ANTIANGIOGENIC THERAPY WITH INTRAVITREAL BEVACIZUMAB FOR RETINOPATHY OF PREMATURITY

ANTIANGIOGENIC THERAPY WITH INTRAVITREAL BEVACIZUMAB FOR RETINOPATHY OF PREMATURITY HUGO QUIROZ-MERCADO, MD,* MARIA A. MARTINEZ-CASTELLANOS, MD,* MYRIAM L. HERNANDEZ-ROJAS, MD,* NELIDA SALAZAR-TERAN, MD,* ROBINSON VERNON PAUL CHAN, MD Purpose: To evaluate the role of antiangiogenic therapy with intravitreal bevacizumab for retinopathy of prematurity (ROP). Methods: In this noncomparative, prospective, interventional case series, bevacizumab was injected into the vitreous of patients with ROP in three different groups: group I, patients with stage IVa or IVb ROP who had no response to conventional treatment; group II, patients with threshold ROP who were difficult to treat with conventional therapy because of poor visualization of the retina; and group III, patients with high-risk prethreshold or threshold ROP. Results: Thirteen patients (18 eyes; mean age SD, 4 3 months; mean follow-up, 6 months) were included in the study. We found neovascular regression in 17 eyes. One patient with stage IVa ROP had spontaneous retinal reattachment after an intravitreal injection of bevacizumab. There were no serious ocular or systemic adverse events. Conclusion: The use of bevacizumab may be promising in the treatment of patients with ROP. Further studies need to be performed to determine the safety and long-term efficacy of intravitreal injection of bevacizumab, either as first-line therapy or after failure of conventional therapy. RETINA 28:S19 S25, 2008 From the *Retina Service, Asociacion Para Evitar La Ceguera en México (APEC), Mexico City, Mexico; and the Retina Service, New York Presbyterian Hospital, Weill Medical College of Cornell University, New York, New York, USA. Partially presented at the Cannes Retina Festival (American Society of Retinal Specialists [ASRS] and EuropeanVitreoretinal Society [EVRS]); Cannes, France; September 13, 2006. Presented at the Association of Paediatric Retinal Surgeons (APRS) Meeting; ST. Thomas, Virgin Islands; January 18 21, 2007. Partially published in Seminars in Ophthalmology (2007;22: 109 125). The authors have no proprietary interest in this study. Reprints requests: Hugo Quiroz-Mercado, MD, Retina Service, Asociacion para Evitar la Ceguera en México, Vicente Garcia Torres No. 46 San Lucas Coyoacan, Mexico City, Mexico 04030; e-mail: retinamex@yahoo.com Retinopathy of prematurity (ROP) is a proliferative disorder of the developing retina that continues to be a major cause of blindness in children. 1 4 In the United States, ROP is the second most common cause of blindness in children younger than 6 years of age, and it is estimated that of the 100,000 Latin American children who are blind, 24,000 have ROP. 1 Although ablation of the retina with cryotherapy or laser photocoagulation (standard treatment of ROP) reduces the progression of disease, the visual outcomes after treatment are often poor. 2 9 The Early Treatment for Retinopathy of Prematurity Cooperative Group found that eyes with high-risk prethreshold disease that underwent treatment had better outcomes than those treated after progression to threshold. 5 12 Even with timely treatment of threshold or highrisk prethreshold ROP, retinal detachment cannot be prevented in all cases. Scleral buckling and vitrectomy have both been shown to have some efficacy for treatment of these patients, but there is still some controversy as to the appropriate treatment and timing of intervention for retinal detachment associated with ROP. 9 S19

S20 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2008 VOLUME 28 NUMBER 3 Table 1. Systemic and Ophthalmologic Examination Schedule Examination Baseline Injection 24 Hours 1 2 4 12 24 38 Complete pediatric evaluation Yes Yes Yes Yes Yes Yes Yes Yes Yes Blood biochemistry analysis Yes No Yes No No Yes No No No Systemic blood pressure Yes Yes Yes Yes No Yes Yes No No determination Vital sign measurement Yes Yes Yes No No No No No No Complete ophthalmic evaluation Yes Yes Yes Yes Yes Yes Yes Yes Yes Fundus photography/video Yes Yes No No No Yes Yes No No (if possible) Fundus drawing Yes No No Yes Yes Yes Yes No No It was previously believed that ROP was associated only with the use of supplemental oxygen in neonates. 13 Currently, however, it is understood that the progression of ROP is multifactorial and may be associated with multiple risk factors. It has also been established that ROP is directly related to the release of angiogenic factors such as vascular endothelial growth factor (VEGF). 14,15 VEGF, which is produced by a variety of cells after hypoxic conditions, is necessary for normal vascular development of the retina and is also a main trigger for neovascularization in proliferative retinopathies. 15 20 During exposure to high levels of oxygen, the premature retina becomes hyperoxic, which reduces the secretion of VEGF, thus inhibiting retinal angiogenesis, particularly in the inner retina where the astrocytes are expressed. When the newborn is returned to normal oxygen exposure, the retina may then become hypoxic subsequent to a lack of normal vessels and the decreased oxygen concentration. Hypoxia induces degeneration of astrocytes that leads to an increase of VEGF; the astrocytes are part of the glia of the internal limiting membrane, and their degeneration allows for the migration of VEGF into the vitreous. Therefore, vascular growth occurs both in the retina and Table 2. Demographic Information for Patients of Patients Demographic Information (n 13) Males 6 (46.15) Females 7 (53.84) Hispanic 13 (100) Birth weight* (g) 1,000 4 (30.76) 1,001 1,499 8 (61.53) 1,500 1 (7.69) Gestational age (wk) 27.0 29.0 8 (61.53) 30.0 32.0 5 (38.46) *Mean, 1,233.3 g. Mean, 29.1 wk. outside the retina into the vitreous cavity. 13,15,21,22 Maximum expression of VEGF generally occurs at the border of vascular and avascular retina. This induces rapid neovascularization with abnormal vessels that lack barrier properties. 20 Although ablation of the retina with cryotherapy or laser photocoagulation inhibits the increase in VEGF by destruction of retinal neurons that are the source of VEGF after hypoxia, many patients still have progression to retinal detachment despite conventional therapy. 8,20 The use of anti-vegf therapy for ROP may be justified because it has been demonstrated that in ROP different than in diseases such as neovascular age-related macular degeneration and diabetic retinopathy there is single burst of VEGF that promotes retinal neovascularization. In contrast, in wet agerelated macular degeneration and diabetic retinopathy, there is constant production of VEGF, likely requiring multiple treatments. 23,24 Several VEGF-specific antiangiogenic agents have been approved for use. Bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA), a monoclonal antibody that inhibits the activity of VEGF, is approved for treatment of metastatic colon cancer and is commercially available in Mexico. 20 Currently, there are an increasing number of reports describing the off-label use of bevacizumab for treatment of various proliferative retinopathies with subsequent good visual and structural outcomes. 25 27 Regardless, there is still concern of significant systemic side effects from the use of intravitreal bevacizumab. After a dose of 4 ml to 16 ml of intravenous bevacizumab, a few patients had sudden death, systemic hypertension, and gastrointestinal bleeding. 20 In the vitreous, 2.5 mg (0.1 ml) of bevacizumab is injected, and it has been shown that the systemic absorption is 7.8 ng/ ml, which is the limit of quantification in plasma. This small amount of bevacizumab in the systemic circulation has not been found to be associated with severe systemic side effects. 28

ANTIANGIOGENIC THERAPY FOR ROP QUIROZ-MERCADO ET AL S21 Table 3. Distribution of Patients in Three Different Groups on the Basis of Stages of ROP, Emphasizing Attachment of the Retina and Macula of Eyes With Totally Attached Retina With Macula Attached With Macula Detached Unable to Grade Group I 4 (30.76) 3 (23.07) 1 (7.6) Group II 5 (38.46) 5 (38.46) Group III 9 (69.23) 9 (69.23) Group I, 3 patients (4 eyes) with stage IVa or IVb ROP in whom there was no response to conventional treatment; group II, 5 patients (5 eyes) with threshold ROP and poor visualization of the retina; group III, 5 patients (9 eyes) who had high-risk prethreshold or threshold ROP. We present our experience with the off-label use of intravitreal bevacizumab for treatment of ROP. Methods We performed an observational, prospective, noncomparative, interventional case series including premature or low-birth-weight infants who had new vessels or abnormal vessels identified and were treated with intravitreal injections of bevacizumab. The infants presented with the following clinical findings by routine ophthalmoscopy 4 weeks to 6 weeks after birth: stage III ROP plus disease, high-risk prethreshold ROP, threshold ROP, stage IVa or IVb ROP, or inadequate dilatation or poor visualization of the fundus. Eyes with new vessels in the vitreous cavity or at the retinal periphery were included as well as eyes with persistent fetal vasculature in the anterior segment. All patients were divided in three different groups: group I, patients with stage IVa or IVb ROP in whom there was no response to conventional treatment (laser photocoagulation or cryotherapy); group II, patients with threshold ROP for whom visualization of the retina was poor, therefore making treatment with laser or cryotherapy difficult; and group III, patients with high-risk prethreshold or threshold ROP. The patients were recruited between September 2005 and July 2006. 29,30 Exclusion criteria included leukokoria not secondary to ROP, presence of other ophthalmologic conditions at the time of diagnosis, and absence of either active new vessels or abnormal vessels. Worsening of the systemic condition led to withdraw from the study. Ocular inflammation or infection at the time of the diagnosis was a relative contraindication. The protocol was reviewed and approved by the Ethics Committee of the Asociacion para Evitar la Ceguera en México (Mexico City), and informed consent was obtained under Mexican law regulations (Norma Oficial Mexicana NOM 168 del Expediente Clinico). The treatment was administered during general anesthesia. Each intravitreal bevacizumab injection was performed as follows. The patient was prepped with sterile povidone iodine solution around the eye to be injected. A lid speculum was used to retract the lids, and two to three drops of 5% povidone iodine were placed on the eye to be injected. Using a Castroviejo caliper (Jorgensen Laboratories, Loreland, CO), 2 mm was measured posterior to the limbus in the inferotemporal quad- ROP Table 4. Group I: Three Patients (Four Eyes) With Stage IVa or IVb ROP Who Had No Response to Conventional Treatment Preinjection, 4 s, 12 s, 38 s, None 2 (15.38) 2 (15.38) Stage I 2 (15.38) Stage II Stage III Stage IVa 3 (23.07) Vitrectomy, 1 (7.6) Retina attached, 1 (7.6) Retina attached, 1 (7.6) Stage IVb 1 (7.6) Vitrectomy, 1 (7.6) Retina attached, 1 (7.6) Retina attached, 1 (7.6) Retinal detachments were classified as stage IVa in 3 eyes (23.07%) and stage IVb in 1 eye (7.6%). Four weeks after intravitreal injection of bevacizumab, 2 eyes (15.38%) had improvement, and at 12 weeks and 38 weeks, complete regression of neovascularization was observed. Two eyes (15.38%) required vitrectomy due to persistent retinal detachment with stage IVa ROP and stage IVb ROP. At subsequent visits, the retina was noted to be reattached in both eyes.

S22 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2008 VOLUME 28 NUMBER 3 ROP Table 5. Group II: Five Patients (Five Eyes) With Threshold ROP and Poor Visualization of the Retina Preinjection, 4 s, 12 s, 38 s, Regressed 5 (38.46) 5 (38.46) Stage I 4 (30.76) Stage II Stage III plus disease, prethreshold, 1 (7.60) and threshold Unable to grade 5 (38.46) Poor visualization was secondary to persistent tunica vasculosa lentis that prevented mydriasis. One week after intravitreal injection, we were able to classify 4 patients (30.76%) as having stage II ROP after complete mydriasis. After 4 weeks, we found that these four patients had had regression of ROP. In all patients in this group, we observed involution of tunica vasculosa lentis. rant; 0.05 ml (1.25 mg) of bevacizumab was then injected at this location with a 27-gauge needle. Anterior chamber paracentesis was performed to achieve appropriate intraocular pressure. 28,31 33 We performed close follow-up of the patients to determine systemic status and ophthalmologic findings (Table 1). Results Thirteen consecutive patients (18 eyes; 6 males and 7 females) were included in the study. Mean gestational age was 29.1 weeks (range, 27 32 weeks), and mean birth weight was 1,233.3 g (range, 900 1,600 g). Mean age at the time of intravitreal treatment was 2.3 months (range, 1 4 months), and minimum follow-up was 6 months. Demographic data are listed in Table 2. The groups were distributed as follows: group I, 3 patients (4 eyes) with stage IVa or IVb ROP in whom there was no response to conventional treatment; group II, 5 patients (5 eyes) with threshold ROP and poor visualization of the retina; and group III, 5 patients (9 eyes) who had high-risk prethreshold or threshold ROP. None of the patients received laser treatment after bevacizumab injection. The distribution of the patients in the different groups is listed in Table 3. We measured neovascularization by clock hours and by zones as indicated in the International Classification of Retinopathy of Prematurity. 29 The structural outcomes of the three groups are listed and explained in Tables 4 6. We found neovascular regression in all patients, with one patient who had stage IVa ROP having spontaneous retinal reattachment after intravitreal injection of bevacizumab (Fig. 1). Fundus photography and video of the fundus were not always possible, but careful drawings of anatomical findings were obtained. Reinjection was not necessary during a 6-month follow up. There were no serious ocular or systemic adverse events in any case. Discussion The main findings of our study are that intravitreal injection of bevacizumab effectively inhibits neovascularization in ROP with no adverse systemic or ocular side effects. We also observed regression of tunica vasculosa lentis 34 (Fig. 2). In an animal model, we observed that antiangiogenic therapy with prolactin (16 kd) induces apoptosis of fetal vasculature. 35 The association of persistent fetal vasculature with ROP is well known, and apoptosis of the vascular system may be affected. 34 In group II, the anatomy of the eye may have been improved by regression of persistent fetal vasculature with induction of apoptosis (Fig. 3). ROP Table 6. Group III: Five Patients (Nine Eyes) Who Had High-Risk Prethreshold or Threshold ROP Preinjection, 4 s, 12 s, 38 s, None 9 (69.23) 9 (69.23) Stage I 9(69.23) Stage II Stage III plus disease, prethreshold, and threshold 9 (69.23) After treatment, we observed that ROP regressed from stage III to stage I 1 week later. At week 12, we found complete vascularization of the avascular retina.

ANTIANGIOGENIC THERAPY FOR ROP QUIROZ-MERCADO ET AL S23 Fig. 1. Case 1. Patient from group I. A, Baseline photograph showing retinal temporal detachment and active neovascularization (arrow). B, One week after treatment, we observed a decrease of neovascularization, but the retina was still detached (arrow). C, At week 4, it was evident only as a nonvascularized fibrous membrane (arrow). D, At week 12, we observed retinal reattachment without neovascularization (arrow). Fig. 2. Case 2. Patient from group II. A, We were not able observe mydriasis due to tunica vasculosa lentis in the anterior segment (arrow). B, Five days after bevacizumab injection, we observed mydriasis; there were still some vessels (arrow), but they involuted completely during week 2.

S24 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES 2008 VOLUME 28 NUMBER 3 Fig. 3. Case 3. Patient who was enrolled in two different groups: group I for the right eye and group II for the left eye. Both eyes presented with regression of pathologic neovascularization and of persistent fetal vasculature in the anterior segment. Two weeks later, he had stage I retinopathy of prematurity. One month later, retinal vessels reached zone 3. ROP is a disease in need of better therapy. 29,30,36,37 It occurs in premature infants and remains a leading cause of vision impairment in children younger than 6 years of age. Although cryotherapy and laser ablation of avascular retina improve the chances for good outcome, 30% of infants with threshold ROP will develop a retinal detachment and blindness. More than 80% of infants who develop threshold ROP have visual acuity of 20/60 or worse, even when the retina remains attached (with an abnormal retinal structure that persists into adulthood). 4 6 With our current knowledge, it would seem that ROP may be an ideal disease model to treat with antiangiogenic therapy because it occurs in early development (occurring only during a short period) and affects an immunologically privileged part of the body. Progression to high-risk prethreshold or threshold ROP occurs during a narrow window of time, and the time from the onset of pathologic neovascularization to devastating retinal detachment is usually measured in weeks. Clearly, for this disease, antiangiogenic therapy has a fighting chance to succeed. 29 There is concern, however, that antiangiogenic therapy for infants may affect abnormal vascular development; however, in an animal model, it has been demonstrated that a single injection of recombinant human VEGF 165 b peptide can significantly reduce preretinal neovascularization without exacerbation of inner retinal ischemia because it permits physiologic angiogenesis to proceed normally and with no interference to the normal development of photoreceptors or other cells in the eye. 36 39 In addition, with the specificity of monoclonal antibody to VEGF, we may very possibly avoid these collateral effects, such as alteration of photoreceptor cell development or downregulation of factors that contribute to healthy blood vessel formation. We believe that the findings of this study move us closer to better treatment of ROP. Intravitreal injection of bevacizumab may be promising for treatment of patients with ROP. However, further studies need to be performed to determine the timing, safety, and long-term efficacy of intravitreal bevacizumab for treatment of ROP either as first-line therapy with or without laser or after failure of conventional therapy. Key words: antiangiogenic therapy, bevacizumab, retinopathy of prematurity, persistent fetal vasculature. References 1. Gilbert C, Rahi J, Eckstein M, et al. Retinopathy of prematurity in middle-income countries. Lancet 1997;350:12 14. 2. Chang-Ling T, Stone J. Retinopathy of prematurity: its origins in the architecture of the retina. Prog Retinal Eye Res 1993;12:155 178. 3. Hutchenson KA, Kelly A. Retinopathy of prematurity. Curr Opin Ophthalmol 2003;14:286 290. 4. Earl A, Palmer MD, John T, et al. Incidence and early course of retinopathy of prematurity. Ophthalmology 1991;98:1628 1639. 5. Early Treatment for Retinopathy of Prematurity Cooperative Group. Revised indications for the treatment of retinopathy of prematurity: results of the Early Treatment for Retinopathy of Prematurity Study Randomized Trial. Arch Ophthalmol 2003;121:1684 1696. 6. Repka Michael X, Tung BMS, Good W. Outcome of eyes developing retinal detachment during the Early Treatment for Retinopathy of Prematurity Study (ETROP). Arch Ophthalmol 2006;124:24 30. 7. Cryotherapy for Retinopathy of Prematurity Cooperative Group. Multicenter trial of cryotherapy for retinopathy of prematurity; ophthalmological outcome 10 years. Arch Ophthalmol 2001;119:1110 1118. 8. Harnet ME, Mc Colm JR. Diode laser for ROP. Ophthalmology 2005;112:1636. 9. Early Treatment for Retinopathy of Prematurity Cooperative Group. Revised indications for the treatment of retinopathy

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