
==== Front
Ophthalmol Ther
Ophthalmol Ther
Ophthalmology and Therapy
2193-8245
2193-6528
Springer Healthcare Cheshire

39133375
1010
10.1007/s40123-024-01010-0
Original Research
Factors Affecting Disease Stability After Intravitreal Brolucizumab Injection for Refractory Neovascular Age-Related Macular Degeneration
Kim Yung-Hwi 12
Moon Tae Kyu 1
http://orcid.org/0000-0001-8480-8952
Ji Yong-Sok yongsok.ji@jnu.ac.kr

1
1 https://ror.org/05kzjxq56 grid.14005.30 0000 0001 0356 9399 Department of Ophthalmology and Research Institute of Medical Sciences, Chonnam National University Medical School and Hospital, 42 Jebong-ro, Dong-gu, Gwangju, 61469 Republic of Korea
2 MSC Research Institute, Parangsae Eye Clinic, Gwangju, Republic of Korea
12 8 2024
12 8 2024
10 2024
13 10 26792695
10 5 2024
24 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc/4.0/.
Introduction

The purpose of this study is to identify the factors affecting neovascular age-related macular degeneration (nAMD) disease stability after brolucizumab treatment.

Methods

We retrospectively analyzed the medical records of 31 patients (31 eyes) with recalcitrant nAMD who were switched to brolucizumab after conventional anti-vascular endothelial growth factor (VEGF) treatment. We divided patients into two groups by treatment extension (TE) period: group 1 with TE < 12 weeks (N = 16) and group 2 with TE ≥ 12 weeks (N = 15). We compared outcomes between the groups at 2, 4, 8, and 12 weeks, including morphological characteristics of choroidal neovascularization (CNV). Logistic regression analysis identified factors associated with TE ≥ 12 weeks.

Results

Group 2 had a significantly greater proportion of patients with dry macula (subretinal and intraretinal fluids absent) than group 1 (60 vs. 12.5%) at 2 weeks (P < 0.05). Best-corrected visual acuity (BCVA) and subfoveal choroidal thickness (SFCT) did not differ significantly between groups at all timepoints. Central subfield retinal thickness (CST) was significantly lower in group 2 at 2 (237.1 vs. 280.8 μm; P < 0.05), 4 (224.0 vs. 262.9 μm; P < 0.05), and 8 weeks (216.8 vs. 331.1 μm; P < 0.05). Group 2 had less vessel area (0.63 vs. 1.27 mm2; P < 0.05) and total vessel length (0.22 vs. 0.42 mm; P < 0.05). Choriocapillaris flow deficit (CCFd) was significantly lower in group 2 (42.7 vs. 48.2%; P < 0.05). Dry macula at 2 weeks (odds ratio [OR] = 8.3; P < 0.05) and a lower CCFd (OR = 0.73; P < 0.05) were associated with TE ≥ 12 weeks.

Conclusions

Early fluid-free status after switching to brolucizumab and choriocapillary function around CNV were prognostic factors for disease stability in nAMD refractory to anti-VEGF treatment.

Keywords

Refractory
nAMD
CNV
Brolucizumab
Factor
Treatment interval
Choriocapillary
Stability
OCT
OCTA
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pmcKey Summary Points

Why carry out this study?	
Brolucizumab had a higher probability of extending the treatment interval to 12 weeks or more compared to aflibercept when treating neovascular age-related macular degeneration (nAMD). However, concerns still remain that, in quite a few patients, disease stability does not last more than 8 weeks, even after brolucizumab injection.	
Using optical coherence tomography (OCT) and OCT angiography (OCTA) imaging analysis, we hypothesized that morphological factors of choroidal neovascularization (CNV) would affect disease stability after switching to brolucizumab in patients with refractory nAMD to previous anti-vascular endothelial growth factor (anti-VEGF) treatment.	
What was learned from this study?	
Early fluid-free status after switching to brolucizumab and choriocapillary function around CNV were important factors determining the stability of nAMD refractory to treatment with other anti-VEGFs.	
Therefore, when switching to brolucizumab in patients with refractory nAMD, treatment options should be personalized depending on the morphological characteristics of CNV and the response to brolucizumab.	

Introduction

Neovascular age-related macular degeneration (nAMD) is a vision-threatening disease among elderly individuals with high prevalence in developed countries [1, 2]. Intravitreal injections of anti-vascular endothelial growth factor (VEGF) have been used in the past few decades and significantly preserve vision and reduce the risk of blindness in nAMD [3–7]. However, response to anti-VEGF treatment varies among individuals, with some patients requiring prolonged treatment to maintain the therapeutic effect [8–11].

Anti-VEGF treatment with bevacizumab (Avastin®; Genentech, San Francisco, CA/Roche, Basel, Switzerland), ranibizumab (Lucentis®; Genentech, San Francisco, CA/Roche, Basel, Switzerland), and aflibercept (Eylea®, Regeneron, Tarrytown, NY) has been widely administered to treat nAMD. Many ophthalmologists use either a treat-and-extend (T&E) or a treat-as-needed (pro re nata [PRN]) regimen to minimize the number of injections and treatment burden [8, 12–14]. Despite these treatment options, controlling disease activity in nAMD is challenging in the real-world setting [15–18].

Brolucizumab (Beovu®, Novartis Pharma GmbH, Nurenberg, Germany) has been introduced as a new anti-VEGF agent for nAMD treatment. Brolucizumab is a single-chain antibody fragment with a molecular weight of 26 kDa and is characterized by high affinity to all forms of VEGF-A and superior tissue penetration [19–21]. HAWK and HARRIER trials have shown that brolucizumab provided visual improvement comparable to that of aflibercept and better fluid resolution. In addition, brolucizumab treatment had a higher probability of extending the treatment interval to 12 weeks compared to aflibercept treatment, promising a lower burden on the eyes of patients with nAMD [19, 20].

The fluid-free state (defined as the absence of intraretinal fluid [IRF] and/or subretinal fluid [SRF]) and the potential to extend the treatment interval are two crucial indicators of treatment response or disease stability in nAMD after intravitreal anti-VEGF injections. Previous studies have shown that baseline optical coherence tomography (OCT) features such as the presence of IRF and SRF can predict persistent disease activity [17]. By analyzing OCT and OCT angiography (OCTA) of choroidal neovascularization (CNV), the authors hypothesize that morphological factor for disease stability would be important to eventually predict the future treatment direction and reduce the treatment burden for patients with nAMD.

To the best of our knowledge, no prior study on factors associated with a treatment interval of ≥ 12 weeks after intravitreal brolucizumab injections in eyes with refractory nAMD has been published. Therefore, we aimed to determine the factors that affect the treatment extension (TE) after switching to brolucizumab in patients with nAMD refractory to previous anti-VEGF treatment.

Methods

This retrospective study analyzed the medical records of patients treated between May 2021 and July 2022 at the Department of Ophthalmology at Chonnam National University Hospital. It adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Boards of Chonnam National University Hospital (IRB No. CNUH-2023-035).

Patients

All patients with nAMD who had received repeat treatment with anti-VEGF agents, including bevacizumab, ranibizumab and aflibercept, and then received a 6-mg intravitreal injection of brolucizumab were enrolled in this study. The diagnosis of nAMD was as follows: active CNV confirmed by fluorescein angiography (FA) and indocyanine angiography (ICG) /SRF or IRF or sub-retinal pigment epithelial (RPE) fluid confirmed by OCT: (1) Type 1 CNV was diagnosed when FA indicates occult leakage and sub-RPE CNV on OCT. Polypoidal choroidal vasculopathy (PCV) was diagnosed when aneurysmal dilations of the branching vascular network is visible on FA. (2) Type 2 CNV was diagnosed when FA indicates classic CNV and the presence of CNV beyond the RPE on OCT. (3) Type 3 CNV was diagnosed when retinal angiomatous proliferation (RAP) occurs within the neurosensory retina and invades into the subretinal space. Refractory or recalcitrant nAMD despite at least three consecutive anti-VEGF injections was defined as persistent fluid (one or more of SRF, IRF, or sub-RPE fluid) and/or early recurrence (TE < 8 weeks). These patients with refractory nAMD were switched to brolucizumab from previous anti-VEGFs. The authors defined a stability of treatment response to brolucizumab as TE of ≥ 12 weeks. Group 1 was defined as eyes which fluid recurred before 12 weeks and the treatment interval could not be extended by more than 12 weeks (TE < 12 weeks). Group 2 was defined as eyes which fluid was absent for more than 12 weeks after the first injection of brolucizumab and the treatment interval could be extended by more than 12 weeks (TE ≥ 12 weeks). All patients underwent T&E regimen after brolucizumab switching. All eyes in group 1 received a second intravitreal injection at 8 weeks after switching to brolucizumab. The day of the first intravitreal brolucizumab injection was considered the baseline visit. Patients visited at 2, 4, 8, and 12 weeks after injection. During each visit, a detailed ocular examination was conducted, including measurement of best-corrected visual acuity (BCVA), intraocular pressure, and examination of the anterior segment using a slit-lamp and the fundus examination following pupil dilation using an indirect ophthalmoscope. Retinal imaging was performed at each visit using spectral-domain OCT (SD-OCT) (SPECTRALIS HRA2 + OCT: Heidelberg Engineering, Heidelberg, Germany) and swept-source OCTA (SS-OCTA) (PLEX Elite 9000: Carl Zeiss Meditec, Dublin, CA, USA). Eyes with geographic atrophy, disciform scar, and other ocular diseases such as diabetic retinopathy, uveitis, glaucoma, and any chorioretinal diseases were excluded.

SD-OCT Imaging Analysis

Extended depth imaging (EDI)-OCT B-scan images were acquired with central 30° × 20° near-infrared reflectance (λ = 820 nm, automatic real time, at least 15 frames). Using automated segmentation of the Heidelberg Eye Explorer software, the authors evaluated the following OCT parameters: (1) central subfield retinal thickness (CST; defined as the mean retinal thickness between the internal limiting membrane and the Bruch’s membrane of the circular area within a 1-mm diameter around the center of the fovea), (2) macular volume (MV; defined as the mean volume of the retina in a circular area within a 6-mm diameter around the fovea), (3) foveal volume (FV; defined as the mean volume of the fovea in a circular area within a 1-mm diameter around the center of the fovea); and (4) subfoveal choroidal thickness (SFCT; defined as the distance from the choroidal-RPE junction to the choroidal-scleral junction. For SFCT measurement, the average of the values measured by two investigators (Y.H.K and T.K.M) was used. For optimal quantifications, if necessary, EDI-OCT B-scans were manually adjusted after careful consideration and agreement among the investigators.

Characteristics of Choroidal Neovascularization (CNV)

Morphological features of CNV in refractory nAMD were assessed using OCTA, with the images acquired at a wavelength of 1060 nm and at 100,000 A scans/s, obtaining two successive B scans in a 6 × 6-mm image with 500 A scans each. To minimize segmentation error, the authors evaluated OCTA images at the most fluid-free time point before switching to brolucizumab. The most fluid-free state was defined as the absence or minimal SRF and IRF on OCT examination following anti-VEGF injection at any time prior to switching to brolucizumab. The automated choriocapillaris slab in which CNV was best visible was defined as a 10-μm-thick slab with an inner and outer boundary located 21 μm and 31 μm below the anatomic location of the Bruch’s membrane, respectively. In this slab, CNV was most visible. Any segmentation error was manually corrected after censoring every case of automatic segmentation to ensure accurate referencing of the Bruch’s membrane. Projection artifacts were removed using the embedded software of PLEX Elite 9000. All en face OCTA images of the choriocapillaris were analyzed using a public domain software (Fiji application version [http://imageJ.net/software/fiji] of ImageJ [National Institute of Health, Bethesda, MD, USA]) [22]. All images were compensated for signal loss and shadowing effects [23]. The outer boundary of the CNV was manually outlined and adjusted after careful agreement among the investigators (Y.H.K and T.K.M). The area inside the boundary was measured to calculate the CNV area. After binarization of the CNV vessels by the Otsu’s thresholding method (Fig. 1) [24], the vessel area, vessel density, total vessel length, and vessel length density were calculated using the “Vessel density” plugin of ImageJ. To quantify CNV complexity, fractal dimension and lacunarity were analyzed using the “FracLac” plugin of ImageJ [25].Fig. 1 Example en face choriocapillaris optical coherence tomography (OCTA) image. Choroidal neovascularization (CNV) lesion manually delineated from the original choriocapillaris slab (A) after binarization using the Otsu’s thresholding method (B). OCTA image after binarization using the Phansalkar’s thresholding method (C). After masking the superficial and large vessels from the binarized image, CNV was highlighted with a yellow line (arrowhead). Concentric 500-µm (arrow) and 1000-µm rings (dotted arrow) were created from the edge of the CNV lesion using the “Enlarge” command in ImageJ. The choriocapillaris flow deficit (CCFd) was then calculated in the area from the border of CNV lesion to the border of the 500- or 1000-μm ring by the “Analyze Particles” command in ImageJ (D)

Choroidal Vascularity Index (CVI) and Choriocapillaris Flow Deficit (CCFd) Analysis

After mounting the 6 × 6-mm B-scan SD-OCT images on ImageJ, the choroidal region could be drawn and adjusted manually using the polygon tool upon agreement among the investigators. The upper border of the region of interest was traced along the choroidal-RPE junction and the lower border along the choroidal–scleral junction. This choroidal area was added to the Region of Interest Manager Tool. The images were converted to 8-bit images to allow the application of Niblack’s thresholding for binarization [26]. As described in the authors’ previous study [27], the choroidal vascularity index (CVI, %), defined as the vascular area divided by the total choroidal area in the selected region of interest, was then calculated.

The choriocapillaris flow deficit (CCFd, %) was defined as the percentage of pixels representing flow deficits relative to all the pixels within a region of interest. CCFd was quantified in a 6 × 6-mm binarized choriocapillaris slab of the OCTA image and specifically analyzed in two concentric 500- and 1000-μm rings outside the CNV lesion (Fig. 1). Phansalkar’s thresholding using a three-pixel radius of 17.6 μm was used for binarization of CCFd [28–31]. A 500-μm ring was created from the edge of the CNV lesion using the “Edit-Selection-Enlarge” command in ImageJ. It automatically creates a border resembling the contour of the CNV lesion, and the selected border of CNV was enlarged by 85.3 pixels corresponding to a 500-μm distance (1024 × 1024 pixels). A 1000-μm ring was created using the same method (enlarged by 170.6 pixels). The CCFd was then calculated as a percentage of the analyzed area (from the border of CNV lesion to the border of the 500- or 1000-μm ring) using the “Analyze Particles” command in ImageJ [31, 32].

Statistical Analysis

Statistical analysis was performed using Statistical Package for Social Sciences (version 22.0) for Windows (SPSS Inc., Chicago, IL, USA). The normal distribution for all variables was assessed using the Kolmogorov–Smirnov test. Data are presented as means ± standard deviations. Differences between the groups for continuous variables were assessed using Student’ t test or Mann–Whitney U test. Differences between the groups for categorical variables were assessed using the Pearson’s chi-square test. Differences between baseline and each timepoint were evaluated using the paired T test or Wilcoxon signed-rank test. Binary logistic regression analysis was performed for factor assessment. P values < 0.05 were considered statistically significant.

Results

Thirty-one eyes with refractory nAMD (22 men, nine women) were included in this study. The mean age was 73.56 ± 6.46 and 70.87 ± 9.33 (P = 0.355) in group 1 and group 2, respectively. The proportion of polypoidal choroidal vasculopathy (PCV) was 18.8% in group 1 and 53.3% in group 2 (P = 0.066). In group 1, eight eyes (50.0%) had type I (occult) CNV, and five eyes (31.3%) had type II (classic) CNV. Only type I (occult) CNV was observed in group 2 (seven eyes, 46.7%). Baseline characteristics did not significantly differ between the groups (Table 1).Table 1 Baseline characteristics of the nAMD patient refractory to previous intravitreal injections of anti-VEGF

	Group 1 (TE < 12 week)
(N = 16)	Group 2 (TE ≥ 12 week)
(N = 15)	P value	
Age	73.56 ± 6.46	70.87 ± 9.33	0.355	
Sex, M/F (%)	12 (75)/4 (25)	10 (66.7)/5 (33.3)	0.704	
HTN, ± (%)	7 (43.8)/9 (56.3)	9 (60.0)/6 (40.0)	0.479	
DM, ± (%)	3 (18.8)/13 (81.3)	4 (26.7)/11 (73.3)	0.685	
CVA history, ± (%)	5 (31.3)/11 (68.8)	4 (26.7)/11 (73.3)	0.371	
Lens status, phakia/pseudophakia (%)	9 (56.3)/7 (43.8)	5 (33.3)/10 (66.7)	0.285	
AMD/PCV (%)	13 (81.3)/3 (18.8)	7 (46.7)/8 (53.3)	0.066	
AMD type (%)	
 Type I (occult)	8 (50)	7 (46.7)	0.026*	
 Type II (classic)	5 (31.3)	0 (0)	
 Type III (RAP)	0 (0)	0 (0)	
 PCV	3 (18.8)	8 (53.3)	
Number of previous anti-VEGF injection	21.25 ± 10.25	18.60 ± 7.86	0.428	
Previous IOI history after injection, ± (%)	4 (25.0)/12 (75.0)	0 (0)/15 (100)	0.101	
nAMD neovascular age-related macular degeneration, VEGF vascular endothelial growth factor, TE extension of treatment interval, M male, F female, HTN hypertension, DM diabetes mellitus, CVA cerebrovascular accident, PCV polypoidal choroidal vasculopathy, RAP retinal angiomatous proliferation, IOI intraocular inflammation

*P < 0.05, compared between the groups

The presence of SRF, IRF, or sub-RPE fluid was analyzed at all timepoints. At baseline, group 1 had 13 (81.3%) and group 2 had 13 (86.7%) eyes with SRF. After switching to brolucizumab, the proportion of eyes with SRF decreased gradually by 4 weeks. At 8 weeks, group 1 had a significantly higher proportion of eyes with SRF (60.0 vs. 9.1% in group 2; P < 0.05; Fig. 2A). At baseline, group 1 and 2 had eight (50.0%) and six (40.0%) eyes with IRF, respectively. After switching to brolucizumab, IRF was not observed in group 2. Group 1 had one (6.25%) eye with IRF at 2 weeks, which increased gradually by 8 weeks. At 8 weeks, group 1 had a significantly higher proportion of eyes with IRF than in group 2 (40.0 vs. 0.0%; P < 0.05; Fig. 2B). At baseline, group 1 and two had 13 (81.3%) and 13 (86.7%) eyes with sub-RPE fluid, respectively. After switching to brolucizumab, the proportion of eyes with sub-RPE fluid decreased gradually by 4 weeks. At 8 weeks, group 1 had a significantly higher proportion of eyes with sub-RPE fluid (80.0 vs. 9.1% in group 2; P < 0.05; Fig. 2C). At 2 weeks after switching to brolucizumab, 12.5% and 60.0% eyes had dry macula in group 1 and group 2, respectively (P < 0.05). At 8 weeks, 13.3% and 90.9% eyes had dry macula in group 1 and group 2, respectively (P < 0.05; Fig. 2D). The mean BCVA of all patients was 0.33 ± 0.39 LogMAR at baseline. By 12 weeks after switching to brolucizumab, the mean BCVA decreased albeit insignificantly. Mean BCVA did not differ between the groups at all timepoints (all P > 0.05; Fig. 3).Fig. 2 The proportion of eyes with fluid was compared between the groups. Compared to group 2, group 1 had a significantly higher proportion of eyes with subretinal fluid (SRF) (A), intraretinal fluid (IRF) (B), and sub-retinal pigment epithelial (RPE) fluid (C) at 8 weeks after switching to brolucizumab in refractory neovascular age-related macular degeneration (nAMD). The percentage of eyes with dry macula was significantly higher in group 2 than in group 1 at 2 and 8 weeks (D). (*P < 0.05, compared between groups)

Fig. 3 Changes in best-corrected visual acuity (BCVA) after switching to brolucizumab for refractory nAMD. Compared to the baseline (before switching to brolucizumab), BCVA improvement was statistically insignificant at all timepoints (all P > 0.05) (A). No significant differences were observed between the groups (all P > 0.05) (B). (*P < 0.05, compared to the baseline; †P < 0.05, compared between groups)

Compared to the baseline (370.9 ± 134.8 μm), the mean CST of all patients significantly decreased at all timepoints (2 weeks: 262.4 ± 54.4 μm; 4 weeks: 246.5 ± 45.0 μm; 8 weeks: 282.7 ± 94.0 μm; 12 weeks: 259.6 ± 70.6 μm; all P < 0.05; Fig. 4A). The mean CST in group 2 was significantly less than that in group 1 at 2 weeks (280.8 ± 64.2 vs. 238.4 ± 31.5 μm), 4 weeks (262.9 ± 54.1 vs. 225.6 ± 23.0 μm), and 8 weeks (331.1 ± 95.7 vs. 218.7 ± 33.8 μm) after switching to brolucizumab (all P < 0.05; Fig. 4B). Compared to the baseline (243.3 ± 134.8 μm), the mean SFCT of all patients significantly decreased at 2 weeks (200.8 ± 94.1 μm), 4 weeks (182.8 ± 83.6 μm), 8 weeks (192.6 ± 85.1 μm), and 12 weeks (203.6 ± 83.5 μm; all P < 0.05; Fig. 4C). Mean SFCT did not significantly differ between groups at all timepoints (Fig. 4D).Fig. 4 Changes in central subfield retinal thickness (CST) and subfoveal choroidal thickness (SFCT) after switching to brolucizumab in refractory nAMD. Compared to the baseline (before switching to brolucizumab), CST (A) and SFCT (C) significantly decreased at all timepoints. Compared to the baseline, CST in group 1 and group 2 were significantly lower at all timepoints, except for group 1 at 8 weeks. CST was significantly lower in group 2 than in group 1 at 2, 4, and 8 weeks (B). Compared to the baseline, SFCT in group 1 and group 2 significantly decreased at all timepoints, except in group 1 at 2 weeks (D). (*P < 0.05, compared to the baseline; †P < 0.05, compared between groups)

Compared to the baseline (8.95 ± 1.05 mm3), the mean MV of all participants significantly decreased at 2 weeks (8.13 ± 0.56 mm3), 4 weeks (7.96 ± 0.44 mm3), 8 weeks (8.12 ± 0.59 mm3), and 12 weeks (7.99 ± 0.55 mm3; all P < 0.05; Fig. 5A). The mean MV in group 2 was significantly lower than that in group 1 at 8 weeks (8.41 ± 0.54 vs. 7.72 ± 0.34 mm3) after switching to brolucizumab (P < 0.05; Fig. 5B). Compared to the baseline (0.27 ± 0.07 mm3), the mean FV of all patients significantly decreased at 2 weeks (0.21 ± 0.04 mm3), 4 weeks (0.20 ± 0.04 mm3), 8 weeks (0.22 ± 0.07 mm3), and 12 weeks (0.21 ± 0.06 mm3; all P < 0.05; Fig. 5C). The mean FV in group 2 was significantly less than that in group 1 at 2 weeks (0.22 ± 0.05 vs. 0.19 ± 0.03 mm3) and 8 weeks (0.26 ± 0.07 vs. 0.18 ± 0.03 mm3) after switching to brolucizumab (P < 0.05; Fig. 5D).Fig. 5 Changes in macular volume (MV) and foveal volume (FV) after switching to brolucizumab in refractory nAMD. Compared to the baseline (before switching to brolucizumab), MV (A) and FV (C) significantly decreased at all timepoints. Compared to the baseline, MV in group 1 and group 2 were significantly lower at all timepoints. MV was significantly lower in group 2 than in group 1 at 8 weeks (B). Compared to the baseline, FV in group 1 and group 2 were significantly lower at all timepoints, except for group 1 at 8 weeks. FV was significantly lower in group 2 than in group 1 at 2 and 8 weeks (D). (*P < 0.05, compared to the baseline; †P < 0.05, compared between groups)

The mean CVI of all patients was 62.0 ± 4.0% at baseline. The mean CVI before and after switching to brolucizumab did not differ. The mean CVI in group 2 was significantly higher than that in group 1 at 8 weeks (61.6 ± 2.0 vs. 64.5 ± 2.4%) and 12 weeks (60.7 ± 2.3 vs. 63.9 ± 3.4%; all P < 0.05; Fig. 6).Fig. 6 Changes in choroidal vascularity index (CVI) after switching to brolucizumab in refractory neovascular age-related macular degeneration (nAMD). Compared to the baseline (before switching to brolucizumab), no significant changes were observed at all timepoints (all P > 0.05) in all patients (A). Compared to the baseline, CVI in group 1 and group 2 did not differ significantly at all timepoints (all P > 0.05). CVI was significantly higher in group 2 than in group 1 at 8 and 12 weeks (B). (†P < 0.05, compared between groups)

Table 2 summarizes the analysis of CNV characteristics. Group 2 had a smaller vessel area than group 1 (0.63 ± 0.54 mm2 vs. 1.27 ± 0.89 mm2; P = 0.031). Group 1 had a longer total vessel length than group 2 (0.42 ± 0.28 vs. 0.22 ± 0.18 mm, respectively; P = 0.047). Both fractal dimension and lacunarity showed no differences between the groups. CCFd at 500 µm in group 2 (42.73 ± 5.67%) was significantly lower than that in group 1 (48.19 ± 4.18%; P = 0.009). In addition, group 2 had a lower CCFd at 1000 µm than group 1 (43.20 ± 3.48 vs. 39.81 ± 5.76%) although the difference was statistically insignificant.Table 2 Comparisons of the morphologic characteristics of CNV between the groups

	Group 1 (TE < 12 week)	Group 2 (TE ≥ 12 week)	P value	
Vessel analysis	
 CNV area, mm2	3.40 ± 2.23	1.93 ± 1.58	0.061	
 Vessel area, mm2	1.27 ± 0.89	0.63 ± 0.54	0.031*	
 Total vessel length, mm	0.42 ± 0.28	0.22 ± 0.18	0.047*	
 Vessel density, %	40.0 ± 13.5	32.6 ± 4.90	0.059	
 Vessel length density, %	12.8 ± 3.11	11.3 ± 1.82	0.184	
Fractal analysis	
 Fractal dimension	1.68 ± 0.08	1.62 ± 0.08	0.070	
 Lacunarity	0.44 ± 0.11	0.48 ± 0.09	0.305	
Choriocapillaris flow deficit (CCFd)	
 CCFd_500 µm, %	48.19 ± 4.18	42.73 ± 5.67	0.009*	
 CCFd_1000 µm, %	43.20 ± 3.48	39.81 ± 5.76	0.093	
CNV choroidal neovascularization, TE extension of treatment interval, CCFd_500 µm choriocapillaris flow deficit (%) of 500 µm outside CNV lesion, CCFd_1000 µm choriocapillaris flow deficit (%) of 1000 µm outside CNV lesion

*P < 0.05, compared between the groups

Multivariate logistic regression analysis was performed to identify the demographic factors and CNV characteristics associated with TE of ≥ 12 weeks (Table 3). TE ≥ 12 weeks was associated with a dry macula state at 2 weeks (odds ratio [OR] = 8.339; 95% confidence interval [CI] = 1.217–57.143; P = 0.032) and a low CCFd at 500 µm (OR = 0.734; 95% CI = 0.549–0.980; P = 0.036). Dry macula at 4 weeks, the ratio of PCV to AMD, CNV area, and total vessel length were not significantly associated.Table 3 Factors affecting extension of treatment interval ≥ 12 weeks

Variables	Univariate	Multivariate	
OR	95% CI	P value	OR	95% CI	P value	
Dry macula at 2 weeks	10.50	1.725–63.913	0.011*	8.339	1.217–57.143	0.032*	
Dry macula at 4 weeks	5.056	0.847–30.176	0.075	1.069	0.107–10.724	0.955	
PCV (vs. AMD)	4.952	0.986–24.875	0.052	3.537	0.463–27.047	0.224	
Vessel area	1.016	0.748–5.173	0.429				
Total vessel length	1.004	0.915–10.187	0.596				
CCFd_500 µm	0.780	0.625–0.976	0.029*	0.734	0.549–0.980	0.036*	
PCV polypoidal choroidal vasculopathy, AMD age-related macular degeneration, CNV choroidal neovascularization, CCFd_500 µm choriocapillaris flow deficit (%) of 500 µm outside CNV lesion, OR odds ratio, CI confidence interval

*P < 0.05, statistically significant

Discussion

Our study demonstrates the short-term efficacy of brolucizumab and factors affecting the TE after switching to brolucizumab for refractory nAMD based on real-world data. Numerous studies have confirmed the anatomical and functional benefits of intravitreal brolucizumab injections in nAMD. Our study found no significant changes in BCVA before and after switching to brolucizumab, which indicates functional outcome. This result is in line with that of other studies [33–35] and can be attributed to chronic SRF, IRF, or sub-RPE fluid in eyes with refractory nAMD that causes functional damage limiting the potential for visual improvement. However, Abdin et al. [36] observed significant visual improvement at week 16, which may be because of intensive therapy along with monthly loading of brolucizumab initially after switching. Given we evaluated up to 12 weeks, long-term visual improvement must be evaluated in future studies.

Regarding anatomical efficacy in our study, we observed significant improvements in SRF, IRF, or sub-RPE fluid after switching to brolucizumab. Specifically, at 2 weeks after switching to brolucizumab, the proportion of patients with a dry macula was significantly higher in the stable group (group 2) than in the unstable group (group 1; 12.5 vs. 60.0%). The decrease in the mean CST in stable group was greater than that in the unstable group at 2 weeks (∆-95 vs. ∆-129 µm). The mean SFCT of the unstable group at 2 weeks did not differ from the baseline and started significantly decreasing after 4 weeks. The mean SFCT of the stable group, however, began to significantly decrease after 2 weeks compared to the baseline. In addition, the mean CST and FV in the stable group were significantly lower than those in the unstable group at 2 weeks. These results indicate that the stable group was more sensitive and responded faster to brolucizumab than the unstable group. At 8 weeks after switching to brolucizumab, however, the proportions of eyes with SRF, IRF, sub-RPE fluid or dry macula in the unstable group were significantly higher than those in the stable group. Furthermore, the mean CST, MV, and FV of the unstable group increased at 8 weeks and were significantly higher than those in the stable group. In addition, the stable group had a higher rate of PCV than the unstable group (53.3 vs. 18.8%). Our results could be explained by findings of previous studies [37–40] showing that functional and anatomical outcomes of PCV are more favorable compared with those of nAMD.

In this study, the mean CVI of all patients did not differ significantly from the baseline. Similarly, subgroup analysis showed no significant differences in mean CVI before and after switching to brolucizumab in both groups. Viggiano et al. [41] reported significant decreases in the total choroidal area (TCA) and luminal choroidal area (LCA) at 4 weeks and 8 weeks after switching to brolucizumab in refractory nAMD. However, the stromal choroidal area did not differ significantly at all timepoints. In our study, the mean SFCT significantly decreased after switching to brolucizumab, indicating reduced choroidal blood flow. It seems that the CVI, the ratio of LCA to TCA, remains unchanged over time by a reduction of choroidal vascular permeability or a constriction of the choroidal vessel itself, not the choroidal stroma.

We also investigated the baseline CNV characteristics through image analysis using OCTA to noninvasively visualize the CNV. Rubsam et al. [42] suggested that the CNV size serves as an early clinical parameter for treatment response to brolucizumab. In the literature, CNV area decreased only in eyes with a better response to brolucizumab, with improvements maintained on a 12-week treatment interval. In eyes with a poorer response, CNV area did not decrease after brolucizumab treatment and an 8-week treatment interval was needed. In addition, eyes on a 12-week treatment interval had a significantly smaller CNV area at the baseline compared to eyes on an 8-week treatment interval (0.54 ± 0.70 vs. 1.98 ± 2.4 mm2). This finding is in line with our results that the vessel area and the total vessel length of the stable group were significantly less than those in the unstable group.

Fractal analysis of OCTA images has been shown to provide quantitative parameters that strongly correlate with the OCTA features of CNV lesions in eyes with nAMD [43, 44]. The fractal dimension quantifies complexity or structural roughness, with higher values indicating higher complexity. In addition, higher lacunarity represents structural heterogeneity. Recent studies using OCTA have demonstrated that anti-VEGFs promote a vascular remodeling characterized by pruning of small caliber capillaries and dilation of central prominent vessels [44–46]. In our study, fractal dimension analysis showed no difference between groups. This is likely because of already remodeled choriocapillary microstructure after several anti-VEGF treatments, resulting in homogenous vessel complexity between groups.

Factor analysis yielded low CCFd at 500 μm and dry macula at 2 weeks as factors strongly associated with disease stability. If the early fluid status at 2 weeks after brolucizumab injection can be confirmed through OCT scan, it will be able to provide additional help in future treatment planning or prognosis prediction. Neither vessel area nor total vessel length were significantly associated with disease stability. As shown in previous studies [47–49], recalcitrant CNV that recurs despite multiple anti-VEGF treatments has a long duration of disease, wherein CNV develops resistance to anti-VEGF treatment including brolucizumab as immature CNV vessels develop into mature CNV vessels irrespective of the CNV size or vessel length. Regarding CCFd, several studies have analyzed the periphery of low-flow dark halo around the CNV in the OCTA [50–54]. Our investigation showed that the perilesional CCFd in the stable group was significantly lower than that in the unstable group. Similarly, Scharf et al. [31] demonstrated that the mean CCFd was significantly higher in both concentric 500-μm and 1000-μm rings in the CNV versus the normal control group and was significantly higher in the inner ring than in the outer ring. The mean CCFd was also greater in both rings in the exudative versus the non-exudative CNV groups. In our study, the periphery of the CNV itself was analyzed, and not the periphery of the dark halo of CNV. Therefore, caution is needed in interpretation because it is possible that the dark halo was included in the area inside the 500-μm ring. However, it is noteworthy that the significantly higher CCFd in the unstable group indicates that the CNV in the unstable group is more active and induces choriocapillary dysfunction by stealing the blood flow adjacent to the CNV.

This is the first study determining the factors for disease stability after switching to brolucizumab in nAMD refractory to previous anti-VEGF treatment. However, this study had some limitations. First, this was a retrospective study and imaging protocol or sequential follow-up were inconsistent, which need to be verified by a standardized-protocol prospective observational study or a quasi-experimental study. Intravitreal injection method before switching to brolucizumab was not consistent and included various types of anti-VEGF agents. Second, the sample size was small. Third, this study had a short-term follow-up. Assessing long-term disease stability after the first 12-weeks after switching to brolucizumab in the larger sample size is essential to yield more generalized and conclusive results. Fourth, because PCV and nAMD were not differentiated in the CNV analysis using OCTA, results should be interpreted with caution. PCV and nAMD should be distinguished separately in future studies. Fifth, image binarization had potential error of over- or underestimation of the designated region of interest. In this study, SFCT was measured only in the subfovea, and CVI was measured in the entire choroidal region in SD-OCT 6 × 6 mm B-scan, so caution is needed in interpreting the results. For more accurate analysis in the future, studies measuring choroidal thickness and CVI below the CNV will be necessary. The ocular surface status at the time of OCT or OCTA examination may potentially affect image quality. Another limitation is the manual demarcation of CNV. Moreover, en face imaging does not precisely represent a three-dimensional CNV structure. As an example, Classic CNV was also included in this study, and CNV analysis was performed only by selecting the choriocapillaris slab (21–31 μm below the Bruch's membrane) where CNV was best visible in OCTA. Therefore, future investigation should focus on three-dimensional analysis of CNV. Sixth, despite the compensation protocol for analyzing the OCTA images, segmentation error may still exist. Despite these limitations, this study provides evidence in favor of assessing for factors affecting extension of treatment interval such as morphological features of CNV or responsiveness to brolucizumab early in the treatment course to predict long-term visual or anatomical improvement.

Conclusions

In conclusion, early fluid-free status after switching to brolucizumab and choriocapillary function around CNV are important factors determining the stability of nAMD refractory to treatment with other anti-VEGFs. Therefore, when switching to brolucizumab in patients with refractory nAMD, treatment options such as T&E, fixed dosing or PRN should be personalized considering the various morphological characteristics of CNV and response to brolucizumab.

Acknowledgements

We would like to thank all the participants of the study.

Author Contributions

Yung-Hwi Kim: conceived and designed the study, collected the data, analyzed and interpreted the data, wrote the main manuscript text, and reviewed the manuscript. Tae Kyu Moon: collected and analyzed the data and reviewed the manuscript. Yong-Sok Ji: conceived and designed the study, analyzed and interpreted the data, and reviewed the manuscript.

Funding

No funding or sponsorship was received for this study or publication of this article. The Rapid Service Fee was funded by the authors.

Data Availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of Interest

Yung-Hwi Kim, Tae Kyu Moon and Yong-Sok Ji have nothing to disclose.

Ethical Approval

This study was approved by the Institutional Review Boards of Chonnam National University Hospital (CNUH-2023-035). All procedures conducted were in accordance with the Aspire IRB and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This was a retrospective study performed at a single site (Gwangju) in the Republic of Korea.

Prior Presentation: Previously presented as a poster presentation at the ARVO Annual Meeting 2023 (Offline, New Orleans, April 23–27).
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