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PDF file, 169KB, Measurement of Syn1 expression following SAHA treatment of murine NSC-M and NSC-MR cells.

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Medulloblastoma is a malignant pediatric brain tumor. Current treatment following patient stratification into standard and high-risk groups using clinical features has improved survival. However, a subset of patients with standard risk features have unanticipated aggressive disease, underscoring the need for a better understanding of tumor biology and the development of novel treatments. Poor differentiation, a hallmark of medulloblastomas is associated with elevated expression levels of the repressor of neuronal differentiation called repressor element 1-silencing transcription factor (REST). Here, we assessed whether elevated REST expression levels had prognostic significance and whether its pharmacologic manipulation would promote neurogenesis and block tumor cell growth. REST levels in patient tumors were measured by immunohistochemistry and stratified into negative, low/moderate- (+/++/+++), and high-REST (+++++) groups. Kaplan–Meier curves revealed that patients with high-REST tumors had worse overall and event-free survival compared with patients with REST-negative or REST-low tumors. Because histone deacetylases (HDAC) are required for REST-dependent repression of neurogenesis, we evaluated a panel of HDAC inhibitors (HDACI) for their effects on growth and differentiation of established and primary REST-positive cell lines. MS-275, trichostatin-A (TSA), valproic acid (VPA), and suberoylanilide hydroxamic acid (SAHA) upregulated expression of the REST-target neuronal differentiation gene, Syn1, suggesting a potential effect of these HDACIs on REST function. Interestingly, VPA and TSA substantially increased histone acetylation at the REST promoter and activated its transcription, whereas SAHA unexpectedly promoted its proteasomal degradation. A REST-dependent decrease in cell growth was also observed following SAHA treatment. Thus, our studies suggest that HDACIs may have therapeutic potential for patients with REST-positive tumors. This warrants further investigation. Mol Cancer Ther; 11(8); 1713–23. ©2012 AACR.

PDF file, 338KB, Table1A. Overview of Database; Table 1B. Event Free Survival (EFS); Table 1C. Overall Survival (OS).

BackgroundPreclinical studies have suggested that mTOR pathway signaling may be a potential therapeutic target for childhood ependymoma.MethodsA 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.ResultsEleven 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).ConclusionsEverolimus does not appear to have activity for children with recurrent or progressive PF-A ependymoma.

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.

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.

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.

Patient: Male, 16-year-old Final Diagnosis: Lobular capillary hemangioma Symptoms: Epistaxis Medication: — Clinical Procedure: — Specialty: Otolaryngology Objective: Rare disease 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.

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.

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.

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.

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.

DICER1 syndrome1 predisposes to a variety of cancers, including pleuropulmonary blastoma (PPB),2 ovarian Sertoli-Leydig cell tumor (SLCT),3 embryonal rhabdomyosarcoma,4 and kidney tumors.5–8 In DICER1 syndrome, most patients bear a germline null mutation in DICER1, and the tumors uniformly bear a second-hit missense substitution at one of five hotspot positions (1705E, 1709D, 1809G, 1810D, and 1813E). A wide range of clinical phenotypes can be seen in DICER1 syndrome5,9; some patients are asymptomatic, whereas others develop multiple tumors. In the classic cases, patients with a germ-line null mutation develop different somatic hotspot mutations in each tumor.7,10–16 However, in some patients, multiple tumors arise from germline mosaicism of the hotspot mutation, with subsequent somatic loss of the wild-type allele.17–19 Here we report a patient with DICER1 syndrome who developed four tumor types at six anatomic sites over the course of 12 years. These tumors harbor four distinct hotspot mutations, which is one of the highest numbers of distinct somatic DICER1 mutations reported in a single patient. By identifying these mutations, we show that the patient’s bilateral renal tumors and bilateral ovarian SLCTs each constituted a new primary tumor. Because we found no other mutations to explain her particularly severe clinical course, we speculate that her subsequent tumors were a product of the intense chemotherapy and radiation regimens she received.

Summary: Sonic hedgehog (Shh) determines cerebellar granule cell (GC) progenitor proliferation and medulloblastoma pathogenesis. However, the pathways regulating GC progenitors during embryogenesis before Shh production by Purkinje neurons and their roles in tumorigenesis remain unclear. The cilium-localized G-protein-coupled receptor Gpr161 suppresses Shh-mediated signaling in the neural tube. Here, by deleting Gpr161 in mouse neural stem cells or GC progenitors, we establish Gpr161 as a tumor suppressor in Shh subtype medulloblastoma. Irrespective of Shh production in the cerebellum, Gpr161 deletion increased downstream activity of the Shh pathway by restricting Gli3-mediated repression, causing more extensive generation and proliferation of GC progenitors. Moreover, earlier deletion of Gpr161 during embryogenesis increased tumor incidence and severity. GC progenitor overproduction during embryogenesis from Gpr161 deletion was cilium dependent, unlike normal development. Low GPR161 expression correlated with poor survival of SHH subtype medulloblastoma patients. Gpr161 restricts GC progenitor production by preventing premature and Shh-dependent pathway activity, highlighting the importance of basal pathway suppression in tumorigenesis. : Shimada et al. identify the ciliary G-protein-coupled receptor Gpr161 as a tumor suppressor in Shh subtype medulloblastoma. The authors suggest that Gpr161 restricts premature Shh pathway activity during granule cell progenitor development, implying that cilium-mediated pathway suppression preceding Shh signaling prevents tumorigenesis. Keywords: cerebellum, granule cell, medulloblastoma, sonic hedgehog, primary cilia, G-protein-coupled receptor, Gpr161, tumor suppressor

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.

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 SHH-α (children 3–16 years) and SHH-β (infants) sub-groups. Unexpectedly, SHH-β tumors display more neuronal maturation compared to SHH-α tumors, yet both are correlated with poor overall patient survival. We studied the contribution of REST to tumorigenesis using a novel transgenic mouse model (REST(TG)). In this model, conditional NeuroD2-controlled REST transgene expression in lineage committed Patched 1 heterozygous (Ptch1(+/−)) cerebellar granule neuron progenitors (CGNPs) caused markedly accelerated tumorigenesis and penetrance and infiltrative disease. Mechanistic studies revealed a neuronal maturation context specific antagonistic interplay between the transcriptional repressor and activator, REST and Gli1, respectively. REST elevation and low expression of an inhibitor of Gli1, β-Arrestin1 (Arrb1), promoted Gli1 activity and Ptch1 expression in proliferating cells, in spite of increased histone H3K9 methylation at the Ptch1 promoter. However, in lineage committed REST(TG) CGNPs, Arrb1 increase and decreased Gli1 activity in conjunction with increased histone H3K9 methylationat the Ptch1 locus led to premature silencing of Ptch1 expression. In human tumors, PTCH1, GLI1 and ARRB1 expression were significantly decreased in SHH-β tumors compared to SHH-α tumors. These findings as well as the synergistic decrease of MB cell proliferation in culture upon pharmacological inhibition of G9a and histone deacetylase (HDAC) activities support a role for REST in MB progression in more lineage committed cells. Lineage committed REST(TG) CGNPs in comparison with proliferating progenitors, also exhibited decreased expression of the phosphatase tensin homolog (Pten), a negative regulator of Akt kinase. Consistent with this, human SHH-β tumors had significantly lower PTEN expression, although an unexpected decrease in its gene expression was seen in SHH-α tumors. Pharmacological blockade of AKT promoted apoptosis in REST-high cells in culture. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT hyperactivation in MB tissues and models reveal potential subgroup specific therapeutic targets to explore for patients with SHH MB.

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-β (infants) subgroups. Neuronal maturation is greater in SHH-β 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 β-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 REST TG 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-β 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.

Background Gangliogliomas are slow-growing, low-grade central nervous system tumors affecting children and young adults. However, some patients will experience tumor recurrence and/or malignant progression. This article reports on the clinical history, molecular findings, and treatment response in a patient with BRAF V600-mutated high-grade glioma arising from ganglioglioma. Methods Hematoxylin-eosin staining and comprehensive genomic profiling via Foundation One were performed on the tumor sample from a male patient undergoing treatment at the Department of Neuro-Oncology at Baylor University Medical Center. Results The patient was eligible for participation in a clinical trial (ClinicalTrials.gov identifier: NCT00916409) of a tumor treatment fields (TTFields) device, NovoTTF-100A, with concurrent radiation and chemotherapy (CCRT). His disease relapsed 4 months after completion of his CCRT, with MRI showing areas of enhancement. Temozolomide was discontinued and he was offered dabrafenib, an oral selective inhibitor of BRAF V600E, with continued use of NovoTTF. At the time of this report, after 2 years of treatment with dabrafenib and TTFields, the patient shows a durable complete response in all areas with no active lesions or new areas of enhancement. Conclusions This report suggests that TTFields delivered in combination with targeted therapy dabrafenib yielded a remarkable clinical and radiologic response in this recurrent high-grade glioma. Targeted therapy matched to genomic alterations combined with TTFields treatment could provide clinical benefit and should be prospectively explored in the near future.

Medulloblastoma (MB) is a malignant pediatric brain tumor characterized by poor neuronal differentiation. The RE1 Silencing Transcription Factor (REST), which negatively regulates neurogenesis by promoting repressive chromatin remodeling via epigenetic modifications, is overexpressed in human MBs. Analyses of a publically available gene expression array data set revealed association of elevated REST expression in patients with Sonic Hedgehog (SHH) medulloblastoma with poor prognosis. Upon closer evaluation, we identified a subset of highly undifferentiated SHHα tumors with elevated REST expression and activity as determined by expression of REST target genes. These patients had an overall survival rate of 50% at 5 years. To understand the underlying mechanisms, we generated a novel mouse model expressing human REST transgene in a conditional manner in granule neural progenitors (GNPs), the cells of origin of SHH driven MB. Mice with constitutive activation of Shh signaling and REST elevation (Ptch(+/-)/REST(TG)) in their GNPs developed tumors with a significant decrease in latency and increase in penetrance. We observed that 100% of Ptch(+/-)/REST(TG) animals developed heterogeneously proliferative and undifferentiated tumors compared to tumor-bearing Ptch(+/-) animals. Mechanistic studies revealed deregulation of the Shh signaling pathway by REST-dependent G9a activity, accompanied by loss of Ptch1 heterozygosity in Ptch(+/-)/REST(TG) tumors. Thus, our studies suggest that REST contributes to mis-regulation of SHH activity and drives an aggressive disease course by deregulating proliferation and differentiation. Ours is also the first study to implicate G9a, a repressive chromatin remodeler, in medulloblastoma pathogenesis and as a potential therapeutic target.

Summary 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.

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.

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.

Sonic hedgehog (Shh) determines cerebellar granule progenitor proliferation and medulloblastoma pathogenesis. However, pathways regulating progenitors during embryogenesis before Shh production by Purkinje neurons and if such regulation affects tumorigenesis is unknown. The cilia-localized G-protein-coupled receptor, Gpr161 suppresses Shh-mediated signaling in neural tube. Here, by deleting Gpr161 in mouse neural stem cells or granule progenitors, we establish Gpr161 as a bona fide tumor suppressor in Shh-subtype medulloblastoma. Tumorigenesis was consequent upon increased generation and proliferation of progenitors from Shh pathway hyperactivity, irrespective of Shh. Earlier deletion increased tumor incidence and severity. Overproduction of progenitors during embryogenesis from premature Shh pathway activity was cilia-dependent and resulted from lack of Gli3 repressor. Normal progenitor development during embryogenesis did not require cilia. Low GPR161 expression correlated with poor survival of SHH-subtype medulloblastoma patients. Together, Gpr161 restricts progenitor production by preventing premature and Shh-dependent pathway activity, highlighting importance of basal pathway suppression in medulloblastoma pathogenesis.

This report presents a series of 5 pediatric patients with disseminated pilocytic astrocytomas and frequent nonfusion activating mutations. Genetic variants in these patients' tumors include BRAF p.Val600Glu, BRAF p.Val600Asp, and KRAS p.Gly60_Gln62ins7. The 2 patients with BRAF-mutated tumors were treated with dabrafenib or a combination of dabrafenib plus trametinib. The patients had either near complete resolution of the primary tumor (BRAF p.Val600Glu) or a stable primary tumor (BRAF p.Val600Asp). Both patients showed improvement in leptomeningeal dissemination without significant toxicity. Genomic testing of disseminated pilocytic astrocytomas, particularly those arising at extracerebellar locations, may result in the identification of mutations associated with ERK/MAPK activation. Patients with these activating mutations may benefit from targeted therapies.

We describe a case of B-cell lymphoblastic lymphoma with an unusual clinical presentation. A 15-year old male with no significant past medical history presented with persistent foot swelling after an ankle sprain. Imaging revealed a mass in the right foot, which was resected and revealed a diffuse infiltrate of B-lymphoblasts. Peripheral blood and bone marrow examinations revealed no abnormalities. These findings were consistent with B-cell lymphoblastic lymphoma, which is an uncommon disease, accounting for only 2% of lymphomas. This case is a reminder that hematolymphoid malignancies can have presentations that mimic sarcomas with no visible peripheral blood or bone marrow involvement. In addition to describing the clinical and histologic features of this case, we also discuss the challenges that can be encountered when establishing a diagnosis of B lymphoblastic lymphoma

DNA methylation is an epigenetic mechanism critical for tissue development and cell specification. Mammalian brains consist of many different types of cells with assumedly distinct DNA methylation profiles, and thus some genomic loci may demonstrate bipolar DNA methylation pattern, i.e. hypermethylated in one cell subset but hypomethylated in others. Currently, how extensive methylation patterns vary among brain cells is unknown and bipolar methylated genomic loci remain largely unexplored. In this study, we implemented a procedure to infer cell-subset specific methylated (CSM) loci from the methylomes of human and mouse frontal cortices at different developmental stages. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Correlated with enhancer-associated histone modifications, putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. Altogether, this study provides a procedure to identify genomic regions showing methylation differences in a mixed cell population and our results suggest that a set of cis-regulatory elements are primed in early postnatal life whose functions may be compromised in human neurological disorders.

Pilocytic astrocytomas are highly vascular, relatively common primary brain tumors in the pediatric population, but their association with a true arteriovenous malformation (AVM) is extremely rare. We describe an eight-year-old girl with a right supratentorial juvenile pilocytic astrocytoma (WHO grade I) with an angiographically documented AVM entangled in the tumor mass who presented with intracranial hemorrhage due to a ruptured anterior choroidal artery pseudoaneurysm encased in the lesion. The AVM nidus as well as the hemorrhage site was embolized with Onyx. A literature review revealed only one previous report of a true intermixture of these two lesions. We hypothesize whether the association of vascular malformations and primary brain tumors are merely coincidental or if they point to the existence of a distinct entity and/or a common etiologic factor.

The purposes of this study were to determine the accuracy of radiology reports in the diagnosis of pediatric pilomatricoma and to describe the characteristic imaging findings in a large cohort.Query of a pathology database between January 2009 and February 2014 yielded 623 specimens from 596 patients. Seventy-four patients (41 girls, 33 boys; mean age, 8.9 years) underwent imaging of the 80 excised lesions. Ninety imaging studies were available (58 ultrasound, 11 CT, 21 MRI). Radiology reports were reviewed. The lesions were evaluated for location, size, and imaging characteristics.Pilomatricoma was included as an indication for two (2.2%) imaging studies. No differential diagnosis was given in 34 (37.8%) imaging reports. In the radiology reports, pilomatricoma was suggested as a possibility for 12 (13.3%) lesions. Foreign body reaction (13.3%), dermoid cyst (10.0%), and sebaceous cyst (8.9%) were other leading suggested diagnoses. In six (6.7%) reports, lesions were described as aggressive tumors. In nine (10.0%) reports additional studies were recommended. In all examinations, the lesions involved both skin and subcutaneous tissues without infiltration of the deep tissues. Hypoechoic rim (82.8%), internal reticulations (65.5%), and flow at Doppler examination (63.2%) were the most common sonographic features. MRI characteristics were enhancement (100.0%), heterogeneous T2-weighted hyperintensity with intermixed hypointensity (76.2%), T1-weighted isointensity in relation to skeletal muscle (66.7%), and absence of restricted diffusion (100.0%).Although pilomatricoma is a common tumor of the skin and subcutaneous tissues, imaging is infrequently used in the diagnosis, and therefore, many radiologists are not aware of the lesion. The imaging features of pilomatricoma are consistent in all imaging modalities.

Context.—Medulloblastomas (MBs) and atypical teratoid/rhabdoid tumors (AT/RTs) arising in infants and children can be difficult to distinguish; however, histologic characterization is prognostically important.Objective.—To determine histologic and phenotypic markers associated with utility with progression-free survival (PFS) and overall survival (OS) in children younger than 3 years with MBs and AT/RTs.Design.—We undertook a histologic and immunophenotypic study of MBs and AT/RTs arising in infants and children younger than 3 years treated in a Pediatric Brain Tumor Consortium study. The 41 girls and 55 boys ranged in age from 2 to 36 months at enrollment. These infants and children exhibited 51 MBs, 26 AT/RTs, and 24 other tumors (not further studied). Median follow-up of the patients was 17.2 months from diagnosis (range: 1.4–93 months).Results.—Infants and children with AT/RT exhibited a statistically significant shorter PFS and OS when compared to infants and children with MBs (both P < .001). A lack of nuclear BAF47 immunohistochemical reactivity proved reliable in identifying AT/RTs. Among MBs, our data suggest an association of nodularity and prolonged PFS and OS, which must be independently confirmed. Anaplasia correlated with OTX2 reactivity and both OTX2 and moderate to severe anaplasia correlated with PFS but not with OS.Conclusion.—Distinguishing AT/RT from MBs is clinically important. For expert neuropathologists, the diagnoses of AT/RT and MB can be reliably made from hematoxylin-eosin stains in the vast majority of cases. However certain rare small cell variants of AT/RT can be confused with MB. We also found that immunohistochemical reactivity for BAF47 is clinically useful in distinguishing MBs from AT/RTs and for identifying certain small cell AT/RTs. Among MBs, nodularity may be an important prognostic factor for improved PFS and OS in infants and children.

Children with diffuse intrinsic pontine glioma (DIPG) have an extremely poor survival. This underscores the desperate need for novel, biology-driven therapies. Here, we investigated the involvement of RE1 Silencing Transcription Factor (REST), a chromatin remodeler and regulator of brain development, in DIPG pathology. We discovered that when compared to normal controls, REST protein is significantly elevated in DIPG patient samples with H3K27M mutation. Compared to isogenic DIPG cell lines with endogenous REST expression, cells engineered to express higher levels of human REST transgene exhibited enhanced tumor growth. Surprisingly, REST elevation also caused an increase in tumor vasculature. In vitro, REST loss in DIPG cells caused a decrease in tube formation by human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs), indicating that REST regulates the DIPG microenvironment. Mechanistically, we identified a bone morphogenic factor (BMP) signaling antagonist and ligand for VEGFR2, Gremlin-1 (GREM-1), as a downstream effector of REST-dependent changes in tumor vasculature. REST-dependent GREM-1 secretion promoted activation of AKT signaling in HUVECs and HBMECs. VEGFA-C levels were not significantly altered between normal and H3K27M mutant DIPGs and did not correlate with REST expression. In summary, our study is the first to demonstrate both tumor intrinsic and tumor extrinsic functions for REST in DIPG. The latter is of particular significance because it opens the door for re-evaluation of anti-angiogenic therapies targeting GREM-1, in DIPGs with REST elevation, possibly in combination with standard of care.

Global loss of DNA methylation has been known for decades as an epigenomic aberration associated with carcinogenesis and cancer progression. Loss of DNA methylation affects predominantly repetitive elements, which encompass >50% of the CpG dinucleotides present in the human genome. Because of the lack of an effective approach, no studies have been conducted to reveal such genome-wide methylation changes at a single-base resolution. To precisely determine the CpG sites with methylation loss during progression of pediatric intracranial ependymomas, we exploited a high-throughput bisulfite sequencing approach that simultaneously generates methylation profiles for thousands of Alu elements and their flanking sequences. Comparison of the methylation profiles of normal and tumor tissues revealed that the methylation status of the majority of CpG sites adjacent to or within Alu repeats remain unaltered, while a small set of CpG sites gain or lose methylation in ependymomas. Compared to the CpG sites with stable methylation level between normal control and ependymomas, the differentially methylated CpG sites are enriched in the sequences with low CpG density in the flanking regions of Alu repeats, rather than within the Alu sequences themselves. In addition, the CpG sites that are hypermethylated in ependymomas are proximal to CpG islands, whereas those that are hypomethylated are overrepresented in intergenic regions. Lastly, aberrant methylation of several genomic loci was confirmed to be associated with the aggressive primary tumors and the relapsed ependymomas.

Object Direct delivery of chemotherapeutic agents for the treatment of brain tumors is an area of focus in the development of therapeutic paradigms because this method of delivery circumvents the blood-brain barrier without causing adverse systemic side effects. Few studies have investigated longitudinal tumor response to this type of therapy. In this study, the authors examined the time course of tumor response to direct delivery of a chemotherapeutic agent in a rodent malignant glioma model. Methods To visualize tumor response to chemotherapy, the authors used bioluminescence imaging in a rodent model. Rat 9L gliosarcoma cells expressing a luciferase gene were inoculated into adult male rat striata. Ten days following surgery the animals were randomly divided into 4 groups. Groups 1 and 2 received 20 and 40 μl carboplatin (1 mg/ml), respectively, via convection-enhanced delivery (CED); Group 3 received 60 mg/kg carboplatin intraperitoneally; and Group 4 received no treatment. Tumor growth was correlated with luminescence levels twice weekly. Results Differential growth curves were observed for the 4 groups. Systemically treated rats showed decreasing photon flux emission at 15.0 ± 4.7 days; rats treated with 20- or 40-μl CED showed decreased emissions at 4.0 ± 2.0 and 3.2 ± 1.3 days after treatment, respectively. Histopathologically, 6 of 12 CED-treated animals exhibited no residual tumor at the end point of the study. Conclusions Direct and systemic delivery of carboplatin was examined to determine how the method of drug delivery affects tumor growth. The present report is one of the first in vivo studies to examine the time course of tumor response to direct drug delivery. The results indicate that direct drug delivery may be a promising option for treating gliomas.

DNA methylation, the only known covalent modification of mammalian DNA, occurs primarily in CpG dinucleotides. 51% of CpGs in the human genome reside within repeats, and 25% within Alu elements. Despite that, no method has been reported for large-scale ascertainment of CpG methylation in repeats. Here we describe a sequencing-based strategy for parallel determination of the CpG-methylation status of thousands of Alu repeats, and a computation algorithm to design primers that enable their specific amplification from bisulfite converted genomic DNA. Using a single primer pair, we generated amplicons of high sequence complexity, and derived CpG-methylation data from 31 178 Alu elements and their 5' flanking sequences, altogether representing over 4 Mb of a human cerebellum epigenome. The analysis of the Alu methylome revealed that the methylation level of Alu elements is high in the intronic and intergenic regions, but low in the regions close to transcription start sites. Several hypomethylated Alu elements were identified and their hypomethylated status verified by pyrosequencing. Interestingly, some Alu elements exhibited a strikingly tissue-specific pattern of methylation. We anticipate the amplicons herein described to prove invaluable as epigenome representations, to monitor epigenomic alterations during normal development, in aging and in diseases such as cancer.

Pediatric small bowel transplant recipients are susceptible to diarrhea due to rejection or infectious enteritis, particularly of viral etiology. The most common causes of viral enteritis in this setting are rotavirus, adenovirus, cytomegalovirus, and Epstein-Barr virus. This study is the first to compare the histologic findings of rotavirus infection with acute cellular rejection in small bowel transplant biopsies. Three patients with small bowel transplants had rapid stool antigen test–proven rotavirus infection. Endoscopic biopsies during infection were examined, including material from the allograft, native small bowel, stomach, and colon. Biopsies from 2 of the patients during unrelated episodes of mild acute cellular rejection were also evaluated. Blunting of villi was the most common finding in rotavirus infection. Additionally, there was a mononuclear infiltrate that was “top heavy,” or denser towards the lumen. There were surface apoptoses but no increase in crypt apoptotic figures. In contrast, during mild acute cellular rejection, there was no villous blunting, the mononuclear infiltrate was diffuse, and there were increased crypt apoptosis. As expected, the changes of acute cellular rejection were confined to the graft, in contrast to rotavirus infection, in which case native bowel often had more pronounced changes. Although the small number of patients limits this study, several histologic features were helpful in identifying rotavirus infection. These were blunting of villi, distribution of the inflammatory infiltrate, number and location of apoptotic bodies, and anatomic location of the effect. A larger follow-up study would be valuable to confirm these findings.

Currently, there is no conclusive treatment for brainstem tumor. To facilitate the development of new treatments, it is essential to establish predictive preclinical in vivo models in which therapeutic modalities can be evaluated. Although a few rodent models have been reported, there is no novel approach that can monitor tumor response qualitatively and quantitatively.Bioluminescence imaging was used to characterize a rat brainstem tumor model. In this model, 9L gliosarcoma cells, transduced with an onco-retroviral vector containing the luciferase coding sequence, were inoculated into Fisher 344 rats.Histopathological assessment showed successful cell implantation into the brainstem. There was a strong correlation between pathological tumor volume and luminescence strength. Longitudinal quantitative responses of the tumor after application of a therapeutic agent were also demonstrated.This study demonstrates a robust rodent model with the ability to monitor brainstem tumor growth and response to chemotherapeutic agents.

Undifferentiated embryonal sarcoma of the liver is a highly malignant and aggressive tumor that occasionally arises within mesenchymal hamartoma of the liver (MHL), a benign tumor that typically occurs in young children. Undifferentiated embryonal sarcoma arising in MHL, as well as uncomplicated MHL, frequently harbor rearrangements of band 19q13.4, including the translocation t(11;19)(q13;q13.4). In this study we report the cloning and DNA sequence analysis of the translocation breakpoints in an undifferentiated embryonal sarcoma arising in MHL known to harbor t(11;19). In this case, the breakpoint at 11q13 occurred in the MALAT1 gene, also known as ALPHA. MALAT1 is rearranged in renal tumors harboring the t(6;11)(p21;q13) translocation, and noncoding MALAT1 transcripts are overexpressed in a number of human carcinomas. The breakpoint at 19q13.4 occurs at a locus we refer to as MHLB1, for Mesenchymal Hamartoma of the Liver Breakpoint 1. Although the MHLB1 locus does not contain a known gene, several human ESTs map to the region (a subset of which show homology to the nuclear RNA export factor (NXF) gene family), and the region is conserved between many mammalian species.

A major goal of our work is to study medulloblastoma biology to facilitate the development of targeted therapy. Here, we have dissected the contribution of epigenetics to tumor development. Epigenetic perturbations are pharmacologically reversible and may have therapeutic potential. The RE1-Silencing Transcription Factor (REST) is an epigenetic modulator, and a repressor of neuronal differentiation genes. Its expression is significantly elevated in human medulloblastomas, and is associated with poor prognosis. REST is a driver of medulloblastoma in mouse orthotopic models. To understand REST's contribution to tumor development, we created a novel genetically engineered mouse model in which REST transgene is conditionally elevated in the cerebellar granule progenitors (GNPs), the cells of origin of some medulloblastoma. Transgene induction caused GNP hyperproliferation, accompanied by a failure to express the cell cycle regulator-p27. Using genetic and biochemical approaches, we attributed the lack of p27 expression to direct REST-mediated repression of a novel deubiquitylase, USP37. We also identified a role for USP37 in p27 protein stabilization, and terminal cell cycle exit. USP37 and p27 levels were significantly correlated in patient samples, and their loss was associated with poor overall patient survival. Importantly, constitutive USP37 expression blocked the tumorigenic potential of high-REST human medulloblastomas, suggesting a tumor suppressive function for USP37. REST binding to the USP37 promoter caused increased histone H3 lysine (K)-9 tri-methylation, an epigenetic mark associated with gene silencing. The histone methyl-transferase G9a, which promotes this modification, is a known REST co-repressor. Importantly, genetic ablation of G9a or pharmacological inhibition of its activity upregulated USP37 expression in a REST-dependent manner, and blocked tumor growth in mice. Our work has provided the first link between epigenetic deregulation of the proteasome and medulloblastoma development, and supports the manipulation of the REST-epigenome for future therapeutic application.

Low-grade gliomas are the commonest brain tumor in children comprising heterogeneous pathological entities. Though the overall prognosis is good, unresectable, and recurrent or progressive tumors in eloquent areas of the brain remain major therapeutic challenge even with advances in chemotherapeutic strategies. With the evolving surge of molecular data, improved understanding of the biology of these tumors is now perceivable that could provide insights into novel therapies. We hope the new era will enable us to profile comprehensive histopathological/molecular classification and prognostic molecular markers in these tumors and guide us to tailor optimal targeted therapy.