Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes.

Astroblastomas (ABs) are rare brain tumors of unknown origin. We performed an integrative genetic and epigenetic analysis of AB-like tumors. Here, we show that tumors traceable to neural stem/progenitor cells (radial glia) that emerge during early to later brain development occur in children and young adults, respectively. Tumors with MN1-BEND2 fusion appear to present exclusively in females and exhibit overexpression of genes expressed prior to 25 post-conception weeks (pcw), including genes enriched in early ventricular zone radial glia and ependymal tumors. Other, histologically classic ABs overexpress or harbor mutations of mitogen-activated protein kinase pathway genes, outer and truncated radial glia genes, and genes expressed after 25 pcw, including neuronal and astrocyte markers. Findings support that AB-like tumors arise in the context of epigenetic and genetic changes in neural progenitors. Selective gene fusion, variable imprinting and/or chromosome X-inactivation escape resulting in biallelic overexpression may contribute to female predominance of AB molecular subtypes.

Cytotoxic synergy between alisertib and carboplatin versus alisertib and irinotecan are inversely dependent on MGMT levels in glioblastoma cells.

INTRODUCTION: Glioblastoma remains difficult to treat and patients whose tumors express high levels of O6-methylguanine DNA methyltransferase (MGMT) usually respond poorly to standard temozolomide chemotherapy. We have previously shown that the selective AURKA inhibitor alisertib potently inhibits growth of glioblastoma cells. METHODS: We used colony formation assays, annexin V binding, and western blotting to examine the effects of alisertib on the antiproliferative capabilities of carboplatin and irinotecan in glioblastoma cells. RESULTS: In colony formation assays, alisertib potentiated the antiproliferative effects of both carboplatin and irinotecan, often synergistically, including against glioblastoma tumor stem-like cells, as demonstrated by Chou-Talalay and Bliss statistical analyses. Western blotting showed that high MGMT expression in cell lines correlated with more pronounced potentiation of carboplatin's growth inhibitory effects by alisertib, while low MGMT expression correlated with stronger potentiation of irinotecan by alisertib. This pattern was also observed when these drug combinations were tested for their ability to induce apoptosis via annexin V binding assays. MGMT knockdown increased apoptosis caused by combined alisertib and irinotecan, while exogenous MGMT overexpression increased apoptosis from alisertib and carboplatin combination treatment. CONCLUSIONS: These results suggest that tumor MGMT expression levels may be predictive of patient response to these drug combinations, and importantly that the combination of alisertib and carboplatin may be selectively effective in glioblastoma patients with high tumor MGMT who are resistant to standard therapy. Since clinical experience with alisertib, carboplatin and irinotecan as single agents already exists, these findings may provide rationale for the design of clinical trials for their use in combination treatment regimens.

Genomic analysis demonstrates that histologically-defined astroblastomas are molecularly heterogeneous and that tumors with MN1 rearrangement exhibit the most favorable prognosis.

Astroblastoma (AB) is a rare CNS tumor demonstrating abundant astroblastomatous pseudorosettes. Its molecular features have not been comprehensively studied and its status as a tumor entity is controversial. We analyzed a cohort of 27 histologically-defined ABs using DNA methylation profiling, copy number analysis, FISH and site-directed sequencing. Most cases demonstrated mutually exclusive MN1 rearrangements (n = 10) or BRAFV600E mutations (n = 7). Two additional cases harbored RELA rearrangements. Other cases lacked these specific genetic alterations (n = 8). By DNA methylation profiling, tumors with MN1 or RELA rearrangement clustered with high-grade neuroepithelial tumor with MN1 alteration (HGNET-MN1) and RELA-fusion ependymoma, respectively. In contrast, BRAFV600E-mutant tumors grouped with pleomorphic xanthoastrocytoma (PXA). Six additional tumors clustered with either supratentorial pilocytic astrocytoma and ganglioglioma (LGG-PA/GG-ST), normal or reactive cerebrum, or with no defined DNA methylation class. While certain histologic features favored one genetic group over another, no group could be reliably distinguished by histopathology alone. Survival analysis between genetic AB subtypes was limited by sample size, but showed that MN1-rearranged AB tumors were characterized by better overall survival compared to other genetic subtypes, in fact, significantly better than BRAFV600E-mutant tumors (P = 0.013). Our data confirm that histologically-defined ABs are molecularly heterogeneous and do not represent a single entity. They rather encompass several low- to higher-grade glial tumors including neuroepithelial tumors with MN1 rearrangement, PXA-like tumors, RELA ependymomas, and possibly yet uncharacterized lesions. Genetic subtyping of tumors exhibiting AB histology, particularly determination of MN1 and BRAFV600E status, is necessary for important prognostic and possible treatment implications.

The CNS penetrating taxane TPI 287 and the AURKA inhibitor alisertib induce synergistic apoptosis in glioblastoma cells.

Glioblastoma is a highly malignant disease in critical need of expanded treatment options. The AURKA inhibitor alisertib exhibits antiproliferative activity against glioblastoma in vitro and in vivo. Unlike current clinically used taxane drugs, the novel taxane TPI 287 penetrates the CNS. We tested for interactions between three selective AURKA inhibitors and TPI 287 against standard U87 and U1242 cells and primary glioblastoma neurospheres using colony formation assays. Bliss and Chou-Talalay analyses were utilized to statistically test for synergism. Morphological analysis, flow cytometry and annexin V binding were employed to examine cell cycle and apoptotic effects of these drug combinations. TPI 287 not only potentiated the cytotoxicity of the AURKA inhibitors alisertib, MLN8054 and TC-A2317, but was often potently synergistic. Morphologic and biochemical analysis of the combined effects of alisertib and TPI 287 consistently revealed synergistic induction of apoptosis. While each agent alone induces a mitotic block, slippage occurs allowing some tumor cells to avoid apoptosis. Combination treatment greatly attenuated mitotic slippage, committing the majority of cells to apoptosis. Alisertib and TPI 287 demonstrate significant synergism against glioblastoma cells largely attributable to a synergistic effect in inducing apoptosis. These results provide compelling rationale for clinical testing of alisertib and/or other AURKA inhibitors for potential combination use with TPI 287 against glioblastoma and other CNS neoplasms.

Morphological and molecular features of astroblastoma, including BRAFV600E mutations, suggest an ontological relationship to other cortical-based gliomas of children and young adults.

BACKGROUND: Astroblastomas (ABs) are rare glial tumors showing overlapping features with astrocytomas, ependymomas, and sometimes other glial neoplasms, and may be challenging to diagnose. METHODS: We examined clinical, histopathological, and molecular features in 28 archival formalin-fixed, paraffin-embedded AB cases and performed survival analyses using Cox proportional hazards and Kaplan-Meier methods. RESULTS: Unlike ependymomas and angiocentric gliomas, ABs demonstrate abundant distinctive astroblastic pseudorosettes and are usually Olig2 immunopositive. They also frequently exhibit rhabdoid cells, multinucleated cells, and eosinophilic granular material. They retain immunoreactivity to alpha thalassemia/mental retardation syndrome X-linked, are immunonegative to isocitrate dehydrogenase-1 R132H mutation, and only occasionally show MGMT promoter hypermethylation differentiating them from many diffuse gliomas. Like pleomorphic xanthoastrocytoma, ganglioglioma, supratentorial pilocytic astrocytoma, and other predominantly cortical-based glial tumors, ABs often harbor the BRAFV600E mutation, present in 38% of cases tested (n = 21), further distinguishing those tumors from ependymomas and angiocentric gliomas. Factors correlating with longer patient survival included age less than 30 years, female gender, absent BRAFV600E , and mitotic index less than 5 mitoses/10 high-power fields; however, only the latter was significant by Cox and Kaplan-Meier analyses (n = 24; P = .024 and .012, respectively). This mitotic cutoff is therefore currently the best criterion to stratify tumors into low-grade ABs and higher-grade anaplastic ABs. CONCLUSIONS: In addition to their own characteristic histological features, ABs share some molecular and histological findings with other, possibly ontologically related, cortical-based gliomas of mostly children and young adults. Importantly, the presence of BRAFV600E mutations in a subset of ABs suggests potential clinical utility of targeted anti-BRAF therapy.

Primary Meningeal Pleomorphic Xanthoastrocytoma With Anaplastic Features: A Report of 2 Cases, One With BRAF(V600E) Mutation and Clinical Response to the BRAF Inhibitor Dabrafenib.

Primary meningeal gliomas are rare tumors composed of a heterogeneous group of neoplasms. We present 2 clinically aggressive cases of primary meningeal pleomorphic xanthoastrocytoma that clinically mimicked meningioma. One case presented in the posterior fossa of a 56-year-old woman; the other centered on the left operculum of a 35-year-old woman. These cases showed many of the classic features of pleomorphic xanthoastrocytoma, except that xanthomatous cells were rare and eosinophilic granular bodies were inconspicuous. Both cases exhibited high proliferative indices and superficially invaded the brain. One case harboring a BRAF mutation disseminated to the thecal sac and showed a clinical response to the targeted BRAF inhibitor dabrafenib. These cases seem to represent an unusual primarily extra-axial presentation of pleomorphic xanthoastrocytoma and may account for at least some of the previously reported cases of primary meningeal glioma and/or glial fibrillary acidic protein-immunoreactive meningioma variants. We suggest that BRAF mutation analysis be considered in all meningeal lesions showing atypical histologic or immunohistochemical profiles, particularly those exhibiting glial differentiation, as a diagnostic aid and possible indication for targeted therapy.

Identification of medium-sized copy number alterations in whole-genome sequencing.

The genome-wide discoveries such as detection of copy number alterations (CNA) from high-throughput whole-genome sequencing data enabled new developments in personalized medicine. The CNAs have been reported to be associated with various diseases and cancers including acute myeloid leukemia. However, there are multiple challenges to the use of current CNA detection tools that lead to high false-positive rates and thus impede widespread use of such tools in cancer research. In this paper, we discuss these issues and propose possible solutions. First, since the entire genome cannot be mapped due to some regions lacking sequence uniqueness, current methods cannot be appropriately adjusted to handle these regions in the analyses. Thus, detection of medium-sized CNAs is also being directly affected by these mappability problems. The requirement for matching control samples is also an important limitation because acquiring matching controls might not be possible or might not be cost efficient. Here we present an approach that addresses these issues and detects medium-sized CNAs in cancer genomes by (1) masking unmappable regions during the initial CNA detection phase, (2) using pool of a few normal samples as control, and (3) employing median filtering to adjust CNA ratios to its surrounding coverage and eliminate false positives.

The vitamin K oxidoreductase is a multimer that efficiently reduces vitamin K epoxide to hydroquinone to allow vitamin K-dependent protein carboxylation.

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH2). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH2, and recent studies have called into question whether VKORC1 reduces K to KH2. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH2 production. Direct detection of the vitamin K reaction products is confounded by KH2 oxidation, and we therefore developed a new assay that stabilized KH2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH2 reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously.

Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation.

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membrane-embedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.