Histological and immunohistochemical characterisation of conjunctival graft vs host disease following haematopoietic stem cell transplantation

University of Freiburg, Freiburg, Baden-Württemberg, Germany
Albrecht von Graæes Archiv für Ophthalmologie (Impact Factor: 1.91). 07/2007; 245(7):1001-7. DOI: 10.1007/s00417-006-0448-6
Source: PubMed


Conjunctival graft vs host disease (cnGvHD) is a complication of haematopoietic stem cell transplantation, in most cases as part of systemic GvHD. Diagnostic biopsies are commonly collected from bulbar conjunctiva only. The aims of our study were to evaluate whether additional biopsies from the tarsal conjunctiva increase sensitivity upon histopathologic evaluation and to investigate the staining profile for common immunohistochemical markers in cnGvHD. We additionally propose an adaptive histological classification for cnGvHD analogous to Lerner's GvHD skin classification for predicting patient survival.
Formalin-fixed and paraffin-embedded conjunctival specimens from 23 post-mortem control eyes and 42 patients after haematopoietic stem cell transplantation (HSCT) were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) stain and with antibodies against CD1a, CD4, CD8, CD25, CD45RO, CD68, Fas ligand, TIA-1, HLA-DRalpha by means of immunohistochemistry. Cell counting took place in ten representative fields at 64.4 microm (length) x 21.2 microm (width). Multifactorial analysis of variance was performed to assess any influence of cnGvHD on the staining pattern for the immunohistochemical markers. Survival times were estimated by the Kaplan-Meier method.
All 42 specimens and none of the controls were diagnosed as cnGvHD. The bulbar specimens were staged according to the modified Lerner classification: grade (G) I: 0; G II: 17 (tarsal with G<or=II, 2; G>II, 8); G III: 12 (tarsal with G<or=III: 2; G>III: 1); G IV: 12 (tarsal with G<or=IV: 6); G V: 1. The number of pairs with either the tarsal or bulbar counterpart being more severely affected was almost equal (10 vs 9). A tendency towards shorter survival in advanced bulbar cnGvHD was demonstrated (G III-V vs G I-II, p =0.06). Staining for the immunohistochemical markers in cnGvHD differed significantly from that in controls (p<0.01). Proposed markers for cnGvHD (e = epithelium, s = stroma; mean cell counts +/- SD; cnGvHD vs controls) are: CD8 s (15.7 +/- 18.4 vs 6 +/- 5.6), CD25 s (2.6 +/- 2.8 vs 0.7 +/- 1.6), CD68 s (8 +/- 9 vs 3.9 +/- 3.5) at the bulbar site and CD1a e (1.2 +/- 1.6 vs 0.3 +/- 0.6) and TIA-1 e (2.2 +/- 2.2 vs 1.1 +/- 1.3) at the tarsal site.
Additional tarsal biopsy does not seem to add relevant diagnostic sensitivity for cnGvHD when the modified Lerner classification is applied. The modified Lerner classification of the bulbar cnGvHD seems to be of prognostic value.

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    • "Four patients with CD8-positive cells were classified as having ocular cGvHD on the basis of their clinical presentation. Although we might have missed CD8-positive cells due to their occurrence in deeper conjunctival layers [6,33], the detection of these lymphocytes by impression cytology may strengthen a clinical suspicion of ocular cGvHD. In addition, a comparison of patients with and without CD8-positive cells showed that CD8-positive cells could be an indicator of more severe dry eye. "
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    ABSTRACT: To assess the expression of human leucocyte antigen (HLA)-DR in epithelial cells and cluster of differentiation (CD8)-positive lymphocytes as possible markers of chronic ocular graft versus host disease (cGvHD) after hematological stem cell transplantation (HSCT). Twenty-seven consecutive patients with dry-eye symptoms following HSCT (24 [89%] with peripheral blood stem cell transplantation and 3 [11%] with bone marrow transplants; 17 [63%] familiar allogenic grafts) and 19 age-matched controls were included. Conjunctival impression cytology specimens were stained for HLA-DR, cytokeratin 19, and CD8. Oxford grading scale, blinking frequency, Schirmer test, tear film break-up time (TBUT), and Ocular Surface Disease Index (OSDI) were also recorded. Wilcoxon nonparametric testing was used to compare controls and HSCT recipients and to assess HSCT recipient subgroups with and without clinical cGVHD. Eighteen patients showed clinical signs of ocular cGVHD. TBUT and Schirmer test scores were significantly lower in patients, while Oxford grades and OSDI were significantly higher than in controls. Epithelial HLA-DR expression was generally higher in HSCT recipients than in controls, but it did not correlate with ocular cGVHD status. CD8-positive lymphocytes were identified in five patients with ocular cGvHD and one control. A strong HLA-DR expression as detected by impression cytology appears to indicate a general HSCT response and fails to predict ocular cGVHD. However, the detection of CD8-positive lymphocytes using impression cytology was frequently associated with ocular cGvHD. Our data warrant further evaluation of CD8 expression in impression cytology, along with comparison to conjunctival biopsies and brush cytology, as impression cytology may offer a less invasive strategy for assessing cGVHD status.
    Molecular vision 07/2013; 19:1492-501. · 1.99 Impact Factor
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    Bone Marrow Transplantation 07/2008; 42(4):289-91. DOI:10.1038/bmt.2008.161 · 3.57 Impact Factor
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    ABSTRACT: In chronic GVHD after BMT, the conjunctiva represents a target organ. GVHD can lead to severe inflammation and dry-eye syndrome (sicca syndrome). The molecular mechanisms are largely unknown. We examined the expression of chemokines in the conjunctiva in cases of chronic GVHD. In this study, we included 10 patients with chronic GVHD and 10 healthy controls. Clinical data were collected and tear film analysis and conjunctival cytology were carried out. Conjunctival biopsies were taken from all participants. Gene expression profiles of chemokines and their corresponding receptors were evaluated by means of quantitative real-time PCR. Chemokine protein expression was analysed by immunohistochemical analyses. Expressions of the Th1-associated chemokines, chemokine (C-X-C motif) ligand (CXCL) 9 (Mig), CXCL10 (IP-10), and their receptor chemokine (C-X-C motif) receptor 3 (CXCR3) were significantly increased in GVHD patients. Immunohistochemical analysis confirmed marked expression of the inflammatory CXCR3 ligands. A total of six patients had a moderate or severe sicca syndrome. Impression cytology revealed a mild keratinisation, moderate keratinisation or severe squamous metaplasia in three patients, respectively. Chronic GVHD of the conjunctiva is characterised by the expression of Th1-associated chemokines. Taken together, our results confirm that the conjunctiva is a target organ in this T cell-mediated process and add to molecular understanding of conjunctival GVHD.
    Bone marrow transplantation 02/2010; 45(8):1340-6. DOI:10.1038/bmt.2009.346 · 3.57 Impact Factor
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