Fibroblast Activation Protein Expressing Mesenchymal Cells Promote Glioblastoma Angiogenesis.

Fibroblast activation protein (FAP) is a membrane-bound protease that is upregulated in a wide range of tumours and viewed as a marker of tumour-promoting stroma. Previously, we demonstrated increased FAP expression in glioblastomas and described its localisation in cancer and stromal cells. In this study, we show that FAP+ stromal cells are mostly localised in the vicinity of activated CD105+ endothelial cells and their quantity positively correlates with glioblastoma vascularisation. FAP+ mesenchymal cells derived from human glioblastomas are non-tumorigenic and mostly lack the cytogenetic aberrations characteristic of glioblastomas. Conditioned media from these cells induce angiogenic sprouting and chemotaxis of endothelial cells and promote migration and growth of glioma cells. In a chorioallantoic membrane assay, co-application of FAP+ mesenchymal cells with glioma cells was associated with enhanced abnormal angiogenesis, as evidenced by an increased number of erythrocytes in vessel-like structures and higher occurrence of haemorrhages. FAP+ mesenchymal cells express proangiogenic factors, but in comparison to normal pericytes exhibit decreased levels of antiangiogenic molecules and an increased Angiopoietin 2/1 ratio. Our results show that FAP+ mesenchymal cells promote angiogenesis and glioma cell migration and growth by paracrine communication and in this manner, they may thus contribute to glioblastoma progression.

Application of denaturing capillary electrophoresis for the detection of prognostic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes in brain tumors.

Malignant transformation in gliomas is frequently supplemented by somatic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes. It has recently emerged that mutations in these genes are associated with prolonged survival and should be used as prognostic factor in management of brain cancer patients. There are several approaches in use for the detection of isocitrate dehydrogenase 1 and 2 mutations; however, these often exhibit shortcomings such as convoluted protocols with long processing time, complex (and costly) dedicated fluorescent probes, and/or demand on amounts of input DNA. Therefore, a simple and rapid method would be highly desired. Here, we present development and validation of simple and reliable isocitrate dehydrogenase 1 and 2 mutation detection assay using denaturing capillary electrophoresis. The detection sensitivity in terms of the limiting mutated allele fraction detectable estimated from a series of dilution runs was 2.9%. The method was validated by comparing to results obtained by a widely accepted detection technique, the multiplex ligation-dependent probe amplification, on a set of 85 brain tumors. The concordance of both methods was 100%, but denaturing capillary electrophoresis assay required fivefold lower input of DNA (1 versus 5 µL of DNA at concentrations typically between 10 and 30 ng/µL).

Primary and recurrent diffuse astrocytomas: genomic profile comparison reveals acquisition of biologically relevant aberrations.

BACKGROUND: Diffuse astrocytomas are characterized by their highly variable biological behavior. The possibility that tumors develop novel aberrations, with relevant biological properties, is often neglected. In this study, we present two cases of diffuse astrocytoma in which additional cytogenetic and epigenetic markers with potential influence on cell proliferation or differentiation were detected at relapse. FINDINGS: The biopsies taken from the primary and recurrent tumors of two patients were analyzed with molecular methods to detect copy number variations (CNVs), gene mutations and epigenetic changes. Both cases were characterized by the R132H mutation in the isocitrate dehydrogenase 1 (IDH1) gene. Features typical of astrocytomas, such as copy-neutral loss of heterozygosity at 17p and the deletion of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, were also detected in both cases. These markers were present in the primary and recurrent lesions. Other aberrations, predominantly deletions or amplifications of chromosomal segments and the hypermethylation of gene promoters, were detected in the recurrent lesions. CONCLUSIONS: The IDH1 mutation was the primary event, as previously reported. According to our observations, the methylation of promoters constituted later events, which may have further disrupted cell proliferation and/or differentiation, together with additional CNVs.

Genetic and epigenetic characterization of low-grade gliomas reveals frequent methylation of the MLH3 gene.

Diffuse astrocytomas and oligodendrogliomas (WHO grade II) are the most common histological subtypes of low-grade gliomas (LGGs). Several molecular and epigenetic markers have been identified that predict tumor progression. Our aim was in detail to investigate the genetic and epigenetic background of LGGs and to identify new markers that might play a role in tumor behavior. Twenty-three patients with oligodendroglioma or oligoastrocytoma (LGO) and 22 patients with diffuse astrocytoma (LGA) were investigated using several molecular-cytogenetic and molecular methods to assess their copy number variations, mutational status and level of promoter methylation. The most frequent findings were a 1p/19q codeletion in 83% of LGO and copy-neutral loss of heterozygosity (CN-LOH) of 17p in 72% of LGA. Somatic mutations in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) genes were detected in 96% of LGO and 91% of LGA. The O-6-methylguanine-DNA-methyltransferase (MGMT) promoter was methylated in 83% of LGO and 59% of LGA. MutL homolog 3 (MLH3) promoter methylation was observed in 61% of LGO and 27% of LGA. Methylation of the MGMT promoter, 1p/19q codeletion, mutated IDH1, and CN-LOH of 17p were the most frequent genetic aberrations in LGGs. The findings were more diverse in LGA than in LGO. To the best of our knowledge, this is the first time description of methylation of the MLH3 gene promoter in LGGs. Further studies are required to determine the role of the methylated MLH3 promoter and the other aberrations detected.

Recognition of anaplastic foci within low-grade gliomas using MR spectroscopy.

BACKGROUND: The recognition of anaplastic foci within low-grade gliomas is of extreme importance in patients under follow-up for Grade II gliomas. We present the algorithm of MR spectroscopy (MRS)-guided brain biopsy and its correlation with tumour histology. METHODS: Twenty-seven patients harbouring suspected Grade II/III glioma were examined on our 3T MR. 2D PRESS-CSI metabolite images of Choline/Creatine, Creatine/N-acetylaspartate and Choline/N-acetylaspartate were calculated and exported to the DICOM format. According to these maps, a stereobiopsy was performed at the point of maximum Choline/Creatine ratio prior to tumour resection. In the case of enhancing tumour, a subsequent biopsy was performed from the point of enhancement. Comparisons were made between the histology of the biopsied specimens and the resected tumours. RESULTS: Eleven tumours were diagnosed as high-grade and sixteen as low-grade lesions. The correlation between main spectroscopic ratios (Cho/Cr and Cho/NAA) was strongly positive at the points of maximum Cho/Cr. Similar results were obtained at the points of contrast enhancement. Comparison of histological parameters of biopsy samples at the points of maximum Cho/Cr and histological examination of the completely resected tumours gives a strong correlation of tumour grade, number of mitoses and Ki-67 expression. The diagnostic accuracy of MRS-guided biopsy was 84%. The absolute value of Cho/NAA was higher in high-grade compared to that of low-grade lesions. The value of Cho/NAA ratio of 0.9 using MRS produced a sensitivity and specificity of 78% in the differentiation between low-grade and high-grade lesions. Combining MRS with structural MR, the sensitivity increased to 86% and the specificity to 80%. CONCLUSIONS: Strong correlation was demonstrated between Cho/Cr and Ch/NAA ratios. Strong correlation was demonstrated between histological parameters of biopsy samples taken using Cho/Cr ratio and those from total tumour examination. Diagnostic accuracy of MRS-guided biopsy was 84%. Sensitivity and specificity of MRS combined with structural MR reaches 86% and 80%.

Intraoperative MR imaging in a case of a cervical spinal cord lesion.

The aim of this article is to describe the feasibility of performing intraoperative MR imaging in patients with spinal cord lesions and the potential value of this technique. The authors report a case involving a 28-year-old man who presented with chronic cervical pain and pain along the ulnar side of the forearms during neck flexion. Findings on clinical examination were normal, but MR imaging revealed a multicystic cervical spinal cord lesion. Surgery was undertaken to open the cysts, evacuate old blood, and search for pathological tissue. Intraoperative MR imaging showed that the caudal cyst was not opened, and surgery was therefore continued. The caudal cyst was fenestrated and a suspected small cavernous malformation was removed. Electrophysiological monitoring was performed both before and after the intraoperative MR imaging. The use of intraoperative MR imaging changed the strategy of the procedure and helped the surgeon to safely enter all the cysts in the cervical cord.

Multifunctional surgical suite (MFSS) with 3.0 T iMRI: 17 months of experience.

The 3T ioMRI in Prague is composed of two independent suites: the operating theatre and the 3T MR suite, both of which can and do work independently. They are connected by a double door and a special transportation system. The whole operating table is moved on rails to and from the MR gantry. Anaesthesiological equipment is built from paramagnetic material, which is also moved to and from the MR suite. The integral parts of the multifunctional surgical suite (MFSS) are the neuronavigation system, electrophysiological monitoring, surgical microscope with availability of indocyanin green angiography and fluorescence-guided glioma resection technique and endoscopy equipment. The operating theatre is equipped in a normal fashion with the exception of a head holder that is paramagnetic. MR radiologist and MR assistants are alerted approximately 30 min before the requested intraoperative and out-patient service is interrupted to clean the MR suite. The ioMRI takes 15-20 min and immediately after the door closes the out patient activity is resumed. Intraoperative MR was performed in 332 surgeries in the first 17 months of operation. The most frequent indications were pituitary adenomas, followed by gliomas. Other indications were less frequent and included meningiomas, cavernomas, aneurysms, epilepsy surgery, intramedullary lesions, non-pituitary sellar lesions, metastases and various other surgeries. In 332 cases no technical or medical complication connected with ioMRI was encountered.

One year experience with 3.0 T intraoperative MRI in pituitary surgery.

A multifunctional surgical suite with intraoperative 3.0 T MRI (ioMRI) has been operating at the Central Military Hospital, Prague since April 2008. Our experiences over the past year and the effect of ioMRI on the extent of pituitary adenoma resection are evaluated. Eighty-six pituitary adenoma resections were performed in 85 patients with ioMRI in the first year of the ioMRI service. Pituitary adenoma suprasellar extension was present in 60 cases, invasion into cavernous sinus in 49 cases, and retrosellar growth in one case. The surgical goal was set before surgery: either a radical resection (49 cases) or a partial resection (37 cases). In the group of patients where a decision for a radical resection was taken the results are as follows: ioMRI confirmed radical resection in 69.4% of the cases; ioMRI disclosed unexpected adenoma residuum and further resection led to radical resection in 22.4%. In the group of patients where a decision for a partial resection was taken, the results are as follows: no further resection was perfomed after ioMRI in 51.3% of the cases and further resection was performed after ioMRI in 48.7% of the cases. ioMRI seems to be a valuable tool to increase the extent of pituitary adenoma resection.

Cytogenetic analyses in 81 patients with brain gliomas: correlation with clinical outcome and morphological data.

Specific gene mutations, loss of heterozygosity, deletions and/or amplifications of entire chromosomal regions and gene silencing have been described in gliomas. 82 samples from 81 patients were investigated to detect the deletion of TP53, RB1, CDKN2A genes, deletion of 1p36 and 19q13.3 region, amplification of EGFR gene, trisomy of chromosome 7 and monosomy of chromosome 10 in glial cells. Dual-colour interphase fluorescence in situ hybridization (I-FISH) with locus-specific and/or chromosome enumeration DNA probes were used for cytogenetic analyses. In the study, molecular cytogenetic analyses were successfully performed in 74 patients (91.3%) and were uninformative in 7 only (8.7%). The cytogenetic analyses were correlated with morphological data and clinical outcome. I-FISH was the essential part of diagnostics. In comparison with the clinical data, the patients' age seems to be a factor more important for the overall survival, rather than cytogenetic findings in glial tumours. The combined deletion of 1p36 and 19q13.3 chromosomal regions predicts longer overall survival for patients with oligodendroglial tumours.