Dorota Denkhaus

Phosphatidylinositol 3′-Kinase/AKT Signaling Is Activated in Medulloblastoma Cell Proliferation and Is Associated with Reduced Expression of PTEN

Purpose: Medulloblastomas represent the most frequent malignant brain tumors of childhood. They are supposed to originate from cerebellar neural precursor cells. Recently, it has been shown that Sonic Hedgehog–induced formation of medulloblastoma in an animal model is significantly enhanced by activation of the phosphatidylinositol 3′-kinase (PI3K) signaling pathway. Experimental Design: To examine a role for PI3K/AKT signaling in the molecular pathogenesis of human medulloblastoma, we did an immunohistochemical study of the expression of Ser473-phosphorylated (p)-AKT protein in 22 medulloblastoma samples: All samples displayed p-AKT expression. To investigate if an activated PI3K/AKT pathway is required for medulloblastoma cell growth, we treated five human medulloblastoma cell lines with increasing concentrations of the PI3K inhibitor LY294002 and analyzed cellular proliferation and apoptosis. The antiproliferative effect could be antagonized by overexpressing constitutively active AKT. As the activation of PI3K/AKT signaling may be associated with alterations of the PTEN gene located at 10q23.3, a chromosomal region subject to frequent allelic losses in medulloblastoma, we screened PTEN for mutations and mRNA expression. Results: Proliferation of all of the medulloblastoma cell lines was dependent on PI3K/AKT signaling, whereas apoptosis was not prominently affected. Allelic loss was detected in 16% of the cases. One medulloblastoma cell line was found to carry a truncating mutation in the PTEN coding sequence. Even more important, PTEN mRNA and protein levels were found to be significantly lower in medulloblastomas compared with normal cerebellar tissue of different developmental stages. Reduction of PTEN expression was found to be associated with PTEN promoter hypermethylation in 50% of the tumor samples. Conclusions: We conclude that activation of the PI3K/AKT pathway constitutes an important step in the molecular pathogenesis of medulloblastoma and that dysregulation of PTEN may play a significant role in this context.

Insulin-Like Growth Factor II Is Involved in the Proliferation Control of Medulloblastoma and Its Cerebellar Precursor Cells

Medulloblastomas (MBs), the most frequent malignant brain tumors of childhood, presumably originate from cerebellar neural precursor cells. An essential fetal mitogen involved in the pathogenesis of different embryonal tumors is insulin-like growth factor II (IGF-II). We screened human MB biopsies of the classic (CMB) and desmoplastic (DMB) variants for IGF2 transcripts of the four IGF2 promoters. We found IGF2 transcription from the imprinted promoter P3 to be significantly increased in the desmoplastic variant compared to the classic subgroup. This was not a result of loss of imprinting of IGF2 in desmoplastic tumors. We next examined the interaction of IGF-II and Sonic hedgehog (Shh), which serves as a critical mitogen for cerebellar granule cell precursors (GCPs) in the external granule cell layer from which DMBs are believed to originate. Mutations of genes encoding components of the Shh-Patched signaling pathway occur in approximately 50% of DMBs. To analyze the effects of IGF-II on Hedgehog signaling, we cultured murine GCP and human MB cells in the presence of Shh and Igf-II. In GCPs, a synergistic effect of Shh and Igf-II on proliferation and gli1 and cyclin D1 mRNA expression was found. Igf-II, but not Shh, induced phosphorylation of Akt and its downstream target Gsk-3beta. In six of nine human MB cell lines IGF-II displayed a growth-promoting effect that was mediated mainly through the IGF-I receptor. Together, our data point to an important role of IGF-II for the proliferation control of both cerebellar neural precursors and MB cells.

Nuclear Exclusion of TET1 Is Associated with Loss of 5-Hydroxymethylcytosine in IDH1 Wild-Type Gliomas

The recent identification of isocitrate dehydrogenase 1 (IDH1) gene mutations in gliomas stimulated various studies to explore the molecular consequences and the clinical implications of such alterations. The Cancer Genome Atlas Research Network showed evidence for a CpG island methylator phenotype in glioblastomas that was associated with IDH1 mutations. These alterations were associated with the production of the oncometabolite, 2-hydroxyglutarate, that inhibits oxygenases [ie, ten-eleven translocation (TET) enzymes involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC)]. We investigated 60 gliomas for 5hmC presence, 5-methylcytosine content, TET1 expression, and IDH1 mutation to gain insight into their relationships on a histological level. Of gliomas, 61% revealed no immunoreactivity for 5hmC, and no correlation was observed between IDH1 mutations and loss of 5hmC. Interestingly, expression of TET1 showed remarkable differences regarding overall protein levels and subcellular localization. We found a highly significant (P = 0.0007) correlation between IDH1 mutations and nuclear accumulation of TET1, but not with loss of 5hmC. Of 5hmC-negative gliomas, 70% showed either exclusive or dominant cytoplasmic expression, or no detectable TET1 protein (P = 0.0122). Our data suggest that the loss of 5hmC is a frequent event in gliomas, independent of IDH1 mutation, and may be influenced by the nuclear exclusion of TET1 from the nuclei of glioma cells.

Supratentorial ependymomas of childhood carry C11orf95-RELA fusions leading to pathological activation of the NF-κB signaling pathway.

Methods). This fusion was not detectable in a large series of infratentorial and spinal ependymomas including myxopapillary ependymomas. It was also not detectable in ependymoblastomas, medulloblastomas or pineoblastomas. 14/19 samples from supratentorial ependymomas expressed this novel fusion transcript resulting in an N-terminal part of C11orf95 encoding 212aa of the hypothetical 678aa protein fused to relA that is thereby uncoupled from its normal upstream regulators. The c-terminal relA part of the putative fusion protein contains almost the full relA sequence; only the first three amino acids (encoded by exon 2) are deleted in-frame. The occurrence of the fusion did not seem to be correlated to a specific histology, although several but not all cases showed clear cell morphology, and the fusionpositive cases showed no predominant location within the supratentorium (supplementary Table 1). Interestingly, all five reLA fusion-negative samples were from female patients (two-tailed Fisher’s exact test, p = 0.03). The physiological function of C11orf95 is unclear. It has been described as a fusion partner of the gene coding the transcription factor Mkl2 in benign chondroid lipomas representing the molecular correlate of the t(11;16) (q13;p13) translocation found in these tumors [3, 5]. The breakpoint in chondroid lipomas is at exon 5, whereas it is at exon 2 in supratentorial ependymomas. RELA encodes relA (NF-κB3), a 65-kDa protein which interacts with IκB and p50 in the central signaling complex in the NF-κB pathway. After activation of cell surface receptors the signal is transmitted by the IKK complex that phosphorylates IκB and thereby controls the translocation of relA/p50 into the nucleus and transcription of specific target genes (reviewed in [4]). The key physiological function of NF-κB signaling is the orchestration of the inflammatory responses to both Clinical observations and studies on genetic alterations and gene expression indicated that supratentorial ependymomas differ from ependymomas of infratentorial or spinal location [6, 8]. To further elucidate the pathogenesis of supratentorial ependymomas, we performed paired-end rNA sequencing in 19 tumor samples from 18 patients (for clinical data, see supplementary Table 1). Mapping of the reads predicted a novel recurrent fusion mrNA between C11orf95, a gene with unknown function, and v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) encoding the relA p65 subunit of the central NF-κB complex. RELA is located approximately 1.9 Mbp telomeric from C11orf95 on the same chromosomal band 11q13. We confirmed the fusion by sanger sequencing of the cDNA (Fig. 1a; primer sequences, see supplementary

Mutations and elevated transcriptional activity of conductin (AXIN2) in hepatoblastomas

Hepatoblastoma (HB) is the most frequent malignant liver tumour of childhood. Most HBs develop sporadically but their incidence is highly elevated in patients with familial adenomatous polyposis coli (FAP). These patients carry germline mutations in the adenomatous polyposis coli (APC) tumour suppressor gene. APC forms a multi‐protein complex involved in the WNT signalling pathway that controls the stability of β‐catenin, the central effector in this cascade. Whereas APC mutations are rare in sporadic HBs, a high frequency of β‐catenin mutations leading to overactivation of WNT signalling was previously found in these tumours. This pathway is negatively controlled by conductin (axin2), representing a further partner in this signalling complex. To investigate whether alterations in conductin may also be involved in the pathogenesis of sporadic HBs, 37 HBs and five HB cell lines were screened for mutations using single‐strand conformation polymorphism (SSCP) analysis, reverse transcription‐polymerase chain reaction (RT‐PCR), and direct sequencing. In two cases, larger deletions (52 and 1624 bp) leading to frameshifts were found. In addition, one HB carried a somatic point mutation. Expression analysis by competitive RT‐PCR in HBs revealed up‐regulation of conductin mRNA compared with adjacent liver samples. This mRNA overexpression resulted in increased conductin protein levels demonstrated by western blot analysis. Tumours with activating β‐catenin mutations revealed higher levels of conductin mRNA transcripts. This finding indicates that conductin is a direct target gene of WNT signalling in HBs, as has been demonstrated in other tissues. In summary, conductin mutations may represent an alternative mechanism leading to activation of WNT signalling in HBs. The overexpression of conductin mRNA in HBs reflects activation of the WNT pathway because conductin represents a target gene of WNT signalling in liver tissue. Copyright

FGFR1 mutations in Rosette-forming glioneuronal tumors of the fourth ventricle.

Rosette-forming glioneuronal tumors (RGNTs) are rare glioneuronal tumors of the fourth ventricle region that preferentially affect young adults. Despite their histologic similarity with pilocytic astrocytomas (PAs), RGNTs do not harbor KIAA1549-BRAF fusions or BRAF mutations, which represent the most common genetic alteration in PAs. Recently, mutations affecting the hotspot codons Asn546 and Lys656 of fibroblast growth factor receptor 1 (FGFR1) have been described in PAs. They are considered to be the most frequent mechanism of mitogen-activated protein kinase activation, alternative to KIAA1549-BRAF fusion and BRAF mutations. To uncover possible molecular similarities between RGNTs and PAs, we performed a mutational study of FGFR1 in 8 RGNTs. An FGFR1 N546K mutation and an FGFR1 K656E mutation were found in the tumors of 2 patients. Notably, the patient with an FGFR1 K656E mutated RGNT had undergone a resection of a diencephalic pilocytic astrocytoma with pilomyxoid features 5 years before the discovery of the fourth ventricle tumor; the mutational analysis uncovered the presence of the same FGFR1 K656E mutation in the diencephalic tumor. These results indicate that, in addition to histologic similarities, at least a subgroup of RGNTs may show close molecular relationships with PAs. Whether FGFR1 mutated RGNTs represent a specific subset of this rare tumor entity remains to be determined.

GNA11 and N-RAS mutations: alternatives for MAPK pathway activating GNAQ mutations in primary melanocytic tumours of the central nervous system.

M. Gessi, J. Hammes, L. Lauriola, E. Dörner, J. Kirfel, G. Kristiansen, A. zur Muehlen, D. Denkhaus, A. Waha and T. Pietsch (2013) Neuropathology and Applied Neurobiology39, 417–425

Genetic Analysis of Diffuse High-Grade Astrocytomas in Infancy Defines a Novel Molecular Entity

Pediatric high‐grade gliomas are considered to be different when compared to adult high‐grade gliomas in their pathogenesis and biological behavior. Recently, common genetic alterations, including mutations in the H3F3A/ATRX/DAXX pathway, have been described in approximately 30% of the pediatric cases. However, only few cases of infant high‐grade gliomas have been analyzed so far. We investigated the molecular features of 35 infants with diffuse high‐grade astrocytomas, including 8 anaplastic astrocytomas [World Health Organization (WHO) grade III] and 27 glioblastomas (WHO grade IV) by immunohistochemistry, multiplex ligation probe‐dependent amplification (MLPA), pyrosequencing of glioma‐associated genes and molecular inversion probe (MIP) assay. MIP and MLPA analyses showed that chromosomal alterations are significantly less frequent in infants compared with high‐grade gliomas in older children and adults. We only identified H3F3A K27M in 2 of 34 cases (5.9%), with both tumors located in the posterior fossa. PDGFRA amplifications were absent, and CDKN2A loss could be observed only in two cases. Conversely, 1q gain (22.7%) and 6q loss (18.2%) were identified in a subgroup of tumors. Loss of SNORD located on chromosome 14q32 was observed in 27.3% of the infant tumors, a focal copy number change not previously described in gliomas. Our findings indicate that infant high‐grade gliomas appear to represent a distinct genetic entity suggesting a different pathogenesis and biological behavior.

PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway in papillary tumors of the pineal region.

Papillary tumors of the pineal region (PTPR) are recognized as a distinct entity in the World Health Organization classification of CNS tumors. Papillary tumors of the pineal region frequently show loss of chromosome 10, but no studies have investigated possible target genes on this chromosome. Chromosome 10 harbors the PTEN (phosphatase and tensin homolog) gene, the inactivation of which, by mutation or epigenetic silencing, has been observed in different brain tumors, including high-grade gliomas. In this study, we investigated copy number changes by molecular inversion probe (MIP) analysis and the mutational status of PTEN in 13 PTPR by direct sequencing. MIP analysis of 5 PTPR showed chromosome 10 loss in all cases. In addition, there were losses of chromosomes 3, 14, 22, and X, and gains of whole chromosomes 8, 9, and 12 in more than 1 case. One case had a homozygous PTEN deletion; and 2 point mutations in exon 7 of PTEN (G251D and Q261stop) were found. Immunohistochemistry revealed decrease or loss of the PTEN protein and increased expression of p-Akt and p-S6. These results indicated that PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway may play a role in the biology of PTPR. This evidence may lead to the possible use of PI3K/Akt/mTOR inhibitors in therapy for patients with PTPR.

Low level of microsatellite instability in paediatric malignant astrocytomas.

Aim: Microsatellite instability (MSI) has been proposed as a possible mechanism in the development of cancer. The aim of the current study was to determine whether MSI is involved in the pathogenesis of paediatric malignant astrocytomas. Methods: We screened a cohort of 126 high‐grade astrocytoma samples for MSI using a sensitive and precise method of DNA analysis including a panel of five mononucleotide repeats, in combination with immunohistochemistry for DNA mismatch repair (MMR) proteins. Results: We identified low level of MSI (MSI‐L) in four of 126 (3.2%) paediatric malignant astrocytic tumours. To analyse the molecular profile associated with MSI‐L positive tumours, we performed immunohistochemistry for protein expression of hMSH6 and p53 as well as mutational analysis of the K‐ras gene. In MSI‐L paediatric malignant astrocytic tumours we detected retained nuclear expression of hMSH6 protein and strong nuclear accumulation of p53 protein indicating possible mutations of TP53. There was no correlation between K‐ras mutational status and frequency of MSI in this patient population. Conclusion: Our results suggest that the MSI‐L phenotype is associated with p53 accumulation and/or mutations. However, this represents only a small subgroup of paediatric gliomas with possible distinct biological features, and the deficiencies of DNA MMR genes do not play a main role in the tumourigenesis of the majority of paediatric malignant astrocytomas.