bevasiranib, vatalanib, pazopanib, TG10081, etc.; Fig. 9).

Pegaptanib selectively blocks the VEGF-A isoform VEGF165 and is nearly obsolete in clinical practice. Bevacizumab and ranibizumab are humanized monoclonal antibody-binding fragments that block all isoforms and biologically active degradation products of VEGF-A, thereby preventing binding of VEGF-A to its receptors VEGFR-1 and VEGFR-2. Whereas ranibizumab is specifically designed for ophthalmic use, bevacizumab was developed for oncology. The average vitreous half-life of ranibizumab in the human eye is reported to be approximately 9 days (Genentech 2010; Novartis 2010). Aflibercept is a soluble decoy receptor fusion protein that has a high binding affinity for VEGF-A, allowing for less frequent dosing compared with ranibizumab. The half-life of aflibercept after intravitreal injection in the human eye is unknown (Heier et al. 2012). Angiogenesis can be mediated by other growth factors than VEGF-A, such as TGF-b and PDGF that are upregulated in injured RPE cells. These growth factors are presumed to contribute to certain ocular wound healing processes and mediate subretinal fibrosis in neovascular AMD and other fibrous diseases (Kent & Sheridan 2003; Friedlander 2007; Friedman et al. 2013). New experimental combination therapies targeting multiple growth factors expressed in neovascular AMD may eventually improve the visual prognosis in AMD (Yamada et al. 1994; Miyazawa et al. 1995; Jo

Fig. 9. Diagram illustrating vascular endothelial growth factor (VEGF) signalling pathways and sites of inhibition by current and emerging anti-VEGF agents. Vatalanib and pazopanib are intracellular inhibitors of the VEGF receptor. Small interfering RNA molecules (sirna-027) and bevasiranib silence the genetic coding of VEGF receptor 1 and VEGF-A, respectively.

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et al. 2006; Zhang & Liu 2012; Ophthotech Corp 2013). Treatment strategy for CNV in Denmark

Guideline criteria in Denmark for initiating ranibizumab treatment are active subfoveal CNV believed to be caused by AMD; BCVA 20/400 (0.05) or better; ability to comply with treatment and follow-up; absence of extensive fibrosis; and a greatest linear lesion dimension no larger than 5400 lm. Predominantly classic CNV lesion types are defined based on FA. Such lesions are considered to be active unless clear signs of involution are present, such as fibrosis, pigmentation and an absence of serous detachment on OCT. Minimally classic and occult lesions are not considered active unless there is evidence of ongoing disease progression, evidenced by recent vision loss, subretinal haemorrhage or an increase in the greatest linear lesion dimension of 10% or more, or if serous detachment or cystoid intraretinal oedema is seen on OCT (Brown et al. 2006; Rosenfeld et al. 2006). The treatment protocol with ranibizumab for neovascular AMD prescribes three initial 0.5 mg ranibizumab injections at intervals of 4 weeks followed by a renewed clinical examination 4 weeks after the third injection. Follow-up visits are not scheduled between the first injection and 16 weeks later. Renewed ranibizumab injection is given in a pro re nata regimen based on monthly disease monitoring, including updating the history of symptoms, assessment of BCVA, transfoveal OCT and colour fundus photography. FA can be made at the investigating physician’s discretion. Renewed ranibizumab injection, usually only once, is ordered if the treating physician observes signs of CNV activity, in which case a new follow-up visit is scheduled 4 weeks after this injection. If no sign of CNV activity is found, a follow-up visit is scheduled 4–12 weeks later depending informally on lesion type and previous response to treatment. Signs of CNV activity are defined as an increase in fluid under the macula compared with any previous visit, an increase in macular thickness of 50 lm or more, persistent thickening and fluid in the macula despite ranibizumab injections, fresh haemorrhage and

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growth of the CNV (Fung et al. 2007). Decreasing BCVA of five ETDRS letters or two lines on a Snellen chart since the most recent visit is considered a potential sign of CNV activity that can motivate additional investigations, but does not, in itself, constitute a criterion for retreatment. Danish guidelines and clinical practice deviate from the European Lucentis label towards more liberal retreatment criteria. Guideline criteria for termination of treatment are (1) BCVA below 20/400 and (2) stable BCVA for 6 months or more without treatment. Atrophy of the foveal photoreceptor layer, GA of the foveal RPE and subfoveal fibrosis indicating the absence of a potential for visual acuity improvement are adjuvant criteria for termination of ranibizumab treatment. Termination of treatment was not considered during the first year in patients who initiated treatment between 1 January and 1 July, 2007.

Methods Local database: BOB (Paper I–III)

Intravitreal treatment with ranibizumab was introduced in Denmark under a fully reimbursed single-payer public healthcare system. A uniform treatment strategy with a common set of guidelines was administered at five designated centres of ophthalmology and their affiliated subcentres. Treatment with ranibizumab for neovascular AMD was administered and funded under a project initiated in May 2006 by the National Board of Health (NBH) to ensure consistent high quality and to observe whether treatment in Denmark could meet the standard of the international clinical studies upon which EMA approval was based. A database (BOBBedre Oftalmologi for Brugere) was developed for the Greater Copenhagen Region to record treatment and outcome parameters in patients with neovascular AMD. The development of this database was led by Sara Brandi Bloch and involved a professional software engineer. The studies included in this thesis involve analysis of data from this database. Demographic and clinical characteristics (age, gender, number of follow-up examinations and ranibizumab injections during 12 and 24 months, time interval from diagnosis to first injection with ranibizumab,

visual acuity measurements in ETDRS letter scores and Snellen acuity scores at baseline and follow-up examinations) of the study population were collected from the BOB database. The BOB database was qualitytested to ensure data reliability. The first 1400 patient’s records were manually inserted in a separate Access database from 2007 and onwards. Data from all patients treated with ranibizumab were continuously updated manually during this period. When BOB was developed and implemented at the department in 2009, data from the Access database were converted into the BOB SQL database. In this conversion, tests revealed conformity between the two databases. Quality testing has been performed at several occasions between the SQL database, the patient administrative system (the national ‘open green system’) and Eyeclinic (a local database used in the surgery section of the Glostrup Hospital and its satellite clinics). Whenever discrepancies were met, a thorough investigation was initiated to ensure an updated and reliable database. National databases: Danish Association of the Blind and the National Bureau of Statistics (Paper III)

Incidence rates of legal blindness were assessed on the basis of admission records of the Danish Association of the Blind (DAB), a private national organization founded in 1911 that provides free membership, counselling, rehabilitation services and selected social benefits for blind and visually impaired residents of Denmark and the Faroe Islands. Admission requires legal blindness defined as BCVA of 20/200 (0.1) or lower in the better-seeing eye, tunnel vision defined as constriction to 5° of eccentricity or less or homonymous hemianopia. Physicians have no formal obligation to advise patients about the option of joining the DAB. Demographic information about the background population was obtained from the Website of the national bureau of statistics (Statistics Denmark 2011). Grading of non-stereoscopic FA and colour fundus photography (Paper I–II)

Baseline fundus photographs and fluorescein angiographs were 30°- and 50°

digital colour and red-free greyscale photographs (ff450; Carl Zeiss Meditec, Jena, Germany). Digital non-stereoscopic fundus photographs and fluorescein angiographs using standardized grading grids and assuming a disc area of 2.54 mm2 were used to measure the patterns, boundaries, compositions and locations of the neovascular lesions (adapted from the Macular Photocoagulation Study and an extension of the Wisconsin agerelated maculopathy grading system; Chamberlin et al. 1989; Klein et al. 1991; Macular Photocoagulation Study Group 1991b; University of Wisconsin Fundus Photographic Reading Center, 1998, unpublished; AREDS Research Group 2001b). The CNV lesion area was defined by the leakage area on early- to mid-phase angiograms (occult lesion component) or early-phase angiograms (classic lesion component). In paper II, lesions were furthermore classified as type 1 CNV lesions for occult and minimally classic lesions and type 2 CNV lesions for classic or predominantly classic lesions (Gass 1994, 1997; Grossniklaus & Gass 1998; Lafaut et al. 2000; Grossniklaus & Green 2004). Subretinal fibrous tissue was identified on colour fundus photographs and defined as whitish material under the retina. No attempt was made in these two studies to distinguish between fibrin and fibrous tissue (AREDS Research Group 2001b). Grading of OCT (Paper II)

Optical coherence tomography was performed using a Topcon spectral domain OCT/fundus camera including 6 radial scans and 2 cross-hair scans (Topcon, Tokyo, Japan). An abbreviated classification of subretinal fibrous tissue was assessed on the basis of standard OCT and colour photographs representing a three-stage grading system (Rogers et al. 2002). A basic assumption was that on OCT, subretinal fibrous tissue has the appearance of a continuous, highly reflective band between the neurosensory retina and Bruch’s membrane, with the reservations that Bruch’s membrane may be invisible if shaded by dense fibrous tissue and that it may be indiscernible if it is embedded in a fibrovascular CNV (Joeres et al. 2007; Keane et al. 2008b, 2012). Stage I was

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Original peer-reviewed publications – summary Paper I – Predictors of 1-year visual outcome in neovascular age-related macular degeneration following intravitreal ranibizumab treatment

Fig. 10. Abbreviated classification of subretinal fibrous tissue based on the study by Rogers et al. (2002) using colour fundus photographs (right) and optical coherence tomograms (left). Stage I (top): minimal subretinal fibrosis with a thin continuous, highly reflective band on optical coherence tomography between a less reflective outer neurosensory retina and Bruch’s membrane, with or without subretinal fluid; fundus photography showing a distinct pale patch of fibrosis. Stage II (middle): prominent subretinal fibrosis with a thick, continuous highly reflective subretinal mass and diffuse thickening of the neurosensory fovea centralis. Stage III (bottom): hyper-reflective subretinal fibrosis with atrophy of the overlying neurosensory retina.

defined as minimal fibrosis with or without subretinal fluid; stage II, as prominent subretinal fibrosis with or without oedema; and stage III, as subretinal fibrosis with atrophy of the

overlying foveal neurosensory retina (Fig. 10). The diagnosis of subretinal fibrous tissue required consistency between colour fundus photographs and OCT.

Fig. 11. Scatter plot showing mean best-corrected visual acuity and standard error of the mean relative to baseline at months 3, 6 and 12. Mean change in early treatment diabetic retinopathy study letters from baseline to 3, 6 and 12 months of follow-up was +4.7 (p < 0.0001), +4.2 (p < 0.0001) and 0.4 (p = 0.667), respectively.

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Purpose: To evaluate visual acuity outcome and describe predictors of visual outcome in patients treated with intravitreal ranibizumab for neovascular AMD. Design: Retrospective, observational case series. Methods: Settings and study population: The study comprised 279 eyes in 279 patients with CNV in AMD who fulfilled the principal MARINA/ANCHOR (Brown et al. 2006; Rosenfeld et al. 2006) study eligibility criteria and were treated with 0.5 mg ranibizumab in a routine clinical setting, beginning with three initial injections at 4-week interval followed by a pro re nata regimen for the subsequent 9 months. Main outcome measures: BCVA and morphological characteristics at baseline, month 3 and after 12 months of ranibizumab treatment. Statistics: Student’s two-sample t-test for means was used to evaluate changes in ETDRS letter scores from baseline to months 3, 6 and 12. Multiple logistic regression analysis with backward elimination was used to identify prognostic factors for visual outcomes at 12 months. Results: BCVA improved significantly from baseline to months 3 and 6, whereas BCVA at month 12 was indistinguishable from baseline (Fig. 11). A supplementary analysis made after publication examined absolute BCVA during the study stratified by relative change in BCVA after 12 months (Fig. 12). A striking feature is that patients who went on to experience stable vision had better baseline visual acuity than patients who experienced significant acuity loss or acuity gain. Of 279 patients, 45 patients (16%) had lost 15 ETDRS letters or more after 12 months of treatment. The average number of administered injections of 5.1 in the first year was comparable in the three groups (p = 0.5784). Another supplementary analysis made after publication stratified patients into quartiles by baseline

Acta Ophthalmologica 2013

Fig. 12. Best-corrected visual acuity during study for patients who after 12 months had gained vision, lost vision or remained stable as expressed in early treatment diabetic retinopathy study letters. Patients with stable vision at month 12 had on average a 5.5 letter higher baseline visual acuity score than patients who had gained or lost acuity (CI95 1.24–9.67, p = 0.0118).

Fig. 13. Best-corrected visual acuity in early treatment diabetic retinopathy study letters at baseline, months 3, 6 and 12 where baseline best-corrected visual acuity has been divided in quartiles.

BCVA and plotted BCVA as a function of time (Fig 13). Mean change in BCVA after 12 months was statistically indistinguishable from baseline in all of the four groups divided by quartile points (p = 0.5343). Nonetheless, the change in visual acuity in the first 6 months had a diverse temporal pattern in the four groups with a more pronounced increase in BCVA during the first 3 months in patients with poorer baseline BCVA compared with patients in the upper quartile. The average number of injections of 4.8 in

the first year of treatment was significantly lower in patients within the lower quartile compared with patients in the upper quartile who were treated on average 5.4 times in the first year (CI95 0.22 to 1.23, p = 0.0053). Delay to first injection had a significant negative effect on the change in BCVA from baseline to months 3 and 12 (Fig. 14). Predictors of BCVA outcome in absolute values after 12 months of treatment with ranibizumab were primarily related to BCVA at baseline and

month 3 (Table 1–3). Thus, patients with BCVA of 35 ETDRS letters or lower at baseline had a 64% chance of having BCVA of 35 ETDRS letters or lower at the conclusion of the study at month 12. Patients with BCVA of 35 ETDRS letters or lower at month 3 had a 74% probability of having BCVA of 35 letters or lower after 12 months. Patients with BCVA of 35 letters or lower at baseline as well as at month 3 had a 94% probability of a poor visual outcome of 35 letters or lower at month 12. In addition to the significant relation between absolute BCVA at 12 months and BCVA at baseline and month 3, total lesion size smaller than 4 DA compared with a large total lesion size had higher odds of achieving BCVA of 70 ETDRS letters or more after 12 months (Table 3). Paper II – Subfoveal fibrosis in eyes with neovascular age-related macular degeneration treated with intravitreal ranibizumab

Purpose: To evaluate baseline and follow-up characteristics of CNV in AMD in relation to the development of subfoveal fibrosis. Design: Retrospective, observational case series. Methods: Settings and study population: The study included 197 treatmentna€ıve eyes in 197 patients of Caucasian ethnicity with CNV in AMD without subfoveal fibrosis at first presentation who were treated with ranibizumab in a pro re nata regimen. Main outcome measure: Subfoveal fibrosis at the conclusion follow-up of 24 months or fewer. Statistics: A general linear model (PROCGLM) was used to assess the association between BCVA and subfoveal fibrous tissue after 24 months of variable ranibizumab treatment. A Cox proportional hazards model (TPHREG) taking length of follow-up into consideration was used to find hazard ratios for subfoveal fibrous tissue depending on treatment and initial lesion characteristics. Results: In this retrospective study, the average period of observation was 1.80 years (CI95 1.75–1.85). In the majority of cases, subfoveal fibrous tissue developed during the first 6 months after initiation of treatment (Fig. 15). The hazard ratio for developing subfoveal fibrosis of any stage

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Fig. 14. Regression line (right) showing a decrease in visual acuity between baseline and month 3 of 0.20 (CI95 0.33 to 0.06, p = 0.0052) early treatment diabetic retinopathy study (ETDRS) letters per day deferred between diagnosis of age-related macular degeneration (AMD) and first administration of 0.5 mg ranibizumab. Regression line (left) showing a decline in visual acuity between baseline and month 12 of 0.15 (CI95 0.29 to 0.02, p = 0.0406) ETDRS letters per day from diagnosis to first injection.

Table 1. Predictive factors for BCVA ≤35 ETDRS letters (0.1) after 12 months of treatment with ranibizumab in age-related macular degeneration with choroidal neovascularization.

Age ≥80 versus age 35 letters Month 3, VA ≤35 letters versus >35 letters Baseline and month 3, VA ≤35 letters versus >35 letters

N

OR (CI95)

p-Value

149 98 23 16 27

0.8 0.6 10.6 16.3 91.6

0.519 0.264

Implementation studies of ranibizumab for neovascular age-related macular degeneration.

The pathogenesis of AMD is associated with age changes plus pathological changes involving oxidative stress and an altered inflammatory response leadi...
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