Molecular pathology of gliomas:
Identification of novel glioma-associated genetic alterations and clinical relevance.

G. Reifenberg

The classification of gliomas is based on the histological assessment of the tumor tissue according to the WHO classification system. However, the clinical course may be quite variable from patient to patient, even among patients with histologically identical gliomas. Therefore, it is of great importance to identify novel biological markers that may supplement and refine the histological brain tumor classification.
The research of our group is primarily focussed on the idenfication of novel glioma-associated tumor suppressor genes and proto-oncogenes. In addition, we are working on the translation of genetic and epigenetic findings into the molecular diagnostics of gliomas and other brain tumors.
To achieve these goals, we are systematically investigating different types of human gliomas for aberrations at the chromosomal, genetic, epigenetic and mRNA levels using focussed analyses of candidate genes and chromosomes, as well as more comprehensive methods such as microarrays for genomic and epigenomic profiling as well as for mRNA expression analysis.
Molecular results obtained by these different approaches are being correlated to histology and clinical outcome on well-documented series of glioma patients.
For the molecular classification of gliomas based on genomic profiles, we have devised a brain tumor-tailored genomic microarray for array-based comparative genomic hybridization analysis. Application of this microarray for the genomic profiling of different types of gliomas revealed that molecular classification of these tumors based on their genomic profiles was largely congruent with histological classification.
Correlation of molecular genetic data with patient survival confirmed significant correlations of certain genetic alterations with overall survival of glioma patients, including combined losses of 1p and 19q in oligodendroglial tumors as well as other changes.
Interestingly 1p/19q losses in oligodendroglial tumors were closely associated with hypermethylation of the MGMT gene and reduced MGMT expression. This finding might in part explain the higher chemosensitivity of 1p/19q deleted oligodendroglial tumors. However, MGMT hypermethylation was also detected in approximately half of the glioblastomas, in which we could confirm the correlation of MGMT hypermethylation with a better response to chemotherapy and longer time to progression. Taken together, our findings clearly indicate that molecular markers provide clinically useful information that will improve the diagnostic assessment of gliomas.

G. Reifenberger
Department of Neuropathology,
Heinrich-Heine-University,
Düsseldorf, Germany

 

Cancer stem-like cells in gliomas

R. Bjerkving

A highly infiltrative cancer stem cell phenotype was established by xenotransplantation of human brain tumors in immuno-deficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis.
The malignant cells expressed neural stem cell markers and showed a migratory behavior similar to normal human neural stem cells. The cells showed self-renewal capacity and gave rise to tumors/ in vivo/. Serial animal passages, gradually transformed the stem cell tumors into an angiogenesis-dependent phenotype.
This process was characterized by a reduction in stem cells markers. Pro-invasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem cell tumors. In contrast, pro-invasive genes were down-regulated in the angiogenesis-dependent tumors, derived from the stem cell tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the cancer stem cells and the cells derived from them respectively, points at two completely independent mechanisms that drive tumor progression. The presented work underlines the need for developing new therapies that specifically target the cancer stem cell pools in tumors.

R. Bjerkvig
NorLux Neuro-Oncology, Department of Biomedicine, University of Bergen,
Bergen, Norway, and CRP-Santé, Luxembourg

Dendritic cells pulsed with glioma extracts induce immunity against syngeneic
intracranial gliomas and increase survival
of tumor-bearing mice.

G. Finocchiaro

In recent years the use of dendritic cells (DC), the most powerful antigen presenting cells, has been proposed for the creation of vaccines against gliomas. This approach has been demonstrated to be safe and non toxic in phase I or I-II trials. As a first step in the preparation of appropriate conditions for a clinical evaluation in Italy we have performed pre-clinical experiments on immune-competent mice injected intra-cerebrally (ic) with syngeneic GL261GBM cells and treated subcutaneously (sc) with DC loaded with a GL261 homogenate.
Murine DC were prepared from the bone marrow (BM) of C57Bl6/J.EGFP transgenic mice (H-2b).
Experiments were based on vaccination with DC pulsed with GL261 tumor extracts. In the first experiment, 56 female C57Bl6 mice (5 weeks) were injected with GL261 on day 0 and treated with three subcutaneous injections on days 0, 7 and 14. To evaluate the effects of tumor-extract-pulsed DC vaccination on the survival of mice with intracranial GL261 cells, 40 mice were injected sc with: PBS (group 1; n=10); Tumor Extracts (group 2; n=10); unpulsed DCs (group 3; n=10); DCs pulsed with tumor extract (group 4; n=10).
Survival rates of vaccinated mice was significantly higher than that of mice treated with PBS (p<0.0005), tumor extract (p=0.001), unpulsed-DC (p=0.05).
Mice injected with DCs pulsed with tumor extract (n=8) or with tumor extracts only (n=8) were also investigated by MRI, JAM assay and histological analysis. The JAM assay was performed by examining the ability of splenocytes (including CD8+ T cells) to lyse autologous tumor cells in vitro.
In mice vaccinated with pulsed DC, MRI showed no evidence of tumor, while robust cytotoxicity was demonstred with JAM assay, and a significant presence of CD3+/CD8+ cell infiltrates into the tumor was evaluated histologically in the same mice.
grouping the second experiment, 20 mice were injected with GL261 GBM cells on day 0 and treated on day 7, 14 and 21 by injection of: PBS (group 1; n=4); Tumor Extracts (group 2; n=8); DCs pulsed with tumor extract (group3; n=8). Survival rates of vaccinated mice was higher than that of mice treated with PBS (p<0.05), or tumor extract alone (p=0.2)
These results support the development of DC-based clinical trials for patients with glioblastomas that do not respond to standard therapies.

S. Pellegatta, D. Corno, L. Poliani, M. Grisoli,
M. Cusimano, F. Baggi, M. G. Bruzzone, G. Finocchiaro
National Neurologic Institute
“Carlo Besta”, Milan, Italy