MGMT CpG island is invariably methylated in adult astrocytic and oligodendroglial tumors with IDH1 or IDH2 mutations.

We have previously identified a region containing 16 CpGs within the MGMT CpG islands which is critical for the transcriptional control of MGMT (Malley, Acta Neuropathol 2011). To investigate the patterns and incidence of MGMT methylation in astrocytic and oligodendroglial tumors, we quantitatively assessed methylation at these 16 CpGs using bisulfite modification followed by pyrosequencing of 362 gliomas not treated with temozolomide, and correlated the findings with previously identified patterns of genetic abnormalities, patients' age and survival. The MGMT gene was considered to be methylated when the mean methylation of the 16 CpGs was 10% or higher. This cut-off value distinguished diffuse astrocytomas with high and low MGMT expression. Within each tumor type, the patterns of methylation were highly variable and also highly heterogeneous across the 16 CpGs. A high incidence of MGMT methylation was observed in all subtypes of gliomas included in this study. Among a subset of 97 tumors where conventional methylation-specific PCR (MSP) was also applied, methylation was detected by both methods in 54 tumors, while the pyrosequencing results identified a further 17 tumors. No additional cases were found using MSP alone, indicating that pyrosequencing is a robust method for methylation analysis. All tumors with IDH1/IDH2 mutations except two had MGMT methylation, while there were many tumors with MGMT methylation, particularly primary glioblastomas, which had no mutations of IDH1/2. We suggest that MGMT methylation may be one of the earliest events in the development of astrocytic and oligodendroglial tumors.

BCR and TLR signaling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas.

The genetics and pathogenesis of splenic marginal zone lymphoma are poorly understood. The lymphoma lacks chromosome translocation, and approximately 30% of cases are featured by 7q deletion, but the gene targeted by the deletion is unknown. A recent study showed inactivation of A20, a "global" NF-κB negative regulator, in 1 of 12 splenic marginal zone lymphomas. To investigate further whether deregulation of the NF-κB pathway plays a role in the pathogenesis of splenic marginal zone lymphoma, we screened several NF-κB regulators for genetic changes by PCR and sequencing. Somatic mutations were found in A20 (6/46=13%), MYD88 (6/46=13%), CARD11 (3/34=8.8%), but not in CD79A, CD79B and ABIN1. Interestingly, these genetic changes are largely mutually exclusive from each other and MYD88 mutation was also mutually exclusive from 7q deletion. These results strongly suggest that deregulation of the TLR (toll like receptor) and BCR (B-cell receptor) signaling pathway may play an important role in the pathogenesis of splenic marginal zone lymphoma.

A distinct region of the MGMT CpG island critical for transcriptional regulation is preferentially methylated in glioblastoma cells and xenografts.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl DNA adducts such as those induced by alkylating agents. Loss of MGMT expression through transcriptional silencing by hypermethylation of its CpG island (CGI) is found in diverse human cancers including glioblastomas. Glioblastomas that have MGMT methylation respond to temozolomide, an alkylating agent, resulting in improved survival. Consequently, assessment of MGMT methylation has become a therapy response and prognostic indicator. However, it is not clear whether the region of the MGMT CGI commonly analysed is the critical region involved in transcriptional control. We measured methylation levels at each CpG site for the entire MGMT CGI using bisulfite modification and pyrosequencing, and compared them with MGMT mRNA expression in glioblastoma cell lines, xenografts and normal brain tissues (41 samples). Two critical regions were identified (DMR1 and DMR2). DMR2 encompasses the commonly analysed region and was always methylated when DMR1 was methylated. A luciferase reporter assay showed that substitutions of several specific CpG sites within DMR2 significantly attenuated the promoter activity of the MGMT CGI. Our results indicate that several CpG sites within DMR2 play a critical role in the transcriptional control of MGMT, making DMR2 the optimal target for methylation testing. However, given the highly variable patterns of MGMT methylation associated with transcriptional silencing observed in this region among the tumours in this study, methylation levels need to be measured at a number of individual CpGs within DMR2 to confidently predict transcriptional silencing and thus sensitivity to alkylating agents.

IDH1 mutations are present in the majority of common adult gliomas but rare in primary glioblastomas.

We screened exon 4 of the gene isocitrate dehydrogenase 1 (NADP+), soluble (IDH1) for mutations in 596 primary intracranial tumors of all major types. Codon 132 mutation was seen in 54% of astrocytomas and 65% of oligodendroglial tumors but in only 6% of glioblastomas (3% of primary and 50% of secondary glioblastomas). There were no mutations in any other type of tumor studied. While mutations in the tumor protein p53 gene (TP53) and total 1p/19q deletions were mutually exclusive, IDH1 mutations were strongly correlated with these genetic abnormalities. All four types of mutant IDH1 proteins showed decreased enzymatic activity. The data indicate that IDH1 mutation combined with either TP53 mutation or total 1p/19q loss is a frequent and early change in the majority of oligodendroglial tumors, diffuse astrocytomas, anaplastic astrocytomas, and secondary glioblastomas but not in primary glioblastomas.

The prognosis of MYC translocation positive diffuse large B-cell lymphoma depends on the second hit.

A proportion of MYC translocation positive diffuse large B-cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double-hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double-hit DLBCL, and whether there is a difference in clinical outcome between the double-hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81 = 74%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R-CHOP (n = 67), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double-hits had the worst overall survival, followed by those with MYC/BCL2 double-hits. In MYC translocation negative DLBCL treated by R-CHOP (n = 101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double-hit DLBCLs from those with an isolated MYC translocation.

Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas.

Brain tumors are the most common solid tumors of childhood, and pilocytic astrocytomas (PA) are the most common central nervous system tumor in 5 to 19 year olds. Little is known about the genetic alterations underlying their development. Here, we describe a tandem duplication of approximately 2 Mb at 7q34 occurring in 66% of PAs. This rearrangement, which was not observed in a series of 244 higher-grade astrocytomas, results in an in-frame fusion gene incorporating the kinase domain of the BRAF oncogene. We further show that the resulting fusion protein has constitutive BRAF kinase activity and is able to transform NIH3T3 cells. This is the first report of BRAF activation through rearrangement as a frequent feature in a sporadic tumor. The frequency and specificity of this change underline its potential both as a therapeutic target and as a diagnostic tool.

A small family of sushi-class retrotransposon-derived genes in mammals and their relation to genomic imprinting.

Ty3/gypsy retrotransposons are rare in mammalian genomes despite their abundance in invertebrate and other vertebrate classes. Here we identify a family of nine conserved mammalian genes with homology to Ty3/gypsy retrotransposons but which have lost their ability to autonomously retrotranspose. Of these, five map to the X chromosome while the remaining four are autosomal. Comparative phylogenetic analyses show them to have strongest homology to the sushi-ichi element from Fugu rubripes. Two of the autosomal gene members, Peg10 and Rtl1, are known to be imprinted, being expressed from the paternally inherited chromosome homologue. This suggests, consistent with the host-parasite response theory of the evolution of the imprinting mechanism, that parental-origin specific epigenetic control may be mediated by genomic "parasitic" elements such as these. Alternatively, these elements may preferentially integrate into regions that are differentially modified on the two homologous chromosomes such as imprinted domains and the X chromosome and acquire monoallelic expression. We assess the imprinting status of the remaining autosomal members of this family and show them to be biallelically expressed in embryo and placenta. Furthermore, the methylation status of Rtl1 was assayed throughout development and was found to resemble that of actively, silenced repetitive elements rather than imprinted sequences. This indicates that the ability to undergo genomic imprinting is not an inherent property of all members of this family of retroelements. Nonetheless, the conservation but functional divergence between the different members suggests that they have undergone positive selection and acquired distinct endogenous functions within their mammalian hosts.