Stefan Burdach

Myeloablative megatherapy with autologous stem-cell rescue versus oral maintenance chemotherapy as consolidation treatment in patients with high-risk neuroblastoma: a randomised controlled trial

BACKGROUNDnMyeloablative megatherapy is commonly used to improve the poor outlook of children with high-risk neuroblastoma, yet its role is poorly defined. We aimed to assess whether megatherapy with autologous stem-cell transplantation could increase event-free survival and overall survival compared with maintenance chemotherapy.nnnMETHODSn295 patients with high-risk neuroblastoma (ie, patients with stage 4 disease aged older than 1 year or those with MYCN-amplified tumours and stage 1, 2, 3, or 4S disease or stage 4 disease and <1 year old) were randomly assigned to myeloablative megatherapy (melphalan, etoposide, and carboplatin) with autologous stem-cell transplantation (n=149) or to oral maintenance chemotherapy with cyclophosphamide (n=146). The primary endpoint was event-free survival. Secondary endpoints were overall survival and the number of treatment-related deaths. Analyses were done by intent to treat, as treated, and treated as randomised.nnnFINDINGSnIntention-to-treat analysis showed that patients allocated megatherapy had increased 3-year event-free survival compared with those allocated maintenance therapy (47% [95% CI 38-55] vs 31% [95% CI 23-39]; hazard ratio 1.404 [95% CI 1.048-1.881], p=0.0221), but did not have significantly increased 3-year overall survival (62% [95% CI 54-70] vs 53% [95% CI 45-62]; 1.329 [0.958-1.843], p=0.0875). Improved 3-year event-free survival and 3-year overall survival were also recorded for patients given megatherapy in the as-treated group (n=212) and in the treated-as-randomised group (n=145). Two patients died from therapy-related complications during induction treatment. No patients given maintenance therapy died from acute treatment-related toxic effects. Five patients given megatherapy died from acute complications related to megatherapy.nnnINTERPRETATIONnMyeloablative chemotherapy with autologous stem-cell transplantation improves the outcome for children with high-risk neuroblastoma despite the raised risk of treatment-associated death.

Immunodominance of Lytic Cycle Antigens in Epstein-Barr Virus-Specific CD4+ T Cell Preparations for Therapy

Background Epstein-Barr virus (EBV) is associated with a number of human malignancies. EBV-positive post-transplant lymphoproliferative disease in solid organ and hematopoietic stem cell transplant recipients has been successfully treated by the adoptive transfer of polyclonal EBV-specific T cell lines containing CD4+ and CD8+ T cell components. Although patients receiving T cell preparations with a higher CD4+ T cell proportion show better clinical responses, the specificity of the infused CD4+ component has remained completely unknown. Methodology/Principal Findings We generated LCL-stimulated T cell lines from 21 donors according to clinical protocols, and analyzed the antigen specificity of the CD4+ component in EBV-specific T cell preparations using a genetically engineered EBV mutant that is unable to enter the lytic cycle, and recombinantly expressed and purified EBV proteins. Surprisingly, CD4+ T cell lines from acutely and persistently EBV-infected donors consistently responded against EBV lytic cycle antigens and autoantigens, but barely against latent cycle antigens of EBV hitherto considered principal immunotherapeutic targets. Lytic cycle antigens were predominantly derived from structural proteins of the virus presented on MHC II via receptor-mediated uptake of released viral particles, but also included abundant infected cell proteins whose presentation involved intercellular protein transfer. Importantly, presentation of virion antigens was severely impaired by acyclovir treatment of stimulator cells, as currently performed in most clinical protocols. Conclusions/Significance These results indicate that structural antigens of EBV are the immunodominant targets of CD4+ T cells in LCL-stimulated T cell preparations. These findings add to our understanding of the immune response against this human tumor-virus and have important implications for the improvement of immunotherapeutic strategies against EBV.

Granulocyte and granulocyte-macrophage colony-stimulating factors for treatment of neutropenia in glycogen storage disease type ib

Two children with glycogen storage disease type Ib associated with numerous recurrent bacterial infections as a result of neutropenia and neutrophil dysfunction were treated with recombinant human granulocyte colony-stimulating factor (G-CSF). One of the two patients was previously treated with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); therapy had to be discontinued because of severe local side effects. Both colony-stimulating factors at dosages of 3 and 8 micrograms/kg/per day, respectively, increased the average neutrophil counts from less than 300 cells/microliters to more than 1200 cells/microliters. Two subpopulations of neutrophils could be identified by their capacity to produce H2O2: one subpopulation generated H2O2 normally and a second was defective in H2O2 production. The doses of G-CSF effectively enhanced and maintained that subpopulation of neutrophils which produced normal amounts of H2O2. Moreover, these colony-stimulating factor-induced neutrophils demonstrated effective phagocytosis of zymosan particles and killing of staphylococci. Chemotaxis was decreased and could not be normalized by treatment with G-CSF. We conclude that maintenance treatment with G-CSF improved the quality of life in both patients: The number and severity of bacterial infections decreased markedly during treatment. Long-term treatment with G-CSF (12 and 10 months, respectively) was well tolerated, and no adverse clinical events were observed.

Efficacy of dendritic cell generation for clinical use: recovery and purity of monocytes and mature dendritic cells after immunomagnetic sorting or adherence selection of CD14+ starting populations.

Immunotherapy with monocyte-derived dendritic cells (Mo-DCs) is applied to an increasing number of patients requiring large-scale production of clinical-grade dendritic cells with standardized Mo-DC generation protocols. In many countries, e.g., in Germany, Mo-DCs are legally considered medicinal products, which must be produced under Good Manufacturing Practice (GMP) conditions by an institution holding an official production license. Plastic adherence, immunomagnetic selection of CD14(+) monocytes and depletion of CD2(+) and CD19(+) cells are used to enrich monocytes for Mo-DC culture. The latter two have received approval of the European Union (CE). However, enrichment by plastic adherence is well-established and commonly used for clinical and research Mo-DC applications. The various plastic materials, nevertheless, have not been officially approved for monocyte selection for clinical use. In the present study therefore, we compared three methods for enrichment of CD14(+) monocytes with regard to efficiency of enrichment, yield of monocyte-derived functional mature dendritic cells, cost effectiveness, and handling. We demonstrate that CD14 selection and CD2 and CD19 depletion yield similar results regarding purity of mature DEs MoDCs (97-99% vs. 64-97%) and their immunostimulatory capacity. However, cell preparations cultured after CD14 selection possessed 91% to 97% CD14(+) cells, whereas CD2-/and DC19-depleted preparations contained only 8% to 57% CD14(+) cells. Thus, positive selection requires smaller culture volumes to generate equal numbers of Mo-DCs. Both methods gave better results than plastic adherence. In conclusion, of the techniques examined, CD14 selection of monocytes gave the best results regarding reproducibility, yield, and purity of the resulting monocytes and mature Mo-DCs.

Glycogen storage disease type Ib: infectious complications and measures for prevention.

A patient with glycogen storage disease (GSD) type Ib, neutrophenia, chronic inflammatory bowel disease and recurrent abscesses was treated with recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF (and also granulocyte colony stimulating factor) therapy markedly increased the neutrophil counts and reduced the frequency of infections and inflammation. We conclude that myeloid growth factors are effective for the treatment and prevention of acute infections and chronic inflammatory complications in patients with GSD Ib.

IDENTIFICATION OF AN IMMUNOGENIC EWS-FLI1-DERIVED HLA-DR-RESTRICTED T HELPER CELL EPITOPE

Immunotherapy with tumor-specific peptide-loaded dendritic cells represents a promising therapeutic approach for patients with multifocal primary or early relapsed Ewing family tumors (EFT). The authors therefore screened a peptide library derived from the fusion region of the EFT-specific chimeric transcription factor EWS-FLI1 for immunogenic peptides. T-cell priming with 10 peptides was evaluated using IFNγ video-assisted automated enzyme-linked immunospot technique. The authors report the identification of the first EFT-specific immunogenic T-cell epitope so far. Its identification will lead to a better understanding of EFT immunology and may improve DC-based immunotherapy.

A complex pattern of chemokine receptor expression is seen in osteosarcoma

BackgroundOsteosarcoma is the most frequent bone tumor in childhood and adolescence. Patients with primary metastatic disease have a poor prognosis. It is therefore important to better characterize the biology of this tumor to define new prognostic markers or therapeutic targets for tailored therapy. Chemokines and their receptors have been shown to be involved in the development and progression of malignant tumors. They are thought to be active participants in the biology of osteosarcoma. The function of specific chemokines and their receptors is strongly associated with the biological context and microenvironment of their expression. In this report we characterized the expression of a series of chemokine receptors in the complex environment that defines osteosarcoma.MethodsThe overall level of chemokine receptor mRNA expression was determined using TaqMan RT-PCR of microdissected archival patient biopsy samples. Expression was then verified at the protein level by immunohistochemistry using a series of receptor specific antibody reagents to elucidate the cellular association of expression.ResultsExpression at the RNA level was found for most of the tested receptors. CCR1 expression was found on infiltrating mononuclear and polynuclear giant cells in the tumor. Cells associated with the lining of intratumoral vessels were shown to express CCR4. Infiltrating mononuclear cells and tumor cells both showed expression of the receptor CCR5, while CCR7 was predominantly expressed by the mononuclear infiltrate. CCR10 was only very rarely detected in few scattered infiltrating cells.ConclusionOur data elucidate for the first time the cellular context of chemokine receptor expression in osteosarcoma. This is an important issue for better understanding potential chemokine/chemokine receptor function in the complex biologic processes that underlie the development and progression of osteosarcoma. Our data support the suggested involvement of chemokines and their receptors in diverse aspects of the biology of osteosarcoma, but also contradict aspects of previous reports describing the expression of these receptors in this tumor.