Alpha Thalassemia/mental Retardation Syndrome X-linked Research Articles

MGMT gene promoter methylation and its correlation with clinicopathological parameters in glioblastomas.

MGMT (O6-methyl guanine DNA methyl transferase) promoter hypermethylation is a prognostic and predictive biomarker for glioblastomas (GBM). To evaluate the frequency of MGMT methylation status in a single institute series of 134 GBMs and correlate it with clinical (age, sex, location, survival) and other molecular parameters [such as p53 expression, alpha thalassemia/mental retardation syndrome X-linked (ATRX) expression, isocitrate dehydrogenase (IDH) 1R132H mutation, and epidermal growth factor receptor (EGFR) gene amplification]. One hundred and thirty-four GBMs were evaluated by methylation-specific polymerase chain reaction (MSP) for MGMT promoter methylation status. The results were correlated with the above mentioned clinicopathological parameters. MGMT gene promoter methylation was identified in 49.2% (66/134) GBMs, and was significantly associated with IDH1R132H mutation (14/66; 21%; P - value, 0.01) and ATRX loss (15/66; 23%; P - value, 0.01). Confluent necrosis was found to be significantly associated with MGMT unmethylation status (P - value: 0.002). Multivariable logistic regression analysis showed confluent necrosis as a single independent predictor (odds ratio [OR], 2.5; confidence interval [CI], 1.0-5.8; P - value, 0.04) of MGMT unmethylation status among all the parameters studied. The frequency of MGMT promoter methylation in GBMs was 49.2%, which was significantly associated with IDHR132H mutation and ATRX loss. In addition, the presence of confluent necrosis was significantly associated with MGMT unmethylation and was found to be an independent predictor of the same.

TMOD-45. DEFINING THE ROLE OF MUTANT IDH IN GLIOMA INITIATION

Gliomas are the most common primary central nervous system tumor but the molecular mechanisms responsible for their development and progression are not completely understood. Genomic analyses identified a mutation at codon 132 in the isocitrate dehydrogenase 1 (IDH1) gene in ~80% of grade II-III gliomas. In patient tumors, IDH1R132H mutations co-exist with TP53 mutations and inactivating alterations of alpha thalassemia/mental retardation syndrome X-linked (ATRX). Some of these tumors also have amplification of loci encoding platelet-derived growth factor receptor A (PDGFRA), cyclin dependent kinase 4 (CDK4), and MYC. Loss of the chromosomal region surrounding phosphatase and tensin homolog deleted on chromosome ten (PTEN) is associated with the progression of low grade glioma to grade IV glioblastoma. The functional requirement of these alterations for glioma initiation has previously been hampered by difficulties in generating autochthonous mouse models harboring mutant IDH. In this study, we used the RCAS/TVA glioma mouse model to evaluate the effect of these clinically-relevant genetic alterations on the growth of astrocytes in vitro and glioma development in vivo. In the context of mutant IDH1 expression, increased proliferation and anchorage-independent growth was observed in astrocytes derived from these mice, relative to wild type IDH-expressing cells. Similar to patient tumors, mutant IDH elevated intracellular (R)-2-hydroxyglutarate (2-HG) levels and reduced NADPH, which results from the conversion of α-KG to 2-HG. Postnatal delivery of IDH1R132H to Nestin-expressing cells in vivo initiated neoplasms that resembled human gliomas harboring mutant IDH1, both genetically and histologically. These results functionally validate the genomic alterations observed in the human disease and suggest that mutant IDH is a bona fide glioma oncogene. This model will aid in furthering our understanding of the biology of mutant IDH1-driven gliomas and is ideally suited for assessing rational therapeutic strategies designed to combat this deadly disease.

ALK Protein Expression Is Related to Neuroblastoma Aggressiveness But Is Not Independent Prognostic Factor.

PurposeIn this study, anaplastic lymphoma kinase (ALK) mutation and amplification, ALK protein expression, loss of the nuclear alpha thalassemia/mental retardation syndrome X-linked (ATRX) protein, and telomerase reverse transcriptase (TERT) protein expressionwere studied to investigate potential correlations between these molecular characteristics and clinical features or outcomes in neuroblastoma.Materials and MethodsSeventy-two patients were enrolled in this study. Polymerase chain reaction amplification and direct sequencing were used for mutation analysis. ALK and MYCN amplifications were detected by fluorescence in situ hybridization. Protein expressionwas evaluated by immunohistochemical (IHC) staining.ResultsALK mutation was found in only two patients (4.1%); ALK amplification was not detected. ALK positivity, loss of nuclear ATRX protein, TERT positivity by IHC were detected in 40 (55.6%), nine (13.0%), and 42 (59.2%) patients, respectively. The incidence of ALK expression increased in accordance with increasing tumor stage (p=0.001) and risk group (p < 0.001). The relapse rate was significantly higher in ALK+ patients compared to that of other patients (47.5% vs. 11.3%, p=0.007). However, there was no significant difference in relapse rate when the survival analysis was confined to the high-risk patients.ConclusionAlthough ALK mutation was rare and no amplification was observed, ALK protein expression was found in a significant number of patients and was correlated with advanced stage and high-risk neuroblastoma. ALK protein expression could be considered as a marker related to the aggressive neuroblastoma, but it was not the independent prognostic factor for the outcome.

New Molecular Considerations for Glioma: IDH, ATRX, BRAF, TERT, H3 K27M

This review will discuss the role of several key players in glioma classification and biology, namely isocitrate dehydrogenase 1 and 2 (IDH1/2), alpha thalassemia/mental retardation syndrome X-linked (ATRX), B-Raf (BRAF), telomerase reverse transcriptase (TERT), and H3K27M. IDH1/2 mutation delineates oligoden-droglioma, astrocytoma, and secondary glioblastoma (GBM) from primary GBM and lower-grade gliomas with biology similar to GBM. Additional mutations including TERT, 1p/19q, and ATRX further guide glioma classification and diagnosis, as well as pointing directions toward individualized treatments for these distinct molecular subtypes. ATRX and TERT mutations suggest the importance of telomere maintenance in gliomagenesis. BRAF alterations are key in certain low-grade gliomas and pediatric gliomas but rarely in high-grade gliomas in adults. Histone mutations (e.g., H3K27M) and their effect on chromatin modulation are novel mechanisms of cancer generation and uniquely seen in midline gliomas in children and young adults. Over the past decade, a remarkable accumulation of knowledge from the genomic study of gliomas has led to reclassification of tumors, new understanding of oncogenic mechanisms, and novel treatment strategies.

The Role of ATRX in the Alternative Lengthening of Telomeres (ALT) Phenotype.

Telomeres are responsible for protecting chromosome ends in order to prevent the loss of coding DNA. Their maintenance is required for achieving immortality by neoplastic cells and can occur by upregulation of the telomerase enzyme or through a homologous recombination-associated process, the alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor cells are not fully understood, although cellular origin may favor one of the other mechanisms that have been found thus far in mutual exclusivity. Specific mutational events influence ALT activation and maintenance: a unifying frequent feature of tumors that acquire this phenotype are the recurrent mutations of the Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) or Death-Domain Associated Protein (DAXX) genes. This review summarizes the established criteria about this phenotype: its prevalence, theoretical molecular mechanisms and relation with ATRX, DAXX and other proteins (directly or indirectly interacting and resulting in the ALT phenotype).

In vivo molecular profiling of human glioma using diffusion kurtosis imaging.

The purpose of this study is to assess the diagnostic performance of diffusion kurtosis imaging (DKI) for in vivo molecular profiling of human glioma. Normalized mean kurtosis (MKn) and mean diffusivity (MDn) metrics from DKI were assessed in 50 patients with histopathologically confirmed glioma. The results were compared in regard to the WHO-based histological findings and molecular characteristics leading to integrated diagnosis (Haarlem Consensus): isocitrate-dehydrogenase (IDH1/2) mutation status, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) expression, chromosome 1p/19q loss of heterozygosity (LOH), and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status. MKn was significantly lower in tumors with IDH1/2 mutation (0.43 ± 0.09) and ATRX loss of expression (0.41 ± 0.11) than in those with IDH1/2 wild type (0.57 ± 0.09, p < 0.001) and ATRX maintained expression (0.51 ± 0.10, p = 0.004), respectively. Regarding the integrated molecular diagnosis, MKn was significantly higher in primary glioblastoma (0.57 ± 0.10) than in astrocytoma (0.39 ± 0.11, p < 0.001) and oligodendroglioma (0.47 ± 0.05, p = 0.003). MK may be used to provide insight into the human glioma molecular profile regarding IDH1/2 mutation status and ATRX expression. Considering the diagnostic and prognostic significance of these molecular markers, MK appears to be a promising in vivo biomarker for glioma. The diagnostic performance of MK seems to fit more with the integrated molecular approach than the conventional histological findings of the current WHO 2007 classification.

A mutation-specific peptide vaccine targeting IDH1R132H in patients with newly diagnosed malignant astrocytomas: A first-in-man multicenter phase I clinical trial of the German Neurooncology Working Group (NOA-16).

TPS2082Background: Mutations in the gene for isocitrate dehydrogenase type 1 frequently occur in diffuse gliomas resulting in a point mutation (IDH1R132H) in most of the cases. Preclinical studies have shown that mutation-specific T helper (Th) cell responses spontaneously occur in patients with IDH1-mutated gliomas and that a peptide vaccine encoding IDH1R132H is therapeutic in a humanized mouse tumor model. Methods: NOA-16 (ClinicalTrials.gov Identifier: NCT02454634) is a multicenter, first-in-man, phase I clinical trial, which is planned to enroll 39 patients with newly diagnosed malignant astrocytomas with IDH1R132H mutations at eight German sites. The target population is molecularly enriched for an unfavorable prognosis within the IDH1-mutated subgroup by mandating (i) absence of co-deletion of 1p/19q and (ii) loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) expression. Patients aged ≥ 18 years with a Karnofsky performance score of ≥ 70 will receive radiotherapy only (cohort 1),...

Compromised Telomeric Heterochromatin Promotes ALTernative Lengthening of Telomeres

Alternative lengthening of telomeres (ALT) is an enigmatic process that allows certain cancers to maintain telomeres in the absence of telomerase. ALT cancers are frequently defective for ATRX/DAXX, a chaperone complex that deposits histone variant H3.3 at telomeres. We propose that mutations in alpha thalassemia-mental retardation syndrome X-linked (ATRX)/death-domain associated protein (DAXX) prime ALT activation by disrupting telomeric heterochromatin.

Immunohistochemical Analysis of ATRX, IDH1 and p53 in Glioblastoma and Their Correlations with Patient Survival.

Glioblastoma (GBM) can be classified into molecular subgroups, on the basis of biomarker expression. Here, we classified our cohort of 163 adult GBMs into molecular subgroups according to the expression of proteins encoded by genes of alpha thalassemia/mental retardation syndrome X-linked (ATRX), isocitrate dehydrogenase (IDH) and TP53. We focused on the survival rate of molecular subgroups, depending on each and various combination of these biomarkers. ATRX, IDH1 and p53 protein expression were evaluated immunohistochemically and Kaplan-Meier analysis were carried out in each group. A total of 15.3% of enrolled GBMs demonstrated loss of ATRX expression (ATRX-), 10.4% expressed an aberrant IDH1 R132H protein (IDH1+), and 48.4% exhibited p53 overexpression (p53+). Survival differences were statistically significant when single protein expression or different combinations of expression of these proteins were analyzed. In conclusion, in the case of single protein expression, the patients with each IDH1+, or ATRX-, or p53- GBMs showed better survival than patients with counterparts protein expressed GBMs. In the case of double protein pairs, the patients with ATRX-/p53-, ATRX-/IDH1+, and IDH1+/p53- GBMs revealed better survival than the patients with GBMs with the remained pairs. In the case of triple protein combinations, the patients with ATRX-/p53-/IDH+ showed statistically significant survival gain than the patients with remained combination of proteins-expression status. Therefore, these three biomarkers, individually and as a combination, can stratify GBMs into prognostically relevant subgroups and have strong prognostic values in adult GBMs.

TERT promoter mutations are a rare event in gastrointestinal stromal tumors.

Recently, the impact of telomere dysregulation on malignant progression has been reported in many cancers. A few studies have examined TERT promoter mutations in gastrointestinal stromal tumors (GISTs). Irregular telomerase activation can be maintained by TERT hot spot alterations and alternative lengthening of telomeres (ALT) characterized by inactivation of either the alpha-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain–associated protein (DAXX). To elucidate the clinicopathological impact of telomere dysregulation in GISTs, we examined 92 cases of GISTs for TERT promoter hot spot mutations along with immunohistochemical analysis of ATRX and DAXX expression, and compared these findings with the clinicopathological features. Univariate clinicopathological analysis revealed that tumor site, smaller tumor size, presence of necrosis, higher mitotic rate (>5/50 high-power fields) and risk classification were prognostic factors for either disease-free survival or overall survival. Two of 92 informative cases (2.2 %) were found to have heterozygous TERT promoter mutations (C228T), and these mutations occurred in a low-risk and a high-risk tumor, respectively. On immunohistochemical analysis for ATRX and DAXX, 16 (17.4 %) and 3 (3.3 %) of 92 cases showed loss of expression of ATRX and DAXX, respectively. Loss of expression of ATRX and DAXX were mutually exclusive except for one case. TERT promoter mutations were also mutually exclusive of the ALT phenotype. Telomere dysregulation was not associated with patient survival; however, telomere dysregulation was frequently observed in tumors of extra-gastric origin, which have an adverse outcome compared to those of gastric origin.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1606-2) contains supplementary material, which is available to authorized users.

Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes.

Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)? Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance. Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote. Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM). GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization. We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging. Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro. The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest.

MPTH-30ATRX CAN OCCUR IN IDH WILD-TYPE GBM

BACKGROUND: ATRX (alpha thalassemia/ mental retardation syndrome X-linked) is a transcriptional regulatory protein which contains an ATPase/helicase domain. In gliomas loss of ATRX expression has been recently described and is mostly restricted to IDH mutant tumors who seem to have a significantly better prognosis. ATRX loss is thought to be extremely rare in IDH wild type (WT) tumors and in that setting one group reported H3F3A mutation. METHODS: Tissue samples of 23 patients with IDH wild-type malignant gliomas were analyzed for ATRX expression using immunohistochemistry. ATRX status was associated with clinical characteristics, histology and other molecular markers (MGMT, BRAF, EGFR). RESULTS: Median age in all patients was 49,3 years. Loss of ATRX expression was detected in 4 out of 23 patient tumors. All ATRX loss patients were primary glioblastomas. Median survival in ATRX mutated gliomas is 237 weeks which was longer than overall survival in gliomas with retained ATRX (88 weeks). All of our patients underwent concurrent chemoradiotherapy with temozolomide. One out of four ATRX loss tumors was BRAF mutated, three out of four ATRX loss patients tested for EGFR were found to be wild type and the two ATRX mutated patients tested for MGMT were methylated. CONCLUSION: ATRX mutation can occur in IDH wild-type patients. In that setting it appears to be associated with a better outcome. Our data is hypothesis generating. Tumors with IDH WT/ ATRX loss should be evaluated prospectively in a larger cohort and possibly checked for H3F3A mutation.

MPTH-01ESTABLISHMENT OF A RAPID AND EFFICIENT GENETIC ANALYSIS SYSTEM OF GLIOMAS

PURPOSE: Of the genetic mutations seen in gliomas, IDH1/2 mutations, chromosome 1p/19q loss, MGMT promoter methylation, and genetic alterations of TERT promoter, BRAF, and H3F3A, have become increasingly important as biomarkers for the diagnosis, prognostication, and treatment response of gliomas. Here we report on the establishment of a system, within our institution, of the efficient and rapid detection of these genetic mutations. METHODS: Tumor-derived DNA and RNA extracted from either frozen or paraffin-embedded histological sections were used as specimens. For the point mutation assay, the following mutations of: IDH1/2, H3F3A, BRAF codon 600, and the TERT promoter were screened using High Resolution Melting (HRM) method, and identified by DNA sequencing. For the MGMT promoter methylation, a quantitative analysis was performed using the methylation-specific HRM method. Real-time PCR was used for determining the presence or absence of the BRAF-KIAA fusion gene, and FISH was used for detecting chromosome 1p/19q loss. Further, immunostaining was performed to analyze loss of ATRX (alpha-thalassemia/mental-retardation syndrome-X-linked) expression. RESULTS: Screening of each of the genetic mutations by the point mutation assay using the HRM method was completed in one run of approximately 70 minutes. Furthermore, all genetic mutation results were obtained within 5 ∼ 72 hours after nucleic acid extraction using our assay system. CONCLUSION: For clinics that specialize in diagnosis and treatment of brain tumors, the genetic biomarker information mentioned above will become indispensible in the future. Outsourcing this testing has issues relating to cost and time to obtaining results, and thus testing in-house is desirable. Utilizing PCR primer settings that allow the screening of multiple genetic mutations in one run, and simple and sensitive/specific assays such as the HRM method, allows efficient and rapid analysis.

Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells.

Mutations in ATRX (alpha thalassemia/mental retardation syndrome X-linked), a chromatin-remodeling protein, are associated with the telomerase-independent ALT (alternative lengthening of telomeres) pathway of telomere maintenance in several types of cancer, including human gliomas. In telomerase-positive glioma cells, we found by immunofluorescence that ATRX localized not far from the chromosome ends but not exactly at the telomere termini. Chromatin immunoprecipitation (ChIP) experiments confirmed a subtelomeric localization for ATRX, yet short hairpin RNA (shRNA)-mediated genetic inactivation of ATRX failed to trigger the ALT pathway. Cohesin has been recently shown to be part of telomeric chromatin. Here, using ChIP, we showed that genetic inactivation of ATRX provoked diminution in the amount of cohesin in subtelomeric regions of telomerase-positive glioma cells. Inactivation of ATRX also led to diminution in the amount of TERRAs, noncoding RNAs resulting from transcription of telomeric DNA, as well as to a decrease in RNA polymerase II (RNAP II) levels at the telomeres. Our data suggest that ATRX might establish functional interactions with cohesin on telomeric chromatin in order to control TERRA levels and that one or the other or both of these events might be relevant to the triggering of the ALT pathway in cancer cells that exhibit genetic inactivation of ATRX.

Classification of gliomas. Current progress and perspectives

The diagnostic subdivision of gliomas is traditionally based on histological features as defined by the World Health Organization (WHO) classification of tumors of the central nervous system. In recent years molecular studies have identified a number of genetic and epigenetic markers that could contribute to an improved tumor classification and better prediction of response to therapy and prognosis in the individual patient. The most important molecular tests with differential diagnostic relevance in patients with astrocytic and oligodendroglial tumors include the detection of genetic mutations in the isocitrate dehydrogenase 1 (IDH1), IDH2, alpha thalassemia/mental retardation syndrome X-linked (ATRX), histone H3.3 (H3F3A) and v-raf murine sarcoma viral oncogene homolog B (BRAF) genes as well as the demonstration of codeletions of chromosomal arms 1p and 19q. Important predictive markers that have been linked to the response to alkylating chemotherapy are O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in glioblastoma patients and 1p/19q codel status in anaplastic glioma patients. Oncogenic c11orf95/RELA fusion gene formation is characteristic for a subgroup of patients with supratentorial ependymoma. In addition to diagnostic testing of individual genes, novel microarray and next generation sequencing (NGS) techniques show promising perspectives in glioma diagnostics. The assessment of DNA methylation profiles using DNA methylation arrays representing 450,000 CpG dinucleotides distributed throughout the human genome (450k array test) now allows the robust molecular classification of gliomas into clinically relevant entities and variants. Moreover, glioma-associated gene panel NGS promises the timely parallel sequencing of relevant diagnostic and predictive marker genes in a single test. It will now be a major task to integrate these novel results and techniques into the conventional histological procedures in the up-coming revision of the WHO classification.

ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of “repressive” histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX’s ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this “methyl/phos” switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

Treatment of anaplastic glioma.

Anaplastic gliomas have received increasing attention over the past years. As opposed to glioblastoma, where the focus has been on the evaluation of novel compounds (with mainly disappointing results), in anaplastic gliomas relevant progress was generated with genotoxic therapies and translational work on biomarkers. Anaplastic gliomas are classified using single biomarkers, namely isocitrate dehydrogenase (IDH) or the related CpG island methylator phenotype (CIMP), alpha-thalassemia/mental retardation syndrome X-linked (ATRX), telomerase reverse transcriptase (TERT), p53, 1p/19q, and O(6)-methylguanine DNA-methyltransferase (MGMT). With these molecular biomarkers, three main prognostically distinct groups have been defined: (i) CIMP-negative anaplastic gliomas, which have a similar prognosis as glioblastoma, (ii) CIMP-positive 1p/19q intact, and (iii) CIMP-positive 1p/19q codeleted gliomas. In the CIMP-negative, mainly IDH wild-type group, MGMT promoter methylation may be used to identify patients who benefit from alkylating chemotherapy. The mutually exclusive ATRX losses and 1p/19q codeletions are used to subcategorize anaplastic tumors with a mixed histology according to microscopic features. This eliminates the biological basis and clinical necessity for the diagnosis of mixed gliomas (anaplastic oligoastrocytomas). Retrospective long-term analysis of the EORTC 26951 and RTOG 9402 trials revealed that patients with tumors harboring 1p/19q codeletions benefit from addition of procarbazine, lomustine, and vincristine (PCV) chemotherapy to primary radiotherapy. RTOG 9402 suggests that this may be the case also for patients with 1p/19q intact tumors, but IDH mutation. Future developments in addition to the ongoing CATNON and CODEL trials, will focus on further refinement of the molecular predictors and development of treatments that not only increase survival but also maintain neurological function, cognition, and health-related quality of life.

Ileal neuroendocrine tumors show elevated activation of mammalian target of rapamycin complex

BackgroundNeuroendocrine tumors (NETs) of the ileum are sporadic tumors derived from submucosal gastrointestinal stem cells. They often show clinical symptoms only after hepatic metastasation when curative therapy is limited or impossible. In this study, we analyzed the expression of the candidate genes mammalian target of rapamycin (mTOR), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and death domain-associated protein (DAXX) to investigate the specific oncogenetics and potential therapeutic options for ileal NETs. MethodsIn a prospective database, all patients who underwent surgical removal of a NET of the ileum between 2001 and 2011 were specified. Expression analysis was performed for mTOR, ATRX, and DAXX by immunohistochemistry of paraffin-embedded tumor samples. To evaluate the results the immunoreactive score was applied. Normal tissue and tumor tissue were analyzed for the comparison of gene expression levels using quantitative-real-time polymerase chain reaction for ATRX and mTOR genes. Results were correlated under pathologic and clinical aspects. ResultsA total of 69 patients were admitted to the study. Positive cytosolic expression of the potential oncogene mTOR was immunohistochemically detected in 76.2% of the human probes. A loss of nuclear ATRX expression was detected in 13.0% of the samples. A nonexpression of the DAXX-protein in cell nuclei was not found (0%). Gene transcript levels did not show a significant alteration in ileal NETs in comparison with normal tissue. ConclusionsmTOR is overexpressed in ileal NETs. Additionally, the loss of ATRX expression was registered, thus underlying a tumorigenic role in a subgroup of these tumors. To enable potential therapeutic application of mTOR inhibitors, further trials with larger study groups are needed.

Abstract A33: Exploring pediatric cancer genomics with the UCSC Cancer Genomics Browser

Abstract The UCSC Cancer Genomics Browser (https://genome-cancer.ucsc.edu) is a set of web-based tools to display, investigate and analyze cancer genomics data and associated clinical data. Experimental quantities such as gene expression levels, copy number variation and somatic mutations are displayed next to clinical features such as age of onset and cancer subtype. Users can interactively group or sort data by clinical features to dynamically explore how genomic aberrations relate to clinical outcomes. Integrated Kaplan–Meier plots help investigators assess how clinical or genomic values impact long-term survival. The browser currently hosts data from 144 cancer studies including TCGA, CCLE and LINCS. It can display data from multiple studies at once, facilitating cross-cancer comparisons. We are currently interested in hosting new pediatric cancer datasets, and can offer either public or protected access as appropriate. Figure 1 illustrates the power of this approach. It compares patterns of somatic mutations in pediatric high-risk neuroblastoma, a cancer of the peripheral nervous system (Pugh, Morozova et al, Nature Genetics 2013) to those in two adult cancers of the central nervous system: lower grade glioma (LGG, TCGA) and glioblastoma multiforme (GBM, TCGA), and contrasts the age of the patient with frequency of mutations in the Alpha Thalassemia/Mental Retardation Syndrome X-linked (ATRX) gene. The data for all three cancers is sorted by the age of the patient, with yellow being the oldest. The age ranges overlap: the oldest neuroblastoma patients are 16, while the youngest LGG and GBM patients are 14 and 21 respectively. While the three cancers show distinct patterns of somatic mutations, all three show frequent mutations in the ATRX gene in older teens or young adults. This suggests that these cancers may share age-related subtypes with similar genomic signatures. These subtypes may indicate the age-dependent importance of ATRX in the development of both central and peripheral nervous systems, and may ultimately highlight common therapeutic avenues for the two groups of diseases. This data set can be explored at the UCSC Cancer Genomics Browser at https://genome-cancer.ucsc.edu/proj/site/hgHeatmap/#?bookmark=pc Citation Format: Melissa Cline, Olena Morozova, Teresa Swatloski, Brian Craft, Mary Goldman, David Haussler, Jingchun Zhu. Exploring pediatric cancer genomics with the UCSC Cancer Genomics Browser. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A33.

O8.06 * ATRX AND TERT EXPRESSION IN RELAPSING LOW GRADE GLIOMA

Mechanisms responsible for telomere maintenance are been recently elucidated: many cancers harbor mutations in alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene resulting in a phenomenon known as alternative lengthening of telomeres (ALT) or point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene leading to an increased telomerase expression. ATRX and TERT mutations have been recently identified also in brain tumors. In gliomas, a molecular stratification based on IDH and ATRX mutational status and 1p/19q codeletion has been suggested. ATRX mutations have been observed mainly in astrocytic tumors and TERT promoter mutations mainly in oligodendroglial tumors. Aim of our study was to evaluate the expression of both ATRX and TERT in WHO grade II gliomas and in their resected recurrences, related to a set of confirmed molecular markers and to clinical data. We performed an immunohistochemical analysis of ATRX and TERT expression in 30 grade II gliomas (astrocytomas, oligoastrocytomas and oligodendroglioma) and in their respective recurrences (grade III-IV). Also evaluation of IDH1 and p53 status and 1p/19q LOH were carried out. Loss of expression of ATRX protein has been associated to the mutated gene and ALT phenotype, while expression of TERT protein could be related to TERT promoter mutations. We found a loss of ATRX expression in 70% of cases; recurrences showed the same pattern of ATRX expression. In the most part of cases ATRX loss was associated to IDH1 and p53 mutation and intact 1p/19q, as previously observed. In the major part of cases with ATRX loss of expression TERT was not detected. TERT high expression was observed in about 30% of grade II gliomas at the first surgery, minor expression of TERT was found in grade III-IV recurrent tumors. In most case high expression of TERT was associated to 1p/19q deletion. We observed a statistically significant correlation between TERT high expression and a better outcome. Our study of protein expression of both ATRX and TERT in low grade gliomas and in their recurrences in relationship with established molecular markers and follow-up data, confirm the possibility of using immunohistochemistry in routine diagnostic procedures for better define this subset of neoplasm.