We utilized the images collected as part of the protocol of a 52-week, randomized, double-masked clinical trial of participants with PCV (ClinicalTrials.gov Identifier: NCT03941587). The study was conducted in accordance with the Declaration of Helsinki. The study protocol received approval from the institutional review board. Details of the trial design and methodology have been published previously. Participants were aged ≥50 years with symptomatic macular PCV and were treatment-naïve in the study eye. For the current analysis, only participants recruited at the main study site (Singapore National Eye Centre), where Plex Elite OCTA (Zeiss, Germany), was performed, were included. In brief, all participants gave written informed consent prior to participating in the trial. Participants were aged 50 and above with symptomatic macular PCV who were treatment-naïve in the study eye. Eligible participants were randomized to their treatment arms with a blocked randomization method using a ratio of 1:1. Both participants and the investigators were masked to the treatment received. At baseline visit, participants in the combination arm received a single treatment of RF-PDT plus intra-vitreal aflibercept; participants in the monotherapy arm received sham RF-PDT plus intra-vitreal aflibercept. For participants in the monotherapy arm, a sham infusion and laser procedure were performed to mimic active RF-PDT, with laser applied at 0 fluence to the target lesion area. After the baseline visit, all participants were assessed every 4 weeks until week 52. Best-corrected visual acuity and optical coherence tomography (OCT) were performed at every visit. In addition, OCTA, fundus fluorescein angiography (FFA), and ICGA were performed at baseline, week 12, and week 52. From week 4 to week 52, participants in either arm could receive additional intra-vitreal aflibercept based on a PRN regimen according to protocol-specific retreatment criteria.

Multimodal imaging, including OCT, OCTA, fundus fluorescein angiography (FFA), and ICGA, was performed at baseline, week 12, and week 52 according to the trial protocol. Best-corrected visual acuity and OCT were performed at every study visit (every 4 weeks).

Swept-source OCTA images were obtained using the PlexElite system (Zeiss, Germany) with 3 × 3 mm and 6 × 6 mm scans centered on the fovea . OCTA was assessed by 2 retinal specialists (KT and GC) who were masked to the findings of the other imaging modalities. A minimum signal strength of 7 was required for image inclusion. Segmentation errors within preset slabs (outer retina to choriocapillaris, avascular, and RPE-Bruch’s membrane) were manually corrected prior to analysis. The OCTA signal within the PL at week 12 was determined using the in-built OCTA viewing software, with confirmation achieved by correlating OCTA signals within sharp-peaked PEDs on OCTA with enface OCTA images. The presence of trunk vessel within the BNN was also assessed, with trunk vessels defined as large-caliber feeder vessels with a diameter at least equivalent to a retinal vein. These vessels were identified qualitatively on en face OCTA based on their larger caliber and linear morphology relative to the surrounding capillary network.

Quantitative OCTA measurements were performed using ImageJ at baseline, week 12 and week 52. Total lesion size (mm ² ) was determined by manual segmentation of the entire lesion complex (BNN + PL), including both the abnormal vascular network and intervening avascular spaces , using the freehand selection tool on enface OCTA images.

Flow-positive signal was defined as bright pixels identified following image binarization. The binarization method used was previously described. In brief, en face OCTA images were binarized using the Otsu thresholding method in ImageJ (National Institutes of Health, Bethesda, MD, USA), which automatically determines an optimal threshold based on minimization of intra-class variance. Following binarization, grayscale attribute filtering (minimum area threshold of 50 pixels, connectivity set at 4) was applied to reduce background noise and remove isolated artefactual signals.

BNN area was defined as the total area of bright pixel in the BNN area. BNN vessel density (VD, %) was quantified as the proportion of bright pixel (flow detected) vessel area relative to the manually segmented BNN area on en face OCTA after binarization.

FFA and ICGA were performed using the Heidelberg Spectralis HRA + OCT system (Heidelberg Engineering, Heidelberg, Germany) following standardized imaging protocols. FFA images were acquired after intravenous injection of fluorescein dye (10%, 5 mL) , capturing early, mid, and late phases to assess vascular leakage and abnormalities. ICGA was performed using a 25 mg intravenous bolus of indocyanine green dye , with sequential image acquisition to visualize the choroidal vasculature , detect PL and BNN at baseline , and assess PL closure at week 52 .

OCT was acquired using the Heidelberg Spectralis OCT system (Heidelberg Engineering, Heidelberg, Germany) with an enhanced-depth imaging (EDI) protocol to allow for detailed choroidal assessment.

FFA, ICGA, and OCT were independently assessed by trained graders at the Singapore National Eye Centre Ocular Reading Center . The diagnosis of PCV was determined using ICGA based on the EVEREST PCV diagnostic criteria. ^, Structural OCT B-scans were analyzed for fluid status, including the presence of subretinal fluid (SRF) and intraretinal fluid (IRF). SRF was defined as a hyporeflective space between the neurosensory retina and the retinal pigment epithelium (RPE) , while IRF was identified as hyporeflective cystic spaces within the retinal layers . Additional quantitative structural OCT measures included central subfield thickness (CST, µm) , measured as the thickness of the central 1-mm macular area, automatically calculated using inbuilt OCT viewing software ( Heyex software, Spectralis, Heidelberg). Subfoveal Choroidal thickness (SFCT, µm) was manually measured from Bruch’s membrane to the choroid-scleral interface at the foveal center.

Baseline and week 12 characteristics and imaging features were compared between patients who achieved PL closure by week 52 and those who did not. Continuous data were summarized using means and SDs (SD), while categorical data were presented as counts and percentages. Comparisons of continuous variables were conducted using independent t-tests or Mann–Whitney U tests , depending on data distribution, while categorical variables were analyzed using chi-square tests or Fisher’s exact tests , as appropriate.

Quantitative OCTA measures, including total lesion size, BNN area, BNN-VD , were analyzed as absolute values at baseline. Structural OCT parameters, including CST, and SFCT, were evaluated as absolute changes. Qualitative features such as the presence or absence of PL, trunk vessels, SRF and IRF at week 12 were included as binary predictors.

Multivariable regression analyses were performed to identify predictors of treatment outcomes at week 52. For continuous outcomes, including change in BCVA and change in CST, multivariable linear regression models were constructed. For the binary outcome of PL closure at week 52, multivariable logistic regression was used. Candidate predictors included baseline demographic factors, baseline BCVA, baseline OCT, OCTA imaging parameters, and treatment exposure. All variables were entered simultaneously into the models, and regression estimates therefore represent effects adjusted for the other covariates included in the model. Regression coefficients (β) with 95% CIs (CI) were reported for linear models, while odds ratios (OR) with 95% CIs were reported for logistic regression. All statistical analyses were conducted using R version 4.3.3, and statistical significance was set at P <.05.

Table 2 and Figure 1 summarize the OCTA features according to treatment arms at baseline, Week-12, and Week-52. The total lesion area at baseline was similar between the two treatment arms, and showed a transient reduction at week-12, but subsequently increased towards baseline at week-52 (combination: 3.72 ± 3.01 mm ² baseline, 2.86 ± 2.50 mm ² week-12, 3.59 ± 3.26 mm ² week-52; monotherapy: 3.77 ± 2.23 mm ² baseline, 3.27 ± 2.36 mm ² week-12, 3.47 ± 2.58 mm ² week-52). The transient reduction was numerically larger in the combination arm but was not statistically significant (−0.86 vs −0.50, P =.55). In contrast, BNN area showed reduction at week 12 in both treatment arms and remained significantly reduced at week 52 compared to baseline (combination 2.29 ± 2.08 mm ² baseline, 1.46 ± 1.36 mm ² week 12, 1.53 ± 1.32 mm ² week 52; monotherapy 2.39 ± 1.85 mm ² baseline, 1.87 ± 1.68 mm ² week 12, 1.82 ± 1.38 mm ² week 52). There were no significant differences in total lesion area and BNN area between treatment arms at any time point. BNN vessel density showed a significant reduction in the combination arm at week-12, but the difference was not statistically significant anymore at week-52 (57 ± 24% baseline, 47 ± 27% week-12, 50 ± 28% week-52). In contrast, BNN VD did not show a significant change at week-12 and week-52 (63 ± 20% baseline, 60 ± 24% week-12, 59 ± 19% week-52). The reduction in BNN VD was significantly higher in the combination arm compared to the monotherapy arm (-10 ± 15 vs-3 ± 12, P =.02) at week-12. Finally, the proportion of eyes with trunk vessels increased progressively from baseline to week-12 and to week-52 in both treatment arms (combination: 35.7% baseline, 59.3% week-12, 67.8% week-52; monotherapy 25.9% baseline, 44.4% week-12, 71.4% week-52). At week 52, the increase was significant compared to baseline in the combination arm (64.3% vs 35.7%, P =.03).

TABLE 2

OCTA Features at Baseline, Week 12, and Week 52 According to Treatment Arms.

	All	Combo (28)	Mono (27)	P
Total Lesion (PL + BNN) area, mm 2, mean (SD)
Baseline	3.75 (2.64)	3.72 (3.01)	3.77 (2.23)	0.94
Week 12	3.06 (2.42)	2.86 (2.50)	3.27 (2.36)	0.54
Week 52	3.67 (4.16)	3.59 (3.26)	3.47 (2.58)	0.73
BL- W12	−0.69 (1.97)	−0.86 (2.25)	−0.50 (1.66)	0.55
BL- W52	−0.08 (2.67)	−0.13 (1.47)	−0.30 (1.42)	0.55
P (W12 vs BL)	<0.01	<0.01	<0.01
P (W52 vs BL)	0.83	0.85	0.28
BNN area, mm 2, mean (SD)
Baseline	2.34 (1.95)	2.29 (2.08)	2.39 (1.85)	0.86
Week 12	1.66 (1.52)	1.46 (1.36)	1.87 (1.68)	0.32
Week 52	1.67 (1.35)	1.53 (1.32)	1.82 (1.38)	0.43
BL- W12	−0.68 (1.32)	−0.84 (1.74)	−0.51 (0.62)	0.36
BL- W52	−0.67 (1.42)	−0.76 (1.76)	−0.56 (0.95)	0.61
P (W12 vs BL)	<0.01	0.02	<0.01
P (W52 vs BL)	<0.01	0.03	<0.01
BNN vessel density (VD), %, mean (SD)
Baseline	60 (22)	57 (24)	63 (20)	0.34
Week 12	53 (27)	47 (27)	60 (24)	0.06
Week 52	54 (24)	50 (28)	59 (19)	0.19
BL- W12	−7 (14)	−10 (15)	−3 (12)	0.02
BL- W52	−6 (18)	−7 (21)	−5 (15)	0.46
P (W12 vs BL)	<0.01	<0.01	0.26
P (W52 vs BL)	0.02	0.08	0.13
Trunk vessel present, n (%)
Baseline	–	10 (35.7%)	7 (25.9%)	0.43
Week 12	–	16 (59.3%)	12 (44.4%)	0.29
Week 52	–	19 (67.8%)	15 (71.4%)	0.37
P (W12 vs BL)	–	0.09	0.16	–
P (W52 vs BL)	–	0.03	0.05	–

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