Human leukocyte antigen distribution in German Caucasians with advanced Ewing's sarcoma.

BACKGROUND: Risk stratification criteria for patients with Ewing's sarcoma family of tumors (ESFT) are still limited. We hypothesized divergent human leukocyte antigen (HLA) patterns in ESFT patients and compared HLA-A, -B and -DR phenotype frequencies of patients with advanced ESFT with those of healthy controls. PATIENTS: HLA types of all German Caucasian patients with advanced ESFT and available HLA-A, -B and -DR data registered in the European Group for Blood and Marrow Transplantation, Paediatric Registry for Stem Cell Transplantation and the MetaEICESS data bases (study group, n=30) were retrospectively compared with HLA types of healthy German stem cell donors (control group, n=8 862 for single HLA frequencies and n=8 839 for allele combinations). Study group patients had been immuno-typed due to eligibility for allogeneic stem cell transplantation for high risk of treatment failure, and thus constituted a selected subgroup of ESFT patients. RESULTS: After Bonferroni correction for multiple testing (PC), phenotype frequencies of HLA-A24 remained significantly higher in the study group compared to controls (PC<0.05). Furthermore, several HLA combinations were significantly more frequent in the study group compared to controls (all PC<0.05). CONCLUSION: We report an increased incidence of circumscribed HLA patterns in German Caucasians with advanced ESFT. The possible clinical significance of this observation has to be re-assessed in prospective trials comprising larger ESFT patient numbers of all risk groups.

High STEAP1 expression is associated with improved outcome of Ewing's sarcoma patients.

BACKGROUND: Ewing's sarcoma (ES) is the second most common bone or soft-tissue sarcoma in childhood and adolescence and features a high propensity to metastasize. The six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is a membrane-bound mesenchymal stem cell marker highly expressed in ES. Here, we investigated the role of STEAP1 as an immunohistological marker for outcome prediction in patients with ES. PATIENTS AND METHODS: Membranous STEAP1 immunoreactivity was analyzed using immunohistochemistry in 114 primary pre-chemotherapy ES of patients diagnosed from 1983 to 2010 and compared with clinical parameters and patient outcome. Median follow-up was 3.85 years (range 0.43-17.51). RESULTS: A total of 62.3% of the ES samples displayed detectable STEAP1 expression with predominant localization of the protein at the plasma membrane. High membranous STEAP1 immunoreactivity was found in 53.5%, which correlated with better overall survival (P=0.021). Accordingly, no or low membranous STEAP1 expression was identified as an independent risk factor in multivariate analysis (hazard ratio 2.65, P=0.036). CONCLUSION: High membranous STEAP1 expression predicts improved outcome and may help to define a specific subgroup of ES patients, who might benefit from adapted therapy regimens.

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells.

BACKGROUND: The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient's immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8(+) T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis. METHODS: Following repetitive CHM1(319) (VIMPCSWWV) and EZH2(666) (YMCSFLFNL) peptide-driven stimulations with HLA-A 0201(+) dendritic cells (DC), allo-restricted HLA-A 0201(-) CD8(+) T cells were stained with HLA-A 0201/peptide multimers, sorted and expanded by limiting dilution. RESULTS: Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A 0201 and killed HLA-A 0201(+) ET lines expressing the antigen while HLA-A 0201(-) ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2(-/-)γ(C)(-/-) mice. Within this context, we identified the CHM1(319) peptide as a new candidate target antigen for ET immunotherapy. CONCLUSION: These results clearly identify the ET-derived antigens, EZH2(666) and CHM1(319), as suitable targets for protective allo-restricted human CD8(+) T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation.

Only therapeutic ICOS:ICOSL blockade alleviates acute graft versus host disease.

Inducible co-stimulator (ICOS) interaction with its ligand (ICOSL) is involved in several T cell effector functions. While blockade of ICOS:ICOSL interaction in chronic graft versus host disease (GVHD) seems beneficial, results for acute GVHD remain controversial. To further elucidate its role in acute GVHD, C57BL/6 mice were reconstituted with allogeneic spleen cells in the absence or presence of ICOSL-blocking mAb. Mice reconstituted with allogeneic spleen cells experienced severe GVHD and died untreated within 6-9 days after transplantation. Mice treated with an anti-ICOSL mAb starting from day 3 after transplantation gained weight again and survived for at least additional 12 days, although the treatment was already stopped at day 11 after transplantation. In contrast, the anti-ICOSL treatment starting from day 0 did not prevent GVHD. The difference between therapeutic (day 3) and prophylactic (day 0) anti-ICOSL treatment was independent of CD25+CD4+ regulatory T cells since their depletion did not abrogate the therapeutic effect of ICOSL blockade. Microarray analysis revealed IFN-gamma and chemokine up-regulation in spleen cells of prophylactically treated mice, emphasizing kinetic dependence of acute GVHD modulation via blockade of ICOS:ICOSL interaction.

CD25 blockade protects T cells from activation-induced cell death (AICD) via maintenance of TOSO expression.

CD25 monoclonal antibody binding to the alpha-chain of the Interleukin-2 (IL-2) receptor, blocks high-affinity IL-2 binding, thereby preventing complete T-cell activation and being of ample importance in transplantation medicine and potentially the treatment of autoimmune disease. However, CD25 antibodies do not only block T-cell activation but also prevent activation-induced cell death (AICD) attributing a dual function to IL-2. In this study, the modulation of the genomic expression profile of human peripheral blood mononuclear cells (PBMC) with therapeutic concentrations of humanized anti-CD25 mAb was investigated. PBMC were stimulated with CD3 antibody OKT-3 together with recombinant IL-2 in the absence or presence of anti-CD25 mAb. RNA was extracted and subjected to microarray analysis on U133A microarrays (Affymetrix). Anti-CD25 treatment inhibited several genes typically expressed during T-cell activation including granzyme B, signalling lymphocyte activation molecule, family member 1 (SLAMF1), CD40-Ligand (CD40-L), IL-9 and interferon (IFN)-gamma. Interestingly, anti-CD25 mAb also blocked the expression of several genes important for susceptibility to apoptosis, such as death receptor 6 (DR6) or reversed IL-2-mediated repression of anti-apoptotic genes, such as Fas apoptotic inhibitory molecule 3 (FAIM3)/TOSO. Functional significance of DR6 and TOSO expression in IL-2-dependent T-cell activation was subsequently evaluated by RNA interference in AICD: While siRNA specifically directed against DR6 did not modulate FAS-L-mediated apoptosis induction in primary T cells, down-regulation of TOSO significantly increased susceptibility to apoptosis, emphasizing an important role for TOSO in IL-2-mediated AICD.