On the Boundary of Exploratory Genomics and Translation in Sequential Glioblastoma.

OMICS methods brought significant advancements to the understanding of tumor cell biology, which transformed the treatment and prognosis of several cancers. Clinical practice and outcomes, however, changed significantly less in the case of glioblastoma (GBM). In this study, we aimed to assess the utility of whole exome (WES) sequencing in the clinical setting. Ten pairs of formalin-fixed, paraffin-embedded (FFPE) GBM specimens were obtained at onset (GBM-P) and at recurrence (GBM-R). Histopathological and molecular features of all samples supported the diagnosis of GBM based on WHO CNS5. WES data were filtered, applying a strict and custom-made pipeline, and occurrence of oncogenic and likely oncogenic variants in GBM-P, GBM-R or both were identified by using the VarSeq program version 2.5.0 (Golden Helix, Inc.). Characteristics and recurrence of the variants were analyzed in our own cohort and were also compared to those available in the COSMIC database. The lists of oncogenic and likely oncogenic variants corresponded to those identified in other studies. The average number of these variants were 4 and 5 out of all detected 24 and 34 variants in GBM-P and GBM-R samples, respectively. On average, one shared oncogenic/likely oncogenic variant was found in the pairs. We assessed the identified variants' therapeutic significance, also taking into consideration the guidelines by the Association for Molecular Pathology (AMP). Our data support that a thorough WES analysis is suitable for identifying oncogenic and likely oncogenic variants in an individual clinical sample or a small cohort of FFPE glioma specimens, which concur with those of comprehensive research studies. Such analyses also allow us to monitor molecular dynamics of sequential GBM. In addition, careful evaluation of data according to the AMP guideline reveal that though therapeutic applicability of the variants is generally limited in the clinic, such information may be valuable in selected cases, and can support innovative preclinical and clinical trials.

[Vanishing white matter disease, a rare leukodystrophy with mutation in the EIF2B5 gene]. / Eltuno fehérállomány-betegség, a leukodystrophiák ritka altípusa az EIF2B5 gén mutációjával.

Background - Leukodystrophies, a hete­ro­­ge­neous group of brain and spinal cord dis­orders, often pose challenges in es­tab­li­shing molecular etiology. Vanishing White Matter Disease (VWMD) is a rare sub­type of leu­ko­dys­trophies presenting with characteristic clinical and MRI features, ne­ver­theless, achieving diag­nostic certainty requires genetic studies.

Case presentation - Our patient is a nine year old girl, who developed progressive gait difficulties at around 3-4 years of age. Her brain MRI showed confluent lesions with in­­creased signal intensity in the cerebral and cerebellar white matter on T2/FLAIR se­quen­ces, within which hypointense regions ap­peared with signal intensity resembling that of the cerebrospinal fluid on T1 sequences. Whole exome sequencing identified a homozygous likely pathogenic variant within the EIF2B5 gene in the proband, which was present in a heterozygous state in both asymptomatic parents. Having the clinical and molecular genetic diagnosis established, we explored therapeutic possibilities for the patient.

Conclusion - VWMD is a severe form of leukodystrophies with little or no disease modifying therapy available until recently. A better understanding of its molecular pathogenesis offers some hope for new inventive therapies. 

Glioblastoma epigenomics discloses a complex biology and potential therapeutic targets.

Background and purpose:

Glioblastoma (GBM), a highly aggressive form of brain tumors, has been extensively studied using OMICS methods, and the most characteristic molecular determinants have been incorporated into the histopathological diagnosis. Research data, nevertheless, only partially have been adopted in clinical practice. Here we aimed to present results of our epige­no­mic GBM profiling to better understand early and late determinants of these tumors, and to share main elements of our findings with practicing professionals.

GBM specimens were surgically obtained after first diagnosis (GBM1) and at recurrence (GBM2). DNA was extracted from 24 sequential pairs of formalin-fixed, paraffin-embedded tumor tissues. The Reduced Representation Bisulfite Sequencing kit was used for library preparation. Pooled libraries were sequenced on an Illumina NextSeq 550 instrument. Methylation controls (MC) were obtained from a publicly available database. Bioinformatic analyses were performed to identify differentially methylated pathways and their elements in cohorts of MC, GBM1 and GBM2.

Several differentially methylated pathways involved in basic intracellular and brain tissue developmental processes were identified in the GBM1 vs. MC and GBM2 vs. MC comparisons. Among differentially me­thylated pathways, those involved in immune regulation, neurotransmitter (particularly dopaminergic, noradrenergic and glutaminergic) responses and regulation of stem cell differentiation and proliferation stood out in the GBM2 vs. GBM1 comparisons.

Our study revealed biological complexity of early and late gliomagenesis encompassing mechanisms from basic intracellular through distorted neurodevelopmental processes to more specific immune and highjacked neurotransmitter pathways in the tumor microenvironment. These findings may offer considerations for therapeutic approaches.

Wnt pathway markers in low-grade and high-grade gliomas.

BACKGROUND AND PURPOSE: Aberrant activation of the Wnt pathway contributes to differentiation and maintenance of cancer stem cells underlying gliomagenesis. The aim of our research was to determine as to what degrees some Wnt markers are expressed in gliomas of different grades, lineages and molecular subtypes. METHODS: Nine grade II, 10 grade III and 72 grade IV surgically removed, formalin-fixed paraffin-embedded glioma specimens were included. Mutation status of IDH1 codon 132 was defined by immunohistochemistry and pyrosequencing in all tumors. Grade II and III astrocytic and oligodendroglial tumors were further tested for the expression of p53 and ATRX by immunohistochemistry, and codeletion of 1p19q by fluorescent in situ hybridization. Expression levels of the non-canonical Wnt5a and Fzd2, and the canonical Wnt3a and beta-catenin Wnt pathway markers were determined by immunohistochemistry, and compared between subgroups stratified according to grade, lineage and the presence or absence of IDH1 R132H/C mutations. RESULTS: In the normal brain - grade II-IV glioma comparisons, a gradual increase was observed for the expressions of Wnt5a, Wnt3a, Fzd2 and beta-catenin. In the astroglial and oligodendroglial lineages of grade II and III gliomas, only the Wnt5a expression was significantly higher in the astroglial subgroup. Stratification according to the IDH1 status resulted in a significant increase of the Wnt3 expression in the wild type grade II-IV gliomas. CONCLUSION: These data extend previous observations and show a correlation of Wnt pathway activity with glioma grade. Further investigations of the Wnt marker expression regulation according to glioma lineage or IDH gene mutational status are in progress by using more exact molecular approaches.

Secretory meningioma with bone infiltration and orbital spreading.

Secretory meningioma is a rare form of meningiomas which differentiates from the meningothelial subtype. It is characterized by significant peritumor edema and distinct immunohistochemical and molecular genetic profiles. We present a middle aged female patient with secretory meningioma infiltrating the orbital bone from the primary cranial base location and causing exophthalmos, features rarely described with this tumor. Surgical resection was challenging because of the associated brain swelling and rich vascularization of the tumor. Imaging and immunohistochemical studies revealed characteristic hallmarks of secretory meningioma. While histologically it was a benign tumor, due to the orbital bone and soft tissue infiltration, postoperative management of neurological sequelae was challenging. This case highlights distinctive clinical, imaging and histological features along with individual characteristics of a rare form of meningiomas.

Isocitrate dehydrogenase mutations in defining the biology of and supporting clinical decision making in glioblastoma.

BACKGROUND AND PURPOSE: Oncogenesis is related to a sequential accumulation of somatic mutations. Comprehensive characterizations of the genomic landscapes have been completed recently for several tumors, glioblastoma being among the first ones. Our own translational research studies have been focused on defining molecular subtypes of glioblastoma in the clinical setting because of an expected prognostic and therapeutic utility of the information. Somatic mutations in genes of the isocitrate dehydrogenase (IDH) enzyme family appear to be among the best-defined biomarkers that also influence tumor behavior and confer clinical utility. METHODS: We have reviewed the literature including our own results to summarize basic science and clinical correlates of IDH mutations. RESULTS: The surveyed data reveal genomic, transcriptomic, epigenomic and biochemical consequences of IDH mutations in the context of glioblastoma biology and phenotype. In addition, a few studies highlight the therapeutic potential of targeting IDH, although thus far all tests have only been conducted in the preclinical setting. CONCLUSION: Somatic mutations in isoforms of IDH genes represent important biomarkers that correlate with biochemical, biological and phenotypic features of glioblastoma, and may also facilitate the development of new therapeutic strategies complementing the currently available approved protocols.

Epigenetic Suppression of the IL-7 Pathway in Progressive Glioblastoma.

BACKGROUND: Immune evasion in glioblastoma (GBM) shields cancer cells from cytotoxic immune response. METHODS: We investigated CpG methylation in promoters, genes, and pathways in 22 pairs of formalin-fixed paraffin-embedded sequential (FFPE) GBM using restricted resolution bisulfite sequencing (RRBS) and bioinformatic analyses. RESULTS: Gene ontology revealed hypermethylation in elements of the innate and adaptive immune system when recurrent GBM samples (GBMrec) were compared to control (CG) and primary GBM samples (GBMprim). Higher methylation levels of the IL-7 signaling pathway and response to IL-7 were found in GBMrec suggesting a progressive blockade of the IL-7 driven T cell response in sequential GBM. Analyses of the Cancer Genome Atlas array-based data confirmed hypermethylation of the IL-7 pathway in recurrent compared with primary GBM. We also quantified DNA CpG methylation in promoter and gene regions of the IL-7 ligand and IL-7 α-receptor subunit in individual samples of a large RRBS-based sequential cohort of GBM in a Viennese database and found significantly higher methylation levels in the IL-7 receptor α-subunit in GBMrec compared with GBMprim. CONCLUSIONS: This study revealed the progressive suppression of the IL-7 receptor-mediated pathway as a means of immune evasion by GBM and thereby highlighted it as a new treatment target.

DNA methylation and protein expression of Wnt pathway markers in progressive glioblastoma.

BACKGROUND: Wnt signaling plays important roles in tumorigenesis, invasiveness and therapeutic resistance of glioblastoma (GBM). METHODS: We simultaneously investigated six Wnt pathway markers (Wnt5a, Fzd-2, beta-catenin, Wnt3a, Wnt7b, Fzd-10) at epigenetic and protein levels in 21 sequential formalin-fixed paraffin-embedded GBM pairs and controls. RESULTS: Expression levels of Wnt5a, beta-catenin and Wnt3a proteins either moderately or significantly increased, while those of Fzd-2, Wnt7b and Fzd-10 decreased in the primary (GBM-P) and recurrent (GBM-R) tumors compared to the controls. Methylation levels within promoters and genes showed corresponding decreases for Wnt5a, beta-catenin and Wnt3a in tumors vs. controls, while that of Fzd-10 was uniformly high. Comparing the GBM-P and GBM-R pairs, proteins of Fzd-2, beta-catenin and Wnt3a were either moderately or significantly up-, while that of Wnt7b was downregulated in GBM-R, but these patterns were not accompanied by inverse methylation patterns in the corresponding promoters and genes over time. No methylation differences were noted within promoters and genes of the same markers in 112 pairs of primary and recurrent GBMs in a database, suggesting that the observed changes in protein expression levels may not be explained by CpG methylation status alone. The promoter and gene methylation rate was the highest for Fzd-10 in the database cohort too, supporting the noted low Fzd-10 protein expression. DISCUSSION: These analyses underscore the relevance of Wnt pathway molecules in the context of their methylation profiles in the development and evolution of GBM, and suggest that Wnt pathway regulation as a potential treatment target merits further studies.

DNA CpG methylation in sequential glioblastoma specimens.

PURPOSE: Glioblastoma is the most aggressive form of brain tumors. A better understanding of the molecular mechanisms leading to its evolution is essential for the development of treatments more effective than the available modalities. Here, we aim to identify molecular drivers of glioblastoma development and recurrence by analyzing DNA CpG methylation patterns in sequential samples. METHODS: DNA was isolated from 22 pairs of primary and recurrent formalin-fixed, paraffin-embedded glioblastoma specimens, and subjected to reduced representation bisulfite sequencing. Bioinformatic analyses were conducted to identify differentially methylated sites and pathways, and biostatistics was used to test correlations among clinical and pathological parameters. RESULTS: Differentially methylated pathways likely involved in primary tumor development included those of neuronal differentiation, myelination, metabolic processes, synapse organization and endothelial cell proliferation, while pathways differentially active during glioblastoma recurrence involved those associated with cell processes and differentiation, immune response, Wnt regulation and catecholamine secretion and transport. CONCLUSION: DNA CpG methylation analyses in sequential clinical specimens revealed hypomethylation in certain pathways such as neuronal tissue development and angiogenesis likely involved in early tumor development and growth, while suggested altered regulation in catecholamine secretion and transport, Wnt expression and immune response contributing to glioblastoma recurrence. These pathways merit further investigations and may represent novel therapeutic targets.

Wnt pathway markers in molecular subgroups of glioblastoma.

Glioblastoma (GBM) is a highly heterogeneous and aggressive brain tumor. Comprehensive genomic and transcriptomic analyses revealed that GBM segregates into three subgroups with characteristic signaling pathways. The Wnt pathway recently received increasing attention with the recognition of its importance in tumor development and recurrence through the promotion of glioma stem cells. As an extension of our previous translational studies, here we tested the possible interactions between key subgroup markers (IDH-1 R132H, EGFRvIII and both cytoplasmic and nuclear loss [c-/n-] of NF-1 expression) and the canonical (Wnt3a and beta-catenin) and non-canonical (Wnt5a and Fzd2) Wnt pathway markers by immunohistochemistry. These analyses revealed increased expression levels of both Wnt pathway markers with significant quantitative differences within, but not among subgroups. Wnt5a and Fzd2 levels were higher than the canonical marker levels in all subgroups. The strongest evidence for correlation between expression levels of the EGFRvIII subgroup marker and the Fzd2 Wnt marker was found, but weaker correlations also could be noted for IDH-1 R132H and NF-1 and some Wnt markers. The correlations detected between markers of the canonical and non-canonical pathways raised the possibility of cross-talk between the two pathways. Analyses of tumors with various NF-1 expression patterns (c+/n-; c-/n+ combined with c+/n+, and c-/n-) revealed that aberrant nuclear accumulation of NF-1 is accompanied by nuclear accumulation of beta-catenin, suggesting that they may act synergistically to define the tumor's biology. Altogether, our study presents expression characteristics of Wnt ligands and receptors, and suggests complex molecular interactions in subgroups of GBM.

Cognitive Enhancer Effects of Low Memantine Doses Are Facilitated by an Alpha7 Nicotinic Acetylcholine Receptor Agonist in Scopolamine-Induced Amnesia in Rats.

Alpha7 nicotinic acetylcholine receptors (nAChRs) play an important role in learning and memory and are promising targets for pharmacological cognitive enhancement. Memantine, an approved substance for Alzheimer's disease treatment, is an antagonist of the N-Methyl-D-aspartate receptor (NMDAR) and also acts as an alpha7 nAChR antagonist. Here, we tested the interaction between an alpha7 nAChR agonist (PHA-543613) and memantine. Efficacy of memantine, PHA-543613, and their co-administration were investigated on the spatial working memory of rats using the spontaneous alternation paradigm in T-maze. Scopolamine-induced transient amnesia was used to model cognitive impairment. First, the dose-response relationship was assessed for memantine, and its lowest effective dose was found to be 0.1 mg/kg. Then, co-administration treatments with subeffective doses of the alpha7 nAChR agonist PHA-543613 and different doses of memantine were tested. The co-administration of subeffective drug doses significantly improved memory performance of the rats and reversed scopolamine-induced deficits. Interestingly, a higher than effective (0.3 mg/kg) dose of memantine did not increase performance in monotreatment, only in co-administration with PHA-543613. However, the co-administration of PHA-543613 did not further increase the efficacy of the previously effective monotreatment doses of memantine. Thus, the efficacy of memantine monotreatment and its co-administration with PHA-543613 converged to create a common ceiling effect, with an additive interaction found in the behavioral effects. These results suggest that memantine and PHA-543613 may exert their cognitive enhancer effects on the same target, possibly on the alpha7 nAChRs. Results also suggest possible benefits of a combination therapy with memantine and alpha7 nAChR agonists.

Contribution of the Wnt Pathway to Defining Biology of Glioblastoma.

Glioblastoma (GBM), a highly lethal brain tumor, has been comprehensively characterized at the molecular level with the identification of several potential treatment targets. Data concerning the Wnt pathway are relatively sparse, but apparently very important in defining several aspects of tumor biology. The Wnt ligands are involved in numerous basic biological processes including regulation of embryogenic development, cell fate determination, and organogenesis, but growing amount of data also support the roles of Wnt pathways in the formation of many tumors, including gliomas. Two main Wnt pathways are distinguished: the canonical (ß-catenin) and non-canonical (planar cell polarity, Wnt/Ca2+) routes. Wnt signaling regulates glioma stem cells (GSCs), thereby defining invasive potential, recurrence, and treatment resistance of GBM. Some observations suggest that the Wnt pathways are differentially active in molecular subtypes of this tumor, thereby may also guide prognostication and novel therapeutic decisions. In this review, we highlight main elements and biological relevance of the Wnt pathways, primarily focusing on the pathogenesis and subtypes of GBM. Finally, we briefly summarize newer therapeutic strategies targeting networks of the Wnt signaling cascades and their molecular associates that appear to be marked contributors to GBM aggressiveness.