An imbalance between proliferation and differentiation underlies the development of microRNA-defective pineoblastoma.

Mutations in the microRNA processing genes DICER1 and DROSHA drive several cancers that resemble embryonic progenitors. To understand how microRNAs regulate tumorigenesis, we ablated Drosha or Dicer1 in the developing pineal gland to emulate the pathogenesis of pineoblastoma, a brain tumor that resembles undifferentiated precursors of the pineal gland. Accordingly, these mice develop pineal tumors marked by loss of microRNAs, including the let-7/miR-98-5p family, and de-repression of microRNA target genes. Pineal tumors driven by loss of Drosha or Dicer1 mimic tumors driven by Rb1 loss, as they exhibit upregulation of S-phase genes and homeobox transcription factors that regulate pineal development. Blocking proliferation of these tumors facilitates expression of pinealocyte maturation markers, with a concomitant reduction in embryonic markers. Select embryonic markers remain elevated, however, as the microRNAs that normally repress these target genes remain absent. One such microRNA target gene is the oncofetal transcription factor Plagl2, which regulates expression of pro-growth genes, and inhibiting their signaling impairs tumor growth. Thus, we demonstrate that tumors driven by loss of microRNA processing may be therapeutically targeted by inhibiting downstream drivers of proliferation.

Heterozygous Kmt2d loss diminishes enhancers to render medulloblastoma cells vulnerable to combinatory inhibition of lysine demethylation and oxidative phosphorylation.

The histone H3 lysine 4 (H3K4) methyltransferase KMT2D (also called MLL4) is one of the most frequently mutated epigenetic modifiers in medulloblastoma (MB) and other types of cancer. Notably, heterozygous loss of KMT2D is prevalent in MB and other cancer types. However, what role heterozygous KMT2D loss plays in tumorigenesis has not been well characterized. Here, we show that heterozygous Kmt2d loss highly promotes MB driven by heterozygous loss of the MB suppressor gene Ptch in mice. Heterozygous Kmt2d loss upregulated tumor-promoting programs, including oxidative phosphorylation and G-protein-coupled receptor signaling, in Ptch-mutant-driven MB genesis. Mechanistically, both downregulation of the transcription-repressive tumor suppressor gene NCOR2 by heterozygous Kmt2d loss and upregulation of the oncogene MycN by heterozygous Ptch loss increased the expression of tumor-promoting genes. Moreover, heterozygous Kmt2d loss extensively diminished enhancer signals (e.g., H3K27ac) and H3K4me3 signature, including those for tumor suppressor genes (e.g., Ncor2). Combinatory pharmacological inhibition of oxidative phosphorylation and the H3K4 demethylase LSD1 drastically reduced tumorigenicity of MB cells bearing heterozygous Kmt2d loss. These findings reveal the mechanistic basis underlying the MB-promoting effect of heterozygous KMT2D loss, provide a rationale for a therapeutic strategy for treatment of KMT2D-deficient MB, and have mechanistic implications for the molecular pathogenesis of other types of cancer bearing heterozygous KMT2D loss.

Current Utilization of Electron Microscopy in the Pediatric Pathology Setting: A Survey by the SPP Practice Committee.

BACKGROUND: Electron microscopy (EM), once an important component in diagnosing pediatric diseases, has experienced a decline in its use. To assess the impact of this, pediatric pathology practices were surveyed regarding EM services. METHODS: The Society of Pediatric Pathology Practice Committee surveyed 113 society members from 74 hospitals. Settings included 36 academic tertiary, 32 free-standing children's, and 6 community hospitals. RESULTS: Over 60% maintained in-house EM services and had more than 2 pathologists interpreting EM while reporting a shortage of EM technologists. Freestanding children's hospitals had the most specimens (100-200 per year) and more diverse specimen types. Hospitals with fewer than 50 yearly specimens often used reference laboratories. Seventeen had terminated all in-house EM services. Challenges included decreasing caseloads due to alternative diagnostic methods, high operating costs, and shortages of EM technologists and EM-proficient pathologists. Kidney, liver, cilia, heart, and muscle biopsies most often required EM. Lung/bronchoalveolar lavage, tumor, skin, gastrointestinal, nerve, platelet, and autopsy samples less commonly needed EM. CONCLUSIONS: The survey revealed challenges in maintaining EM services but demonstrated its sustained value in pediatric pathology. Pediatric pathologists may need to address the centralization of services and training to preserve EM diagnostic proficiency among pathologists who perform ultrastructural interpretations.

Cachexia, chorea, and pain in chronic nonbacterial osteitis and inflammatory bowel disease: a case report.

BACKGROUND: Inflammatory bowel disease is an inflammatory disorder that primarily impacts the gastrointestinal tract, leading to malnutrition and chronic microscopic intestinal blood loss. Uncontrolled systemic inflammation can impact other parts of the body, known as extraintestinal manifestations. Up to 25% of patients with inflammatory bowel disease are reported to have these complications in their skin, joints, bones, eyes, liver, lung, and pancreas (Rogler et al. in Gastroenterology 161(4):1118-1132, 2021). Neurologic involvement as extraintestinal manifestations are less common, reported at 3-19%, including neuropathies, demyelination, and cerebrovascular events (Morís in World J Gastroenterol. 20(5):1228-1237, 2014). CASE PRESENTATION: A 13-year-old Caucasian boy presented with 1 month of progressive lower-extremity pain, weakness, and weight loss. His physical examination was notable for cachexia, lower-extremity weakness, and chorea. Labs revealed normocytic anemia and systemic inflammation. Imaging revealed symmetric abnormal marrow signal in the pelvis and upper femurs. Pathologic examination of the bone revealed chronic inflammation consistent with chronic nonbacterial osteitis. Endoscopy revealed colonic inflammation consistent with inflammatory bowel disease. CONCLUSIONS: Children and adolescents with musculoskeletal pain lasting more than 2 weeks with systemic signs or symptoms like weight loss should prompt evaluation for systemic inflammatory disorders such as chronic nonbacterial osteitis, which can occur in isolation or associated with inflammatory bowel disease. This patient also had a nonspecific neurologic abnormality, chorea, which resolved with treatment of underlying inflammatory disorder. These extraintestinal manifestations may be concurrent with or precede intestinal inflammation, requiring a high index of suspicion when investigating nonspecific systemic inflammation.

Phase II study of everolimus for recurrent or progressive pediatric ependymoma.

Background: Preclinical studies have suggested that mTOR pathway signaling may be a potential therapeutic target for childhood ependymoma. Methods: A phase II clinical trial (ClinicalTrials.gov identifier: NCT02155920) of single-agent everolimus was performed to test the hypothesis that mTOR pathway inhibition would result in tumor responses for children with recurrent and/or progressive ependymomas. Results: Eleven subjects [sex: 4 females (36.4%); median age: 8 years (range: 2-15 years); race: 9 white; prior therapies: median 6 (range: 3-9)] were enrolled on the study. Ten primary tumors were located in the posterior fossa and one primary tumor was located in the spinal cord. Eight of 9 tumors were PF-A subtype epenydmomas. All subjects were treated with oral everolimus 4.5 mg/m2/day (each cycle = 28 days) that was titrated to achieve serum trough levels of 5-15 ng/ml. Overall, everolimus was well tolerated; except for a single event of grade 3 pneumonia, all adverse events were grade 1-2. No objective tumor responses were observed. Participating subjects experienced tumor progression and discontinued therapy after a median of 2 cycles of therapy (1 cycle = 2; 2 cycles = 6; 3, 4, and 8 cycles = 1 each). Conclusions: Everolimus does not appear to have activity for children with recurrent or progressive PF-A ependymoma.

Obstructive hydrocephalus and intracerebral mass secondary to Epicoccum nigrum.

Here we report a case of a 14-week-old girl with a history of intrauterine drug exposure and hypoxic ischemic encephalopathy secondary to cardiac arrest requiring prolonged resuscitation at birth presented with irritability and a bulging anterior fontanelle. After neurosurgical resection, pathologic examination showed fungal hyphae, and Epicoccum nigrum was detected by fungal PCR and sequencing. To our knowledge, this is the first reported case of a central nervous system infection due to Epicoccum nigrum.

Pediatric paraneoplastic neuromyelitis optica spectrum disorder associated with ovarian teratoma.

Neuromyelitis optica spectrum disorder is an inflammatory condition of the central nervous system typically manifesting as myelitis, optic neuritis, and/or area postrema syndrome. Here, we present a pediatric patient who developed symptoms consistent with area postrema syndrome with positive anti-aquaporin-4 (AQP4) antibodies who was also found to have an ovarian teratoma. Pathological specimens revealed the presence of aquaporin-4. This was felt to be the antigenic trigger that led to the patient's condition. She suffered no further clinical attacks and seroconverted to negative AQP4 status upon teratoma removal. This case varies from others, in that the paraneoplastic presentation occurred in a pediatric patient and in that the patient has not required maintenance immunotherapy after teratoma removal.

Recurrent Epistaxis and Unilateral Intranasal Mass in A Teenager.

BACKGROUND Epistaxis in children is a common problem encountered in outpatient clinics and emergency departments. A wide variety of conditions may cause recurrent epistaxis in children. We describe clinical, radiologic, and histologic features of a lobular capillary hemangioma presenting as a rapidly growing intranasal mass in a child with recurrent epistaxis. CASE REPORT A 16-year-old male presented with a 2-month history of recurrent unilateral epistaxis requiring multiple visits to the emergency department. The child had nasal obstruction, snoring, no recurrent sinus infections, no anosmia nor hyposmia, no weight loss, no night sweats, no fever, no decreased activity, and no easy bruising. He denied any history of local trauma. On physical examination, a fleshy violaceous mass was found, protruding from and obliterating the right nasal cavity. Magnetic resonance imaging documented an avidly enhancing mass centered at the right nasal vestibule. Upon resection, histologic evaluation indicated a pyogenic granuloma. At the 2-month followup, the surgical site was healed with no evidence of recurrent lesion. CONCLUSIONS Lobular capillary hemangioma, although uncommon, should be considered in the differential diagnosis of recurrent epistaxis and intranasal mass in children.

AIF3 splicing switch triggers neurodegeneration.

BACKGROUND: Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain. METHODS: AIF splicing induction in brain was determined by multiple approaches including 5' RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration. RESULTS: We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2-4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice. CONCLUSIONS: We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

Chronic Histiocytic Intervillositis With Trophoblast Necrosis Is a Risk Factor Associated With Placental Infection From Coronavirus Disease 2019 (COVID-19) and Intrauterine Maternal-Fetal Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission in Live-Born and Stillborn Infants.

CONTEXT.­: The number of neonates with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is increasing, and in a few there are reports of intrauterine infection. OBJECTIVE.­: To characterize the placental pathology findings in a preselected cohort of neonates infected by transplacental transmission arising from maternal infection with SARS-CoV-2, and to identify pathology risk factors for placental and fetal infection. DESIGN.­: Case-based retrospective analysis by a multinational group of 19 perinatal specialists of the placental pathology findings from 2 cohorts of infants delivered to mothers testing positive for SARS-CoV-2: live-born neonates infected via transplacental transmission who tested positive for SARS-CoV-2 after delivery and had SARS-CoV-2 identified in cells of the placental fetal compartment by molecular pathology, and stillborn infants with syncytiotrophoblast positive for SARS-CoV-2. RESULTS.­: In placentas from all 6 live-born neonates acquiring SARS-CoV-2 via transplacental transmission, the syncytiotrophoblast was positive for coronavirus using immunohistochemistry, RNA in situ hybridization, or both. All 6 placentas had chronic histiocytic intervillositis and necrosis of the syncytiotrophoblast. The 5 stillborn/terminated infants had placental pathology findings that were similar, including SARS-CoV-2 infection of the syncytiotrophoblast, chronic histiocytic intervillositis, and syncytiotrophoblast necrosis. CONCLUSIONS.­: Chronic histiocytic intervillositis together with syncytiotrophoblast necrosis accompanies SARS-CoV-2 infection of syncytiotrophoblast in live-born and stillborn infants. The coexistence of these 2 findings in all placentas from live-born infants acquiring their infection prior to delivery indicates that they constitute a pathology risk factor for transplacental fetal infection. Potential mechanisms of infection of the placenta and fetus with SARS-CoV-2, and potential future studies, are discussed.

A Case of Dubin-Johnson Syndrome Presenting as Neonatal Cholestasis With Paucity of Interlobular Bile Ducts.

Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder that typically manifests in young adulthood as jaundice with conjugated hyperbilirubinemia. We report a case presenting as neonatal cholestasis with the unexpected histologic finding of paucity of interlobular bile ducts, a feature that is not typically seen in DJS. The diagnosis was confirmed by absent canalicular multidrug-resistance-associated protein 2 (MRP2) immunohistochemical staining on liver biopsy tissue and molecular genetic testing that demonstrated heterozygous mutations in the ATP-Binding Cassette Subfamily C Member 2 (ABCC2) gene, including a novel missense mutation. This report describes a case of DJS with atypical clinicopathologic findings and suggests that DJS should be considered in patients with neonatal cholestasis and bile duct paucity.

Intrauterine Transmission of SARS-COV-2 Infection in a Preterm Infant.

We present a preterm infant who developed a fever and mild respiratory disease on the second day of life. Infant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nasopharyngeal testing was positive at 24 and 48 hours of life. Placenta histopathology revealed SARS-CoV-2 infection by electron microscopy and immunohistochemistry. Further understanding of the risk factors that lead to in utero transmission of SARS-CoV-2 infection is needed.

Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities.

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

Transcriptional repressor REST drives lineage stage-specific chromatin compaction at Ptch1 and increases AKT activation in a mouse model of medulloblastoma.

In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-ß (infants) subgroups. Neuronal maturation is greater in SHH-ß than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model (RESTTG ) wherein conditional NeuroD2-controlled REST transgene expression in lineage-committed Ptch1 +/- cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context-specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 at Ptch1 Expression of Arrb1, which encodes ß-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committed RESTTG cells compared with wild-type proliferating CGNPs. Lineage-committed RESTTG cells also had decreased GLI1 activity and increased histone H3K9 methylation at the Ptch1 locus, which correlated with premature silencing of Ptch1 These cells also had decreased expression of Pten, which encodes a negative regulator of the kinase AKT. Expression of PTCH1 and GLI1 were less, and ARRB1 was somewhat greater, in patient SHH-ß than SHH-α MBs, whereas that of PTEN was similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.

MLL4 Is Required to Maintain Broad H3K4me3 Peaks and Super-Enhancers at Tumor Suppressor Genes.

Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes.

Pan-Trk Immunohistochemistry Identifies NTRK Rearrangements in Pediatric Mesenchymal Tumors.

Activating neurotrophic receptor kinase (NTRK) fusions define certain pediatric mesenchymal tumors, including infantile fibrosarcoma and cellular mesoblastic nephroma. Traditionally, molecular confirmation of these fusions has included either fluorescent in situ hybridization for ETV6 rearrangements or reverse-transcriptase polymerase chain reaction for the classic ETV6-NTRK3 fusion. However, these methods overlook variant NTRK rearrangements, which are increasingly appreciated as recurrent events in a subset of pediatric mesenchymal tumors. New therapeutic agents successfully target these fusions and may prevent morbid surgeries in very young children, making recognition of tumors harboring NTRK rearrangements of increasing importance. We evaluated the performance of immunohistochemical (IHC) staining using pan-Trk and TrkA antibodies in 79 pediatric mesenchymal tumors. Negative controls included pediatric mesenchymal tumors not harboring (n=28) or not expected to harbor (n=22) NTRK fusions. NTRK rearrangements were detected predominantly by DNA-based next-generation sequencing assays, specifically UW OncoPlex and UCSF500 Cancer Gene Panel. Pan-Trk IHC (EPR17341) was 97% sensitive and 98% specific for the presence of an NTRK rearrangement, and TrkA IHC (EP1058Y) was 100% sensitive and 63% specific for the presence of an NTRK rearrangement. Tumors with NTRK1 or NTRK2 rearrangements showed cytoplasmic staining, whereas tumors with NTRK3 rearrangements showed nuclear +/- cytoplasmic staining. We conclude that pan-Trk IHC is a highly sensitive and specific marker for NTRK rearrangements in pediatric mesenchymal tumors.

REST upregulates gremlin to modulate diffuse intrinsic pontine glioma vasculature.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive glial tumor that occurs in children. The extremely poor median and 5-year survival in children afflicted with DIPG highlights the need for novel biology-driven therapeutics. Here, we have implicated the chromatin remodeler and regulator of brain development called RE1 Silencing Transcription Factor (REST), in DIPG pathology. We show that REST protein is aberrantly elevated in at least 21% of DIPG tumors compared to normal controls. Its knockdown in DIPG cell lines diminished cell growth and decreased their tumorigenicity in mouse intracranial models. DIPGs are vascularized tumors and interestingly, REST loss in DIPG cells also caused a substantial decline in tumor vasculature as measured by a decrease in CD31 and VEGFR2 staining. These observations were validated in vitro, where a significant decline in tube formation by human umbilical vein endothelial cells (HUVEC) was seen following REST-loss in DIPG cells. Mechanistically, REST controlled the secretion of a pro-angiogenic molecule and ligand for VEGFR2 called Gremlin-1 (GREM-1), and was associated with enhanced AKT activation. Importantly, the decline in tube formation caused by REST loss could be rescued by addition of recombinant GREM-1, which also caused AKT activation in HUVECs and human brain microvascular endothelial cells (HBMECs). In summary, our study is the first to demonstrate autocrine and paracrine functions for REST in DIPG development. It also provides the foundation for future investigations on anti-angiogenic therapies targeting GREM-1 in combination with drugs that target REST-associated chromatin remodeling activities.

Adult Brainstem Gliomas With H3K27M Mutation: Radiology, Pathology, and Prognosis.

Adult brainstem gliomas are difficult to classify based on radiologic and histologic features. A K27M mutation in histone 3 has been described to identify high-grade midline gliomas associated with a particularly unfavorable prognosis. While initially considered a pediatric entity, it is now known that H3K27M-mutant brainstem gliomas occur in all age groups, but they are less well understood in adults. We studied clinical, radiologic, and pathologic features of 25 brainstem gliomas diagnosed at our institution between 1994 and 2017 in subjects at least 18 years old. Seven tumors (28%) were positive for the H3K27M mutation, and their median overall survival was significantly shorter than in the H3-wildtype group (p = 0.004). Although the mutation was invariably associated with a poor prognosis, our study also illustrates the radiologic and pathologic heterogeneity in this molecular tumor subtype. The results showed that H3K27M-mutant status and clinically aggressive course cannot be ruled out based on low-grade histology on the initial biopsy, exophytic growth, only focal or minimal enhancement or an extrapontine location, such as midbrain or medulla. These results favor an integrated approach employing a combination of clinical, radiologic, histologic features as well as H3K27M immunohistochemistry for the diagnostic subclassification of adult brainstem gliomas.