H3 K27M-altered glioma and diffuse intrinsic pontine glioma: Semi-systematic review of treatment landscape and future directions

Abstract

H3 K27M-mutant diffuse glioma is a recently identified brain tumor associated with poor prognosis. As of 2016, it is classified by the World Health Organization as a distinct form of grade IV glioma. Despite recognition as an important prognostic and diagnostic feature in diffuse glioma, radiation remains the sole standard of care and no effective systemic therapies are available for H3K27M mutant tumors. This review will detail treatment interventions applied to diffuse midline glioma and diffuse intrinsic pontine glioma (DIPG) prior to the identification of the H3 K27M mutation, the current standard-of-care for H3 K27M-mutant diffuse glioma treatment, and ongoing clinical trials listed on www.clinicaltrials.gov evaluating novel therapeutics in this population. Current clinical trials were identified using clinicaltrials.gov, and studies qualifying for this analysis were active or ongoing interventional trials that evaluated a therapy in at least 1 treatment arm or cohort comprised exclusively of patients with DIPG and H3 K27M-mutant glioma. Forty-one studies met these criteria, including trials evaluating H3 K27M vaccination, chimeric antigen receptor T-cell therapy, and small molecule inhibitors. Ongoing evaluation of novel therapeutics is necessary to identify safe and effective interventions in this underserved patient population.

Key Points.

• H3 K27M-mutant diffuse glioma, including diffuse intrinsic pontine glioma (DIPG), has a poor prognosis and no effective treatments; there is a significant unmet need for effective therapies.

• A variety of novel investigational approaches are now being explored for DIPG.

Importance of the Study.

This comprehensive review provides an up-to-date account of ongoing clinical trials for the treatment of H3K27M-mutant and diffuse intrinsic pontine glioma. As such, this work can guide current management and future studies for improved clinical care of patients with this disease.

The presence of the H3 K27M mutation is an important diagnostic feature in diffuse midline glioma (DMG), which can occur in the thalamus, pons, cerebellum, or spinal cord, and includes diffuse intrinsic pontine glioma (DIPG). In these tumors, with a median overall survival of 10.1 to 14.4 months from diagnosis, its presence is associated with inferior prognosis compared to patients with H3 wild-type tumors.^1,2 The 2021 World Health Organization (WHO) central nervous system (CNS) classification criteria defines H3 K27-altered DMG as a separate category of high-grade glioma (HGG), regardless of histological features.³ However, before the identification of the mutation, biopsy was not commonly performed due to the risk associated with surgical intervention, especially in the brainstem. As a result, the existence of molecularly and/or morphologically distinct subsets of DMG and pediatric DIPGs was not fully appreciated.⁴ Before the significance of the H3 K27M mutation in subsets of DMG and DIPG was understood, multiple treatments including various chemotherapy regimens that previously demonstrated efficacy in adult glioma patients were investigated in these patients; however, no significant improvement in survival was demonstrated. This review will focus on treatments for H3 K27M-mutant glioma and DIPG, including a brief review of therapies that were evaluated before the identification of this alteration.

Historical Treatment Approaches for DMG and DIPG

In this narrative portion of the review, we highlight notable publications that helped define the historical strategy for DMG and DIPG treatment. Therefore, this section, while not intended to be exhaustive, is meant to present the historical approaches to DMG and DIPG management in order to provide context for understanding the current treatment landscape and advances in the treatment of DMG and DIPG over the past decade that follows.

Radiation therapy is a key component of the treatment of glioma, with a standard dose of 54–60 Gy that temporarily alleviates symptoms.⁵ Prior to the identification of the H3 K27M mutation, various radiation schedules were attempted in an effort to optimize efficacy in these patients, with little gain in survival. In pediatric patients with newly diagnosed DIPG, hypofractionated radiotherapy, delivered at 13 fractions of 3 Gy or 6 fractions of 5.5 Gy over 3 weeks, resulted in a median OS of only 8.6 months⁶; similarly, another study in DIPG found that a total dose of 45 Gy, delivered as daily 3 Gy for 3 weeks, had a median overall survival (OS) of 7.6 months.⁷ Re-irradiation has been administered to patients with progressive disease. For example, in a retrospective chart review of 26 patients, a schedule of 20 Gy in 10 daily fractions of intensity-modulated radiation therapy was administered in patients with DIPG undergoing re-irradiation, which was well tolerated without observed treatment toxicity.⁸ A separate phase I/II study of 12 patients evaluated 3 dose levels in patients with DIPG undergoing re-irradiation and determined that a course of 24 Gy in 12 fractions was the most appropriate regimen.⁹ Importantly, a Canadian study demonstrated the safety and feasibility of re-irradiation in 16 DIPG patients, with neurological improvement observed in all but 3.¹⁰

Various chemotherapy schedules and combinations were assessed for efficacy in patients with DMG and DIPG, following or in combination with radiotherapy, largely without encouraging results. A regimen including cisplatin, etoposide, vincristine, and combination cisplatin, etoposide, and ifosfamide, provided no improvement in OS in pediatric patients with pontine tumors (n = 37) or lesions outside the pons with residual tumor following resection, relative to historical controls.¹¹ Another trial assessed neo-adjuvant frontline chemotherapy (alternating carmustine and methotrexate cycles) in 23 pediatric patients with diffuse brainstem tumors who had progressed after chemotherapy; while median OS in this molecularly unselected population of patients was 17 months, the 3-year OS rate was only 4% and treatment was associated with serious complications such as septicemia, which required hospitalization.¹² Furthermore, in 63 patients with newly diagnosed DIPG, combined chemo-irradiation with temozolomide, also failed to improve OS compared to the older regimen (1-year OS 40%).¹³ Similar single-arm studies on pediatric populations with DIPG also showed no evidence of improved survival with combined radiotherapy and temozolomide, compared to historical controls.^14,15

A variety of targeted therapies were also attempted in pediatric glioma patients; however, clinically meaningful improvements were not observed in those with brainstem gliomas. Tipifarnib, a farnesyltransferase inhibitor, given concurrently with radiotherapy achieved a 1-year OS rate of 36.4% (SE 16.7%) in pediatric patients with newly diagnosed diffuse brainstem glioma, compared to a historical control of 30.0% ± 3%.¹⁶ The tyrosine kinase inhibitor (TKI) imatinib together with radiotherapy achieved a 1-year event-free survival of 45.0% ± 11.1% in newly diagnosed pediatric patients with DIPG, however, treatment was associated with an increased risk of intratumoral hemorrhage.¹⁷ The EGFR inhibitors gefitinib and erlotinib were also associated with intratumoral hemorrhage; median OS with gefitinib in pediatric patients with newly diagnosed brain stem glioma or incompletely resected supratentorial malignant glioma was 4.1–12 months, depending on dose level.^18,19 Vandetanib, a vascular endothelial growth factor receptor (VEGFR) inhibitor, given concurrent with radiotherapy, achieved 1- and 2-year OS rates of 37.5% and 21.4%, respectively, in pediatric patients with newly diagnosed DIPG.²⁰ Last, a phase II clinical trial was conducted examining nimotuzumab and radiotherapy for the treatment of newly diagnosed DIPG.²¹ In this trial, concomitant treatment with RT and nimotuzumab was found to be feasible in an outpatient setting, with comparable PFS and OS to RT and intensive chemotherapy in a hospitalized setting.

Convection-enhanced delivery (CED) is a technique designed to enhance the availability of targeted therapies within the brain, as both the blood–brain barrier (BBB) and the midline location of tumors such as DIPG may present obstacles to achieving therapeutic concentrations within the tumor.²² When administered via CED, treatment is delivered via catheters to interstitial areas of the brain, thereby providing the treatment to an extracellular space more proximal to the tumor.²³ While still experimental, this technique was recently evaluated in 7 pediatric patients with DIPG following radiation, in which the anti-CD276 antibody ¹²⁴l-omburtamab was administered into the brainstem using CED. The treatment was generally well tolerated, with no grade 3 or higher adverse events observed, and was considered feasible by the investigators.²⁴ Future research, including ongoing phase I trials of ¹³¹I-omburtama²⁵ and a gold nanoparticle formulation of the histone deacetylase (HDAC) inhibitor panobinostat²⁶ delivered using CED, will help determine the efficacy of CED in the treatment of midline tumors.

Identification of H3 K27M Mutation Defines a Disease

H3 K27M-mutant glioma is a recently identified entity. The discovery of the somatic H3 K27M mutation was reported in 2 independent publications in 2012, using exome sequencing of pediatric glioblastomas²⁷ and whole-genome sequencing of DIPG samples.²⁸ Subsequent studies have demonstrated that the H3 K27M mutation may occur in H3.1 or H3.3 isoforms of the protein and is generally a founder mutation that is retained throughout the course of the disease and throughout the tumor, though occasional mosaic immunostaining throughout a tumor has been reported in case studies.^29–31 While the presence of H3 K27M mutation co-occurs with many other genetic alterations (for example, H3.3 with p53 mutation and H3.1 with ACVR1)^32–35 it is mutually exclusive with IDH and EGFR mutations.³⁶ Studies have shown that K27M-mutant tumors are almost always MGMT promoter unmethylated, explaining the lack of benefit observed when temozolomide was added to radiotherapy for DIPG treatment.¹³ Importantly, H3 K27M-mutant tumors are notable for a global loss of H3 K27 trimethylation and an attendant gain of H3K27Ac (acetylation), rendering silencing of tumor suppressor genes and activation of tumor oncogenes.³⁷ This loss of H3K27me3 is also incorporated into current neuropathological assessments of biopsies using immunohistochemistry.

Further underscoring the appreciation of this alteration as a disease-defining mutation, the 2016 WHO classification of CNS tumors subsequently categorized H3 K27M-mutant DMG as a distinct form of Grade IV glioma, regardless of other histological features. In the 2021 update to this classification, the WHO refined this classification as H3 K27-altered, due to recognition of other pathways that may also underlie the pathogenesis of these tumors.³

The presence of the H3 K27M mutation is associated with dismal survival, although the reported prognostic magnitude relative to gliomas without the H3 K27M mutation depends on many variables.^38–43 Although the H3 K27M mutation was discovered in DIPG and is often thought to be a pediatric disease, young adult patients are also affected. Reports indicate up to 90% of pediatric DIPG cases are H3 K27M-mutant. Among adults with DMG, the H3 K27M mutation occurs in 15%–60% of cases. In a study of young adult and adult patients with thalamic tumors, the mutation was absent in all patients over the age of 50, whereas 91% of patients under the age of 50 (range, 17–46 years) were H3 K27M-mutant.⁴⁴

Current Standard of Care for H3 K27M Glioma

At present, the standard of care for patients with H3 K27M disease is radiation (total, 54–60 Gy; 2 Gy per daily fraction over 6 weeks); in patients with DIPG, 54 Gy is usually given due to the lower radiation tolerance of the brainstem.^5,40 This is the only treatment that has shown even a modest effect on survival. Given the unique biology of H3 K27M-mutant gliomas, its poor prognosis, the absence of effective systemic treatments, and the historical lack of success in applying previously developed interventions to this population, the need for new therapeutic approaches for this disease remains high.

Clinical Trials Currently Assessing Potential Therapies for H3 K27M-mutant Glioma

To identify current treatments being tested in H3 K27M-mutant glioma and DIPG, several searches were conducted on clinicaltrials.gov on July 18, 2023, with results limited to interventional studies that were not yet recruiting or recruiting. Searches were conducted using the following terms for Condition or Disease: DIPG, K27-altered, or K27M. Results for expanded access protocols were included where available. Following de-duplication, 57 unique trials remained. Fourteen of these trials were excluded due as they did not include analysis of K27M-mutant diffuse glioma or DIPG separately from other qualifying tumors (eg, solid tumors, all gliomas, etc.). An additional 2 studies were excluded, as K27M-mutant disease was excluded (n = 1) or because the subject of the study was a stratification method, rather than a treatment (n = 1). The remaining 41 studies were included in this review (Supplementary Figure S1; Supplementary Table S1).

Chemotherapy

Historically, efforts to treat DIPG with chemotherapy have proven unsuccessful, despite over 45 years of clinical trials of a wide variety of agents and regimens.⁴⁵ This could be due to the fact that many chemotherapeutic agents do not cross the BBB into tumor tissue in sufficient concentration. Two active chemotherapy trials were identified for the treatment of DIPG. The first is a single-site, open-label, early-phase I study of gemcitabine (Table 1, NCT02992015).⁴⁶ Patients aged 3–17 years may participate in this study if newly diagnosed (radiographic or histologic) with DIPG. All patients on-study receive 2 100 mg/m2 IV gemcitabine over 30 min, within 4 h of planned tumor biopsy. PK testing for levels of gemcitabine, its metabolite difluorodeoxyuridine, and gemcitabine nucleotides in peripheral blood and DIPG tumor tissue will be performed using LC-MS/MS, in order to determine if gemcitabine is able to cross the BBB.

Table 1.

Summary of Studies on ClinicalTrials.gov for Patients With H3 K27M-Mutant or H3K27-Altered Disease and/or DIPG

NCT number	Phase	Study title	Study status	Therapy type	Age
Chemotherapys
NCT03257631	II	A study of pomalidomide monotherapy for children and young adults with recurrent or progressive primary brain tumors	Active, not recruiting	Chemotherapy	Child, adult
NCT02992015	I	Gemcitabine in newly diagnosed diffuse intrinsic pontine glioma	Recruiting	Chemotherapy	Child
Re-irradiation
NCT03126266	II	Re-irradiation of progressive or recurrent DIPG	Recruiting	Radiation	Child, adult, older adult
Vaccination
NCT02960230	I/II	H3.3K27M peptide vaccine with nivolumab for children with newly diagnosed DIPG and other gliomas	Active, not recruiting	Vaccine: K27M	Child, adult
NCT04749641	I	Neoantigen vaccine therapy against H3.3-K27M diffuse intrinsic pontine glioma	Recruiting	Vaccine: K27M	Child, adult, older adult
NCT04808245	I	A MultIceNTER phase I peptide vaccine trial for the treatment of H3-mutated gliomas	Recruiting	Vaccine: K27M	Adult, older adult
NCT05096481	II	PEP-CMV vaccine targeting CMV antigen to treat newly diagnosed pediatric HGG and DIPG and recurrent medulloblastoma	Not yet recruiting	Vaccine: CMV (pp65)	Child, adult
NCT04943848	I	rHSC-DIPGVax plus checkpoint blockade for the treatment of newly diagnosed DIPG and DMG	Recruiting	Vaccine: neo-epitopes	Child, adult
Adoptive transfer
NCT05478837	I	Genetically modified cells (KIND T cells) for the treatment of HLA-A*0201-positive patients with H3.3K27M-mutated glioma	Not yet recruiting	Adoptive transfer: CAR-T	Child, adult
NCT05544526	I	CAR T cells to target GD2 for DMG	Not yet recruiting	Adoptive transfer: CAR-T	Child
NCT04185038	I	Study of B7-H3-specific CAR T-cell locoregional immunotherapy for diffuse intrinsic pontine glioma/diffuse midline glioma and recurrent or refractory pediatric CNS tumors	Recruiting	Adoptive transfer: CAR-T	Child, adult
NCT05768880	I	Study of B7-H3, EGFR806, HER2, And IL13-Zetakine (Quad) CAR T-cell locoregional immunotherapy for pediatric diffuse intrinsic pontine glioma, diffuse midline glioma, and recurrent or refractory CNS tumors	Recruiting	Adoptive transfer: CAR-T	Child, adult
NCT04196413	I	GD2 CAR T cells in diffuse intrinsic pontine gliomas (DIPG) and spinal diffuse midline glioma (DMG)	Recruiting	Adoptive transfer: CAR-T	Child, adult
NCT03396575	I	Brain stem gliomas treated with adoptive cellular therapy during focal radiotherapy recovery alone or with dose-intensified temozolomide (phase I)	Recruiting	Adoptive transfer: non-CAR-T	Child, adult
NCT04837547	I	PEACH TRIAL- precision medicine and adoptive cellular therapy	Recruiting	Adoptive transfer: non-CAR-T | vaccine: tumor Ag-pulsed DCs	Child, adult
Monoclonal antibodies
NCT03389802	I	Phase I study of APX005M in pediatric CNS tumors	Active, not recruiting	Monoclonal antibodies: anti-CD40	Child, adult
NCT02359565	I	Pembrolizumab in treating younger patients with recurrent, progressive, or refractory high-grade gliomas, diffuse intrinsic pontine gliomas, hypermutated brain tumors, ependymoma or medulloblastoma	Recruiting	Monoclonal antibodies: anti-PD1	Child, adult
Small molecules
NCT05099003	I/II	A study of the drug selinexor with radiation therapy in patients with newly diagnosed diffuse intrinsic pontine (DIPG) glioma and high-grade glioma (HGG)	Recruiting	Small molecule: selective inhibitor of nuclear export (SINE)	Child, adult
NCT02644460	I	Abemaciclib in children with DIPG or recurrent/refractory solid tumors	Recruiting	Small molecule: CDK4/6 inhibitor	Child, adult
NCT05843253	II	Study of ribociclib and everolimus in HGG and DIPG	Not yet recruiting	Small molecule: CDK4/6 inhibitor | amall molecule: mTOR inhibitor	Child, adult
NCT02420613	I	Vorinostat and temsirolimus with or without radiation therapy in treating younger patients with newly diagnosed or progressive diffuse intrinsic pontine glioma	Active, not recruiting	Small molecule: mTOR inhibitor	Child, adult
NCT04870944	I/II	CBL0137 for the treatment of relapsed or refractory solid tumors, including CNS tumors and lymphoma	Active, not recruiting	Small molecule: FACT sequesteration	Child, adult
NCT03893487	I	Fimepinostat in treating brain tumors in children and young adults	Active, not recruiting	Small molecule: HDAC and PI3K inhibitor	Child, adult
NCT02717455	I	Trial of panobinostat in children with diffuse intrinsic pontine glioma	Active, not recruiting	Small molecule: HDAC inhibitor	Child, adult
NCT04341311	I	Phase I study of marizomib + panobinostat for children with DIPG	Active, not recruiting	Small molecule: proteasome inhibitor	Child, adult
NCT05518838	EAP	Expanded access to OKN-007 for Patients with DMG, H3 K27-altered	Available	Small molecule: HIF1-alpha and VEGFR2 expression	Child, adult
NCT04049669	II	Pediatric trial of Indoximod with chemotherapy and radiation for relapsed brain tumors or newly diagnosed DIPG	Recruiting	Small molecule: indoleamine 2,3-dioxygenase (IDO) inhibitor	Child, adult
NCT02525692	II	Oral ONC201 in recurrent GBM, H3 K27M glioma, and midline glioma	Active, not recruiting	Small molecule: Imipridone	Child, adult, older adult
NCT03295396	II	ONC201 in adults with recurrent H3 K27M-mutant glioma	Active, not recruiting	Small molecule: Imipridone	Adult, older adult
NCT03416530	I	ONC201 in pediatric H3 K27M gliomas	Active, Not Recruiting	Small molecule: Imipridone	Child, adult
NCT04617002	EAP	Intermediate-size expanded access to ONC201 for patients with H3 K27M-mutant and/or Midline Gliomas	Available	Small molecule: Imipridone	Child, adult, older adult
NCT05476939	III	Biological medicine for diffuse intrinsic pontine glioma (DIPG) eradication 2.0	Recruiting	Small molecule: Imipridone	Child, adult, older adult
NCT05580562	III	ONC201 in H3 K27M-mutant diffuse glioma following radiotherapy (the ACTION study)	Recruiting	Small molecule: imipridone	Child, adult, older adult
Enhanced delivery
NCT04804709	I	Non-invasive focused ultrasound (FUS) with oral panobinostat in children with progressive diffuse midline glioma (DMG)	Active, not recruiting	FUS	Child, adult
NCT05123534	I/II	A phase I/II study of sonodynamic therapy using SONALA-001 and Exablate 4000 Type 2 in patients With DIPG	Recruiting	FUS	Child, adult, older adult
NCT05615623	I/II	Blood brain barrier (BBB) disruption using exablate focused ultrasound with doxorubicin for treatment of pediatric DIPG	Recruiting	FUS	Child, adult
NCT05630209	I/II	Blood brain barrier (BBB) disruption using exablate focused ultrasound with doxorubicin for treatment of pediatric DIPG	Recruiting	FUS	Child, adult
NCT05762419	I	FUS etoposide for DMG—a feasibility study	Recruiting	FUS	Child, adult
Other therapies
NCT04771897	I	A study of BXQ-350 in children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) or diffuse midline glioma (DMG)	Recruiting	Novel/unclassed: SapC-nanovesicle	Child, adult
NCT04758533	I/II	Clinical trial to assess the safety and efficacy of AloCELYVIR with newly diagnosed diffuse intrinsic pontine glioma (DIPG) in combination with radiotherapy or medulloblastoma in monotherapy	Recruiting	Oncolytic virus	Child, adult
NCT03033992	I/II	Optune for children with high-grade glioma or ependymoma, and optune with radiation therapy for children with DIPG	Active, not recruiting	Tumor-treating field	Child, adult

A second novel chemotherapy study is designed to investigate the efficacy of pomalidomide monotherapy as a treatment for recurrent or progressive DIPG or HGG (Table 1, NCT03257631).⁴⁷ Pomalidomide is an immunotherapeutic agent known to enhance T cell- and natural killer cell-mediated immunity.⁴⁸ It also inhibits production of proinflammatory cytokines, such as TNF-α and IL-6, by monocytes, and has antiangiogenic properties.⁴⁸ In this multi-site, open-label phase II trial, patients 1–21 years of age will be treated with oral pomalidomide, where objective response rate (ORR) and long-term stable disease rate will be measured as primary outcomes.

Re-irradiation

Fractionated radiation of approximately 60 Gy is the mainstay of DIPG therapy. However, while this may provide relief of symptoms for approximately 6 months. A Canadian consortium is currently investigating the efficacy of re-irradiation for patients of any age with progressive or recurrent DIPG (Table 1, NCT03126266).⁴⁹ Patients may qualify for this multisite study if they have evidence of progression or recurrence after receiving a cumulative radiation dose of ≥60 Gy at least 180 days prior to re-irradiation. Participants receive 30.6 Gy or 36 Gy as a second course of radiation therapy and are evaluated for second PFS as the primary outcome measure, with OS as a secondary outcome measure.

Vaccination

H3K27M Vaccines

Midline gliomas are often characterized as immunologically cold due to the absence of tumor-infiltrating lymphocytes (TILs). This is reflected by immune-oncology (IO) agents so far having failed to demonstrate efficacy in this disease.⁵⁰ In an effort to transform the tumor microenvironment into an immunologically hot state, in which immune cells detect and kill cancer cells, vaccines utilizing tumor antigens are being evaluated in many forms of cancer to determine if they can engender a tumor-specific immune response. Three early-phase clinical trials were identified on clinicaltrials.gov that are evaluating vaccination in patients with H3 K27M-mutant disease. One additional study is designed to examine the efficacy of a novel peptide vaccine in treating newly diagnosed pediatric HGG and DIPG, while a final study investigates a neo-epitope vaccine for the treatment of newly diagnosed DMG and DIPG.

A synthetic H3 K27M peptide (H3.3K27M_26–35) was identified as a potential binder to HLA-A*0201 using MHC-binding algorithms. In murine models, this peptide induced a mutation-specific immune response, including both cytotoxic and T-helper-1-cell mediated immune responses.⁵¹ In a murine xenograft model of glioma, adoptively transferred CD8 + T-cells transduced with a H3.3K27M_26–35-specific T-cell receptor (TCR) resulted in a significant delay in disease progression.⁵² To evaluate the feasibility and efficacy of vaccination with the K27M synthetic peptide in patients, a phase I/II peptide vaccine trial with polyIC ± nivolumab is currently underway in pediatric patients with newly diagnosed H3 K27M-mutant DIPG or nonpontine DMG (Table 1, NCT02960230); as this vaccine targets a human leukocyte antigen (HLA)-restricted epitope, all patients must be HLA-A2 (02:01)+. In their publication on this ongoing trial, Mueller, et al. (2020) reported that among 18 patients with DIPG and available data, 6 demonstrated a H3 K27M-reactive immune response. The vaccine was well tolerated; the most common treatment-related adverse event (TRAE) was injection site reaction.⁵³

Also utilizing a synthetic H3.3K27M peptide vaccine, the phase I trial INTERCEPT H3 trial is designed to evaluate the H3 K27M vaccine in combination with radiotherapy and the anti-PD-L1 antibody atezolizumab in adults with H3 K27M-mutant DMG (Table 1; NCT04808245). Immune checkpoint inhibitors are antibody therapeutics that prevent inhibition of T-cell proliferation, potentially boosting immune responses against tumor cells, thereby amplifying immune-mediated anticancer effects resulting from the cancer vaccine. Atezolizumab, an anti-PD-L1 monoclonal antibody, has previously demonstrated safety in adults with glioblastoma; however, small trials failed to give evidence of clinical activity. This phase I trial will evaluate the efficacy of 11 doses of the H3 K27M vaccine in patients receiving standard radiation followed by a course of atezolizumab delivered once every 3 weeks. The primary endpoints include safety and assessment of immunogenicity. This study is currently open and enrolling patients.

ENACTING, a phase I study of the H3.3-K27M targeted neoantigen peptide in combination with standard-of-care radiotherapy, is being conducted in pediatric and adult patients with H3 K27M-mutant DIPG with the HLA-A2 subtype (Table 1, NCT04749641). The primary objectives are safety and a 1-year OS rate. This trial is currently enrolling patients.

Importantly, other research groups have failed to demonstrate that vaccination results in endogenous processing and presentation of the H3 K27M epitope by cancer cells. With these early-phase clinical trials ongoing, the role of vaccination in H3 K27M-mutant disease will be better understood in due time.

PEP-CMV Vaccine

A multicenter phase II study is being conducted to determine the efficacy of a novel peptide vaccine, PEP-CMV, which targets CMV antigen in the treatment of patients aged 3–25 years with newly diagnosed pediatric HGG and DIPG (Table 1, NCT05096481).⁵⁴ In this study, patients must enroll within 6 weeks of their final dose of standard-of-care radiation therapy (required for DIPG patients), with or without chemotherapy. Participants will receive standard chemotherapy with temozolomide for 5 days, followed by the study vaccine, intradermal PEP-CMV on day 21. The primary outcome measure is the 1-year OS for both HGG and DIPG.

Neo-epitopes

The rHSC-DIPGVax is a neo-antigen heat shock protein containing 16 peptides reflecting neo-epitopes found in the majority of DIPG and DMG tumors. A multicenter trial testing vaccination targeting these neo-epitopes in pediatric DMG and DIPG is currently recruiting (Table 1, NCT04943848). This study is a phase I clinical trial evaluating the safety and tolerability of rHSC-DIPGVax in combination with Balstilimab and Zalifrelimab (immune checkpoint inhibitors). Newly diagnosed patients with DIPG and DMG who have completed radiation 6–10 weeks prior to enrollment are eligible for this study.

Adoptive Transfer

Chimeric Antigen Receptor T-cell Therapy

Chimeric antigen receptor T-cell (CAR-T) therapy is a treatment that entails isolating autologous T cells from a patient’s blood and transducing them to express a TCR specific for a cancer-associated antigen. When these CAR-T cells are reintroduced to the patient’s system, these antigen-specific T cells help steer anticancer immune responses, potentially resulting in the clearance of cancer cells while leaving healthy cells intact. Currently approved CAR-T therapies are limited to B-cell malignancies; however, ongoing work is evaluating the application of CAR-T therapy in patients with solid tumors.⁵⁵ On clinicaltrials.gov website, 3 phase I trials are active or planned to determine the safety, efficacy, and feasibility of CAR-T therapy in patients with H3 K27M-mutant disease.

It was previously established that patient-derived H3 K27M-mutant glioma cell cultures over-express the disialoganglioside GD2, and subsequent in vitro studies demonstrated the anticancer efficacy of anti-GD2 CAR-T cells in murine xenograft models.⁵⁶ Currently, early-phase trials are assessing the feasibility of anti-GD2 CAR-T cell therapy in patients with H3 K27M-mutant disease. A phase I trial assessing the feasibility, safety, and efficacy of GD2-CAR T cell therapy in patients with H3 K27M-mutant DIPG/spinal DMG is currently recruiting (Table 1; NCT04196413). Patients with tumor involvement in the cerebellar vermis or hemisphere, thalamus, or supratentorial area are not eligible. The study will evaluate 3 dose levels and compare intravenous and intracerebroventricular delivery, with primary endpoints of successful manufacture of GD2-CAR-T cells, maximum tolerated dose, and safety. An assessment of the first 4 patients treated in the study found that 3 patients experienced radiographic and clinical improvement, including improved motor function. Additionally, the presence of TIL in the tumor suggests a conversion to an immunologically hot state. However, safety remains a concern, as immune responses invariably are associated with inflammation, which could present an issue in the brainstem region. As a precaution, all patients enrolled on trial were therefore required to undergo placement of a ventricular reservoir, allowing rapid monitoring of intracranial pressure and removal of cerebrospinal fluid to mitigate swelling. All 4 patients experienced cytokine release syndrome. Toxicity was reversible but required intensive supportive inpatient care.⁵⁷

Additionally, the phase I CARMIGO trial will assess the feasibility, safety, and efficacy of GD2-CAR T cell therapy in a single-center trial (Table 1; NCT05544526). This study, which has not yet begun recruiting, will enroll pediatric and young adult patients with H3 K27M-mutant DMG, and will require placement of a ventricular reservoir prior to treatment. Initial therapy will be delivered intravenously; patients who do not demonstrate a response 28 days following administration and who have not experienced intolerable toxicity will be eligible to receive a second intraventricular administration of CAR-T cells.

CAR-T therapy is also under investigation using a H3 K27M-specific TCR in place of the GD2-specific trials described above. A phase I dose-escalation trial will evaluate the dosage and safety of administration of autologous anti-H3.3K27M TCR-expressing CAR-T cells (KIND cells) in HLA-A*02:01-positive pediatric and young adult patients with H3.3K27M-mutated DMG. Treatment will be delivered intravenously. This trial is not yet recruiting. However, similar to the concerns raised around the ultimate viability of the H3 K27M synthetic peptide vaccine, there is some concern that the lack of endogenous processing of the H3 K27M peptide limits the efficacy of therapies relying on immune responses to this antigen.

Last, an innovative trial, SC-CAR4BRAIN, is underway to evaluate a quadrivalent CAR-T cell therapy against B7-H3, EGFR806, HER2, and IL13-zetakine. Given the design against 4 distinct antigens, this trial is of great interest to the neuro-oncology community. This study also requires an indwelling ventricular access device and is open to patients with pontine and nonpontine DMG (NCT05768880).

Non-CAR-T Cell Adoptive Transfer Therapies

Adoptive transfer is a technology by which the transfer of a patient’s own immune cells back into their bloodstream is performed following ex vivo manipulation. Patients are usually induced to be lymphopenic (lymphodepletion) prior to reintroduction via administration of myeloablative and non-myeloablative chemotherapy, as this has been shown to result in dramatic T cell expansion and potent immunologic responses. There are currently 2 trials utilizing this approach for the treatment of DMG. The first is the BRAVO trial, designed to investigate the efficacy of TMZ, which produces profound lymphopenia in children with CNS tumors, as an adjuvant therapy during and following radiotherapy for newly diagnosed brainstem gliomas (Table 1, NCT03396575).⁵⁸ In this study, patients receive tumor-specific lymphocytes, expanded ex vivo with the use of TTRNA-pulsed dendritic cells (DCs), that preferentially expand in this lymphopenic environment following TMZ administration, and serve as a source of responder cells to subsequent DC vaccination. The second study utilizing this approach is the PEACH trial (Table 1, NCT04837547).⁵⁹ This is a phase I open-label, multicenter study, to evaluate the safety, feasibility, and MTD of DIPG treatment with adoptive cell therapy (Total tumor mRNA-pulsed autologous DCs [TTRNA-DCs], Tumor-specific ex vivo expanded autologous lymphocyte transfer [TTRNA-xALT] and Autologous G-CSF mobilized Hematopoietic Stem Cells [HSCs]).

Monoclonal Antibodies

One increasingly popular cancer immunotherapy approach is to use monoclonal antibodies to target ligands on immune cells in order to stimulate an immune response to attack cancer cells.⁶⁰ Two trials were identified that test this approach for pediatric brain tumor treatment, including HGG and DIPG.

Anti-CD40

First, APX005M is a CD40 agonistic monoclonal antibody that binds to CD40, triggering B cell, monocyte, and DC activation, and stimulating cytokine release from lymphocytes and monocytes. CD40 is also expressed on many human tumor cells, hence APX005M can mediate a direct cytotoxic effect on CD40 + tumor cells. A multicenter, nonrandomized open-label Phase I trial (Table 1, NCT03389802) is currently underway to evaluate the safety and tolerability of APX005M in children with recurrent or refractory primary malignant CNS tumors including HGG or newly diagnosed DIPG.⁶¹

Anti-PD-1

Pembrolizumab (MK-3475) is an anti-PD-1 monoclonal antibody that acts as an immune checkpoint inhibitor by binding its ligands, PD-L1 and PD-L2. This drug has been investigated as a potential treatment for a variety of high-grade pediatric brain tumors, initially including DIPG, through a multicenter, phase I open-label study supported by the Pediatric Brain Tumor Consortium (Table 1, NCT02359565).⁶² Unfortunately, the DIPG arm closed early due to worse OS and the rapid decline of the patients.⁶³ In this study, patients aged 1–30 years with a recurrent, progressive, or refractory DIPG, following radiation therapy with or without chemotherapy, were treated with Pembrolizumab. Primary outcome measures the included incidence of AEs within 30 days of treatment, sustained objective response (partial response + complete response) within 12 cycles (approximately 9 months), and the change in the percentage of CD8 + T cells that are PD-1 + due to treatment with pembrolizumab via serial serum testing.

Small Molecules

Despite the prevalence of small molecules for a variety of cancers, few compounds are being evaluated in DIPG and H3 K27M-mutant-exclusive populations. Our search identified 16 clinical trials evaluating small molecules in patients with H3 K27M-mutant disease or DIPG. These studies are designed to investigate a wide variety of treatment strategies, from immunotherapy to epigenetic-targeted agents, as described subsequently.

Selinexor

Selinexor is an inhibitor of exportin-1 (XPO-1), a nuclear export protein that is over-expressed in multiple solid tumors, including gliomas, where its upregulation is associated with a worse prognosis.⁶⁴ Selinexor was previously approved by the FDA for relapsed/refractory multiple myeloma and relapsed/refractory diffuse large B-cell lymphoma.⁶⁵

In preclinical models of glioblastoma, selinexor demonstrated anticancer efficacy in cell lines and murine models.⁶⁶ A subsequent phase II, open-label study of molecularly unselected adults with recurrent glioblastoma evaluated the safety, efficacy, and pharmacokinetics of selinexor monotherapy. Biopsy of patients undergoing resection indicated that intratumor concentrations of selinexor reached therapeutic levels. In a trial on recurrent glioblastoma, the 6-month PFS varied in the various study arms between 7.7 and 17%; further trials are ongoing.⁶⁷ As preclinical research has indicated, selinexor may act as a radiosensitizer in models of GBM.⁶⁸ A phase I/II study of selinexor in combination with radiotherapy is currently being conducted through the Children’s Oncology Group in children and young adults with DIPG or H3 K27M-mutant DMG (Table 1; NCT05099003). Patients will receive standard-of-care radiation in combination with selinexor, followed by maintenance selinexor after completion of radiotherapy. The primary objectives include maximum tolerated dose, event free survival, OS, and overall response rate. From the study design and protocol information publicly available, it appears that a group of patients in Step 1 will be required to have H3 K27M-mutant disease; however, broader populations of patients with H3 K27-WT HGG or DIPG will also be evaluated.

CDK 4/6 Inhibitors

The next category of small molecule inhibitors includes 2 studies examining the effects of cyclin D-cyclin-dependent kinase (CDK4/6) pathway inhibition. The CDK4/6-p16-retinoblastoma (Rb) pathway is commonly disrupted in cancers, including HGG, resulting in abnormal cell proliferation causing tumorigenesis.⁶⁹ Chemotherapeutic agents targeting this CDK4/6 pathway were shown to have antitumor effects in multiple clinical studies of a variety of tumors, and are currently used in combination with hormone therapy to treat some breast cancers. One such CDK4/6 inhibitor, abemaciclib, is being investigated in a nonrandomized parallel assignment, phase I clinical trial in children and young adults with newly diagnosed DIPG (Table 1, NCT02644460).⁷⁰ In this study, DIPG patients between 2 and 25 years of age will receive RT in 30–33 fractions over approximately 6 weeks. Treatment with oral abemaciclib is initiated with RT, and continues twice daily during and after RT, for a maximum treatment duration of 2 years. Primary outcome measures include the determination of the MTD of abemaciclib in DIPG patients, as well as PK properties.

The efficacy of a second CDK4/6 inhibitor, ribociclib, is being evaluated alone and in combination with everolimus, an mTOR inhibitor (see following sections) for the treatment of HGG and DIPG (NCT05843253).⁷¹ The rationale for this approach is that CDK4/6 inhibitors alone have not shown a response in HGG.⁶⁹ In this multicenter, international, phase II study, patients aged 12 months to 30 years old with newly diagnosed with HGG or DIPG that harbors alterations of the cell cycle and/or PI3K/mTOR pathways will receive a combination of oral ribociclib and everolimus after receiving radiotherapy (RT). Efficacy (primary outcome measure) is defined by progression-free survival (PFS) in HGG patients (Stratum A), and OS in DIPG patients (Stratum B).

mTOR Inhibition

Dysregulation of mTOR signaling, including PI3K amplification/mutation and PTEN loss of function, has been noted in a variety of cancers. As a result, mTOR inhibitors have been explored alone and in combination with anticancer therapy, including HGG. In addition to the above study examining ribociclib (CDK4/6 inhibitor) with everolimus (mTOR inhibitor), a second mTOR inhibitor, temsirolimus, is currently under clinical trial investigation (Table 1, NCT02420613) in combination with vorinostat, an HDAC inhibitor (see below).⁷² Patients 7 months to 20 years old with newly diagnosed or progressive DIPG may enroll. In this single-center phase I, nonrandomized, dose-escalation study. The primary goals of the study are to examine the MTD and potential side effects (AEs) of increasing the dosage of temsirolimus in combination with vorinostat for DIPG treatment.

FACT Complex-targeting Curaxin CBL0137

Curaxin CBL0137 is a molecule that regulates p53 and NF-κB to exert antitumor activity in multiple cancers, including HGG. It is thought that CBL0137 works by binding to tumor DNA through the facilitates chromatin transcription (FACT) complex, targeting cancer stem cell proliferation.⁷³ Currently, 1 trial is designed to investigate the efficacy of CBL0137 for the treatment of CNS tumors, including H3K27M mutant DMG and DIPG (Table 1, NCT04870944). The goal of this phase II study is to determine the MTD of CBL0137 and to determine the ORR in children with progressive/recurrent DIPG and other H3 K27M-mutant DMG.

HDAC Inhibition

Because of the distinct epigenetic events driving DMG and H3K27M mutant glioma formation, studies to investigate multiple different epigenetic targeted therapies have been performed or are currently underway. One such strategy includes inhibition of a family of enzymes known as HDAC, which effectively maintains an acetylated histone state to promote the expression of tumor suppressor genes and inhibit oncogene expression. Although a phase I/II study of vorinostat and radiotherapy in patients with newly diagnosed DIPG failed to demonstrate improved outcome measures relative to historical controls,⁷⁴ 2 clinical trials of HDAC inhibition in DIPG and/or H3K27M mutant glioma were identified by our search.

The first is a multicenter, early-phase I (target validation) study examining fimepinostat in children and young adults with newly diagnosed DIPG, recurrent medulloblastoma, or recurrent HGG (NCT03893487).⁷⁵ In this study, patients 3–39 years old will receive oral fimepinostat, and then drug and metabolite concentration will be measured in primary tumor tissue post-treatment to confirm penetration of fimepinostat across the BBB (primary objective).

The second trial is a study of the efficacy of panobinostat, a HDAC inhibitor, for the treatment of DIPG (Table 1, NCT02717455). Panobinostat also induces expression of cell-cycle control genes including CDKN1A (p21), and selectively inhibits tumor cell proliferation in DIPG cell cultures and orthotopic xenograft models. This phase I study of panobinostat in children with recurrent/progressive DIPG has been accruing subjects since 2016, with the goal of determining the MTD and PK properties of the drug.

Proteasome Inhibition

A variety of proteaosome inhibitors are currently under clinical trial for different cancer types, including glioma. It is generally understood that proteasome inhibitors prevent the degradation of proapoptotic molecules, including p53, thereby promoting apoptosis. One such molecule is Marizomib, which is currently under investigation for the treatment of newly diagnosed DIPG (NCT04341311) in combination with Panobinostat (see HDAC inhibition section).⁷⁶ This single-center, phase I, nonrandomized trial for DIPG patients up to 21 years old consists of 2 parts. First, the MTD of IV Marizomib will be determined. Next, subjects who tolerate IV Marizomib alone will be treated with IV Marizomib and oral Panobinostat, but at a lower dose of Marizomib than the dose of marizomib when given alone. Primary outcome measures are the determination of the MTD and dose-limiting toxicity (DLT) for Marizomib alone and Marizomib in combination with Panobinostat, as well as pharmacokinetic properties.

HIF1-alpha and VEGFR2 Expression Targeting

OKN-007 is a small molecule agent that inhibits both HIF-1 protein expression and VEGFR2 protein expression in glioma, thereby impeding tumor angiogenesis.⁷⁷ OKN-007 has also been shown to increase glioma cell apoptosis.⁷⁷ Given these properties, OKN-007 has been evaluated in murine models of DIPG,⁷⁸ and is now being evaluated in recurrent glioblastoma; expanded access for DIPG patients is currently underway (Table 1, NCT05518838).

Indoleamine 2,3-dioxygenase (IDO) Inhibition

Amongst immunotherapeutic strategies, Indoleamine 2,3-dioxygenase (IDO) inhibition has been investigated for a variety of cancer types, including glioma. IDO is a tryptophan-degrading enzyme that has been found to be expressed by cancer cells. Tumor cell expression of IDO has an immunosuppressive effect, increasing apoptosis of cytotoxic T lymphocytes and converting naïve T cells into inducible immunosuppressive regulatory T cells (Tregs; CD4 + CD25 + FoxP3 +).⁷⁹ As such, targeting this metabolic pathway can enhance the immune response against tumor cells. One trial of this strategy for brain tumor treatment, including DIPG, is currently being conducted (NCT04049669).⁸⁰ This phase II, nonrandomized, open-label, multicenter trial is designed to investigate the efficacy of brain tumor treatment with the oral IDO inhibitor Indoximod and radiation therapy (RT) in combination with temozolomide, with etoposide, cyclophosphamide, and/or lomustine for salvage therapy. This investigation consists of 4 distinct cohorts of patients: those with progressive glioblastoma, medulloblastoma, or ependymoma, and those with newly diagnosed DIPG (no prior radiation or other therapy, biopsy not required). For DIPG patients, the primary outcome measure of this study 12-month of OS.

ClpP/DRD2 Targeting

ONC201 is a first-in-class small molecule first-in-class dopamine receptor D2 (DRD2) antagonism and caseinolytic protease P (ClpP) agonist.⁸¹ ONC201 has been evaluated in several glioma trials and recently, an integrated analysis of 5 open-label studies found that the monotherapy ORR assessed by blinded independent central review using RANO-HGG and RANO-LGG criteria among pediatric and adult patients with recurrent H3 K27M DMG was 30.0%, with no treatment-related grade 4 TEAEs, deaths, or discontinuations reported; median duration of response and time to response were 11.2 months (95% CI, 3.8, not reached) and 8.3 months (range, 1.9–15.9), respectively.⁸² Previous research has confirmed that oral ONC201 administration results in therapeutic intratumoral concentrations,⁸³ efficacy in nonmidline H3 K27M-mutant diffuse gliomas⁸⁴, reduction in H3 K27M circulating tumor DNA (ctDNA) in cerebrospinal fluid following ONC201 treatment, and reversal of H3 K27 trimethylation loss, which is a hallmark characteristic of the H3 K27M mutation and independently associated with negative prognosis.⁸⁵

There are currently 5 active clinical trials evaluating ONC201 in at least 1 treatment arm or cohort that proactively requires the presence of the H3 K27M mutation; of these, an expanded access protocol and the phase III trial are currently recruiting patients, and the remaining 3 are closed to further enrollment. The expanded access protocol (Table 1; NCT04617002) is available for pediatric and young adult patients with H3 K27M-mutant glioma who previously received radiotherapy and are not eligible for other ongoing ONC201 trials. ONC201 will be taken once weekly; adult patients will receive 625 mg and pediatric patients will receive a dose scaled by body weight.

A phase I trial of ONC201 in pediatric patients with newly diagnosed DIPG and relapsed/refractory pediatric glioma (Table 1, NCT03416530) is currently assessing several ONC201 formulations and treatment schedules, including twice-weekly dosing on consecutive days.⁸⁶ The primary objective is determination of the recommended phase II dose. Requirements for H3 K27M status vary by cohort; some arms of the trial require H3 K27M-mutant glioma.

A phase II trial of young adult and adult patients (≥16 years old) is assessing ONC201 in recurrent HGG (Table 1; NCT02525692). This study has several treatment arms, including one that requires H3 K27M mutant disease. The primary objective is a 6-month PFS.

A phase II trial is assessing ONC201 in adults with recurrent H3 K27M-mutant HGG (Table 1; NCT03295396). All treatment arms require H3 K27M-mutant disease. The primary endpoint is the best overall response.

Finally, ACTION, a phase III, randomized, placebo-controlled study of ONC201 following radiotherapy in adult and pediatric patients with newly diagnosed, H3 K27M-mutant diffuse glioma is currently enrolling (Table 1; NCT05580562). Eligible patients who are 2–6 weeks from prior radiation are randomized 1:1:1 to receive twice-weekly ONC201, once weekly ONC201, or placebo. Pontine and spinal tumors are excluded from this study. The primary endpoints are overall survival and progression-free survival by RANO-HGG criteria per BICR. ACTION is currently enrolling at global sites, and the study design and eligibility criteria are discussed in detail in Arrillaga-Romany et al. (2023).⁸⁷

Of note, a recent report by Cantor et al demonstrates the feasibility of cell-free tumor DNA tracking to predict ONC201 treatment response and disease progression in DMG.⁸⁸ This approach has great potential to improve the clinical care and outcomes of these patients via treatment stratification and longitudinal response monitoring, without the need to repeat tissue biopsy.

Delivery Enhancement

Focused Ultrasound

An area of novel research is an investigation of the use of focused ultrasound (FUS) to open the BBB, to improve delivery, and thereby efficacy, of chemotherapeutics targeted to H3K27M mutant disease and DIPG. FUS is a technique whereby microbubbles and neuronavigator-controlled sonication opens the BBB noninvasively. This technique is currently being applied in clinical trials for the treatment of a variety of brain tumor types, including glioblastoma and DMG, after being demonstrated to be safe and effective in mouse models of disease.^89–92 A total of 4 distinct clinical trials investigating the use of FUS for H3K27M mutant disease and/or DIPG or DMG treatment were identified on clinicaltrials.gov using the current described search of the literature.

The first study is being conducted by Wu and colleagues at Columbia University in New York, NY in order to examine the efficacy of oral Panobinostat administration with the use of FUS (Table 1, NCT04804709). The purpose of this study is to evaluate the safety and feasibility of BBB opening with FUS in young patients with progressive/recurrent DMG. In this study, patients aged 4–21 years with clinical and/or radiographic progression of DMG (defined as a radiological diagnosis of with tumor involving the pons, thalami, and/or histological confirmation of H3K27M mutation in a pontine or thalamic glioma), are being actively enrolled. All patients will be treated with FUS, followed by administration of 15 mg of oral Panobinostat. The trial then will follow a 3 + 3 Number of Tumor Sites (NOTS) escalation design, with the NOTS defined as the number of openings created in the BBB using FUS, increased incrementally from 1 to 3 if no DLTs are observed. The primary outcome measure is the number of adverse events measured up to 90 days after the end of the last FUS treatment.

A second trial by the same investigators is being conducted to examine the feasibility of FUS-enhanced delivery of etoposide chemotherapy for DMG patients (Table 1, NCT05762419). Patients aged 4–21 years with DMG are being actively enrolled. Subjects will receive FUS followed by once daily oral etoposide (50 mg) taken every day for 21 days, followed by 1 week of rest. In the first cycle, etoposide will be administered immediately following radiographic (MRI) confirmation of BBB opening, which must be performed within 4 h of FUS treatment. For subsequent cycles (maximum 4), etoposide will be administered immediately following the FUS procedure. Primary outcome measures include the number of adverse events AEs measured up to 90 days after the end of the last FUS treatment, and the number of patients with successful opening of the BBB as measured by contrast-enhanced MRI.

Another study is being conducted (Table 1, NCT05615623)⁹³ to investigate the safety, feasibility, and preliminary efficacy of FUS in pediatric patients with DIPG undergoing Doxorubicin chemotherapy. In this prospective, single-arm, nonrandomized feasibility study, patients 5–18 years old with DIPG will undergo 3 treatment cycles, approximately 4–6 weeks apart. The primary outcome measure for these studies is any adverse event after treatment until the conclusion of the study (2 years).

Last, a multicenter study is currently being conducted to investigate the safety and tolerability of sonodynamic therapy using SONALA-001 and Exablate Type-2 device and to determine the maximum tolerated dose (MTD) or recommended phase II dose (RP2D) of MR-Guided Focused Ultrasound (MRgFUS) energy in combination with SONALA-001 (ALA) in subjects with DIPG (Table 1, NCT05123534).⁹⁴ To be eligible for this nonrandomized, dose-escalation study, patients aged 5 years or older must have newly diagnosed, radiographically typical DIPG with or without histologic confirmation. MTD of SONALA-001 and Exablate 4000 Type-2 in combination will be determined. SONALA-001 will be given 6–9 h prior to receiving 1 of 3 energy levels of MRgFUS. Primary outcome measures include safety and tolerability of dose escalation, as measured by the frequency and severity of dose-limiting toxicities (DLTs), AEs, and the MTD of MRgFUS energy in combination with SONALA-001. Additional secondary outcomes include: the mechanical performance of Exablate 4000 Type 2.0 device, and the PK of ALA and Protoporphyrin IX (PpIX) following IV dosing with SONALA-001.

Other Novel Interventions

There remain 3 additional trials for H3K27M glioma and DIPG treatment that warrant mention here. These studies all employ novel approaches to anticancer therapy. The first is an investigation of the safety and efficacy of BXQ-350 for the treatment of newly diagnosed DIPG or DMG (Table 1, NCT04771897). BXQ-350 is a novel agent configured from 2 components: Saposin C (SapC), an expressed (human) lysosomal protein, and the phospholipid²⁶ oleoyllphosphatidyl-serine (DOPS), a cell membrane phospholipid. This phase I study will focus on establishing the MTD of BXQ-350, and then measuring drug concentrations in biopsied DMG tissue, including DIPG.

The second study of interest is an investigation of the utility of an oncolytic virus, AloCELYVIR, in treating newly diagnosed DIPG in combination with radiotherapy (Table 1, NCT04758533).⁹⁵ In this open, nonrandomized, single-center phase I clinical trial, patients aged 1–21 years with previously untreated DIPG are treated with AloCELYVIR infusion (bone marrow-derived allogenic mesenchymal allogenic cells infected with an oncolytic adenovirus) ICOVIR-5, a potent and selective oncolytic adenovirus-based on the pRB pathway weekly over the course of 8 weeks, with DLT determination at 1 month as a primary outcome measure.

The last is the application of tumor-treating fields using the Optune device in combination with radiation therapy for the treatment of DIPG (NCT03033992). The Optune System produces alternating electrical fields, called tumor treatment fields (TTFields) via 4 transducer arrays placed on the shaved scalp. The very low intensity, intermediate frequency electric fields impair the growth of tumor cells by arresting cell division and inducing apoptosis. Preclinical studies have demonstrated TTFields synergistically enhance the efficacy of irradiation in glioma cell lines, therefore in this trial children with DIPG will also receive concurrent standard RT. This single-arm trial will focus on the feasibility of wearing the Optune device for ≥18 h per day, and the safety and efficacy of Optune treatment with standard RT for DIPG patients.

Conclusion

Despite the recognition of H3 K27M as a molecular alteration defining a class of HGG,^3,96 to date this has failed to improve the established treatment paradigm. Several clinical trials are currently ongoing that focus specifically on this disease subset, as well as on the treatment of DMGs of the brainstem that may or may not carry this mutation (DIPG). At writing, 642 planned, recruiting, or active interventional brain cancer trials were indexed on clinicaltrials.gov. In contrast, a search for H3 K27M-mutant disease and/or DIPG identified only 41 such trials that proactively included treatment arms or cohorts to evaluate therapeutics in an exclusive population of patients with these conditions. The main 3 categories of therapeutics currently under clinical evaluation for this patient population are chemotherapy, vaccines, adoptive transfer therapy, small molecules, and FUS.

Our summary of clinical trials conducted in H3 K27M-mutant disease and DIPG has limitations. The intention of the search strategy was to identify trials focusing on H3 K27M-mutant glioma and DIPG. However, due to the variation in detail provided on clinicaltrials.gov, the structure of some analyses and groups is not universally detailed. In our selection of trials for inclusion here, many studies were excluded, as they did not detail in their listing a treatment arm or cohort evaluating the intervention exclusively in a population of patients with H3 K27M or DMG, including DIPG. However, it is possible that some trials intend to evaluate interventions in this disease subset, but did not provide sufficient detail in the clincialtrials.gov listing for qualification for this summary. Of note, additional resources exist, including the DIPG Registry and DIPG Trials Toolbox, which are designed to help pair families with existing clinical trials for DIPG. Some notable analyses and treatments that may have relevance for the treatment of H3 K27M-mutant diffuse glioma were therefore omitted, including a retrospective analysis of panobinostat in adults with H3 K27M-mutant DMG, which demonstrated tolerability and a median overall survival of 42 months,⁹⁷ and the Biological Medicine for Diffuse DIPG Eradication (BIOMEDE trials). The first BIOMEDE trial evaluated erlotinib, dasitinib, and everolimus in pediatric patients with diffuse pontine glioma, and BIOMEDE 2.0 (Supplementary Table S1, NCT05476939) evaluates everolimus, ONC201, and radiotherapy in patients with DIPG or H3 K27M-mutant DMG. Additionally, a phase II trial evaluating ONC201 in combination with panobinostat or paxalisib is currently underway in pediatric and young adult patients with DMG; while H3 K27M is not required for eligibility, it is permitted in several cohorts (NCT05009992).

A final major development in the care and management of DMGs that warrants mention is the advent of stereotactic tumor biopsy for histological diagnosis and molecular analysis of these deep-seated, unresectable tumors. To this end, an important multicenter study utilizing upfront biology-guided treatment, including tumor biopsy tissue as well as liquid biopsy specimens, was recently completed.⁹⁸ In this study, DIPG patients underwent tumor biopsy, and tissue was submitted for whole-exome and mRNA sequencing. After undergoing standard radiotherapy treatment, molecular data was reviewed by a tumor board and patients were subsequently assigned up to 4 FDA-approved drugs. H3K27M-mutant ctDNA was also longitudinally measured throughout diagnosis and treatment in blood serum, predicting response to therapy and disease recurrence. Although not included in our systematic analysis (due to having been completed), this groundbreaking study demonstrates the feasibility of molecular subtyping for treatment stratification and lays the foundation for future liquid biopsy for clinical correlates of treatment response and resistance.

Despite strides in understanding of the molecular underpinnings of DMG and H3 K27M-mutant disease, few therapies have demonstrated efficacy in this population. There is a significant unmet need for treatments effective in patients with H3 K27M-mutant disease. Due to the relatively rare patient population, enrollment of patients in active clinical trials will be vital in the development and eventual approval of novel therapies in this indication.

Conflict of Interest

M.v.d.B., M.G., and E.F. have no relevant conflicts of interest to declare. A.M.S. is a paid consultant of Chimerix, Inc.

Supplement sponsorship

This article appears as part of the supplement “H3 K27M-mutant Glioma: Disease State Overview,” sponsored by Chimerix, Inc.

Author Contributions

Study Design: MvdB, MG, EF, ASM. Data Collection: non-applicable. Statistical Analyses: non-applicable. Manuscript Preparation: MvdB, MG, EF, ASM. Manuscript Review: MvdB, MG, EF, ASM.