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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.