If Artificial In Vitro Microenvironment Can Influence Tumor Drug Resistance Network via Modulation of lncRNA Expression?-Comparative Analysis of Glioblastoma-Derived Cell Culture Models and Initial Tumors In Vivo.

The tumor resistance of glioblastoma cells in vivo is thought to be enhanced by their heterogeneity and plasticity, which are extremely difficult to curb in vitro. The external microenvironment shapes the molecular profile of tumor culture models, thus influencing potential therapy response. Our study examines the expression profile of selected lncRNAs involved in tumor resistance network in three different glioblastoma-derived models commonly utilized for testing drug response in vitro. Differential expression analysis revealed significant divergence in lncRNA profile between parental tumors and tumor-derived cell cultures in vitro, including the following particles: MALAT1, CASC2, H19, TUSC7, XIST, RP11-838N2.4, DLX6-AS1, GLIDR, MIR210HG, SOX2-OT. The examined lncRNAs influence the phenomenon of tumor resistance via their downstream target genes through a variety of processes: multi-drug resistance, epithelial-mesenchymal transition, autophagy, cell proliferation and viability, and DNA repair. A comparison of in vivo and in vitro expression identified differences in the levels of potential lncRNA targets, with the highest discrepancies detected for the MDR1, LRP1, BCRP and MRP1 genes. Co-expression analyses confirmed the following interrelations: MALAT1-TYMS, MALAT1-MRP5, H19-ZEB1, CASC2-VIM, CASC2-N-CAD; they additionally suggest the possibility of MALAT1-BCRP, MALAT1-mTOR and TUSC7-PTEN interconnections in glioblastoma. Although our results clearly demonstrate that the artificial ex vivo microenvironment changes the profile of lncRNAs related to tumor resistance, it is difficult to anticipate the final phenotypic effect, since this phenomenon is a complex one that involves a network of molecular interactions underlying a variety of cellular processes.

microRNA interaction with MAPK and AKT pathways in paediatric brain tumours - preliminary results and review of the literature.

Nowadays molecular investigations have a significant impact on the understanding of primary brain tumour biology,as well as on their classification and progress in the treatment modalities. Among novel type of biomarkers with potential therapeutic value, microRNAs (miRNAs) are considered in some cases. miRNAs are small molecules regu-lating gene expression, including genes encoding key proteins involved in signalling pathways responsible for growth and cell survival during tumour formation. Incorrectly hyperactivated pathways implicated in brain tumour development are inter alia the PI3K/AKT/mTOR and RAS/MAPK/ERK cascades associated with worse prognosis and decreased patient survival. This work presents relationships between changes in the expression of individual miRNAs and the genes involved in the regulation of PI3K/AKT/mTOR and RAS/MAPK/ERK signalling pathways in primary brain tumours. Herein we present the preliminary results of miR-17-5p and miR-20a (key representatives of the miR-17-92 oncogenic cluster) expression analysis and their connection with signalling pathway activation in two of the most frequent paediatric tumours: medulloblastoma and ependymoma. Our study was performed using the microarray and qPCR techniques and showed PI3K/AKT/mTOR and RAS/MAPK/ERK among the forefront of the list of pathways with the largest number of genes involved in their activation compared to the control. Predicted target analysis indicated the agents from miR-17-92 cluster within miRNAs regulating activity of PI3K/AKT/mTOR and RAS/MAPK/ERK deregulated genes. The expression level of key representatives of the oncogenic cluster, miR-17-5p, and miR-20a, increased with the WHO grade of the analysed cases; the highest levels were found in medulloblastomas.

Artificial microenvironment of in vitro glioblastoma cell cultures changes profile of miRNAs related to tumor drug resistance.

Purpose: The in vitro environment can influence not only the molecular background of glioblastoma drug-resistance and treatment efficiency, but also the mechanisms and pathways of cell death. Both crucial molecular pathways and the deregulation of miRNAs are thought to participate in tumor therapy-resistance. The aim of our study is to examine the potential influence of ex vivo conditions on the expression of miRNAs engaged in the machinery of tumor-drug resistance, since in vitro models are commonly used for testing new therapeutics. Methods: Glioblastoma-derived cells, cultured under three different sets of conditions, were used as experimental models in vitro. The expression of 84 miRNAs relevant to brain tumorigenesis was evaluated by multi-miRNA profiling for initial tumors and their corresponding cultures. Finally, the expression of selected miRNAs related to temozolomide-resistance (miR-125b, miR-130a, miR-21, miR-221, miR-222, miR-31, miR-149, miR-210, miR-181a) was assessed by real-time PCR for each tumor and neoplastic cells in cultures. Results: Our results demonstrate significant discrepancies in the expression of several miRNAs between tumor cells in vivo and in vitro, with miR-130a, miR-221, miR-31, miR-21, miR-222, miR-210 being the most marked. Also differences were observed between particular models in vitro. The results of computational analysis revealed the interplay between examined miRNAs and their targets involved in processes of glioblastoma chemosensitivity, including the genes relevant to temozolomide response (MGMT, PTEN, MDM2, TP53, BBC3A). Conclusion: The artificial environment may influence the selective proliferation of cell populations carrying specific patterns of miRNAs and/or the phenotype of neoplastic cells (eg differentiation) by the action of molecular events including miRNAs. These phenomena may influence the tumor-responsiveness to particular drugs, disturbing the evaluation of their efficacy in vitro, with unpredictable results caused by the interdependency of molecular pathways.

Sensitive detection of FGFR1 N546K mosaic mutation in patient with encephalocraniocutaneous lipomatosis and pilocytic astrocytoma.

Encephalocraniocutaneous lipomatosis (ECCL) is a rare neurocutaneous disorder, with only about 100 cases reported worldwide. It is characterized by congenital lesions of the eye, skin, and central nervous system. Only recently, potential causative FGFR1 point mutations have been identified in brain tumors and cultured skin biopsies from patients with this condition. Here, we analyzed the molecular status of a patient with ECCL and a coexisting pilocytic astrocytoma with detected FGFR1 N546K mutation. The presence of the alteration in both affected and unaffected tissues has been evaluated using Sanger sequencing and droplet digital polymerase chain reaction (ddPCR) technique. The ddPCR analysis showed differential distribution of the alteration in all specimens, including unaffected and untreated samples. Therefore, we confirm that FGFR1 N546K is a plausible causative mutation of ECCL patients and could be associated with a risk of brain tumor development. We also show the usefulness of sensitive ddPCR method for detection of low levels of autosomal mosaic mutation in blood or swabs. We suggest that utilization of this method may improve the diagnostic process, especially when targeted therapies are considered.

Expression-based decision tree model reveals distinct microRNA expression pattern in pediatric neuronal and mixed neuronal-glial tumors.

BACKGROUND: The understanding of the molecular biology of pediatric neuronal and mixed neuronal-glial brain tumors is still insufficient due to low frequency and heterogeneity of those lesions which comprise several subtypes presenting neuronal and/or neuronal-glial differentiation. Important is that the most frequent ganglioglioma (GG) and dysembryoplastic neuroepithelial tumor (DNET) showed limited number of detectable molecular alterations. In such cases analyses of additional genomic mechanisms seem to be the most promising. The aim of the study was to evaluate microRNA (miRNA) profiles in GGs, DNETs and pilocytic asytrocytomas (PA) and test the hypothesis of plausible miRNA connection with histopathological subtypes of particular pediatric glial and mixed glioneronal tumors. METHODS: The study was designed as the two-stage analysis. Microarray testing was performed with the use of the miRCURY LNA microRNA Array technology in 51 cases. Validation set comprised 107 samples used during confirmation of the profiling results by qPCR bioinformatic analysis. RESULTS: Microarray data was compared between the groups using an analysis of variance with the Benjamini-Hochberg procedure used to estimate false discovery rates. After filtration 782 miRNAs were eligible for further analysis. Based on the results of 10 × 10-fold cross-validation J48 algorithm was identified as the most resilient to overfitting. Pairwise comparison showed the DNETs to be the most divergent with the largest number of miRNAs differing from either of the two comparative groups. Validation of array analysis was performed for miRNAs used in the classification model: miR-155-5p, miR-4754, miR-4530, miR-628-3p, let-7b-3p, miR-4758-3p, miRPlus-A1086 and miR-891a-5p. Model developed on their expression measured by qPCR showed weighted AUC of 0.97 (95% CI for all classes ranging from 0.91 to 1.00). A computational analysis was used to identify mRNA targets for final set of selected miRNAs using miRWalk database. Among genomic targets of selected molecules ZBTB20, LCOR, PFKFB2, SYNJ2BP and TPD52 genes were noted. CONCLUSIONS: Our data showed the existence of miRNAs which expression is specific for different histological types of tumors. miRNA expression analysis may be useful in in-depth molecular diagnostic process of the tumors and could elucidate their origins and molecular background.

Morphometric analysis of mitochondria in lymphocytes of patients with exacerbations of chronic obstructive pulmonary disease - pilot study.

Introduction: Exacerbations of chronic obstructive pulmonary disease (ECOPD) are important events in the course of the disease, negatively influencing health status and disease progression. Therefore, there is a strong need for deeper understanding of the pathology of ECOPD to elaborate new therapeutic approaches and ameliorate prognoses. Contributions of mitochondria to pathobiology of COPD are still under investigation, although growing evidence suggests their important role in this disease. The aim of our study was to assess the morphometric parameters of mitochondria in lymphocytes of patients with ECOPD. Patients and methods: Lymphocytes were isolated from the peripheral blood of patients with COPD. Transmission electron microscopy was used to assess absolute number of mitochondria per cell, mitochondrial content, and morphometric parameters of individual mitochondria. We also counted indexes for elongation and interconnectivity. Results: Eighteen patients (9 with ECOPD and 9 in the stable period of the disease) were analyzed. We observed significantly lower length of mitochondrion (P=0.03) and significant decrease both in elongation (P=0.03) and interconnectivity indexes (P=0.04) in ECOPD patients. Conclusions: The morphometric parameters of mitochondria in lymphocytes derived from patients during the early period of ECOPD requiring hospitalization are altered in comparison to patients in the stable period of the disease. This suggests their contribution to pathobiology of ECOPD. These preliminary outcomes should be further validated in larger size samples.

Glioblastoma-derived cells in vitro unveil the spectrum of drug resistance capability - comparative study of tumour chemosensitivity in different culture systems.

Resistance to cancer drugs is a complex phenomenon which could be influenced by in vitro conditions. However, tumour-derived cell cultures are routinely used for studies related to mechanisms of drug responsiveness or the search for new therapeutic approaches. The purpose of our work was to identify the potential differences in drug resistance and response to treatment of glioblastoma with the use of three in vitro models: traditional adherent culture, serum-free spheroid culture and novel adherent serum-free culture.The experimental models were evaluated according to 'stemness state' and epithelial-to-mesenchymal transition (EMT) status, invasion capability and their expression pattern of genes related to the phenomenon of tumour drug resistance. Additionally, the response to drug treatments of three different culture models was compared with regard to the type of cell death.Multi-gene expression profiling revealed differences between examined culture types with regard to the expression pattern of the selected genes. Functionally, the examined genes were related to drug resistance and metabolism, DNA damage and repair and cell cycle control, and included potential therapeutic targets.Cytotoxicity analyses confirmed that environmental factors can influence not only the molecular background of glioblastoma drug-resistance and efficiency of treatment, but also the mechanisms/pathways of cell death, which was reflected by a distinct intensification of apoptosis and autophagy observed in particular culture models. Our results suggest that parallel exploitation of different in vitro experimental models can be used to reveal the spectrum of cancer cell resistance capability, especially regarding intra-heterogeneous glioblastomas.

Altered Ca2+ homeostasis induces Calpain-Cathepsin axis activation in sporadic Creutzfeldt-Jakob disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent form of human prion disease and it is characterized by the presence of neuronal loss, spongiform degeneration, chronic inflammation and the accumulation of misfolded and pathogenic prion protein (PrPSc). The molecular mechanisms underlying these alterations are largely unknown, but the presence of intracellular neuronal calcium (Ca2+) overload, a general feature in models of prion diseases, is suggested to play a key role in prion pathogenesis.Here we describe the presence of massive regulation of Ca2+ responsive genes in sCJD brain tissue, accompanied by two Ca2+-dependent processes: endoplasmic reticulum stress and the activation of the cysteine proteases Calpains 1/2. Pathogenic Calpain proteins activation in sCJD is linked to the cleavage of their cellular substrates, impaired autophagy and lysosomal damage, which is partially reversed by Calpain inhibition in a cellular prion model. Additionally, Calpain 1 treatment enhances seeding activity of PrPSc in a prion conversion assay. Neuronal lysosomal impairment caused by Calpain over activation leads to the release of the lysosomal protease Cathepsin S that in sCJD mainly localises in axons, although massive Cathepsin S overexpression is detected in microglial cells. Alterations in Ca2+ homeostasis and activation of Calpain-Cathepsin axis already occur at pre-clinical stages of the disease as detected in a humanized sCJD mouse model.Altogether our work indicates that unbalanced Calpain-Cathepsin activation is a relevant contributor to the pathogenesis of sCJD at multiple molecular levels and a potential target for therapeutic intervention.

Expression of SOX11, PAX5, TTF-1 and ISL-1 in medulloblastoma.

The aim of our study was to evaluate the immunohistochemical expression of SOX11, PAX5, TTF-1 and ISL-1 in medulloblastoma (MB) to investigate their diagnostic usefulness. METHODS: Immunohistochemical expression of PAX5 (two antibodies: Dako, DAK-Pax5; and BD, clone 24), TTF-1 (Dako, 8G7G3/1), SOX11 (CL0142; Abcam) and ISL-1 (1 H9, Abcam) was analyzed using the h-score and Remmele score in 25 cases of MB. RESULTS: There were 18 MBs of classic and 7 of desmoplastic type. SOX11 was strongly expressed in all tumors. The expression of PAX5 was higher and more frequent in a case of DAK-Pax5 clone (25/25) than clone 24 (6/25). ISL-1 was positive in 11 (44%) and TTF-1 in 3 (12%) cases. ISL-1 expression correlated positively (p<0.001), while TTF-1 correlated negatively with the age of patients (p=0.039). PAX5 expression correlated with ISL-1 (p=0.039) and showed a trend toward higher expression in the desmoplastic subtype (p=0.069). CONCLUSIONS: SOX11 is strongly and robustly expressed in MBs. PAX5 expression pattern differs substantially among two antibody clones. TTF-1 and ISL-1 is associated with the age of patients.

Recurrence-associated chromosomal anomalies in meningiomas: Single-institution study and a systematic review with meta-analysis.

Complete removal of a meningioma (MG) does not guarantee relapse-free survival. Alterations on several chromosomes responsible for MG recurrence were suggested, although their role was not validated by a systematic review. Following the analysis of own 161 cases, all previously published data has been collected for evidence synthesis. Based on own series, WHO grade >I (odds ratio (OR)=92.0; 95%CI: 19.1-443.5) and a combination of loss of heterozygosity (LOH) on 1p and 14q (OR=10.2; 95%CI: 19-55.7) were the independent recurrence-specific prognosticators. The deleterious role of LOH on 1p/14q was demonstrated in a subset of parasagittal and falcine MGs. A total of 742 cases and 10 studies were pooled for the Individual Patient Data and Aggregate Data models of meta-analysis, respectively. The prognostic role of WHO classification (OR=90.4) and anomaly of chromosome 14 (OR=3.5) was confirmed. LOH on 14 showed lesser impact on recurrence than suggested by the WHO grading (area under the curve 0.65 for LOH vs. 0.74 for WHO). Fixed effect model of meta-analysis provided high summarized OR values for 1p (OR=5.4; 95%CI: 3.6-8.1) and 14q (OR=7.6; 95%CI: 4.3-13.6), and low for chromosome 22 (OR=1.6; 95%CI: 1.1-2.4). Final appraisal of recurrence-associated chromosomal alterations indicated that arms 1p and 14q deserve attention while predicting MG recurrence.

The correlation of clinical and chromosomal alterations of benign meningiomas and their recurrences.

Meningiomas (MGs) are the frequent benign intracranial tumors. Their complete removal does not always guarantee relapse-free survival. Recurrence-associated chromosomal anomalies in MGs haves been proposed as prognostic factors in addition to the World Health Organisation (WHO) grading, tumor size and resection rate. The aim of this study was to evaluate the frequency of deletions on chromosomes in sporadic MGs and to correlate them with the clinical findings and tumor behaviour. Along with survival, the tumor recurrence was the main endpoint. Chromosomal loss of heterozygosity (LOH) was studied. 46 benign MGs were subjected to the analysis, complete tumor resection was intended and no early mortalities were observed. Incomplete removal was related to parasagittal location and psammomatous hisptopathology (p<0.01). Chromosomal alterations were present in 82.6% of cases; LOH at 22q (67.4%) and 1p (34.8%) were the most frequent and associated with male sex (p=0.04). Molecular findings were not specific for any of the histopathologic grade. Tumor recurrence (14 of 46) correlated with tumor size (≥35mm), LOH at 1p, 14q, coexistence of LOH at 1p/14q, 10q/14q, 'complex karyotype' status (≥2 LOHs excluding 22q), patient age (younger <35), and Simpson grading of resection rate (≥3 of worse prognosis). The last 3 variables were independent significant prognostic factors in multivariate analysis and of the same importance in recurrence prediction (Receiver Operating Characteristic curves comparison p>0.05). Among the cases of recurrence, tumor progression was observed in 3 of 14. In 2 cases, LOH on 1p and/or coexistence of LOH 1p/14q correlated with anaplastic transformation.

The molecular pattern of histopathological progression to anaplastic meningioma - A case report.

Meningiomas (MGs) are the most frequent primary tumours of the central nervous system (CNS) and exhibit a large spectrum of histological types and clinical phenotypes. The WHO classification of CNS tumours established strict diagnostic criteria of the benign (Grade 1), atypical (Grade 2) and anaplastic (Grade 3) subtypes. Combined with the resection rate, WHO grading has the most crucial role as the prognostic factor. Additionally, such biomarkers as Ki-67/MIB-1, progesterone receptors and phosphor-histone H3 were correlated with MG progression. Recently, it was suggested that the aggressive behaviour of some MGs is attributed to molecular alterations, regardless of their histopathology. The analysis of loss of heterozygosity (LOH) at chromosomes 1, 9, 10, 14 and 22 was performed. The presented case of WHO Grade 2 MG initially exhibited LOH at chromosomes 10, 14 and 22. In the first recurrence, the tumour genetic profiling revealed additional LOH at chromosome 1p and atypical histopathology. During the second recurrence, an aggressive phenotype was observed and tumour progressed to an anaplastic form. Considering the appearance of the tumour relapses, the set of molecular changes overtook the histopathological progression. The genetic and histopathological imbalance in the tumour progression in secondary anaplastic MGs has not been previously described. The evolution of genetic and histopathological changes was presented in the same patient. In the future, the individualised therapy of potentially more aggressive forms of MGs could be based on certain chromosome aberrations.

Altered MicroRNA Expression Is Associated with Tumor Grade, Molecular Background and Outcome in Childhood Infratentorial Ependymoma.

BACKGROUND: Ependymal tumors are the third most common group of brain tumors in children, accounting for about 10% of all primary brain neoplasms. According to the current WHO classification, they comprise four entities with the most frequent ependymoma and anaplastic ependymoma. The most of pediatric tumors are located within the posterior fossa, with a tendency to infiltrate the vital brain structures. This limits surgical resection and poses a considerable clinical problem. Moreover, there are no appropriate outcome prognostic factors besides the extent of surgical resection. Despite definition of molecular subgroups, the majority of childhood ependymomas present a balanced genome, which makes it difficult to establish molecular prognostic factors. METHODS: The purpose of our study was to explore whether miRNA expression could be used as prognostic markers in pediatric infratentorial ependymomas. We also performed a mRNA expression pattern analysis of NELL2 and LAMA2 genes, with immunohistochemical illustrations of representative cases. The miRNA and mRNA expression was measured in 53 pediatric infratentorial ependymomas using a real-time quantitative PCR. RESULTS: Three miRNAs were shown to efficiently differentiate between grade II and III ependymomas: miR-17-5p, miR-19a-3p, and miR-106b-5p. Survival analysis showed that the probabilities of overall (p = 0.036) and event-free survival (p = 0.002) were reduced with higher than median miRNA expression levels of miR-17-5p. Using multivariate analysis adjusted for patient's age, sex, tumor grade and localization, we showed statistically significant associations with event-free survival (p = 0004) and borderline statistical significance with overall survival (p = 0.057) for miR-17-5p. Correlation analysis of miR-19a, miR-17-5p, miR-106b revealed that their expression levels were significantly correlated with EZH2 expression, suggested marker of PFA ependymomas. Furthermore, lower expression level of LAMA2 mRNA was shown to be associated with an increased risk of death in covariate-adjusted analyses. CONCLUSIONS: Our data provide a better understanding of pediatric ependymoma and suggests the presence of plausible molecular biomarkers connected with the outcome.

Germline and somatic FGFR1 abnormalities in dysembryoplastic neuroepithelial tumors.

Dysembryoplastic neuroepithelial tumor (DNET) is a benign brain tumor associated with intractable drug-resistant epilepsy. In order to identify underlying genetic alterations and molecular mechanisms, we examined three family members affected by multinodular DNETs as well as 100 sporadic tumors from 96 patients, which had been referred to us as DNETs. We performed whole-exome sequencing on 46 tumors and targeted sequencing for hotspot FGFR1 mutations and BRAF p.V600E was used on the remaining samples. FISH, copy number variation assays and Sanger sequencing were used to validate the findings. By whole-exome sequencing of the familial cases, we identified a novel germline FGFR1 mutation, p.R661P. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in the tumor samples and further evidence for functional relevance was obtained by in silico modeling. The FGFR1 p.K656E mutation was confirmed to be in cis with the germline p.R661P variant. In 43 sporadic cases, in which the diagnosis of DNET could be confirmed on central blinded neuropathology review, FGFR1 alterations were also frequent and mainly comprised intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis (25/43; 58.1 %) while BRAF p.V600E alterations were absent (0/43). In contrast, in 53 cases, in which the diagnosis of DNET was not confirmed, FGFR1 alterations were less common (10/53; 19 %; p < 0.0001) and hotspot BRAF p.V600E (12/53; 22.6 %) (p < 0.001) prevailed. We observed overexpression of phospho-ERK in FGFR1 p.R661P and p.N546K mutant expressing HEK293 cells as well as FGFR1 mutated tumor samples, supporting enhanced MAP kinase pathway activation under these conditions. In conclusion, constitutional and somatic FGFR1 alterations and MAP kinase pathway activation are key events in the pathogenesis of DNET. These findings point the way towards existing targeted therapies.

Transcriptional profiles of pilocytic astrocytoma are related to their three different locations, but not to radiological tumor features.

BACKGROUND: Pilocytic astrocytoma is the most common type of brain tumor in the pediatric population, with a generally favorable prognosis, although recurrences or leptomeningeal dissemination are sometimes also observed. For tumors originating in the supra-or infratentorial location, a different molecular background was suggested, but plausible correlations between the transcriptional profile and radiological features and/or clinical course are still undefined. The purpose of this study was to identify gene expression profiles related to the most frequent locations of this tumor, subtypes based on various radiological features, and the clinical pattern of the disease. METHODS: Eighty six children (55 males and 31 females) with histologically verified pilocytic astrocytoma were included in this study. Their age at the time of diagnosis ranged from fourteen months to seventeen years, with a mean age of seven years. There were 40 cerebellar, 23 optic tract/hypothalamic, 21 cerebral hemispheric, and two brainstem tumors. According to the radiological features presented on MRI, all cases were divided into four subtypes: cystic tumor with a non-enhancing cyst wall; cystic tumor with an enhancing cyst wall; solid tumor with central necrosis; and solid or mainly solid tumor. In 81 cases primary surgical resection was the only and curative treatment, and in five cases progression of the disease was observed. In 47 cases the analysis was done by using high density oligonucleotide microarrays (Affymetrix HG-U133 Plus 2.0) with subsequent bioinformatic analyses and confirmation of the results by independent RT-qPCR (on 39 samples). RESULTS: Bioinformatic analyses showed that the gene expression profile of pilocytic astrocytoma is highly dependent on the tumor location. The most prominent differences were noted for IRX2, PAX3, CXCL14, LHX2, SIX6, CNTN1 and SIX1 genes expression even within different compartments of the supratentorial region. Analysis of the genes potentially associated with radiological features showed much weaker transcriptome differences. Single genes showed association with the tendency to progression. CONCLUSIONS: Here we have shown that pilocytic astrocytomas of three different locations can be precisely differentiated on the basis of their gene expression level, but their transcriptional profiles does not strongly reflect the radiological appearance of the tumor or the course of the disease.

Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR.

Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.

Astrocytoma-associated antigens - IL13Rα2, Fra-1, and EphA2 as potential markers to monitor the status of tumour-derived cell cultures in vitro.

BACKGROUND: The molecular heterogeneity of high-grade astrocytomas underlies the difficulties in the development of representative and valuable in vitro experimental models for their studies. The purpose of our study was to estimate the value of astrocytoma-associated antigens (AAAs) - IL13Rα2, Fra-1, EphA2 - and the most common molecular aberrations typical for astrocytomas as potential markers to screen the status of tumour-derived cell cultures in vitro. METHODS: The tumour-derived cell cultures were established from high-grade astrocytomas. The expression analyses of the tested genes were performed via semi-quantitative real-time PCR and subsequently verified by immunohistochemical and immunocytochemical technique. The analyses of molecular aberrations at DNA level included gene dosage status evaluation based on real-time PCR, sequencing analysis, and loss of heterozygosity (LOH) assay. RESULTS: The expression analyses based on semi-quantitative real-time PCR showed that in the final stage of culture the expression level of all tested AAAs was significantly higher or at least comparable to that of primary tumours; however, two expression patterns were observed during cell culture establishment. Analysis at the single cell level via immunocytochemistry also demonstrated an increase of the level of tested proteins and/or selection of tumour cell populations strongly positive for AAAs vs. other cell types including admixed non-tumoural cells. Confrontation of AAA expression data with the results of molecular analyses at DNA level seems to support the latter, revealing that the expression pattern of astrocytoma-associated antigens in tumour-derived cells in subsequent stages of culture is convergent with changes in the molecular profile of examined cell populations. CONCLUSIONS: The consistency of the obtained results seems to support the use of the selected AAAs, in particular IL13Rα2 and Fra-1, as tools facilitating the establishment of tumour-derived cultures. However, the intratumoural heterogeneity of high-grade astrocytomas may require further detailed characterisation of the molecular profile of a tumour in order to evaluate the value of the experimental model in relation to the individual context of particular studies.

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein.

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.

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.

Novel gene expression model for outcome prediction in paediatric medulloblastoma.

Medulloblastoma is the most frequent type of embryonal tumour in the paediatric population. The disease progression in patients with this tumour may be connected with the presence of stem/tumour-initiating cells, but the precise source and characteristics of such cells is still a subject of debate. Thus, we tried to analyse biomarkers for which a connection with the presence of stem/tumour-initiating cells was suggested. We evaluated the transcriptional level of the ATOH1, FUT4, NGFR, OTX1, OTX2, PROM1 and SOX1 genes in 48 samples of medulloblastoma and analysed their usefulness in the prediction of disease outcome. The analyses showed a strong correlation of PROM1, ATOH1 and OTX1 gene expression levels with the outcome (p ≤ 0.2). On the basis of the multivariate Cox regression analysis, we propose a three-gene model predicting risk of the disease, calculated as follows: RS(risk score) =( 0:81 x PROM1) + (0:18 x OTX1) + (0:02 x ATOH1). Survival analysis revealed a better outcome among standard-risk patients, with a 5-year survival rate of 65 %, compared to the 40 % rate observed among high-risk patients. The most promising advantage of such molecular analysis consists in the identification of molecular markers influencing clinical behaviour, which may in turn be useful in therapy optimization.