Hilko Ardon

Adjuvant dendritic cell-based tumour vaccination for children with malignant brain tumours

A large experience with dendritic cell (DC)‐based vaccination for malignant brain tumours has been gained in adults. Here we focus on the results obtained in children with relapsed malignant brain tumours.

Dendritic Cell Therapy of High-Grade Gliomas

The prognosis of patients with malignant glioma is poor in spite of multimodal treatment approaches consisting of neurosurgery, radiochemotherapy and maintenance chemotherapy. Among innovative treatment strategies like targeted therapy, antiangiogenesis and gene therapy approaches, immunotherapy emerges as a meaningful and feasible treatment approach for inducing long‐term survival in at least a subpopulation of these patients. Setting up immunotherapy for an inherent immunosuppressive tumor located in an immune‐privileged environment requires integration of a lot of scientific input and knowledge of both tumor immunology and neuro‐oncology. The field of immunotherapy is moving into the direction of active specific immunotherapy using autologous dendritic cells (DCs) as vehicle for immunization. In the translational research program of the authors, the whole cascade from bench to bed to bench of active specific immunotherapy for malignant glioma is covered, including proof of principle experiments to demonstrate immunogenicity of patient‐derived mature DCs loaded with autologous tumor lysate, preclinical in vivo experiments in a murine orthotopic glioma model, early phase I/II clinical trials for relapsing patients, a phase II trial for patients with newly diagnosed glioblastoma (GBM) for whom immunotherapy is integrated in the current multimodal treatment, and laboratory analyses of patient samples. The strategies and results of this program are discussed in the light of the internationally available scientific literature in this fast‐moving field of basic science and translational clinical research.

Technical advancement in regulatory T cell isolation and characterization using CD127 expression in patients with malignant glioma treated with autologous dendritic cell vaccination.

We have successfully treated over two hundred high-grade glioma (HGG) patients with immunotherapy consisting of vaccination with autologous dendritic cells (DCs) loaded with autologous tumour lysate. It has been documented that regulatory T cells (Treg) can counteract anti-tumour immune responses. Therefore, monitoring of Treg in these patients is essential. Up till now, Treg have been characterized based on the expression of the transcription factor Foxp3. Here, we validated IL-7 receptor alpha subunit (CD127)dim expression as a marker for human Treg within HGG patients, as a less laborious assay for routine use in tumour vaccination trials. We noted a strong positive correlation between Foxp3 expression and CD127dim expression in CD4+CD25+ and CD4+ cells. The suppressive function of CD4+CD127dim cells was assessed in an allogeneic mixed lymphocyte reaction (MLR). We conclude that CD127 staining is a fast, well-suited and reproducible Treg monitoring tool in HGG patients treated with immunotherapy.

Aquaporin-4 in glioma and metastatic tissues harboring 5-aminolevulinic acid-induced porphyrin fluorescence

INTRODUCTION Aquaporin channels (AQPs) are a group of integral membrane proteins that regulate the transport of water through cell membranes. Previous studies have shown that up-regulation of AQP1 and AQP4, two of the predominant AQPs in the human brain, in high grade glial tumors contribute to cerebral edema. Others link AQPs to the regulation of human glioma cell migration and invasion. The aim of this study was to determine AQPs expression in tumor tissue harboring 5-aminolevulinic acid (ALA)-induced porphyrin fluorescence with flow cytometry and compare it to the expression in normal brain tissue. METHODS Tissue samples were obtained from fluorescing brain tumors of 26 patients treated with ALA prior to surgery (20 mg/kg b.w.). Expression levels of aquaporin channels were measured in primary tissue cultures using a FACS CANTO I flow cytometer. A control group consisted of four non-fluorescing tissue samples, the C6 and the U87 cell line. RESULTS Nineteen gliomas (14 high grade, 5 low grade) and 7 metastases were analyzed. On the 4th post-operative day, expression levels of AQP4 channels, but not of AQP1 channels, were significantly increased in samples from fluorescing tissue compared to non-fluorescing tissue. In addition we could see how ALA induces fluorescence in metastases. CONCLUSION Flow cytometry appears to be an auspicious method for the analysis of porphyrins and AQPs in primary brain cell tumor cultures. ALA fluorescing tissue showed higher AQP4 expression compared to normal brain tissue. The demonstrated expression in a context with ALA could open a targeted therapeutic spectrum, for example to selectively target AQP4.

Paraganglioma of the cauda equina region: A report of three cases.

Background: Cauda equina paragangliomas (CEP) are rare tumors. Low back pain and sciatica are the main presenting symptoms. Magnetic resonance imaging (MRI) is the study of choice and treatment consists of total excision when feasible. Definitive diagnosis can only be made after immunohistochemical investigation. CEP is classified as grade I WHO and after total removal the prognosis is excellent. Nonetheless, after subtotal removal, tumor recurrence can occur. Case Description: We present 3 cases of CEP, preoperatively diagnosed as an intradural mass on MRI and suspected to be ependymoma. All 3 patients presented with low back pain and variable sciatic pain. Total resection of the tumor was performed after which all patients fully recovered. There is no recurrence after 13, 11, and 5 years, respectively. Conclusion: CEP is a rare tumor. We diagnosed 3 paragangliomas out of a series of 105 intradural extramedullary tumors in adults (1994–2005). No recurrence was seen after total resection. In retrospect, both the intraoperative appearance and the MR image were not completely typical for schwannoma or ependymoma, but final diagnosis can only be made histologically.

Biochemical Differentiation of Gestational Compartments in the Midgestational Fetal Rabbit

Objectives: The fetal rabbit at midgestation is increasingly being used as a model in fetal diagnosis and therapy. In this study, we aimed to establish a reliable method for identification of the origin of sampled extra-embryonic fluids based on selected biochemical components. Methods: In 6 pregnant does at 22 days of gestation, 18 gestational sacs were sampled for amniotic, allantoic and exocoelomic fluid. These fluids, as well as matching maternal and fetal blood samples, were assayed for levels of sodium, potassium, chloride, bicarbonate, total protein, alkaline phosphatase, γ-glutamyl transferase and progesterone. Results: Levels of sodium and potassium were, respectively, lower and higher in the allantoic fluid when compared to other extra-embryonic spaces. Amniotic fluid had a significantly lower total protein content and higher level of alkaline phosphatase when compared to the exocoelomic fluid. Significant levels of progesterone could only be detected in maternal blood. Conclusions: In the midgestational rabbit, a combined assay of potassium, alkaline phosphatase and progesterone can determine the gestational cavity of origin of the sampled fluid. The obtained gradients for these markers suggest compartment-specific production and/or inter-cavity transfer mechanisms.

High-Grade Gliomas: Dendritic Cell Therapy

The relationship between the development of a tumor and the immune system is depicted by the fundamental model of “cancer immunoediting”: a tumor will only grow if the immune system is not capable of eliminating immune-resistant tumor cells (“tumor immune escape”). Both the adaptive and innate immunity play a key-role in this process. Multiple tumor-associated antigens have been described in gliomas and the expression of these antigens is very heterogeneous with important inter- and intra-individual differences. This is in contrast with other tumors, such as malignant melanoma, where tumor-associated antigens are almost universally expressed in all tumor cells. Several studies have shown that antigens can drain from the central nervous system to the cervical lymph nodes, where antigen-specific T cells can be activated by antigen-presenting cells. These activated T cells can subsequently migrate into the brain parenchyma to the sites of antigen challenge. This points to possible, naturally occurring immune responses eventually also against tumors, and indicates that the central nervous system is not an “immune-silent” environment. Therefore, it seems that the immune system can provide us with some interesting tools to treat gliomas. Several types of immunotherapy have been used and the most promising strategy for the induction of durable immune responses seems to be active, specific immunotherapy, or the so-called tumor vaccination. Further research has to be done, however, to establish the value of this form of immunotherapy in the treatment of gliomas.