A clinical-immunopathological-surgical A clinical-immunopathological-surgical

outcome correlation of cases with clinical outcome correlation of cases with clinical

Limbal Stem Cell Deficiency (LSCD)Limbal Stem Cell Deficiency (LSCD)

1 Tennent Insitute of Ophthalmology, Glasgow , UK2Pathology Department, Western Infirmary, Glasgow, UK

The authors have no financial interest in the subject matter of this poster

Maria Elena GregoryMaria Elena Gregory11, Elisabeth CA McDonald, Elisabeth CA McDonald11, ,

Yvonne ReiveYvonne Reive22, Fiona Roberts, Fiona Roberts22, Kanna Ramaesh, Kanna Ramaesh11

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;102:1476-85.

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.

METHODS:Panel of antibodies

CYTOKERATIN CLONE ANTIGEN RETRIEVAL METHOD (Incubation time, min)

BOND DILUTION

POSITIVE CONTROL TISSUE

CK 2 AE3 H1 (30) 1:400 Skin

CK 3 AE5 E1 (10) 1:5000 Cornea

CK 7 RN7 H1 (30) 1:100 Breast

CK 10 LHP1 E1 (10) 1:100 Skin

CK 12 H-60 E1 (10) 1:50 Cornea

CK 19 RCK108 H2 (20) 1:100 Colon

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 -ve CK 19 -ve

CK 3 -ve CK 10 +ve CK 19 +ve

A B C D E

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 (19 - 71 months)

RESULTS

PATIENT No

PRIMARYPATHOLOGY

MANAGEMENT HISTOLOGY(EPITHELIUM)

ICC STAINING OUTCOME

1 Aniridia keratopathy

PK I: MMF, Pred

Goblet cell +veAtrophic

CK 3 –veCK 12 +ve (patchy)CK 19 +ve

Epithelium stable at 20 months

2 Rosacea keratitis

DLK + AMG I: Pred

Goblet cell –vehalf atrophic, half hyperplastic

Atrophic half:CK3-ve, 12 –ve, 19+ve Hyperplastic half:CK3+ve, 12–ve, 19+ve

Epithelium stable at 52 months

3 Oculo cicatricial pemphigoid

Pseudopterygium excision, conjuncti-val autograft + AMG I: Pred

Goblet cell –ve Irregular epithelium

CK 3 –veCK 12 -ve CK 19 +ve (patchy)

Epithelium stable at 71 months

4 Chemical injury Keratectomy + AMGI: Pred

Goblet cell –ve Squamous & cuboidal stratified

CK 3 +ve (focal)CK 12 +ve CK 19 +ve (diffuse)

Epithelium stable at 19 months

5 Aniridia keratopathy

Superficial keratectomy

Goblet cell –veSquamous & cuboidal

CK 3 -veCK 12 -veCK 19 +ve

Epithelium stable at 26 months

6 Keratoglobus PK I: MMF, Pred

Goblet cell +veOedematous

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

PRIMARYPATHOLOGY

MANAGEMENT HISTOLOGY(EPITHELIUM)

ICC STAINING OUTCOME

7 Steven’s Johnson Syndrome

DLKI: Pred

Goblet cell –ve Atrophic

CK 3 +ve (superficial)CK 12 -ve CK 19 +ve (diffuse)

Epithelium stable at31 months

8 Pseudophakic bullous keratopathy

PK Peripheral epithelium Goblet cell +ve

CK 3+ve CK 12 –ve peripherallly, CK 12 +ve centrallyCK 19 +ve (diffuse)

Epithelium stable at 24 months, subsequent failure secondary to infection

9 Congenital glaucoma

PKI: Pred, T

Goblet cell –veOedematous

CK 3 +veCK 12 +veCK 19 +ve

Epithelium stable at 28 months

10 Aniridia keratopathy

DLK + AMGI: Pred

Goblet cell +veAtrophic

CK 3 +veCK 12 -veCK 19 +ve

Epithelium stable at 53 months

11 Rosacea keratitis

Keratectomy + AMGI: MMF, Pred

Goblet cell –vePart cuboidal, part squamous

CK 3 +veCK 12 +ve (basal)CK 19 +ve (basal)

Epithelium stable at 30 months

12 Childhood injury PK Goblet cell –veAtrophic

CK 3 +veCK 12 +ve (patchy)CK 19 +ve (patchy)

Epithelium stable at 59 months

13 Atopic kerato-conjunctivitisPrevious failed DLK

PK I: MMF, Pred

Goblet cell –veOdematous

CK 3 +veCK 12 +ve (superficial)CK 19 +ve (superficial)

Epithelium stable at 32 monthssubsequent 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 100 CK12 X 100 CK19 X 100

Patient 4: IHC staining: CK 3 +ve (focal), CK 12 +ve, CK 19 +ve (diffuse)

CK3 X 100 CK19 X 100

Patient 10: H&E staining showing atrophic epithelium IHC staining: CK 3 +ve, CK 12 -ve, CK 19 +ve

H&E X 40 CK3 X 40 CK12 X 40 CK19 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).

A B

Patient 4: LSCD secondary to chemical injury (A) Preoperatively, (B) Stable epithelium 21 months post amniotic membrane graft (AMG)

BAA

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 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

CONCLUSION

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.