Pediatric Hemispheric High-Grade Gliomas and H3.3-G34 Mutation: A Review of the Literature on Biological Features and New Therapeutic Strategies.

Pediatric high-grade glioma (pHGG) encompasses a wide range of gliomas with different genomic, epigenomic, and transcriptomic features. Almost 50% of pHGGs present a mutation in genes coding for histone 3, including the subtype harboring the H3.3-G34 mutation. In this context, histone mutations are frequently associated with mutations in TP53 and ATRX, along with PDGFRA and NOTCH2NL amplifications. Moreover, the H3.3-G34 histone mutation induces epigenetic changes in immune-related genes and exerts modulatory functions on the microenvironment. Also, the functionality of the blood-brain barrier (BBB) has an impact on treatment response. The prognosis remains poor with conventional treatments, thus eliciting the investigation of additional and alternative therapies. Promising molecular targets include PDGFRA amplification, BRAF mutation, EGFR amplification, NF1 loss, and IDH mutation. Considering that pHGGs harboring the H3.3-G34R mutation appear to be more susceptible to immunotherapies (ITs), different options have been recently explored, including immune checkpoint inhibitors, antibody mediated IT, and Car-T cells. This review aims to summarize the knowledge concerning cancer biology and cancer-immune cell interaction in this set of pediatric gliomas, with a focus on possible therapeutic options.

Cryo-EM structure and biochemical analyses of the nucleosome containing the cancer-associated histone H3 mutation E97K.

The Lys mutation of the canonical histone H3.1 Glu97 residue (H3E97K) is found in cancer cells. Previous biochemical analyses revealed that the nucleosome containing the H3E97K mutation is extremely unstable as compared to the wild-type nucleosome. However, the mechanism by which the H3E97K mutation causes nucleosome instability has not been clarified yet. In the present study, the cryo-electron microscopy structure of the nucleosome containing the H3E97K mutation revealed that the entry/exit DNA regions of the H3E97K nucleosome are disordered, probably by detachment of the nucleosomal DNA from the H3 N-terminal regions. This may change the intra-molecular amino acid interactions with the replaced H3 Lys97 residue, inducing structural distortion around the mutated position in the nucleosome. Consistent with the nucleosomal DNA end flexibility and the nucleosome instability, the H3E97K mutation exhibited reduced binding of linker histone H1 to the nucleosome, defective activation of PRC2 (the essential methyltransferase for facultative heterochromatin formation) with a poly-nucleosome, and enhanced nucleosome transcription by RNA polymerase II.

Chromatin structure related to oncogenesis.

Chromatin is the fundamental structure of genomic DNA in eukaryotic cells. The nucleosome, the primary unit of chromatin, consists of DNA and histone proteins, and is important for the maintenance of genomic DNA. Histone mutations are present in many types of cancers, suggesting that chromatin and/or nucleosome structures could be closely related to cancer development. Histone modifications and histone variants are also involved in regulating chromatin and nucleosome structures. Chromatin structures are dynamically changed by nucleosome binding proteins. In this review article, we discuss the current progress toward understanding the relationship between chromatin structure and cancer development.

Acidic patch histone mutations and their effects on nucleosome remodeling.

Structural and biochemical studies have identified a histone surface on each side of the nucleosome disk termed 'the nucleosome acidic patch' that acts as a regulatory hub for the function of numerous nuclear proteins, including ATP-dependent chromatin complexes (remodelers). Four major remodeler subfamilies, SWI/SNF, ISWI, CHD, and INO80, have distinct modes of interaction with one or both nucleosome acidic patches, contributing to their specific remodeling outcomes. Genome-wide sequencing analyses of various human cancers have uncovered high-frequency mutations in histone coding genes, including some that map to the acidic patch. How cancer-related acidic patch histone mutations affect nucleosome remodeling is mainly unknown. Recent advances in in vitro chromatin reconstitution have enabled access to physiologically relevant nucleosomes, including asymmetric nucleosomes that possess both wild-type and acidic patch mutant histone copies. Biochemical investigation of these substrates revealed unexpected remodeling outcomes with far-reaching implications for alteration of chromatin structure. This review summarizes recent findings of how different remodeler families interpret wild-type and mutant acidic patches for their remodeling functions and discusses models for remodeler-mediated changes in chromatin landscapes as a consequence of acidic patch mutations.

Clinicopathologic and molecular features of denosumab-treated giant cell tumour of bone (GCTB): Analysis of 21 cases.

GCTB is an osteolytic, locally-aggressive, rarely-metastasizing tumour, characterized by abundance of osteoclast-like giant cells, induced by neoplastic mononuclear cells expressing high-levels of the receptor activator of nuclear factor Kappa-B ligand (RANKL), a mediator of osteoclast activation. Although the mainstay of treatment is complete tumour removal with preservation of bone, therapy with denosumab, an inhibitor of RANKL, has been introduced for selected cases. OBJECTIVES: Denosumab-treated GCTB (DT-GCTB) was reported to show a wide spectrum of histological changes such as depletion of osteoclast-like giant cells and intralesional bone deposition, which may lead to diagnostic difficulties. We investigated clinicopathologic and molecular features of DT-GCTB, matched with pre-therapy samples. PARTICIPANTS: 21 cases were included (13 females, 8 males), aged 15 to 64 (median, 30 years). RESULTS: DT-GCTB showed development of sclerotic neocortex and varying degrees of osteosclerosis radiographically. Marked depletion of giant cells, different degree of ossification, fibrosis, and proliferation of mononuclear cells was observed. Staining for H3.3G34W was positive in mononuclear cells in 19 cases (90.5%), while one negative case was positive for H3.3G34V. H3F3A G34W mutation was confirmed in 17 of 19 cases (89.5%), corresponding to nuclear staining with H3.3 G34W antibody. G34L mutation was detected in one G34W negative case, in which H3.3 G34V nuclear positive staining was observed, possibly due to cross-reaction. CONCLUSIONS: Post-therapy tumours still exhibit a similar mutation profile, while significantly differing from classic GCTB morphologically. Correlation with history of denosumab administration, awareness of features of DT-GCTB, IHC and molecular studies for histone H3 mutations are important in its assessment.

Histone H2B Mutations in Cancer.

Oncohistones have emerged as a new area in cancer epigenetics research. Recent efforts to catalogue histone mutations in cancer patients have revealed thousands of histone mutations across different types of cancer. In contrast to previously identified oncohistones (H3K27M, H3G34V/R, and H3K36M), where the mutations occur on the tail domain and affect histone post-translational modifications, the majority of the newly identified mutations are located within the histone fold domain and affect gene expression via distinct mechanisms. The recent characterization of the selected H2B has revealed previously unappreciated roles of oncohistones in nucleosome stability, chromatin accessibility, and chromatin remodeling. This review summarizes recent advances in the study of H2B oncohistones and other emerging oncohistones occurring on other types of histones, particularly those occurring on the histone fold domain.

Pediatric Gliomas: Molecular Landscape and Emerging Targets.

Next-generation sequencing of pediatric gliomas has revealed the importance of molecular genetic characterization in understanding the biology underlying these tumors and a breadth of potential therapeutic targets. Promising targeted therapies include mTOR inhibitors for subependymal giant cell astrocytomas in tuberous sclerosis, BRAF and MEK inhibitors mainly for low-grade gliomas, and MEK inhibitors for NF1-deficient BRAF:KIAA fusion tumors. Challenges in developing targeted molecular therapies include significant intratumoral and intertumoral heterogeneity, highly varied mechanisms of treatment resistance and immune escape, adequacy of tumor penetrance, and sensitivity of brain to treatment-related toxicities.

Surprising phenotypic diversity of cancer-associated mutations of Gly 34 in the histone H3 tail.

Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.

Overview of Histone Modification.

Epigenetics is the epi-information beyond the DNA sequence that can be inherited from parents to offspring. From years of studies, people have found that histone modifications, DNA methylation, and RNA-based mechanism are the main means of epigenetic control. In this chapter, we will focus on the general introductions of epigenetics, which is important in the regulation of chromatin structure and gene expression. With the development and expansion of high-throughput sequencing, various mutations of epigenetic regulators have been identified and proven to be the drivers of tumorigenesis. Epigenetic alterations are used to diagnose individual patients more accurately and specifically. Several drugs, which are targeting epigenetic changes, have been developed to treat patients regarding the awareness of precision medicine. Emerging researches are connecting the epigenetics and cancers together in the molecular mechanism exploration and the development of druggable targets.

Integrated Metabolic and Epigenomic Reprograming by H3K27M Mutations in Diffuse Intrinsic Pontine Gliomas.

H3K27M diffuse intrinsic pontine gliomas (DIPGs) are fatal and lack treatments. They mainly harbor H3.3K27M mutations resulting in H3K27me3 reduction. Integrated analysis in H3.3K27M cells, tumors, and in vivo imaging in patients showed enhanced glycolysis, glutaminolysis, and tricarboxylic acid cycle metabolism with high alpha-ketoglutarate (α-KG) production. Glucose and/or glutamine-derived α-KG maintained low H3K27me3 in H3.3K27M cells, and inhibition of key enzymes in glycolysis or glutaminolysis increased H3K27me3, altered chromatin accessibility, and prolonged survival in animal models. Previous studies have shown that mutant isocitrate-dehydrogenase (mIDH)1/2 glioma cells convert α-KG to D-2-hydroxyglutarate (D-2HG) to increase H3K27me3. Here, we show that H3K27M and IDH1 mutations are mutually exclusive and experimentally synthetic lethal. Overall, we demonstrate that H3.3K27M and mIDH1 hijack a conserved and critical metabolic pathway in opposing ways to maintain their preferred epigenetic state. Consequently, interruption of this metabolic/epigenetic pathway showed potent efficacy in preclinical models, suggesting key therapeutic targets for much needed treatments.

MR imaging features of diffuse intrinsic pontine glioma and relationship to overall survival: report from the International DIPG Registry.

BACKGROUND: This study describes imaging features of diffuse intrinsic pontine glioma (DIPG) and correlates with overall survival (OS) and histone mutation status in the International DIPG Registry (IDIPGR). METHODS: Four hundred cases submitted to the IDIPGR with a local diagnosis of DIPG and baseline MRI were evaluated by consensus review of 2 neuroradiologists; 43 cases were excluded (inadequate imaging or alternative diagnoses). Agreement between reviewers, association with histone status, and univariable and multivariable analyses relative to OS were assessed. RESULTS: On univariable analysis imaging features significantly associated with worse OS included: extrapontine extension, larger size, enhancement, necrosis, diffusion restriction, and distant disease. On central review, 9.5% of patients were considered not to have DIPG. There was moderate mean agreement of MRI features between reviewers. On multivariable analysis, chemotherapy, age, and distant disease were predictors of OS. There was no difference in OS between wild-type and H3 mutated cases. The only imaging feature associated with histone status was the presence of ill-defined signal infiltrating pontine fibers. CONCLUSIONS: Baseline imaging features are assessed in the IDIPGR. There was a 9.5% discordance in DIPG diagnosis between local and central review, demonstrating need for central imaging confirmation for prospective trials. Although several imaging features were significantly associated with OS (univariable), only age and distant disease were significant on multivariable analyses. There was limited association of imaging features with histone mutation status, although numbers are small and evaluation exploratory.

Significance of H3K27M Mutation in "Nonmidline" High-Grade Gliomas of Cerebral Hemispheres.

BACKGROUND: "Diffuse midline glioma H3K27M-mutant" (a mutation gene encoding H3 histone variants) represents a defined pathologic entity since the 2016 World Health Organization Central Nervous System classification and includes midline structures, namely the thalamus, brainstem, and spinal cord. H3K27M-mutant glioma responds more poorly and is associated with worse clinical outcome. Mutation detection is now diagnostic for "diffused midline gliomas." We report on a case arising in an atypical nonmidline localization with an exceedingly adverse course. The present case raises the question of the possible meaning of the H3K27M-mutation in high-grade gliomas arising from the cerebral hemispheres and its implications from prognostic and therapeutic views. CASE DESCRIPTION: A 48-year-old woman was admitted with a mass arising from the splenium of the corpus callosum with bilateral hemispheric extension, displaying magnetic resonance imaging characteristics typical of a high-grade glioma. Needle biopsy confirmed the diagnosis of "diffuse astrocytic neoplasm, World Health Organization grade IV." Immunostaining of the tumor revealed the H3K27M-mutant nuclear expression. The patient was referred to adjuvant therapy and died 3 months after biopsy. CONCLUSIONS: This experience highlights the possible negative prognostic impact of H3K27M-m and possibility that H3K27M-m could be a reliable prognostic indicator to be considered in not only diffuse midline gliomas but also gliomas from the cerebral hemispheres. Because of the rarity and limited knowledge of this specific mutation in this experience, reports should be encouraged.

Methylation array profiling of adult brain tumours: diagnostic outcomes in a large, single centre.

The introduction of the classification of brain tumours based on their DNA methylation profile has significantly changed the diagnostic approach for cases with ambiguous histology, non-informative or contradictory molecular profiles or for entities where methylation profiling provides useful information for patient risk stratification, for example in medulloblastoma and ependymoma. We present our experience that combines a conventional molecular diagnostic approach with the complementary use of a DNA methylation-based classification tool, for adult brain tumours originating from local as well as national referrals. We report the frequency of IDH mutations in a large cohort of nearly 1550 patients, EGFR amplifications in almost 1900 IDH-wildtype glioblastomas, and histone mutations in 70 adult gliomas. We demonstrate how additional methylation-based classification has changed and improved our diagnostic approach. Of the 325 cases referred for methylome testing, 179 (56%) had a calibrated score of 0.84 and higher and were included in the evaluation. In these 179 samples, the diagnosis was changed in 45 (25%), refined in 86 (48%) and confirmed in 44 cases (25%). In addition, the methylation arrays contain copy number information that usefully complements the methylation profile. For example, EGFR amplification which is 95% concordant with our Real-Time PCR-based copy number assays. We propose here a diagnostic algorithm that integrates histology, conventional molecular tests and methylation arrays.

Biopsying a spinal cord lesion: A diagnostic dilemma. Case report and review of literature.

Spinal cord biopsy is a difficult procedure fraught with the risk of false-negative results or even misdiagnosis in up to 30% of cases. Differential diagnoses of spinal cord lesions include a wide range of inflammatory, infectious and neoplastic diseases. Given the importance of correctly managing these pathologies, it is crucial to avoid delays in making the correct diagnosis in order to improve the patient's outcome. We present here the case of a 21-year-old male with rapidly progressing sphincter and lower limb motor dysfunctions up to complete paraplegia with evidence of thoracic spinal cord lesion on magnetic resonance imaging. None of the blood and cerebrospinal fluid tests pointed to a diagnosis, while a first spinal cord biopsy revealed an inflammatory necrotic process. After several weeks of empirical treatments and clinical stability, the patient started having focal structural seizures that became generalized with local progression of the lesion and diffuse leptomeningeal spread on magnetic resonance imaging. A second spinal cord biopsy found a grade IV glioblastoma with H3 K27M histone mutation. Unfortunately the patient passed away before any treatment could be initiated. In this report, the authors analyze the difficulty of making the rapid, correct diagnosis of a highly malignant intrinsic spinal cord lesion, discussing also possible strategies to avoid diagnostic delays and to improve the outcome of these difficult patients.

Cancer-driving H3G34V/R/D mutations block H3K36 methylation and H3K36me3-MutSα interaction.

Somatic mutations on glycine 34 of histone H3 (H3G34) cause pediatric cancers, but the underlying oncogenic mechanism remains unknown. We demonstrate that substituting H3G34 with arginine, valine, or aspartate (H3G34R/V/D), which converts the non-side chain glycine to a large side chain-containing residue, blocks H3 lysine 36 (H3K36) dimethylation and trimethylation by histone methyltransferases, including SETD2, an H3K36-specific trimethyltransferase. Our structural analysis reveals that the H3 "G33-G34" motif is recognized by a narrow substrate channel, and that H3G34/R/V/D mutations impair the catalytic activity of SETD2 due to steric clashes that impede optimal SETD2-H3K36 interaction. H3G34R/V/D mutations also block H3K36me3 from interacting with mismatch repair (MMR) protein MutSα, preventing the recruitment of the MMR machinery to chromatin. Cells harboring H3G34R/V/D mutations display a mutator phenotype similar to that observed in MMR-defective cells. Therefore, H3G34R/V/D mutations promote genome instability and tumorigenesis by inhibiting MMR activity.

Molecular pathology of paediatric central nervous system tumours.

Advances in our understanding of the biology of paediatric central nervous system (CNS) tumours have encouraged pathologists to use molecular markers alongside histopathological analysis for disease classification or prognostication and treatment stratification. In this article, we review molecular genetic alterations in paediatric CNS tumours, including those in low-grade and high-grade gliomas, ependymomas, and embryonal tumours. Some of these molecular changes with clinicopathological utility have been used for the first time in the most recent edition of the World Health Organization (WHO) classification of CNS tumours to define entities like ependymoma, RELA fusion-positive or diffuse midline glioma, H3 K27M-mutant. The classification of paediatric CNS tumours is entering a new era when histopathologists must work with molecular genetic data and their molecular pathology colleagues to provide an optimal diagnostic evaluation for their patients and clinical colleagues. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mutations in chromatin machinery and pediatric high-grade glioma.

Pediatric central nervous system tumors are the most common solid tumor of childhood. Of these, approximately one-third are gliomas that exhibit diverse biological behaviors in the unique context of the developing nervous system. Although low-grade gliomas predominate and have favorable outcomes, up to 20% of pediatric gliomas are high-grade. These tumors are a major contributor to cancer-related morbidity and mortality in infants, children, and adolescents, with long-term survival rates of only 10 to 15%. The recent discovery of somatic oncogenic mutations affecting chromatin regulation in pediatric high-grade glioma has markedly improved our understanding of disease pathogenesis, and these findings have stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. We review the current perspective on pediatric high-grade glioma genetics and epigenetics, and discuss the emerging and experimental therapeutics targeting the unique molecular abnormalities present in these deadly childhood brain tumors.