Five-year outcomes of photodynamic therapy combined with intravitreal injection of ranibizumab or aflibercept for polypoidal choroidal vasculopathy.

We report 5-year visual and anatomical outcomes after combination therapy of photodynamic therapy (PDT) and intravitreal injection of ranibizumab or aflibercept for polypoidal choroidal vasculopathy (PCV) and predictive factors for visual outcomes at 5-year and time to recurrence. Medical charts were retrospectively reviewed for 43 consecutive eyes with PCV treated with combination therapy of PDT and intravitreal injection of ranibizumab(n = 13) or aflibercept(n = 30) and completed 5-year follow-up. The variants of ARMS2 A69S and CFH I62V were genotyped using TaqMan assay. Best corrected visual acuity (BCVA) significantly improved at 5-year (P = 0.01) with 20% reduction of subfoveal choroidal thickness irrespective of presence or absence of recurrence. Visual improvement was associated with baseline shorter greatest linear dimension (GLD) (P = 1.0×10-4). Mean time to recurrence was 28.6±23.1 months (95% CI: 21.5-35.7, Median:18.0) and time to recurrence was associated with G allele (protective allele) of ARMS2 A69S and GLD (P = 4.0×10-4 and 1.0×10-2, respectively). Multiple regression analysis revealed that time to recurrence extended by 15.5 months when the G allele of ARMS2 A69S increased by one allele (TT: 15.7±17.0, TG: 30.8±23.5, GG: 41.1±22.6 months). The combination therapy resulted in a favorable visual outcome for PCV during 5-year follow-up.

Introduction

Polypoidal choroidal vasculopathy (PCV) often exhibits serosanguinous detachment of retinal pigment epithelium and neurosensory retina and is characterized by aneurysmal dilation with or without branching vascular network on indocyanine green angiography (ICGA).[1, 2]

Several treatment options have been reported to treat PCV including photocoagulation, photodynamic therapy (PDT), intravitreal injection of anti- vascular endothelial growth factor (VEGF) agent and combination therapy of PDT and intravitreal injection of anti-VEGF agent.[3-6] Of these treatment options, combination therapy of PDT and intravitreal injection of anti-VEGF agents including ranibizumab and aflibercept is one of the prevalent treatment options along with intravitreal injection of anti-VEGF agent monotherapy. A prospective randomized clinical trial EVERESTII study demonstrated that PDT combined with intravitreal ranibizumab was superior to intravitreal ranibizumab monotherapy in terms of occlusion of polypoidal lesion and visual improvement.[7] Since the follow-up period was short in most studies reporting the combination therapy,[8-12] there has been few reports investigating 5-year outcome of combination therapy of PDT and intravitreal injection of anti-VEGF agent for PCV.[13]

In the present study, we report 5-year visual and anatomical outcomes after combination therapy of PDT and intravitreal injection of ranibizumab or aflibercept for PCV and the predictive factors for visual outcomes at 5 years and time to recurrence during the follow-up.

Materials and methods

This study was approved for institutional review board of Yamanashi University (Approval No.1957) and was followed in accordance with Declaration of Helsinki. Written informed consent was obtained from each participant. All participants were recruited from the Macular Clinic of the Department of Ophthalmology at University of Yamanashi Hospital between August 2011 and August 2014.

Treatment and follow-up

Prior to initial treatment, all participants received comprehensive ophthalmic examination including measurement of best-corrected visual acuity(BCVA) using Landolt chart and intraocular pressure, biomicroscopy with or without a 78 diopter lens, color fundus photography, fluorescein and indocyanine green angiography(FA/ICGA) (HRA-2, Heidelberg Engineering, Dossenheim, Germany), spectral-domain optical coherence tomography(SD-OCT) (Spectralis ver5.4 HRA+OCT). All SD-OCT images were obtained by both horizontal and vertical lines through the fovea using EDI-mode. Central retinal thickness was defined as a vertical distance from bottom of RPE to inner limiting membrane at the fovea and subfoveal choroidal thickness was defined as a vertical distance from bottom of RPE to choroidoscleral border at the fovea as measured by SD-OCT.

Diagnosis of PCV was made as previously described.[14] All PCV cases exhibited solitary or clusters of polypoidal dilation with or without abnormal vascular networks in the superficial choroid on ICGA and irregular RPE elevations with serous and/or hemorrhagic detachment of neurosensory retina or RPE on SD-OCT.

Exclusion criteria was the case with massive subretinal hemorrhage with or without vitreous hemorrhage, dense cataract, history of recent thromboembolic events, or pregnancy.

All participants received intravitreal injection of ranibizumab (0.05mg/0.05ml) or aflibercept (0.2mg/0.05ml) 1 week before PDT (1 injection and 1PDT). The initial treatments were chosen according to time period. From August 2011 to 2012 December, combination therapy of PDT and intravitreal ranibizumab injection was administrated and from January 2013 to August 2014 combination therapy of PDT and intravitreal aflibercept was administrated. Standard PDT (a laser light at 689 nm delivered at a dose of 50J/cm2 at an intensity of 600mW/cm2 over 83 sec with verteporfin 6mg/m2) was conducted. Greatest linear dimension (GLD) was determined as maximum length to cover the polypoidal lesion and branching vascular networks. Spot size was defined as the length of 1000μm added to GLD.

Follow-up examination included assessment of BCVA using Landolt chart, intraocular pressure, biomicroscopy with or without a 76 D lens, and SD-OCT, and was performed every 3 months until recurrent exudation developed. Recurrence was defined as newly developed hemorrhage on fundoscopy or subretinal fluid detected by SD-OCT. Additional FA/ICGA was performed when recurrent exudation was seen. When ICGA showed residual or recurrent polypoidal lesion, additional combination therapy (1injection and 1 PDT) was administrated in the same fashion as the initial combination therapy. When ICGA exhibited abnormal vascular network without polyp, additional intravitreal injection of anti-VEGF agent was administrated. After first recurrence, patients were followed every month and PRN treatment was performed until exudation including subretinal fluid and intraretinal fluid was completely absorbed.

Genotyping

Genomic DNA was obtained from peripheral blood using PURE GENE Isolation Kit (Gentra Systems, Minneapolis, US). The genotyping of ARMS2 A69S (rs10490924) and CFH I62V (rs800292) was performed for all participants using TaqMan assays on ABI7300/7500 Real Time PCR System (Applied Biosystems, Foster City, US) as we recently described.[15]

Statistical analysis

Statistical analysis was conducted using DR. SPSS (IBM, Tokyo, Japan). A chi-square test was used to evaluate differences of categorical variables between 2 groups. Mann-Whitney U test was used to evaluate differences of continuous variables between 2 groups. Decimal BCVA was converted to log MAR BCVA for statistical analysis. A paired t-test was used to compare log MAR BCVA before and after treatment. Cox-regression analysis was performed to investigate risk factors for recurrence and multiple regression analysis was performed to investigate the factors associated with time to recurrence. A p-value less than 0.05 was considered a statistical significance.

Results

During the study period, 43 eyes from 43 patients were included in the present study. Table 1 shows the baseline characteristics in 43 subjects. Table 2 shows the baseline characteristics comparison of 2 treatment groups. There were no significant differences between 2 treatment groups but central retinal thickness, in which IVR+PDT group had greater CRT than IVA+PDT group at baseline. Fig 1 shows changes of BCVA (Fig 1A) and changes of BCVA gains (Fig 1B) following combination therapy. After combination therapy, BCVA significantly improved (p<0.01) compared with baseline value throughout 5-year follow-up. Mean logMAR BCVA improved from 0.55±0.28 at baseline to 0.40±0.40 at 60 month. Mean logMAR BCVA gains at 2-year from baseline were greatest throughout 5-year follow-up. Compared with BCVA at 2-year, those values at 4-year and 5-year were significantly worse (p = 0.005 and p = 0.001, respectively). Fig 2 shows BCVA gains from baseline at 5-year in each treatment group. There were no significant differences in BCVA gains between the 2 treatment groups throughout 5-year follow-up. Factors at 60 months multivariate linear regression analysis revealed that shorter GLD (p = 1.0×10−4) were associated with better BCVA at 60 months while other factors such as age, type of anti-VEGF agent, and central retinal thickness were not associated with improved BCVA (Table 3). Another multivariate linear regression analysis associated with the BCVA gains from baseline to 5-year revealed that shorter GLD(p = 1.0×10−4) and worse baseline BCVA(p = 7.0×10−4) was associated with greater BCVA gains (Table 4). T allele (risk allele) of ARMS2 A69S was the only variable associated with recurrence of exudation from PCV (P = 0.002, Table 5). Fig 3(A), 3(B) and 3(C) demonstrates recurrence-free proportion according to ARMS2 A69S genotypes(A), CFH I62V genotypes(B), and anti-VEGF agents(C). ARMS2 A69S genotype TT demonstrated a lower proportion of recurrence-free eyes compared with genotypes TG and GG (p = 0.019). Across CFH genotypes and anti-VEGF agents no statistically significant difference in recurrence free proportions were found (p = 0.95 and p = 0.48).

Table 1

Clinical and genetic characteristics of the subjects.

Age	72.8±7.6(57–92)
Male gender	30(69.8%)
Baseline log MAR BCVA	0.55±0.27(0.10–1.22)
Greatest linear dimension (μm)	1666±797(450–3600)
Central Retinal Thickness (μm)	368.1±99.9(178–610)
Subfoveal Choroidal Thickness (μm)	257.0±87.7(95–480)
Initial Anti-VEGF agent
Ranibizumab	13(30.2%)
Aflibercept	30(69.8%)
Number of Polyps	1.79±1.15(1.0)1-5
ARMS2 A69S(rs10490924)	Genotype frequency
TT	13(30.2%)
TG	20(46.5%)
GG	10(23.3%)
CFH I62V(rs800292)	Genotype frequency
GG	25(58.1%)
GA	16(37.2%)
AA	2(4.7%)
Lens Status (phakic eye)	38(88.4%)
Current Smoker	5(11.6%)

ARMS: age-related maculopathy susceptibility, BCVA: best-corrected visual acuity

CFH: complement factor H, GLD: greatest linear dimension, log MAR: logarithm of the minimal angle resolution, BCVA: best-corrected visual acuity, VEGF: vascular endothelial growth factor

Table 2

Baseline characteristics comparison of 2 treatment groups.

	IVR+PDT(n = 13)	IVA+PDT(n = 30)	P value
Age	71.0±5.3(62–80)	73.6±8.3(57–92)	0.23
Male gender	10(76.9%)	20(66.7%)	0.50
Baseline log MAR BCVA (range)	0.56±0.25(0.22–1.00)	0.55±0.29(0.10–1.22)	0.72
Greatest linear dimension (μm)(range)	1492±881(600–3600)	1742±761(450–3500)	0.20
Central retinal thickness (μm) (range)	422±97(283–610)	345±93(178–588)	0.01
Subfoveal choroidal thickness (μm)(range)	275±96(145–480)	249±85(95–419)	0.48
Number of polyps(range)	2.00±1.22 (1–5)	1.70±1.12 (1–4)	0.32
ARMS2 A69S(rs10490924)			0.32
TT	4(30.8%)	9(30%)
TG	8(61.6%)	12(40%)
GG	1(7.6%)	9(30%)
CFH I62V(rs800292)			0.10
GG	5(38.5%)	20(66.7%)
GA	7(53.8%)	9(30%)
AA	1(7.7%)	1(3.3%)
Current Smoker	2(15.4%)	3(10%)	0.61
Lens Status (phakic eye)	12(92.3%)	26(86.7%)	0.60

ARMS: age-related maculopathy susceptibility, BCVA: best-corrected visual acuity

CFH: complement factor H, GLD: greatest linear dimension, log MAR: logarithm of the minimal angle resolution, BCVA: best-corrected visual acuity, VEGF: vascular endothelial growth factor

Fig 1

(A) Changes of best-corrected visual acuity after the combination therapy. (B) Changes of best-corrected visual acuity gains after the combination therapy.

Fig 2

Best-corrected visual acuity gain after the combination therapy according to anti-VEGF agents.

Table 3

Results of multivariate linear regression analyses associated with the logarithm of the minimal angle resolution best-corrected visual acuity at 60 months.

Variables	β-coefficient	p-value
Age	0.004	0.58
Sex	0.23	0.05
Baseline BCVA (log MAR)	0.33	0.07
Greatest linear dimension (μm)	2.8×10−4	1.0×10−4
Central retinal thickness (μm)	-0.001	0.07
Subfoveal choroidal thickness (μm)	-8.0×10−4	0.15
Initial anti-VEGF agent	-0.12	0.27
(1: aflibercept, 0: ranibizumab)
Current smoker	0.13	0.39
ARMS2 A69S T-allele	0.11	0.09
CFH I62V G-allele	0.06	0.51
Number of Polyps	-0.02	0.61
Lens status (0: phakic, 1: pseudophakic)	0.03	0.83

BCVA: best corrected visual acuity

Log MAR: logarithm of the minimal angle resolution

VEGF: vascular endothelial growth factor

ARMS: age-related maculopathy susceptibility

CFH: complement factor H

Table 4

Results of multivariate linear regression analyses associated with the logarithm of the minimal angle resolution best-corrected visual acuity gains from baseline to at 60 months.

Variables	β-coefficient	p-value
Age	0.004	0.58
Sex	0.23	0.05
Baseline BCVA (log MAR)	-0.67	7.0×10−4
Greatest linear dimension (μm)	2.8×10−4	1.0×10−4
Central retinal thickness (μm)	-0.001	0.066
Subfoveal choroidal thickness (μm)	-8.0×10−4	0.15
Initial anti-VEGF agent	-0.12	0.27
(1: aflibercept, 0: ranibizumab)
Current smoker	0.13	0.39
ARMS2 A69S T-allele	0.11	0.09
CFH I62V G-allele	0.05	0.51
Number of Polyps	-0.02	0.61
Lens status (0: phakic, 1: pseudophakic)	0.03	0.83

BCVA: best corrected visual acuity

Log MAR: logarithm of the minimal angle resolution

Table 5

Cox regression analysis of factors associated with recurrence.

Variables	p-value	Hazard ratio	95% confidence interval
Age	0.23	1.05	0.97–1.13
Male gender	0.14	2.23	0.77–6.49
Baseline logMAR BCVA	0.68	0.68	0.11–4.34
Greatest linear dimension	0.13	1.00	1.00–1.00
Central retinal thickness	0.25	1.00	0.99–1.00
Subfoveal choroidal thickness	0.58	1.00	0.99–1.00
Aflibercept	0.23	0.53	0.19–1.50
Current smoker	0.66	0.71	0.15–3.26
ARMS2 A69S T allele	6.0×10–4	3.38	1.68–6.79
CFH I62V G allele	0.57	1.24	0.59–2.63
Number of Polyps	0.13	1.34	0.91–1.98
Lens status(0:phakic, 1:pseudophakic)	0.07	0.21	0.04–1.16

ARMS2: age-related maculopathy susceptibility, BCVA: best-corrected visual acuity, CFH: complement factor H, logMAR: logarithm of the minimal angle resolution

Fig 3

Kaplan-Meier survival curve associated with recurrence-free proportion after the initial combination therapy.

Recurrence-free proportion after the initial combination therapy among ARMS2 A69S genotypes.

Mean time to recurrence was 28.6 months (95%CI: 21.5–35.7, median:18 months) in all subjects. Time to recurrence was significantly different among ARMS2 A69S genotypes (P = 0.019, log-rank test). Mean time to recurrence was 41.1±22.6 months in GG genotype (95%CI:24.9–57.3, median:54 months), 30.8±23.5 months in TG genotype (95%CI:19.7–41.8, median:24 months), 15.7±17.0 months in TT genotype (95%CI:5.4–26.0, median:9 months).

Recurrence-free proportion after the initial combination therapy among CFH I62V genotypes.

Time to recurrence was not significantly different among CFH I62V genotypes (P = 0.95, log-rank test). Mean time to recurrence was 31.5±40.3 months in AA genotype (95%CI:3–60,median:30 months), 29.3±23.9 months in GA genotype (95%CI:16.5–42.0,median:27 months), 28.0±22.5 months in GG genotype (95%CI:18.7–37.3, median:18 months).

Recurrence-free proportion after the initial combination therapy depending on anti-VEGF agents.

Time to recurrence was not significantly different depending on anti-VEGF agents (P = 0.48, log-rank test). Mean time to recurrence was 29.4±24.2 months in IVA group (95%CI:20.4–38.4, median:18 months), 26.8±21.2 months (95%CI:14.0–39.6, median:30 months) in IVR group.

GG genotypes at A69S of ARMS2 recurred less frequently compared with other genotypes. Multivariate linear regression analyses (backward stepwise selection method, Table 6) revealed that increased time to recurrence was associated with G allele (protective allele) of ARMS2 A69S and shorter GLD and time to recurrence was estimated as follows: 39.2+15.5× (number of G allele)-0.01× GLD (μm) (months). Thus, for each G allele the recurrence free time increased by 15.5 months on average. Annual number of additional treatments including anti-VEGF injection and the combination therapy was shown in Table 7. Mean number of additional injections and the combination therapy during 5-year follow-up was 7.5 and 0.5, respectively. Table 8 shows changes of subfoveal choroidal thickness depending on presence or absence of recurrence. Subfoveal choroidal thickness significantly decreased throughout the follow-up period. Irrespective of presence or absence of recurrence, subfoveal choroidal thickness decreased by approximately 20% at 60 months. Fig 4 shows changes of central macular thickness after the initial combination therapy. Compared with baseline value, central macular thickness significantly decreased (p = <0.001) throughout the follow-up period and mean CRT decreased from 368±100 μm at baseline to 217±94 μm at 60 month.

Table 6

Results of multivariate linear regression analyses associated with time to recurrence (backward stepwise selection method).

Variables	β-coefficient	p-value
	39.2
Sex	-13.2	0.053
ARMS2 A69S G-allele	15.5	4.0×10−4
Greatest linear dimension (μm)	-0.01	0.01

ARMS: age-related maculopathy susceptibility

Age, sex, smoking status, baseline best-corrected visual acuity, baseline central retinal thickness, baseline subfoveal choroidal thickness, initial anti-VEGF agent, and CFH I62V were eliminated in this analysis.

Table 7

Annual number of additional injection/combination therapy in all patients.

Months after initial treatment	0-12M	13-24M	25-36M	37-48M	49-60M	Total (0-60M)
mean(median) number of additional injections	0.47±0.93 (0.0)	1.09±1.60 (0.0)	1.84±2.34 (1.0)	1.93±2.09 (1.0)	2.19±2.22 (2.0)	7.51±7.25 (7.0)
mean(median) number of additional combination therapies	0.12±0.32 (0.0)	0.12±0.32 (0.0)	0.16±0.43 (0.0)	0.09±0.29 (0.0)	0.02±0.15 (0.0)	0.51±1.01 (0.0)

Table 8

Changes of subfoveal choroidal thickness in patients with or without recurrence.

Months after initial treatment	0M (baseline)	12M	24M	36M	48M	60M
SCT (n = 43, all patient)	257.0±87.7	222.5±87.4	218.1±81.9	218.1±98.9	210.6±95.0	206.3±92.7
P value (vs baseline)		7.1×10−5	3.2×10−5	4.0×10−5	1.6×10−5	1.7×10−6
SCT (n = 31, recurrence group)	256.3±75.3	225.2±80.3	220.6±73.3	220.6±93.4	210.0±90.3	207.3±88.7
P value (vs baseline)		3.6×10−3	2.2×10−3	3.1×10−3	8.0×10−4	2.0×10−4
SCT (n = 12, non-recurrence group)	258.9±117.8	215.6±107.4	211.7±104.3	211.5±116.1	212.2±110.6	203.7±106.5
P value (vs baseline)		4.3×10−3	2.4×10−3	9.0×10−4	2.5×10−3	1.5×10−3

SCT: subfoveal choroidal thickness

Fig 4

Changes of central macular thickness after the initial combination therapy.

Compared with baseline value (368±100),central macular thickness significantly decreased to 199±77 (P = 2.5×10−12), 189±64 (P = 1.2×10−14), 218±87 (P = 2.8×10−11), 216±85 (P = 3.9×10−15), and 217±94 (P = 2.7×10−11) at 12-month, 24-month, 36-month, 48-month, and 60-month, respectively.

Discussion

We investigated long-term visual and anatomical outcomes after the combination therapy involving PDT and intravitreal injection of ranibizumab or aflibercept for PCV. In the present study, significant visual gain was obtained at 1 year and was maintained through 5-years. Several studies reported long-term results of the combination therapy involving PDT and anti-VEGF agents for PCV.[13, 16, 17] Miyata et al. reported 5-year results after combination therapy involving PDT and intravitreal injection of ranibizumab for 20 eyes with PCV. In their report, BCVA significantly improved at 1 year, but thereafter BCVA declined and returned to baseline value at 5 year. Similarly, in the present study, there has been the trend of BCVA deterioration after 3 years from initial treatment, but the significant BCVA improvement was maintained through 5 years. One possible reason for the differences of visual outcome between the 2 studies is that, in this study, mean age of the patients was younger than that in the Miyata’s report. We reported that in the previous study, older age was associated with recurrence of PCV lesions after the combination therapy.[18] The difference of mean age between the 2 studies might affect the visual outcome. Differences in baseline characteristic between the 2 studies might result in different results. To the best of our knowledge, this is the first report demonstrating long-term results of combination therapy involving IVA and PDT for PCV. We compared the long-term visual outcome between IVA+PDT group and IVR+PDT group; however, there were no significant differences in visual outcomes between the 2 groups. In this study, predictive factors of visual gain at 5 years was shorter GLD. Regarding the association between visual improvement and shorter GLD, Tsujikawa et al.[19] demonstrated that eyes with a smaller lesion size (less than one-disc diameter) determined by ICGA have a better visual prognosis than eyes with larger lesion size (larger than one-disc diameter) over 24-month follow up. In addition to the report, several studies showed that GLD is one of the determinant factors for BCVA after treatment for PCV.[20-22]

Regarding morphological changes during the follow-up period, central retinal thickness and subfoveal choroidal thickness significantly decreased at 1 year and maintained throughout 5-year. Several studies demonstrated that subfoveal choroidal thickness reduced after PDT in eyes with PCV.[22, 23] Maruko et al.[23] reported that subfoveal decreased choroidal thickness increased and returned to the baseline level when recurrent exudation occurred in eye with PCV. On the basis of the previous report, we subdivided eyes with PCV into recurrent group or non-recurrent group during 5-year follow up. Subfoveal choroidal thickness decreased by approximately 20% from baseline to 5-year in both groups. However, there was not a significant difference of subfoveal choroidal thickness changes between the 2 groups.

We genotyped two major genetic variants susceptible to PCV including ARMS2 A69S and CFH I62V. Especially, variants of ARMS2 A69S have been reported to be associated with various clinical phenotype including lesion size, subfoveal choroidal thickness and bilateral involvement.[24-26] In addition to the association of PCV phenotypes, several investigators reported the relationship between ARMS2 A69S and treatment response for PCV including PDT monotherapy, intravitreal anti-VEGF treatment monotherapy and combination therapy.[18, 22, 27–31] Most studies focused on visual outcomes or recurrence after treatment and demonstrated that at-risk allele homozygosity (TT genotype) at A69S of ARMS2 were more likely to recur compared with other genotypes. In addition to revealing that recurrence was more frequently seen in risk allele of ARMS2 A69S dependent fashion, multiple regression analysis revealed that time to recurrence extended by 15.5 months when G allele (non-risk allele) of ARMS2 A69S increased by one allele. To the best of our knowledge, this is the first report revealing that non-risk variants of the ARMS2 gene are associated with time to recurrence as well as chance of recurrence. These finding might be helpful for both physicians and patients when considering an optimal follow-up duration.

In the present study, intravitreal injection of anti-VEGF agents was administrated a week before PDT. The reason why we chose this method was that, PDT causes photochemical thrombotic occlusion of polypoidal lesions, while also induces the upregulation of VEGF as an adverse side effect. Initial injection of anti-VEGF agents is expected to decrease the high intraocular concentration of VEGF, which causes exudation of both intraretinal and subretinal fluid.

There are several limitations in the current study. Major limitation of this study is a relatively small sample size and a retrospective nature of analysis. Secondly, ICGA was performed only when recurrent exudation developed though all patients received ICGA prior to the initial treatment. Therefore, we cannot discuss the progression or regression of branching vascular network and polypoidal lesion during the follow-up period. Thirdly, early ICGA images were not performed for all patients, therefore we could not judge presence or absence of feeder vessels of polypoidal lesions. For this reason, we did not distinguish the PCV subtypes. A large-scale prospective study would be needed to confirm the present tentative conclusion.

In summary, the combination therapy of PDT and intravitreal injection of ranibizumab or aflibercept is an effective treatment for PCV over 5-year follow-up. Time to recurrence is associated with G allele of ARMS2 A69S and GLD. When G allele increases by one allele, it is estimated that time to recurrence extends by 15.5 months.

(XLSX)

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31 Dec 2019

PONE-D-19-34381

Five-year outcomes of photodynamic therapy combined with intravitreal injection of ranibizumab or aflibercept for polypoidal choroidal vasculopathy

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**********

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Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: The article is well written, and the first article regarding long-term results of combination of PDT and IVA for PCV. The article can accepted after major revision.

Comments:

1. A table should be added regarding Baseline characteristics comparison of 2 combination therapy. Are they matched in age, gender, etc..?

2. A table should be added about “multivariate linear regression analyses associated with the logarithm of the minimal angle resolution best-corrected visual acuity gains from baseline to at 60 months”

3. Lens status and polyp number should be added as an analyzed factor (phakic or pseudophakic) in either baseline characteristics, final VA, and VA gains

4. Each combination therapy included one PDT with one injection or two injections? After recurrence, how many injections were given? One injection or 3 monthly injections? If SRF or IRC persisted after injection or combination therapy , how did you do?

5. It seems BCVA at year 1, 2, and 3 were better than that at year 4 and 5. Can you compare the difference of BCVA at each year? Maybe there is still the trend of VA deterioration after long-term follow-up, which similar to the prior results of the study.

6. BCVA of PDT+IVA and PDT+IVR should be drawn in another figure and compared each year results. It will be an interesting result because this was the first long-term report about PDT+IVA for PCV. The authors should emphasize this unique point.

7. Are there any cases presenting with massive subretinal hemorrhage with or without vitreous hemorrhage? Do you still use combination therapy at the first place, instead of use of intravitreal tPA or gas? Or you exclude this kind of cases? If so, you should add exclusion criteria.

8. If the case with dense cataract and PCV as the first presentation, how did you do? Or you exclude this kind of cases?

9. When you use intravitreal anti-VEGF, do you exclude the cases with recent thromboembolic events or pregnancy?

10. Table 1 did not have the range of Age, Baseline log MAR BCVA, Greatest linear dimension (μm), Central Retinal Thickness (μm), and Subfoveal Choroidal Thickness

11. Can you list in a table or describe in the article about final log MAR BCVA, Greatest linear dimension (μm), Central Retinal Thickness (μm), and Subfoveal Choroidal Thickness

Reviewer #2: It is useful to publish the data presented as the long term (5 year) outcomes are valuable information. The combination of anti-VEGF + PDT is at present a treatment of choice for PCV. I have the following remarks:

1. Line 44: Please leave out the statement starting with "To date PCV has been considered ...". This is not generally true.

2. In the description of the PDT important parameters are left out. Over what time was the injection performed? How long was the delay between injection and light application?

3. At the bottom of page 5: Please describe more precisely how the follow-up treatments were done. When was only anti-VEGF applied, at what frequency, and what conditions? When combination therapy was applied in the follow-up, did anti-VEGF precede PDT, etc.

4. There are very important differences in the outcome when compared with the data of reference 13 (Miyata et. al.). This needs to be discussed in some detail.

5. The literature reports are rather incomplete. The literature reported is to a significant extent self-citation. See for instance P. Jain et. al. Indian J. Ophthalmol. 2018, Jingyuan Yang et. al. BMC Ophthalmology December 2019, and Qian T. et. al. Eur. J. Clin. Invest. 2018. among others.

6. Discussion of the influence of the ARMS2 gene as done by the present authors is of interest. Why not also discuss the results in terms of the subtypes PCV1 AND PCV2.

7. Other authors have done this combination therapy by treating first with PDT and the following up with anti-VEGF. Please discuss your choice, and why you think it is preferable.

**********

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Reviewer #1: No

Reviewer #2: No

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28 Jan 2020

Dear Editor:

We thank you, and the reviewer for timely and constructive feedback on our manuscript. We appreciate the opportunity to respond.

Sincerely

Yoichi Sakurada, M.D., Ph.D

Reviewer #1: The article is well written, and the first article regarding long-term results of combination of PDT and IVA for PCV. The article can be accepted after major revision.

Reply: We thank you for your constructive feedback and insightful comments.

Comments:

1. A table should be added regarding Baseline characteristics comparison of 2 combination therapy. Are they matched in age, gender, etc.?

Reply: Based on the suggestion, we added a new table as Table 2, and we added the explanation of the table in the Results section as follows " Table 2 shows the baseline characteristics comparison of 2 treatment groups. There were no significant differences between 2 treatment groups but central reinal thickness, in which IVR+PDT group had greater CRT than IVA+PDT group at baseline."

2. A table should be added about “multivariate linear regression analyses associated with the logarithm of the minimal angle resolution best-corrected visual acuity gains from baseline to at 60 months”

Reply: On the basis of the suggestion above, we added a new table as Table 4, and we added the explanation of the table in the Results section as follows "Another multivariate linear regression analysis associated with the BCVA gains from baseline to 5-year revealed that shorter GLD(p=1.0×10-4) and worse baseline BCVA(7.0×10-4) was associated with greater BCVA gains(Table 4)."

3. Lens status and polyp number should be added as an analyzed factor (phakic or pseudophakic) in either baseline characteristics, final VA, and VA gains

Reply: Thank you for the advice. We added lens status and number of polyps as additional factors to the baseline characteristics and described the results in Table 1, Table2, Table 3 and Table 4 in the Results Section.

4. Each combination therapy included one PDT with one injection or two injections? After recurrence, how many injections were given? One injection or 3 monthly injections? If SRF or IRC persisted after injection or combination therapy, how did you do?

Reply: We added the detailed explanation regarding the retreatment in the Materials and Methods section as follows "Recurrence was defined as newly developed hemorrhage on fundoscopy or subretinal fluid detected by SD-OCT. Additional FA/ICGA was performed when recurrent exudation was seen. When ICGA showed residual or recurrent polypoidal lesion, additional combination therapy (1 injection and 1 PDT) was administrated in the same fashion as the initial combination therapy. When ICGA exhibited abnormal vascular network without polyp, additional intravitreal injection of anti-VEGF agent was administrated. After first recurrence, patients were followed every month and PRN treatment was performed until exudation including subretinal fluid and intraretinal fluid was completely disappeared."

5. It seems BCVA at year 1, 2, and 3 were better than that at year 4 and 5. Can you compare the difference of BCVA at each year? Maybe there is still the trend of VA deterioration after long-term follow-up, which similar to the prior results of the study.

Reply: Thank you for the advice. We added a new figure showing the BCVA gains in each year as Fig 1B and described the result in the Results section as follows " Mean log MAR BCVA improved from 0.55±0.28 at baseline to 0.40±0.40 at 60-month. Mean log MAR BCVA gains at 2-year from baseline were greatest thoughout 5-year follow-up. Compared with BCVA at 2-year, those values at 4-year and 5-year were significantly worse (p=0.005 and p=0.001, respectively)."

6. BCVA of PDT+IVA and PDT+IVR should be drawn in another figure and compared each year results. It will be an interesting result because this was the first long-term report about PDT+IVA for PCV. The authors should emphasize this unique point.

Reply: Thank you for the positive comments. On the basis of your suggestion, we added a new figure as Fig 2 and described the result in the Results section as follows "Fig 2 shows BCVA gains from baseline at 5 years in each treatment group. There were no significant differences in BCVA gains between the 2 treatment groups throughout 5-year follow-up." We also added the new sentences in the Discussion section as follows " To the best of our knowledge, this is the first report demonstrating long-term results of combination therapy involving IVA and PDT for PCV. We compared the long-term visual outcome between IVA+PDT group and IVR+PDT group; however, there were no significant differences in visual outcomes between the 2 groups."

7. Are there any cases presenting with massive subretinal hemorrhage with or without vitreous hemorrhage? Do you still use combination therapy at the first place, instead of use of intravitreal tPA or gas? Or you exclude this kind of cases? If so, you should add exclusion criteria.

8. If the case with dense cataract and PCV as the first presentation, how did you do? Or you exclude this kind of cases?

9. When you use intravitreal anti-VEGF, do you exclude the cases with recent thromboembolic events or pregnancy?

Reply to the question 7-9: We added new sentences in the Materials and Methods section as follows " Exclusion criteria was the case with massive subretinal hemorrhage with or without vitreous hemorrhage, dense cataract, history of recent thromboembolic events, or pregnancy."

10. Table 1 did not have the range of Age, Baseline log MAR BCVA, Greatest linear dimension (μm), Central Retinal Thickness (μm), and Subfoveal Choroidal Thickness

Reply: Thank you for the advice. we revised Table 1 adding the range of Age, Baseline log MAR BCVA, GLD, CRT, and SCT.

11. Can you list in a table or describe in the article about final log MAR BCVA, Greatest linear dimension (μm), Central Retinal Thickness (μm), and Subfoveal Choroidal Thickness

Reply: We added new sentences to point out the final BCVA in the Results section as follows " Mean log MAR BCVA improved from 0.55±0.28 at baseline to 0.40±0.40 at 60-month." We also added new sentences in the Results section to point out the final CRT as follows "Fig 4 shows changes of central macular thickness after the initial combination therapy. Compared with baseline value, central macular thickness significantly decreased (p=<0.001) throughout the follow-up period and mean CRT decreased from 368±100 µm at baseline to 217±94 µm at 60-month." The final mean SCT was shown in Table 8.

Reviewer #2: It is useful to publish the data presented as the long term (5 year) outcomes are valuable information. The combination of anti-VEGF + PDT is at present a treatment of choice for PCV. I have the following remarks:

Replay: Thank you for your valuable comments and a positive feedback.

1. Line 44: Please leave out the statement starting with "To date PCV has been considered ...". This is not generally true.

Reply: Thank you for the advice. We deleted these sentences.

2. In the description of the PDT important parameters are left out. Over what time was the injection performed? How long was the delay between injection and light application?

Reply: We added new sentences in the Materials and Methods section to describe the combination therapy as follows " All participants received intravitreal injection of ranibizumab (0.05mg/0.05ml) or aflibercept (0.2mg/0.05ml) 1 week before PDT(1 injection and 1 PDT)."

3. At the bottom of page 5: Please describe more precisely how the follow-up treatments were done. When was only anti-VEGF applied, at what frequency, and what conditions? When combination therapy was applied in the follow-up, did anti-VEGF precede PDT, etc.

Reply: Thank you for the comments. We described the follow-up treatments in detail in the Materials and Methods section as follows "Follow-up examination included assessment of BCVA using Landolt chart, intraocular pressure, biomicroscopy with or without a 76 D lens, and SD-OCT, and was performed every 3 months until recurrent exudation developed. Recurrence was defined as newly developed hemorrhage on fundoscopy or subretinal fluid detected by SD-OCT. Additional FA/ICGA was performed when recurrent exudation was seen. When ICGA showed residual or recurrent polypoidal lesion, additional combination therapy (1 injection and 1 PDT) was administrated in the same fashion as the initial treatment. When ICGA exhibited abnormal vascular network without polyp, additional intravitreal injection of anti-VEGF agent was administrated. After first recurrence, patients were followed every month and PRN treatment was performed until exudation including subretinal fluid and intraretinal fluid was completely absorbed."

4. There are very important differences in the outcome when compared with the data of reference 13 (Miyata et. al.). This needs to be discussed in some detail.

Reply: Thank you for the valuable comments. We added new sentences in the Discussion section as follows "Miyata et al. reported 5-year results after combination therapy involving PDT and intravitreal injection of ranibizumab for 20 eyes with PCV. In their report, BCVA significantly improved at 1 year, but thereafter BCVA declined and returned to baseline value at 5 year. Similarly, in the present study, there has been the trend of BCVA deterioration after 3 years from initial treatment, but the significant BCVA improvement was maintained through 5 years. One possible reason for the differences of visual outcome between the 2 studies is that, in this study, mean age of the patients was younger than that in the Miyata's report. We reported that in the previous study, older age was associated with recurrence of PCV lesions after the combination therapy. The difference of mean age between the 2 studies might affect the visual outcome. Differences in baseline characteristic between the 2 studies might result in different results.[18] "

5. The literature reports are rather incomplete. The literature reported is to a significant extent self-citation. See for instance P. Jain et. al. Indian J. Ophthalmol. 2018, Jingyuan Yang et. al. BMC Ophthalmology December 2019, and Qian T. et. al. Eur. J. Clin. Invest. 2018. among others.

Reply: Thank you for the advice. On the basis of your suggestions, we added the articles suggested above. Line 272-274,” Several studies reported long-term results of the combination therapy involving PDT and anti-VEGF agents for PCV. [13,16,17]”

6. Discussion of the influence of the ARMS2 gene as done by the present authors is of interest. Why not also discuss the results in terms of the subtypes PCV1 AND PCV2.

Reply: Thank you for the suggestion. To reply this point, we added new sentences in the Discussion section as follows " Thirdly, early ICGA images were not performed for all patients, therefore we could not judge presence or absence of feeder vessels of polypoidal lesions. For this reason, we did not distinguish the PCV subtypes. A large-scale prospective study would be needed to confirm the present tentative conclusion."

7. Other authors have done this combination therapy by treating first with PDT and the following up with anti-VEGF. Please discuss your choice, and why you think it is preferable.

Reply: Thank you for the advice. In order to discuss our choice about the initial combination therapy, we added new sentences in the Discussion section as follows " In the present study, intravitreal injection of anti-VEGF agents was administrated a week before PDT. The reason why we chose this method was that, PDT causes photochemical thrombotic occlusion of polypoidal lesions, while also induces the upregulation of VEGF as an adverse side effect. Initial injection of anti-VEGF agents is expected to decrease the high intraocular concentration of VEGF, which causes exudation of both intraretinal and subretinal fluid."

Submitted filename: Reply to reviewer comments plos (1).docx

Click here for additional data file.

3 Feb 2020

Five-year outcomes of photodynamic therapy combined with intravitreal injection of ranibizumab or aflibercept for polypoidal choroidal vasculopathy

PONE-D-19-34381R1

Dear Dr. Sakurada,

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Academic Editor

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The questions were properly answered. The paper can be accepted. The outcome demonstrated combined PDT and IVA can be long-term as effective as PDT and IVR.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

5 Feb 2020

PONE-D-19-34381R1

Five-year outcomes of photodynamic therapy combined with intravitreal injection of ranibizumab or aflibercept for polypoidal choroidal vasculopathy

Dear Dr. Sakurada:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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