Just a test

The goal of this initiative is to obtain sufficient genomic data and a reference data set from retrospective studies of a large number of stored human DC samples to understand the genetic basis of inter-donor variability in responses to DC maturation stimuli. ‘Illumina’ microarrays rather than the ‘Affymetrix’ platform will be used for the necessary transcriptional profiling and subsequent data analysis. Jointly, P2 and P6 identified a large number of stored samples of DC from melanoma patients (who had been treated in clinical trials of DC-based therapy ...

Currently there is no knowledge about the relevance of MDSC in ovarian cancer patients. However, it is known that both Tregs and suppressive macrophages are increased in this malignancy and have been suggested to correlate with worse prognosis. In order to obtain data, we are collecting blood and ascitic fluid of ovarian cancer patients to study the presence of immature myeloid populations as compared to healthy donor blood. We have identified two candidate populations that will be tested for the ability to suppress T cells. We also aim to characterize these cel...

Data has been obtained on the phenotype and function of myeloid derived suppressor cells in melanoma in patients. Accumulation of myeloid derived suppressor cells (MDSC) with the ability to suppress T cells has been observed in serveral types of cancer, including melanoma. Using blood from melanoma patients we are investigating phenotypic markers that can be used to further characterize MDSC, focusing on molecules that are related to their suppressive function. As it is believed that recruitment from the bone marrow by tumor derived factors leads to increased pre...

Data has been obtained on the BRAF/NRAS mutation status and expression of COX-2 and iNOS comparing their prognostic value for overall survival in stage III malignant melanoma. New prognostic markers for malignant melanoma are urgently needed. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. We have examined the BRAF/NRAS mutation status and expression of COX-2 and iNOS to compare their progno...

Based on the data we recently reported concerning the role of GM-CSF in regulating human DC differentiation (Conti et al., EJI 2008), we will perform a detailed analysis of mRNA and microRNA expression in immature and TLR ligand-stimulated GM-DCs in order to identify specific mechanisms that control the generation/activation of these cells. Data obtained in this study will become available. Moreover, the role of microRNAs in controlling the switch of GM-CSF-exposed monocyte precursors toward DCs or macrophages will also be investigated.

Data on the role of STAT3 signalling in directing DC-mediated responses toward immunogenicity or tolerance will be generated. To this purpose, we will identify (i) genes and (ii) micro-RNA selectively controlled by STAT3 in human MDDCs treated with proinflammatory or tolerogenic stimuli (including TLR ligands, cytokines and vitamin D3). This will shed light on the role of STAT3 signalling.

BruCells is collaborating with Professor De Witte from ULB for the preclinical study in which circulating tumour-specific CD8+ T-cells will be detected in the blood of newly diagnosed glioblastoma patients and from which data will be obtained. In order to obtain the blood samples and tumour biopsies from the glioblastoma patients, a file has been submitted to the Ethics Committee of the Erasme Hospital and has been recently approved.

This test, described for glioblastoma, performed with PBMC from newly diagnosed glioblastoma patients will verify the ability of tumour-specific T-cells to recognize the fusion vaccine. Data will be made available. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

This test will verify whether the fusion vaccine possesses the properties of both parental cell types for inducing primary CD8+ T-cell responses (data will become available). Detection of circulating tumour-specific CD8+ T-cells. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

Data will be available from in-vitro pre-clinical tests on the pharmacodynamic activity of the fusion vaccines. This test will verify the capacity of the fusion vaccine to present certain tumour-antigen derived peptides corresponding to antigens present in the fused cancer cells. BruCells has met with the Dutch MEB for a Scientific Advice Meeting on January 10, 2008 in The Hague. The Dutch authorities consider that the proposed in-vitro pre-clinical tests are adequate to check the pharmacodynamic activity of the fusion vaccines and that no animal model tests would be necessary.

We found that Indium111 labelled DC should not be loaded with tumor antigens if applied in patients, which may result in loss of specific immune responses and are testing this in-vivo. Analysis of DC migration in vivo in a first patient by i.v. application of indium labelled E/L-S DC (without antigen loading) is scheduled for March 2009. Data will become available.

We explored for the first time transfection of CD4(+) and CD8(+) T cells with mRNA encoding recombinant immunoreceptors for use in the adoptive immunotherapy of cancer and obtained data from this study. CD4(+) and CD8(+) T cells were efficiently transfected with immunoreceptors specific for ErbB2 and CEA. The immunoreceptor expression was transient with half-maximal expression at day 2 and no detectable immunoreceptor expression at day 9 after electroporation. Immunoreceptor-transfected T cells were specifically activated upon coincubation with ErbB2(+) and CEA(+...

Pathways-based analysis will be done in collaboration with P15 (Cavalieri) to identify the intracellular target of Japanin and data will become available from this study.

Collaborative work with P34 (Sica) has been started to study the effects of Japanin on DC exposed to hypoxia (below) and to perform transcriptional profiling of the treated cells. This work, which could not have been undertaken at this stage without support from DC-THERA, has now led to a joint publication in 2008 (Mancino et al.). Transcriptional data are available.

A worldwide first randomized adjuvant trial in a subgroup of high risk melanoma patients (monosomy 3) using autologous DC transfected with amplified total tumor RNA derived from patients' uvea melanomas was to be started in 2009 and data will become available.

We can provide Illumina microarray data for four experiments composed of 4 microarrays each on DCs stimulated with pathogenic and non pathogenic fungi. RNA preparation, labeling, hybridization on a HT12 array (Illumina), and scanning were performed according to Illumina instructions by Genomics Lab, Wellcome Trust Centre for Human Genetics (Univeristy of Oxford).

Affymetrix and Illumina microarray data is available on 3X106 DCs that were either not stimulated or stimulated with different stimuli. Cells were harvested after 4 hours of stimulation. RNA extraction was done with TRIzol reagent (Invitrogen). microarray hybridization were performed on the basis of technology used. Affymetrix array data files (CEL files) were pre-processed and normalized using the Robust Multichip Average procedure (RMA). Annotations were updated following a procedure devised by Dai and co-workers (Dai et al., 2005). Computation was performed wit...

High-throughput data is available on tumour-associated antigen (TAA) specific T-cells in breast cancer patients using the Becton Dickinson Lyoplate™ technology. Therefore we have established multiparameter assessments of antigen-specific CD4+ and CD8+ T-cells enabling a simultaneous quantitative and qualitative analysis.

We have data available from a comparison of the use of fresh peripheral blood mononuclear cells (PBMC) versus fresh heparinised whole blood. Only when peptide pools as an antigen source are used PBMC exhibited a slightly higher stimulation capacity compared to whole blood. However, slightly higher background was observed when using PBMC to stimulate with antigen. Lowest background was observed when either peptides or protein were used for stimulation enabling assessment also of rare antigen-specific Th-cells below frequencies of 0.05% of peripheral CD4+ Th-cell...