Acta Ophthalmologica 2014
Circulating anti-retinal antibodies in response to anti-angiogenic therapy in exudative age-related macular degeneration Agnieszka Kubicka-Trzaz ska,1 Joanna Wila nska,2 Bo_zena Romanowska-Dixon1 and Marek Sanak2 1
Department of Ophthalmology and Ocular Oncology, Jagiellonian University Medical College, Krakow, Poland Division of Molecular Biology and Clinical Genetics, Jagiellonian University Medical College, Krakow, Poland
2
ABSTRACT. Purpose: To determine changes in anti-retinal antibodies (ARAs) during antiVEGF therapy in patients with exudative age-related macular degeneration (AMD) and to assess the correlations between ARAs and disease activity. Methods: The study comprised 98 patients treated with intravitreal bevacizumab. The ophthalmic examination included best corrected visual acuity (BCVA), slit lamp biomicroscopy, fundoscopy, fluorescein angiography (FA), and optical coherence tomography (OCT). Serum ARAs levels were assessed by indirect immunofluorescence (IIF) on normal monkey retina substrate. These studies were repeated at 4 week intervals within 8 months of a follow-up. The sera of 50 sex- and age-matched healthy subjects were used as controls. Results: At baseline examination, 94 (95.5%) of the 98 patients were positive for ARAs. The ARAs titres were significantly higher (p = 0.0000) than in controls. A positive correlation was found between titres of ARAs and the diameter of choroidal neovascularization (CNV) as measured by FA (p = 0.0000), and central retinal thickness (CRT) assessed by OCT (p = 0.0000). A positive correlation was also found between the diameter of CNV, CRT and the complexity of circulating ARAs. Following treatment all patients demonstrated significant decrease in ARAs levels as well as improvement of BCVA, reduction of subretinal fluid on OCT and decreased leakage on FA. Conclusion: Changes in serum ARAs levels occurred in parallel with clinical outcomes of anti-VEGF therapy. Treatment reduced serum levels of ARAs, with the greatest reduction occurring during the ‘loading’ phase. This study demonstrated that ARAs may act as a serum biomarker of the efficacy of anti-VEGF therapy. Key words: age-related macular degeneration – anti-retinal autoantibodies – anti-vascular endothelial growth factor therapy – bevacizumab
Acta Ophthalmol. 2014: 92: e610–e614 ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
doi: 10.1111/aos.12435
Introduction Age-related macular degeneration (AMD)is the leading cause of blindness in people over the age of 50 years in developed countries. Despite extensive
e610
experimental and clinical studies, its pathogenesis is still not fully understood (Fine et al. 2000). However, there is growing evidence that autoimmunity against retinal antigens might
be involved in the disease process (Karlstetter & Langmann 2012; Yu et al. 2012; Whitcup et al. 2013). It has been known that ocular diseases in which inflammatory and autoimmune components play an important role, may be associated with the presence of circulating ARAs. Several studies have shown that ARAs are present in the sera of patients with exudative AMD (Penfold et al. 1990; Gurne et al. 1991; Patel et al. 2005; Cherepanoff et al. 2006; Joachim et al. 2007; KubickaTrzaz ska & Wila nska 2009; KubickaTrzaz ska et al. 2012). The precise role of these antibodies in the disease process is unclear. It has been suggested that circulating ARAs may be an epiphenomenon developing in response to retinal damage or alternatively, they could be involved in the pathogenesis of AMD (Penfold et al. 1990; Gurne et al. 1991; Patel et al. 2005). Although the presence of serum ARAs in patients with AMD is thought to be a significant disease marker, to our knowledge no data on the behaviour of circulating ARAs in response to anti-VEGF therapy is currently available. The aim of this study was to assess the presence and profile of serum ARAs in patients with exudative AMD, and to establish the correlations between the immunofluorescence patterns and levels of ARAs, and disease activity. The second aim was to determine changes in ARAs during intravitreal bevacizumab therapy and to assess the correlations between circulating ARAs and the characteristics of disease activity,
Acta Ophthalmologica 2014
notably BCVA and CRT. This study also attempts to determine whether ARAs could act as a serum biomarker of the efficacy of anti-VEGF therapy.
Materials and Methods Ninety eight patients (108 eyes) with exudative AMD were treated with intravitreal bevacizumab (Avastin, Roche Pharma AG, Mannheim, Germany). There were 38 males (39%) and 60 females (61%) aged 55–93 years (mean: 76 years). Exclusion criteria included the following: other retinal diseases, uveitis and/or retinal vasculitis, paraneoplastic retinopathies, glaucoma, diabetes and diabetic retinopathy, the use of systemic steroid and/or immunosupressive agents and associated autoimmune systemic disease. Fifty age- and sex-matched healthy subjects with no history of AMD or any chronic eye disease, requiring surgery for agerelated cataracts served as controls. The control group consisted of 20 males (40%) and 30 females (60%) aged 53– 91 years (mean: 75.2 years). Baseline examination in all cases included BCVA, slit lamp and fundus examination, OCT and FA. Follow-up examination was performed monthly and was the same as the baseline examination, except for angiography. All patients were treated with intravitreal bevacizumab 1.25 mg/0.05 ml every 4 weeks during the first 3 months (‘loading’ phase) and then the decision concerning reinjection was based on the results of BCVA and OCT (‘maintenance’ phase). In all patients serum ARAs were determined using indirect immunofluorescence (IIF) on frozen sections of normal monkey retina as antigens substrate and fluorescein isothiocyanate labelled goat’s anti-human IgA, G, M serum as the secondary antibody (Euroimmun AG, Lubeck, Germany). Blood samples were collected from all subjects at the beginning of the study, and thereafter at monthly intervals. The samples were centrifuged at 1000 g for 10 min and stored at 80°C in aliquots until tested by IIF. The thawed sera from patients and controls were diluted in a series of titres: 1:10, 1:20, 1:40, 1:80 etc., according to the standard protocol attached by the producer of the kit. IIF incubations times and washings were as recommended by the manufacturer’s
protocol. Control sera were also processed. All immunohistochemical slides were evaluated by two independent observers using fluorescence microscopy (Axioscope fl equipped with HBO 100 lamp). The independent observers, who were unaware of the diagnosis and the stage of disease, recorded the level of staining in all layers of the retina as well as the highest serum dilution which still gave a positive result. During the follow-up period in patients whose sera demonstrated more than one type of immunofluorescence pattern of reactivity against retinal tissue, only that which occurred at the highest titres at the baseline examination was further evaluated and used to monitor the effects of anti-VEGF therapy. Sera with a positive reaction within the outer and inner nuclear layers of retina were screened for the presence of antinuclear antibodies (ANA) using the IIF method and HEp-2 cell lines as a substrate (Euroimmun AG). Serum ARAs were assessed at the beginning of the study and then immunohistochemical studies were repeated at four weekly intervals. The total follow-up period was 8 months. Approval by the Jagiellonian University Ethical Committee (KBET/165/ B/2009) was obtained as well as informed consent of all participating subjects. Statistical analysis was performed using the nonparametric Mann-Whitney U test for ordered data and Chi-square test for frequencies. The Pearson’s linear correlation coefficient was used to measure the strength of dependence between two variables. Type I statistic error p-value
Circulating anti-retinal antibodies in response to anti-angiogenic therapy in exudative age-related macular degeneration Agnieszka Kubicka-Trzaz ska,1 Joanna Wila nska,2 Bo_zena Romanowska-Dixon1 and Marek Sanak2 1
Department of Ophthalmology and Ocular Oncology, Jagiellonian University Medical College, Krakow, Poland Division of Molecular Biology and Clinical Genetics, Jagiellonian University Medical College, Krakow, Poland
2
ABSTRACT. Purpose: To determine changes in anti-retinal antibodies (ARAs) during antiVEGF therapy in patients with exudative age-related macular degeneration (AMD) and to assess the correlations between ARAs and disease activity. Methods: The study comprised 98 patients treated with intravitreal bevacizumab. The ophthalmic examination included best corrected visual acuity (BCVA), slit lamp biomicroscopy, fundoscopy, fluorescein angiography (FA), and optical coherence tomography (OCT). Serum ARAs levels were assessed by indirect immunofluorescence (IIF) on normal monkey retina substrate. These studies were repeated at 4 week intervals within 8 months of a follow-up. The sera of 50 sex- and age-matched healthy subjects were used as controls. Results: At baseline examination, 94 (95.5%) of the 98 patients were positive for ARAs. The ARAs titres were significantly higher (p = 0.0000) than in controls. A positive correlation was found between titres of ARAs and the diameter of choroidal neovascularization (CNV) as measured by FA (p = 0.0000), and central retinal thickness (CRT) assessed by OCT (p = 0.0000). A positive correlation was also found between the diameter of CNV, CRT and the complexity of circulating ARAs. Following treatment all patients demonstrated significant decrease in ARAs levels as well as improvement of BCVA, reduction of subretinal fluid on OCT and decreased leakage on FA. Conclusion: Changes in serum ARAs levels occurred in parallel with clinical outcomes of anti-VEGF therapy. Treatment reduced serum levels of ARAs, with the greatest reduction occurring during the ‘loading’ phase. This study demonstrated that ARAs may act as a serum biomarker of the efficacy of anti-VEGF therapy. Key words: age-related macular degeneration – anti-retinal autoantibodies – anti-vascular endothelial growth factor therapy – bevacizumab
Acta Ophthalmol. 2014: 92: e610–e614 ª 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
doi: 10.1111/aos.12435
Introduction Age-related macular degeneration (AMD)is the leading cause of blindness in people over the age of 50 years in developed countries. Despite extensive
e610
experimental and clinical studies, its pathogenesis is still not fully understood (Fine et al. 2000). However, there is growing evidence that autoimmunity against retinal antigens might
be involved in the disease process (Karlstetter & Langmann 2012; Yu et al. 2012; Whitcup et al. 2013). It has been known that ocular diseases in which inflammatory and autoimmune components play an important role, may be associated with the presence of circulating ARAs. Several studies have shown that ARAs are present in the sera of patients with exudative AMD (Penfold et al. 1990; Gurne et al. 1991; Patel et al. 2005; Cherepanoff et al. 2006; Joachim et al. 2007; KubickaTrzaz ska & Wila nska 2009; KubickaTrzaz ska et al. 2012). The precise role of these antibodies in the disease process is unclear. It has been suggested that circulating ARAs may be an epiphenomenon developing in response to retinal damage or alternatively, they could be involved in the pathogenesis of AMD (Penfold et al. 1990; Gurne et al. 1991; Patel et al. 2005). Although the presence of serum ARAs in patients with AMD is thought to be a significant disease marker, to our knowledge no data on the behaviour of circulating ARAs in response to anti-VEGF therapy is currently available. The aim of this study was to assess the presence and profile of serum ARAs in patients with exudative AMD, and to establish the correlations between the immunofluorescence patterns and levels of ARAs, and disease activity. The second aim was to determine changes in ARAs during intravitreal bevacizumab therapy and to assess the correlations between circulating ARAs and the characteristics of disease activity,
Acta Ophthalmologica 2014
notably BCVA and CRT. This study also attempts to determine whether ARAs could act as a serum biomarker of the efficacy of anti-VEGF therapy.
Materials and Methods Ninety eight patients (108 eyes) with exudative AMD were treated with intravitreal bevacizumab (Avastin, Roche Pharma AG, Mannheim, Germany). There were 38 males (39%) and 60 females (61%) aged 55–93 years (mean: 76 years). Exclusion criteria included the following: other retinal diseases, uveitis and/or retinal vasculitis, paraneoplastic retinopathies, glaucoma, diabetes and diabetic retinopathy, the use of systemic steroid and/or immunosupressive agents and associated autoimmune systemic disease. Fifty age- and sex-matched healthy subjects with no history of AMD or any chronic eye disease, requiring surgery for agerelated cataracts served as controls. The control group consisted of 20 males (40%) and 30 females (60%) aged 53– 91 years (mean: 75.2 years). Baseline examination in all cases included BCVA, slit lamp and fundus examination, OCT and FA. Follow-up examination was performed monthly and was the same as the baseline examination, except for angiography. All patients were treated with intravitreal bevacizumab 1.25 mg/0.05 ml every 4 weeks during the first 3 months (‘loading’ phase) and then the decision concerning reinjection was based on the results of BCVA and OCT (‘maintenance’ phase). In all patients serum ARAs were determined using indirect immunofluorescence (IIF) on frozen sections of normal monkey retina as antigens substrate and fluorescein isothiocyanate labelled goat’s anti-human IgA, G, M serum as the secondary antibody (Euroimmun AG, Lubeck, Germany). Blood samples were collected from all subjects at the beginning of the study, and thereafter at monthly intervals. The samples were centrifuged at 1000 g for 10 min and stored at 80°C in aliquots until tested by IIF. The thawed sera from patients and controls were diluted in a series of titres: 1:10, 1:20, 1:40, 1:80 etc., according to the standard protocol attached by the producer of the kit. IIF incubations times and washings were as recommended by the manufacturer’s
protocol. Control sera were also processed. All immunohistochemical slides were evaluated by two independent observers using fluorescence microscopy (Axioscope fl equipped with HBO 100 lamp). The independent observers, who were unaware of the diagnosis and the stage of disease, recorded the level of staining in all layers of the retina as well as the highest serum dilution which still gave a positive result. During the follow-up period in patients whose sera demonstrated more than one type of immunofluorescence pattern of reactivity against retinal tissue, only that which occurred at the highest titres at the baseline examination was further evaluated and used to monitor the effects of anti-VEGF therapy. Sera with a positive reaction within the outer and inner nuclear layers of retina were screened for the presence of antinuclear antibodies (ANA) using the IIF method and HEp-2 cell lines as a substrate (Euroimmun AG). Serum ARAs were assessed at the beginning of the study and then immunohistochemical studies were repeated at four weekly intervals. The total follow-up period was 8 months. Approval by the Jagiellonian University Ethical Committee (KBET/165/ B/2009) was obtained as well as informed consent of all participating subjects. Statistical analysis was performed using the nonparametric Mann-Whitney U test for ordered data and Chi-square test for frequencies. The Pearson’s linear correlation coefficient was used to measure the strength of dependence between two variables. Type I statistic error p-value
