A clinical- immunopathological -surgical outcome correlation of cases with clinical Limbal Stem Cell Deficiency (LSCD)

1. A clinical-immunopathological-surgical outcome correlation of cases with clinical Limbal Stem Cell Deficiency (LSCD) 1TennentInsitute of Ophthalmology, Glasgow , UK 2Pathology Department, Western Infirmary, Glasgow, UK The authors have no financial interest in the subject matter of this poster Maria Elena Gregory1, Elisabeth CA McDonald1, Yvonne Reive2, Fiona Roberts2, Kanna Ramaesh1

2. INTRODUCTION • 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: • Characterize the shift in cytokeratin profiles of conjunctival and corneal epithelial cells exposed to chronic inflammation. • Correlate the clinical appearance of patients with LSCD, their corneal epithelial cytokeratin profile and surgical outcomes. 1 Puangsricharern V, Tseng SC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 1995;102:1476-85.

3. 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):241-62.

4. METHODS: Panel of antibodies 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.

5. RESULTS: Shift in cytokeratin profiles • 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

6. Figure 1: Shift in cytokeratin profiles A B C D E 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 7 -ve CK 19 -ve CK 3 +ve CK 10 +ve OCP cornea specimen with keratinising squamous metaplasia; lost the CK 3 and CK12 and gained CK10 CK 3 -ve CK 12 -ve CK 10 +ve CK 19 +ve

7. RESULTS • 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: • superficial keratectomy (n=1) • Penetrating/deep lamellar keratoplasty (n=7) • amniotic membrane graft (AMG) (n=3) • 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 (19 - 71 months)

8. 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.

9. Correlation of clinical findings, surgical outcome and immunopathological analysis

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

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

12. CONCLUSION • 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 phenotype3 • We hypothesise that detection of CK19 in corneas with clinically diagnosed LSCD may signal metaplasia of corneal epithelial cells rather than conjunctival cell migration4 • 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 epithelium5 3 Lugo M, Putong PB. Metaplasia. An overview. Arch Pathol Lab Med 1984;108(3):185-9. 4 Elder MJ, Hiscott P, Dart JK. Intermediate filament expression by normal and diseased human corneal epithelium. Hum Pathol 1997;28:1348-54. 5 Miri A, Alomar T, Yeung AM, Said DG, Dua HS. The role of limbal stem cells in corneal epithelial maintenance: testing the Dogma.