FACTORS ASSOCIATED WITH REACTIVATION AFTER INTRAVITREAL BEVACIZUMAB OR RANIBIZUMAB THERAPY IN INFANTS WITH RETINOPATHY OF PREMATURITY

Abstract

Purpose: 

To investigate the efficacy and risk factors of intravitreal antivascular endothelial growth factor injection (anti-VEGF therapy) for retinopathy of prematurity (ROP).

Methods: 

We retrospectively reviewed 80 consecutive eyes of 43 patients with Type 1 ROP or worse who received anti-VEGF therapy during January 2012–February 2018. Patients were divided into those who were injected with 0.25 mg of bevacizumab (IVB group, 37 eyes) and 0.25 mg of ranibizumab (IVR group, 43 eyes). Serum VEGF concentrations of 18 patients were measured before and after IVR.

Results: 

Antivascular endothelial growth factor injection therapy reduced ROP activity in all eyes; however, 14 eyes (17.5%) exhibited reactivation. The reactivation rates of the IVB and IVR groups were 13.5% and 20.9%, respectively (P = 0.556). Multivariate logistic regression analysis showed that postmenstrual age ≤35 weeks at anti-VEGF therapy (P = 0.014) and aggressive posterior ROP (P = 0.044) was significantly associated with reactivation. Serum VEGF was significantly suppressed at Days 1 (P < 0.001) and 7 (P = 0.012) after IVR and returned to the preinjection level by Day 14 (P = 0.210).

Conclusion: 

Both IVR and IVB seemed effective in reducing ROP activity. Reactivation after anti-VEGF therapy may be associated with younger postmenstrual age at anti-VEGF therapy and aggressive posterior ROP.

In Brief

Antivascular endothelial growth factor therapy was effective for retinopathy of prematurity. Reactivation after this therapy may be associated with younger postmenstrual age at therapy and aggressive posterior retinopathy of prematurity.

Retinopathy of prematurity (ROP), caused by abnormal development of the retinal vessels of preterm infants,¹ is the leading cause of infant blindness in both developed and developing countries.² The largest randomized clinical trial that compared the off-label use of antivascular endothelial growth factor (VEGF) (bevacizumab) monotherapy with laser therapy found a significantly lower reactivation rate of ROP after intravitreal bevacizumab than after laser photocoagulation in Zone I ROP.³ However, several studies have reported that bevacizumab escapes from the vitreous into the systemic circulation and suppresses serum VEGF for weeks to months.^4,5 This prolonged suppression of systemic VEGF in the early newborn period may potentially affect neurodevelopmental growth.

In contrast to bevacizumab, ranibizumab is rapidly cleared from the systemic circulation⁶ and hence is considered to cause less systemic effects in premature infants. Previous reports compared intravitreal injection of bevacizumab (IVB) and ranibizumab (IVR) in the treatment of ROP and found similar efficacies but with a higher reactivation rate for ranibizumab than for bevacizumab.^7–10 Generally, when ROP is reactivated, progression occurs on a weekly basis, and if the patient is not examined at appropriate intervals, intractable retinal detachment may develop. Reactivated eyes may require additional treatment, including second anti-VEGF therapy, laser therapy, and vitrectomy, to prevent further progression of the disease. However, little is known about the timing and risk factors of reactivation after anti-VEGF therapy in the real-world clinical setting.

The purpose of this study is to evaluate the results of IVB and IVR for ROP in infants with anti-VEGF monotherapy, a history of laser therapy, and previtrectomy adjunctive therapy. We will also examine risk factors for reactivation after anti-VEGF therapy and changes in serum VEGF.

Methods

Patients and Examinations

Medical records of consecutive patients with ROP treated with 0.25 mg of IVB or 0.25 mg of IVR at Kindai University Hospital between January 2012 and February 2018 were retrospectively reviewed. None of the ROP cases treated by anti-VEGF therapy during the study period were excluded. The study was approved by the Institutional Review Board of Kindai University Hospital (#26-251) and adhered to the tenets of the Declaration of Helsinki.

At initial examination, fundus photographs and fluorescein angiograms were taken with a RetCam 3 digital fundus camera (Natus, San Carlo, CA). These examinations and diagnoses of the stage and zone were performed by two pediatric retinal specialists (K.K. and S.K.), both of whom have more than 20 years' experience of ROP examination and treatment. Retinopathy of prematurity stage and zone were evaluated based on the International Classification of ROP.¹¹ Ophthalmic examinations were performed before and 1, 7, 14, and 28 days after IVB or IVR therapy at our hospital and biweekly or monthly thereafter at referral hospitals, depending on the fundus findings. The efficacy of anti-VEGF therapy was evaluated by improvements in tortuosity and dilation of the retinal vessels and dilation of the tunica vasculosa lentis. Reactivation was defined as the reappearance of vascular dilation, tortuosity, or new/recurrent neovascularization that required further treatment. We performed fluorescein angiograms in four eyes with reactivation before treatment (vitrectomy for two eyes and reinjection for two eyes) and found evidence of fluorescein leakage from neovascularization. In cases of reactivation of ROP without tractional retinal detachment, we offered parents two kinds of treatment, namely, laser treatment and anti-VEGF therapy, and we let parents decide which treatment to be used. Vitrectomy was performed in cases of ROP reactivation with tractional retinal detachment. Antivascular endothelial growth factor injection and laser therapy were performed by two pediatric retinal specialists (K.K. and S.K.), and vitrectomy was performed by one surgeon (S.K.).

Intravitreal Injections of Antivascular Endothelial Growth Factor

Antivascular endothelial growth factor injection therapy was performed for eyes with Type 1 ROP or worse. Because 41 of 43 patients were outborn and transferred to our hospital for further treatment, there were eyes with or without a history of laser treatment and eyes with Stage 4A ROP. Indications for intravitreal anti-VEGF injection included the following three patterns of administration: monotherapy, salvage therapy, or adjunctive therapy before vitrectomy. Anti-VEGF was administered as monotherapy for treatment-naive patients, and additional therapy was provided to treat reactivation or persistent disease after laser therapy (salvage therapy) or as adjunctive therapy before vitrectomy (previtrectomy adjunctive therapy) for eyes with severe Stage 3 or Stage 4A ROP. All patients who received anti-VEGF as adjunctive therapy or salvage therapy underwent laser therapy as the initial treatment before IVB or IVR.

The choice of IVB or IVR depended on the treatment period. Patients treated between January 2012 and June 2015 received IVB (0.25 mg/0.01 mL), and patients treated between July 2015 and February 2018 received IVR (0.25 mg/0.025 mL). All parents or guardians were well informed about the efficacy and possible complications before IVB or IVR, and written informed consent was obtained from each patient's parents or guardians. Antivascular endothelial growth factor drugs were injected with a 30-gauge needle intravitreally, 0.5 to 1.0 mm away from the limbus, in the neonatal intensive care unit under topical anesthesia.

Measurement of Vascular Endothelial Growth Factor after Intravitreal Injection of Ranibizumab

Serum VEGF concentrations were measured in patients whose parents or guardians agreed to blood sample collection. Patients who received whole-blood transfusions before or after IVR were excluded. Blood samples were collected before and 1, 7, 14, and 28 days after administration of IVR. The blood samples were collected in sterile tubes by neonatologists and centrifuged at 5,000 rpm for 10 minutes until a clear separation between serum and cell components was seen. The serum was transferred to sterile tubes and stored at −80°C until assay. The serum concentration of VEGF was measured with an enzyme-linked immunosorbent assay kit for human anti-VEGF (R & D Systems, Minneapolis, MN) according to the manufacturer's protocol. The anti-VEGF kit can detect the 121 and 165 isoforms of VEGF. The minimum detectable level of the test was 9.0 pg/mL for VEGF. Optical density was determined at 450 nm by an absorption spectrophotometer with the correction wavelength set at 540 nm. The assays were performed in duplicate.

Statistical Analysis

Statistical analyses were performed using JMP version 13.0 for Windows (SAS Institute, Cary, NC). The medical records of the patients were reviewed for sex, birth weight, gestational age (GA), stage and zone of ROP, previous treatment, postmenstrual age (PMA) at the time of intravitreal anti-VEGF injection, and indication of anti-VEGF injection (monotherapy, salvage therapy, or previtrectomy adjunctive therapy). Data are presented as means and SDs, unless otherwise stated. Statistical analyses of continuous variables were performed using Student's t-test or the Wilcoxon rank-sum test, as appropriate. Categorical variables were compared using the Mann–Whitney test or Fisher's exact test, as appropriate. To verify the factors that contributed significantly to reactivation of ROP after intravitreal injection, multivariate logistic regression analysis was performed, with the factors that showed a significant difference in the univariate analysis as independent variables. The odds ratio and its 95% confidence interval for each possible risk factor were also calculated. A P value of less than 0.05 was considered to indicate statistical significance.

Results

Eighty eyes of 43 consecutive patients with ROP received intravitreal anti-VEGF therapy. Patient demographics are listed in Table 1. The mean GA of the patients was 24.8 ± 1.9 weeks (range, 22.1–30.8 weeks), and the mean birth weight was 667.8 ± 259.1 g (range, 365–1,685 g). Fifty-five eyes were ROP Stage 3, 13 eyes were ROP Stage 4A, and the remaining 12 eyes were aggressive posterior ROP (AP-ROP). There were no significant differences between the IVB and IVR groups in sex, PMA at the time of intravitreal anti-VEGF injection, or indications of intravitreal anti-VEGF injection. The follow-up period was significantly longer in the IVB group than that in the IVR group because of the different treatment periods.

Table 1. - Demographic Characteristics of Patients Treated With Intravitreal Injection of Bevacizumab or Ranibizumab

Characteristic	IVB Group	IVR Group	P
No. of eyes (patients)	37 (21)	43 (22)
Boys/girls (eyes)	15/22	25/18	1.00
Gestational age, weeks	24.7 ± 1.5	25.0 ± 2.2	0.398
Birth weight, g	629.9 ± 216.2	714.1 ± 291.7	0.162
ROP stage			1.00
3	26	29	1.00
4A	7	6
AP-ROP	4	8
Previous treatment (eyes)
Laser	29	29	1.00
None	8	14
PMA at IVI, weeks	33.7 ± 3.3	36.9 ± 2.6	0.475
Indications
Salvage therapy	19	22
Monotherapy	8	14
Previtrectomy adjunctive therapy	10	7
Follow-up period, months	39.0 ± 15.0	10.6 ± 6.6	0.01

AP-ROP, aggressive posterior retinopathy of prematurity; IVI, intravitreal injection; ROP, retinopathy of prematurity.

Of 80 eyes, 22 eyes without a history of ROP treatment received intravitreal anti-VEGF monotherapy. In the remaining 58 eyes, 41 eyes received intravitreal anti-VEGF injection as additional therapy for the treatment of reactivated or persistent disease activity after laser therapy, and 17 eyes received intravitreal anti-VEGF injection as adjunctive therapy before vitrectomy; the surgery was scheduled within 2 to 9 days (mean, 5.4 ± 2.4 days) after intravitreal anti-VEGF injection. All eyes in both groups showed regression of tortuosity and dilation of the retinal vessels and tunica vasculosa lentis after intravitreal injections. No ocular complications related to intravitreal injection were noted.

Baseline data and treatments of reactivation are shown in Table 2. Reactivation occurred in five eyes (13.5%) in the IVB group and nine eyes (20.9%) in the IVR group (P = 0.556). The period between intravitreal injection and reactivation was 8.3 ± 5.2 and 7.3 ± 1.9 weeks in the IVB and IVR groups, respectively (P = 0.947). Additional laser therapy was performed in 5 eyes in the IVB group and 5 eyes in the IVR group that developed reactivation. Two eyes in the IVR group received a second IVR, and the remaining two eyes in the IVR group underwent vitrectomy because of the development of tractional retinal detachment. All eyes except one eye in the IVR group achieved final retinal reattachment.

Table 2. - Reactivation After Intravitreal Injection of Bevacizumab or Ranibizumab

Variable	IVB Group (n = 37)	IVR Group (n = 43)	P
No. of eyes	5	9
Ratio of reactivation (%)	13.5	20.9	0.556
Period after IVI, weeks	8.3 ± 5.2 (3.9–17.0)	7.3 ± 1.9 (4.4–10.3)	0.947
Treatment of reactivation
Vitrectomy	0	2
Laser	5	5
IVR	0	2
Final retinal reattachment	5	8

IVI, intravitreal injection.

To identify the risk factors for reactivation, associations of eight explanatory variables with reactivation (sex, GA, birth weight, AP-ROP, ROP stage, previous treatment, type of anti-VEGF, and PMA at anti-VEGF therapy) were individually examined. Simple logistic regression analysis identified low-birth weight, AP-ROP, no previous treatment, and younger PMA (35 weeks or earlier) at anti-VEGF therapy as factors associated with reactivation. In addition, stepwise multivariate logistic regression analysis identified younger PMA at anti-VEGF therapy and AP-ROP as predictors of reactivation (Table 3).

Table 3. - Univariate and Multivariate Logistic Regression Analyses of Risk Factors of Reactivation of ROP

Risk Factor	Reactivation	No Reactivation	Univariate			Multivariate
			OR	95% CI	P	OR	95% CI	P
Gender
Boy, no. (%)	8 (18.6)	35 (81.4)	1
Girl, no. (%)	6 (16.2)	31 (83.8)	0.590	0.179–1.950	0.384
GA, weeks, mean ± SD	24.4 ± 1.7	24.9 ± 2.0	0.976	0.931–1.024	0.319
BW, g, mean ± SD	540.1 ± 151.9	398.9 ± 271.2	0.674	0.461–0.985	0.042	0.759	0.503–1.143	0.138
AP-ROP, no. (%)	6 (50.0)	6 (50.0)	7.500	1.942–28.95	0.004	5.532	1.049–29.17	0.044
Stage
3, no. (%)	6 (10.7)	50 (89.3)	1
4, no. (%)	2 (16.7)	10 (83.3)	0.600	0.105–3.413	0.565
Previous treatment
Laser, no. (%)	6 (10.3)	52 (89.7)	1
None, no. (%)	8 (36.3)	14 (63.7)	4.952	1.474–16.64	0.010	1.978	0.443–8.831	0.372
Treatment
IVB, no. (%)	5 (13.5)	32 (86.5)	1
IVR, no. (%)	9 (20.9)	34 (79.1)	0.590	0.179–1.950	0.384
PMA at IVI, weeks
≤35, no. (%)	11 (55.0)	9 (45.0)	1
>35, no. (%)	3 (5.0)	57 (95.0)	8.433	2.121–33.53	0.003	7.524	1.494–37.89	0.014

AP-ROP, aggressive posterior retinopathy of prematurity; BW, birth weight; CI, confidence interval; IVI, intravitreal injection; OR, odds ratio.

The serum VEGF concentrations were measured in 18 patients in the IVR group. The average serum VEGF concentrations before and 1, 7, 14, and 28 days after IVR were 1,092.6 ± 689.7, 390.2 ± 257.8, 588.4 ± 392.5, 800.3 ± 596.4, and 1,010.4 ± 738.2 pg/mL, respectively (Figure 1). Serum VEGF concentrations decreased significantly 1 day and 7 days after IVR compared with those before IVR therapy (P < 0.001, P = 0.012, respectively). Serum VEGF concentrations returned to the preinjection level by 14 days (P = 0.021). Among these 18 patients, reactivation of ROP occurred in 5 patients. The average serum VEGF concentrations of patients with reactivation before and at 1, 7, 14, and 28 days after IVR were 1,073.6 ± 138.9, 405.3 ± 50.0, 471.2 ± 156.0, 1,036.3 ± 201.6, and 995.6 ± 232.0 pg/mL, respectively. The average serum VEGF concentrations of patients without reactivation before and 1, 7, 14, and 28 days after IVR were 1,097.4 ± 141.5, 386.5 ± 85.8, 617.8 ± 72.1, 741.3 ± 112.8, and 1,014.1 ± 144.8 pg/mL, respectively. There were no significant differences in serum VEGF concentrations between the two groups at any of the time points (P = 0.910, 0.863, 0.447, 0.274, and 0.950). In patients without reactivation, serum VEGF levels at 1, 7, and 14 days after IVR decreased significantly as compared with baseline serum VEGF levels (P < 0.001, <0.001, and <0.001, respectively). By contrast, in patients with reactivation, a significant decrease of the serum VEGF level compared with baseline was observed only at 1 day after IVR (P = 0.015) (Figure 2).

Fig. 1.: Serum levels of VEGF after intravitreal injection of ranibizumab. Changes in the serum level of VEGF during 28 days after intravitreal injection of ranibizumab (n = 18) (mean ± SEM). Serum VEGF was significantly suppressed at 1 day and 7 days after IVR and returned to the preinjection level by 14 days. *P < 0.05 versus baseline.

Fig. 2.: Comparison of serum levels of VEGF in patients with and without reactivation. Changes in the serum level of VEGF during 28 days after intravitreal injection of ranibizumab in eyes with reactivation and those without reactivation (mean ± SEM). The serum level of VEGF was significantly decreased at 1 day, 7 days, and 14 days from baseline in eyes without reactivation and at 1 day from baseline in eyes with reactivation. *P < 0.05, **P < 0.01 versus baseline.

Discussion

This study investigated the clinical outcomes of intravitreal anti-VEGF therapy, including monotherapy, salvage therapy, and previtrectomy adjunctive therapy, in a real-world clinical setting in Japan. The results demonstrated that both IVR and IVB were effective in reducing disease activity in all eyes. All 37 eyes in the IVB group and 42 of 43 eyes in the IVR group achieved final retinal attachment. In total, the final attachment rate was 98.8%. However, 14 of 80 eyes (17.5%) developed reactivation of ROP and required additional treatment. Because the study included monotherapy, salvage therapy, and previtrectomy adjunctive therapy groups, simple comparison with previous studies was not possible. However, we found that the reactivation rate was comparable with those in previous studies. We also identified AP-ROP and younger PMA at anti-VEGF therapy as significant risk factors associated with the reactivation of ROP. Serum VEGF was significantly suppressed within 7 days after IVR and returned to the preinjection level by 14 days.

The BEAT-ROP study was the first randomized clinical trial that compared the use of bevacizumab with conventional laser therapy, showing that bevacizumab reduced the risk of reactivation of Zone 1+ retinopathy by five times compared with conventional laser therapy evaluated at 54 weeks of PMA.³ Since then, the off-label use of bevacizumab therapy for ROP in clinical practice has increased, despite concerns about potential systemic side effects of anti-VEGF therapy. Ranibizumab is an alternative anti-VEGF agent with a shorter half-life and potentially less systemic toxicity. Its use in the treatment of ROP has been reported recently in several case reports and in randomized controlled trials.^7,9,12,13 Both IVB and IVR are highly effective at inducing ROP regression; however, reactivation of ROP after anti-VEGF therapy is not uncommon. Previous studies assessing the clinical outcome of IVB therapy in ROP eyes have found reactivation rates of 6% to 10%.^3,9,14–17 By contrast, there was considerable variation in the reactivation rate after IVR therapy, which varied from 0% to 80%.^{7–10,12,18–22} This significant variation was presumed to be due to the small sample size and differences in screening methods, follow-up schedules of patients with ROP in various countries, dosage of anti-VEGF agents, ROP characteristics especially between developed and developing countries, and definitions of reactivation. Several studies have warned the possibility of higher rates of reactivation of ROP in eyes treated with IVR than in eyes treated with IVB.^7–10 Similarly, the reactivation rate in our study tended to be higher in the IVR group (20.9%) than that in the IVB group (13.5%), although the difference was not statistically significant (P = 0.556). In our study, the reactivation rate after IVB was slightly higher than the values (6%–10%) reported in previous research^3,9,14–17 in which 0.625 mg of bevacizumab was used, but they were slightly lower than the reactivation rate of 18% after an injection of 0.25 mg of bevacizumab reported by Wallace et al.²³

Previously reported risk factors for reactivation after IVR or IVB monotherapy include lower birth weight, lower GA, longer duration of hospitalization, extensive retinal neovascularization, requirement for supplemental oxygen, and preretinal hemorrhage before injection.^14,18,24,25 Ling et al ¹⁰ reported that ROP was reactivated in 44 of 340 eyes (12.9%) with Type 1 ROP treated by either laser photocoagulation, IVB monotherapy, or IVR monotherapy; the risk factors for reactivation were early PMA at initial treatment, Zone I ROP, low Apgar score, and multiple births. Using multivariate logistic regression analysis, our study also demonstrated that younger PMA at treatment and AP-ROP were possible risk factors for reactivation. The average GA at anti-VEGF therapy in our patients with reactivation was 34.0 weeks, which was the median age at detection of Stage 1 ROP in the BEAT-ROP study, suggesting a greater degree of immaturity in our patients.³ Moreover, the mean birth weight in our study (667.8 ± 259.1 g) was lower than that in the report by Ling et al (857.4 ± 231.3 g), which may indicate the possibility of more severe ROP in our population. AP-ROP is a more virulent form of ROP that is observed in more immature babies. As these two factors are risk factors for reactivation, immaturity may be associated with the underlying mechanism of reactivation. Interestingly, in our study that includes anti-VEGF monotherapy, salvage therapy, and previtrectomy adjunctive therapy, univariate logistic regression analysis revealed that monotherapy was another possible risk factor for reactivation. Eyes without previous laser therapy had a significantly higher reactivation rate than eyes with previous laser therapy. As for complications of anti-VEGF therapy, posterior atypical tractional retinal detachment due to fibrovascular contraction was reported after anti-VEGF monotherapy for ROP.²⁵ Transforming growth factor beta, a profibrotic cytokine, is elevated in the vitreous of eyes with ROP²⁶ and also after anti-VEGF therapy,²⁷ which might be associated with the development of tractional retinal detachment. Thus, eyes receiving anti-VEGF monotherapy without previous laser therapy might be at a higher risk for reactivation and progression to tractional retinal detachment. Careful monitoring of patients with ROP after anti-VEGF monotherapy seems mandatory.

In this study, 17 eyes received IVR or IVB as previtrectomy adjunctive therapy. The reactivation rate in this group of patients was 11.8% (2 of 17 eyes). Vitrectomy for eyes with high vascular activity should be avoided because it is often associated with bleeding during and/or after surgery and severe postoperative inflammation, and hence poor surgical outcomes.²⁸ We therefore used anti-VEGF therapy as previtrectomy adjunctive therapy for eyes with high vascular activity. We previously reported a decreased fluorescein leakage of the neovascular membrane, regression of the tunica vasculosa lentis, and improvement of vascular dilation and tortuosity after IVB for severe ROP.²⁹ Xu et al³⁰ reported that IVB before early vitrectomy for vascularly active Stage 4 ROP resulted in reduced surgical time, reduced postoperative complications, increased lens preservation, and better recovery of vision. Although both IVB monotherapy and IVR monotherapy are effective for the treatment of ROP, the efficacy of IVR before vitrectomy has not been described. In this study, seven eyes received IVR before vitrectomy, resulting in reduced vascular activity without apparent ocular or systemic adverse effects. All seven eyes had a history of laser treatment before IVR. Although it is difficult to make a conclusion based on a small number of patients, IVR, like IVB, seemed to be effective as previtrectomy adjunctive therapy for vascularly active ROP.

Vascular endothelial growth factor injection may enter the systemic circulation and decrease systemic VEGF levels after intravitreal injection. We previously reported that bevacizumab could reduce serum VEGF levels for at least 2 weeks after IVB in infants with ROP.⁴ Wu et al⁵ reported that the decrease in serum VEGF levels started as early as 1 week after IVB. Our results showed that serum VEGF concentrations decreased significantly at 1 day and 7 days after IVR and returned to preinjection levels by 14 days. Hoerster et al³¹ reported that serum VEGF levels after administration of 0.2 mg of ranibizumab were suppressed for 2 to 3 weeks after injection. The RAINBOW study, with 151 participants, is the largest study to measure VEGF levels after 0.2 or 0.1 mg of ranibizumab, and it showed no evidence of plasma VEGF suppression by IVR.¹³ However, only three measurements of VEGF levels were taken as follows: 24 hours before, 14 days after, and 28 days after IVR. Therefore, information on serum VEGF concentrations within 13 days after IVR was lacking. Regarding the VEGF levels 14 days after injection, the RAINBOW study obtained the same result we did, although the dosage (0.1 or 0.2 mg) was different from that used in our study. Chen et al investigated serum VEGF concentrations before and 1, 3, and 7 days after IVR and reported that the levels were completely suppressed from 1 day to 7 days after IVR,³² which is consistent with our findings. By contrast, Zhou et al³³ reported that plasma VEGF suppression started as early as 1 day after IVR and was normalized 1 week after IVR. The dose of ranibizumab did not differ among these two studies: all patients received IVR 0.25 mg per eye. The differences in the results of these studies may be due to differences in patients' PMA, treatments before injection, and methods of measurement.

This study has several limitations. First, the condition of ROP at the time of anti-VEGF therapy and the method of anti-VEGF therapy, i.e., monotherapy, salvage therapy, or previtrectomy adjunctive therapy, were not uniform because most of the patients were referred to our hospital. Second, because of the small number of cases for each treatment, adequate statistical analysis could not be performed for each treatment and lack of generalization. Third, dosing of IVB of 0.25 mg used in our study is not generalizable because it was different from the most commonly used dosage of 0.625 mg. The strengths of the study are its consecutive design and the results on the efficacy and risk for reactivation after IVB or IVR, including eyes with a history of laser therapy and eyes treated with adjunctive therapy before vitrectomy in the real-world clinical setting.

In conclusion, IVR, as well as IVB, seemed effective in reducing ROP. However, approximately 20% of the cases exhibited reactivation, and close monitoring and/or early additional treatment is recommended for high-risk patients of reactivation, such as AP-ROP and younger PMA at anti-VEGF therapy, after anti-VEGF therapy. Ranibizumab was recently approved by the U.S. Food and Drug Administration for the treatment of ROP and no longer needs to be used off-label for this condition. Therefore, ranibizumab, rather than bevacizumab, is expected to become the mainstay of ROP treatment or at least a major drug for anti-VEGF therapy for ROP in the future. However, it is necessary to be aware of the risk for reactivation, especially in infants who require the treatment at a younger PMA.