Association of loss of heterozygosity with shorter survival in primary glioblastoma patients.

Loss of heterozygosity (LOH) co-deletion 1p/19q, MGMT promoter methylation and/or IDH1 mutation generally signify a better prognosis for patients with glioma. However, the influence of 1p/19q co-deletion and the LOH on other chromosomes in primary glioblastoma on survival is still debatable. The aim of our study was to identify LOH on chromosomes 1p, 19q, 9p, 10q, 13q, and 17p, and evaluate their impact either alone or 1p/19q co-deletion or by groups of LOH on the overall survival of 42 primary glioblastoma patients without an oligodendroglial component. These patients were additionally molecularly characterized for EGFR amplification, IDH1 mutations and TP53 mutations. We assessed their influence on the overall survival of glioblastoma patients. LOH in at least one of the loci on all examined chromosomes was detected in 65% of cases and was significantly associated with shorter overall survival (hazard ratio 3.07; 95% CI: 1.29-7.31, p = 0.006). 1p/19q co-deletion was infrequent (7.14%) and had no impact on overall survival. Our results indicate that in primary glioblastoma a specific LOH group analysis may be important for the prognosis. LOH 1p/19q co-deletion is rare in glioblastoma without an oligodendroglial component and has no impact on patient survival.

Magnetic resonance spectroscopy in intracranial tumours of glial origin.

BACKGROUND AND PURPOSE: To determine in vivo magnetic resonance spectroscopy (MRS) characteristics of intracranial glial tumours and to assess MRS reliability in glioma grading and discrimination between different histopathological types of tumours. MATERIAL AND METHODS: Analysis of spectra of 26 patients with glioblastomas, 6 with fibrillary astrocytomas, 4 with anaplastic astrocytomas, 2 with pilocytic astrocytoma, 3 with oligodendrogliomas, 3 with anaplastic oligodendrogliomas and 17 control spectra taken from healthy hemispheres. RESULTS: All tumours' metabolite ratios, except for Cho/Cr in fibrillary astrocytomas (p = 0.06), were statistically significantly different from the control. The tumours showed decreased Naa and Cr contents and a high Cho signal. The Lac-Lip signal was high in grade III astrocytomas and glioblastomas. Reports that Cho/Cr ratio increases with glioma's grade whereas Naa/Cr decreases were not confirmed. Anaplastic astrocytomas compared to grade II astrocytomas had a statistically significantly greater mI/Cr ratio (p = 0.02). In pilocytic astrocytomas the Naa/Cr value (2.58 ± 0.39) was greater, whilst the Cho/Naa ratio was lower (2.14 ± 0.64) than in the other astrocytomas. The specific feature of oligodendrogliomas was the presence of glutamate/glutamine peak Glx. However, this peak was absent in two out of three anaplastic oligodendrogliomas. Characteristically, the latter tumours had a high Lac-Lip signal. CONCLUSIONS: MRS in vivo cannot be used as a reliable method for glioma grading. The method is useful in discrimination between WHO grade I and WHO grade II astrocytomas as well as oligodendrogliomas from other gliomas.

Differential diagnosis of intracranial meningiomas based on magnetic resonance spectroscopy.

BACKGROUND AND PURPOSE: To determine in vivo magnetic resonance spectroscopy (MRS) characteristics of intracranial meningiomas and to assess MRS reliability in meningioma grading and discrimination from tumours of similar radiological appearance, such as lymphomas, schwannomas and haemangiopericytomas. MATERIAL AND METHODS: Analysis of spectra of 14 patients with meningiomas, 6 with schwannomas, 2 with lymphomas, 2 with haemangiopericytomas and 17 control spectra taken from healthy hemispheres. RESULTS: All the patients with meningiomas had a high Cho signal (long TE). There were very low signals of Naa and Cr in the spectra of 10 patients. A reversed Ala doublet was seen only in 2 cases. Four patients had a negative Lac signal, whereas 3 had high Lac-Lip spectra. Twelve spectra showed high Cho signals (short TE). In one case the Cho signal was extremely low. All spectra displayed a very low Cr signal, but high Glx and Lac-Lip signals. Ala presence was found only in 3 patients. The mean Cho/Cr ratio (PRESS) was 5.97 (1.12 in normal brain, p < 0.05). Lac-Lip was present in all the meningiomas (STEAM). The Ala signal was seen only in 2 spectra with long TE and in 3 sequences of the short TE sequences. There were both ß/γ-Glx and α-Glx/glutathione signals in all 14 meningiomas. CONCLUSIONS: MRS is unable to discriminate low and high grade meningiomas. The method seems to be helpful in discriminating lymphomas (absent Glx signal), schwannomas (mI signal in the short TE sequences) and haemangiopericytomas (presence of mI band) from meningiomas.

Glioma cells showing IDH1 mutation cannot be propagated in standard cell culture conditions.

BACKGROUND: It has recently been reported by several sources that original (i.e., present in vivo) glioma cell phenotypes or genotypes cannot be maintained in vitro. For example, glioblastoma cell lines presenting EGFR amplification cannot be established. METHODS AND RESULTS: IDH1 sequencing and loss of heterozygosity analysis was performed for 15 surgery samples of astrocytoma and early and late passages of cells derived from those and for 11 archival samples. We were not able to culture tumour cells presenting IDH1 mutations originating from currently proceeded 10 tumours; the same results were observed in 7 samples of archival material. CONCLUSION: The IDH1 mutation is expected to be almost mutually exclusive with EGFR amplification, so glioma cells with IDH1 mutations seem to represent a new group of tumour cells, which cannot be readily analysed in vitro because of their elimination. The reasons for this intriguing phenomenon should be investigated since its understanding can help to define a new therapeutic approach based on simulating in vivo conditions, responsible for tumour cells elimination in vitro. Moreover, a new model for culturing glioma cells in vitro should be designed since the current one does not provide conditions corresponding to in vivo growth.

BCR expression is decreased in meningiomas showing loss of heterozygosity of 22q within a new minimal deletion region.

Neurofibromin 2 (NF2), located on chromosome arm 22q, has been established as a tumor suppressor gene involved in meningioma pathogenesis. In our study, we investigated 149 meningiomas to determine whether there are additional tumor suppressor genes localized on chromosome 22q, apart from NF2, that might be involved in meningioma pathogenesis. The LOH analysis on chromosome 22q identified two regions of deletion: the first one, which is limited to the NF2 gene locus, and the second one, which is outside this location. The new minimal deletion region (MDR) included the following genes: BCR (breakpoint cluster region), RAB36 (a member of RAS oncogene family), GNAZ [guanine nucleotide binding protein (G protein), alpha-z polypeptide], and RTDR1 (rhabdoid tumor deletion region gene 1). The expression levels of all these genes, including NF2, were subsequently analyzed by quantitative real-time polymerase chain reaction. We observed a significantly lowered expression level of NF2 in meningiomas with 22q loss of heterozygosity (LOH) within NF2 region compared to the one in meningiomas with 22q retention of heterozygosity (ROH, P<0.05). Similarly, BCR showed a significantly lowered expression in meningiomas with 22q LOH within the new MDR compared to cases with 22q ROH (P<0.05). Our data, together with the already published information considering BCR function suggest that BCR can be considered as a candidate tumor suppressor gene localized on chromosome 22q which may be involved in meningioma pathogenesis.

Molecular heterogeneity of meningioma with INI1 mutation.

BACKGROUND: INI1 (hSNF5) mutations are linked to rhabdoid tumours, but mutations in meningiomas with hot spot mutations in position 377 have also been reported. AIMS: To analyse the INI1 gene in meningioma. METHODS: Exons 1, 4, 5, and 9 of the INI1 gene were analysed by the polymerase chain reaction and direct sequencing in 80 meningiomas. For all cases, western blotting of the INI1 protein was performed. RESULTS: Only one of the 80 samples showed a cytosine insertion in codon 376. This mutation changed the open reading frame in almost the whole exon 9 and resulted in a longer hSNF5 protein. Complex analysis of the above described tumour sample by western blotting, DNA sequencing, and loss of heterozygosity (LOH) analysis showed that this particular meningioma consisted of heterogeneic cellular components. One of these components had a mutated INI1 gene, whereas in the other component INI1 was intact. CONCLUSIONS: INI1 mutation is a rare event in the molecular pathology of meningiomas. It is possible for the INI1 gene to be mutated in only a proportion of meningioma cells.

Online Internet-based robotic telepathology in the diagnosis of neuro-oncology cases: a teleneuropathology feasibility study.

This feasibility study examined the diagnostic accuracy of Internet-based dynamic-robotic telepathology using neuropathology cases. Randomly, 83 cases were selected from the routine diagnostic workload of the Neurosurgical Pathology Laboratory in Poznan, Poland. Telepathology diagnoses were compared with conventional paraffin section diagnosis. The neuropathologists, operating a robotically controlled motorized microscope over the Internet from 3 different Polish cities, individually reviewed the cases using computer workstations. Viewing times ranged from 2 minutes 54 seconds to 32 minutes 12 seconds per case. The mean diagnostic accuracy for telepathology diagnosis was 95%, with 2 of 3 observers achieving 100% diagnostic accuracy. Image quality was judged to be sufficient for correct evaluation, and the viewing times required to establish a final diagnosis by remote video microscopy were acceptable. Generally, user acceptance of robotic telepathology was high.

Determining the feasibility of diagnosing meningiomas using static teleneuropathy images transmitted electronically.

The feasibility of using still images sent via the Internet for remote neuropathological consultation was examined. We assessed the diagnostic agreement between three groups of neuropathologists and a reference laboratory. All groups independently evaluated pictorial data exchanged by e-mail or ftp server. In the group of benign meningothelial meningioma agreement was reached in 100%, 100%, 92% of cases. In three cases of malignant meningioma agreement was achieved in 100%, 33%, 33% of cases, and in ten cases of atypical meningioma agreement was achieved in 50%, 30%, 40% of cases. Average concordance for all three groups was 83.33%, 54.33%, 57.67%. Our experiment showed that there were no discrepancies in the typical cases of benign meningothelial meningiomas. Disagreement related to the grade II and III lesions i.e. atypical and malignant meningioma. Atypical as well as malignant meningioma seems to need more direct discussion to achieve consensus. The main problem was the subjectivity of the local pathologist who overstated the informational content of the images, and an insufficient number of pictures. This may be prevented in the future by employment of dynamic teleneuropathology using a very high-resolution camera.

Real-time teleneuropathology for a second opinion of neurooncological cases.

A teleneuropathology system directed by Java programs through a standard Internet browser was evaluated in the present study. Assessment of neurooncological cases was done by remote microscope through the "Case Study" Web page at the Department of Pathology Web site (http:¿ampat.amu.edu.pl). The site was used to control a remote automatic microscope, the Axioplan 2 (Zeiss), which was connected to a computer that acts as an Internet server. The Java program for the control of the microscope server is automatically downloaded and started if the user selects the Web site of the corresponding microscope server. The microscope server receives microscope operation commands from the telemicroscopy clients, executes them, and distributes the new microscope image to all of the connected telemicroscopy clients. Fifteen cases were evaluated over several weeks. The percentage of correctly classified cases sent by remote consultation was 100%. Since the system does not require specialized software for the remote side and since the number of possible discussion partners is unlimited, this system may help to overcome obstacles to the practice of teleneuropathology.

[Evaluation of CD44 adhesion molecule, nm23 gene product expression and intensity of angiogenesis in patients with laryngeal cancer]. / Ocena ekspresji czasteczki adhezyjnej CD44, produktu genu nm23 i intensywnosci angiogenezy u chorych z rakiem krtani.

One of the most important factor in prognosis of the patients with laryngeal cancer is presence of the metastases in lymph nodes of the neck. The main purpose of the paper was the evaluation of CD34 and FVIII antigens as angiogenesis markers, and nm23 protein and CD44 antigen expression as metastasis potential markers and description of their role in the tumour progression and making metastasis in the patients with laryngeal cancer. Paraffin-embedded tissue sections from 89 patients with laryngeal cancer were stained with a monoclonal antibody raised against CD34 and FVIII antigens, against nm 23 protein and against CD44 antigen. Measuring the density of the microvasculature in tumour was investigated. We found significant dependence between intensity of angiogenesis (IA) and pT, nodal metastasis, histological grading and survival. There were also significant correlation between nm23 protein expression and nodal metastasis, and between CD44 antigen expression and pT, nm23 protein expression and FVIII antigen expression. Evaluation of mentioned markers allowed to asses the aggressiveness of tumour cells and anticipate neck metastasis in the patients with laryngeal cancer.

N-acetylcysteine and ambroxol inhibit endotoxin-induced phagocyte accumulation in rat lungs.

UNLABELLED: We have investigated whether pretreatment with N-acetylcysteine (NAC) and/or ambroxol (Amb), drugs known as reactive oxygen species (ROS) scavengers, would minimize lipopolysaccharide (LPS)-induced leucocyte accumulation in rat lung microvasculature and protect lungs from damage and the effect of these drugs on chemotactic peptide (fMLP)-induced chemiluminescence of human polymorphonuclear leukocytes (PMNs). Animals were injected ip with NAC (27.6 mg/kg, n=8), ambroxol (70 mg/kg, n=8), combination NAC+ambroxol (n=8), or 1 ml buffer alone (n=8), once a day for 3 consecutive days. Then animals were injected with LPS (17 mg/kg), and killed 3 h later. In each of another four groups eight rats were used as a control, and received the same drug treatment but LPS was replaced with 0.9% NaCl. PMNs and macrophages (Ms) were counted in histologic slides of lung tissue. Using computer image analysis we measured the area of alveolar profiles. Luminol-enhanced chemiluminescence was measured in PMNs suspensions obtained from healthy volunteers. Chemiluminescence intensity was measured in resting and fMLP-stimulated cells, and compared between cells incubated with Amb, NAC or distilled water. We observed significant differences in the number of PMNs and Ms, alveolar profile area between control and LPS-treated animals (P<0.01). PMNs and Ms were numerous in lungs of LPS-administered animals (PMNs: Median (M)=137.5 per 6 high power fields range (r)=54.0; Ms: M=123.0 r=11.0), less numerous in ambroxol-treated group (PMNs: M=101.5 r=32.0 and Ms:53.5 r=36.0), not abundant in NAC (PMNs:M=56.0 r=28.0 and Ms:M=20.5 r=13.0) and in NAC+ambroxol treated rats (PMNs:M=53.5 r=21.0 and Ms:M=29.0 r=9.0), and rare in LPS+drugs-untreated control group (PMNs:M=40.5 r=19.0 and Ms:M=18.5 r=15.0). Chemiluminescence assay revealed that 100 micro;M ambroxol stimulated fMLP-induced PMNs chemiluminescence and NAC of the same concentration had no significant effect. CONCLUSION: In our experiment we showed that pretreatment with NAC and ambroxol may inhibit phagocyte influx to rat lung and may protect it from damage. We also revealed that NAC at dose 27.6 mg/kg has stronger protective properties than ambroxol at dose 70 mg/kg and this may result from enhancing effect of ambroxol on fMLP-provoked PMNs chemiluminescence.

Programmed cell death (apoptosis) in Alzheimer's disease and Creutzfeldt-Jakob disease.

The aim of our study was the estimation of the apoptosis process using in situ-end labelling of DNA breaks method on paraffin sections in 5 human cases of Alzheimer's disease (AD), 6 of Creutzfeldt-Jakob disease (CJD) and in 25 mice infected experimentally with the Fujisaki strain of CJD, killed sequentially at one-week intervals. The numbers of apoptotic cells in CJD-infected mice in the later stages of the disease and in terminally ill mice were progressively higher that at the early stage of the disease. Further, we found a correlation between the intensity of apoptosis and major lesions hallmark of disease--the intensity of spongiform changes in the cerebral cortex but not in the accumulation of PrP in CJD infected mice. The number of A beta-amyloid plaques in AD was not related to apoptotic index. Our study showed that apoptosis is a very important event in these neurodegenerative diseases and may become a basic mechanism in loss of neurons.

Primitive neuroectodermal tumors: ultrastructural and immunohistochemical studies.

We report here ultrastructural and immunohistochemical studies of neuroblastic differentiation in the retrospective (n = 17) and prospective (n = 26) series of primitive neuroectodermal tumors (PNETs). By electron microscopy, neuritelike structures containing parallel-oriented microtubules, adhesive plaque junctions, and pleomorphic dense-core vesicles were found in the majority of tumor specimens while synaptic specializations were very rare. By immunohistochemistry, synaptophysin appeared to be the most reliable marker for neuroblastic differentiation present in the most reliable marker for neuroblastic differentiation present in the majority of tumors, while 200 kDa neurofilament protein was immunovisualized in a lower proportion of tumors. Glial fibrillary acidic protein (GFAP) was expressed in both reactive astrocytes and in a small proportion of otherwise typical neoplastic cells. We conclude that the majority of PNETs revealed diverse differentiation and that electron microscopy is still the most reliable tool for its detection followed by immunohistochemistry for synaptophysin.

Prevention of chronic relapsing experimental autoimmune encephalomyelitis by soluble tumor necrosis factor receptor I.

We have evaluated the effect of the type I (p-55, type beta) soluble tumor necrosis factor receptor (sTNFrI) in an animal model of multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) was induced in SJL/J mice by adoptive transfer of T lymphocytes sensitized to myelin basic protein (MBP). sTNFrI completely blocked both clinical signs of disease and pathological changes that included CNS demyelination and inflammatory cell infiltration. Effective inhibition of disease expression was obtained using several different regimens of subcutaneous (s.c.) injection. These included daily doses starting at day 0, every other day injections starting at day 0, daily doses starting on day 4, and two doses separated by 12 h on day 1 and 2. Furthermore, treatment with sTNFrI for 15 days completely protected these animals from the recurrent episodes of disease normally associated with adoptively transferred EAE. These findings suggest that TNF plays a major causative role in EAE and that the sTNFrI may prove to be a useful therapeutic approach in multiple sclerosis.