Young Eun Lee, MD 1,2, Eui Seok Han, MD 1,2, Mee Kum Kim, MD 1,2, Won Ryang Wee, MD 1,2 Mee Kum Kim, MD 1,2, Won Ryang Wee, MD 1,2 Risk factor Analysis for Long-term Outcome of Keratolimbal Allografts 1 Department of Ophthalmology, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Korea 2 Laboratory of corneal regenerative medicine and ocular immunology, Seoul Artificial Eye Center, Seoul National University Hospital Clinical Research Institute, Seoul, Korea The authors have no financial interest in the subject matter of this poster

Introduction & Purpose Keratolimbal allograft (KLAL) Several successful short-term reports in the 1990s 1-4 Long-term results were not satisfactory. 5,6 Survival of KLAL: 23.7%/21.2% at 5 years (2002 reports) 5,6 Several conditions influencing KLAL success 7-11 Chronic conjunctival inflammation Tear film deficiency Symblepharon Rejection Because there have been few documented studies on a large number of KLAL patients, the important prognostic factors influencing KLAL success are unknown. So, we retrospectively reviewed our cases to elucidate the prognostic factors for KLAL success and the long-term outcomes of KLAL in our series over 10-year period. Pupose

Patients and Methods Recipient superficial keratectomy Symblepharon dissection, if present PKP or cataract surgery, if required Preparation of ring-shaped donor graft (trephination and 1/3 thickness dissection) Limbo- scleral side suture with 8-0 Vicryl Limbo- corneal side suture with 10-0 Nylon Transient amniotic membrane transplantation (AMT) 24 eyes of 22 patients Partial or total KLAL surgery From 2000 to 2009 Follow-up ≥ 1 year Limbal deficiency diagnosis Corneal vascularization or Conjunctivalization or Persistent epithelial defect or Goblet cell on the corneal surface Corneal vascularization or Conjunctivalization or Persistent epithelial defect or Goblet cell on the corneal surface PatientsSurgical procedures

Patients and Methods Delayed epithelialization: full epithelialization after 2 months postoperatively Infectious keratitis: proved when culture (+) Increased intraocular pressure (IOP) ≥ 21mmHg Graft failure: Corneal vascularization or Conjunctivalization or Persistent epithelial defect or Goblet cell on the corneal surface Corneal vascularization or Conjunctivalization or Persistent epithelial defect or Goblet cell on the corneal surface If rejection, Oral CSA ↑ or add MMF 1g bid Oral Corticosteroid ↑ Corticosteroid eyedrop ↑ Postoperative careRejection care Immunosuppression Corticosteroid 30mg/day for 2weeks Cyclosporin A (CSA) 2.5~5.0mg/kg for 6 months/ dose-adjustment for 100~150ng/ml trough level Mycophenolate mofetil (MMF) 1g bid in high-risk Ocular care Ofloxacin Corticosteroid Autoserum Artificial tears Therapeutic contact lens Definition of complications

Patients and Methods KLAL success No persistent epithelial defect No conjunctivalization No neovascularization PKP success No corneal opacity No Persistent, irreversible rejection Best corrected visual acuity (BCVA) Before surgery After surgery Last follow-up Primary failure Refractory corneal epithelial defect Irreversible rejection Demographics Age Gender Diagnosis Stevens Johnson syndrome (SJS) Chemical burn Others Previous history Graft rejection KLAL PKP Concurrent surgery Partial or total KLAL PKP AMT Preoperative state BCVA ≥ 0.1 or not Lid deformity Conjunctival inflammation High IOP Symblepharon Postoperative state Initial CSA dose High IOP Epithelialization within 2 months Interval for full epithelialization Clinical outcome evaluationPrognostic factor evaluation

Patients and Methods Clinical outcome evaluation Statistics of prognostic factor Evaluation of KLAL survival Kaplan-Meier survival curve Statistically significance Multivariate analysis Cox proportional hazards survival regression Univariate analysis Chi-square testMann-Whitney U test P < 0.10 P < 0.05

Result (Clinical outcome) Patients characteristics M:F (17:5), 24 eyes of 22 patients Age: 39.4 ± 17.4 years Follow-up: 47.3 ± 22.0 months (17-114) Diagnosis SJS 6 Chemical burn 5 Others 13 Total KLAL: 46 times (1 outside) Simultaneous PKP: 11 eyes Delayed epithelialization: 3 eyes (5months on average) Newly developed epithelial defect: 8 (33.3%) Increased IOP: 9 (37.5%), 6 medically controlled. Corneal melting: 2 (8.3%) Bacterial keratitis: 3 Fungal keratitis: 1 Herpetic keratitis: 1 KLAL failure (n=16, 66.7%) Primary failure 3 (12.5%) Persistent epithelial defect 8 (33.3%) Conjunctivalization 4 (16.6%) Corneal neovascularization 7 (29.2%) PKP failure (n=7, 63.6%) Corneal opacity by limbal insufficiency 5 (45.5%) Irreversible rejection with opacity 2 (18.2%) Reversibility of KLAL and PKP rejection Only KLAL rejection: 6 eyes (all reversible) Only PKP rejection: 5 eyes (all reversible) KLAL+PKP rejection: 4 eyes (2 irreversible) PatientsCauses of KLAL & PKP failureRejectionsComplications

Result (Clinical outcome and survival) Repeated KLAL Rapid decline until postoperative 10 months SJS: Stevens-Johnson syndrome CB: chemical burn O: others 114 months Long-term survival (+) after KLAL once Postoperative months

Result (Clinical outcome and survival) ABC FED Postoperative BCVA means the BCVA at the last follow-up, which was almost the same or better than preoperative BCVA KLAL failure (Ocular cicatrical pemphigoid) KLAL success (thermal burn) Preop1 month4 months Preop1 month4 months Postoperative months

Result (Prognostic factor analysis) KLAL success (n=13) KLAL failure (n=32) P value a ( b ) Demographics and diagnosis Age (year)43.5± ± Gender (Male)11 (85%)19 (59%)0.165 Diagnosis SJS Chemical burn35 Others816 Previous history of Graft rejection2 (22%)3 (23%)1.000 PKP5 (38%)8 (25%)0.473 KLAL6 (46%)16 (50%)1.000 Preoperative state BCVA ≥ 0.13 (23%)5 (15%)0.672 Lid deformity3 (23%)20 (63%) (0.244) Conjunctival inflammation 7 (54%)26 (81%) (0.109) High IOP0 (0%)3 (9%)0.546 Symblepharon2 (15%)23 (72%) (0.010) c KLAL success (n=13) KLAL failure (n=32) P value a ( b ) Concurrent surgery Partial KLAL 8 (62%)16 (50%)0.528 PKP 7 (54%)14 (44%)0.743 AMT 5 (38%)10 (31%)0.732 Postoperative data Initial CSA dose 2.5mg d 2 (15%)7 (26%)0.690 High IOP within 1week 6 (55%)13 (50%)1.000 Epithelialization within 2 months 13 (100%)20 (63%) (0.737) Interval for full epithelialization (day) 10.4± ± (0.858) PKP = penetrating keratoplasty; BCVA = best corrected visual acuity; IOP = intraocular pressure; AMT = amniotic membrane transplantation; CSA = cyclosporine A. a Univariate analysis was performed using the chi-square or Mann-Whitney U test. b Variables which were associated with risk of KLAL failure on univariate analysis (p<0.1) were analyzed in the multivariate Cox regression survival model. c 95% confidence interval: d Initial CSA dose was 5.0mg/kg daily in usual.

Discussion The overall success of KLAL was 33.3% at 4 years, which is similar to two previous reports of the long-term outcome of KLAL. 5-6 Our study included a relatively high portion of patients with SJS and OCP (37.5%), which are known to lead to a relatively poor outcome. 11 Simultaneous PKP and KLAL were performed because of the limited availability of donor corneal tissues. The success of PKP was highly dependent on the success of KLAL, because grafted cornea often becomes opaque due to persistent epithelial defect or neovascularization following KLAL failure. Our KLAL rejection rate was 41.7% with strong systemic immune suppression, lower than the 87.5% in eyes with CSA alone 6 but higher than the 17% rate in eyes receiving mycophenolate mofetil and tacrolimus. 7 All the rejection in KLAL alone was reversible, whereas concurrent rejection in both KLAL and PKP was irreversible in half cases. A more potent combination of immunosuppressants should be considered in concurrent KLAL/PKP rejection.

Discussion Preoperative symblepharon is the most significant prognostic factor. –Forniceal contracture usually leads to frequent falling off of the contact lens, resulting in an unstable surface. –Combining lid entropion due to shortage of the fornix induces surface irritation and epithelial erosion.. Limitations –It is a retrospective study and has limited variable factors that could not be controlled –The protocol of immunosuppressant administration and the surgeon’s skill likely changed over 9 years. 1. Thoft RA. The role of the limbus in ocular surface maintenance and repair. Acta Ophthalm ol Suppl. 1989;192: Tsai RJ, Tseng SC. Human allograft limbal transplantation for corneal surface reconstructi on. Cornea. 1994;13(5): Tan DT, Ficker LA, Buckley RJ. Limbal transplantation. Ophthalmology. 1996;103(1): Tsubota K, Toda I, Saito H, Shinozaki N, Shimazaki J. Reconstruction of the corneal epith elium by limbal allograft transplantation for severe ocular surface disorders. Ophthalmology. 1995;102(10): Tsubota K, Satake Y, Ohyama M, et al. Surgical reconstruction of the ocular surface in ad vanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome. Am J Ophthalmol ;122(1): Solomon A, Ellies P, Anderson DF, et al. Long-term outcome of keratolimbal allograft with or without penetrating keratoplasty for total limbal stem cell deficiency. Ophthalmology ;109(6): Ilari L, Daya SM. Long-term outcomes of keratolimbal allograft for the treatment of severe ocular surface disorders. Ophthalmology. 2002;109(7): Liang L, Sheha H, Tseng SC. Long-term outcomes of keratolimbal allograft for total limbal stem cell deficiency using combined immunosuppressive agents and correction of ocular sur face deficits. Arch Ophthalmol. 2009;127(11): Maruyama-Hosoi F, Shimazaki J, Shimmura S, Tsubota K. Changes observed in keratolim bal allograft. Cornea. 2006;25(4): Samson CM, Nduaguba C, Baltatzis S, Foster CS. Limbal stem cell transplantation in chr onic inflammatory eye disease. Ophthalmology. 2002;109(5): Shimazaki J, Shimmura S, Fujishima H, Tsubota K. Association of preoperative tear func tion with surgical outcome in severe Stevens-Johnson syndrome. Ophthalmology. 2000;107( 8): Tsubota K, Satake Y, Kaido M, et al. Treatment of severe ocular-surface disorders with c orneal epithelial stem-cell transplantation. N Engl J Med. 1999;340(22): In conclusion, KLAL has been partly successful for reconstruction of limbal stem cell deficiency, and symblepharon has been identified as a significant prognostic factor for KLAL survival