Zoltán Hanzély

Molecular Profiling Identifies Prognostic Subgroups of Pediatric Glioblastoma and Shows Increased YB-1 Expression in Tumors

PURPOSE Pediatric glioblastoma (pGBM) is a rare, but devastating brain tumor. In contrast to GBM in adults (aGBM), little is known about the mechanisms underlying its development. Our aim is to gain insight into the molecular pathways of pGBM. MATERIALS AND METHODS Thirty-two pGBM and seven aGBM samples were investigated using biochemical and transcriptional profiling. Ras and Akt pathway activation was assessed through the phosphorylation of downstream effectors, and gene expression profiles were generated using the University Health Network Human 19K cDNA arrays. Results were validated using real-time polymerase chain reaction and immunohistochemistry and compared with existing data sets on aGBM. RESULTS There are at least two subsets of pGBM. One subset, associated with Ras and Akt pathway activation, has very poor prognosis and exhibits increased expression of genes related to proliferation and to a neural stem-cell phenotype, similar to findings in aggressive aGBM. This subset was still molecularly distinguishable from aGBM after unsupervised and supervised analysis of expression profiles. A second subset, with better prognosis, is not associated with activation of Akt and Ras pathways, may originate from astroglial progenitors, and does not express gene signatures and markers shown to be associated with long-term survival in aGBM. Both subsets of pGBM show overexpression of Y-box-protein-1 that may help drive oncogenesis in this tumor. CONCLUSION Our work, the first study of gene expression profiles in pGBM, provides valuable insight into active pathways and targets in a cancer with minimal survival, and suggests that these tumors cannot be understood exclusively through studies of aGBM.

Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours

Background:Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location.Methods and results:Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 – 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 – 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 – 62.5%) and is rare in hemispheric JPA (1 of 7 – 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation.Conclusions and interpretation:Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

Gene Expression Profiling from Formalin-Fixed Paraffin-Embedded Tumors of Pediatric Glioblastoma

Purpose: Gene expression profiling has proved crucial for understanding the biology of cancer. In rare diseases, including pediatric glioblastoma (pGBM), the lack of readily available fresh frozen (FF) material limits the feasibility of this analysis, as well as its validation, on independent data sets, a step needed to ensure relevance, mandating the use of alternate RNA sources. To overcome the limitation of material number and to validate results we obtained on FF pGBM, we did microarray analysis on RNA extracted from formalin-fixed, paraffin-embedded archival samples from pGBM and control brains, wherein we had no control on the fixation process. Experimental Design: RNA from 16 pGBM and 3 control brains was extracted and linearly amplified. Reverse transcription–PCR on housekeeping and formerly identified tumor-associated genes and microarray analysis were done on this RNA source. Results were validated by immunohistochemistry. Results: Despite extensive RNA degradation, microarray analysis was possible on 16 of 19 samples and reproduced the pattern of results obtained on FF pGBM. Gene lists and ontology subgrouping were highly concordant in both sample types. Similar to the findings on FF samples, we were able to identify two subsets of pGBM based on their association/lack of association with evidence consistent with an active Ras pathway. Conclusions: Archival formalin-fixed, paraffin-embedded tissues are an invaluable resource as they are the most widely available materials often accessible in conjunction with clinical and follow-up data. Gene expression profiling on this material is feasible and may represent a significant advance for understanding the biology of rare human diseases.

Histopathological findings in a surgically resected thalamic cavernous hemangioma 1 year after 40-Gy irradiation

✓ Stereotactic radiosurgery is a controversial treatment modality in the management of cerebral cavernous hemangiomas (CHs), and results vary from center to center. Even the interpretation of treatment failure is controversial. It is suggested that the systematic pathological investigation of irradiated specimens could help to resolve the controversy. A hemorrhagic lesion in the posterior part of the thalamus had been diagnosed as a tumor and was treated with 40-Gy fractionated radiotherapy. One year after this treatment the case was reconsidered based on new imaging evidence, and the lesion was removed by conventional craniotomy. Histopathological examination revealed a CH with postirradiation changes. Compared with nonirradiated control CH tissue samples, there was endothelial cell destruction and marked fibrosis with scar tissue formation in the stroma of the treated lesion. The histopathological findings in this specimen were similar to those described in arteriovenous malformations after gamma knife surgery. The results of light microscopic investigations suggest that the ionizing effect of radiation energy evokes vascular and connective tissue stroma changes in CHs as well.

Expression of invasion-related extracellular matrix molecules in human glioblastoma versus intracerebral lung adenocarcinoma metastasis.

Tumor cell invasion into the surrounding brain tissue is mainly responsible for the failure of radical surgical resection, with tumor recurrence in the form of microdisseminated disease. Extracellular matrix (ECM)-related molecules and their receptors predominantly participate in the invasion process, including cell adhesion to the surrounding microenvironment and cell migration. The extent of infiltration of the healthy brain by malignant tumors strongly depends on the tumor cell type. Malignant gliomas show much more intensive peritumoral invasion than do metastatic tumors. In this study, the mRNA expression of 30 invasion-related molecules (twenty-one ECM components, two related receptors, and seven ECM-related enzymes) was investigated by quantitative reverse transcriptase-polymerase chain reaction. Fresh frozen human tissue samples from glioblastoma (GBM), intracerebral lung adenocarcinoma metastasis, and normal brain were evaluated. Significant differences were established for 24 of the 30 molecules. To confirm our results at the protein level, immunohistochemical analysis of seven molecules was performed (agrin, neurocan, syndecan, versican, matrix metalloproteinase 2 [MMP-2], MMP-9, and hyaluronan). Determining the differences in the levels of invasion-related molecules for tumors of different origins can help to identify the exact molecular mechanisms that facilitate peritumoral infiltration by glioblastoma cells. These results should allow the selection of target molecules for potential chemotherapeutic agents directed against highly invasive malignant gliomas.

Expression and Prognostic Examination of Heat Shock Proteins (HSP 27, HSP 70, and HSP 90) in Medulloblastoma

Expression of heat shock proteins (HSPs) is of prognostic significance in several tumor types. HSP expression levels were determined in medulloblastomas and tested whether HSPs expression was associated with prognostic parameters. Expression of antiapoptotic HSP 27, HSP 70, and HSP 90 was investigated by immunohistochemistry, on paraffin-embedded sections from 65 patients. Expression of HSPs was validated on internal vascular controls and by Western blotting analysis. Sample evaluation was based on the estimated percentage of HSP positive tumor cells. For survival analysis Kaplan-Meier method, for statistical analysis χ2 test, univariate analysis, and log rank test were applied. Expression of HSPs varied in medulloblastomas. On the basis of the average expression rate of HSPs, at HSP 27 and HSP 90 with a 10% cut off, and at HSP 70 with a 70% cut off 2 groups were created. The amount of expression of any of the HSP types was not significantly associated with known prognostic factors (age of patient, extent of resection, presence of metastasis) and histologic subtype. After an average follow-up period of 4.30 years, no significant difference was observed in survival depending on the expression of HSP 27 or HSP 70 or HSP 90. The high expression of HSPs indicates that these proteins are potential therapeutic targets.

[Expression pattern of invasion-related molecules in brain tumors of different origin].

Tumor cell invasion into the surrounding brain tissue is mainly responsible for the failure of radical surgical resection and successful treatment, with tumor recurrence as microdisseminated disease. Epidermal growth factor receptors (EGFRs), integrins and their ligands in the extracellular matrix (ECM) predominantly participate in the invasion process, including the cell adhesion to the surrounding microenvironment and cell migration. The extent of infiltration of the surrounding brain tissue by malignant tumors strongly depends on the tumor cell type. Malignant gliomas show much more intensive peritumoral invasion than do metastatic tumors. In this study, the mRNA expression of 29 invasion-related molecules (18 cell membrane receptors or receptor subunits (EGFRs and integrins) and 11 ECM components: collagens, laminins and fibronectin) was investigated by quantitative reverse transcriptase-polymerase chain reaction. Fresh frozen human tissue samples from glioblastoma (GBM) and intracerebral bronchial adenocarcinoma metastases (five pieces from each) were evaluated. Significant differences were established in six of the 29 molecules (ErbB1, 2, 3, integrins alpha3, 7 and beta1). To confirm our results at the protein level, immunohistochemical analysis of nine molecules was performed. The staining intensity differed definitely in the case of ErbB1, 2 and integrins alpha3 and beta1. Determining the differences in invasion-related molecules in tumors of different origin can help identify the exact molecular mechanisms that facilitate peritumoral infiltration by glioblastoma cells. These results should allow the selection of target molecules for potential chemotherapeutic agents directed against highly invasive malignant gliomas.

Introduction: the contribution of pathology to radiosurgery.

The term radiosurgery signifies any kind of application of ionizing radiation energy, in experimental biology or clinical medicine, aiming at the precise and complete destruction of chosen target structures containing healthy and/or pathological cells, without significant concomitant or late radiation damage to adjacent tissues. The goal of this study is to explore the short- and long-term pathophysiological effects of high-dose focused irradiation on neural tissue and its pathologies with histological, electron-microscopical tissue culture and biological-biochemical methods. Radiosurgical pathology focuses its scope and microscope on tissue, cellular, genetic and molecular changes in the human organism and experimental animals, or in cell lines and other in vitro experiments, generated by the ionizing radiation delivered from radiosurgical devices.

Pathological Considerations to Irradiation of Cavernous Malformations

Stereotactic radiosurgery is a controversial treatment modality in the management of cerebral cavernous malformations (CVMs). Systematic pathological studies of irradiated specimens probably could help to resolve the controversy. Light microscopic investigation of a surgically resected thalamic CVM 1 year after 40-Gy irradiation revealed endothelial cell destruction in the cavernous channels, and marked fibrosis with scar tissue formation in the connective stroma of the lesion. These histopathological findings were similar to those described in arteriovenous malformations after Gamma Knife surgery, and suggest that the ionizing effect of radiation energy evokes vascular and connective tissue stroma changes in CVMs as well.

Effect of somatostatin analogue octreotide in medulloblastoma in xenograft and cell culture study.

Background: The effect and possible timing of nonradiolabeled somatostatin analogue octreotide are still not determined in the treatment of medulloblastoma, while the presence of somatostatin receptor type-2 (SSTR2) is proved in the majority of medulloblastoma by several authors. Procedures: Daoy, SSTR2A positive medulloblastoma cell culture was tested with octreotide in monotherapy and combined with cisplatin, etoposide, and vincristine. Daoy medulloblastoma mice xenograft was treated with octreotide alone. Results: In monolayer cell culture high-dose octreotide (44 μM) resulted in mitotic inhibition with parallel increment of apoptosis. Combination with cytostatic drugs did not result in additive or synergistic effect, but vincristine was partially antagonized. In medulloblastoma xenograft, octreotide monotherapy (100 μg/kg/day for 10 days) resulted in partial tumor growth inhibition. Conclusions: High concentration of nonradiolabeled octreotide may have role in the treatment of medulloblastoma by long-term administration. Concomitant administration of octreotide with widely used cytostatic drugs against medulloblastoma will not have beneficial impact.