Recurrence,risk,factors,of,intravitreal,ranibizumab,monotherapy,in,retinopathy,of,prematurity:,a,retrospective,study,at,one,center

Feng-Yue Wu, Wen-Ting Yu, Dai-Xin Zhao, Wei Pu, Xue Zhang, Chun-Liu Gai

1Department of Ophthalmology, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China

2Department of Neonatology, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China

3Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China

Abstract

● KEYWORDS: retinopathy of prematurity; ranibizumab;recurrence; visual acuity; refractive error

Retinopathy of prematurity (ROP) is a proliferative disorder of the developing retina, which is one of the leading causes of childhood blindness worldwide[1‐2]. Although the etiology of ROP is multifactorial and not fully understood,vascular endothelial growth factor (VEGF) is a key factor in the progression of ROP[3]. In phase 1, both relative hyperoxia and decreased levels of VEGF led to delayed physiological retinal vascular development, resulting in a peripheral avascular area of the retina. In phase 2, the relative hypoxia and increased levels of VEGF led to vasoproliferation in the form of intravitreal angiogenesis[4].

Conventional laser treatment aims to destroy avascular retina cells, which produce VEGF. However, it has several disadvantages, such as ametropia and loss of the peripheral visual field. Anti‐VEGF agents reverse pathological angiogenic changes, and induce the progression of a physiological retinal vasculature. With increasing use of anti‐VEGF agents to treat ROP, some studies have reported that there was a significantly decreased rate of myopia and very high myopia compared with laser treatment[5‐8]. However, intravitreal anti‐VEGF therapy increases the risk of intraocular infection, delayed or incomplete vascularization, and potential local and systemic developmental effects; therefore, patients need a longer and more frequent follow‐up.

The Bevacizumab Eliminates the Angiogenic Threat of Retinopathy of Prematurity (BEAT‐ROP) study, which was the first multicenter randomized prospective trial, showed a lower recurrence of ROP following intravitreal bevacizumab,when compared with laser therapy in zone I stage 3+ ROP[9].Bevacizumab is a full‐size monoclonal anti‐VEGF antibody,which contains an Fc fragment, while ranibizumab is a monoclonal antibody Fab fragment lacking the Fc region.The Fc fragment may facilitate large molecule transport across the blood‐brain barrier[10]. A study in rabbits found that ranibizumab was cleared from the vitreous with a terminal half‐life of approximately 2.88d, which was shorter than the bevacizumab half‐life of 4.32d[11]. These results showed rapid clearing from the vitreous, theoretically making ranibizumab a better option because of its lower systemic side effects to premature infants during neurodevelopment[11‐12]. One study showed that the efficacy of ranibizumab in the treatment of ROP was significantly better than treating with laser therapy alone[13]. However, in the treatment of ROP, ranibizumab may be associated with a higher incidence of recurrence compared with other anti‐VEGF agents[14‐17]. To completely determine vascularization, a longer follow‐up schedule is important for patients treated with ranibizumab. Limited studies have reported the associations between perinatal factors and the recurrence of ROP in patients using anti‐VEGF agents[14,18‐20].However, the risk factors for ROP recurrence in patients with intravitreal ranibizumab (IVR) after long‐term follow‐up have not been clearly identified. Furthermore, visual acuities of preschool patients and refractive errors of ROP patients treated with IVR have not been reported[21]. In this study, we therefore identified risk factors for recurrence in ROP patients with IVR monotherapy after long‐term follow‐ups. We also reported visual outcomes of preschool age patients in a part of patients.

Ethical ApprovalEthical approval (Ethical Committee No.2021PS657K) was provided by the Institutional Research and Ethics Committee of the Shengjing Hospital Affiliated China Medical Universtiy in Shenyang, China. Informed consent from all legal guardians of the patients was obtained.This study adhered to the contents of the Declaration of Helsinki. This trial is registered with www.chictr.org.cn/index.aspx, ChiCTR2100051203.

Figure 1 Flow diagram of the study IVR: Intravitreal ranibizumab.

Subject RecruitmentWe retrospectively studied 25 patients who were referred to the neonatal intensive care units and underwent initial IVR monotherapy at our center between April 1, 2016 and December 31, 2017. Six patients were excluded from the study because of loss of follow‐ups, missing data, or neurodevelopmental impairment. In total, 19 patients were included in the final analysis (Figure 1).

ROP screening was according to the Chinese screening criteria[22].Infants were screened if they were born at gestational age (GA)of less than 32wk, or their birth weight (BW) was less than 2000 g, or if they had an unstable clinical course as determined by the infant’s neonatologist. The diagnosis and treatment standards were in accordance with the ETROP trial[23], and the international classification of ROP revisited[24]. Treatment was performed in all cases of type 1 ROP. Patients who were treated with laser therapy or other intravitreal treatment initially and those who had a follow‐up lasting less than 4wk after the initial IVR treatment were excluded.

InterventionTreatment was performed within 72h, once type 1 ROP was diagnosed. Intravitreal injection of ranibizumab(Lucentis; Novartis, Basel, Switzerland) was performed using topical anesthesia in a standard ophthalmic operating room.Mydriasis before the injection was achieved by using eye drops containing 0.5% tropicamide and 0.5% phenylephrine,with one drop to the eyes, three times at 10min intervals.Topical anesthesia was administered using oxybuprocaine hydrochloride eye drops (Benoxil; 80 mg oxybuprocaine hydrochloride/20 mL; Santen, Osaka, Japan). Disinfection of the injection side was conducted using 5% betadine. The eye was stabilized using toothed forceps while a dose of 0.25 mg/0.025 mL ranibizumab was injected into the vitreous cavity using a 30 gauge needle, aiming the needle directly toward the direction of the optic nerve, through the conjunctiva,approximately 1.5 mm behind the corneoscleral junction.Ophthalmic antibiotic eye drops (Tobrex; 15 mg tobramycin/5 mL;Novartis Pharma, Vilvoorde, Belgium) were administered four times per day for 7d after treatment. Mydriasis before the fundus examination and cycloplegic refraction was conducted by administering eye drops containing 0.5% tropicamide and 0.5% phenylephrine, with one drop to the eyes, for three times at 10min intervals.

OutcomesType 1 ROP was defined as any stage of ROP with plus disease or stage 3 ROP in zone I, or stage 2 or 3 ROP with plus disease in zone II, based on the ETROP Trial study[23].ROP recurrences were defined as the reappearance of ridge and/or plus disease in eyes after an initial resolution of ROP. Any recurrence in one or both eyes was defined as a ROP recurrence.Potential risk factors obtained from clinical characteristics were the patient GA, gender, BW, side, multiple gestations,Apgar score, postmenstrual age (PMA) of IVR treatment,factors during pregnancy including gestational diabetes mellitus[25]and pregnancy‐induced hypertension, patient comorbidities including surfactant (pulmonary surfactant was used at least once after birth), blood transfusion (red blood cell transfusion), anemia (hemoglobin＜110 g/L), sepsis (positive blood cultures), intraventricular hemorrhage[26], respiratory distress syndrome[27], carbohemia (PaCO2＞50 mm Hg), and congenital heart defect. The diagnosis of intraventricular hemorrhage was according to clear and accepted disease definitions using magnetic resonance imaging[26]. Hemorrhage before or after treatments involved preretinal or vitreous hemorrhage, and none of the eyes involved the visual axis or required vitrectomy. Pregnancy‐induced hypertension was defined as diastolic blood pressure of 90 mm Hg or higher and/or systolic blood pressure of 140 mm Hg or higher,when measured during the gestation period[28]. The refractive error data involved spherical power, cylindrical power, and spherical equivalent. Cycloplegic refraction was performed using an automatic computer optometer (AR‐1; Nidek, Tokyo,Japan). Snellen uncorrected visual acuity was measured and converted to the logarithm of the maximum angle of resolution(logMAR).

Follow-UpPhotographic documentation was conducted using Retcam3 (Clarity Medical Systems, Pleasanton, CA,USA) or Optos ultrawide‐field retinal imaging (Optos PLC,Dunfermline, UK). Binocular indirect ophthalmoscopy with scleral indentation was performed as needed. Each infant was independently examined by two experienced retina specialists, and eligibility was confirmed by both specialists.All patients were re‐examined the following day, the third day after treatment, 1wk after treatment, the second week after treatment, 1mo after treatment, and then depending on regression of ROP and the status of vascularization of the avascular retina.

Statistical AnalysisData were analyzed using SPSS statistical software for Windows, version 25.0 (SPSS., Chicago, IL,USA). Continuous variables with non‐normal distributions were expressed as the median (interquartile range), and;categorical variables were reported as numbers (percentages).Univariate analysis to determine the association between risk factors and the recurrence was conducted using univariate proportional Cox regression. Baseline variables with a value ofP＜0.15 or clinically relevant in the univariate analysis were included in a multivariate proportional Cox regression model.Adjusted hazard ratios (HRs) and 95% confidence intervals were also determined after adjusting for potential confounders using multivariate proportional Cox regression. A value ofP＜0.05 was considered statistically significant.

A total of 33 eyes of 19 infants were treated with IVR.Patient demographics and characteristics are listed in Table 1.Recurrences occurred in 12 eyes at a mean of 8.6±2.0wk(4.9-11.3wk) after initial IVR treatment (between 37.0wk and 50.1wk at the PMA). Ten out of 12 eyes required a second intravitreal injection (83.3%). Two eyes of one patient (16.7%)progressed to retinal detachment and were treated with vitrectomies 5wk after the initial IVR treatment (43.4wk at the PMA), which resolved the retinal detachment.

Eyes with recurrences had a lower GA (26.4vs28.0wk,P=0.049),when compared with eyes without recurrences. A higher percentage of females (66.7%vs19.0%,P=0.021), single gestation (47.6%vs30.7%,P=0.014), zone I disease (16.7%vs0,P=0.024), and mothers with gestational diabetes mellitus(50.0%vs0,P=0.001) were noted in eyes of patients with ROP recurrences, when compared with those without recurrences. All these variables were significant, based on univariate analysis and were then included in the multivariate analysis (Table 2). There were two independent risk factors for recurrence, based on multivariate proportional Cox regression:Zone I (ⅡvsI, HR: 0.056,P=0.003) and gestational diabetes mellitus (novsyes, HR: 0.095,P＜0.001; Table 2).

We observed that retinal blood vessels continued to develop,but not all blood vessels from infants reached zone III.Two eyes of one female patient received third intravitreal injections because of the second recurrences at 54.1wk at PMA(11.6wk after recurrence). Her post‐treatment photographs even showed terminated vascularization of the peripheral retina for up to 4y (Figure 2). At the last follow‐up, no infant developed endophthalmitis, vitreous hemorrhage, or cataract,and none of these eyes developed a recurrence. At the most recent follow‐up (43-58.9mo after the initial IVR treatment),uncorrected visual acuity could be measured in 14 of 33 eyes (eight infants), showing a mean 0.46 (0.19, 0.70, range:0.1-1.0) logMAR. There was no significant difference in visual outcomes (spherical power, cylindrical power, spherical equivalent, uncorrected visual acuity, and strabismus) between the recurrence eyes and those without recurrences (Table 3).No abnormality using slit‐lamp microscope was seen in these 14 eyes.

Figure 2 Fundus photographs taken from one patient and her twin brother in the study The female’s post-treatment photographs show stopped vascularization of the peripheral retina. The male’s post-treatment photographs show continued vascularization of the peripheral retina. A: Left retina of one female patient before initial intravitreal ranibizumab (IVR) [32.9wk at postmenstrual age (PMA)]; B: Left retina of the female patient after a second intravitreal injection because of recurrence (44.7wk at PMA); C: Four years after primary IVR treatment, the fundus photograph of the left retina of the female patient after three intravitreal injections because of another recurrence; D: Right retina of the male patient before initial IVR (32.9wk at PMA); E: Right retina of the male patient after initial IVR (45.7wk at PMA); F: Four years after primary IVR, the fundus photograph of the right retina of the male patient after one IVR without recurrence. Arrowheads indicate the extent of vascularization at the time of primary IVR. Black arrows indicate identical retinal points for comparison before and after treatment, and white arrows indicate the extent of vascularization at each time point.

Table 1 Demographics and characteristics of patients in this cohort n (%)

Table 2 Multivariate proportional Cox regression of recurrence

Table 3 Refractive errors and visual acuity of patients in this cohort

Compared with other anti‐VEGF agents, ranibizumab is a monoclonal antibody Fab fragment lacking the Fc region. A short half‐life and rapid clearing from the vitreous and the absence of a Fc region theoretically makes it a better option for ROP treatment, because of its lower systemic side effects to premature infants during neurodevelopment[11‐12]. However,IVR treatment for ROP may be associated with a higher incidence of recurrence, when compared with other anti‐VEGF agents[14‐17,29]. Recurrence rates after IVR monotherapy varied greatly in previous retrospective studies, which ranged from 0-83% with different recurrence definitions, inclusion criteria,IVR dosages, and follow‐up schedules[16,19,30‐32]. Moreover, the risk factors for ROP recurrence in patients with IVR after long‐term follow‐up are scarce.

In the present study, zone I disease and gestational diabetes mellitus were found to be independent risk factors for recurrence after IVR monotherapy. In our experience, IVR was effective in curing type 1 ROP for extended periods of time; however, successful treatment could not be achieved in all eyes with a single injection. The overall recurrence after IVR monotherapy was 36.4%, and recurrences occurred before 50.1wk at PMA (11.3wk following the initial IVR treatment). Two eyes of one patient had a second recurrence 54.1wk at PMA (11.6wk after recurrence). A previous study also reported that follow‐up in the first 12wk after IVR treatment was important for timely identification of ROP recurrences[19]. Several late recurrence cases (even 2.5y later)have shown the need for long‐term examinations after anti‐VEGF agent monotherapy for ROP[33‐35]. Thus, after the initial IVR treatment, it is important to conduct follow‐ups in the first 12wk for timely identification of ROP recurrences.

Zone I consists of a circle, the radius of which extends from the center of the optic disc to twice the distance from the center of the optic disc to the center of the macula[24]. It has been an important predictor for prognosis of ROP, and is associated with a high risk of adverse anatomic outcomes[36‐38].Aggressive posterior ROP has been most commonly observed in zone I[24,39], and has been identified as a risk factor for recurrence after intravitreal bevacizumab treatment[12], which is consistent with our results. Lyuet al[19]reported that zone I did not correlate with ROP recurrence. One of the reasons for the discrepancy may be that they enrolled only patients with severe forms of type 1 ROP and poor systemic conditions.

Another independent factor for ROP recurrence in our study was gestational diabetes mellitus. Based on the pathogenesis,ROP and diabetic retinopathy are both retinal vascular diseases, in which there is leakage and/or neovascularization from damaged retinal vessels, based on retinal ischemia.Moreover, Oparaet al[40]have suggested that maternal diabetes is associated with ROP, with the strength of association increasing with increasing severity of ROP. Linget al[16],however, reported that gestational diabetes did not correlate with ROP recurrences after IVR treatments. The mean GA(26.2±1.6wk) and mean BW (827.9±187.3 g) in their study were lower, whereas our study patients had an older GA of 28.0 (26.1, 28.1)wk and higher BW 993.0 (926.0, 1,114.0) g.These infants with lower GA and BW may have been more ill and with more advanced ROP.

Most retinal vessels of patients progressed anteriorly within the retina after IVR treatments. As an additional word of caution, two eyes of one patient had second recurrences 54.1wk at PMA, leaving the partially stopped vascularization retina. We recommend that follow‐up examinations need to continue for several months after each injection, because of the lack of standard follow‐up recommendations, especially for those patients with initial zone I disease and/or mothers with gestational diabetes mellitus.

After the anti‐VEGF agent treatment for ROP, persistent avascular areas in the peripheral retina were common. How to deal with the avascular area is still debatable. Although it was associated with shallower anterior chamber depths and more refractive errors[5], some studies have used laser ablation,to inhibit late recurrence[19,41]. However, we did not find recurrence after 54.1wk at PMA for even 5y when only using anti‐VEGF therapy.

Our study reported visual outcomes of a subgroup of eight patients, which made statistical comparisons difficult. Visual rehabilitation is the final objective of ROP treatment. Although depth increases with age, amblyopia remains treatable until 60mo, with a decline in treatment effectiveness after an age of 5y[42]. Eyewear corrections are often needed for associated refractive errors and strabismus or amblyopia. During this study, amblyopia trainings was already conducted by a local ophthalmologist for one child. At the end of the study, one child was ready for strabismus surgery.

Huet al[18]reported that preretinal hemorrhage before treatment was an important risk factor that was associated with the recurrence of ROP, but hemorrhages before treatment were not associated with the recurrence of ROP in our study. The study by Huet al[18]excluded zone I disease. Other possible reasons for the discrepancy may be that ROP specialists defined plus disease differently, but they tended to be internally consistent[43], thus resulting in different treatment criteria for ROP and clinical diagnoses of ROP recurrence.

There were several limitations to this study. First, it was a retrospective study with a small sample size. Second, some variables that may be related to ROP recurrence were not included, such as oxygen requirement, necrotizing enterocolitis,and hospital duration. Further, fluorescein angiography was not performed for all patients, and we only used an automatic computer optometer for cycloplegic refraction. Additionally,the possibility that screening and treatment of our population consisted of Chinese patients with older GAs and higher BWs than most other clinical trials may have influenced the clinical findings.

We give special thanks to all the teachers at the Department of Ophthalmology and professor Song Bai at the Department of Urology of Shengjing Hospital for their help and support.We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.The authors would like to thank all the study participants.

Conflicts of Interest: Wu FY,None;Yu WT,None;Zhao DX,None;Pu W,None;Zhang X,None;Gai CL,None.