Wielisław Papierz

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.

High incidence of MGMT promoter methylation in primary glioblastomas without correlation with TP53 gene mutations

O(6)-methylguanine DNA methyltransferase (MGMT) reduces cytotoxicity of therapeutic or environmental alkylating agents. MGMT promoter methylation has been associated with TP53 G: C to A:T transition mutations in various types of cancers, and with poor prognosis in patients who did not receive chemotherapy. Mutations of TP53 are more frequent in secondary than in primary glioblastoma, thus the expected MGMT promoter methylation was low in primary glioblastoma. Glioblastoma patients with MGMT promoter methylation showed better response to chemotherapy based on alkylating agents and longer survival than patients without MGMT methylation. We examined 32 primary glioblastomas, treated with radiotherapy and surgery, for TP53 mutation by direct sequencing and MGMT promoter methylation by methylation-specific PCR. MGMT promoter methylation and TP53 mutations were detected in 72% and 31% of primary glioblastoma, respectively. Although not statistically significant, the frequency of TP53 G:C to A:T mutations were higher in cases with (26%) than without (11%) MGMT promoter methylation (p=0.376). MGMT promoter methylation had no impact on patient survival. Our data indicate that MGMT promoter methylation occurs frequently in primary glioblastoma, but does not lead to G:C to A:T TP53 mutations, has no independent prognostic value and is not a predictive marker unless glioblastoma patients are treated with chemotherapy.

Synucleinopathy with features of both multiple system atrophy and dementia with Lewy bodies.

Alpha-synuclein is a main component of neuronal inclusions in Parkinson’s disease, dementia with Lewy bodies (DLB) and glial and neuronal inclusions in multiple system atrophy (MSA) – as a group often referred to as alpha-synucleinopathies [1,2]. Dementia with Lewy bodies is the second most frequent neurodegenerative dementing disorder in the elderly after Alzheimer’s disease. Clinically, it is associated with fluctuating cognition and hallucinations in association with neurological features of Parkinsonism. DLB is neuropathologically characterized by the presence of Lewy bodies (LBs) in the brain [3]. Classically, LBs appear as one or more intraneuronal eosinophilic spherical structures with a dense core. They are often surrounded by a halo, although in the neocortex they usually lack the halo and may be inconspicuous in routine staining. Multiple system atrophy is a sporadic neurodegenerative disease clinically characterized by varying degrees of Parkinsonism, cerebellar ataxia and/or features of autonomic failure. The term MSA was coined to unify three separate entities: striatonigral degeneration, olivopontocerebellar atrophy and the Shy–Drager syndrome, which may overlap clinically and pathologically [4]. However, it was not until 1989, when Papp and Lantos described filamentous inclusions in the cytoplasm of oligodendroglial cells in the brains of patients with MSA, irrespective of clinical symptoms, that the existence of MSA as a clinicopathological entity was confirmed [5]. Glial cytoplasmic inclusions (GCIs), also called Papp–Lantos bodies, were more recently shown also to be composed mainly of alpha-synuclein [1,6]. Similar inclusions were later identified in neuronal cytoplasm (neuronal cytoplasmic inclusions, or NCIs) and in the nuclei of neurones and oligodendrocytes [7,8]. The clinical phenotype depends on the neuronal systems predominantly involved; hence, the two clinical presentations are now designated MSAcerebellar type and MSA-Parkinsonism. We report here an archival case of a 51-year-old woman with progressive cognitive deficits, psychotic symptoms (hallucinations) and muscle rigidity, who died of unrelated gastric haemorrhage a year after the onset of neurological and psychiatric signs. According to the archival files, the patient presented on admission with delirium superposed on progressive dementia. Neuroleptic sensitivity reaction (extrapyramidal signs) occurred during the course of the disease. Owing to the lack of detailed clinical data, thorough analysis of the clinical phenotype of this patient could not be performed in this retrospective study. Brain autopsy was performed after 14 days of fixation in 4% formaldehyde. Recently, the archival paraffin blocks were re-examined. Fivemicrometre-thick sections were obtained from each tissue block for routine stainings, conventional immunohistochemistry and double immunofluorescent labelling. Immunohistochemistry was performed using a panel of antibodies (Table 1) according to the producers’ protocols. As a secondary system we used the ChemMateTM detection kit Dako, Glostrup, Denmark. For immunofluorescent labelling and multichannel confocal microscopy we used mouse anti-microtubuleassociated protein (MAP)-2 monoclonal antibody (Chemicon International, Inc. Temecula, CA, USA, dilution 1:250) and rat anti-a-synuclein monoclonal antibody (Alexis Bichemicals, Lausen, Switzerland, dilution 1:100). The fluorescent-labelled secondary antibody for the antia-synuclein antibody was Alexa Fluor 488 rabbit anti-rat IgG (Molecular Probes, Eugene, USA, 1:200) and for antiMAP-2 Alexa Fluor 546 goat anti-mouse IgG (Molecular Probes, 1:200). To visualize cell nuclei, Sytox orange stain was used (Dako, Glostrup, Denmark). Immunofluorescence labelling was evaluated using a Zeiss LSM 510 laser scanning confocal microscope. Material for electron microscopy was retrieved from formalin-fixed, paraffin-embedded sections and reprocessed for electron microscopy. On gross neuropathological examination, moderate and diffuse brain atrophy was observed, along with slight depigmentation of the substantia nigra. Microscopic examin-

Accuracy of a remote quantitative image analysis in the whole slide images

The rationale for choosing a remote quantitative method supporting a diagnostic decision requires some empirical studies and knowledge on scenarios including valid telepathology standards. The tumours of the central nervous system [CNS] are graded on the base of the morphological features and the Ki-67 labelling Index [Ki-67 LI]. Various methods have been applied for Ki-67 LI estimation. Recently we have introduced the Computerized Analysis of Medical Images [CAMI] software for an automated Ki-67 LI counting in the digital images.Aims of our study was to explore the accuracy and reliability of a remote assessment of Ki-67 LI with CAMI software applied to the whole slide images [WSI].The WSI representing CNS tumours: 18 meningiomas and 10 oligodendrogliomas were stored on the server of the Warsaw University of Technology. The digital copies of entire glass slides were created automatically by the Aperio ScanScope CS with objective 20x or 40x. Aperios Image Scope software provided functionality for a remote viewing of WSI. The Ki-67 LI assessment was carried on within 2 out of 20 selected fields of view (objective 40x) representing the highest labelling areas in each WSI. The Ki-67 LI counting was performed by 3 various methods: 1) the manual reading in the light microscope - LM, 2) the automated counting with CAMI software on the digital images – DI , and 3) the remote quantitation on the WSIs – as WSI method. The quality of WSIs and technical efficiency of the on-line system were analysed. The comparative statistical analysis was performed for the results obtained by 3 methods of Ki-67 LI counting. The preliminary analysis showed that in 18% of WSI the results of Ki-67 LI differed from those obtained in other 2 methods of counting when the quality of the glass slides was below the standard range. The results of our investigations indicate that the remote automated Ki-67 LI analysis performed with the CAMI algorithm on the whole slide images of meningiomas and oligodendrogliomas could be successfully used as an alternative method to the manual reading as well as to the digital images quantitation with CAMI software. According to our observation a need of a remote supervision/consultation and training for the effective use of remote quantitative analysis of WSI is necessary.

Selection of reference genes for gene expression studies in astrocytomas

This study was aimed to test a panel of six housekeeping genes (GAPDH, HPRT1, POLR2A, RPLP0, ACTB, and H3F) so as to identify and validate the most suitable reference genes for expression studies in astrocytomas. GAPDH was the most stable and HPRT1 was the least stable reference gene. The effect of reference gene selection on quantitative real-time polymerase chain reaction data interpretation was demonstrated, normalizing the expression data of a selected gene of interest. Thus, GAPDH may be recommended for data normalization in gene expression studies in astrocytomas. Nevertheless, a preliminary validation of reference gene stability is required prior to every study.

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.

Screening for EGFR Amplifications with a Novel Method and Their Significance for the Outcome of Glioblastoma Patients

Glioblastoma is a highly aggressive tumour of the central nervous system, characterised by poor prognosis irrespective of the applied treatment. The aim of our study was to analyse whether the molecular markers of glioblastoma (i.e. TP53 and IDH1 mutations, CDKN2A deletion, EGFR amplification, chromosome 7 polysomy and EGFRvIII expression) could be associated with distinct prognosis and/or response to the therapy. Moreover, we describe a method which allows for a reliable, as well as time- and cost-effective, screening for EGFR amplification and chromosome 7 polysomy with quantitative Real-Time PCR at DNA level. In the clinical data, only the patient’s age had prognostic significance (continuous: HR = 1.04; p<0.01). At the molecular level, EGFRvIII expression was associated with a better prognosis (HR = 0.37; p = 0.04). Intriguingly, EGFR amplification was associated with a worse outcome in younger patients (HR = 3.75; p<0.01) and in patients treated with radiotherapy (HR = 2.71; p = 0.03). We did not observe any difference between the patients with the amplification treated with radiotherapy and the patients without such a treatment. Next, EGFR amplification was related to a better prognosis in combination with the homozygous CDKN2A deletion (HR = 0.12; p = 0.01), but to a poorer prognosis in combination with chromosome 7 polysomy (HR = 14.88; p = 0.01). Importantly, the results emphasise the necessity to distinguish both mechanisms of the increased EGFR gene copy number (amplification and polysomy). To conclude, although the data presented here require validation in different groups of patients, they strongly advocate the consideration of the patient’s tumour molecular characteristics in the selection of the therapy.

Reduced expression of ELAVL4 in male meningioma patients

Meningioma is a frequently occurring tumor of the central nervous system. Among many genetic alternations, the loss of the short arm of chromosome 1 is the second most frequent chromosomal abnormality observed in these tumors. Here, we focused on the previously described and well-established minimal deletion regions of chromosome 1. In accordance with the Knudson suppressor theory, we designed an analysis of putative suppressor genes localized in the described minimal deletion regions. The purpose was to determine the molecular background of the gender-specific occurrence of meningiomas. A total of 149 samples were examined for loss of heterozygosity (LOH). In addition, 57 tumor samples were analyzed using real-time polymerase chain reaction. We examined the association between the expression of selected genes and patient age, gender, tumor grade and presence of 1p loss. Furthermore, we performed an analysis of the most stable internal control for real-time analysis in meningiomas. LOH analysis revealed gender-specific discrepancies in the frequency of 1p aberrations. Moreover, statistical correlation between the gene expression level and gender was significant for the ELAVL4 gene as we found it to be lower in males than in females. We conclude that meningiomas present different features depending on patient gender. We suggest that ELAVL4 can be involved in the pathogenesis of meningiomas in male patients.

Ultrastructural heterogeneity of gangliogliomas.

Gangliogliomas are rare brain tumors, composed of neuronal and glial cells mixed in a different proportion. The basic histopathological pattern of gangliogliomas is well recognized but the variable microscopic appearance still can pose a challenge to the neuropathologist. The authors reanalyzed their series of gangliogliomas in the files of two departments of neuropathology. All analyzed tumors fulfilled the WHO histological criteria of ganglioglioma. Seven tumors were examined by electron microscopy. The following ultrastructural features were graded: presence of dense-cored vesicles, synaptic vesicles, synapses and intermediate filaments, abundant basal membranes, dystrophic neurites, autophagic vacuoles, and multivesicular bodies. Most of the neoplastic neurons were large, polyglonal or oval with well-developed subcellular organelles, round nuclei, and prominent nucleoli. In most cases there were abundant dense core vesicles, observed in both the tumor cell bodies as well as in their processes. Synapses were typically observed. Intermediate filaments were abundant in all tumors. The most intriguing ultrastructural finding was abundant presence of autophagic vacuoles. In 4 cases, multivesicular bodies were observed. All of the tumors with multivesicular bodies also contained abundant autophagic vacuoles.

Supratentorial neurenteric cyst--a case report.

Supratentorial neurenteric cyst is a rare congenital lesion. We report here a case of a 33-year-old female who presented with seizures. A multicystic lesion in the left premotor cortex with moderate contrast enhancement was demonstrated with MRI. Microscopically, the lesion showed small cystic structures filled with a proteinaceous fluid. The wall of the cysts was lined with a single layer of ciliated columnar or cuboidal epithelium on a basement membrane. Glandular structures resembling gastrointestinal glands were also present. The cells of the cyst lining and glandular structures revealed strongly positive immunoreactions for epithelial markers (cytokeratin and epithelial membrane antigen).