Graefes Arch Clin Exp Oplahalol (2000) 247:1701-1706 OE 10,1007SC0417-009411 7047 CORNEA Avastin use in high risk corneal transplantation Petja Ivanova Vassileva + ‘Tatyana Georgieva Hergeldzhieva Roocived: 28 February 2009 /Revisod: 24 July 2008 / Accepted: 27 July 2009 Published cone: 13 Aust 2009 (© Springst-Verlaz 2009 Abstract Background Comeal neovascularization is a major risk factor for graft failure after comeal transplantation, The purpose of this study was to investigate the effect of subconjunctival, perilimbal, ancVor intrastromal bevaciai- mab (Avastin®) on comeal neovascularization in patients ‘with penetrating keratoplasty (PKP), ‘Methods Fourteen eyes of 14 patients with high risk ‘comeal transplantation and comeal neovascularization after PKP (nine men and five women with a mean age of 68 years) were included in this non-comparative intervene tional case series, Indications for PKP were: vascularized Teucomas after herpetic keratitis and chemical bum, advanced pseudophakic bullous keratopaday with superti- cial and deep comeal vascularization, keratooomus, severe infection in hereditary comeal dystrophy, and failed comeal ‘gaafis Suboonjunctival, perilimbal, and/or inwrastromal bevaciamab of dose of 2.5 mg! 0.1 ml’ per affected quadrant was injected at the site of neovascularization in cach patient at the end of surgery andr at follow up visits. ‘One or two injections were applied. At each visit a fall eye examination with photo documentation was performed. Follow-up period was 2 to 8 months (mean 7.1 months) Presented in part as oral presentation atthe 106th Anmal Meeting of the Gaman Society of Opthalmology, Bern, September 2008 ‘The authors have fll conmol ofall primary daa, and aur w allow Graefes Archive far Clinical and Experimental Ophthalmology to review our daa upon request PL Vasiloa TG. Hewsidzhieva =), University Eye Hospital “Prof. Pashew", 51 Eman Vaskidovich Stoct, Sofia 1517, Bulgaria emma: enia_het@abv-by ‘email: tnia_hor hotmail.com Results Decrease of comeal neovascularization was ob- served in eleven patients (78.6%). Regression of neo- vascularization with fading of small vessels was demonstrated. Despite high- risk patient pool, twelve grafts (85.7%) remained transparent for the period of cbservation, ‘and patients maintained good visual acuity. In two patients ‘with intial erat rejection and vascularization, subconjunctival and perilimbal application of bevacizamab was beneficial in ‘overcoming the comeal inflammation and intial rejection, No adverse reactions have been detected to date in patients with subconjunctival, perilimbal, and/or inuzstromal injection of bevacizumab. Conclusions Bevacizsmab is an efficient and safe additional treatment option for improvement of prognosis in highstisk ‘comeal transplantation with pre- and postoperative comeal neovescularization, Keywords Comeal transplantation - Penetrating keratoplasty -Comeal neovascularization + Subconiunctival Avastin Introduction ‘Comeal transplantation was the first tissue transplantation performed in medicine, and it is still the most common allotransplantation, Comeal graft remains clear in majority of patients in low-risk keratoplasties. This sucoess relies on site-specific characteristics, such as comeal inmmune privilege and comeal avascularity [1]. On the other hand, graft rejection rates of more than S0% have been reported in patients with high-risk keratoplasties with pre-existing, ‘comeal neovascularization (NV), Comeal NV occurs when the balance between angiogenic and antiangiogenic factors shifts toward angiogenic factors [2], Vascular endothelial D springerme Gracfis Arch Clin Exp Oplahalmal (2009) 247:1 701-1706 ‘goth factor (VEGF) has been found to be a significant angiogenic factor in comeal NV in human and animal models [3]. Inflammatory prooesses may stimulate varying degrees of vascubaization. Comeal NV is a major risk factor for graft failure after penetating keratoplasty, Vsculeization of the graft may be the rsuit ofa poorly prepared recipient 6 the expression of intolerance tothe suture material, ora defense mechanism against a suture abscess or graft infection [4], Vasculaization of the emft is also a sign of immune reaction, Inhibition of VEGF may contribute to the treatment of patients with vascularized comeal grafts. Evidence on efficacy and safety of locally applied bevacizamal on ‘comeal NVas an offabel treatment was obtained in human, and animal models [5-7]. Bevacizumab (Avastin; Roche, Weluyn Garden City, UK) is a recombinant, humanized, monoclonal antibody against VEGF-A. Recently, small case series and isolated ‘ase reports have been discussed in the literature, demon- stating safety and good results after off-label application of | bevacizumal for comeal neovascularization. “The purpose of this study was to investigate the effect (of subcenjunctival, perlimbal, andor intasromal bevaciar- mab on comeal NV in pationts with penetrating keratoplasty (PKP). ‘Material and methods ‘The study was approved by the Institutional Review Board, Al patients were asked to sign a writen informative ‘consent before any treatment had bean initiated, Fourteen eyes of 14 patients, nine men and five women, with a mean age of 634 years (range 30-83) with a highe risk comeal transplantation and comeal NV after PKP were included in this non-comparative interventional case series (table 1). Indications for penetrating keratoplasty included: vascu- Jarizad leucoma after herpetic keratitis in three patients and chanical bum in two patients, advanced pseudophakic bullous keratopathy with superficial and deep vasculatizae tion of the recipient comea in four patients, keratoconus in ‘wo patients, graft failure in two patients, and severe infection in hereditary comeal dystrophy in one patient, Patients with advanced PBK were referred to our hospital 3 to 5 years after cataract extraction, Suboonjunctival, perilimbal, and intastromal bevacize ab of a dose of 2.5 mg/ 0.1 ml per affected quadrant was injocted at the site of NV before PKP in two pationts with ‘afi failure, and subconjunctivally and perilimbally at the end of PKP in ten andlor at follow-up visits in four patients. One to two injections were applied depending an progres- sion of vascularization, D springer Special instructions for regular examinations and report ‘on possible adverse events were given to all participants. AIL PKP patients were treated with topical cortioo- steroids and artificial tears. We did not change the routine corticosteroid topical regiment depending on injection, ‘The effect of bevacizmab application was accessed on the first and seventh day after the injection, as well as every month thereafter. At each vist a full eye examination with Photo documentation was performed. Comeal photographs ‘were analysed by the same reviewer, who was not masked, ‘The state of neovascularization before and after interven tion was determined by comparing the size, number and centricity of vessels as well as the number of affected quadrants. The effect of bevacizmab application on comeal NV was assessed as mld in cases with only temporal effect on superficial and deep vessels when they eventually invade the graft despite treatment, good in cases with partial regression of vascularization and a need for ‘another injection, and excellent when both superficial and deep vascularization disappeared! and no additional injec tion of the medication was indicated. All PKPs and bevacizumab injections were performed! by the same surgeon, ‘The follow-up period was 2 to 8 months (mean 7.1 months). Results A majority of patients (71.4%) had both superficial and deep comeal NV. A decrease of comeal NV was observed in 11 patients (78.6%), with regression of NV and fading of small vessels (Figs. 1,2 and 3). In nine patients (64.3%) the result of bevacizumab application was excellent, and there ‘was no need for another injection. In these cases, new vessels were not observed 10 cross the graft-host interface and invade the comeal graft afier the intervention, Two Patients (14.3%) showed inital good results with decreased size of the new vessels, but since the effect was temporary ‘and comeal neovascularization progressed again, another bevacizumab injection was performed. In three patients (21.4%) there was little change in comeal NV. ‘Two patients with vascularized failed comeal grafts, one after PKP for PBK, and the other one after comeal ‘transplantation for keratopathy as a result of radial keratotomy, were included in this series, Bevacizumab ‘was applied 1 and 2 months respectively before re- penetrating keratoplasty. Anti-VEGF therapy was per formed before comeal re-ransplantation, in order to induce regression of existing NV and improve prognosis for graft survival. The effect on vessel regression was excellent in patient #12 and good in patient #13. Patient #13 needed ‘another bevacizumab injection because superficial vessels,repunlicogns ys Kurromny paper yey Sone Fumeapend yy “ipl soqIMy SpRlopNONT yer URS fT py SHA se KUNA “Hy Sigdonsip simueus 9 1 Pau Key MAAMADS LH Se aE seuss z Awmloresyy GASKET 95 EL SOuPURR, simuous Ir emg A ANA AMES ERTL soupeayg ee simu g 1 swooefa yeid wom Sr fT soup ee seu 1 wont yes POE TUE fT 381 3 sous z ANE WMA WT AIUD 96 savour 1 Sopa rome 98 simuou Ir wa 9 WL siauous 1 SOU ROME OL SIREN simuou Ir opalny nye Hunn pores. 19 NS sammy seu 1 opal Dye MUON POMC. GW sR seus 1 opacioy sym wenn porwr. SE used yrocorn “te simuou Ir EUDYP AY! MUNN PPS LNT ou ft ny simu 8 TREES ESO CRE! — damp -FPYRKNS —HUNIP.KyE MUON perms, 6ST opty sony wom sumpmb oye wo sido PVE AU JON, 09S _ Jo‘ pur wypmyekiojo sum, oa sofia sf ERED WN Graefes Arch Clin Exp Oplihalmol (2008) 247:1701-1706 womeet nome 1 Rae, springer asGracfis Arch Clin Exp Oplahalmal (2009) 247:1 701-1706 Fig. 1 Photgmph of patient #7 with PBK prior w PKP and bxaciaumab applicaion—marked noovasculrization is seat, espe cally superiorly and nasally progressed and invaded the graft in one quadrant, Avastin injection led to marked regression of these vessels, In ten patients, bevacizamab was applied at the end of the penetrating keratoplasty. In six of these patients, both deep and superficial vascularization were suppressed, and lid not appear in the graft for the period of observation. In four patients, bevacizamab was applied at different time periods after PKP depending on vascularization, with a ‘varying effect on the vessels, In two of these patients with initial graft rejection and vascularization (patients #10 and, 1), bevacizumab was beneficial in overcoming the comeal inflammation and initial rejection, Case #10 had mycotic keratitis and inflamed comeal ulcer at the time of comeal ‘transplantation, About a month after PKP a recurrent uloer developed close to the graft-host junction, with initial graft rejection. Topical antismycotic preparation was used in this patient also. In case #11 the keratoconus was advanced, ‘ith severe thinning of the comea, which led to permanent ‘comeal defect for more than a month, Mild inflammation Fig. 3 Photoweph of the same patient 8 months afer PKP and bovaciaumab applicaion, showing sable wsulis conscming comcal noovascularizaion and wensparency with invading vessels was observed. Ammiotic membrane ‘transplantation was performed in both patients. The graft ‘cedema disappeared, and the rejection line did not advance in patient #10 and the comeal defect healed in patient #11. Despite application of amniotic membrane in both patients, superficial new vessels advanced to the graft, and we decided to use bevacizimab application, In both patients, ‘comeal neovascularization regressed afier a single injection (Figs. 4 and 5 of patient #10), Comeal transplants have remained clear without signs of rejection for more than 8 months, Despite a high-risk patient pool, 12 grafts (85.7%) remained transparent for the period of observation, and. Patients maintained good visual acuity. No adverse reactions have been detected to date in patients with subconjunctival, petilimbal, andlor intrastwo- mal injection of bevacizamab, Fig. 2 Phowgrpph of the same patent 2 months ater PKP and bevacizumab application, showing dramatic duction of comeal noovascularization D springer Fig. 4 Phowgraph of patent #10 with inital grat rejection and superficial vascularization advancing towans the prafhost junction, bfore bevacizumab applicationGraefes Arch Clin Exp Oplihalmol (2008) 247:1701-1706 1s Fig. § Phoogzph of the same patient 1 month alfer bevacizumab applicaion, showing regessol grat ondms, no progression of the ‘pci line, and dssppeaance of comeal noovascularivation Discussion ‘Comeal neovascularization is a major risk factor for graft failure after penetrating keratoplasty. Its prevention and ‘treatment may improve the prognosis for graft transpar- ency and visual rehabilitation of patients with comeal ‘wansplantation, All patients with penetrating keratoplasty are strictly instructed 10 immediately report symptoms of graft rejec= tion, such as decrease of visual acuity, redness, pain, initation, and photophobia. Meticulous examination for limbal injection, postoperative infection, new vessels threatening or invading the graft, epithelial defects, graft ‘cedema, stromal infiltrates or sizns of endothelial rejection (Khodadoust line, keratic precipitates, anterior chamber reaction, etc.) is performed at follow-up visits in all cases with PKP at our hospital, Since patients with graft rejection may be asymptomatic, they are asked 10 come for regular examination after hospital discharge on the Tih, 4th and 30th postoperative day, once monthly in the first 6 months afier PKP and every 2 months till the end of the first year. Afier this period, observation continues with an individual approach but generally at Jonger time intervals. Different treatment modalities have been used to manage ‘comeal neovascularization (argon laser photocoagulation, Photodynamic therapy, pharmacologic agents, etc), with ‘varying success, Our observations conceming the short= tem effect of subconjunctival, perilimbal, and/or intrae stromal injection of bevacizamab on comeal new vessels ‘are similar to those reported by other investigators. The matication is well4olerated, and associated with a partial regression of comeal NV [8] For many years, ophthalmologists have been using local and systemic corticosteroids as a powerfil and effective ‘treatment against vascularization ofthe graft. The dosage of ‘corticosterDids should be inoreased in cases with vascularizae tion of recipien’s comea advancing towards the grafi-host interface and the graft. Adverse effects such as secondary ‘glaucoma and cataract formation are common complications in these cases. Other immunosuppressive drugs such as azathioprine and cyclosporine have permitted a signifi- ‘cant reduction in the corticosteroid dosage, and a greatly reduced incidence of graft vascularization. All patients with PKP at our hospital are treated with topical corticosterbids and artificial tears. Application of bevacia mab may be considered as an additional stratezy for management of comeal neovascularization, Other authors have published similar results [9]. A possible role of bevaciumab in the management of ‘graft rejection with vascularization may be discussed, as stated by other authors as well [10]. ‘One disadvantage of our investigation is the lack of a control group. As a referral hospital, our patient pool consists mostly of young patients with bilateral blindness or monocular patients, Sometimes these people live in remote areas, with no possibility for frequent examinations, We decided to inject bevacizumab in cases with vascularization instead of just observing these patients, ‘Superficial vascularization in the recipient comea tends to disappear after penetrating keratoplasty. Even so, there is a greater risk for graft failure in cases with advanced superficial vascularization in all quacrants of the comea. ‘That is why we consider this indication for intervention and application of bevacizamab. Since penetrating keratoplasty is not a planned surgical procedure in our country, the period between bevacizumab application and PKP differs in the two patients with pre-existing NV and failed comeal grafts, Anti-VEGF therapy was performed before comeal re- transplantation in order to induce regression of existing, NV and improve prognosis for graft survival. Neverthe- Jess, graft failure was diagnosed in ane of these patients 8 months afier comeal transplantation. Other factors in addition to comeal vascularization are of great impor- tance in transplantation immunobiology. Data on topical application of bevacizuma are contro versal, Further controlled and long-term studies are needed to evaluate the effect of this new treatment, Based on our observations, bevacizumab (Avastin®) may be considered as an efficient and safe additional ‘treatment option for improvement of prognesis in high-risk ‘comeal transplantation with pro- and postoperative comeal neovascularization, Conflict oF interest None D springer1706 References 1, George Al, Larkin DF @004) Come wansplamatien: the fonvodten graf. Am J Tensplant 452678685 2, Folkman J (1995) Angiouenesis in cancer, vascular, sheumoid ‘and ether disease. Nat Med 127-31 {3 Philipp W, Speicher L, Humpel C @c00) Expression of vascular axotheld growth ctor ane ts reogptors in inflamed and ‘vascularized human comeas. Invest Ophthalmol Vis Sci 4125142502 4 Barcuer J, Rutllan J (1984) Mirosursery ofthe comes an Atlas an Textbook. Ediciones Sciba, Baraons ‘5, Marvano RP, Peyman GA, Khan P, Canounis PE, Kivileim M, Ran M, Lake JC, ChévexBarrios P 2007) inhibition of ‘experimental comeal nowascularsaton by bevaciauma (Aves in), BrJ Ophthalmol 91(6)804-807 D springer Gracfis Arch Clin Exp Oplahalmal (2009) 247:1 701-1706 6, Barus LE, Belfor R Jr (2007) The effets of the sutoanjunctval injection oF bovacizmab (Avasin) on angiogenesis in the ra comet. An Acad Bras Ciene 79(3)389-304 7. Tabingen Bevaciamab Sty Group, Yoorek E, Ziemssen F, Henke-Fahle S, Taar 0, Tura A, Grisan S, Baru-Sclmidt KU, ‘Samm P (2008) Safty, penctration and cicacy af topically applicd bevacizumab: cxalution of eyedkops in comeal noo ‘vascularization after chesnical bum. Acta Opkuhalmol 863)322— 228 8. Balur 1, Kaisermun 1, MeAllum P, Roctman D, Slomovic A (2008) Subciunetival bevacizma injcton for comcal noo ‘vascularization. Comea 272) 142-147 9, Exdummus M, Tot Y 2007) Subconjunetival bevaciauma for comeal namascularization, Graefes Arch Clin Exp Ophthalmol A910 ;1STEISTO 10, Avadein A 2007) Subconjunctval bevacizumab for vascularized rejected come gga, J Cataract Reffact Suz 331119911995,Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.