3020

H3F3A

H3.3A|H3F3|H3F3B;H3 histone, family 3A;H3F3A;H3 histone, family 3A

histone H3.3

1q42.12

protein-coding

Eleven pediatric brain tumors were studied for the histone H3, Vimentin and MYC gene expression. H3, an S phase cell cycle-related gene (ccr), was found prevalently expressed in tumors with a high mitotic index (MI). Vimentin gene, which contributes to maintaining the cell structure but is also demonstrated to be an early responder gene to growth stimulation was found variously expressed. The different expression of Vimentin gene in the examined samples suggests the active proliferation of the tumor cells. Analysis of MYC gene expression was found increased only in a mesenchymal chondrosarcoma while in other samples MYC mRNA was undetectable. Medulloblastoma, chondrosarcoma, and choroid plexus carcinoma have high S phase H3 gene expression associated with a high MI. Differently an astrocytoma shows a low MI associated with high H3 gene expression. This first preliminary report of H3, Vimentin and MYC gene expression in brain tumors demonstrates that malignant cells are characterized by a different gene expression and different growth potentials.

pediatric glioblastomas recently have been exon sequenced with evidence that approximately 30 % of cases harbour mutations of the histone H3.3 gene. Althoughstudies to determinate their role in risk stratification are on-going, it remains to be determined whether H3.3 mutations could be found in other tumors such as pediatric primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) and whether the presence of H3.3 mutations in glioblastomas could beused as diagnostic tool in their differential diagnosis with CNS-PNETs. We performed a large mutational pyrosequencing-based screening on 123 pediatric glioblastomas and 33 CNS-PNET. The analysis revealed that 39/123 (31.7 %) glioblastomas carry H3.3 mutations. The K27M (AAG --> ATG, lysine --> methionine) mutation was found in 33 glioblastomas (26 %); the G34R (GGG --> AGG, glycine --> arginine) was observed in 6 glioblastomas (5.5 %). However, we also identified 4of 33 cases (11 %) of CNS-PNETs harbouring a H3.3 G34R mutation. Multiplex ligation-dependent probe amplification analysis revealed PDGFR-alpha amplification and EGFR gain in two cases and N-Myc amplification in one case of H3.3 G34R mutated CNS-PNET. None of H3.3 mutated tumors presented a CDKN2A loss.In conclusion, because pediatric patients with glioblastoma and CNS-PNET are treated according to different therapeutic protocols, these findings may raise further concerns about the reliability of the histological diagnosis in the caseof an undifferentiated brain tumor harbouring G34R H3.3 mutation. In this view, additional studies are needed to determine whether H3.3 G34 mutated CNS-PNET/glioblastomas may represent a defined tumor subtype.

H3F3A mutations are seen in approximately 30% of pediatric glioblastoma (GBMs) and involve either the lysine residue at position 27 (K27M) or glycine at position 34 (G34R/V). Sixteen genes encode histone H3, each variant differing inonly a few amino acids. Therefore, how mutations in a single H3 gene contribute to carcinogenesis is unknown. H3F3A K27M mutations are predicted to alter methylation of H3K27. H3K27me3 is a repressive mark critical to stem cell maintenance and is mediated by EZH2, a member of the polycomb-group (PcG) family. We evaluated H3K27me3 and EZH2 expression using immunohistochemistry in 76 pediatric brain tumors. H3K27me3 was lowered/absent in tumor cells but preservedin endothelial cells and infiltrating lymphocytes in six out of 20 GBMs. H3K27me3 showed strong immunoreactivity in ALL other tumor subtypes. Sequencing of GBMs showed H3F3A K27M mutations in ALL six cases with lowered/absent H3K27me3. EZH2 expression was high in GBMs, but absent/focal in other tumors. However, no significant differences in EZH2 expression were observed between H3F3A K27M mutant and wild type GBMs, suggesting that EZH2 mediated trimethylation of H3K27is inhibited in GBM harboring K27M mutations. Our results indicate that H3F3A K27M mutant GBMs show decreased H3K27me3 that may be of both diagnostic and biological relevance.#CI- (c) 2013 International Society of Neuropathology.

Brain tumors are one of the most common childhood malignancies. Diffuse high-grade gliomas represent approximately 10% of pediatric brain tumors. Exon sequencing has identified a mutation in K27M of the histone H3.3 gene (H3F3A K27M and G34R/V) in about 20% of pediatric glioblastomas, but it remains to be seen whether these mutations can be considered specific for pediatric diffuse high-grade astrocytomas or also occur in other pediatric brain tumors. We performed a pyrosequencing-based analysis for the identification of H3F3A codon 27 and codon 34 mutations in 338 pediatric brain tumors. The K27M mutation occurred in 35 of 129 glioblastomas (27.1%) and in 5 of 28 (17.9%) anaplastic astrocytomas. None of the other tumor entities showed H3F3A K27M mutation. Because H3F3A K27M mutations occur exclusively in pediatric diffuse high-grade astrocytomas, analysis of codon 27 mutational status could be useful in the differential diagnosis of these neoplasms.

Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60% of high-grade pediatric glioma cases. The median survival of this group of patientsafter diagnosis is approximately 1 yr. Here we show that the levels of H3K27 di-and trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient samples due to the expression of the H3.3K27M mutant allele. Remarkably,we also observed that H3K27me3 and Ezh2 (the catalytic subunit of H3K27 methyltransferase) at chromatin are dramatically increased locally at hundreds of gene loci in H3.3K27M patient cells. Moreover, the gain of H3K27me3 and Ezh2 at gene promoters alters the expression of genes that are associated with various cancer pathways. These results indicate that H3.3K27M mutation reprograms epigenetic landscape and gene expression, which may drive tumorigenesis.

Epigenetic studies include the investigation of DNA methylation, histone modifications, chromatin remodeling and gene regulation by noncoding RNAs (ncRNAs). Epigenetic alterations are critical for early developmental processes,the silencing of the inactive X-chromosome and tissue-specific gene regulation. A comprehensive picture of epigenetic patterns in normal cells is now emerging; these patterns are disturbed in human diseases such as cancer. In this review, we highlight some of the most recent advances and discoveries in the field. First, while DNA methylation is known for many years, we are just beginning to learn about novel modifications of the DNA such as 5-hydroxymethylation and the enzymes that establish and remove these marks (e.g. TET1, TET2, TET3). Furthermore, altered epigenetic patterns in diseases might be linked to recurrent mutations within enzymes required for the establishment, maintenance and editing of these patterns. Examples are mutations in the gene encoding chromatin remodeling factor SMARCB1 in rhabdoid tumors or mutations in one of the three histone H3.3-encoding genes, H3F3A, in pediatric glioblastomas. A further focus in this review will beon recent findings in the field of ncRNAs as exemplified by the long noncoding RNA CTBP1-AS involved in prostate cancer and circular RNA CDR1as which captures and negatively regulates microRNA mir-7. Finally, we will highlight some of the novel technologies that have recently emerged in the field and will help in the profiling of disease genomes by allowing the use of small cell numbers and a higher resolution.