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Spinal muscular atrophy type 1 (SMA1) is a debilitating neurodegenerative disease resulting from survival motor neuron 1 gene (SMN1) deletion/mutation. Onasemnogene abeparvovec (formerly AVXS-101) is a gene therapy that restores SMN production via one-time systemic administration. The present study demonstrates widespread biodistribution of vector genomes and transgenes throughout the central nervous system (CNS) and peripheral organs, after intravenous administration of an AAV9-mediated gene therapy. Two symptomatic infants with SMA1 enrolled in phase III studies received onasemnogene abeparvovec. Both patients died of respiratory complications unrelated to onasemnogene abeparvovec. One patient had improved motor function and the other died shortly after administration before appreciable clinical benefit could be observed. In both patients, onasemnogene abeparvovec DNA and messenger RNA distribution were widespread among peripheral organs and in the CNS. The greatest concentration of vector genomes was detected in the liver, with an increase over that detected in CNS tissues of 300–1,000-fold. SMN protein, which was low in an untreated SMA1 control, was clearly detectable in motor neurons, brain, skeletal muscle and multiple peripheral organs in treated patients. These data support the fact that onasemnogene abeparvovec has effective distribution, transduction and expression throughout the CNS after intravenous administration and restores SMN expression in humans. Biodistribution analysis of two patients with spinal muscular atrophy shows widespread onasemnogene abeparvovec DNA, mRNA and SMN protein throughout the central nervous system and peripheral organs following intravenous gene therapy administration.

Persistent activation of signal transducer and activator of transcription 3 (STAT3) is frequently reported in cancers and plays important roles in tumor progression. Therefore, directly targeting persistent STAT3 signaling is an attractive cancer therapeutic strategy. The aim of this study is to test the inhibitory efficacy of novel STAT3 small molecule inhibitors, LLY17 and LLL12B, in combination with irradiation in human medulloblastoma cells. Both LLY17 and LLL12B inhibit the IL-6-induced and persistent STAT3 phosphorylation in human medulloblastoma cells. Irradiation using 4 Gy alone exhibits some inhibitory effects on medulloblastoma cell viability, and these effects are further enhanced by combining with either STAT3 inhibitor. Irradiation alone also shows certain inhibitory effects on medulloblastoma cell migration and invasion and the combination of LLY17 or LLL12B with irradiation further demonstrates greater inhibitory effects than monotherapy. STAT3 inhibitor alone or irradiation alone exhibits some suppression of medulloblastoma tumorsphere growth, and the combination of LLY17 or LLL12B and irradiation exhibits greater suppression of tumorsphere growth than monotherapy. Combining either STAT3 inhibitor with irradiation reduces the expression of STAT3 downstream targets, Cyclin D1 and Survivin, and induces apoptosis in medulloblastoma cells. These results support that combination of a potent STAT3 inhibitor such as LLY17 or LLL12B with irradiation is an effective and novel therapeutic approach for medulloblastoma.

Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor and an oncogene product, which plays a pivotal role in tumor progression. Therefore, targeting persistent STAT3 signaling directly is an attractive anticancer strategy. The aim of this study is to test the efficacy of a novel STAT3 small molecule inhibitor, LLL12B, in suppressing medulloblastoma cells in vitro and tumor growth in vivo. LLL12B selectively inhibited the induction of STAT3 phosphorylation by interleukin-6 but not induction of STAT1 phosphorylation by INF-γ. LLL12B also induced apoptosis in human medulloblastoma cells. In addition, LLL12B exhibited good oral bioavailability in vivo and potent suppressive activity in tumor growth of medulloblastoma cells in vivo. Besides, combining LLL12B with cisplatin showed greater inhibition of cell viability and tumorsphere formation as well as induction of apoptosis comparing to single agent treatment in medulloblastoma cells. Furthermore, LLL12B and cisplatin combination exhibited greater suppression of medulloblastoma tumor growth than monotherapy in vivo. The present study supported that LLL12B is a novel therapeutic agent for medulloblastoma and the combination of LLL12B with a chemotherapeutic agent cisplatin may be an effective approach for medulloblastoma therapy.

Epilepsy is characterized as recurrent seizures, and it is one of the most prevalent disorders of the human nervous system. A large and diverse profile of different syndromes and conditions can cause perturbations in neural networks that are associated with epilepsy. Advances in neuroimaging and electrophysiological monitoring have enhanced our ability to localize the neuropathological lesions that alter the neural networks giving rise to epilepsy, whereas advances in surgical management have resulted in excellent seizure control in many patients following resections. Histopathologic study using a variety of special stains, molecular analysis, and functional studies of these resected tissues has facilitated the neuropathological characterization of these lesions. Here, we review the neuropathology of common structural lesions that cause epilepsy and are amenable to neurosurgical resection, such as hippocampal sclerosis, focal cortical dysplasia, and its associated principal lesions, including long-term epilepsy-associated tumors, as well as other malformations of cortical development and Rasmussen encephalitis.

Septo-optic dysplasia (SOD) is defined by the presence of 2 or more features in a diagnostic triad: (1) optic nerve hypoplasia, (2) pituitary dysfunction, and (3) midline forebrain anomalies. SOD arises due to diverse pathogenetic mechanisms including acquired and genetic factors, and it shows considerable clinical and phenotypic variability. Our knowledge of SOD is incomplete in part because of a paucity of published neuropathology data, so we reviewed the autopsy neuropathology of 4 SOD patients. All patients met SOD criteria according to the triad. Additional neuropathologic findings included malformations involving non-forebrain structures and possible secondary phenomena. Autopsies demonstrate that SOD patients often have additional neuropathologic findings beyond the triad and we feel that use of the term SOD-complex appropriately underscores this diversity and its likely clinical impact. This study suggests that autopsies enhance our understanding of SOD and may be an asset in performing needed clinical and phenotypic correlation studies.

ROS1 is a transmembrane receptor tyrosine kinase proto-oncogene that has been shown to have rearrangements with several genes in glioblastoma and other neoplasms, including intrachromosomal fusion with GOPC due to microdeletions at 6q22.1. ROS1 fusion events are important findings in these tumors, as they are potentially targetable alterations with newer tyrosine kinase inhibitors; however, whether these tumors represent a distinct entity remains unknown. In this report, we identify 3 cases of unusual pediatric glioma with GOPC-ROS1 fusion. We reviewed the clinical history, radiologic and histologic features, performed methylation analysis, whole genome copy number profiling, and next generation sequencing analysis for the detection of oncogenic mutation and fusion events to fully characterize the genetic and epigenetic alterations present in these tumors. Two of 3 tumors showed pilocytic features with focal expression of synaptophysin staining and variable high-grade histologic features; the third tumor aligned best with glioblastoma and showed no evidence of neuronal differentiation. Copy number profiling revealed chromosome 6q22 microdeletions corresponding to the GOPC-ROS1 fusion in all 3 cases and methylation profiling showed that the tumors did not cluster together as a single entity or within known methylation classes by t-Distributed Stochastic Neighbor Embedding.

Background:Medulloblastoma is the most common malignant brain tumor in children. Despite improvement in overall survival rate, it still lacks an effective targeted treatment strategy. The Janus family of cytoplasmic tyrosine kinases (JAKs) and Src kinases, upstream protein kinases of signal transducer and activator of transcription 3 (STAT3), play important roles in medulloblastoma pathogenesis and therefore represent potential therapeutic targets.Methods:In this report, we examined the inhibitory efficacy of the JAK1/2 inhibitor, ruxolitinib, the JAK3 inhibitor, tofacitinib and two Src inhibitors, KX2-391 and dasatinib.Results:These small molecule drugs significantly reduce cell viability and inhibit cell migration and colony formation in human medulloblastoma cells in vitro. Src inhibitors have more potent efficacy than JAK inhibitors in inhibiting medulloblastoma cell migration ability. The Src inhibitors can inhibit both phosphorylation of STAT3 and Src while JAK inhibitors reduce JAK/STAT3 phosphorylation. We also investigated the combined effect of the Src inhibitor, dasatinib with cisplatin. The results show that dasatinib exerts synergistic effects with cisplatin in human medulloblastoma cells through the inhibition of STAT3 and Src.Conclusion:Our results suggest that the small molecule inhibitors of STAT3 upstream kinases, ruxolitinib, tofacitinib, KX2-391, and dasatinib could be novel and attractive candidate drugs for the treatment of human medulloblastoma.

Background We report a patient with primary central nervous system mixed malignant germ cell tumor (GCT) who presented with recurrent malignant germinomatous infiltration of the retina. Case Description A 10-year-old girl initially presented with a large suprasellar mixed malignant GCT with a near-complete response after initial induction of chemotherapy and irradiation. Three and a half years after initial therapy, she presented with progressively worsening vision in her left eye. Magnetic resonance imaging showed infiltrative changes within the left optic nerve but no discrete mass. Serum and cerebrospinal fluid tumor markers were not elevated and cerebrospinal fluid cytology was negative. Left optic nerve biopsy confirmed the presence of mature teratoma and pure germinoma components. She was treated with gross-total resection of the left eye and optic nerve and chemotherapy. Histopathologic evaluation of the optic nerve showed only mature teratoma elements, but with pure germinoma cells infiltrating the inner layers of the retina. Conclusions Loco-regional extension of suprasellar GCT to the optic nerve is not uncommon; however, to the best of our knowledge, infiltration of the tumor into the retina is not reported in the literature. Early detection of optic pathway involvement and proper delineation of the irradiation field may prevent GCT infiltration of the retina with subsequent vision loss.

Phosphatase and tensin homologue (PTEN) regulates cell growth and survival through inhibition of the mammalian target of rapamycin (MTOR) signalling pathway. Germline genetic variation of PTEN is associated with autism, macrocephaly and PTEN hamartoma tumour syndromes. The effect of developmental PTEN somatic mutations on nervous system phenotypes is not well understood, although brain somatic mosaicism of MTOR pathway genes is an emerging cause of cortical dysplasia and epilepsy in the paediatric population. Here we report two somatic variants of PTEN affecting a single patient presenting with intractable epilepsy and hemimegalencephaly that varied in clinical severity throughout the left cerebral hemisphere. High-throughput sequencing analysis of affected brain tissue identified two somatic variants in PTEN. The first variant was present in multiple cell lineages throughout the entire hemisphere and associated with mild cerebral overgrowth. The second variant was restricted to posterior brain regions and affected the opposite PTEN allele, resulting in a segmental region of more severe malformation, and the only neurons in which it was found by single-nuclei RNA-sequencing had a unique disease-related expression profile. This study reveals brain mosaicism of PTEN as a disease mechanism of hemimegalencephaly and furthermore demonstrates the varying effects of single- or bi-allelic disruption of PTEN on cortical phenotypes.

Retinoblastoma is a childhood cancer of the retina involving germline or somatic alterations of the RB Transcriptional Corepressor 1 gene, RB1. Rare cases of sellar-suprasellar region retinoblastoma without evidence of ocular or pineal tumors have been described. A nine-month-old male presented with a sellar-suprasellar region mass. Histopathology showed an embryonal tumor with focal Flexner-Wintersteiner-like rosettes and loss of retinoblastoma protein (RB1) expression by immunohistochemistry. DNA array-based methylation profiling confidently classified the tumor as pineoblastoma group A/intracranial retinoblastoma. The patient was subsequently enrolled on an institutional translational cancer research protocol and underwent comprehensive molecular profiling, including paired tumor/normal exome and genome sequencing and RNA-sequencing of the tumor. Additionally, Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) sequencing was performed from comparator normal and disease-involved tissue to resolve complex structural variations. RNA-sequencing revealed multiple fusions clustered within 13q14.1-q21.3, including a novel in-frame fusion of RB1-SIAH3 predicted to prematurely truncate the RB1 protein. SMRT sequencing revealed a complex structural rearrangement spanning 13q14.11-q31.3, including two somatic structural variants within intron 17 of RB1. These events corresponded to the RB1-SIAH3 fusion and a novel RB1 rearrangement expected to correlate with the complete absence of RB1 protein expression. Comprehensive molecular analysis, including DNA array-based methylation profiling and sequencing-based methodologies, were critical for classification and understanding the complex mechanism of RB1 inactivation in this diagnostically challenging tumor.

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is remarkable for its degree of both morphologic and genomic heterogeneity. In children and adults, as defined by the World Health Organization (WHO), GBM is a grade IV neoplasm with a diffusely infiltrative growth pattern populated by cells showing predominately astrocytic differentiation. As discussed in previous chapters, GBM features prominent nuclear atypia and cellular pleomorphism, as well as high tumor cell mitotic activity accompanied by microvascular proliferation and/ or necrosis. As its former name indicates, GBM may include one or many morphologic patterns within a single tumor. In some tumors, this reflects underlying clonal evolution with newly acquired genetic changes in tumor cell subpopulations. Even more variable than GBM morphology is the range of genomic heterogeneity, creating multiple molecular signatures that define tumor behavior, prognosis and treatment response independent of tumor histopathology. Furthermore, while pediatric and adult glioblastoma share many histopathologic similarities, they are unequivocally biologically distinct neoplasms. In many pediatric and adult gliomas, the molecular subgroup is a better predictor of tumor behavior than histologic grade. In particular, WHO grade II or III diffuse astrocytomas lacking morphologic features associated with GBM (microvascular proliferation and necrosis), but with certain defined molecular alterations, should be considered GBM for prognostic and therapeutic purposes due to their expected WHO grade IV-like behavior. The focus of this chapter centers on the genomic heterogeneity and pathobiology of aggressive pediatric and adult diffuse astrocytomas with WHO grade IV behavior independent of morphologic features.

Embryonal tumors of the pediatric central nervous system are challenging clinically and diagnostically. These tumors are aggressive, and patients often have poor outcomes even with intense therapy. Proper tumor classification is essential to patient care, and this process has undergone significant changes with the World Health Organization recommending histopathologic and molecular features be integrated in diagnostic reporting. This has especially impacted the workup of embryonal tumors because molecular testing has resulted in the identification of clinically relevant tumor subgroups and new entities. This review summarizes recent developments and provides a framework to workup embryonal tumors in diagnostic practice.

ObjectiveMany genetic studies of intractable epilepsy in pediatric patients primarily focus on inherited, constitutional genetic deficiencies identified in patient blood. Recently, studies have revealed somatic mosaicism associated with epilepsy in which genetic variants are present only in a subset of brain cells. We hypothesize that tissue-specific, somatic mosaicism represents an important genetic etiology in epilepsy and aim to discover somatic alterations in epilepsy-affected brain tissue.MethodsWe have pursued a research study to identify brain somatic mosaicism, using next-generation sequencing (NGS) technologies, in patients with treatment refractory epilepsy who have undergone surgical resection of affected brain tissue.ResultsWe used an integrated combination of NGS techniques and conventional approaches (radiology, histopathology, and electrophysiology) to comprehensively characterize multiple brain regions from a single patient with intractable epilepsy. We present a 3-year-old male patient with West syndrome and intractable tonic seizures in whom we identified a pathogenic frameshift somatic variant in SLC35A2, present at a range of variant allele fractions (4.2%–19.5%) in 12 different brain tissues detected by targeted sequencing. The proportion of the SLC35A2 variant correlated with severity and location of neurophysiology and neuroimaging abnormalities for each tissue.ConclusionsOur findings support the importance of tissue-based sequencing and highlight a correlation in our patient between SLC35A2 variant allele fractions and the severity of epileptogenic phenotypes in different brain tissues obtained from a grid-based resection of clinically defined epileptogenic regions.

Mowat–Wilson syndrome (MWS) is a syndromic form of Hirschsprung disease that is characterized by variable degrees of intellectual disability, characteristic facial dysmorphism, and a diverse set of other congenital malformations due to haploinsufficiency of ZEB2. A variety of brain malformations have been described in neuroimaging studies of MWS patients, and the role of ZEB2 in the brain has been studied in a multitude of genetically engineered mouse models that are now available. However, a paucity of autopsy information limits our ability to correlate data from neuroimaging studies and animal models with actual MWS patient tissues. Here, we report the autopsy neuropathology of a 19-year-old male patient with MWS. Autopsy neuropathology findings correlated well with the reported MWS neuroimaging data and are in keeping with data from genetically engineered MWS mouse models. This autopsy enhances our understanding of ZEB2 function in human brain development and demonstrates the reliability of MWS murine models.

Meningiomas are common in adults (~35% of brain tumors) but rare in children, where they exhibit unique clinical, pathological and molecular features compared to adult counterparts. Thus, data generated from adult cohorts may be imperfectly suited to guiding diagnostic, prognostic and treatment decisions for children. We studied 50 meningioma patients ≤18 years with available clinical and pathological data to address the need for data obtained in the pediatric setting. As previously described, we noted a slight bias toward male patients and a higher proportion of spinal tumors compared to adults. Thirty-eight of 50 specimens were further analyzed by next generation sequencing. Loss-of-function mutations in NF2 and chromosome 22 losses were common, but pathogenic variants in other genes (SMARCB1, FUBP1, BRAF, TERT promoter, CHEK2, SMAD and GATA3) were identified in a minority of cases. Copy number variants outside of chromosomes 22 and 1 were infrequent. H3K27 hypomethylation, a useful biomarker in adult tumors, was not found in our cohort. In exploring the correlation between mitotic count and recurrence-free survival, we found a threshold of six mitoses per 10 high powered fields as the optimal cutoff in predicting recurrence-free survival. If independently validated in larger studies, adjusted grading thresholds could enhance the clinical management of pediatric meningiomas.

This paper will address students who have the diagnosis of Down syndrome. It will encompass the overall understanding of what a child with Down syndrome experiences in and outside of the classroom, as well as some of the physical characteristics that can manifest as a result of a genetic mutation resulting in the diagnosis of Down syndrome.

When educating boys and girls with Autism Spectrum Disorders (ASD), it is vital that teachers know how to best support their students within the classroom as well as how to support their families with community activities. Knowing the definition, etiology, and causes of ASD as well as specific strategies will strengthen educational programming around the world.

Introduction: X-linked myotubular myopathy (XLMTM) is a severe, frequently fatal, type of congenital myopathy for which only supportive care is currently available. XLMTM is due to MTM1 mutations t...

Centronuclear myopathies (CNM) are a subtype of congenital myopathies (CM) characterized by skeletal muscle weakness and an increase in the number of central myonuclei. We have previously identified three CNM probands, two with associated dilated cardiomyopathy, carrying striated preferentially expressed gene (SPEG) mutations. Currently, the role of SPEG in skeletal muscle function is unclear as constitutive SPEG-deficient mice developed severe dilated cardiomyopathy and died in utero. We have generated a conditional Speg-KO mouse model and excised Speg by crosses with striated muscle-specific cre-expressing mice (MCK-Cre). The resulting litters had a delay in Speg excision consistent with cre expression starting in early postnatal life and, therefore, an extended lifespan up to a few months. KO mice were significantly smaller and weaker than their littermate-matched controls. Histopathological skeletal muscle analysis revealed smaller myofibers, marked fiber-size variability, and poor integrity and low number of triads. Further, SPEG-deficient muscle fibers were weaker by physiological and in vitro studies and exhibited abnormal Ca(2+) handling and excitation-contraction (E-C) coupling. Overall, SPEG deficiency in skeletal muscle is associated with fewer and abnormal triads, and defective calcium handling and excitation-contraction coupling, suggesting that therapies targeting calcium signaling may be beneficial in such patients.

Objective Hepcidin, a mediator of innate immunity, binds the iron exporter ferroportin, leading to functional hypoferremia through intracellular iron sequestration. We explored hepcidin–ferroportin interactions in neonates clinically diagnosed with early-onset neonatal sepsis (EONS). Study Design Hepcidin and interleukin (IL)-6 were quantified by enzyme-linked immunosorbent assay (ELISA) in 92 paired cord blood–maternal blood samples in the following groups: “Yes” EONS (n = 41, gestational age [GA] 29 ± 1 weeks) and “No” EONS (n = 51, GA 26 ± 1 weeks). Placental hepcidin and ferroportin expression were evaluated by immunohistochemistry and real-time-polymerase chain reaction (RT-PCR). Liver hepcidin and ferroportin expression patterns were ascertained in autopsy specimens of neonates (n = 8) who died secondary to culture-proven sepsis. Results Cord blood hepcidin was significantly elevated (GA corrected, p = 0.018) and was positively correlated with IL-6 (r = 0.379, p = 0.001) in EONS. Hepcidin localized at syncytiotrophoblast and fetal vascular endothelium. Placental ferroportin, but not hepcidin mRNA correlated with cord blood hepcidin levels (r = 0.46, p = 0.039) and funisitis severity (r = 0.50, p = 0.018). Newborns who died from sepsis (n = 4) had higher hepatic hepcidin and iron sequestration, but lower ferroportin staining than those who died of nonsepsis causes (n = 4). Conclusion Premature fetuses with EONS have elevated circulating hepcidin, likely related to lower placenta and liver ferroportin expression. Fetal hepcidin–ferroportin interaction appears to play a role in EONS pathophysiology independent of maternal response to intrauterine inflammation.

Purpose We previously reported the cases of 3 children with diffuse intrinsic pontine glioma (DIPG) in whom noncontiguous treatment-related abnormalities (NCTRAs) developed in the brain after expanded-field radiation therapy (RT). To investigate the occurrence and putative mechanism of NCTRAs, we reviewed brain magnetic resonance imaging studies of patients with DIPG treated in 2 consecutive phase I clinical trials (trials 1 and 2). Methods and Materials The 55 children included in these trials received small-molecule inhibitors: vandetanib in trial 1 (n=32; mean age 6.4 years) and vandetanib and dasatinib in trial 2 (n=23; mean age 5.8 years). The patients also received conformal 3-dimensional RT (cumulative dose 54 Gy). For patients enrolled in trial 1, the clinical target volume (CTV) was expanded by 1 cm from the gross tumor volume. In trial 2, the expansion to form the CTV was 2 to 3 cm. A review of imaging studies was performed from the initial diagnosis through the end of progression-free survival. The imaging findings were grouped into 5 categories according to the presence, absence, location, extent, and putative mechanism of NCTRAs. Statistical analysis was performed to evaluate the association between covariates and NCTRA, cohort characterization, and survival comparisons. Results Overall survival was similar in both studies ( P =.74). NCTRAs developed in 9 patients (39%) treated in trial 2 but in none treated in trial 1. The NCTRAs included T2-weighted hyperintensities with (n=3; radiation necrosis) or without (n=5) contrast uptake, supratentorial leukoencephalopathy (n=2), and ischemic stroke (n=1). All NCTRAs, except for 1, occurred within the CTV. Compared with nonaffected patients, patients with a NCTRA were younger ( P =.003) and had had larger relative brain volumes exposed to doses >20 Gy. Conclusions The imaging features of NCTRAs suggest that their development is secondary to synergistic steno-occlusive vascular effects induced by the combination of RT, an expanded CTV, potent antiangiogenic therapy, young age, and, in 1 case, a genetic predisposition.

OBJECTIVESporadic meningiomas have been classified in many different ways. Radiographically, these lesions can be described as occurring in either typical or atypical locations. The purpose of this study was to determine if there are any histopathological differences between sporadic meningiomas that arise in these varying locations in children.METHODSThe neuroimaging, histopathological findings, and clinical records in patients with sporadic pediatric meningiomas not associated with neurofibromatosis Type 2 or prior radiation therapy were retrospectively reviewed. Tumors were classified by radiological findings as either typical or atypical, and they were categorized histopathologically by using the latest WHO nomenclature and grading criteria.RESULTSFifteen sporadic meningiomas in pediatric patients were biopsied or resected at the authors’ institution between 1989 and 2013. Five (33%) were typical in radiographic appearance and/or location and 10 (67%) were atypical. Four (80%) typical meningiomas were WHO Grade I tumors. Most (60%) of the atypical meningiomas were WHO Grade II or III.CONCLUSIONSThis study is the largest series of sporadic pediatric meningiomas in atypical locations to date. Although sporadic meningiomas are relatively infrequent in children, those with atypical imaging, specifically those with apparently intraparenchymal and intraosseous locations, may be more common than previously recognized. In this study, pediatric sporadic meningiomas arising in atypical locations, in particular intraparenchymal meningiomas, may be of higher histopathological grade. The authors’ findings should alert clinicians to the potential for more aggressive clinical behavior in these tumors.

// Kellie B. Haworth 1, 2 , Michael A. Arnold 3, 4 , Christopher R. Pierson 3, 4, 5 , Kwangmin Choi 6 , Nicholas D. Yeager 1 , Nancy Ratner 6 , Ryan D. Roberts 1, 2 , Jonathan L. Finlay 1 and Timothy P. Cripe 1, 2 1 Division of Hematology, Oncology, Blood and Marrow Transplant, Department of Pediatrics, Nationwide Children’s Hospital, Columbus, Ohio, USA 2 Center for Childhood Cancer and Blood Diseases, The Research Institute, Nationwide Children’s Hospital, Columbus, Ohio, USA 3 Division of Anatomic Pathology, Department of Pathology and Laboratory Medicine, Nationwide Children’s Hospital, Columbus, Ohio, USA 4 Department of Pathology, The Ohio State University College of Medicine, Columbus, Ohio, USA 5 Division of Anatomy, Department of Biomedical Education and Anatomy, The Ohio State University College of Medicine, Columbus, Ohio, USA 6 Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA Correspondence to: Kellie B. Haworth, email: kellie.haworth@nationwidechildrens.org Keywords: NF1, MPNST, neurofibromas, immunotherapy, immunophenotype Received: December 14, 2016 Accepted: April 16, 2017 Published: May 30, 2017 ABSTRACT Successful treatment of neurofibromatosis type 1 (NF1)-associated tumors poses a significant clinical challenge. While the primary underlying genetic defect driving RAS signaling is well described, recent evidence suggests immune dysfunction contributes to tumor pathogenesis and malignant transformation. As immunologic characterizations, prognostic and predictive of immunotherapeutic clinical response in other cancers, are not fully described for benign and malignant NF1-related tumors, we sought to define their immunologic profiles. We determined the expression of human leukocyte antigen (HLA)-A/-B/-C, β-2-microglobulin (B2M), and T cell inhibitory ligands PD-L1 and CTLA-4 by microarray gene analysis and flow cytometry. We examined HLA-A/-B/-C, B2M, and PD-L1 expression on thirty-six NF1-associated tumor samples by immunohistochemistry, and correlated these with tumoral CD4 + , CD8 + , FOXP3 + , CD56 + , and CD45RO + lymphocytic infiltrates. We evaluated several tumors from a single patient, observing trends of increasing immunogenicity over time, even with disease progression. We observed similarly immunogenic profiles for malignant peripheral nerve sheath tumors (MPNST) and nodular and plexiform neurofibromas, contrasting with diffuse neurofibromas. These studies suggest that while immunotherapies may offer some benefit for MPNST and nodular and plexiform neurofibromas, tumor heterogeneity might pose a significant clinical challenge to this novel therapeutic approach.

Pediatric brain tumors including medulloblastoma and atypical teratoid/rhabdoid tumor are associated with significant mortality and treatment-associated morbidity. While medulloblastoma tumors within molecular subgroups 3 and 4 have a propensity to metastasize, atypical teratoid/rhabdoid tumors frequently afflict a very young patient population. Adjuvant treatment options for children suffering with these tumors are not only sub-optimal but also associated with many neurocognitive obstacles. A potentially novel treatment approach is oncolytic virotherapy, a developing therapeutic platform currently in early-phase clinical trials for pediatric brain tumors and recently US Food and Drug Administration (FDA)-approved to treat melanoma in adults. We evaluated the therapeutic potential of the clinically available oncolytic herpes simplex vector rRp450 in cell lines derived from medulloblastoma and atypical teratoid/rhabdoid tumor. Cells of both tumor types were supportive of virus replication and virus-mediated cytotoxicity. Orthotopic xenograft models of medulloblastoma and atypical teratoid/rhabdoid tumors displayed significantly prolonged survival following a single, stereotactic intratumoral injection of rRp450. Furthermore, addition of the chemotherapeutic prodrug cyclophosphamide (CPA) enhanced rRp450’s in vivo efficacy. In conclusion, oncolytic herpes viruses with the ability to bioactivate the prodrug CPA within the tumor microenvironment warrant further investigation as a potential therapy for pediatric brain tumors.

BACKGROUND The optimal management for children with recurrent choroid plexus carcinoma (CPC), is not established. We report two children with germline TP53 mutations, whose CPC relapses were managed with marrow-ablative chemotherapy and oral biologically-targeted therapies. PATIENTS: Patient A: A 17 months old male presented with non-metastatic bilateral CPC. A de novo mosaic germline TP53 mutation was identified. After near-total resections, 16 months of standard chemotherapy were administered; 18 months later, localized tumor growth developed, again near-totally resected. Two cycles of re-induction chemotherapy were administered followed by three cycles of thiotepa/carboplatin with autologous hematopoietic cell rescue (AuHCR) and subsequently 21 months of sirolimus and thalidomide, continuing without residual or recurrent disease. Patient B: A 30 months old male presented with left lateral ventricular non-metastatic CPC. A de novo TP53 germline mutation was identified. Following sub-total resection, craniospinal irradiation with boost was administered followed by eight cycles of standard chemotherapy; 18 months later, localized recurrence developed; gross total resection was followed by 15 months of standard dose chemotherapies; four months thereafter, a second local recurrence developed, again gross totally resected. He then received one cycle of high-dose cyclophosphamide followed by three cycles of thiotepa/carboplatin with AuHCR. Subsequently he received sirolimus and thalidomide for 12 months, complicated by progressive pancytopenia. A small localized CPC recurrence was noted, gross totally resected, concomitant with myelodysplastic syndrome; he underwent an allogeneic matched unrelated donor marrow transplantation. CONCLUSIONS Marrow-ablative chemotherapy with post-transplant targeted biological therapy may afford durable survival for select children with recurrent CPC.

Metastatic disease at initial presentation of intracranial ependymoma is an uncommon occurrence with only rare reports of survival and is reportedly more prevalent in the youngest of children. Clinical and molecular characteristics associated with metastatic presentation, their prognostic implications, as well as optimal treatment options for such patients, have not been identified. CASE REPORT: A seven months old child presented with posterior fossa anaplastic ependymoma; following sub-total resection of primary tumor, a spine MRI revealed leptomeningeal dissemination along the cervical spinal cord and nerve roots of the cauda equina. The patient was successfully treated with five cycles of intensive induction chemotherapy (as per Head Start with high-dose methotrexate) followed by three sequential cycles of marrow-ablative chemotherapy and autologous hematopoietic progenitor cell rescue (AuHPCR) without irradiation; he is currently without evidence of the disease now 60 months following initial diagnosis. MOLECULAR/GENOMIC RESULTS: The patient was enrolled on a patient-centric comprehensive molecular profiling protocol, which included paired tumor-normal whole-exome sequencing, RNA sequencing of the disease-involved tissue, and DNA methylation classification. The genomic profile of the tumor was relatively unremarkable, revealing only a terminal gain of chromosome 3p and a terminal deletion of chromosome 22q, suggestive of an unbalanced translocation. Using RNA sequencing, we identified a novel SPECC1L-RAF1 gene fusion. The tumor harbors unique transcriptomic and DNA methylation profiles, failing to discretely classify with well-established ependymoma subgroups. CONCLUSION: Use of genomic profiling techniques provides meaningful information for disease characterization allowing for further expansion of the molecular spectrum associated with malignant disease.

BACKGROUND Wnt-MB infers an excellent prognosis, and metastatic disease is rare. However, specific treatment strategies and patterns of failure for patients with recurrent Wnt-MB are unknown. We report two cases of recurrent beta-catenin nucleopositive Wnt-MBs treated with an irradiation-sparing strategy, incorporating HDCx/AuHPCR. PATIENT 1: A nine-year-old female experienced local recurrence of a non-metastatic Wnt-MB nine months after gross total resection (GTR) followed by 18Gy craniospinal irradiation (CSI) with primary site boost to 54Gy, accompanied by weekly vincristine, followed by a maintenance regimen of nine cycles of cisplatin/lomustine/vincristine alternating with cyclophosphamide/vincristine every third cycle. GTR of the relapsed tumor was followed by three cycles of HDCx/AuHPCR. She is disease-free for over three years following relapse treatment. PATIENT 2: A 17-year-old male initially underwent GTR, followed by 23.4Gy CSI with 54Gy posterior fossa boost with concomitant weekly vincristine, followed by a maintenance regimen that included nine alternating cycles of vincristine/lomustine/cisplatin and cyclophosphamide/vincristine. Isolated right frontal horn metastatic recurrence developed 19 months post-treatment; three cycles of irinotecan/temozolomide/bevacizumab and gamma-knife radiosurgery produced complete response. A second isolated metastatic recurrence within the left frontal horn occurred 13 months post-treatment, which was treated with two cycles of cyclophosphamide/etoposide followed by two cycles of HDCx/AuHPCR. MRI of the brain showed no residual tumor one month post-treatment. He currently awaits follow-up stereotactic radiosurgery. CONCLUSION Patients with recurrent Wnt-MB may be treated with curative intent using a multi-disciplinary approach that includes HDCx/AuHPCR, and minimization or avoidance of re-irradiation.

BACKGROUND We report a unique case of spinal ependymoma with classic histology and aggressive clinical behavior which harbored a focal MYC (8q24) amplification. CASE REPORT: A-12-year old male presented with a three months history of back pain and acute onset weakness with ataxia. A spine MRI revealed an avidly enhancing intradural, extramedullary mass occupying the dorsal spinal canal from C6 through T2. The tumor demonstrated mild diffusion restriction and was associated with severe cord compression and mild edema. He underwent gross total resection. Pathological diagnosis was classic grade II ependymoma. Eleven months later, he re-presented with acute onset lower extremity paresthesia and left-handed weakness. Spine MRI demonstrated tumor recurrence extending from C2 through T1-T2 with resultant severe cord compression, again demonstrating avid enhancement and restricted diffusion. He underwent subtotal resection of the mass and focal proton beam irradiation. MOLECULAR CHARACTERISTICS: The patient was enrolled on an institutional comprehensive genomic profiling protocol. The tumor’s copy number profile was complex, including homozygous loss of 17p and notably, amplification of the MYC oncogene. Using fluorescence in situ hybridization, we identified >20 copies of MYC in interphase cells, confirming the gene amplification, while two copies of MYCN (2p24) were seen. DNA methylation further classified this tumor as clustering near posterior fossa group A (score=0.6073) tumors. CONCLUSION We report a unique case of an adolescent male with aggressive spinal ependymoma harboring focal MYC amplification. Testing for MYC amplification may be reasonable in newly-diagnosed spinal ependymomas to aid in characterization.

Background Glioblastoma (GBM) remains one of the least successfully treated cancers. It is essential to understand the basic biology of this lethal disease and investigate novel pharmacological targets to treat GBM. The aims of this study were to determine the biological consequences of elevated expression of ΔNp73, an N-terminal truncated isoform of TP73, and to evaluate targeting of its downstream mediators, the angiopoietin 1 (ANGPT1)/tunica interna endothelial cell kinase 2 (Tie2) axis, by using a highly potent, orally available small-molecule inhibitor (rebastinib) in GBM. Methods ΔNp73 expression was assessed in glioma sphere cultures, xenograft glioblastoma tumors, and glioblastoma patients by western blot, quantitative reverse transcription PCR, and immunohistochemistry. Immunoprecipitation, chromatin immunoprecipitation (ChiP) and sequential ChIP were performed to determine the interaction between ΔNp73 and E26 transformation-specific (ETS) proto-oncogene 2 (ETS2) proteins. The oncogenic consequences of ΔNp73 expression in glioblastomas were examined by in vitro and in vivo experiments, including orthotopic zebrafish and mouse intracranial-injection models. Effects of rebastinib on growth of established tumors and survival were examined in an intracranial-injection mouse model. Results ΔNp73 upregulates both ANGPT1 and Tie2 transcriptionally through ETS conserved binding sites on the promoters by interacting with ETS2. Elevated expression of ΔNp73 promotes tumor progression by mediating angiogenesis and survival. Therapeutic targeting of downstream ΔNp73 signaling pathways by rebastinib inhibits growth of established tumors and extends survival in preclinical models of glioblastoma. Conclusion Aberrant expression of ΔNp73 in GBM promotes tumor progression through autocrine and paracrine signaling dependent on Tie2 activation by ANGPT1. Disruption of this signaling by rebastinib improves tumor response to treatment in glioblastoma.

BACKGROUND The phenotypic spectrum of the skeletal muscle voltage-gated sodium channel gene (SCN4A) mutations has been expanding dramatically with advancements in genetic testing. Previously only known to cause autosomal dominant myotonia or periodic paralysis, now recessive mutations have been found causing congenital myopathies and congenital myasthenic syndromes. CASE PRESENTATION A 27-year-old woman who was born with Arnold-Chiari malformation, hydrocephalus, high-arched palate, bilateral hip dysplasia, and severe scoliosis presented for evaluation of episodic muscle stiffness and weakness. Electrodiagnostic studies revealed myopathy and widespread myotonia. Muscle histopathology showed marked fiber size variability, type I fiber predominance with minimal scattered necrosis and regeneration which was typical of a congenital myopathy with an additional finding of a lobulated structural pattern in type I fibers. Sequential individual gene testing revealed a novel de novo heterozygous c.2386 C > G, p.Leu796Val missense mutation in the SCN4A gene. DISCUSSION To the best of our knowledge, this is the first report of a dominant, heterozygous mutation in SCN4A causing a complex phenotype of congenital myopathy and myotonia with multiple congenital anomalies and unique muscle pathology findings. This case is another addition to the ever expanding phenotype of SCN4A mutations.

“Head Start” 4 (HS 4) is a prospective randomized clinical trial that tailors treatment based on medulloblastoma molecular subgroups and response to induction chemotherapy to compare the efficacy of one versus three (tandem) cycles of myeloablative therapy. Advances in RNA and DNA profiling have identified four core molecular subgroups of medulloblastoma with prognostic significance: Sonic Hedgehog (SHH) subtype, WNT subtype, Group 3, and Group 4. In HS 4 trial, we utilize a combination of histopathology and immunohistochemistry (pathology/IHC), as well as chromosomal microarray analysis (CMA) utilizing OncoScanTM (Thermo Fisher) to classify medulloblastoma samples into either SHH, WNT, or non-WNT/non-SHH (Group 3/4) subgroups at the time of diagnosis. NanoString based 22-gene assay is performed retrospectively to test concordance. We have pathology/IHC, CMA, and NanoString data on 26 infants and young children with medulloblastoma enrolled on HS 4. Pathology/IHC was able to assign samples to SHH, WNT, and non-WNT/non-SHH subgroups in all but two cases: one case was classified as Group 3, and the second as SHH by both CMA and NanoString. CMA was indeterminate in six cases, of which, pathology/IHC was able to assign all six samples aforementioned three subgroups. NanoString was indeterminate in two cases: one case was classified as SHH by CMA and pathology/IHC, and the second case was indeterminate by CMA but was assigned as non-WNT/non-SHH on pathology/IHC. There is excellent correlation between NanoString and combination of histopathology and CMA for core medulloblastoma subgrouping on HS 4. Methylation studies are ongoing.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been indicated as a novel cancer drug target, since it plays an important role in diverse oncogenic processes including survival, cell proliferation and migration. Emerging STAT3 inhibitors have demonstrated efficacy in cancer cells and animal tumor models. It is well known that most solid tumors are characterized by hypoxia, but it is not clear if hypoxic conditions affect activity of STAT3 inhibitors. To examine this, two STAT3 inhibitors were tested to investigate their inhibitory efficacy in cancer cells grown under hypoxic conditions compared with those without hypoxia. Cell proliferation, colony formation and western blot assays were performed to examine the differences in the cell viability, proliferation and proteins in the STAT3 pathway. Under hypoxic conditions, the half-maximal inhibitory concentration values for both STAT3 inhibitors were increased compared to normoxic conditions in human pancreatic cancer, medulloblastoma and sarcoma cell lines. In addition, the ability of both STAT3 inhibitors to inhibit colony formation in pancreatic cancer, medulloblastoma and sarcoma cell lines was reduced under hypoxic conditions when compared to cells under normoxic conditions. Furthermore, there was an increase in phosphorylated STAT3 levels in cancer cells under hypoxic conditions, suggesting this may be one of the mechanisms of resistance. In summary, the results presented here provide a novel finding of STAT3 inhibitor activity under hypoxic conditions and indicate that under such low oxygen conditions, the anticancer efficacy of STAT3 inhibitors was indeed hampered. These results highlight the need to develop new therapeutic strategies to overcome the resistance of cancer cells to STAT3 inhibitors under hypoxic conditions.

Improved survival rates in premature infants and more sensitive neuroimaging techniques have expanded the scope of recognized neurodevelopmental disabilities in this vulnerable population and have implicated a role for cerebellar pathology in their origin. Although supratentorial pathologies are well studied, cerebellar pathology has been under-recognized in premature infants. The purpose of this review is to provide a concise description of established acquired cerebellar pathologies in premature infants including cerebellar atrophy/hypoplasia, hemorrhage, and infarction. The cerebellum develops over an extended period during which time cerebellar injury tends to occur with the potential to derail the cerebellum from its expected growth trajectory and perturb the establishment of cerebellar neural circuitry. The occurrence of cerebellar injury in this vulnerable period may have life-long implications that extend beyond the immediate damage sustained by the cerebellum, all of which needs to be considered as we research the causes and effects of neurodevelopmental disabilities in these patients.

Rhabdoid phenotype and loss of SMARCB1 expression in a brain tumor are characteristic features of atypical teratoid/rhabdoid tumors (ATRT). Rare non-rhabdoid brain tumors showing cribriform growth pattern and SMARCB1 loss have been designated cribriform neuroepithelial tumor (CRINET). Small case series suggest that CRINETs may have a relatively favorable prognosis. However, the long-term outcome is unclear and it remains uncertain whether CRINET represents a distinct entity or a variant of ATRT. Therefore, 10 CRINETs were clinically and molecularly characterized and compared with 10 ATRTs of each of three recently described molecular subgroups (i.e. ATRT-TYR, ATRT-SHH and ATRT-MYC) using Illumina Infinium HumanMethylation450 arrays, FISH, MLPA, and sequencing. Furthermore, outcome was compared to a larger cohort of 27 children with ATRT-TYR. Median age of the 6 boys and 4 girls harboring a CRINET was 20 months (range 10-129 months). On histopathological examination, all CRINETs demonstrated a cribriform growth pattern and distinct tyrosinase staining. On unsupervised cluster analysis of methylation data, all CRINETs examined exclusively clustered within the ATRT-TYR molecular subgroup. As ATRT-TYR, CRINETs mainly showed large heterozygous 22q deletions (9/10) and SMARCB1 mutations of the other allele. In two patients, SMARCB1 mutations were also present in the germline. Estimated mean overall survival in patients with CRINETs was 125 months (95% confidence interval 100-151 months) as compared to only 53 (33-74) months in patients with ATRTs of the ATRT-TYR subgroup (Log-Rank P

Individuals with Down syndrome (DS) have an increased risk of acute leukemia compared to a markedly decreased incidence of solid tumors. Medulloblastoma, the most common malignant brain tumor of childhood, is particularly rare in the DS population, with only one published case. As demonstrated in a mouse model, DS is associated with cerebellar hypoplasia and a decreased number of cerebellar granule neuron progenitor cells (CGNPs) in the external granule cell layer (EGL). Treatment of these mice with sonic hedgehog signaling pathway (Shh) agonists promote normalization of CGNPs and improved cognitive functioning. We describe a 21-month-old male with DS and concurrent desmoplastic/nodular medulloblastoma (DNMB)—a tumor derived from Shh dysregulation and over-activation of CGNPs. Molecular profiling further classified the tumor into the new consensus SHH molecular subgroup. Additional testing revealed a de novo heterozygous germ line mutation in the PTCH1 gene encoding a tumor suppressor protein in the Shh pathway. The developmental failure of CGNPs in DS patients offers a plausible explanation for the rarity of medulloblastoma in this population. Conversely, patients with PTCH1 germline mutations experience Shh overstimulation resulting in Gorlin (Nevoid Basal Cell Carcinoma) syndrome and an increased incidence of malignant transformation of CGNPs leading to medulloblastoma formation. This represents the first documented report of an individual with DS simultaneously carrying PTCH1 germline mutation. We have observed a highly unusual circumstance in which the PTCH1 mutation appears to “trump” the effects of DS in causation of Shh-activated medulloblastoma.

BACKGROUND Rhabdoid predisposition syndrome is characterized by germline alterations in SMARCB1 or SMARCA4, leading to synchronous or metachronous central nervous system (CNS) and extra-CNS rhabdoid tumors. Rare survivors have been reported to date. METHODS We describe the molecular profiling and treatment regimen of three patients with synchronous atypical teratoid/rhabdoid tumor (ATRT) and malignant rhabdoid tumor of the kidney (MRT-K). All patients underwent radical nephrectomy of the kidney, and gross total resection of the primary CNS tumor was achieved for two patients. An intensive chemotherapy regimen was administered; an induction phase based on the modified Third Intergroup Rhabdomyosarcoma Study (IRS-III) for ATRT followed by a consolidation phase with three cycles of high-dose chemotherapy and autologous hematopoietic progenitor cell rescue, without irradiation. All three patients were enrolled on an institutional comprehensive genomic profiling protocol. RESULTS A germline focal 22q deletion, including SMARCB1, was detected in two patients, while the third patient had a maternally-inherited heterozygous frameshift variant in SMARCB1. Somatic loss of heterozygosity of 22q was identified in all patients, resulting in biallelic inactivation of SMARCB1. Divergent tumor subgroups were described using DNA methylation. The three MRT-K samples were classified as MYC subtype. One ATRT was classified as SHH while the other as TYR. One patient is currently three years off-therapy without evidence of disease, while the other two patients have completed the consolidation phase without recurrent disease. CONCLUSION Molecular profiling of CNS and extra-CNS rhabdoid tumors revealed different epigenetic subgroups. An intensive multimodal therapeutic approach without irradiation may achieve prolonged survival.

BACKGROUND Pediatric brain tumor classification has undergone significant evolution over the last decade requiring a high-level of expertise and diagnostic techniques. Such advances have created challenges for pathologists particularly in low-to-middle income countries (LMIC). We conduct weekly pediatric neuro-oncology teleconferences linking global pediatric neuro-oncologists from high-income countries (HIC) to review patients with pediatric subspecialists from Latin America. METHODS Three to five patients are discussed weekly and second neuropathology review is offered when a high-level of suspicion emerges of a questionable diagnosis based on clinical and radiographical information. Nationwide Children’s Hospital (NCH) provides second neuropathology review at no cost to institutions in Latin America that fulfill these criteria. RESULTS From July 2015 to December 2019 NCH reviewed 54 pathology samples from eleven Latin American countries. Of these, 33 (61.1%) cases resulted in diagnostic changes, of which 28 (51.8%) were significant, impacting treatment plans and overall patient outcomes. The remaining 21 (38.9%) confirmed institutional diagnosis; however, in eight of these 21 cases additional molecular information and/or further tumor subtyping unavailable in their home country at the time (eg: BRAF, RELA-fusion, medulloblastoma subtyping) was provided. CONCLUSIONS This study highlights the importance of centralized pathology review by institutions with the proper equipment, infrastructure and expertise in pediatric neuropathology. Furthermore, this documents the beneficial impact of teleconferencing for subspecialists in LMIC who must treat a wide variety of pediatric cancers with few resources and support. Additionally, our findings underscore the need for pediatric subspecialty training in LMIC.

Klippel-Feil syndrome (KFS) is an exceerlingly rare constitutional disorder in which a paucity of knowledge exists about the disease and its associated morbidity and mortality. We present a 4-year-old male with KFS, who notably was also diagnosed with large-cell anaplastic medulloblastoma. We evaluated the genetic basis of co-occurring KFS and medulloblastoma and the role of MYO18B as related to medulloblastoma. Constitutional and somatic variant and copy number analyses were performed from DNA-based exome studies, along with RNA-sequencing of tumor tissue, to elucidate the genetic etiology of the co-existing disease states. We identified novel constitiitional compound heterozygous frameshift variants (NM_032608.5: p.Leu2257SerfsTerl6 and p.Arg2220SerfsTer74) each encoding a premature stop of translation in MYO18B, consistent with a diagnosis of KFS. We did not identify any somatic variants of known relevance or disease-relevant therapeutic targets in the tumor. The somatic copy number profile was suggestive of Group 3γ medulloblastoma. Relative to pediatric brain tremors, medulloblastoma, particularly, Group 3, had increased gene expression of MYO18B. In summary, coexisting constitutional and somatic diagnoses in this patient enabled the elucidation of the genetic etiology of KFS and provided support for the role of MYO18B in tumor suppression.

BACKGROUND: Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas. METHODS: We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes. RESULTS: Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma. CONCLUSIONS: The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma. (Funded by the National Institutes of Health.)

Elevated levels of the pro-inflammatory cytokine interleukin-6 (IL-6) have tumor-promoting activity and are associated with poor survival outcomes in many cancers. Additionally, the IL-6/GP130/STAT3 axis has been widely studied due to its pivotal role in tumor development and maintenance in a number of tissue types, including the cerebellum. However, the connection between IL-6 signaling and medulloblastoma progression is largely unexplored. In the present study, we observed that IL-6 induced medulloblastoma cell viability, cell proliferation and glycolysis. Furthermore, it also upregulated the expression of phosphorylated STAT3, indicating that the IL-6/GP130/STAT3 pathway plays a central role in medulloblastoma. The FDA-approved drug bazedoxifene, a blocker of the formation of the hexameric IL-6/IL-6R/GP130 complex, was re-purposed in this study to inhibit the IL-6/GP130/STAT3 signaling pathway. Bazedoxifene not only inhibited IL-6 mediated cell viability and cell proliferation, and increased phosphorylated STAT3 expression, but it also decreased cell glycolysis, demonstrating a certain level of therapeutic efficacy in vitro. Collectively, our findings offer new insight into the molecular mechanism underlying the biological aggressiveness of medulloblastoma, the roles of IL-6 in these processes and a possible efficacious adjuvant therapy for medulloblastoma.

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans. The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Experimental autoimmune encephalomyelitis (EAE) has been used as an animal model of multiple sclerosis to identify pathogenic cytokines that could be therapeutic targets. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is the only cytokine reported to be essential for EAE. We investigated the role of GM-CSF in EAE in C3HeB/FeJ mice that uniquely exhibit extensive brain and spinal cord inflammation. Unexpectedly, GM-CSF-deficient C3HeB/FeJ mice were fully susceptible to EAE because IL-17 activity compensated for the loss of GM-CSF during induction of spinal cord-targeted disease. In contrast, both GM-CSF and IL-17 were needed to fully overcome the inhibitory influence of IFN-γ on the induction of inflammation in the brain. Both GM-CSF and IL-17 independently promoted neutrophil accumulation in the brain, which was essential for brain-targeted disease. These results identify a GM-CSF/IL-17/IFN-γ axis that regulates inflammation in the central nervous system and suggest that a combination of cytokine-neutralizing therapies may be needed to dampen central nervous system autoimmunity.

Spinal vascular malformations are rare vascular lesions that most frequently present with back pain, radiculopathy, and/or myelopathy. Neurological decline is typically secondary to progressive radiculopathy, myelopathy, venous thrombosis, and stroke. Few case reports have described thoracolumbar spinal vascular malformations that present with both subarachnoid and intraventricular hemorrhage. This is the first reported case of a thoracolumbar spinal vascular malformation presenting with isolated intraventricular hemorrhage on initial imaging followed by acute and fatal rehemorrhage.

Cancer treatment strategies targeting the tumor microenvironment and corresponding immune cell infiltrate (i.e., immunotherapy) have recently proven effective against many solid tumors. In pediatric brain cancers, there is little known about the immune tumor microenvironment. We aim to characterize immune cell populations of pediatric brain tumors, which will better inform us about immune cell infiltration in different brain cancer subtypes and may indicate potential for patient response to immunotherapy. The Institute for Genomic Medicine at Nationwide Children’s Hospital has recently initiated a translational protocol to evaluate tumor specimens from pediatric patients with rare or refractory brain cancers in a focused N-of-1 manner. We performed RNA-seq and NanoString™ PanCancer Immune expression profiling on extracted tumor RNA from 22 patients comprising eight distinct brain cancer subtypes. We used CIBERSORT algorithm for deconvolution of RNA-seq global gene expression data and NanoString™ expression profiling to investigate methods of quantifying tumor-infiltrating leukocytes (TIL) and expression of immune genes. Our analyses identified a subset of pediatric brain tumors with evidence of immune infiltration. Using CIBERSORT, four gliomas were predicted to harbor significant TIL populations. Overall, we observed high correlation (Pearson R2 = 0.884) between expression values derived from RNA-seq and NanoString™ methods. Similarly, the two methods produced comparable predictions for TIL composition in the glioma tumors. Further genomic analysis identified one patient, with a diagnosis of glioblastoma and Neurofibromatosis type 1 (NF1), with marked infiltration of macrophages and neutrophils. Overexpression of tumor-associated macrophage/microglia genes (CD14, CD163, CD68, ITGAM, PTPRC) and other immune markers (PD-L1, CCL2/MCP-1, IL1B) was observed relative to all other samples. More specifically, overexpression of CD204, CD206, and CD163 suggested the infiltrating macrophages represent the M2/protumor phenotype. CIBERSORT allows more “granularity” in macrophage population prediction and indicated an equal representation of M2 and M0/uncommitted macrophages. Given the M2 phenotype, it is likely that the infiltrating macrophages contributed to tumor progression and radiation resistance observed in this patient. If confirmed, the presence of M0 macrophages and/or overexpression of PD-L1 could indicate potential for immunotherapy in this patient (e.g., oncolytic viral therapy, strategies focusing on M0 to M1/antitumor polarization, or combination targeted therapy with PD-L1 inhibitor). In summary, we identified a patient with progressive glioblastoma tumor growth and significant macrophage tumor infiltration as a likely candidate for immunotherapy. Citation Format: Katherine E. Miller, Kathleen Schieffer, James Fitch, Vincent Magrini, Amy Wetzel, Anthony R. Miller, Daniel R. Boue, Jeffrey Leonard, Jonathan L. Finlay, Diana S. Osorio, Mohamed S. AbdelBaki, Christopher R. Pierson, Annie Drapeau, Jonathan Pindrik, Kristen Leraas, Elizabeth Varga, Devon Dishman, Lauren Shoemaker, Nicole Ross, Jeremy Pitts, Julie Gastier-Foster, Peter White, Catherine E. Cottrell, Richard K. Wilson, Elaine R. Mardis. Expression profiling-based characterization of immune cell populations in pediatric brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4551.

The efficacy of rituximab treatment in multiple sclerosis has renewed interest in the role of B cells in CNS autoimmunity. In this study, we show that B cells are the predominant MHC class II+ subset in the naive CNS in mice, and they constitutively express proinflammatory cytokines. Incidence of experimental autoimmune encephalomyelitis induced by adoptive transfer was significantly reduced in C3HeB/Fej μMT (B cell–deficient) mice, suggesting an important role for CNS B cells in initiating inflammatory responses. Initial T cell infiltration of the CNS occurred normally in μMT mice; however, lack of production of T cell cytokines and other immune mediators indicated impaired T cell reactivation. Subsequent recruitment of immune cells from the periphery driven by this initial T cell reactivation did not occur in μMT mice. B cells required exogenous IL-1β to reactivate Th17 but not Th1 cells in vitro. Similarly, reactivation of Th1 cells infiltrating the CNS was selectively impaired compared with Th17 cells in μMT mice, causing an increased Th17/Th1 ratio in the CNS at experimental autoimmune encephalomyelitis onset and enhanced brain inflammation. These studies reveal an important role for B cells within the CNS in reactivating T cells and influencing the clinical manifestation of disease.

Children with high-grade glioma, including diffuse intrinsic pontine glioma (DIPG), have a poor prognosis despite multimodal therapy. Identifying novel therapeutic targets is critical to improve their outcome. We evaluated prognostic roles of telomere maintenance mechanisms in children with HGG, including DIPG. A multi-institutional retrospective study was conducted involving 50 flash-frozen HGG (35 non-brainstem; 15 DIPG) tumors from 45 children (30 non-brainstem; 15 DIPG). Telomerase activity, expression of hTERT mRNA (encoding telomerase catalytic component) and TERC (telomerase RNA template) and alternative lengthening of telomeres (ALT) mechanism were assayed. Cox Proportional Hazard regression analyses assessed association of clinical and pathological variables, TERC and hTERT levels, telomerase activity, and ALT use with progression-free or overall survival (OS). High TERC and hTERT expression was detected in 13/28 non-brainstem HGG samples as compared to non-neoplastic controls. High TERC and hTERT expression was identified in 13/15 and 11/15 DIPG samples, respectively, compared to controls. Evidence of ALT was noted in 3/11 DIPG and 10/19 non-brainstem HGG specimens. ALT and telomerase use were identified in 4/19 non-brainstem HGG and 2/11 DIPG specimens. In multivariable analyses, increased TERC and hTERT levels were associated with worse OS in patients with non-brainstem HGG, after controlling for tumor grade or resection extent. Children with HGG and DIPG, have increased hTERT and TERC expression. In children with non-brainstem HGG, increased TERC and hTERT expression levels are associated with a worse OS, making telomerase a promising potential therapeutic target in pediatric HGG.

Maternal inflammation is a risk factor for preterm birth, and premature infants are often exposed to supplemental oxygen as a life-sustaining therapy. While more immature neonates are surviving, rates of neurodevelopmental impairment are not improving. We developed a novel mouse model with clinically relevant exposures to test the hypothesis that systemic maternal inflammation with transient neonatal hyperoxia exposure will induce a phenotype similar to diffuse periventricular leukomalacia (PVL) like that observed in premature human infants.Timed-pregnant C3H/HeN mice received intraperitoneal injections of lipopolysaccharide (LPS) or saline on embryonic day 16. Newborn pups were placed in room air (RA) or 85% oxygen (O2) for 14 days, followed by 14 days in RA recovery. Oligodendroglial and microglial populations were evaluated at 14 and 28 days.Brain weight to body weight ratios were lower in mice exposed to LPS. Oligodendrocyte numbers were decreased significantly in the cerebral cortex and hippocampus in groups exposed to LPS or LPS/O2 at 14 days, and persisted in the cerebral cortex at 28 days for LPS/O2 mice. At day 14, cleaved caspase 3 was increased and numbers of microglia were elevated in the cerebral cortex and hippocampus of LPS/O2 animals.These data indicate that combining systemic maternal LPS and neonatal hyperoxic exposure impairs myelination, and suggests that this novel mouse model may represent a subtle, diffuse form of periventricular white matter injury that could provide a clinically relevant platform for further study of perinatal brain injury.

Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and anaplastic astrocytoma (AA), typically have a dismal prognosis. The 5-year progression-free survival (PFS) rates reported from the Children's Cancer Group (CCG)-945 phase III study, which compared the outcomes in children with newly diagnosed pHGG randomized to receive treatment with radiation and chemotherapy consisting of either prednisone, lomustine, and vincristine (pCV) or the 8-in-1 drug regimen, were 23% for AA and 16% for GBM, with no statistical difference between treatment arms.1 Most recently, the Children's Oncology Group (COG) ACNS0126 study, a phase II trial of radiation and concurrent temozolomide followed by adjuvant temozolomide in newly diagnosed pHGG, found no improvement in the 3-year event-free survival (EFS) rate, compared with that previously reported in the CCG-945 study.2 The lack of success in the treatment of pHGG is multifactorial but is believed to be attributable in part to the limited drug delivery across the blood-brain barrier coupled with inherent tumor resistance to radiation and chemotherapy.3,4 Thus, targeting tumor-associated angiogenesis, a distinguishing feature of HGG, has become a major focus of clinical investigation in the treatment of this disease because of the potential for this strategy to circumvent these principal mechanisms of resistance. Integrins are the primary cell adhesion molecules that integrate angiogenic signals between the extracellular matrix (ECM) and intracellular signaling pathways.5 In particular, αv integrins mediate signaling through binding the Arg-Gly-Asp (RGD) sequence in their respective ECM substrates.5,6 It has been demonstrated that appropriate ligation of the integrin αvβ3, which is selectively expressed on neo-endothelium, is important for sustaining angiogenesis.5,6 GBMs strongly express αv integrins and their ECM ligands.6,7 Cilengitide, a cyclic pentapeptide (cyclic-RGD) antagonist of the integrins αvβ3 and αvβ5, successfully blocks angiogenesis and induces the regression of orthotopic GBM in vivo.8–10 In clinical trials, cilengitide has demonstrated activity as a single agent in recurrent adult GBM11–14 and in a phase I pediatric study of refractory CNS tumors.15 Therefore, the COG ACNS0621 phase II study was conducted to evaluate whether cilengitide is active as a single agent in the treatment of children with refractory pHGG.

The cytological examination of cerebrospinal fluid (CSF) and cytological specimens prepared from central nervous system (CNS) lesions plays a pivotal role in the diagnosis and management of CNS disorders, including tumors. The proper evaluation of CNS tumors typically includes a cytological touch preparation or squash preparation, used in conjunction with a frozen section during an intraoperative consult. Considering the potential wealth of information obtained, these cytological examinations constitute relatively simple and inexpensive investments in time and resources. Prompt evaluation and accurate interpretations are essential as patients can be acutely ill with infectious, neoplastic, or other disorders. The goal of this chapter is to provide a practical and illustrated approach to the interpretation of CSF and CNS cytology specimens. The first part of the chapter includes guidelines for the processing and examination of CSF and detailed descriptions of the microscopic findings, while the second part focuses on the practical aspects of brain tumor smear cytology. Smears of brain tumor tissue highlight important cytological features, such as nuclear detail and the presence of fibrillary extensions, that are critical to recognize when formulating a diagnosis and may be under recognized on frozen section; thus, as illustrated in this chapter, cytological smears are a valuable complement to frozen sections during intraoperative consultations.

Background. Astroblastomas (ABs) are rare glial tumors showing overlapping features with astrocytomas, ependymomas, and sometimes other glial neoplasms, and may be challenging to diagnose. Methods. We examined clinical, histopathological, and molecular features in 28 archival formalin-fixed, paraffin-embedded AB cases and performed survival analyses using Cox proportional hazards and Kaplan–Meier methods. Results. Unlike ependymomas and angiocentric gliomas, ABs demonstrate abundant distinctive astroblastic pseudorosettes and are usually Olig2 immunopositive. They also frequently exhibit rhabdoid cells, multinucleated cells, and eosinophilic granular material. They retain immunoreactivity to alpha thalassemia/mental retardation syndrome X-linked, are immunonegative to isocitrate dehydrogenase-1 R132H mutation, and only occasionally show MGMT promoter hypermethylation differentiating them from many diffuse gliomas. Like pleomorphic xanthoastrocytoma, ganglioglioma, supratentorial pilocytic astrocytoma, and other predominantly cortical-based glial tumors, ABs often harbor the BRAFV600E mutation, present in 38% of cases tested (n = 21), further distinguishing those tumors from ependymomas and angiocentric gliomas. Factors correlating with longer patient survival included age less than 30 years, female gender, absent BRAFV600E, and mitotic index less than 5 mitoses/10 high-power fields; however, only the latter was significant by Cox and Kaplan–Meier analyses (n = 24; P = .024 and .012, respectively). This mitotic cutoff is therefore currently the best criterion to stratify tumors into low-grade ABs and higher-grade anaplastic ABs. Conclusions. In addition to their own characteristic histological features, ABs share some molecular and histological findings with other, possibly ontologically related, cortical-based gliomas of mostly children and young adults. Importantly, the presence of BRAFV600E mutations in a subset of ABs suggests potential clinical utility of targeted anti-BRAF therapy.