We identified eight patients diagnosed with pHGG between 2015 and 2022. In the same period, a total number of 1.339 patients with grade 4 glioma were diagnosed at our institution.

Table 1 provides an overview of patient characteristics.

Table 2 summarizes preoperative MR-imaging features of all our cases.

Figure 1 shows a t-distributed stochastic neighbor embedding (t-SNE) analysis of DNA methylation data of the eight diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype together with a reference cohort of 19 different molecular tumor classes (n = 650).

T-distributed stochastic neighbor embedding (t-SNE) analysis of DNA methylation data of the eight cases of diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype in this series together with a reference cohort of 19 different molecular tumor classes (n = 650). Reference methylation classes: a IDH HG astrocytoma, IDH-mutant, high grade (41 cases); A IDH LG astrocytoma, IDH-mutant, low grade (81 cases); AG MYB angiocentric glioma, MYB/MYBL1-altered (14 cases); DLGNT 1 diffuse leptomeningeal glioneuronal tumor, subtype 1 (6 cases); DMG K27 diffuse midline glioma, H3 K27M mutant (74 cases); GBM MES ATYP glioblastoma, IDH-wildtype, mesenchymal subtype, subclass B (novel) (3 cases); GBM MES TYP Glioblastoma, IDH-wildtype, mesenchymal subtype (52 cases); GBM RTK1 glioblastoma, IDH-wild-type, subclass RTK1 (69 cases); GBM RTK2 glioblastoma IDH-wildtype, subclass RTK2 (134 cases); O IDH oligodendroglioma, IDH-mutant and 1p/19q-codeleted (79 cases); pedHGG A diffuse paediatric-type high grade glioma, H3-wildtype and IDH-wildtype, subtype A (2 cases); pedHGG B diffuse paediatric-type high grade glioma, H3-wildtype and IDH-wildtype, subtype B (2 cases); pedHGG MYCN diffuse paediatric-type high grade glioma, MYCN subtype (19 cases); pedHGG RTK1A diffuse paediatric-type high grade glioma, RTK1 subtype, subclass A (2 cases); pedHGG RTK1B diffuse paediatric-type high grade glioma, RTK1 subtype, subclass B (3 cases); pedHGG RTK1B Diffuse paediatric-type high grade glioma, RTK1 subtype, subclass C (11 cases); pedHGG RTK2A diffuse paediatric-type high grade glioma, RTK2 subtype, subclass A (10 cases); pedHGG RTK2B diffuse paediatric-type high grade glioma, RTK2 subtype, subclass B (4 cases); PXA (anaplastic) Pleomorphic xanthoastrocytoma (44 cases)

Figure 2 shows the hematoxylin–eosin stainings and copy number plots of the eight tumors.

Hematoxylin–eosin stainings and copy number plots of the eight tumor tissues. EGFR epidermal growth factor receptor, PDGFRA platelet-derived growth factor receptor alpha

Figure 3 illustrates each patient’s clinical course, including preoperative radiologic imaging.

Illustration of patient’s clinical course, including preoperative MR-imaging. ADM adriamycin, CCNU lomustine, CTX cyclophosphamide, DWI diffusion-weighted imaging, Gd gadolinium, Gy gray, MRI magnetic resonance imaging, n native, OS overall survival, PFS progression-free survival, RCTX radiochemotherapy, RTX radiotherapy, T1w T1 weighted, T2w T2 weighted, TMZ temozolomide, VCR vincristine, y years

Table 2 summarizes preoperative imaging features of all cases: all lesions appear hyperintense on T2 weighted (T2w) MRI imaging. Diffusion-weighted imaging (DWI) shows no diffusion restriction in any case. Other imaging phenomenology in pHGG is inconsistent. Some cases exhibit rim enhancement (patients 1, 3, 4, 5), others do not. Three out of eight show no enhancement at all (patients 6, 7, 8). Lesions present hypo- to isointense on native T1-weighted (T1w) MRI. In five cases tumor margins are sharp (patients 1, 3, 4, 5, 8) whereas three patients have a diffusely infiltrating tumor (three patients 2, 6, 7). Perilesional edema is present in seven cases (patients 1, 2, 3, 4, 5, 6, 7). Three cases demonstrate a cystic tumor (patients 1, 3, 5), all other cases display no signs of cysts or necrosis.

In October 2015, a 51-year-old male presented with right-sided hemiparesis. Cerebral MRI showed a left central necrotic lesion with perifocal edema (see Fig. 3A, B). Neuropathological examination confirmed the diagnosis of a glioblastoma multiforme, MGMT-promoter not methylated. The tumor was later identified as diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (pHGG RTK1; 12.5 classifier score 0.70). The patient received adjuvant radiochemotherapy according to the Stupp protocol [5] until July 2016. At the end of January 2017, imaging revealed a multifocal relapse. The patient underwent a second course of radiotherapy (single doses of 1.8 Gy up to a total dose of 45 Gy), metronomic temozolomide (20 mg/m2 b.i.d.) and tumor-treating fields (TTF). Six months later, in September 2017, MRI again showed multifocal tumor progression. The patient wished to continue metronomic temozolomide and TTF. Two months later his general condition deteriorated. He was taken to the intensive care unit due to a septic shock caused by an erysipelas of the right lower leg. The patient died in a hospice, 28 months after the initial diagnosis.

In February 2016 an 8-year-old girl was externally diagnosed based on histomorphology with a disseminated leptomeningeal oligodendroglial tumor after a tonic–clonic seizure. The patient received carboplatin- and vincristine-based chemotherapy. Almost one year later, in January 2017, she experienced a progressive gait disorder and paresthesia in the right hand. MRI demonstrated local tumor progression as well as intramedullary spinal metastases (see Fig. 3C, D). Neuropathological examination after a stereotactic biopsy now showed an anaplastic astrocytoma, IDH-wildtype with molecular features of glioblastoma, IDH-wildtype. DNA-methylation analysis was performed and the tumor was re-classified as glioblastoma, IDH-wildtype, subclass midline (brain tumor classifier v11b4 Score 0.75). Later, in the 12.5 version of the brain tumor classifier the tumor was re-classified as diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (pHGG, RTK1; 12.5 classifier score 1.0).

Postoperatively, the patient suffered from high intracranial pressure and the insertion of an external ventricle drain was performed. After prolonged convalescence, the girl was able to return to school but reported mild difficulties with writing and concentration. Dose-dense temozolomide (50 mg/m2/day, 21-days on/7-days off) and valproic acid (30 mg/kg/day) were administered from March 2017 to February 2018, when imaging again showed intracranial tumor progression. Chemotherapy was changed to temozolomide (140 mg/day, 5 days on/23 days off) and radiotherapy was administered (total dose of 45 Gy in single doses of 1.8 Gy). In June 2018, the girl presented with progredient hydrocephalus, fever, and neutropenia. Treatment included shunt revision and antibiotics. The patient seemed gradually more apathetic, even though imaging showed no signs of progression. Soon temozolomide had to be paused due to myelotoxicity. The patient was alive at last follow-up, 79 months after the initial diagnosis.

In May 2022 a 45-year-old male was admitted to our emergency department due to a sudden onset of severe headache, amnestic aphasia and a latent hemiparesis. MRI showed two multicystic, post-contrast rim-enhancing lesions in the right frontal lobe with extensive perifocal edema and signs of increased intracranial pressure (see Fig. 3E, F). The two lesions were microsurgically resected. Neuropathological examination revealed a glioblastoma, IDH-wildtype, MGMT-promoter methylated. DNA-methylation analysis was performed and the tumor was re-classified as glioblastoma, IDH-wildtype (brain tumor classifier v11b4 score 0.99). Later, in the 12.5 version of the brain tumor classifier, the tumor reached a classifier score of 0.89 for a diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (pHGG RTK2). The patient underwent radiochemotherapy with single fractions of 2 Gy up to a total dose of 50 Gy, followed by a 10 Gy heavy ion boost in 2 Gy single fractions and concomitant temozolomide and lomustine (CCNU) [6]. During the fourth cycle of adjuvant chemotherapy in November 2022, the patient developed epileptic seizures. He was alive at the last follow-up, five months after surgery without imaging signs of tumor progression.

In January 2021, a 22-year-old female patient presented herself at our institution. After a diagnosis of a right-parietal glioblastoma, IDH-wildtype in July 2020 (see Fig. 3G, H), she had undergone concomitant radiochemotherapy according to the Stupp regimen [5]. Presentation at our institution was made during the third cycle of adjuvant temozolomide. The patient had no clinical signs of disease, but fluoroethyl-l-tyrosine (18F)-positron emission tomography (FET-PET)-MRI showed an area of increased tracer uptake in the resection cavity. We agreed on the observation of the suspicious lesion and recommended the continuation of temozolomide. Four months later, in April 2021, the patient underwent re-resection of multifocal tumor relapse. Neuropathological analysis confirmed the diagnosis of glioblastoma, IDH-wildtype, MGMT-promoter not methylated. Because of a nuclear loss of PMS2 staining there was the suspicion of a diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype [1]. DNA-methylation analysis was performed and the tumor was classified as diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype in the 12.5 version of the Brain Tumor Classifier (pHGG MYCN, 12.5 classifier score 0.91). A second radiochemotherapy (median total dose of 40.05 Gy with single doses of 2.67 Gy) with metronomic temozolomide (20 mg/m2 b.i.d.) was initiated.

Four months later, the patient presented with a right hypoglossal nerve palsy. FET-PET-MRI revealed a multifocal tumor progression. Re-resection was performed in October 2021, which was well tolerated and adjuvant regorafenib was initiated [7]. After the first cycle, the patient presented with intermittent focal seizures, spastic hemiplegia on the left side and Cushing syndrome. MRI showed tumor progression. In February 2022, the patient was transferred to the palliative care unit, regorafenib treatment was terminated and best supportive care was initiated. The patient died 19 months after the initial diagnosis.

In August 2017, a 71-year-old male patient presented with sudden onset motoric dysfunctions and disorientation. MRI showed a large right temporoparietal hemorrhagic, cystic lesion (see Fig. 3I, J). The tumor was subtotally resected. Post-surgical MRI exhibited residual disease. Neuropathological examination revealed a glioblastoma, IDH-wildtype, MGMT-promotor methylated. The tumor profile was not classifiable in the 11b4 version of the brain tumor classifier. In the 12.5 version, the profile was classified as diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (pHGG RTK1; 12.5 classifier score 0.90) We initiated radiochemotherapy in an accelerated hyperfractionated scheme with single doses of 1.6 Gy b.i.d. to a total dose of 59.2 Gy and concomitant temozolomide, followed by one cycle of temozolomide, five cycles of combined temozolomide/CCNU and TTF [6]. Twenty-two months later, in June 2019, MRI showed tumor relapse in the right frontal and pa*****rietal lobe. Re-resection and adjuvant hypofractionated therapy via robotic radiosurgery (3 × 8 Gy, prescribed to the 70% isodose) was performed. Metronomic temozolomide (20 mg/m2 b.i.d.) was prescribed after radiotherapy. Five months later, in November 2019, the patient was admitted to our emergency department with left-sided hemiparesis, fever, elevated infection parameters and general malaise. MRI revealed massive tumor progression. Antibiotic treatment of the infection led to rapid reconstitution. The patient was released into outpatient care and died in February 2020, shortly after the initiation of bevacizumab treatment, 30 months after the initial diagnosis.

In February 2022, a 29-year-old female patient was admitted with new onset of high frequent seizures. MRI showed a left insular lesion, interpreted as focal cortical dysplasia (see Fig. 3K, L). FET-PET displayed an increased tracer-uptake suggestive of a high-grade glioma. Two months later, in April 2022, the patient underwent microsurgical resection. DNA-methylation analysis revealed glioblastoma, IDH-wildtype (v11b4 classifier score 0.93) and later, in the 12.5 classifier version, a diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (pHGG RTK2; 12.5 classifier score 1.0). Next-generation sequencing using the Illumina TruSight Oncology (TSO500) Panel showed five pathogenic mutations and one potential pathogenic mutation (CBL c.1096-1G > T [NM_005188.3]; NF1 c.372T > A; p.C124 [NM_001042492.2]; ANKRD11 c.4300G > T; p.E1434 [NM_013275.5]; BCOR c.3621dup; p.Q1208fs, [NM_001123385.1]; TERT c.G228A; NF1 c.5534T > G; p.I1845S [NM_001042492.2]). Adjuvant therapy comprised radiochemotherapy in an accelerated hyperfractionated schema with single doses of 1.6 Gy b.i.d. up to a total dose of 59.2 Gy and concomitant temozolomide, followed by a cyclic regimen according to Stupp et al. [5].

After initiation of the cyclic temozolomide treatment in July 2022, the patients' overall condition started to deteriorate when she developed pancytopenia and a severe SARS-CoV2-infection. Seizure medication had to be escalated and temozolomide had to be paused after the second cycle due to severe nausea, vertigo and headaches. MRI from November 2022, however, showed no tumor progression. The patient was alive at last follow-up, 6 months after surgery.

In August 2019, a 15-year-old female patient was referred to our neurosurgery department due to tumor relapse of a right fronto-temporal lesion (see Fig. 3M, N) which had been primarily resected abroad in 2015. At the time, external histological examination suspected a spindle cell carcinoma or gliosarcoma. Until 2016, the patient had received radiochemotherapy comprising CCNU, vincristine, cyclophosphamide and adriamycin. For the next two years, she experienced several tonic–clonic seizures. In June 2019, MRI showed a tumor relapse. External stereotactic biopsy was interpreted as spindle-cell astrocytic glioma with desmoplasia. In August 2019 we performed microsurgical re-resection. DNA-methylation analysis revealed a glioblastoma, IDH-wildtype, subclass MYCN glioblastoma (v11b4 classifier score 0.99, subclass score 0.98). The MGMT-promoter was not methylated. The case later scored 1.0 for the methylation class diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (pHGG MYCN) in the 12.5 classifier version. Adjuvant therapy was administered abroad and the patient was lost to follow-up.

A 52-year-old female patient was admitted to our hospital with the chief complaint of severe headache. MRI demonstrated a T1 hypointense and FLAIR hyperintense bithalamic lesion (right > left) with no contrast-enhancement (see Fig. 3O, P). In July 2022, a stereotactic biopsy was performed. DNA-methylation analysis showed a diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype, subtype A&B (v12.5 classifier score 0.96). Immunohistochemical stains with antibodies against H3 K27M and H3 p.27me3 were performed during routine diagnostics. H3 K27M mutation was not present and H3 trimethylation was retained. A TERT promoter C228T mutation was detected by Sanger sequencing. Next-generation sequencing using the Illumina TruSight Oncology (TSO500) Panel revealed two additional, likely pathogenic mutations (BCOR c.4050C > A; p.Y1350 [NM_001123385.1]; CREBBP c.4337G > A; p.R1446H [NM_004380.2]) and an EGFR exon 18–25 kinase domain duplication (EGFR-KDD). Interdisciplinary tumor board recommended radiochemotherapy and TTF. Further treatment took place at an external hospital where it was agreed to postpone radiochemotherapy and try a watch and scan approach as the patient had no clinical signs of disease (Karnofsky performance score (KPS) 100%). MRI from November 2022 showed no tumor progression. The patient was alive at the last follow-up, four months after the initial diagnosis.