Clinical manifestations

Presentation and course

Symptoms vary based on involvement of different midline structures and generally develop over 1 to 2 months (30; 15; 45). Cranial neuropathies can cause expected symptomatology: diplopia, ptosis, facial pain or numbness, facial palsy, tinnitus, hearing loss, vertigo, dysarthria, and dysphagia. Involvement of motor and sensory pathways causes weakness, hypoesthesia, hyperesthesia, and gait abnormalities. High CSF pressure causes headache, nausea, and vomiting. Other CNS symptoms include seizures and persistent hiccups.

H3 K27M mutant diffuse midline gliomas have an incidence rate of 0.06 per 100,000 population and a median diagnosis age of 14 years (42). H3K27M mutant diffuse midline gliomas have been extensively described in children and are less frequently seen in adults (58). In children, H3 K27M-mutant gliomas occur frequently within the brainstem, particularly the pons, whereas in adults they occur more frequently within the thalamus and spinal cord.

In a retrospective study of adult midline gliomas, the H3 K27M mutation was identified in 15% of cases (55). In this series, the most common midline locations for H3 K27M-mutated tumors were midbrain, pons, and cerebellum. In the pediatric population, this mutation is identified in approximately 27% of gliomas (35).

H3 G34-mutant diffuse hemispheric gliomas are reported to occur in less than 1% of all gliomas and in 15% of high-grade gliomas. The median age at diagnosis is 15.8 years, with a male to female ratio of 1.46:1 (11). These tumors are commonly seen in the frontal lobe.

Prognosis and complications

H3 K27M-mutant midline gliomas are associated with poor prognosis and are designated as a grade 4 entity in the 2016 WHO Classification (30). However, there is heterogeneity in the behavior of gliomas with H3 K27M mutation. Prognosis for circumscribed gliomas, thalamic, or diffuse non-midline gliomas with H3 K27M mutation remains uncertain (16; 39; 41; 43).

However, prognosis for H3 K27M-mutant midline gliomas remains dismal even with timely and optimal treatment; overall survival is typically 7 to 11 months in children and 8 to 28 months in adults (24; 52; 58; 68). Overall survival with standard care is slightly longer compared to patients with IDH wildtype glioblastoma but shorter compared to patients with WHO grade 4 IDH-mutant astrocytomas (32). K27M mutation is a significant independent prognostic marker for poor outcome among pediatric glioblastomas (26). Furthermore, as compared to H3.1K27M mutation, H3.3K27M mutation is associated with shorter survival, higher metastatic relapses, and poorer response to radiotherapy (65; 07).

Patients with H3 G34-mutant gliomas have a better prognosis than H3K27M mutant tumors. Median time to progression is approximately 10 months, median overall survival is approximately 17 months, and median time from progression to death is approximately 5 months (11). The 1-, 2-, and 3-year survival estimates are 70%, 39%, and 21%. Survival was better in patients over 18 years old who underwent near or gross-total resection. G34V-mutant tumors had significantly worse overall survival when compared to G34R-mutant tumors (11; 63). Many patients were noted to have distant recurrence (26%).

A retrospective study of H3 K27-altered diffuse midline gliomas or H3 G34-mutant diffuse hemispheric glioma found a higher risk of developing leptomeningeal disease in each of these subgroups (14). In this study, median time from tumor diagnosis to leptomeningeal disease was 12.9 months for H3 K27 patients and 5.6 months for H3 G34 patients.

Biological basis

Etiology and pathogenesis

Frequency of IDH mutation in the midline region has been reported to be low (60).

H3 K27M-mutant gliomas are IDH-wildtype, lack 1p/19q co-deletion, and are defined by the presence of K27M mutation in the H3F3A or HIST1H3B/C genes, which encode the histone H3 variants H3.3 and H3.1 in both children and adults (66; 58). These histone mutations are mutually exclusive from the other common mutations that define distinct infiltrating glioma types.

Histones are basic nuclear proteins responsible for the nucleosome structure within eukaryotic cells. Among the five classes of histones, some are expressed only during the S phase, whereas others are replacement histones that are replication-independent and expressed in quiescent or terminally differentiated cells.

Supratentorial high-grade gliomas have mutations in H3F3A (located on chromosome 1q) encoding histone H3.3. H3.3 is a replacement histone subclass that is encoded by two distinct genes, H3.3A (H3F3A) and H3.3B. This leads to the creation of oncohistones.

Oncohistones in high-grade gliomas have specific associated mutations and brain locations. G34R/V is restricted to hemispheric tumors, while K27M occurs in the midline (24; 60; 65; 10). K27M mutations occur in two main histone variants H3.3 and H3.1 and result in lysine to methionine exchange at position 27 of H3F3A or HIST1H3B/C genes. G34 mutations of H3.3 result in glycine to arginine exchange at codon 34 of H3F3A (57; 65).

The H3 K27M-mutated midline gliomas are further divided into two subgroups, H3.1 K27M and H3.3 K27M (07).

H3.3 K27M mutation. H3.3 K27M mutation occurs at the median age of 7.5 years. Patients with this mutation have a poor prognosis and frequent leptomeningeal dissemination; they are likelier to have an oligodendroglial histologic phenotype, poor response to radiation, and median survival of 9 months (10). H3.3 K27M mutant pediatric high-grade glioma are enriched for PDGFRA amplification (60). These tumors are seen all over the midline structures (66; 07). Approximately 20% of tumors previously categorized as pediatric glioblastomas harbor this mutation (18).

H3.1 K27M mutation. H3.1 K27M mutation occurs at the median age of 5.5 years. Patients with this mutation are likelier to have an astroglial differentiation, a good response to radiation, and a median survival of 15 months (10). H3.1 K27M tumors are associated with ACVR1 mutations (01). These tumors are almost exclusive to the pons (66; 07).

H3 K27M-mutant diffuse midline gliomas frequently share genetic alterations, including tumor protein 53 (TP53) overexpression, alpha-thalassemia/mental retardation X-linked (ATRX) loss, monosomy 10, activin A receptor type 1 (ACVR) mutation, poly ADP-ribose polymerase (PARP1) overexpression, cyclin D1-3(CCND1-3), and cyclin-dependent kinase (CDK4/CDK6) mutations (58; 15). TP53 mutations occur in about 40% of diffuse intrinsic pontine gliomas and represent the second most frequent mutation, correlated with worse overall survival (05).

H3 K27M mutation is mutually exclusive or rarely co-occurs with IDH1 mutation, telomerase reverse transcriptase (TERT) promoter mutation, epidermal growth factor receptor (EGFR) amplification, and BRAF-V600E mutation (58; 34; 15). Prototypic alterations of adult primary glioblastoma IDHwt (eg, EGFR amplification, CDKN2A/B homozygous deletions, PTEN mutations) are infrequent in childhood (47; 60; 48; 26). O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is found in 75% of G34-mutant subtype tumors and in 3% of K27 mutant tumors (26; 34).

Molecular profiling has revealed differences between adult and pediatric H3 K27M-mutant diffuse midline gliomas. Higher frequencies of ATRX loss and H3.3 mutation are found in adult than in pediatric H3 K27M-mt diffuse midline gliomas (68). TERT promoter mutations and O6-methylguanine DNA methyltransferase promoter methylation were not detected in pediatric patients but are present in a few adult patients.

H3 K27 wild type occurs at a median age of 6 years, with a median survival of 9 months (10).

Diffuse glioma, H3.3 G34-mutant are a distinct grade 4 entity described in the WHO 2021 classification (31). H3 G34-mutant diffuse hemispheric glioma was first described in 2012. Point mutations in the H3F3A gene, encoding for the histone variant H3.3, result in amino acid substitution at codon 34 from either glycine-to-arginine (G34R) or, more rarely, glycine-to-valine (G34V) (11). Co-occurring molecular alterations include ATRX mutation in 93%, TP53 mutation in 88%, PDGFA point mutation in 45%, PDGFRA amplification in 11%, and MGMT promoter methylation in 70% (63).

Diagnostic workup

Diagnosis is made with both imaging and histologic confirmation. Radiographic criteria alone are used for tumors with midline locations associated with serious procedural complications (22). Imaging primarily includes a brain MRI with and without gadolinium. MRI abnormalities include diffuse, T2 expansile signal changes in the midline structures.

In most cases, little or no enhancement is noted after the administration of gadolinium at initial diagnosis. Occasionally, areas of necrosis with surrounding serpiginous contrast enhancement can be present. A retrospective study to characterize imaging features of 24 diffuse midline gliomas found distinguishing features in H3K27-mutant midline gliomas as compared to their H3wild-type counterparts (01). In H3K27-mutant gliomas, there was an absence of perilesional edema at low frequency of strong contrast enhancement. Contrast enhancement of intermediate or higher and rim enhancement was associated with microscopically detected microvascular proliferation. Necrosis on MRI is less frequently seen than in the H3wt high-grade midline gliomas (01). H3G34 tumors demonstrate contrast enhancement and diffusion restriction on MRI.

PET imaging with amino acid tracers, such as 18-F-dihydroxy-phenylalanine (F-DOPA) is able to discriminate H3 K27M-mutant from wild-type diffuse midline gliomas and has demonstrated correlation of higher tracer uptake with worse prognosis (69; 49).

Traditional WHO grading for diffuse intrinsic pontine gliomas was not prognostic. Outcomes for grade 2, 3, and 4 tumors are equally poor (05). Next-generation sequencing can help accurately classify these tumors, improve understanding of unique tumor biology, and provide important insights into prognosis and management. Reports have shown that biopsy of these tumors is safe (22). Modern neuronavigation tools allow accurate 3D localization of deep structures in the brain and identification of major vessels prior to surgery, which greatly minimizes surgical complications (22).

Once tissue is obtained, both the macroscopic and microscopic evaluation can provide insight into the underlying disease. Macroscopically, these tumors cause enlargement and distortion of anatomical structures by diffuse infiltration. Microscopically, they typically have an astrocytic morphology or an oligodendroglial pattern. Mitotic activity is present in most cases but is not necessary for diagnosis (30). Features such as microvascular proliferation and necrosis may be seen. Immunohistochemistry reveals expression of neural cell adhesion molecule 1 (NCAM1), S100, oligodendrocyte transcription factor 2 (OLIG2) on tumor cells (30). Microtubule-associated protein 2 (MAP2) expression is common. Immunoreactivity of glial fibrillary acidic protein (GFAP) is variable, and synaptophysin can be focal. Chromogranin A and neuronal nuclei (NeuN) are not typically expressed (30).

Biopsy carries the risk of neurologic injury due to anatomical location of midline glioma. Minimally invasive techniques like liquid biopsy to detect biomarkers of H3 K27M in CSF, blood, and urine for diagnosis and monitoring treatment response are under investigation (25; 46). Two secreted proteins, cyclophilin A (CypA) and dimethylarginase 1 (DDAH1), are upregulated in CSF samples from patients with diffuse intrinsic pontine gliomas (54). Detecting tumor-derived circulating tumor DNA (ctDNA) from CSF samples can help with analysis of the tumor’s mutational profile.

Management

Anatomic location of the tumor severely limits any opportunity for meaningful surgical resection. Treatment usually consists of standard fractionated radiation to a dose of 54-59Gy (over 30 fractions) (10).

Radiation is the mainstay of treatment. Photon-based radiotherapy to a range of 54 to 59.4 Gy given in 30 to 33 fractions of 1.8 Gy daily for 6 weeks, as well as hypo-fractionated radiation (39 to 45 Grays given in 13 fractions of 3.0 Grays over 3 to 4 weeks), has been utilized with similar survival rates (37; 23; 67). Monotherapy and combination chemotherapy have been trialed, with uniformly poor results (21; 62). Temozolomide has limited value in treating pediatric H3 K27M-mutant gliomas, likely due to a higher likelihood of unmethylated MGMT (09; 53). It is still often used in the treatment of adult tumors. It is likely that the pivotal trials that established the standard of care for adult high-grade gliomas included histone mutated tumors, although this was not specifically evaluated. Pre-radiation chemotherapy has shown improvement in overall survival and progression-free survival in a small series of patients (64; 19), although how to best interpret these data in the current WHO 2021 era is uncertain. In a series of adult patients, surgery was followed by concurrent radiation and temozolomide and, subsequently, maintenance temozolomide (56). At recurrence, a combination of re-radiation, bevacizumab, and lomustine were utilized, similar to what is typically used in recurrent glioblastoma IDHwt.

Future directions

Novel ongoing treatment trials include convection-enhanced therapies, targeted therapies, and immunotherapy (vaccine therapies, CAR-T cells).

Convection-enhanced delivery. Convection-enhanced delivery is a therapeutic delivery strategy that allows for targeted treatment of a specific region via a cannula that can be placed in difficult-to-access areas and allows for direct intraparenchymal infusion of drugs at a steady rate over a prolonged period of time. This approach is not limited to a single group of therapeutic agents. Studies have shown safe placement of convection-enhanced delivery catheters into the brainstem in humans (02; 59).

Immunotherapy.

Peptide-based vaccine therapy. Glioma-associated antigens interleukin-13 receptor alpha 2 (IL-13Rα2), EphA2, and survivin are commonly overexpressed in pediatric gliomas (40). A pilot study of subcutaneous vaccination with glioma-associated antigen epitope peptides in HLA-A2-positive children with newly diagnosed brainstem and high-grade gliomas was well tolerated and has preliminary evidence of immunologic and clinical responses (51).

Dendritic cell vaccines reactivate tumor-specific T cells in both clinical and preclinical settings. Autologous dendritic cell vaccine administered intradermally is safe and able to generate a diffuse intrinsic pontine glioma-specific immune response detected in peripheral blood mononuclear cells and CSF (04).

The disialoganglioside GD2 is highly expressed in H3 K27M-mutant glioma cells. Preclinical studies have demonstrated GD2 as a novel immunotherapy target in H3 K27M mutant diffuse midline gliomas, and antitumor efficacy of GD2-CAR T-cells delivered systemically (36). The first-in-human phase I clinical trial included four patients with H3 K27M-mutant diffuse intrinsic pontine glioma or diffuse midline glioma treated with GD2-CAR T cells administered intravenously, with subsequent intraventricular infusions (33). GD2-CAR T therapy produced toxicities that were largely related to tumor location and reversible with intensive supportive care. Radiographic response was seen in three patients but was not durable. Proinflammatory cytokines were increased in plasma and CSF. These preliminary cases suggest that GD2-CAR T-cell therapy holds significant promise for H3K27M+ diffuse intrinsic pontine gliomas or diffuse midline gliomas.

A single-center trial of newly diagnosed pediatric patients with diffuse intrinsic pontine glioma who underwent oncolytic viral therapy (DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells) followed by radiation revealed changes in T-cell activity and stabilization to the reduction of tumor size (17). Adverse events related to virotherapy were largely grade 1 or grade 2, with only one event of grade 3 and no grade 4 or 5 events (17).

Targeted therapies. K27M mutation drives gliomagenesis by alteration of an important site of posttranslational modification in the histone H3 variants and leads to altered DNA methylation and gene expression profiles (60; 03). Ongoing efforts aim to study the efficacy of therapeutics targeting histone-modifying enzymes for midline gliomas with histone H3 mutations.

Panobinostat. Panobinostat is a general histone deacetylase inhibitor that has shown good in vitro efficacy against diffuse intrinsic pontine gliomas (20; 50). Panobinostat synergizes with histone demethylase inhibitor GSKJ4 in H3.3K27M mutant diffuse intrinsic pontine gliomas cells (20). Together, these data suggest a promising therapeutic strategy for diffuse intrinsic pontine gliomas. Compassionate use of panobinostat in four adult patients with H3 K27M-mutant glioma revealed good tolerability without any serious adverse effects (38).

ONC201. ONC201 is a small-molecule selective antagonist of dopamine receptor D2/3 (DRD2/3) that crosses the blood-brain barrier and exhibits P53 independent anticancer efficacy. DRD2 expression within the central nervous system is highest in midline structures of the brain. DRD2 is a G protein-coupled receptor that promotes tumor growth and has emerged as a therapeutic target for gliomas that overexpress this receptor (27; 06). H3 K27M-mutant diffuse midline gliomas have been reported to exhibit elevated expression and dependency on DRD2 as a downstream epigenetic consequence of the mutation (08). It has an exceptional safety profile and has been reported to have clinical and radiographic responses in both recurrent and frontline settings (08). ONC206 is another drug in the same class actively undergoing investigation. An international phase 3 randomized, double-blind, placebo-controlled study (ACTION study) is currently evaluating the benefit of ONC201 after frontline radiation on overall survival and progression-free survival.

Cdk4/6 inhibitors. CDK alterations are described in about 30% to 40% of diffuse midline gliomas. Cdk4/6 inhibitors (palbociclib, ribociclib, and abemaciclib), tyrosine kinase inhibitors, and other agents that more specifically target these mutations are being explored in patients with diffuse midline gliomas (13; 61; 44). Other potential therapeutic targets tested with in vivo studies include BET bromodomain inhibitors (50).

mTOR inhibitors. PI3K/AKT/mTOR pathway is dysregulated in more than 50% of diffuse midline gliomas. A single-center study from Italy reported significant benefit in overall survival with utilization of personalized treatment based on tumor molecular alterations. mTOR inhibition with everolimus was associated with the best overall survival (12).

Multiple phase I and II trials are currently ongoing to test novel agents to treat midline gliomas with histone mutation in a frontline and recurrent setting (Table 2). An understanding of molecular pathology and ongoing efforts to improve diagnostic tools and therapy holds promise for future advances in prognosis.

Table 2. Ongoing Trials to Test Novel Agents for Diffuse Midline Glioma in a Frontline and Recurrent Setting