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In an experimental model we demonstrated that endothelial cells of all organs are targets of the alloimmune reaction. Here, in 68 digestive biopsies, we found endothelial lesions by immunohistochemistry and ultrastructure in patients with severe acute graft-versus-host disease (GVHD). In contrast, no such endothelial cell alterations were found either in patients without GVHD or in nongrafted controls. In the biopsies with severe GVHD lesions, ultrastructure showed rupture of the capillary basal membrane and extravased red blood cells. These pericapillary hemorrhages were highly correlated with GVHD severity. In a separate cohort of 39 patients who underwent an allogeneic transplantation after a nonmyeloablative conditioning, 8 patients had intestinal biopsies. Three of these latter patients had both severe pathologic lesions of GVHD and similar endothelial lesions, thus, strengthening the concept that endothelial lesions are linked to GVHD severity and not to the intensity of the conditioning regimen. (Blood. 2004;103:4681-4684)

Digestive tract damage during graft-versus-host reaction (GVHR) causes high morbidity and mortality. Diagnosis is often late because biopsies are performed when clinical signs are severe and pathologic markers of early inflammatory lesions are lacking. Eosinophils are inflammatory cells, cytotoxic in vitro to digestive epithelium; they are found in biopsy specimens taken during acute flare-ups of inflammatory bowel disease. We performed systematic duodenal biopsies immediately after digestive symptoms occurred and found a digestive GVHR incidence of 73.1% (n = 93), higher than that found when digestive biopsies were performed immediately after severe clinical signs. Eosinophils were only present when there were histologic signs of GVHR; eosinophil presence correlated with GVHR severity. Electron microscopy with immunogold staining showed pathologic signs of in situ eosinophil activation, such as cytoplasmic granule alterations, and eosinophil peroxidase release in all patients. Interleukin-5 presence in activated eosinophils suggests eosinophil recruitment in digestive GVHR is an autocrine mechanism. Eosinophil density also correlated with GVHR severity, whether in acute or chronic clinical phases. Tissue eosinophils could thus be a marker of acute inflammatory flare-ups in GVHR. Systematic duodenal biopsy performed at the onset of digestive symptoms should allow early GVHR detection, and pathologic signs of GVHR, together with eosinophil density, might help modulate immunosuppressive therapy.

Donor origin of epithelial intestinal cells has been studied in animals and humans after transplantation and has been used as evidence of hematopoietic stem cell (HSC) plasticity. However, in the human gastrointestinal tract, no study used X- or Y-chromosome detection by fluorescence in situ hybridization (FISH) coupled with immunologic stainings to characterize cell types on the same tissue section. Here, we combined these techniques on the same section of duodenal epithelium in 6 patients with acute graft-versus-host disease. Donor-derived lymphoid cells were detected in the epithelium and the lamina propria, as expected. However, using our stringent criteria, no donor-derived cells could be proven to be epithelial.

The gastrointestinal (GI) tract is a major target in graft-versus-host disease (GvHD). In rodents both tumor necrosis factor (TNF) and Fas-dependent apoptosis have been shown to play a major role in GvHD lesions, but data in humans on TNF and Fas in situ expression are scarce. More recently, the role of non-T cells as GvHD effectors has also been suggested in experimental models. Here we report a detailed quantitative pathologic analysis in 95 patients who underwent gastroduodenal biopsy. This analysis included characterization and quantification of the cellular infiltrate, TNF, TNF receptors, and Fas in situ expression analyses and quantification of apoptotic cell numbers. TNF was expressed in all biopsies and it was highly specific for acute GvHD. In multivariate analysis, including pathologic factors only, increased early transplantrelated mortality (TRM) was associated with the presence of more than 20 neutrophils per field. Factors affecting early and late TRM were then assessed by multivariate analyses including both pathologic and clinical factors. Increased day-90 TRM was associated with the presence of more than 5 apoptotic bodies per field within the cellular infiltrate, and with stage II or higher acute liver GvHD. One-year TRM associated with the same 2 factors and with chronic GvHD.

Abstract 2339 Introduction: Graft versus host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Gastrointestinal (GI) GVHD occurs in 10 to 40% of patients. Its diagnosis is based upon histological findings of per-endoscopic mucosal biopsies. The aim of our study was to compare endoscopic appearance of the mucosa and histological results and assess the outcomes of transplanted patients who underwent duodenal biopsies according histological and endoscopic findings. Patients and methods: We performed a retrospective analysis including allografted patients who underwent duodenal biopsies because of digestive GVHD suspicion. For each patient we recorded clinical data, endoscopic signs, and then we reviewed histological findings. Diagnosis of GVHD was histologically defined as the presence of single-cell necrosis. The severity of disease was graded on a four-point scale from 0 (absent) to 4 (very severe), according the number of single-cell necrosis, the degree of mucosal infiltration, the presence of neutrophils and eosinophils, epithelial rupture, and vascular involvement. Endoscopic findings were described and graded as normal mucosa (0), presence of erythema or oedema with congestive mucosa (1), ulceration (2), and extensive mucosal involvement (3). Agreement between endoscopy and histology was measured by to kappa statistics. Outcomes were assessed by Kaplan-Meyer survival curves. Results: Between 1995 and 2008, 270 duodenal biopsies in 228 patients have been performed in patients. We focused only on the first one. Indication for HSCT was acute leukaemia or myelodysplastic syndrome in 133 patients (58.3%), NHL/HD in 35 (15.4%), CML or myeloproliferative disease in 35 (15.4%), other diagnosis in 25 (10.9%). Donor was identical sibling in 48% of cases, unrelated donor in 52% (HLA matched 21% and mismatched 31%). Stem cell source was bone marrow in 46%, peripheral blood in 42% and cord blood in 12%. Finally most of patients (72%) received a myeloablative conditioning whereas the others had reduced intensity conditioning. The median time from transplantation to biopsy was 28 days. More than two thirds of patients (77%) presented an acute GVHD occurring before day 100. Upper digestive endoscopy was performed at the onset of digestive symptoms; nausea or vomiting in 25% of cases, diarrhea in 75%. Pathological GVHD was diagnosed in 77% of patients, graded minimal (1) in 29%, mild (2) in 25%, severe (3) in 10% and very severe (4) in 12%. Concerning endoscopic findings, normal macroscopic aspect was noted in 80% of cases (n=176), erythema or oedema was reported for 12% of patients, mucosal ulceration in 6%, and extensive mucosal involvement in 2%. There was no agreement between histological diagnosis and endoscopic findings with a kappa coefficient of -0.08, [95%CI: -0.17;-0.01], even for severe histological stages. Then 80% of patients with normal endoscopy had a diagnosis of GVHD, which was graded as histologically severe or very severe in 39%. The median follow up was 47 months (31-68), the 4 years overall survival was 47% [IC95% 41%; 54%]. Severe (n=24) and very severe (n=27) histological GVHD was significantly associated with mortality, with respectively HR 3.23 [95%CI: 1.82; 5.72] p Conclusion: Since digestive GVHD is a pan-intestinal process, systematic duodenal biopsies in allografted patients suffering from high or low digestive symptoms permit a diagnosis of GI GVHD in more than three quarters of cases. The early realization of endoscopic examination leads most often to a normal macroscopic aspect of small bowel mucosa and there is no agreement between histological and endoscopic findings. Mucosal injury could be more likely linked to a viral involvement. Nevertheless if endoscopic findings do not lead to GVHD diagnosis, presence of ulceration and extensive involvement is predictive of mortality, as well as histological severity of GVHD. Disclosures: No relevant conflicts of interest to declare.