Magdalena Zakrzewska

Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Hotspot Mutations in H3F3A and IDH1 Define Distinct Epigenetic and Biological Subgroups of Glioblastoma

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

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.

Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors

BackgroundAlthough features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed.MethodsFollowing methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed.ResultsIn vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum.ConclusionOur results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

Atypical molecular background of glioblastoma and meningioma developed in a patient with Li–Fraumeni syndrome

We observed three neoplasms with completely different histologies: malignant fibrous histiocytoma (MFH), atypical meningioma (AM), and glioblastoma (GB), developing in a patient with Li–Fraumeni syndrome. By using a combined molecular approach we performed molecular characterization of all three tumours. Data obtained showed an interesting molecular background of the AM and GB. AM showed TP53mutations and a 22q loss of heterozygosity (LOH). GB showed epidermal growth factor receptor (EGFR) amplification and TP53 mutations, whereas P16, PTEN, Rbwere intact in terms of LOH and/or multiplex PCR (polymerase chain reaction) analysis. Additionally, GB has a 1q LOH, which is an extremely rare alteration in glioblastomas. Identical 1q LOH was also observed in MFH.

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.

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.

cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

BackgroundRecently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers.MethodsTo this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry.ResultsWe found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations.ConclusionIn terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.

Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells

BackgroundAlthough extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation – allowing for an increase in percentage yield of neuronal cells.ResultsUncommitted GFAP and SOX2 positive neural progenitors – so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100β-, SOX2-], or mixture of neural and non-neural cells.In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells – i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively.ConclusionWe suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells – such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.

Prevalence of mutated TP53 on cDNA (but not on DNA template) in pleomorphic xanthoastrocytoma with positive TP53 immunohistochemistry

Pleomorphic xanthoastrocytoma (PXA) is a tumor of astrocytic lineage that may have anaplastic features; such a phenotype usually correlates with a less favorable outcome. The molecular profile of PXA differs from other astrocytic tumors, but the molecular mechanisms of its formation and progression are still undefined. We analyzed a loss of heterozygosity and mutations of the SMARCB1 (also known as SNF5 or INI1) and TP53 genes in four cases of PXA. In just one case a TP53 mutation (Cys238Tyr) was readily detectable at the mRNA level, but it was almost undetectable during DNA sequencing. This case was also the only one exhibiting anaplastic features and TP53 overexpression as defined by immunohistochemistry. This observation supports our earlier findings on discrepancies in TP53 status in glioblastomas. We suggest that the incidence of TP53 mutations in pleomorphic xanthoastrocytoma may be underestimated and that molecular approaches should be used for greater diagnostic precision.