A clinical-immunopathological-surgical outcome correlation of cases with clinical Limbal Stem Cell Deficiency (LSCD) 1 Tennent Insitute of Ophthalmology, Glasgow, UK 2 Pathology Department, Western Infirmary, Glasgow, UK The authors have no financial interest in the subject matter of this poster Maria Elena Gregory 1, Elisabeth CA McDonald 1, Yvonne Reive 2, Fiona Roberts 2, Kanna Ramaesh 1

 Corneal changes in LSCD are considered to be due to loss of barrier function and conjunctival cell migration with acquisition of CK19 and goblet cells in the corneal epithelium. 1  The aim of the study is to: A.Characterize the shift in cytokeratin profiles of conjunctival and corneal epithelial cells exposed to chronic inflammation. B.Correlate the clinical appearance of patients with LSCD, their corneal epithelial cytokeratin profile and surgical outcomes. INTRODUCTION 1 Puangsricharern V, Tseng SC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 1995;102:

METHODS:  Paraffin-embedded tissue samples were stained with antibodies against cytokeratin (CK) 3, 12 (cornea) 7,19 (conjunctiva) and 2, 10 (dermal). 2  Samples included normal conjunctiva, cornea, eyelid (n=28), and corneal and conjunctival tissue from chronic ocular surface disease eg OCP, SJS (n=32).  A clinical-immunopathological-surgical outcome correlation of selected cases was performed. 2 Pitz S, Moll R. Intermediate-filament expression in ocular tissue. Prog Retin Eye Res 2002;21(2):

METHODS: Panel of antibodies CYTOKERATINCLONEANTIGEN RETRIEVAL METHOD (Incubation time, min) BOND DILUTION POSITIVE CONTROL TISSUE CK 2AE3H1 (30)1:400Skin CK 3AE5E1 (10)1:5000Cornea CK 7RN7H1 (30)1:100Breast CK 10LHP1E1 (10)1:100Skin CK 12H-60E1 (10)1:50Cornea CK 19RCK108H2 (20)1:100Colon CK = Cytokeratin Heat induced epitope retrieval H1: solution containing citrate-based buffer and surfactant; H2: solution containing EDTA-based buffer and surfactant. Enzyme digestion E1: 7ml Bond enzyme diluent (Tris-buffered saline) &1 drop Bond enzyme concentrate (Proteolytic enzyme – Proteinase K); E2: 7ml Bond enzyme diluent & 2 drops Bond enzyme concentrate.

 Chronically inflamed conjunctiva lost the normal CK7, CK19 profile, and gained CK3, 10, 12  Corneal epithelium from chronic surface inflammation, lost CK12 and gained 19 expression  Some corneal epithelia also expressed CK10 RESULTS: Shift in cytokeratin profiles

Figure 1: Shift in cytokeratin profiles Normal epidermis staining positive for CK10 (A); normal conjunctiva staining positive for CK19 (B) and negative for CK3 (C) Normal cornea staining positive for CK 3 (D) and CK 12 (E). OCP conjunctiva with squamous metaplasia; lost the normal CK 7 and CK19 profile and gained CK3 and CK10 CK 3 +veCK 7 -veCK 19 -ve CK 3 -veCK 10 +veCK 19 +ve ABCDE OCP cornea specimen with keratinising squamous metaplasia; lost the CK 3 and CK12 and gained CK10 CK 10 +ve CK 12 -ve

 13 patients with ocular surface disease had clinically diagnosed LSCD  8 of 13 specimens (61.5%) coexpressed CK 12 (cornea-specific) and CK 19 (conjunctiva-specific)  Surgical interventions: 1.superficial keratectomy (n=1) 2.Penetrating/deep lamellar keratoplasty (n=7) 3.amniotic membrane graft (AMG)(n=3) 4.AMG + keratoplasty (n=2)  10 of 13 cases received systemic immunosuppression  Corneal epithelium recovered and remained stable in all eyes at mean follow up of 34 months ( months) RESULTS

PATIENT No PRIMARY PATHOLOGY MANAGEMENTHISTOLOGY (EPITHELIUM) ICC STAININGOUTCOME 1Aniridia keratopathy PK I: MMF, Pred Goblet cell +ve Atrophic CK 3 –ve CK 12 +ve (patchy) CK 19 +ve Epithelium stable at 20 months 2Rosacea keratitis DLK + AMG I: Pred Goblet cell –ve half atrophic, half hyperplastic Atrophic half: CK3-ve, 12 –ve, 19+ve Hyperplastic half: CK3+ve, 12–ve, 19+ve Epithelium stable at 52 months 3Oculo cicatricial pemphigoid Pseudopterygium excision, conjuncti- val autograft + AMG I: Pred Goblet cell –ve Irregular epithelium CK 3 –ve CK 12 -ve CK 19 +ve (patchy) Epithelium stable at 71 months 4Chemical injuryKeratectomy + AMG I: Pred Goblet cell –ve Squamous & cuboidal stratified CK 3 +ve (focal) CK 12 +ve CK 19 +ve (diffuse) Epithelium stable at 19 months 5Aniridia keratopathy Superficial keratectomy Goblet cell –ve Squamous & cuboidal CK 3 -ve CK 12 -ve CK 19 +ve Epithelium stable at 26 months 6KeratoglobusPK I: MMF, Pred Goblet cell +ve Oedematous CK 3 +ve (patchy) CK 12 +ve (superficial) CK 19 +ve (patchy) Epithelium stable at 20 months Correlation of clinical findings, immunopathological analysis (IHC), and surgical outcome PK = penetrating keratoplasty, DLK = deep lamellar keratoplasty, I = Immunosuppression, MMF = mycophenolate mofetil, Pred = prednisolone, CK = cytokeratin, +ve = positive, - ve = negative.

PATIENT No PRIMARY PATHOLOGY MANAGEMENTHISTOLOGY (EPITHELIUM) ICC STAININGOUTCOME 7Steven’s Johnson Syndrome DLK I: Pred Goblet cell –ve Atrophic CK 3 +ve (superficial) CK 12 -ve CK 19 +ve (diffuse) Epithelium stable at 31 months 8Pseudophakic bullous keratopathy PKPeripheral epithelium Goblet cell +ve CK 3+ve CK 12 –ve peripherallly, CK 12 +ve centrally CK 19 +ve (diffuse) Epithelium stable at 24 months, subsequent failure secondary to infection 9Congenital glaucoma PK I: Pred, T Goblet cell –ve Oedematous CK 3 +ve CK 12 +ve CK 19 +ve Epithelium stable at 28 months 10Aniridia keratopathy DLK + AMG I: Pred Goblet cell +ve Atrophic CK 3 +ve CK 12 -ve CK 19 +ve Epithelium stable at 53 months 11Rosacea keratitis Keratectomy + AMG I: MMF, Pred Goblet cell –ve Part cuboidal, part squamous CK 3 +ve CK 12 +ve (basal) CK 19 +ve (basal) Epithelium stable at 30 months 12Childhood injuryPKGoblet cell –ve Atrophic CK 3 +ve CK 12 +ve (patchy) CK 19 +ve (patchy) Epithelium stable at 59 months 13Atopic kerato- conjunctivitis Previous failed DLK PK I: MMF, Pred Goblet cell –ve Odematous CK 3 +ve CK 12 +ve (superficial) CK 19 +ve (superficial) Epithelium stable at 32 months subsequent failure secondary to infection Correlation of clinical findings, surgical outcome and immunopathological analysis

Figure 2: Histological assessement of patients with LSCD Patient 1: PAS stain showing goblet cells, IHC staining CK 3 –ve, CK 12 +ve, CK 19 +ve CK3 X 100PAS X 100CK12 X 100CK19 X 100 Patient 4: IHC staining: CK 3 +ve (focal), CK 12 +ve, CK 19 +ve (diffuse) CK3 X 100CK19 X 100 Patient 10: H&E staining showing atrophic epithelium IHC staining: CK 3 +ve, CK 12 -ve, CK 19 +ve H&E X 40CK3 X 40CK12 X 40CK19 X 40

Figure 3: Surgical outcomes Patient 3: LSCD secondary to OCP Preoperatively (A) and 48 months following conjunctival autograft and amniotic membrane graft (B). AB Patient 4: LSCD secondary to chemical injury (A) Preoperatively, (B) Stable epithelium 21 months post amniotic membrane graft (AMG) B A Patient 9: 17 months following penetrating keratoplasty Patient 10: 33 months following deep lamellar keratoplasty & AMG

 By acquiring CK10, conjunctival and corneal epithelial cytokeratin profiles in ocular surface disease shifted from normal to epidermal type, hence undergoing metaplasia  The co-expression of corneal and conjunctiva specific cytokeratins suggests metaplasia from corneal to conjunctival phenotype 3  We hypothesise that detection of CK19 in corneas with clinically diagnosed LSCD may signal metaplasia of corneal epithelial cells rather than conjunctival cell migration 4  The recovery and maintenance of the corneal epithelium in the absence of a limbal stem cell graft, suggests a limited role of limbal epithelial stem cells in physiological haemostasis of the corneal epithelium 5 CONCLUSION 3 Lugo M, Putong PB. Metaplasia. An overview. Arch Pathol Lab Med 1984;108(3): Elder MJ, Hiscott P, Dart JK. Intermediate filament expression by normal and diseased human corneal epithelium. Hum Pathol 1997;28: Miri A, Alomar T, Yeung AM, Said DG, Dua HS. The role of limbal stem cells in corneal epithelial maintenance: testing the Dogma.