Integration and Intersection of Cancer Metabolism with Epigenetic Pathways in Gliomas.

Isocitrate dehydrogenase 1 (IDH1) and Succinate dehydrogenase (SDH) mutant tumors may have increased levels of hypoxia inducible factor (HIF), which leads to a tumoral pseudohypoxic profile. Increased HIF levels may promote tumor progression by the activation of numerous cellular processes including resistance to apoptosis, vascular remodeling and angiogenesis. A high frequency of IDH1 mutations has been detected in adult but not in pediatric gliomas. However, loss of SDH expression has not been investigated in gliomas. To further explore the profile and possible contribution of IDH1 and SDHB to glioma tumorigenesis, we investigated the expression of IDH1-R132 mutant and SDHB in a series of 94 histologically confirmed pediatric and adult gliomas. There were 13 diffuse intrinsic pontine gliomas and 81 adult gliomas: 10 lowgrade astrocytomas 8 oligodendrogliomas, 4 ependymomas and 59 glioblastomas (GBMs). Pediatric brain stem gliomas were negative for mutant IDH1 (0/13) and showed preserved SDHB expression (13/13). IDH1 mutant tumors included 6/10 astrocytomas, 5/8 oligodendrogliomas, 0/4 ependymomas and 13/59 GBMs. No adult or pediatric glioma exhibited loss of SDHB expression. IDH1 mutations are frequently present in low- and high-grade gliomas, but not in pediatric brain stem gliomas. It appears that IDH1, but not SDHB, contributes to HIF stabilization in adult gliomas. However, IDH1 or SDHB alterations do not appear to play a role in pediatric gliomagenesis.

Recently, I was discussing the treatment of a pediatric patient who had gliomatosis cerebri. A pediatric neuro-oncologist, one of my respectable friends, insisted on chemotherapy with temozolomide. Because the histologic type was anaplastic astrocytoma (as determined by open biopsy), many would consider radiotherapy the primary option; thus, there was an exciting discussion. On another occasion, the same colleague and I were discussing a pediatric patient with a symptomatic diffuse astrocytoma of the posterior thalamus; this time as well, he insisted on a trial of chemotherapy with carboplatin and vincristine. My colleague's philosophy is that because pediatric gliomas are different from adult gliomas, chemotherapy to defer or avoid radiotherapy is reasonable in cases of low-grade and sometimes even high-grade glioma. Maybe my colleague is right regarding the difference between pediatric and adult gliomas. There have been numerous reports showing substantial differences in the molecular features underlying pediatric and adult high-grade gliomas. Pediatric and adult glioblastomas were shown to be clearly distinguished by frequent gain of chromosome 1q and lower frequency of chromosome 7 gain and 10q loss in the pediatric form. Platelet-derived growth factor receptor-α (PDGFR-α) was found to be the predominant gene subject to focal amplification in childhood high-grade gliomas.1,2 Among the pediatric low-grade gliomas, pilocytic astrocytoma is the predominant histologic type and diffuse astrocytomas are quite rare. In this issue of Neuro-Oncology, Stokland et al. from the Children's Brain Tumour Research Centre, University of Nottingham, UK, report on 639 pediatric patients with low-grade gliomas, 63.7% of which were pilocytic astrocytomas and only 5.9% of which were diffuse fibrillary astrocytomas.3 The predominance of the former type is consistent with other evidence. For instance, in the Brain Tumor Registry of Japan, the incidence of pilocytic astrocytomas peaks in patients between 5 and 9 years old, while that of diffuse astrocytomas peaks in patients between 40 and 44 years old. In the Central Brain Tumor Registry of the United States (CBTRUS), 1,834 pediatric patients (those between 0 and 19 years old) with pilocytic astrocytomas are registered, while only 101 cases of diffuse astrocytoma are found in the pediatric population. Therefore, pediatric low-grade gliomas are mostly pilocytic astrocytomas, and adult low-grade gliomas are mostly diffuse astrocytomas. Pilocytic astrocytomas and diffuse astrocytomas are classified as WHO grades I and II, respectively. However, pilocytic astrocytoma is not a precursor of diffuse astrocytoma, as there is a distinct difference between the molecular features of the two. There appears to be no role for either TP53 mutations or aberrant PDGF signaling in the development of pilocytic astrocytomas, in contrast to the role of TP53 mutations and increased expression of PDGF-A and PDGFR-α as common, early events in the formation of diffuse astrocytomas.4 Pilocytic astrocytomas also differ from diffuse astrocytomas in their altered and increased expression of immune response genes. Hierarchical clustering analysis using a set of 1,176 genes distinguished pilocytic astrocytomas from diffuse astrocytomas and oligodendrogliomas.5 Pilocytic astrocytomas are also different from the diffuse astrocytomas clinically, as evidenced by the fact that histology is one prognostic factor in pediatric low-grade gliomas.3 Fear of radiation-induced adverse effects has been used to justify the use of chemotherapy for unresectable pilocytic astrocytomas. A number of chemotherapy regimens have been used to delay the need for radiotherapy, including carboplatin and vincristine. A recent randomized trial, Children's Oncology Group A9952, compared the combination of carboplatin and vincristine with the combination of procarbazine, thioguanine, lomustine, and vincristine and reported 5-year event-free survival rates of 40%-50%. More than 400 low-grade gliomas were registered, and 83% of them were pilocytic astrocytomas.6 However, although a small study is under way, the role of chemotherapy in the management of pediatric diffuse astrocytomas remains unclear. In adult diffuse astrocytomas, a randomized study comparing radiotherapy and temozolomide is now ongoing in Europe and North America. Yes, pediatric and adult gliomas are different. Pediatric and adult high-grade gliomas are biologically different. On the other hand, differences between pediatric and adult low-grade gliomas are mainly due to the difference in their histologies. Whether pediatric diffuse astrocytomas are chemosensitive remains unproven. The UK study reported in this issue also suggests that, because of the small number of diffuse astrocytomas in the pediatric population, international cooperation will be needed to address the issue of the optimal management of this entity.

Gliomas are the most common primary brain tumors and a major source of mortality and morbidity in adults and children. Recent genomic studies have identified multiple molecular subtypes; however metabolic characterization of these tumors has thus far been limited. We performed metabolic profiling of 114 adult and pediatric primary gliomas and integrated metabolomic data with transcriptomics and DNA methylation classes. We identified that pediatric tumors have higher levels of glucose and reduced lactate compared to adult tumors regardless of underlying genetics or grade, suggesting differences in availability of glucose and/or utilization of glucose for downstream pathways. Differences in glucose utilization in pediatric gliomas may be facilitated through overexpression of SLC2A4, which encodes the insulin-stimulated glucose transporter GLUT4. Transcriptomic comparison of adult and pediatric tumors suggests that adult tumors may have limited access to glucose and experience more hypoxia, which is supported by enrichment of lactate, 2-hydroxyglutarate (2-HG), even in isocitrate dehydrogenase (IDH) wild-type tumors, and 3-hydroxybutyrate, a ketone body that is produced by oxidation of fatty acids and ketogenic amino acids during periods of glucose scarcity. Our data support adult tumors relying more on fatty acid oxidation, as they have an abundance of acyl carnitines compared to pediatric tumors and have significant enrichment of transcripts needed for oxidative phosphorylation. Our findings suggest striking differences exist in the metabolism of pediatric and adult gliomas, which can provide new insight into metabolic vulnerabilities for therapy.

Temozolomide constitutes current standard of care for adult patients with high-grade gliomas. However, results for pediatric gliomas are rather disappointing. In order to investigate the molecular differences between pediatric and adult gliomas that could affect sensitivity to temozolomide, we studied 23 pediatric non-ependymal, non-pilocytic gliomas in comparison to 59 consecutive adult gliomas for the expression of O6-methylguanine methyltransferase (MGMT) and the DNA mismatch repair protein, mutS homolog 6 (MSH6) by immunohistochemistry, as well as for the presence or absence of promoter methylation of the MGMT gene by methylation-specific PCR. The expression of MGMT in pediatric gliomas was significantly higher than in adult gliomas, as shown by immunohistochemistry (p=0.00004). This association was conserved if statistical analysis was carried out only in astrocytic tumors (diffuse astrocytoma, anaplastic astrocytoma and glioblastoma, p=0.00007), or in oligodendroglial tumors (oligodendroglioma and anaplastic oligodendroglioma, p=0.020). Although methylation-specific PCR was successfully performed only in 15 pediatric gliomas, it also showed a trend toward less frequent methylation in pediatric as opposed to adult gliomas (p=0.242). MSH6 was almost equally expressed in pediatric and adult gliomas. Pediatric gliomas appear to have a distinct molecular profile associated with resistance to temozolomide. Higher expression of MGMT and a trend toward less frequent methylation of the promoter region of MGMT gene may partly account for relative resistance to temozolomide in pediatric gliomas as compared to adult gliomas.

Simple SummaryGliomas are major causes of worldwide cancer-associated deaths in children. Generally, paediatric gliomas can be classified into low-grade and high-grade gliomas. They differ significantly from adult gliomas in terms of prevalence, molecular alterations, molecular mechanisms and predominant histological types. The aims of this review article are: (i) to discuss the current updates of biomarkers in paediatric low-grade and high-grade gliomas including their diagnostic and prognostic values, and (ii) to discuss potential targeted therapies in treating paediatric low-grade and high-grade gliomas. Our findings revealed that liquid biopsy is less invasive than tissue biopsy in obtaining the samples for biomarker detections in children. In addition, future clinical trials should consider blood-brain barrier (BBB) penetration of therapeutic drugs in paediatric population.Paediatric gliomas categorised as low- or high-grade vary markedly from their adult counterparts, and denoted as the second most prevalent childhood cancers after leukaemia. As compared to adult gliomas, the studies of diagnostic and prognostic biomarkers, as well as the development of therapy in paediatric gliomas, are still in their infancy. A body of evidence demonstrates that B-Raf Proto-Oncogene or V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) and histone H3 mutations are valuable biomarkers for paediatric low-grade gliomas (pLGGs) and high-grade gliomas (pHGGs). Various diagnostic methods involving fluorescence in situ hybridisation, whole-genomic sequencing, PCR, next-generation sequencing and NanoString are currently used for detecting BRAF and histone H3 mutations. Additionally, liquid biopsies are gaining popularity as an alternative to tumour materials in detecting these biomarkers, but still, they cannot fully replace solid biopsies due to several limitations. Although histone H3 mutations are reliable prognosis biomarkers in pHGGs, children with these mutations have a dismal prognosis. Conversely, the role of BRAF alterations as prognostic biomarkers in pLGGs is still in doubt due to contradictory findings. The BRAF V600E mutation is seen in the majority of pLGGs (as seen in pleomorphic xanthoastrocytoma and gangliomas). By contrast, the H3K27M mutation is found in the majority of paediatric diffuse intrinsic pontine glioma and other midline gliomas in pHGGs. pLGG patients with a BRAF V600E mutation often have a lower progression-free survival rate in comparison to wild-type pLGGs when treated with conventional therapies. BRAF inhibitors (Dabrafenib and Vemurafenib), however, show higher overall survival and tumour response in BRAF V600E mutated pLGGs than conventional therapies in some studies. To date, targeted therapy and precision medicine are promising avenues for paediatric gliomas with BRAF V600E and diffuse intrinsic pontine glioma with the H3K27M mutations. Given these shortcomings in the current treatments of paediatric gliomas, there is a dire need for novel therapies that yield a better therapeutic response. The present review discusses the diagnostic tools and the perspective of liquid biopsies in the detection of BRAF V600E and H3K27M mutations. An in-depth understanding of these biomarkers and the therapeutics associated with the respective challenges will bridge the gap between paediatric glioma patients and the development of effective therapies.

BackgroundPediatric gliomas, the most common solid childhood neoplasm, manifest unique molecular signatures that distinguish them from adult gliomas. Unfortunately, most studies have focused on adult gliomas and extrapolate the findings to treat pediatric gliomas. In this study, we assessed the efficacy of Targetin, a folate conjugated analogue of Noscapine, on the treatment of pediatric low and high grade gliomas.MethodAn assortment of standard cancer assays were used with different drug doses and experimental durations.ResultsWe found that pediatric glioma cells are more susceptible to lower doses of Targetin than parental Noscapine. Targetin functions by disrupting the microtubule network, and can likewise perturb DNA synthesis, delay the cellular transition within the S and G2M cell cycle phases, diminish anchorage independent growth and the migratory/invasiveness of pediatric glioma cells. Moreover, Targetin impairs the expression of several regulators of cancer progression belonging to prominent signalling pathways in pediatric gliomas; including Platelet Derived Growth Factor alpha and some members of the Mitogen Activated Protein Kinase cascade.ConclusionTargetin has an excellent anti-neoplastic profile and functions to modulate the expression of several genes belonging to key cancer progression pathways in pediatric gliomas. Collectively, findings from this study highlight the usefulness of Targetin for the treatment of pediatric high and low grade gliomas.

Malignant gliomas are a leading cause of tumor-related mortality in children. Current therapies often have significant adverse effects on brain development and are largely ineffective against aggressive gliomas. The development of rational therapies has been limited by poor information regarding the cell of origin for pediatric glioma and the relevant underlying genetic alterations. Recent findings now reveal that mutations existing in pediatric glioma are different than those seen in adult gliomas. For example, BRAFV600E activating mutations are associated with deletion of the tumor suppressor gene, p16 (Ink4a-Arf), in about 15% of pediatric gliomas. We have generated a faithful genetic mouse model in which there is concomitant activation of BRAFV600E and deletion of Ink4a-Arf in oligodendrocyte progenitor cells (OPCs) leading to the generation of pediatric high-grade glioma. OPCs have been shown to be a potential cell of origin for glioma and expression of Olig2 is found in 100 percent of human pediatric and adult high-grade gliomas. Given that Olig2 is also required for normal myelin development, targeting all Olig2-expressing cells with small molecule inhibitors would likely have detrimental effects. We have recently found that Olig2 protein is present in a phosphorylated form that promotes proliferation of adult glioma cells. In this study, we demonstrate that phospho-Olig2 is expressed in tumorigenic oligodendrocyte progenitor cells and is required for pediatric glioma generation. In addition, we found that phospho-Olig2 and Olig2 interact with different proteins, some of which are feasible drug targets. This study gives insights into new therapies by understanding key protein interactions in normal oligodendrocyte development as well as in tumorigenic oligodendrocyte progenitor cells. Citation Format: Amelie GRIVEAU, An-Chi TIEN, David H. Rowitch. Role of Olig2 phosphorylation in pediatric glioma formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-206. doi:10.1158/1538-7445.AM2014-LB-206

To assess frequency of neural stem cell compartment (NSC) involvement in adult and pediatric gliomas [World Health Organization (WHO) grades 1-4], and to assess whether NSC involvement at presentation impacts on survival, recurrence rates, and/or transformation from low grade (WHO grade 1-2) to high grade disease (WHO grades 3-4). Cranial MRIs for 154 pediatric and 223 adult glioma patients treated from 2000 to 2012 were reviewed. NSC involvement was documented. Tumors were stratified by age (adult vs. pediatric), histology, tumor grade, tumor location, and involvement of midline structures. Odds ratios (OR) for death were calculated based on NSC status at presentation. Rates of transformation and recurrence rates (ORR) were compared using Fisher's Exact Test. Time to recurrence (TTR) was calculated using student t test. Among recurrent and transformed tumors, we also assessed the rate of NSC involvement at time of recurrence or transformation. 74.8 % of tumors had NSC involvement. Higher rates of NSC involvement were seen among adult (p = .0001); high grade (p = .0001)); grade 2 versus grade 1 (p = .0001) and other grade 1 histologies (p = .0001) versus JPA (juvenile pilocytic astrocytoma) patients); grade 2-4 tumors (p = .0001); and supratentorial tumors (p < .0001). No transformation was noted among pediatric low grade tumors or adult grade 1 tumors. 22/119 (18.5 %) adult grade 2 tumors transformed. Rates of transformation were not impacted by NSC status (p = .47). ORR was 15.1 %, and was greater for NSC+ tumors at presentation (p = .05). 36/41 recurrences (87.8 %) involved NSC at time of recurrence. OR for death was 2.62 (1.16-5.9), p = .02 for NSC+ tumors at presentation. Adult and pediatric gliomas (all grades) frequently involve NSC at presentation, although rates are lower in pediatric JPA and all infratentorial tumors. NSC involvement at presentation increases OR death and reduces TTR for pediatric gliomas (all grades) and adult low grade gliomas, and shows a strong trend toward increased ORR.

S-adenosylmethionine (SAMe) therapy in liver disease: A review of current evidence and clinical utility

Recent genomic studies of pediatric high-grade gliomas (pHGGs) have unraveled important new clues into the pathogenesis of these tumors. One novel insight that was previously unappreciated is the role of epigenetic alterations in pediatric gliomagenesis. This was realized when mutations in histone 3.3/3.1 (H3.3/H3.1) were identified in these tumors. A related concept is that certain genetic alterations only occur in a region-specific manner within the central nervous system. As an example, K27M H3.3/H3.1 mutations are present in high-grade gliomas that arise along the midline of the central nervous system while G34R and G34V H3.3 mutations are present in high-grade gliomas that arise in the cerebral cortex. In addition, the realization that gliomas that harbor histone mutations are mutually exclusive of isocitrate dehydrogenase (IDH) mutant gliomas, which primarily arise in adults, has reinforced the notion that pediatric gliomas and adult gliomas are biologically disparate diseases. Incorporating this new data within the framework of our existing knowledge of the genetic alterations of pHGGs gives new hope that we will successfully identify effective therapies for these tumors in the upcoming decade.KeywordsPediatric high-grade gliomasPediatric astrocytomasBrain tumorsHistone mutations

Introduction: Pediatric brainstem glioma (BSG) is one of the most difficult cancers to treat accounting for 10-20% of all pediatric central nervous system (CNS) tumors. BSGs may occur throughout the brainstem and are categorized into two main groups: diffuse intrinsic pontine gliomas (DIPGs) and focal brainstem gliomas. DIPGs represent about 80% of BSG and have a peak onset of six to nine years of age. DIPGs invade throughout the pons and may spread to other portions of brainstem. To better understand the pathophysiology of the disease, a genetically engineered (PDGFα-expressing) BSG mouse and a xenograft model have been established. Results: We recently reported proteome profiling of DIPG CSF and formalin fixed specimens. To expand our molecular studies of the disease, we generated complete protein profiles of specimens obtained from BSG mouse tumor and aged-matched healthy controls. Neuroglia 2 (NG2), also known as chondroitine sulfate proteoglycan 4 (CSPG4) was selected for further analysis based on its exclusive expression in BSG specimens. Although NG2 has been previously implicated in adult gliomas, its role in pediatric gliomas has not been investigated and currently there are no publications on the role of NG2 in DIPGs. Recently, NG2 expression has been shown to play a significant role in neoplastic transformation of glioma precursor cells. Gliomas are thought to originate from cells including astrocytes, stem cells, and glioma progenitor cells. Glial progenitor cells are specifically important since several groups have reported detection of oligodendrocyte markers including NG2, PDGFRα, and Olig-2 in gliomas. Our preliminary data shows high expression of NG2 in murine model of brainstem glioma as well as 80% of pediatric DIPG specimens tested. We show that shRNA-mediated knockdown of NG2 reduces cellular migration in vitro. NG2 expression is defective (symmetric) in dividing cells in vitro and in vivo. The defective NG2 expression is consistent with a recent observation in adult high grade gliomas. Injection of NG2 expressing neurospheres (NS) into brainstems of 2 day old mice (P2) results in highly aggressive brainstem tumors resulting in death within 3-7 weeks post-injection. Therefore the NS injected mouse model of brainstem glioma provides a solid model for testing therapeutics and evaluating interventions. Furthermore, we show selective delivery of liposomal nanoparticles to brainstem of our robust BSG mouse model. We also show that nanoparticle-mediated delivery of doxorubicin will induce apoptosis in tumor and not the adjacent normal region. Conclusion: We introduce a robust murine model of brainstem glioma that is developed using NG2 expressing cells. High expression of NG2 in a subset of DIPGs and its defective expression may provide novel approaches for treating DIPGs and BSGs. Citation Format: Sridevi Yadavilli, Madhuri Kambhampati, Oren J. Becher, Tobey MacDonald, Ravi Bellamkonda, Roger J. Paker, Javad Nazarian. NG2 upregulation and its defective asymmetric distribution in pediatric brainstem glioma and diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5004. doi:10.1158/1538-7445.AM2013-5004

IDH1 (isocitrate dehydrogenase 1) mutation might be encounter in the low-grade glioma occurs in early stages of development and directs the progression of the tumor to a higher grade. Aim of the study was to assess the frequency of IDH1 mutation in Iraqi patients with gliomas by immunohistochemical study, to correlate its immunoreactivity with some clinicopathological parameters. The study did on formalin fixed, paraffin embedded tumor tissue from 66 patients with different grades of intracranial gliomas of both gender and all age groups in the Baghdad city were collected in this retrospective and prospective randomized study. Ten normal brain tissue samples in form of paraffin blocks took from forensic medicine unit. New technique used, which is manual tissue microarray Immunohistochemical detection of IDH1 antibodies did by Dako autostainer link 48. Positive cytoplasmic IDH1 staining was found in 38 (57.6%) of cases of glioma. In adult gliomas, secondary glioblastoma multiforme, low grade astrocytoma and oligodendroglial tumors had the greatest values of IDH1 positivity (87%, 80% and 72.72% respectively) followed by anaplastic astrocytoma (42%), then primary glioblastoma multiforme (26%). Males and females expressed the IDH1 equally. The conclusions from the work were IDH1 mutation commonly existed in adult gliomas, low-grade gliomas and secondary glioblastoma, it had no role in pediatric gliomas, and it could be a diagnostic and prognostic marker.

BACKGROUND:IDH1 (isocitrate dehydrogenase 1) mutation might be encounter in the low grade glioma and directs the progression of the tumor to a higher grade.OBJECTIVE:To assess the frequency of IDH1 mutations in gliomas and to correlate the IDH1 positivity with the type and grade of tumors, the age and sex of the patients.MATERIAL AND METHODS:A retro– and prospective case series study. One hundred and nine cases of intracranial gliomas were collected between 2008 and 2014 from Mosul Private Laboratories and Al-Jamboree Teaching Hospitals in Mosul. IDH1 mutations were assessed immunohistochemically using anti-IDH1 R132H mouse monoclonal antibody.RESULTS:IDH1 mutation was perceived in 34.86% of gliomas. In adult gliomas, the secondary glioblastoma and the low-grade astrocytoma had the greatest values of IDH1 positivity (88.88% and 62.5% respectively), followed by oligoastrocytoma/oligodendroglioma (50.0%), and anaplastic astrocytoma (47.36%). The primary glioblastomsa showed 17.64% IDH1 positivity. Males and females expressed the IDH1 equally. While, there was no role of IDH1 in pediatric gliomas.CONCLUSION:IDH1 mutation is commonly present in adult gliomas particularly in low-grade gliomas, and secondary glioblastoma, with equal sex distribution, but it has no role in pediatric gliomas.

423P - Characterization and pre-clinical modeling of genetic aberrations in pediatric gliomas

Pediatric and adult IDH-wildtype gliomas (GBM) differ in both their clinical and molecular characteristics. However, whether distinct metabolic vulnerabilities exist between these two types of malignant brain tumors is unknown. Here, we performed integrated molecular and lipidomic profiling of both adult IDHwt and pediatric gliomas to identify distinguishable lipid phenotypes and consequent vulnerabilities. This analysis revealed IDHwt gliomas have heightened de novo lipid biosynthetic programs with an elevated abundance of triacylglycerides (TAGs). By contrast, pediatric gliomas exhibited lower lipid biosynthetic capacity and contained a higher enrichment for phospholipids. As a consequence of this unique metabolic program, pediatric gliomas relied of uptake of environmental complex fatty acids derived from normal resident brain cells for growth. Accordingly, therapeutic abrogation of lipid scavenging pathways selectively inhibited proliferation of pediatric gliomas compared to adult IDHwt counterparts. Together, these studies identify distinct metabolic programs among adult and pediatric gliomas, revealing new therapeutically actionable targets to slow pediatric glioma growth.