Histone H3 K27M Mutation Research Articles

Imaging Characteristics of Pediatric Diffuse Midline Gliomas with Histone H3 K27M Mutation.

The 2016 World Health Organization Classification of Tumors of the Central Nervous System includes "diffuse midline glioma with histone H3 K27M mutation" as a new diagnostic entity. We describe the MR imaging characteristics of this new tumor entity in pediatric patients. We retrospectively reviewed imaging features of pediatric patients with midline gliomas with or without the histone H3 K27 mutation. We evaluated the imaging features of these tumors on the basis of location, enhancement pattern, and necrosis. Among 33 patients with diffuse midline gliomas, histone H3 K27M mutation was present in 24 patients (72.7%) and absent in 9 (27.3%). Of the tumors, 27.3% (n = 9) were located in the thalamus; 42.4% (n = 14), in the pons; 15% (n = 5), within the vermis/fourth ventricle; and 6% (n = 2), in the spinal cord. The radiographic features of diffuse midline gliomas with histone H3 K27M mutation were highly variable, ranging from expansile masses without enhancement or necrosis with large areas of surrounding infiltrative growth to peripherally enhancing masses with central necrosis with significant mass effect but little surrounding T2/FLAIR hyperintensity. When we compared diffuse midline gliomas on the basis of the presence or absence of histone H3 K27M mutation, there was no significant correlation between enhancement or border characteristics, infiltrative appearance, or presence of edema. We describe, for the first time, the MR imaging features of pediatric diffuse midline gliomas with histone H3 K27M mutation. Similar to the heterogeneous histologic features among these tumors, they also have a diverse imaging appearance without distinguishing features from histone H3 wildtype diffuse gliomas.

Co-occurrence of histone H3 K27M and BRAF V600E mutations in paediatric midline grade I ganglioglioma.

Ganglioglioma (GG) is a grade I tumor characterized by alterations in the MAPK pathway, including BRAF V600E mutation. Recently, diffuse midline glioma with an H3 K27M mutation was added to the WHO 2016 classification as a new grade IV entity. As co-occurrence of H3 K27M and BRAF V600E mutations has been reported in midline tumors and anaplastic GG, we searched for BRAF V600E and H3 K27M mutations in a series of 54 paediatric midline grade I GG (midline GG) to determine the frequency of double mutations and its relevance for prognosis. Twenty-seven patients (50%) possessed the BRAF V600E mutation. The frequency of the co-occurrence of H3F3A/BRAF mutations at diagnosis was 9.3%. No H3 K27M mutation was detected in the absence of the BRAF V600E mutation. Double-immunostaining revealed that BRAF V600E and H3 K27M mutant proteins were present in both the glial and neuronal components. Immunopositivity for the BRAF V600E mutant protein correlated with BRAF mutation status as detected by massARRAY or digital droplet PCR. The median follow-up of patients with double mutation was 4 years. One patient died of progressive disease 8 years after diagnosis, whereas the four other patients were all alive with stable disease at the last clinical follow-up (at 9 months, 1 year and 7 years) without adjuvant therapy. We demonstrate in this first series of midline GGs that the H3 K27M mutation can occur in association with the BRAF V600E mutation in grade I glioneuronal tumors. Despite the presence of H3 K27M mutations, these cases should not be graded and treated as grade IV tumors because they have a better spontaneous outcome than classic diffuse midline H3 K27M-mutant glioma. These data suggest that H3 K27M cannot be considered a specific hallmark of grade IV diffuse gliomas and highlight the importance of integrated histomolecular diagnosis in paediatric brain tumors.

Rapid and fulminant leptomeningeal spread following radiotherapy in diffuse intrinsic pontine glioma

A 4-year-old male presented with rapid-onset cranial nerve palsy and ataxia. Brain magnetic resonance imaging (MRI) revealed a pontine mass lesion with discordant conventional and advanced imaging. A stereotactic core biopsy revealed glioblastoma with immunostaining suggestive of histone H3K27M and TP53 mutation, consistent with diffuse intrinsic pontine glioma. MRI 3 months after radiotherapy revealed extensive new leptomeningeal metastatic disease involving both the supra- and infratentorial brain, as well as the imaged portion of the spine. Tissue procured at the time of needle biopsy has undergone striking in vivo expansion as an orthotopic xenograft.

Prognostic impact of the 2016 WHO classification of diffuse gliomas in the French POLA cohort.

The new WHO classification of diffuse gliomas has been refined and now includes the 1p/19q codeletion, IDH1/2 mutation, and histone H3-K27M mutation. Our objective was to assess the prognostic value of the updated 2016 WHO classification in the French POLA cohort. All cases of high-grade oligodendroglial tumors sent for central pathological review and included into the French nationwide POLA cohort were reclassified according to the updated 4th WHO classification. In total, 1041 patients were included, with a median age at diagnosis of 50.4years (range 17.1-84.4). Based on the new histomolecular classification, diagnoses included anaplastic oligodendroglioma IDH mutant and 1p/19q-codeleted (32.5%), anaplastic astrocytoma IDH mutant (IDH (mut)) (11.0%), anaplastic astrocytoma IDH wild type (IDH (wt)) (5.3%), glioblastoma IDH (mut) (17.1%), and glioblastoma IDH (wt) (33.2%). Ten patients presented with a diffuse midline tumor, H3 K27M mutant. The new WHO classification was prognostic for progression-free survival (PFS) and overall survival (OS) (p<0.001). We did not find prognosis differences between grades III and IV for IDH (mut) 1p/19q intact and IDH (wt) gliomas in univariate and multivariate analyses. Among anaplastic astrocytoma IDH (wt), cases with chromosome arm 7p gain and 10q loss (55%) had shorter PFS than the others (p=0.027). In conclusion, the new WHO histomolecular classification of diffuse gliomas presented with high prognostic value. Grading was not discriminant between grade III and IV high-grade gliomas.

Validation of the new glioma WHO classification in the french POLA network: Analysis of 1041 cases.

2015 Background: The upcoming WHO classification of gliomas will be refined taking into account the robust molecular alterations of gliomagenesis: the 1p19q codeletion and IDH1/2mutations. Methods: All cases of high-grade oligodendroglial tumors sent for central pathological review and included into the French nation-wide POLA network were reclassified according the upgrading of the 4th WHO classification that integrates pathological features and molecular data. Immunohistochemical expression of IDH1R132H, and ATRX, 1p19q codeletion, chromosome 7p gain and chromosome 10q loss by genomic-array and IDH1/2and histone K27M mutational statuses by sequencing were evaluated. Results: 1041 patients were included with a median age at diagnosis of 50.4 years (range, 17.1-84.4). Based on the new histo-molecular classification, diagnoses were: anaplastic oligodendroglioma with 1p19q codeletion (32.5%), anaplastic astrocytoma IDH mutated (IDHmt) (11.0%), anaplastic astrocytoma IDHwild-type (IDHwt) (5.3%), glioblastoma IDHmt (17.1%) and glioblastoma IDHwt (33.2%). Ten patients presented a midline tumor with the histone H3-K27M mutation. Both new and oldest WHO classifications were predictive for Progression-Free Survival (PFS) and Overall Survival (OS) but the new histo-molecular classification was more discriminant with higher hazard ratio for PFS (1.603, 1.848, 5.025 and 6.135 versus 1.495, 2.705, 2.799 and 4.933) and for OS (3.588, 3.493, 11.020 and 14.708 versus 2.259, 4.340, 4.214 and 7.534). Grading (III versus IV) was not prognostic for IDHmt non 1p19q codeleted gliomas in univariate analyses (PFS, p=0.5; OS, p=0.8) and multivariate analysis (adjusted by age, type of surgery and first line treatment: PFS, p=0.13; OS, p=0.38). Among anaplastic astrocytoma IDHwt, cases presenting with 7p gain and 10q loss (55%) had a worse prognosis that the others for PFS (p=0.027), suggesting that all anaplastic astrocytoma IDHwt should not be considered as glioblastoma IDHwt. Conclusions: WHO histo-molecular classification of gliomas presented with high predictive and discriminative value, allowing the validation of three main molecular subgroups for the future neuro-oncological trials.

Abstract B01: Biological consequences of histone mutations in the mammalian brain

Abstract Pediatric high-grade gliomas (HGGs) are devastating diseases with poor prognosis. The two-year survival for pediatric HGG ranges from 30%, for tumors arising in the cerebral cortex, to less than 10% for diffuse intrinsic pontine gliomas (DIPGs), which arise in the brainstem. Recurrent somatic mutations in histone H3 found in pediatric HGGs are rare in adult HGGs and may represent genetic drivers of disease. Mutually exclusive somatic heterozygous H3F3A or HIST1H3B mutations encoding a K27M variant of histone H3.3 or H3.1, respectively, are found in nearly 80 percent of DIPGs and 22 percent of non-brainstem HGG, where they occur predominantly in thalamic and other midline tumors. Strikingly, heterozygous H3F3A mutations encoding histone H3.3 G34R or G34V occur in approximately 14 percent of non-brainstem HGG, and are associated predominantly with cortical tumors arising in older children through young adulthood, suggesting a distinct developmental origin compared to the majority of tumors harboring K27M mutations. DNA methylation signatures and gene expression patterns distinguish G34R/V and K27M tumors, but the oncogenic mechanisms and brain region selectivity of these mutations are still uncertain. To study these mutations in the developing mammalian brain, we generated conditionally activated, epitope tagged H3f3a knock-in mice to express K27M, G34R or non-mutated H3.3 proteins. When activated in neural and glial precursor cells, extensive H3f3a expression was observed throughout the embryonic brain, and H3.3-K27M induced loss of H3K27me3, consistent with observations in K27M mutant DIPG tumor samples. Moreover, neural stem cells expressing H3.3-K27M demonstrated an in vitro growth advantage compared to H3.3-G34R or H3.3-WT expressing neural stem cells. We will present an analysis of the epigenetic landscape and gene expression signatures associated with the histone H3 K27M and G34R mutations in different brain regions as well as phenotypic analysis of the mutant mice. Citation Format: Jon D. Larson, Lawryn H. Kasper, Helen R. Russell, Chunxu Qu, Xiaoyan Zhu, Peter J. McKinnon, Suzanne J. Baker. Biological consequences of histone mutations in the mammalian brain. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B01.

Diffuse Midline Gliomas with Histone H3-K27M Mutation: A Series of 47 Cases Assessing the Spectrum of Morphologic Variation and Associated Genetic Alterations.

Somatic mutations of the H3F3A and HIST1H3B genes encoding the histone H3 variants, H3.3 and H3.1, were recently identified in high-grade gliomas arising in the thalamus, pons and spinal cord of children and young adults. However, the complete range of patients and locations in which these tumors arise, as well as the morphologic spectrum and associated genetic alterations remain undefined. Here, we describe a series of 47 diffuse midline gliomas with histone H3-K27M mutation. The 25 male and 22 female patients ranged in age from 2 to 65 years (median = 14). Tumors were centered not only in the pons, thalamus, and spinal cord, but also in the third ventricle, hypothalamus, pineal region and cerebellum. Patients with pontine tumors were younger (median = 7 years) than those with thalamic (median = 24 years) or spinal (median = 25 years) tumors. A wide morphologic spectrum was encountered including gliomas with giant cells, epithelioid and rhabdoid cells, primitive neuroectodermal tumor (PNET)-like foci, neuropil-like islands, pilomyxoid features, ependymal-like areas, sarcomatous transformation, ganglionic differentiation and pleomorphic xanthoastrocytoma (PXA)-like areas. In this series, histone H3-K27M mutation was mutually exclusive with IDH1 mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression, ATRX loss (except in pontine gliomas), and monosomy 10.

TMOD-05MODELING ACVR1 AND HIST1H3B MUTATIONS IN DIFFUSE INTRINSIC PONTINE GLIOMAIN VIVOUSING THE SLEEPING BEAUTY SYSTEM

Diffuse intrinsic pontine glioma (DIPG) is a brainstem tumor that occurs in the pons in children; median age 6-7 years. The most frequent DIPG mutations affect the N-terminal tail of histone H3.3 and histone H3.1 and result in the change of a lysine to methionine at residue 27. In addition, six recurrent somatic activating mutations in ACVR1, which encodes for the bone morphogenetic protein (BMP) receptor kinase, have also been identified in DIPG tumors. We aim to use the sleeping beauty transposase system to generate spontaneous in vivo tumor models that will allow us to test the contributions of these genes to the pathogenesis of DIPG tumors. We have cloned ACVR1 G328V, and HIST1H3B K27M sequences into a plasmid that contains flanking inverted repeats (IR) which contain binding sequences for the sleeping beauty transposase, which will excise DNA between the IR sites and insert it into the host genome. Plasmids encoding a short hair pin for p53, oncogenic NRAS, the sleeping beauty transposase/luciferase were injected with group 1: ACVR1 G328V; group 2: HIST1H3B K27M; or group 3: ACVR1 G328V and HIST1H3B K27M into the subventricular zone of neonatal mice. Tumor development in mice is monitored by measuring luciferase activity in vivo; animals that have reached moribund stage are perfused and processed for histology. Our long-term goal is to understand the interplay between the ACVR1 activating mutation and the histone H3K27M mutation. The ACVR1 mutations result in constitutive BMP pathway activation and the H3K27M mutation has been shown to globally alter H3K27 methylation. Through this study we hope to elucidate how ACVR1 mutations and H3K27 mutations contribute to tumor progression in order to uncover novel therapeutic targets for DIPG which would improve prognosis for DIPG patients.

Abstract A15: Variant histone H3 mutations associate with histone modification, DNA methylation, and gene expression changes in pediatric high-grade gliomas

Abstract High-grade gliomas (HGG) account for twenty percent of primary brain tumors in children and have a devastating outcome with a 2-year survival rate of less than 30%. While adult HGG arise frequently in the cerebral cortex, pediatric HGG can form in cerebral cortex as well as midline regions including thalamus and brainstem, and are genetically distinct from adult tumors. Brainstem HGGs, referred to as diffuse intrinsic pontine glioma (DIPG), are mostly exclusive to children and confer a dismal less than 10% 2-year survival rate. Recent genome-wide sequence analysis of pediatric DIPG and non-brainstem HGG revealed a predominance of mutually exclusive somatic heterozygous H3F3A or HIST1H3B mutations encoding a K27M variant of histone H3.3 or H3.1, respectively. These mutations were found in 78% of DIPG patients and 22% of non-brainstem pediatric HGG. An additional mutually exclusive somatic heterozygous H3F3A mutation encoding histone H3.3 G34R was identified in 14% of non-brainstem HGG. We established xenografts from human non-brainstem HGGs carrying wild-type H3 or the H3.3 G34R mutant, and DIPGs with H3.3 or H3.1 K27M mutations. H3 K27M mutant xenografts showed a marked reduction in H3K27 trimethylation (H3K27me3), while tumors with wild-type or H3.3 G34R mutations retain H3K27me3 as demonstrated by immunohistochemistry and Western blot analyses. To determine if this was a dominant effect of K27M mutation or a developmental signature of DIPG we transduced primary astrocyte cultures derived from p53-deficient neonatal mouse cortex or brainstem with retrovirus expressing wild-type, K27M, K27A or G34R H3 mutant variants. Low-level expression of exogenous H3.3 K27M compared to endogenous H3 was sufficient to recapitulate prominent H3K27me3 reduction observed in the xenografts. Furthermore, we evaluated correlation between H3 mutation and H3K27me3 status in a large cohort of primary pediatric HGG including DIPG. From this we identified a significant relationship between H3 K27M mutation and H3K27me3 loss in DIPG. However, we also observed rare wild-type H3 DIPG cases exhibiting reduced H3K27me3 indicating a potential alternative mechanism for this methylation state. In addition, we evaluated the association of H3K27me3 and K27M mutations with genome-wide DNA methylation patterns, and gene expression signatures. Together, these results implicate histone H3 K27M and G34R mutations as unique, dominant genetic drivers in distinct subtypes of pediatric HGG. Current efforts focus on delineating the divergent pathological roles of H3 K27M and G34R mutations during DIPG and non-brainstem HGG tumorigenesis. Citation Format: Jon D. Larson, Troy A. McEachron, Chunxu Qu, Xiaoyan Zhu, Alexander K. Diaz, David W. Ellison, Brent A. Orr, Suzanne J. Baker. Variant histone H3 mutations associate with histone modification, DNA methylation, and gene expression changes in pediatric high-grade gliomas. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A15.