Thomas S. Jacques

Purpose: Focal cortical dysplasias (FCD) are localized regions of malformed cerebral cortex and are very frequently associated with epilepsy in both children and adults. A broad spectrum of histopathology has been included in the diagnosis of FCD. An ILAE task force proposes an international consensus classification system to better characterize specific clinicopathological FCD entities. Methods: Thirty-two Task Force members have reevaluated available data on electroclinical presentation, imaging, neuropathological examination of surgical specimens as well as postsurgical outcome. Key Findings: The ILAE Task Force proposes a three-tiered classification system. FCD Type I refers to isolated lesions, which present either as radial (FCD Type Ia) or tangential (FCD Type Ib) dyslamination of the neocortex, microscopically identified in one or multiple lobes. FCD Type II is an isolated lesion characterized by cortical dyslamination and dysmorphic neurons without (Type IIa) or with balloon cells (Type IIb). Hence, the major change since a prior classification represents the introduction of FCD Type III, which occurs in combination with hippocampal sclerosis (FCD Type IIIa), or with epilepsy-associated tumors (FCD Type IIIb). FCD Type IIIc is found adjacent to vascular malformations, whereas FCD Type IIId can be diagnosed in association with epileptogenic lesions acquired in early life (i.e., traumatic injury, ischemic injury or encephalitis). Significance: This three-tiered classification system will be an important basis to evaluate imaging, electroclinical features, and postsurgical seizure control as well as to explore underlying molecular pathomechanisms in FCD.

Summary Hippocampal sclerosis ( HS ) is the most frequent histopathology encountered in patients with drug‐resistant temporal lobe epilepsy ( TLE ). Over the past decades, various attempts have been made to classify specific patterns of hippocampal neuronal cell loss and correlate subtypes with postsurgical outcome. However, no international consensus about definitions and terminology has been achieved. A task force reviewed previous classification schemes and proposes a system based on semiquantitative hippocampal cell loss patterns that can be applied in any histopathology laboratory. Interobserver and intraobserver agreement studies reached consensus to classify three types in anatomically well‐preserved hippocampal specimens: HS International League Against Epilepsy ( ILAE ) type 1 refers always to severe neuronal cell loss and gliosis predominantly in CA 1 and CA 4 regions, compared to CA 1 predominant neuronal cell loss and gliosis ( HS ILAE type 2), or CA 4 predominant neuronal cell loss and gliosis ( HS ILAE type 3). Surgical hippocampus specimens obtained from patients with TLE may also show normal content of neurons with reactive gliosis only (no‐ HS ). HS ILAE type 1 is more often associated with a history of initial precipitating injuries before age 5 years, with early seizure onset, and favorable postsurgical seizure control. CA 1 predominant HS ILAE type 2 and CA 4 predominant HS ILAE type 3 have been studied less systematically so far, but some reports point to less favorable outcome, and to differences regarding epilepsy history, including age of seizure onset. The proposed international consensus classification will aid in the characterization of specific clinicopathologic syndromes, and explore variability in imaging and electrophysiology findings, and in postsurgical seizure control.

Detailed neuropathological information on the structural brain lesions underlying seizures is valuable for understanding drug-resistant focal epilepsy.We report the diagnoses made on the basis of resected brain specimens from 9523 patients who underwent epilepsy surgery for drug-resistant seizures in 36 centers from 12 European countries over 25 years. Histopathological diagnoses were determined through examination of the specimens in local hospitals (41%) or at the German Neuropathology Reference Center for Epilepsy Surgery (59%).The onset of seizures occurred before 18 years of age in 75.9% of patients overall, and 72.5% of the patients underwent surgery as adults. The mean duration of epilepsy before surgical resection was 20.1 years among adults and 5.3 years among children. The temporal lobe was involved in 71.9% of operations. There were 36 histopathological diagnoses in seven major disease categories. The most common categories were hippocampal sclerosis, found in 36.4% of the patients (88.7% of cases were in adults), tumors (mainly ganglioglioma) in 23.6%, and malformations of cortical development in 19.8% (focal cortical dysplasia was the most common type, 52.7% of cases of which were in children). No histopathological diagnosis could be established for 7.7% of the patients.In patients with drug-resistant focal epilepsy requiring surgery, hippocampal sclerosis was the most common histopathological diagnosis among adults, and focal cortical dysplasia was the most common diagnosis among children. Tumors were the second most common lesion in both groups. (Funded by the European Union and others.).

Chordomas are malignant tumours that occur along the spine and are thought to derive from notochordal remnants. There is significant morphological variability between and within chordomas, with some showing prominent areas of chondroid differentiation. Our microarray data from a broad range of connective tissue neoplasms indicate that, at the transcriptional level, chordomas resemble cartilaginous neoplasms. Here we show that chordomas express many genes known to be involved in cartilage development, but they also uniquely express genes distinguishing them from chondroid neoplasms. The brachyury transcription factor, known to be involved in notochordal development, is only expressed by chordomas. Using a polyclonal antibody, we show that brachyury is expressed in the embryonic notochord and in all 53 chordomas analysed, labelling both chondroid and chordoid areas of these tumours. In contrast, the protein was not detected in over 300 neoplasms, including 163 chondroid tumours. Brachyury was not detected in the nucleus pulposus, arguing against the hypothesis that this tissue derives directly from the notochord. These data provide compelling evidence that chordomas derive from notochord and demonstrate that brachyury is a specific marker for the notochord and notochord-derived tumours. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Although sepsis is the major cause of mortality and morbidity in the critically ill, precise mechanism(s) causing multiorgan dysfunction remain unclear. Findings of impaired oxygen utilization in septic patients and animals implicate nitric oxide-mediated inhibition of the mitochondrial respiratory chain. We recently reported a relationship between skeletal muscle mitochondrial dysfunction, clinical severity, and poor outcome in patients with septic shock. We thus developed a long-term, fluid-resuscitated, fecal peritonitis model utilizing male Wistar rats that closely replicates human physiological, biochemical, and histological findings with a 40% mortality. As with humans, the severity of organ dysfunction and eventual poor outcome were associated with nitric oxide overproduction and increasing mitochondrial dysfunction (complex I inhibition and ATP depletion). This was seen in both vital (liver) and nonvital (skeletal muscle) organs. Likewise, histological evidence of cell death was lacking, suggesting the possibility of an adaptive programmed shutdown of cellular function. This study thus supports the hypothesis that multiorgan dysfunction induced by severe sepsis has a bioenergetic etiology. Despite the well-recognized limitations of laboratory models, we found clear parallels between this long-term model and human disease characteristics that will facilitate future translational research.

<h2>Summary</h2><h3>Background</h3> International consensus recognises four medulloblastoma molecular subgroups: WNT (MB_WNT), SHH (MB_SHH), group 3 (MB_Grp3), and group 4 (MB_Grp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions. <h3>Methods</h3> In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Children's Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0–16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3–16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent. <h3>Findings</h3> Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MB_WNT remained unchanged and each remaining consensus subgroup was split in two. MB_SHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MB_SHH-Infant; n=65) and childhood patients (≥4·3 years; MB_SHH-Child; n=38). MB_Grp3 and MB_Grp4 were each split into high-risk (MB_Grp3-HR [n=65] and MB_Grp4-HR [n=85]) and low-risk (MB_Grp3-LR [n=50] and MB_Grp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82–100]); standard risk (50 [23%] patients; 81% survival [70–94]); high-risk (82 [38%] patients; 42% survival [31–56]); and very high-risk (29 [13%] patients; 28% survival [14–56]). <h3>Interpretation</h3> The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations. <h3>Funding</h3> Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

Sox2(+) adult mouse pituitary cells can self-renew and terminally differentiate in vitro, but their physiological role in vivo and possible contribution to oncogenesis remain largely unknown. Using genetic lineage tracing, we show here that the Sox2(+) cell compartment of both the embryonic and adult pituitary contains stem/progenitor cells that are able to differentiate into all hormone-producing lineages and contribute to organ homeostasis during postnatal life. In addition, we show that targeted expression of oncogenic β-catenin in Sox2(+) cells gives rise to pituitary tumors, but, unexpectedly, the tumor mass is not derived from the Sox2(+) mutation-sustaining cells, suggesting a paracrine role of Sox2(+) cells in pituitary oncogenesis. Our data therefore provide in vivo evidence of a role for Sox2(+) stem/progenitor cells in long-term physiological maintenance of the adult pituitary, and highlight an unexpected non-cell-autonomous role for these cells in the induction of pituitary tumors.

Wingless (Wnt)/β-catenin signaling plays an essential role during normal development, is a critical regulator of stem cells, and has been associated with cancer in many tissues. Here we demonstrate that genetic expression of a degradation-resistant mutant form of β-catenin in early Rathke's pouch (RP) progenitors leads to pituitary hyperplasia and severe disruption of the pituitary-specific transcription factor 1-lineage differentiation resulting in extreme growth retardation and hypopituitarism. Mutant mice mostly die perinatally, but those that survive weaning develop lethal pituitary tumors, which closely resemble human adamantinomatous craniopharyngioma, an epithelial tumor associated with mutations in the human β-catenin gene. The tumorigenic effect of mutant β-catenin is observed only when expressed in undifferentiated RP progenitors, but tumors do not form when committed or differentiated cells are targeted to express this protein. Analysis of affected pituitaries indicates that expression of mutant β-catenin leads to a significant increase in the total numbers of pituitary progenitor/stem cells as well as in their proliferation potential. Our findings provide insights into the role of the Wnt pathway in normal pituitary development and demonstrate a causative role for mutated β-catenin in an undifferentiated RP progenitor in the genesis of murine and human craniopharyngioma.

Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism-dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.

In this case report, severe encephalitis with no established cause developed in a child with SCID. Through deep sequencing, human coronavirus OC43 was identified in brain tissue.

Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5' arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 "hits" occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis.

Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM).To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency.We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM.Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies.This work was funded or supported by grants from the AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, the L'Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research Institute (US).

Human IFNAR2 deficiency causes fatal susceptibility to live viral vaccines.

Surgery is a widely accepted treatment option for drug-resistant focal epilepsy. A detailed analysis of longitudinal postoperative seizure outcomes and use of antiepileptic drugs for different brain lesions causing epilepsy is not available. We aimed to analyse the association between histopathology and seizure outcome and drug freedom up to 5 years after epilepsy surgery, to improve presurgical decision making and counselling.In this retrospective, multicentre, longitudinal, cohort study, patients who had epilepsy surgery between Jan 1, 2000, and Dec 31, 2012, at 37 collaborating tertiary referral centres across 18 European countries of the European Epilepsy Brain Bank consortium were assessed. We included patients of all ages with histopathology available after epilepsy surgery. Histopathological diagnoses and a minimal dataset of clinical variables were collected from existing local databases and patient records. The primary outcomes were freedom from disabling seizures (Engel class 1) and drug freedom at 1, 2, and 5 years after surgery. Proportions of individuals who were Engel class 1 and drug-free were reported for the 11 main categories of histopathological diagnosis. We analysed the association between histopathology, duration of epilepsy, and age at surgery, and the primary outcomes using random effects multivariable logistic regression to control for confounding.9147 patients were included, of whom seizure outcomes were available for 8191 (89·5%) participants at 2 years, and for 5577 (61·0%) at 5 years. The diagnoses of low-grade epilepsy associated neuroepithelial tumour (LEAT), vascular malformation, and hippocampal sclerosis had the best seizure outcome at 2 years after surgery, with 77·5% (1027 of 1325) of patients free from disabling seizures for LEAT, 74·0% (328 of 443) for vascular malformation, and 71·5% (2108 of 2948) for hippocampal sclerosis. The worst seizure outcomes at 2 years were seen for patients with focal cortical dysplasia type I or mild malformation of cortical development (50·0%, 213 of 426 free from disabling seizures), those with malformation of cortical development-other (52·3%, 212 of 405 free from disabling seizures), and for those with no histopathological lesion (53·5%, 396 of 740 free from disabling seizures). The proportion of patients being both Engel class 1 and drug-free was 0-14% at 1 year and increased to 14-51% at 5 years. Children were more often drug-free; temporal lobe surgeries had the best seizure outcomes; and a longer duration of epilepsy was associated with reduced chance of favourable seizure outcomes and drug freedom. This effect of duration was evident for all lesions, except for hippocampal sclerosis.Histopathological diagnosis, age at surgery, and duration of epilepsy are important prognostic factors for outcomes of epilepsy surgery. In every patient with refractory focal epilepsy presumed to be lesional, evaluation for surgery should be considered.None.

Background. An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy.

Congenital melanocytic naevi (CMN) are a known risk factor for melanoma, with the greatest risk currently thought to be in childhood. There has been controversy over the years about the incidence of melanoma, and therefore over the clinical management of CMN, due partly to the difficulties of histological diagnosis and partly to publishing bias towards cases of malignancy. Large cohort studies have demonstrated that melanoma risk in childhood is related to the severity of the congenital phenotype. New understanding of the genetics of CMN offers the possibility of improvement in diagnosis of melanoma, identification of those at highest risk, and new treatment options. We review the world literature and our centre's experience over the last 25 years, including the molecular characteristics of melanoma in these patients and new melanoma incidence and outcome data from our prospective cohort. Management strategies are proposed for presentation of suspected melanoma of the skin and the central nervous system in patients with CMN, including use of oral mitogen-activated protein kinase kinase inhibitors in NRAS-mutated tumours.

We undertook a comprehensive clinical and biological investigation of serial medulloblastoma biopsies obtained at diagnosis and relapse. Combined MYC family amplifications and P53 pathway defects commonly emerged at relapse, and all patients in this group died of rapidly progressive disease postrelapse. To study this interaction, we investigated a transgenic model of MYCN-driven medulloblastoma and found spontaneous development of Trp53 inactivating mutations. Abrogation of p53 function in this model produced aggressive tumors that mimicked characteristics of relapsed human tumors with combined P53-MYC dysfunction. Restoration of p53 activity and genetic and therapeutic suppression of MYCN all reduced tumor growth and prolonged survival. Our findings identify P53-MYC interactions at medulloblastoma relapse as biomarkers of clinically aggressive disease that may be targeted therapeutically.

The HERBY trial was a phase II open-label, randomized, multicenter trial evaluating bevacizumab (BEV) in addition to temozolomide/radiotherapy in patients with newly diagnosed non-brainstem high-grade glioma (HGG) between the ages of 3 and 18 years. We carried out comprehensive molecular analysis integrated with pathology, radiology, and immune profiling. In post-hoc subgroup analysis, hypermutator tumors (mismatch repair deficiency and somatic POLE/POLD1 mutations) and those biologically resembling pleomorphic xanthoastrocytoma ([PXA]-like, driven by BRAF_V600E or NF1 mutation) had significantly more CD8+ tumor-infiltrating lymphocytes, and longer survival with the addition of BEV. Histone H3 subgroups (hemispheric G34R/V and midline K27M) had a worse outcome and were immune cold. Future clinical trials will need to take into account the diversity represented by the term "HGG" in the pediatric population.

Abstract Pediatric tumors are uncommon, yet are the leading cause of cancer-related death in childhood. Tumor types, molecular characteristics, and pathogenesis are unique, often originating from a single genetic driver event. The specific diagnostic challenges of childhood tumors led to the development of the first World Health Organization (WHO) Classification of Pediatric Tumors. The classification is rooted in a multilayered approach, incorporating morphology, IHC, and molecular characteristics. The volume is organized according to organ sites and provides a single, state-of-the-art compendium of pediatric tumor types. A special emphasis was placed on “blastomas,” which variably recapitulate the morphologic maturation of organs from which they originate. Significance: In this review, we briefly summarize the main features and updates of each chapter of the inaugural WHO Classification of Pediatric Tumors, including its rapid transition from a mostly microscopic into a molecularly driven classification systematically taking recent discoveries in pediatric tumor genomics into account.

ABSTRACT Proliferation and tangential migration of neural precursor cells are essential determinants of CNS development. We have established cell culture models of both these processes using neural precursor cells grown as neurospheres. The pattern of migration that we observe in these cells is homotypic and occurs in the absence of a glial or neuronal scaffold, and is therefore equivalent to that previously described as chain migration. To determine the role of integrins in proliferation and migration, we have analysed the expression pattern of integrins on neurosphere cells and then performed blocking peptide and antibody experiments. Neurosphere cells express five major integrins, α5β1, α6Aβ1, αvβ1, αvβ5 and αvβ8 and, in addition, express low levels of α6Bβ1. Chain migration is inhibited by blocking the α6β1 integrin. Proliferation, by contrast, is inhibited by blocking the other β1 integrins, αvβ1 and α5β1. These results show that integrins are important regulators of neural precursor cell behaviour, with distinct β1 integrins regulating proliferation and migration. They also demonstrate a novel role for the α6β1 integrin in the cell-cell interactions underlying homotypic chain migration.

We report the clinical, genetic, and neuropathological findings of a seven generation-spanning pedigree with 196 individuals, 25 of whom had levodopa-responsive parkinsonism. Genetic analyses indicated Parkin mutations in 77 subjects. Among the 25 patients, 5 carried compound heterozygous mutations and met criteria for definite Parkinson's disease (PD) according to UK PD Society Brain Bank guidelines; 8 subjects carried only a heterozygous Parkin mutation. The mutational status of five deceased patients was unknown, and seven PD patients had no Parkin mutation. Survival analyses showed a significant difference in the age-at-onset distribution between patients with compound heterozygous mutations and the groups of heterozygous carriers and subjects without detectable Parkin mutations. Autopsy of a 73-year-old patient, who carried two mutant Parkin alleles (delExon7 + del1072T), showed PD-type cell loss, reactive gliosis, and alpha-synuclein-positive Lewy bodies in the substantia nigra and locus ceruleus. Surviving neurons were reactive with antibodies to the N terminus of Parkin but not the In-Between-RING ("IBR") domain, which had been deleted by both mutations. This large Parkin pedigree represents a unique opportunity to prospectively study the role of heterozygous Parkin mutations as a PD risk factor, to identify additional contributors to the expression of late-onset PD in heterozygous carriers, and to reexamine the role of Parkin in inclusion formation.

To compare PGP9.5 and transient receptor potential vanilloid receptor (TRPV1) suburothelial immunoreactivity between controls and patients with spinal neurogenic detrusor overactivity (NDO) before and after treatment with intravesical resiniferatoxin, as suburothelial PGP9.5-staining nerve fibres decrease in patients with spinal NDO who respond to intravesical capsaicin, and TRPV1 is present on these suburothelial nerve fibres in normal and overactive human urinary bladder.Patients with refractory NDO were enrolled in a prospective, randomized, parallel-group, double-blind, placebo-controlled trial using escalating doses of resiniferatoxin to a maximum of 1 micro mol/L. Flexible cystoscopic bladder biopsies obtained at baseline, 4 weeks after each instillation and at the time of maximum clinical response were compared with biopsies taken from control subjects. Frozen sections were incubated with rabbit antibodies to TRPV1 and PGP9.5, and assessed using standard immunohistochemical methods. PGP9.5 nerve density was analysed using a nerve-counting graticule by an observer unaware of sample origin. Another two independent observers unaware of each other's results used a random grading scale to evaluate TRPV1 nerve fibre density and intensity. The immunohistochemistry results were compared with histology findings (haematoxylin-eosin), and the Mann-Whitney test used to assess any differences (P < 0.05 significant) and the Pearson test for correlation.There were eight controls and 20 patients with spinal NDO, 14 (five clinical responders and nine not) who received the maximum dose of resiniferatoxin. There were more PGP9.5 and TRPV1 nerve fibres in patients with NDO than in controls (P = 0.007 and 0.002, respectively). Immunoreactivity before resiniferatoxin was similar in both groups for both PGP9.5 and TRPV1. In responders there were fewer PGP9.5 and TRPV1-positive fibres after treatment (P = 0.008 for each) but no change in those not responding. Changes after treatment for TRPV1 correlated well with those for PGP9.5 (r = 0.88, P < 0.001).The decrease of PGP9.5 and TRPV1 immunoreactive nerve fibres in responders to resiniferatoxin (to levels in control tissues) suggests that the increased numbers of nerve fibres in patients with NDO are mainly of sensory origin and express TRPV1. As baseline nerve fibre values were similar in responders and nonresponders, an additional factor may account for the difference in treatment outcome.

Article19 November 2009free access Combinations of genetic mutations in the adult neural stem cell compartment determine brain tumour phenotypes Thomas S Jacques Thomas S Jacques Neural Development Unit, UCL-Institute of Child Health and Department of Histopathology, Great Ormond Street Hospital, London, UK Search for more papers by this author Alexander Swales Alexander Swales Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UK Search for more papers by this author Monika J Brzozowski Monika J Brzozowski Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UKPresent address: Proximagen Limited, Hodgkin Building, King's College London, Guy's Campus, London SE1 1UL, UK Search for more papers by this author Nico V Henriquez Nico V Henriquez Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Jacqueline M Linehan Jacqueline M Linehan Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UK Search for more papers by this author Zaman Mirzadeh Zaman Mirzadeh Department of Neurosurgery and Developmental and Stem Cell Biology Program, University of California, San Francisco, CA, USA Search for more papers by this author Catherine O' Malley Catherine O' Malley Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UK Search for more papers by this author Heike Naumann Heike Naumann Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UKPresent address: Max-Delbruck Centre, AG Spagnoli, Robert Roessle Str. 10, Berlin 13125, Germany Search for more papers by this author Arturo Alvarez-Buylla Arturo Alvarez-Buylla Department of Neurosurgery and Developmental and Stem Cell Biology Program, University of California, San Francisco, CA, USA Search for more papers by this author Sebastian Brandner Corresponding Author Sebastian Brandner Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Thomas S Jacques Thomas S Jacques Neural Development Unit, UCL-Institute of Child Health and Department of Histopathology, Great Ormond Street Hospital, London, UK Search for more papers by this author Alexander Swales Alexander Swales Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UK Search for more papers by this author Monika J Brzozowski Monika J Brzozowski Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UKPresent address: Proximagen Limited, Hodgkin Building, King's College London, Guy's Campus, London SE1 1UL, UK Search for more papers by this author Nico V Henriquez Nico V Henriquez Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Jacqueline M Linehan Jacqueline M Linehan Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UK Search for more papers by this author Zaman Mirzadeh Zaman Mirzadeh Department of Neurosurgery and Developmental and Stem Cell Biology Program, University of California, San Francisco, CA, USA Search for more papers by this author Catherine O' Malley Catherine O' Malley Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UK Search for more papers by this author Heike Naumann Heike Naumann Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UKPresent address: Max-Delbruck Centre, AG Spagnoli, Robert Roessle Str. 10, Berlin 13125, Germany Search for more papers by this author Arturo Alvarez-Buylla Arturo Alvarez-Buylla Department of Neurosurgery and Developmental and Stem Cell Biology Program, University of California, San Francisco, CA, USA Search for more papers by this author Sebastian Brandner Corresponding Author Sebastian Brandner Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK Search for more papers by this author Author Information Thomas S Jacques1, Alexander Swales2, Monika J Brzozowski3, Nico V Henriquez3, Jacqueline M Linehan2, Zaman Mirzadeh4, Catherine O' Malley2, Heike Naumann2, Arturo Alvarez-Buylla4 and Sebastian Brandner 3 1Neural Development Unit, UCL-Institute of Child Health and Department of Histopathology, Great Ormond Street Hospital, London, UK 2Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, London, UK 3Division of Neuropathology and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK 4Department of Neurosurgery and Developmental and Stem Cell Biology Program, University of California, San Francisco, CA, USA *Corresponding author. Division of Neuropathology, Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK. Tel.: +44 20 7676 2188; Fax: +44 20 7676 2157; E-mail: [email protected] The EMBO Journal (2010)29:222-235https://doi.org/10.1038/emboj.2009.327 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info It has been suggested that intrinsic brain tumours originate from a neural stem/progenitor cell population in the subventricular zone of the post-natal brain. However, the influence of the initial genetic mutation on the phenotype as well as the contribution of mature astrocytes to the formation of brain tumours is still not understood. We deleted Rb/p53, Rb/p53/PTEN or PTEN/p53 in adult subventricular stem cells; in ectopically neurografted stem cells; in mature parenchymal astrocytes and in transplanted astrocytes. We found that only stem cells, but not astrocytes, gave rise to brain tumours, independent of their location. This suggests a cell autonomous mechanism that enables stem cells to generate brain tumours, whereas mature astrocytes do not form brain tumours in adults. Recombination of PTEN/p53 gave rise to gliomas whereas deletion of Rb/p53 or Rb/p53/PTEN generated primitive neuroectodermal tumours (PNET), indicating an important role of an initial Rb loss in driving the PNET phenotype. Our study underlines an important role of stem cells and the relevance of initial genetic mutations in the pathogenesis and phenotype of brain tumours. Introduction Brain tumours are classified according to the type of normal tissue they most closely resemble. However, in most cases, it is not known which cell type has given rise to the tumour. Furthermore, it is unclear to what extent the type and behaviour of the tumour is determined by its cell of origin or by the genetic events that occurred in that cell. One possible origin of CNS tumours are endogenous neural stem cells (B type) or transit amplifying cells (C type) that derive from them (Oliver and Wechsler-Reya, 2004; Sanai et al, 2005; Stiles and Rowitch, 2008). After the major phase of brain development is complete, a population of stem cells persists close to the walls of lateral ventricles in an extensive germinal zone known as the sub-ventricular zone (SVZ; Doetsch et al, 1999). These cells continue to proliferate and retain the capacity to generate neurons and glial cells. The SVZ is a complex but well-defined niche containing stem cells (type B cells), transient amplifying precursors (type C cells) and young neuroblasts (type A cells). A number of mouse models have suggested that deregulation of proliferation within the SVZ can lead to hyperplasia or tumour-like masses (Conover et al, 2000; Doetsch et al, 2002; Zhu et al, 2005a, 2005b). A recent study has shown that stimulation of the PDGFRα-expressing B-type neural stem cell induces the formation of hyperplasia resembling oligodendrogliomas next to the SVZ (Jackson et al, 2006). However, in contrast to spontaneous tumours, this hyperplasia regresses after withdrawal of the growth factor. Activation of Ras and Akt in nestin-expressing progenitors (but not in GFAP-expressing SVZ stem cells) induces glioblastoma, a malignant astrocytoma (Holland et al, 2000), and nestin-expressing GFAP-negative progenitor cells deficient in INK4a/ARF and Bmi1, isolated in vitro, can give rise to low-grade diffuse astrocytomas (Bruggeman et al, 2007). GFAP-cre-mediated inactivation of Nf1 and p53 in neural progenitor and in neural stem/progenitor cells of the SVZ induces glia progenitor proliferation and ultimately malignant astrocytomas (Zhu et al, 2005a, 2005b), which is accelerated by additional haploinsufficiency for PTEN (Kwon et al, 2008). In keeping with these observations, GFAP-cre-mediated inactivation of PTEN and p53 in progenitor cells resulted in the formation of malignant astrocytomas (Zheng et al, 2008). A targeted approach, in which only adult nestin-expressing cells were recombined, was used to inactivate Nf1, p53 or Nf1, p53 and PTEN, resulting in the formation of malignant gliomas (Alcantara Llaguno et al, 2009). In a similar approach, Wang et al (2009) introduced mutant p53 alongside a p53 deletion on the other allele, in addition to a deletion of Nf1 in GFAP-expressing cells, again generating glial tumours. Although these models indicate that nestin-expressing cells contained within the adult CNS can give rise to brain tumours, they do not determine whether these tumours derive from stem cells or other precursor/progenitor cells. Most models using a GFAP-cre-mediated approach to target cells expressing GFAP during development, including neural progenitor cells and their progeny and hence do not strictly target only GFAP-expressing adult stem cells and mature astrocytes. Moreover, we do not know how different tumour suppressor genes contribute to the tumour phenotype. We set out to investigate (i) whether the stem cell population of the SVZ can give rise to brain tumours, (ii) which tumours can arise from these cells, (iii) to what extent the phenotype of the tumour is determined by the initial combination of genetic mutations, and (iv) whether mature astrocytes can contribute to the formation of gliomas. This may ultimately answer the questions: why certain brain tumours show reproducible patterns of genetic mutations and why alterations in certain pathways are often associated with certain types of tumours. Results Adeno-cre and adeno GFAP-cre target stem/progenitor cells in the subventricular zone In this study, we used mice bearing conditional alleles (flanked by LoxP sites) of Rb, p53 and PTEN, in different combinations and used an adenovirus expressing cre recombinase (either Adeno-cre or Adeno GFAP-cre) to delete these genes in stem/progenitor cells of the SVZ of adult mice. First, we determined the distribution of cells that underwent recombination after intracerebroventricular (i.c.v.) injection of Adeno-cre into ROSA26 Lox reporter mice (R26R), which express β-galactosidase on Cre-mediated recombination (Soriano, 1999). Controls were wild-type mice injected with an adenovirus expressing green fluorescent protein (Adeno-GFP). Between 4 and 14 days after i.c.v. injection, mice were killed and their brains were analysed by histochemistry (Figure 1A and C), immunohistochemistry or immunofluorescence (Figure 1B) for β-galactosidase. Recombination and expression of β-galactosidase occurred bilaterally in a thin periventricular strip that included the ependyma and the SVZ (Figure 1A–C) up to 4–5 cell diametres deep to the ependymal surface. In Adeno-GFP injected controls, GFP expression co-localized with nestin and GFAP—markers expressed by progenitor and stem cells in the SVZ (Figure 1K–N). Figure 1.Intra-ventricular injections of adenovirus target a thin layer deep to the ependymal wall that includes the progenitor cells. (A) Coronal section through a ROSA26Lox mouse 7 days after intracerebro-ventricular injection with Adeno-cre. Recombined cells are restricted to a thin periventricular region. (B) Superimposed confocal and phase contrast image of the lateral wall of the lateral ventricle after immunostaining against β-gal, confirming that recombination is limited to a thin layer of cells beneath the ependyma. (C) LacZ histochemical stain on a vibratome section shows recombination in the SVZ. (D, E) Recombined neurospheres derived from ROSA26Lox mice following intraventricular injection with Adeno-cre (D); β-gal histochemistry on neurospheres. (E) Increasing proportion of positive cells during time in vitro. (F) In vivo-recombined, in vitro-expanded neural stem cells grow from single cells to neurospheres, demonstrating their ability to self-renew. Each image shows the same cell/sphere during self-renewal and growth, and on day 9 before (F9) and after LacZ staining (F, day 9, LacZ). (G–J) In vitro differentiation of in vivo recombined stem cells after clonal expansion from a single cell. Neurospheres were differentiated in vitro and double labelled for β-galactosidase (green) to detect cre-mediated recombination and a marker of differentiation (nestin, (G); MAP-2 (H); GFAP (I) and O4(J)), indicating their capability to differentiate into neuronal, glial and oligodendroglial lineages. (K–N) Confocal images of coronal sections of the lateral ventricle of wild-type mice after injection by Adenovirus–GFP: (K, L) triple-labelled images; (M, N) co-localization of GFP and nestin (M) or GFAP (N). The same cell population is targeted with Adeno-GFAP-GFP (O–R). Scale bar 200 μm (K–R). Download figure Download PowerPoint As B-type of SVZ cells are considered stem cells and distinguished from other SVZ progenitor cells by their expression of GFAP (Doetsch et al, 1999), we also targeted these cells using an adenovirus expressing Cre (or GFP) under the control of the GFAP promoter. Adeno-GFAP–GFP injection labels a population of SVZ cells that expressed GFAP (Figure 1O–R). Stem and progenitor cells in the SVZ can be cultured as neurospheres. To confirm that SVZ-derived progenitor cells were among cells that underwent cre-mediated recombination, we cultured neurospheres from R26R reporter mice 4 days, 1 or 2 weeks after intraventricular Adeno-cre injection. In three preparations, 8, 12, and 40% of neurospheres contained recombined cells when isolated (Figure 1D and E) but the number of spheres containing recombined cells expanded with increasing passage number, suggesting that recombined cells underwent self-renewal, a property to be expected if the recombined cells included stem cells (Figure 1E). Self-renewal of the SVZ cells was confirmed by isolation and in vitro propagation of the recombined cells grown as neurospheres (Jacques et al, 1998, 1999). In three separate experiments, 1508 cells were plated in total, of which 291 were a single cell in each well. Of these single cells, 68 (23%) self-renewed and formed a neurosphere, of which 51 (18% of all single cells) were uniformly X-gal positive. A representative example of a single cell forming an X-gal-positive neurosphere is shown in Figure 1F. Such neurospheres could be differentiated into neuronal, glial, and oligodendrocyte lineages (Figure 1G–J) and were further propagated, demonstrating their pluripotency and self-renewal capacity. This experiment further confirms that SVZ-derived progenitor cells were among cells that underwent cre-mediated recombination and that these cells can be propagated in vitro. In conclusion, Adeno-cre-mediated recombination targets SVZ cells that can be propagated in vitro and can self renew. Inactivation of tumour suppressor genes in the stem cell compartment of the SVZ causes brain tumours To determine which tumours could arise from these periventricular cells, we injected Adeno-cre into the ventricles of mice with homozygous floxed alleles of the key tumour suppressor genes Rb, p53 (Marino et al, 2000) and PTEN (Marino et al, 2002), as well as the reporter gene R26R (Soriano, 1999) in the combinations Rb/p53, Rb/p53/PTEN, or p53/PTEN. We chose to target genes that are frequently altered in human brain tumours and are fundamental suppressors of neoplasia in a range of tissues (Collins, 2002). Each combination of tumour suppressors produced specific and reproducible types of tumour after a latency determined by the genotype of the mice (Figures 2 and 3, Supplementary Figure S1, Supplementary Table S1). Figure 2.The phenotype of brain tumours is influenced by the initial genetic deletion: left column: tumours in Rb/p53 mice: (A, D, G, J, M, P); middle column: Rb/p53/PTEN tumour (B, E, H, K, N, Q, R); and right column: tumour derived from p53/PTEN mice (C, F, I, L, O, S). Panels A–I are stained with haematoxylin and eosin. (D) Arrowheads point to the border between tumour and the adjacent non-neoplastic brain. (E) Arrowheads indicate thin septae of meningeal origin that separate tumour cells into trabeculae. (F) The area indicated by arrowheads is the ill-defined border between tumour (left) and brain (right; ‘infiltration zone’). (G–I) High-power magnification shows the morphological differences between PNET (G, H) and glioma (I), with rosette formation (G, inset) in the PNETs. (J–L) Synaptophysin immunohistochemistry shows strong positive labelling of all tumour cells in (J, K), and no expression in gliomas (L). (M–O) GFAP immunohistochemical labelling with no expression in PNET (M, N). The small rim on the right in (N) is adjacent brain that shows strong reactive gliosis, and (O) shows GFAP expression in all glioma cells. (P–S) The additional deletion of PTEN in a Rb/p53 background (Q, R) results in extensive desmoplastic reaction/meningeal infiltration: PTEN/Rb/p53 tumours show reticulin and epithelial membrane antigen (EMA)-positive structures separating nests of tumour cells (Q, R). No desmoplastic reaction in Rb/p53 PNET (P) or in PTEN/p53 gliomas (S). Scale bar: 5.2 mm (A–C), 440 μm (D–E), 110 μm (G–I) and 220 μm (J–S). Download figure Download PowerPoint Figure 3.(A) The genotype determines the latency of tumour formation. Mean time (days to tumour presentation±95% confidence intervals). Rb/p53/PTEN has a significantly shortened latency. P<0.05 ANOVA, post-hoc Bonferroni, box plot with 95% of all samples. All tumour types were included. (B) The genotype determines the proliferation rate of the tumours: the graph shows mean mitotic counts per 10 high-power fields (× 40 objective)±95% confidence intervals, PTEN/p53 differs significantly from the other genotypes (P<0.05 ANOVA, post-hoc Bonferroni). All tumours examined were gliomas in PTEN/p53mice and PNET in two other genotypes. (C) The tumour latency is independent of the age at injection. The graph shows the relationship between age at injection and the tumour incubation time (development of neurological signs). Polynomial regression analysis was performed. None of the genotypes showed a significant correlation between age at injection and latency. Rb/p53: r=−0.22, P=0.55; Rb/p53/PTEN: r=−0.05, P=0.38; PTEN/p53: r=0.8, P=0.17. Solid blue line, linear fit; dotted blue line, 95% confidence interval; dotted green line, 95% prediction interval. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Rb/p53 mice developed malignant tumours after a relatively constant latency of approximately nine months (274.42±27.42 days (95% CI)) (Figure 3A). An autopsy of 101 mice revealed extensive, well-demarcated forebrain tumours in 20 mice (19.8%; Figure 2A and D), typically involving lateral ventricles. They were mainly of one histological type, characterized by diffuse sheets of closely packed, undifferentiated and malignant cells (Figure 2G), which in approximately half of the tumours formed rosettes (analogous to Homer Wright rosettes (Figure 2G inset), corresponding to primitive neuroectodermal tumours (PNETs) in humans. These tumours were strongly positive for the neuronal marker, synaptophysin (Figure 2J), but negative for NeuN and neurofilament, both markers of more mature neuronal differentiation, for glial markers, GFAP (Figure 2M) and S100β, and for epithelial markers, cytokeratin and epithelial membrane antigen (EMA). The administration of Adeno-Cre into mice with additional conditional alleles of PTEN, (Rb/p53/PTEN mice) resulted in a considerably shorter latency before the tumours developed (100.65±6.09 days (95% CI); P<0.001, ANOVA with Bonferroni post-hoc testing; Figure 3A). An analysis of 80 mice showed the presence of a PNET in 41 animals (51.3%). The PTEN/Rb/p53 tumours had an appearance similar to Rb/p53 tumours (Figure 2B, E and H), and they showed similar mitotic rates (Figure 3B) and identical immunohistochemical properties; with positive staining for synaptophysin and negative staining for glial markers (Figure 2K and N). Furthermore, they were found in a similar location, typically involving lateral ventricles and extending into the forebrain. However, in contrast to the diffuse architecture of the Rb/p53 tumours, Rb/p53/PTEN tumours had a more distinctive architecture with formation of nodules and clusters of cells separated by fibrous bundles visualized by reticulin silver staining (Figure 2Q), which were also positive for the meningeal (arachnoidal) marker, EMA (Figure 2R). These data suggest that the nodular phenotype is associated with extensive invasion into the meninges. Finally, p53/Rb/PTEN tumours showed necrosis less frequently than those from Rb/p53 mice (P=0.001, χ2 test). Targeted deletion of PTEN and p53 in SVZ cells resulted in neoplasms with an intermediate latency (225.07±18.44 days (95% CI)) slightly shorter than that of Rb/p53 tumours (P<0.001 ANOVA with Bonferroni post-hoc testing; Figure 3A) but importantly, they had an entirely different histological phenotype. These tumours were infiltrative and expanded from the ventricular wall laterally into the corpus callosum, striatum and cortex in a diffuse manner (Figure 2C and F). A histological analysis of 116 mice showed 28 such tumours (27%, Supplementary Table S1). They showed a biphasic pattern with cells with small dense nuclei and small amounts of eosinophilic cytoplasm, along with another population with larger, bright and vesicular nuclei and indistinct cytoplasm, set against a myxoid background (Figure 2I). Areas of micro-vascular proliferation and a type of necrosis characteristic of human high-grade gliomas (‘palisading necrosis’; Louis et al, 2007) were also observed in some of these tumours. The PTEN/p53 tumours had a considerably lower mitotic count (Figure 3B) compared with tumours in Rb/p53 or Rb/p53/PTEN mice (P<0.05, ANOVA with Bonferroni post-hoc testing). In contrast to Rb/p53 and Rb/p53/PTEN tumours, all p53/PTEN tumours contained a population of small neoplastic glial cells that strongly expressed GFAP (Figure 2O), but no neuronal markers (NeuN, neurofilament or synaptophysin; Figure 2L), similar to human anaplastic astrocytomas or anaplastic oligoastrocytomas, corresponding to WHO Grade III. In addition to the tumours mentioned above, we occasionally observed other tumours, such as choroid plexus tumours (n=13; 12.8%; mean latency of 370.6±20.4 days (95% CI); Supplementary Figure S1) in Rb/p53 mice that survived beyond the time at which most PNET-like tumours would have formed. Other tumours included osteogenic skull tumours in PTEN/p53 mice (n=21; 18.1%; Supplementary Figure S1) and meningiomas (predominantly in Rb/P53/PTEN mice; n=20; 25%). In one instance, there was a diffuse infiltrative tumour resembling high-grade glioma together with the more ‘typical’ PNETs that are commonly found in those mice (in two Rb/p53/PTEN mice; Supplementary Figure S2, Supplementary Table S1). As the number of actively proliferating SVZ stem cells gradually decreases during lifetime (Alvarez-Buylla and Temple, 1998), the age at injection may influence the type and number of targeted cells. We therefore analysed whether the age at injection correlates with the latency of tumour development. Using polynomial regression analysis, we found that none of the genotypes showed a significant correlation between age at injection and latency of tumour development (Figure 3C). Microneoplasia arises from recombined SVZ cells and precedes brain tumours To determine where the tumours arose from, we killed Rb/p53, Rb/p53/PTEN and PTEN/p53 mice after intra-ventricular delivery of Adeno-cre or Adeno-GFP before expected tumour formation. We noted small collections of cells with atypical nuclear features, perivascular collections of atypical cells or well-formed nodules of tumour cells at the dorsolateral angles of the lateral ventricles. These areas contain large collections of stem and progenitor cells in the adult brain (Figure 4). Instead, such lesions were observed in the medial wall only in one instance. All lesions arose as a result of cre-mediated recombination, as they expressed the recombination marker β-galactosidase (Figure 5D and G). In contrast, we did not observe β-galactosidase expression or SVZ microneoplasia when Adeno-cre was injected in different areas, such as striatum or hippocampus (Figure 6J–M). Although nestin- and GFAP-positive stem/progenitor cells may also exist in the medial wall (Figure 1K–R; Alvarez-Buylla et al, 2008), the more predominant presence of precursor lesions in the lateral wall suggests that permissive stem/progenitor cells may be more abundant in the lateral wall and that the predominance of lesions in the later wall reflect the stochastic events of cre-mediated recombination. To demonstrate that tumours originated from cells that underwent Adeno-cre mediated recombination, and to address the issue that cre-mediated recombination may not be equally effective at different lox sites (Soriano, 1999; Loonstra et al, 2001; Vooijs et al, 2001), we microdissected a series of tumours derived from mice of all genotypes and verified recombination by PCR (Marino et al, 2000). All tumours showed recombination suggesting that these tumours arose independently from complete recombination of stem or progenitor cells (Figure 5A–C) and that cre-mediated recombination was consistent and complete. To further identify recombined precursor lesion and tumour cells, their relationship to the SVZ and the infiltration pattern into non-recombined surrounding CNS parenchyma, we stained for the recombination marker β-galactosidase as surrogate marker for the genes p53lox, Rblox and PTENlox, and observed that it was expressed in all precursor lesions and tumours (Figure 5D–I). In keeping with the localization of adenovirus-mediated recombination (Figure 1A–C), the SVZ also showed β-galactosidase labelling along the lateral, dorsal and medial walls, but only one instance of precursor lesions in the medial wall (Figure 4F). This suggests that tumours arise from specific populations, that is, the neurogenic portion of the lateral SVZ, and it is unlikely that other cell populations, such as ependymal cells, contribute to tumour formation. Figure 4.Small neoplastic lesions (‘microneoplasia’) after intraventricular Adeno-cre injection precede brain tumours. Upper row (A–C): Histological appearance of small neoplastic lesions located beneath the ependymal layer of the lateral wall of the ventricle. Occasionally, perivascular spread was seen in PTEN/p53 mice (C). (D–I) Schematic representation of precursor lesions: cumulative maps showing the localization of small lesions. Each dot represents microneoplasia or a focal cluster of tumour cells (approximately 200 μm). Each colour represents an individual animal. The map summarizes lesions in 14 Rb/p53 mice, 6 Rb/p53/PTEN mice and 14 PTEN/p53 mice. The upper schematic panel (D–F) summarizes lesions in the anterior ventricular region (Bregma −1.3 to 0.0) and the lower panel (G–I) represents the posterior region (Bregma −0.5 to −0.7). The microneoplastic lesions are almost exclusively localized beneath the lateral wall of the ventricles and in the lateral corner of the ventricle, and may occasionally protrude to the opposite wall. Although Rb/p53 and Rb/p53/PTEN lesions tend to remain locally clustered, PTEN/p53 lesions often spread laterally and into the corpus callosum, in keeping with the more infiltrative nature of these tumours (F). One small lesion in an Rb/p53 animal (D, yellow dot) was observed extending to the medial side but may not have arisen there and one small lesion in a PTEN/p53 animal arose from the medial surface (F, blue dot), indicating a very strong preference for the lateral/dorsolateral walls, consistent with the presumed localization of SVZ stem cells. Cx, cortex; CC, corpus callosum; LV, lateral ventricle; SN, septal nuclei; FH, fimbria hippocampi and Th, thalamus. Download figure Download PowerPoint Figure 5.Gene recombination in experimentally induced brain tumours: (A–C) recombination PCR on microdissected tumours demonstrates recombination of floxed genes in brain tumours: Lanes 1 and 2: rare instance of two histologically distinct tumour phenotypes in the same brain (mouse genotype Rb/p53), both showing recombination of Rb (A) and p53 (B, C). Lanes 3, 4, 7 and 8 are further examples of recombination in PNETs, whereas lane 6 shows recombination in other tumour types and no recombination is seen in control tissue. Lane 8 shows glioma with recombination of p53. PTEN recombination was not te

Dravet syndrome is an epilepsy syndrome of infantile onset, frequently caused by SCN1A mutations or deletions. Its prevalence, long-term evolution in adults and neuropathology are not well known. We identified a series of 22 adult patients, including three adult post-mortem cases with Dravet syndrome. For all patients, we reviewed the clinical history, seizure types and frequency, antiepileptic drugs, cognitive, social and functional outcome and results of investigations. A systematic neuropathology study was performed, with post-mortem material from three adult cases with Dravet syndrome, in comparison with controls and a range of relevant paediatric tissue. Twenty-two adults with Dravet syndrome, 10 female, were included, median age 39 years (range 20-66). SCN1A structural variation was found in 60% of the adult Dravet patients tested, including one post-mortem case with DNA extracted from brain tissue. Novel mutations were described for 11 adult patients; one patient had three SCN1A mutations. Features of Dravet syndrome in adulthood include multiple seizure types despite polytherapy, and age-dependent evolution in seizure semiology and electroencephalographic pattern. Fever sensitivity persisted through adulthood in 11 cases. Neurological decline occurred in adulthood with cognitive and motor deterioration. Dysphagia may develop in or after the fourth decade of life, leading to significant morbidity, or death. The correct diagnosis at an older age made an impact at several levels. Treatment changes improved seizure control even after years of drug resistance in all three cases with sufficient follow-up after drug changes were instituted; better control led to significant improvement in cognitive performance and quality of life in adulthood in two cases. There was no histopathological hallmark feature of Dravet syndrome in this series. Strikingly, there was remarkable preservation of neurons and interneurons in the neocortex and hippocampi of Dravet adult post-mortem cases. Our study provides evidence that Dravet syndrome is at least in part an epileptic encephalopathy.

Abstract A variety of analyses, including fluorescence in situ hybridization (FISH), quantitative PCR (qPCR) and array CGH (aCGH), have been performed on a series of chordomas from 181 patients. Twelve of 181 (7%) tumours displayed amplification of the T locus and an additional two cases showed focal amplification; 70/181 (39%) tumours were polysomic for chromosome 6, and 8/181 (4.5%) primary tumours showed a minor allelic gain of T as assessed by FISH. No germline alteration of the T locus was identified in non‐neoplastic tissue from 40 patients. Copy number gain of T was seen in a similar percentage of sacrococcygeal, mobile spine and base of skull tumours. Knockdown of T in the cell line, U‐CH1, which showed polysomy of chromosome 6 involving 6q27, resulted in a marked decrease in cell proliferation and morphological features consistent with a senescence‐like phenotype. The U‐CH1 cell line was validated as representing chordoma by the generation of xenografts, which showed typical chordoma morphology and immunohistochemistry in the NOD/SCID/interleukin 2 receptor [IL2r] $\gamma^{\rm{null}}$ mouse model. In conclusion, chromosomal aberrations resulting in gain of the T locus are common in sporadic chordomas and expression of this gene is critical for proliferation of chordoma cells in vitro . Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

Activating mutations in the gene encoding β-catenin have been identified in the paediatric form of human craniopharyngioma (adamantinomatous craniopharyngioma, ACP), a histologically benign but aggressive pituitary tumour accounting for up to 10% of paediatric intracranial tumours. Recently, we generated an ACP mouse model and revealed that, as in human ACP, nucleocytoplasmic accumulation of β-catenin (β-cat(nc)) and over-activation of the Wnt/β-catenin pathway occurs only in a very small proportion of cells, which form clusters. Here, combining mouse genetics, fluorescence labelling and flow-sorting techniques, we have isolated these cells from tumorigenic mouse pituitaries and shown that the β-cat(nc) cells are enriched for colony-forming cells when cultured in stem cell-promoting media, and have longer telomeres, indicating shared properties with normal pituitary progenitors/stem cells (PSCs). Global gene profiling analysis has revealed that these β-cat(nc) cells express high levels of secreted mitogenic signals, such as members of the SHH, BMP and FGF family, in addition to several chemokines and their receptors, suggesting an important autocrine/paracrine role of these cells in the pathogenesis of ACP and a reciprocal communication with their environment. Finally, we highlight the clinical relevance of these findings by showing that these pathways are also up-regulated in the β-cat(nc) cell clusters identified in human ACP. As well as providing further support to the concept that pituitary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours.

Most published articles are not cited and citation rates depend on many variables. We hypothesized that specific features of journal titles may be related to citation rates.We reviewed the title characteristics of the 25 most cited articles and the 25 least cited articles published in 2005 in general and specialist medical journals including the Lancet, BMJ and Journal of Clinical Pathology. The title length and construction were correlated to the number of times the papers have been cited to May 2009.Retrospective review of a scientific database.None.Citation rate.The number of citations was positively correlated with the length of the title, the presence of a colon in the title and the presence of an acronym. Factors that predicted poor citation included reference to a specific country in the title.These data suggest that the construction of an article title has a significant impact on frequently the paper is cited. We hypothesize that this may be related to the way electronic searches of the literature are undertaken.

The aim of this study was to define the frequency and associated clinical phenotype of anti-MDA5 autoantibodies in a large UK based, predominantly Caucasian, cohort of patients with juvenile dermatomyositis (JDM).Serum samples and clinical data were obtained from 285 patients with JDM recruited to the UK Juvenile Dermatomyositis Cohort and Biomarker Study. The presence of anti-MDA5 antibodies was determined by immunoprecipitation and confirmed by ELISA using recombinant MDA5 protein. Results were compared with matched clinical data, muscle biopsies (scored by an experienced paediatric neuropathologist) and chest imaging (reviewed by an experienced paediatric radiologist).Anti-MDA5 antibodies were identified in 7.4% of JDM patients and were associated with a distinct clinical phenotype including skin ulceration (P = 0.03) oral ulceration (P = 0.01), arthritis (P <0.01) and milder muscle disease both clinically (as determined by Childhood Myositis Assessment Score (P = 0.03)) and histologically (as determined by a lower JDM muscle biopsy score (P <0.01)) than patients who did not have anti-MDA5 antibodies. A greater proportion of children with anti-MDA5 autoantibodies achieved disease inactivity at two years post-diagnosis according to PRINTO criteria (P = 0.02). A total of 4 out of 21 children with anti-MDA5 had interstitial lung disease; none had rapidly progressive interstitial lung disease.Anti-MDA5 antibodies can be identified in a small but significant proportion of patients with JDM and identify a distinctive clinical sub-group. Screening for anti-MDA5 autoantibodies at diagnosis would be useful to guide further investigation for lung disease, inform on prognosis and potentially confirm the diagnosis, as subtle biopsy changes could otherwise be missed.

Migrating partial seizures of infancy, also known as epilepsy of infancy with migrating focal seizures, is a rare early infantile epileptic encephalopathy with poor prognosis, presenting with focal seizures in the first year of life.A national surveillance study was undertaken in conjunction with the British Paediatric Neurology Surveillance Unit to further define the clinical, pathological and molecular genetic features of this disorder.Fourteen children with migrating partial seizures of infancy were reported during the 2 year study period (estimated prevalence 0.11 per 100 000 children).The study has revealed that migrating

We examined, for the first time in a prospective study, the histological changes in the urothelium and suburothelium of patients with neurogenic (NDO) or idiopathic detrusor overactivity (IDO) after one or repeat treatments with intradetrusor BoNTA.Flexible cystoscopic bladder biopsies were obtained from patients with urodynamically proven intractable spinal NDO or IDO before and 4 and 16 wk after one or repeat treatments with intradetrusor injections of BOTOX1 (NDO 300 U, IDO 200 U). Specimens were stained for haematoxylin-eosin and analysed blindly for inflammatory changes, fibrosis, hyperplasia, and dysplasia in the urothelium and suburothelium. Statistical comparisons were significant at p values less than 0.05.Signs of chronic inflammation were found in 59.1% of baseline biopsies (65.6% of NDO vs. 50% of IDO, p=0.049), 67.6% of post-first biopsies and 86.4% after repeat injections. The two groups were comparable for degree of baseline inflammation, which did not change significantly after first injection and up to 16 wk after a third injection. Mild fibrosis was found in 2.2% of biopsies examined, equally before and after treatment, but not after repeat injections. No dysplasia or hyperplasia was identified. Eosinophils were identified more frequently in biopsies taken after repeat injections compared with the post-first injection and baseline biopsies (chi2=8.23, p=0.018). No difference existed between NDO and IDO bladders.BoNTA injections do not appear to be producing significant inflammatory changes, fibrosis, or dysplastic changes in human bladder urothelium/suburothelium after a single injection and in a limited number of repeat treatment biopsies. The presence of eosinophils might be treatment-related, because they were mostly found in post-treatment biopsies.

Rapid developments in molecular genetic technology and research have swiftly advanced our understanding of neuro-oncology. As a consequence, the WHO invited their expert panels to revise the current classification system of brain tumours and to introduce, for the first time, a molecular genetic approach for selected tumour entities, thus setting a new gold standard in histopathology. The revised 5th edition of the 'blue book' was released in May 2016 and will have a major impact in stratifying diagnosis and treatment. However, low-grade neuroepithelial tumours that present with early-onset focal epilepsy and are mostly seen in children and young adults (previously designated as long-term epilepsy-associated neuroepithelial tumours, LEAT) lack such innovative clinicopathological and molecular genetic tools. The Neuropathology Task Force of the International League against Epilepsy will critically discuss this issue, and will offer perspectives on how to decipher and validate clinically meaningful LEAT entities using the current WHO approach that integrates clinicopathological and genetic classification systems.

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

The “isomorphic subtype of diffuse astrocytoma” was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of MYBL1 or MYB (13/25, 52%). Gene fusions of MYBL1 or MYB with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective MYB-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated MYBL1 (54%) or MYB (23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of MYB/MYBL1-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent MYBL1 and MYB alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric MYB/MYBL1-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification.

Senescent cells may promote tumour progression through the activation of a senescence-associated secretory phenotype (SASP), whether these cells are capable of initiating tumourigenesis in vivo is not known. Expression of oncogenic β-catenin in Sox2+ young adult pituitary stem cells leads to formation of clusters of stem cells and induction of tumours resembling human adamantinomatous craniopharyngioma (ACP), derived from Sox2- cells in a paracrine manner. Here, we uncover the mechanisms underlying this paracrine tumourigenesis. We show that expression of oncogenic β-catenin in Hesx1+ embryonic precursors also results in stem cell clusters and paracrine tumours. We reveal that human and mouse clusters are analogous and share a common signature of senescence and SASP. Finally, we show that mice with reduced senescence and SASP responses exhibit decreased tumour-inducing potential. Together, we provide evidence that senescence and a stem cell-associated SASP drive cell transformation and tumour initiation in vivo in an age-dependent fashion.

Juvenile dermatomyositis (DM) is a rare and severe autoimmune condition characterized by rash and proximal muscle weakness. While some patients respond to standard treatment, others do not. This study was carried out to investigate whether histopathologic findings and myositis-specific autoantibodies (MSAs) have prognostic significance in juvenile DM.Muscle biopsy samples (n = 101) from patients in the UK Juvenile Dermatomyositis Cohort and Biomarker Study were stained, analyzed, and scored for severity of histopathologic features. In addition, autoantibodies were measured in the serum or plasma of patients (n = 90) and longitudinal clinical data were collected (median duration of follow-up 4.9 years). Long-term treatment status (on or off medication over time) was modeled using generalized estimating equations.Muscle biopsy scores differed according to MSA subgroup. When the effects of MSA subgroup were accounted for, increased severity of muscle histopathologic features was predictive of an increased risk of remaining on treatment over time: for the global pathology score (histopathologist's visual analog scale [hVAS] score), 1.48-fold higher odds (95% confidence interval [95% CI] 1.12-1.96; P = 0.0058), and for the total biopsy score (determined with the standardized score tool), 1.10-fold higher odds (95% CI 1.01-1.21; P = 0.038). A protective effect was identified in patients with anti-Mi-2 autoantibodies, in whom the odds of remaining on treatment were 7.06-fold lower (95% CI 1.41-35.36; P = 0.018) despite muscle biopsy scores indicating more severe disease. In patients with anti-nuclear matrix protein 2 autoantibodies, anti-transcription intermediary factor 1γ autoantibodies, or no detectable autoantibody, increased histopathologic severity alone, without adjustment for the effect of MSA subtype, was predictive of the risk of remaining on treatment: for the hVAS global pathology score, 1.61-fold higher odds (95% CI 1.16-2.22; P = 0.004), and for the total biopsy score, 1.13-fold higher odds (95% CI 1.03-1.24; P = 0.013).Histopathologic severity, in combination with MSA subtype, is predictive of the risk of remaining on treatment in patients with juvenile DM and may be useful for discussing probable treatment length with parents and patients. Understanding these associations may identify patients at greater risk of severe disease.

Immune-therapy is an attractive alternative therapeutic approach for targeting central nervous system (CNS) tumors and the constituency of the Tumor Immune Microenvironment (TIME) likely to predict patient response. Here, we describe the TIME of >6000 primarily pediatric CNS tumors using a deconvolution approach (methylCIBERSORT). We produce and validate a custom reference signature defining 11 non-cancer cell types to estimate relative proportions of infiltration in a panCNS tumor cohort spanning 80 subtypes. We group patients into three broad immune clusters associated with CNS tumor types/subtypes. In cohorts of medulloblastomas (n = 2325), malignant rhabdoid tumors (n = 229) and pediatric high-grade gliomas (n = 401), we show significant associations with molecular subgroups/subtypes, mutations, and prognosis. We further identify tumor-specific immune clusters with phenotypic characteristics relevant to immunotherapy response (i.e. Cytolytic score, PDL1 expression). Our analysis provides an indication of the potential future therapeutic and prognostic possibilities of immuno-methylomic profiling in pediatric CNS tumor patients that may ultimately inform approach to immune-therapy.

Abstract Background Malignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the epidermal growth factor receptor gene (EGFR) have recently been identified in a distinct subset of pediatric-type bithalamic gliomas with a unique DNA methylation pattern. Methods Here, we investigated an epigenetically homogeneous cohort of malignant gliomas (n = 58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in comparison with this recently identified subtype of pediatric bithalamic gliomas. Results EGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; 5 of them co-occurring with EGFR amplification). Additionally, 8 of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (6 of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of overexpression of the EZH inhibitory protein (EZHIP) in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations. Conclusions Our findings present a distinct molecular class of pediatric-type malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.

Defects of mitochondrial dynamics are emerging causes of neurological disease. In two children presenting with severe neurological deterioration following viral infection we identified a novel homozygous STAT2 mutation, c.1836 C>A (p.Cys612Ter), using whole exome sequencing. In muscle and fibroblasts from these patients, and a third unrelated STAT2-deficient patient, we observed extremely elongated mitochondria. Western blot analysis revealed absence of the STAT2 protein and that the mitochondrial fission protein DRP1 (encoded by DNM1L) is inactive, as shown by its phosphorylation state. All three patients harboured decreased levels of DRP1 phosphorylated at serine residue 616 (P-DRP1(S616)), a post-translational modification known to activate DRP1, and increased levels of DRP1 phosphorylated at serine 637 (P-DRP1(S637)), associated with the inactive state of the DRP1 GTPase. Knockdown of STAT2 in SHSY5Y cells recapitulated the fission defect, with elongated mitochondria and decreased P-DRP1(S616) levels. Furthermore the mitochondrial fission defect in patient fibroblasts was rescued following lentiviral transduction with wild-type STAT2 in all three patients, with normalization of mitochondrial length and increased P-DRP1(S616) levels. Taken together, these findings implicate STAT2 as a novel regulator of DRP1 phosphorylation at serine 616, and thus of mitochondrial fission, and suggest that there are interactions between immunity and mitochondria. This is the first study to link the innate immune system to mitochondrial dynamics and morphology. We hypothesize that variability in JAK-STAT signalling may contribute to the phenotypic heterogeneity of mitochondrial disease, and may explain why some patients with underlying mitochondrial disease decompensate after seemingly trivial viral infections. Modulating JAK-STAT activity may represent a novel therapeutic avenue for mitochondrial diseases, which remain largely untreatable. This may also be relevant for more common neurodegenerative diseases, including Alzheimer's, Huntington's and Parkinson's diseases, in which abnormalities of mitochondrial morphology have been implicated in disease pathogenesis.

Focal cortical dysplasia (FCD) is a major cause of difficult-to-treat epilepsy in children and young adults, and the diagnosis is currently based on microscopic review of surgical brain tissue using the International League Against Epilepsy classification scheme of 2011. We developed an iterative histopathological agreement trial with genetic testing to identify areas of diagnostic challenges in this widely used classification scheme.Four web-based digital pathology trials were completed by 20 neuropathologists from 15 countries using a consecutive series of 196 surgical tissue blocks obtained from 22 epilepsy patients at a single center. Five independent genetic laboratories performed screening or validation sequencing of FCD-relevant genes in paired brain and blood samples from the same 22 epilepsy patients.Histopathology agreement based solely on hematoxylin and eosin stainings was low in Round 1, and gradually increased by adding a panel of immunostainings in Round 2 and the Delphi consensus method in Round 3. Interobserver agreement was good in Round 4 (kappa = .65), when the results of genetic tests were disclosed, namely, MTOR, AKT3, and SLC35A2 brain somatic mutations in five cases and germline mutations in DEPDC5 and NPRL3 in two cases.The diagnoses of FCD 1 and 3 subtypes remained most challenging and were often difficult to differentiate from a normal homotypic or heterotypic cortical architecture. Immunohistochemistry was helpful, however, to confirm the diagnosis of FCD or no lesion. We observed a genotype-phenotype association for brain somatic mutations in SLC35A2 in two cases with mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Our results suggest that the current FCD classification should recognize a panel of immunohistochemical stainings for a better histopathological workup and definition of FCD subtypes. We also propose adding the level of genetic findings to obtain a comprehensive, reliable, and integrative genotype-phenotype diagnosis in the near future.

<h2>Summary</h2><h3>Background</h3> Marked variation exists in the use of genomic data in tumour diagnosis, and optimal integration with conventional diagnostic technology remains uncertain despite several studies reporting improved diagnostic accuracy, selection for targeted treatments, and stratification for trials. Our aim was to assess the added value of molecular profiling in routine clinical practice and the impact on conventional and experimental treatments. <h3>Methods</h3> This population-based study assessed the diagnostic and clinical use of DNA methylation-based profiling in childhood CNS tumours using two large national cohorts in the UK. In the diagnostic cohort—which included routinely diagnosed CNS tumours between Sept 1, 2016, and Sept 1, 2018—we assessed how the methylation profile altered or refined diagnosis in routine clinical practice and estimated how this would affect standard patient management. For the archival cohort of diagnostically difficult cases, we established how many cases could be solved using modern standard pathology, how many could only be solved using the methylation profile, and how many remained unsolvable. <h3>Findings</h3> Of 484 patients younger than 20 years with CNS tumours, 306 had DNA methylation arrays requested by the neuropathologist and were included in the diagnostic cohort. Molecular profiling added a unique contribution to clinical diagnosis in 107 (35%; 95% CI 30–40) of 306 cases in routine diagnostic practice—providing additional molecular subtyping data in 99 cases, amended the final diagnosis in five cases, and making potentially significant predictions in three cases. We estimated that it could change conventional management in 11 (4%; 95% CI 2–6) of 306 patients. Among 195 historically difficult-to-diagnose tumours in the archival cohort, 99 (51%) could be diagnosed using standard methods, with the addition of methylation profiling solving a further 34 (17%) cases. The remaining 62 (32%) cases were unresolved despite specialist pathology and methylation profiling. <h3>Interpretation</h3> Together, these data provide estimates of the impact that could be expected from routine implementation of genomic profiling into clinical practice, and indicate limitations where additional techniques will be required. We conclude that DNA methylation arrays are a useful diagnostic adjunct for childhood CNS tumours. <h3>Funding</h3> The Brain Tumour Charity, Children with Cancer UK, Great Ormond Street Hospital Children's Charity, Olivia Hodson Cancer Fund, Cancer Research UK, and the National Institute of Health Research.

The fifth edition of the World Health Organization Classification of Tumours of the Central Nervous System (WHO CNS5) published in 2021 builds on the 2016 edition and incorporates output from the Consortium to Inform Molecular and Practical Approaches to CNS Tumour Taxonomy (cIMPACT-NOW). WHO CNS5 introduces fundamental changes to brain tumour classification through the introduction of new tumour families and types, especially in the paediatric population, and a revision of diagnostic criteria for some of the existing neoplasms. Neuroradiologists are central to brain tumour diagnostics, and it is therefore essential that they become familiar with the key updates. This review aims to summarise the most relevant updates for the neuroradiologist and, where available, discuss the known radiophenotypes of various new tumour types to allow for increased accuracy of language and diagnosis. Of particular importance, WHO CNS5 places greater emphasis on organising tumours by molecular type to reflect biology, as well as to allow for better planning of treatment. The principal updates in adult tumours concern the molecular definition of glioblastoma, restructuring of diffuse gliomas, and the introduction of several new tumour types. The updates to the paediatric classification are protean, ranging from the introduction of new types to establishing separate tumour families for paediatric-type gliomas. This review summarises the most significant revisions and captures the rationale and radiological implications for the major updates.

Background Radiogenomics of pediatric medulloblastoma (MB) offers an opportunity for MB risk stratification, which may aid therapeutic decision making, family counseling, and selection of patient groups suitable for targeted genetic analysis. Purpose To develop machine learning strategies that identify the four clinically significant MB molecular subgroups. Materials and Methods In this retrospective study, consecutive pediatric patients with newly diagnosed MB at MRI at 12 international pediatric sites between July 1997 and May 2020 were identified. There were 1800 features extracted from T2- and contrast-enhanced T1-weighted preoperative MRI scans. A two-stage sequential classifier was designed—one that first identifies non-wingless (WNT) and non–sonic hedgehog (SHH) MB and then differentiates therapeutically relevant WNT from SHH. Further, a classifier that distinguishes high-risk group 3 from group 4 MB was developed. An independent, binary subgroup analysis was conducted to uncover radiomics features unique to infantile versus childhood SHH subgroups. The best-performing models from six candidate classifiers were selected, and performance was measured on holdout test sets. CIs were obtained by bootstrapping the test sets for 2000 random samples. Model accuracy score was compared with the no-information rate using the Wald test. Results The study cohort comprised 263 patients (mean age ± SD at diagnosis, 87 months ± 60; 166 boys). A two-stage classifier outperformed a single-stage multiclass classifier. The combined, sequential classifier achieved a microaveraged F1 score of 88% and a binary F1 score of 95% specifically for WNT. A group 3 versus group 4 classifier achieved an area under the receiver operating characteristic curve of 98%. Of the Image Biomarker Standardization Initiative features, texture and first-order intensity features were most contributory across the molecular subgroups. Conclusion An MRI-based machine learning decision path allowed identification of the four clinically relevant molecular pediatric medulloblastoma subgroups. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Chaudhary and Bapuraj in this issue.

<h2>Abstract</h2><h3>Introduction</h3> Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal region. We provide a comprehensive analysis of this malignancy with molecular and clinical trial data on a subset of our cohort to report on the potential efficacy of somatostatin receptor 2 (SSTR2)-targeting imaging and therapy. <h3>Methods</h3> We conducted a retrospective analysis of 404 primary, locally recurrent, and metastatic olfactory neuroblastoma (ONB) patients from 12 institutions in the United States of America, United Kingdom and Europe. Clinicopathological characteristics and treatment approach were evaluated. SSTR2 expression, SSTR2-targeted imaging and the efficacy of peptide receptor radionuclide therapy [PRRT](¹⁷⁷Lu-DOTATATE) were reported in a subset of our cohort (LUTHREE trial; NCT03454763). <h3>Results</h3> Dural infiltration at presentation was a significant predictor of overall survival (OS) and disease-free survival (DFS) in primary cases (n = 278). Kadish-Morita staging and Dulguerov T-stage both had limitations regarding their prognostic value. Multivariable survival analysis demonstrated improved outcomes with lower stage and receipt of adjuvant radiotherapy. Prophylactic neck irradiation significantly reduces the rate of nodal recurrence. 82.4% of the cohort were positive for SSTR2; treatment of three metastatic cases with SSTR2-targeted peptide-radionuclide receptor therapy (PRRT) in the LUTHREE trial was well-tolerated and resulted in stable disease (SD). <h3>Conclusions</h3> This study presents pertinent clinical data from the largest dataset, to date, on ONB. We identify key prognostic markers and integrate these into an updated staging system, highlight the importance of adjuvant radiotherapy across all disease stages, the utility of prophylactic neck irradiation and the potential efficacy of targeting SSTR2 to manage disease.

Medulloblastoma is currently subclassified into distinct DNA methylation subgroups/subtypes with particular clinico-molecular features. Using RNA sequencing (RNA-seq) in large, well-annotated cohorts of medulloblastoma, we show that transcriptionally group 3 and group 4 medulloblastomas exist as intermediates on a bipolar continuum between archetypal group 3 and group 4 entities. Continuum position is prognostic, reflecting a propensity for specific DNA copy-number changes, and specific switches in isoform/enhancer usage and RNA editing. Examining single-cell RNA-seq (scRNA-seq) profiles, we show that intratumoral transcriptional heterogeneity along the continuum is limited in a subtype-dependent manner. By integrating with a human scRNA-seq reference atlas, we show that this continuum is mirrored by an equivalent continuum of transcriptional cell types in early fetal cerebellar development. We identify distinct developmental niches for all four major subgroups and link each to a common developmental antecedent. Our findings show a transcriptional continuum arising from oncogenic disruption of highly specific fetal cerebellar cell types, linked to almost every aspect of group 3/group 4 molecular biology and clinico-pathology.

Tumours of the anterior part of the pituitary gland represent just 1% of all childhood (aged <15 years) intracranial neoplasms, yet they can confer high morbidity and little evidence and guidance is in place for their management. Between 2014 and 2022, a multidisciplinary expert group systematically developed the first comprehensive clinical practice consensus guideline for children and young people under the age 19 years (hereafter referred to as CYP) presenting with a suspected pituitary adenoma to inform specialist care and improve health outcomes. Through robust literature searches and a Delphi consensus exercise with an international Delphi consensus panel of experts, the available scientific evidence and expert opinions were consolidated into 74 recommendations. Part 1 of this consensus guideline includes 17 pragmatic management recommendations related to clinical care, neuroimaging, visual assessment, histopathology, genetics, pituitary surgery and radiotherapy. While in many aspects the care for CYP is similar to that of adults, key differences exist, particularly in aetiology and presentation. CYP with suspected pituitary adenomas require careful clinical examination, appropriate hormonal work-up, dedicated pituitary imaging and visual assessment. Consideration should be given to the potential for syndromic disease and genetic assessment. Multidisciplinary discussion at both the local and national levels can be key for management. Surgery should be performed in specialist centres. The collection of outcome data on novel modalities of medical treatment, surgical intervention and radiotherapy is essential for optimal future treatment. Evidence and guidance for the care of pituitary adenomas in children and adolescents is currently lacking. Part 1 of this two-part consensus guideline presents 17 general recommendations for diagnosis and management of pituitary adenomas in children and young people under 19 years of age.

Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase-derived prostaglandin (PG) H(2) to PGD(2), which is dehydrated to cyclopentenone PGs, including 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). PGD(2) acts through two receptors (DP1 and DP2/CRTH2), whereas 15d-PGJ(2) can activate peroxisome proliferator-activated receptors or inhibit a range of proinflammatory signaling pathways, including NF-kappaB. Despite eliciting asthmatic and allergic reactions through the generation of PGD(2), it is not known what role hPGD(2)S plays in T helper (Th)1-driven adaptive immunity. To investigate this question, the severity and duration of a delayed type hypersensitivity reaction was examined in hPGD(2)S knockout and transgenic mice. Compared with their respective controls, knockouts displayed a more severe inflammatory response that failed to resolve, characterized histologically as persistent acute inflammation, whereas transgenic mice had little detectable inflammation. Lymphocytes isolated from inguinal lymph nodes of hPGD(2)S(-/-) animals showed hyperproliferation and increased IL-2 synthesis effects that were rescued by 15d-PGJ(2), but not PGD(2), working through either of its receptors. Crucially, 15d-PGJ(2) exerted its suppressive effects through the inhibition of NF-kappaB activation and not through peroxisome proliferator-activated receptor signaling. In contrast, lymph node cultures from transgenics proliferated more slowly and synthesized significantly less IL-2 than controls. Therefore, contrary to its role in driving Th2-like responses, this report shows that hPGD(2)S may act as an internal braking signal essential for bringing about the resolution of Th1-driven delayed type hypersensitivity reactions. Consequently, hPGD(2)S-derived cyclopentenone PGs may protect against inflammatory diseases, where T lymphocytes play a pathogenic role, as in rheumatoid arthritis, atopic eczema, and chronic rejection.

Pontocerebellar hypoplasia (PCH) represents a group of neurodegenerative disorders with prenatal onset. Eight subtypes have been described thus far (PCH1-8) based on clinical and genetic features. Common characteristics include hypoplasia and atrophy of the cerebellum, variable pontine atrophy, and severe mental and motor impairments. PCH1 is distinctly characterized by the combination with degeneration of spinal motor neurons. Recently, mutations in the exosome component 3 gene (EXOSC3) have been identified in approximately half of the patients with PCH subtype 1.We selected a cohort of 99 PCH patients (90 families) tested negative for mutations in the TSEN genes, RARS2, VRK1 and CASK. Patients in this cohort were referred with a tentative diagnose PCH type 1, 2, 4, 7 or unclassified PCH. Genetic analysis of the EXOSC3 gene was performed using Sanger sequencing. Clinical data, MR images and autopsy reports of patients positive for EXOSC3 mutations were analyzed.EXOSC3 mutations were found in twelve families with PCH subtype 1, and were not found in patients with other PCH subtypes. Identified mutations included a large deletion, nonsense and missense mutations. Examination of clinical data reveals a prolonged disease course in patients with a homozygous p.D132A mutation. MRI shows variable pontine hypoplasia in EXOSC3 mediated PCH, where the pons is largely preserved in patients with a homozygous p.D132A mutation, but attenuated in patients with other mutations. Additionally, bilateral cerebellar cysts were found in patients compound heterozygous for a p.D132A mutation and a nonsense allele.EXOSC3 mediated PCH shows clear genotype-phenotype correlations. A homozygous p.D132A mutation leads to PCH with possible survival into early puberty, and preservation of the pons. Compound heterozygosity for a p.D132A mutation and a nonsense or p.Y109N allele, a homozygous p.G31A mutation or a p.G135E mutation causes a more rapidly progressive course leading to death in infancy and attenuation of the ventral pons.Our findings imply a clear correlation between genetic mutation and clinical outcome in EXOSC3 mediated PCH, including variable involvement of the pons.

•Post mortem MRI (PMMRI) has a sensitivity of >87% for detecting cerebral malformations, intracranial bleeds and neurological cause of death. •PMMRI provides important diagnostic information in >50% of fetuses where conventional brain autopsy is non-diagnostic. •PMMRI is currently poor at reliably identifying ischaemic brain injury, especially in fetuses. Aim To compare the diagnostic accuracy of non-invasive cerebral post-mortem magnetic resonance imaging (PMMRI) specifically for cerebral and neurological abnormalities in a series of fetuses and children, compared to conventional autopsy. Materials and methods Institutional ethics approval and parental consent was obtained. Pre-autopsy cerebral PMMRI was performed in a sequential prospective cohort (n = 400) of fetuses (n = 277; 185 ≤ 24 weeks and 92 > 24 weeks gestation) and children <16 years (n = 123) of age. PMMRI and conventional autopsy findings were reported blinded and independently of each other. Results Cerebral PMMRI had sensitivities and specificities (95% confidence interval) of 88.4% (75.5 to 94.9), and 95.2% (92.1 to 97.1), respectively, for cerebral malformations; 100% (83.9 to 100), and 99.1% (97.2 to 99.7) for major intracranial bleeds; and 87.5% (80.1 to 92.4) and 74.1% (68 to 79.4) for overall brain pathology. Formal neuropathological examination was non-diagnostic due to maceration/autolysis in 43/277 (16%) fetuses; of these, cerebral PMMRI imaging provided clinically important information in 23 (53%). The sensitivity of PMMRI for detecting significant ante-mortem ischaemic injury was only 68% (48.4 to 82.8) overall. Conclusions PMMRI is an accurate investigational technique for identifying significant neuropathology in fetuses and children, and may provide important information even in cases where autolysis prevents formal neuropathological examination; however, PMMRI is less sensitive at detecting hypoxic–ischaemic brain injury, and may not detect rarer disorders not encountered in this study. To compare the diagnostic accuracy of non-invasive cerebral post-mortem magnetic resonance imaging (PMMRI) specifically for cerebral and neurological abnormalities in a series of fetuses and children, compared to conventional autopsy. Institutional ethics approval and parental consent was obtained. Pre-autopsy cerebral PMMRI was performed in a sequential prospective cohort (n = 400) of fetuses (n = 277; 185 ≤ 24 weeks and 92 > 24 weeks gestation) and children <16 years (n = 123) of age. PMMRI and conventional autopsy findings were reported blinded and independently of each other. Cerebral PMMRI had sensitivities and specificities (95% confidence interval) of 88.4% (75.5 to 94.9), and 95.2% (92.1 to 97.1), respectively, for cerebral malformations; 100% (83.9 to 100), and 99.1% (97.2 to 99.7) for major intracranial bleeds; and 87.5% (80.1 to 92.4) and 74.1% (68 to 79.4) for overall brain pathology. Formal neuropathological examination was non-diagnostic due to maceration/autolysis in 43/277 (16%) fetuses; of these, cerebral PMMRI imaging provided clinically important information in 23 (53%). The sensitivity of PMMRI for detecting significant ante-mortem ischaemic injury was only 68% (48.4 to 82.8) overall. PMMRI is an accurate investigational technique for identifying significant neuropathology in fetuses and children, and may provide important information even in cases where autolysis prevents formal neuropathological examination; however, PMMRI is less sensitive at detecting hypoxic–ischaemic brain injury, and may not detect rarer disorders not encountered in this study.

Summary Purpose: An International League Against Epilepsy (ILAE) consensus classification system for focal cortical dysplasias (FCDs) has been published in 2011 specifying clinicopathologic FCD variants. The aim of the present work was to microscopically assess interobserver agreement and intraobserver reproducibility for FCD categories among an international group of neuropathologists with different levels of experience and access to epilepsy surgery tissue. Methods: Surgical FCD specimens covering a broad histopathology spectrum were retrieved from 22 patients with epilepsy. Three surgical nonepilepsy specimens served as controls. A total of 188 slides with routine or immunohistochemical stainings were digitalized with a slide scanner to allow Internet‐based microscopy review. Nine experienced neuropathologists were invited to review these cases twice at a time gap of 3 months and different orders of case presentation. The 2011 ILAE FCD consensus classification served as instruction. Kappa analysis was calculated to estimate interobserver and intraobserver agreement levels. In a third evaluation round, 21 additional neuropathologists with different experience and access to epilepsy surgery reviewed the same case series. Key Findings: Interobserver agreement was good (κ = 0.6360), with 84% consensus of diagnoses during the first evaluation (21 of 25 cases). Kappa values increased to 0.6532 after reevaluation, and consensus was obtained in 24 (96%) of 25 cases. Overall intraobserver reproducibility was also good (κ = 0.7824, ranging from 0.4991 to 1.000). Fewest changes in the classification were made in the FCD type II group (2.2% of 225 original diagnoses), whereas the majority of changes occurred in FCD type III (13.7% of 225 original diagnoses). In the third evaluation round, interobserver agreement was reflected by the level of experience of each neuropathologist, with κ values ranging from moderate (0.5056; high level of experience &gt;40 cases/year) to low (0.3265; low level of experience &lt;10 cases/year). Significance: Our study achieved a good and reliable interobserver agreement among the group of expert neuropathologists originally involved in the ILAE FCD consensus classification system. Intraobserver reproducibility in this group was even more robust. These results showed considerable improvement compared to a previous study evaluating the 2004 Palmini FCD classification. Agreement levels were lower in our second group of neuropathologists and were related to their level of access and experience with epilepsy surgery specimens. These results suggested that the more precise ILAE definition of FCD histopathology patterns improves operational procedures in the diagnosis of FCDs. On the other hand, microscopic assessment of FCD is a challenge and requires sustained experience and teaching. The virtual slide review system allowed testing of this hypothesis and reached a widespread group of participating colleagues from different centers all over the world. We propose to further use this tool as a teaching device and also to address other epilepsy‐associated entities still difficult to classify such as hippocampal sclerosis, long‐term epilepsy‐associated tumors, or mild malformations of cortical development (mMCDs), which were not yet covered by current ILAE classification systems.

Brown-Vialetto-Van Laere syndrome represents a phenotypic spectrum of motor, sensory, and cranial nerve neuropathy, often with ataxia, optic atrophy and respiratory problems leading to ventilator-dependence. Loss-of-function mutations in two riboflavin transporter genes, SLC52A2 and SLC52A3, have recently been linked to Brown-Vialetto-Van Laere syndrome. However, the genetic frequency, neuropathology and downstream consequences of riboflavin transporter mutations are unclear. By screening a large cohort of 132 patients with early-onset severe sensory, motor and cranial nerve neuropathy we confirmed the strong genetic link between riboflavin transporter mutations and Brown-Vialetto-Van Laere syndrome, identifying 22 pathogenic mutations in SLC52A2 and SLC52A3, 14 of which were novel. Brain and spinal cord neuropathological examination of two cases with SLC52A3 mutations showed classical symmetrical brainstem lesions resembling pathology seen in mitochondrial disease, including severe neuronal loss in the lower cranial nerve nuclei, anterior horns and corresponding nerves, atrophy of the spinothalamic and spinocerebellar tracts and posterior column-medial lemniscus pathways. Mitochondrial dysfunction has previously been implicated in an array of neurodegenerative disorders. Since riboflavin metabolites are critical components of the mitochondrial electron transport chain, we hypothesized that reduced riboflavin transport would result in impaired mitochondrial activity, and confirmed this using in vitro and in vivo models. Electron transport chain complex I and complex II activity were decreased in SLC52A2 patient fibroblasts, while global knockdown of the single Drosophila melanogaster riboflavin transporter homologue revealed reduced levels of riboflavin, downstream metabolites, and electron transport chain complex I activity. This in turn led to abnormal mitochondrial membrane potential, respiratory chain activity and morphology. Riboflavin transporter knockdown in Drosophila also resulted in severely impaired locomotor activity and reduced lifespan, mirroring patient pathology, and these phenotypes could be partially rescued using a novel esterified derivative of riboflavin. Our findings expand the genetic, clinical and neuropathological features of Brown-Vialetto-Van Laere syndrome, implicate mitochondrial dysfunction as a downstream consequence of riboflavin transporter gene defects, and validate riboflavin esters as a potential therapeutic strategy.

Pediatric adamantinomatous craniopharyngioma (ACP) is a highly solid and cystic tumor, often causing substantial damage to critical neuroendocrine structures such as the hypothalamus, pituitary gland, and optic apparatus. Paracrine signaling mechanisms driving tumor behavior have been hypothesized, with IL-6R overexpression identified as a potential therapeutic target. To identify potential novel therapies, we characterized inflammatory and immunomodulatory factors in ACP cyst fluid and solid tumor components. Cytometric bead analysis revealed a highly pro-inflammatory cytokine pattern in fluid from ACP compared to fluids from another cystic pediatric brain tumor, pilocytic astrocytoma. Cytokines and chemokines with particularly elevated concentrations in ACPs were IL-6, CXCL1 (GRO), CXCL8 (IL-8) and the immunosuppressive cytokine IL-10. These data were concordant with solid tumor compartment transcriptomic data from a larger cohort of ACPs, other pediatric brain tumors and normal brain. The majority of receptors for these cytokines and chemokines were also over-expressed in ACPs. In addition to IL-10, the established immunosuppressive factor IDO-1 was overexpressed by ACPs at the mRNA and protein levels. These data indicate that ACP cyst fluids and solid tumor components are characterized by an inflammatory cytokine and chemokine expression pattern. Further study regarding selective cytokine blockade may inform novel therapeutic interventions.

Despite the importance of the RAS-RAF-MAPK pathway in normal physiology and disease of numerous organs, its role during pituitary development and tumourigenesis remains largely unknown. Here, we show that the over-activation of the MAPK pathway, through conditional expression of the gain-of-function alleles BrafV600E and KrasG12D in the developing mouse pituitary, results in severe hyperplasia and abnormal morphogenesis of the gland by the end of gestation. Cell-lineage commitment and terminal differentiation are disrupted, leading to a significant reduction in numbers of most of the hormone-producing cells before birth, with the exception of corticotrophs. Of note, Sox2+ stem cells and clonogenic potential are drastically increased in the mutant pituitaries. Finally, we reveal that papillary craniopharyngioma (PCP), a benign human pituitary tumour harbouring BRAF p.V600E also contains Sox2+ cells with sustained proliferative capacity and disrupted pituitary differentiation. Together, our data demonstrate a crucial function of the MAPK pathway in controlling the balance between proliferation and differentiation of Sox2+ cells and suggest that persistent proliferative capacity of Sox2+ cells may underlie the pathogenesis of PCP.

Glioneuronal tumours are an important cause of treatment-resistant epilepsy. Subtypes of tumour are often poorly discriminated by histological features and may be difficult to diagnose due to a lack of robust diagnostic tools. This is illustrated by marked variability in the reported frequencies across different epilepsy surgical series. To address this, we used DNA methylation arrays and RNA sequencing to assay the methylation and expression profiles within a large cohort of glioneuronal tumours. By adopting a class discovery approach, we were able to identify two distinct groups of glioneuronal tumour, which only partially corresponded to the existing histological classification. Furthermore, by additional molecular analyses, we were able to identify pathogenic mutations in BRAF and FGFR1, specific to each group, in a high proportion of cases. Finally, by interrogating our expression data, we were able to show that each molecular group possessed expression phenotypes suggesting different cellular differentiation: astrocytic in one group and oligodendroglial in the second. Informed by this, we were able to identify CCND1, CSPG4, and PDGFRA as immunohistochemical targets which could distinguish between molecular groups. Our data suggest that the current histological classification of glioneuronal tumours does not adequately represent their underlying biology. Instead, we show that there are two molecular groups within glioneuronal tumours. The first of these displays astrocytic differentiation and is driven by BRAF mutations, while the second displays oligodendroglial differentiation and is driven by FGFR1 mutations.

Abstract: The recently published 2016 World Health Organization (WHO) classification of tumours of the Central Nervous System (CNS) introduces a number of significant changes from the previous edition. Based on an improved understanding of the genetic and molecular basis of tumorigenesis there has been a shift towards defining tumours by means of these characteristics in addition to their histological features, thus providing an integrated diagnosis. In this article, we will provide a concise overview of the salient changes in the 2016 WHO classification of tumours of the CNS that are of relevance to the paediatric neuroradiologist when it comes to day-to-day reporting.

Objective. We aimed to establish the prevalence and clinical associations of anti-HMG-CoA-reductase (anti-HMGCR) in a large UK cohort with juvenile myositis. Methods. There were 381 patients investigated for anti-HMGCR using ELISA. Results. Anti-HMGCR autoantibodies were detected in 4 patients (1%). These children had no or minimal rash and significant muscle disease. Muscle biopsies were considered distinctive, with widespread variation in fiber size, necrotic fibers, and chronic inflammatory cell infiltrates; all had prolonged elevation of creatine kinase and all ultimately received biologic therapies. Conclusion. Anti-HMGCR in UK children with myositis are associated with severe disease that is poorly responsive to standard treatment.

Low-grade epilepsy-associated brain tumours (LEAT) are the second most common cause for drug-resistant, focal epilepsy, that is ganglioglioma (GG) and dysembryoplastic neuroepithelial tumours (DNT). However, molecular pathogenesis, risk factors for malignant progression and their frequent association with drug-resistant focal seizures remain poorly understood. This contrasts recent progress in understanding the molecular-genetic basis and targeted treatment options in diffuse gliomas. The Neuropathology Task Force of the International League Against Epilepsy examined available literature to identify common obstacles in diagnosis and research of LEAT. Analysis of 10 published tumour series from epilepsy surgery pointed to poor inter-rater agreement for the histopathology diagnosis. The Task Force tested this hypothesis using a web-based microscopy agreement study. In a series of 30 LEAT, 25 raters from 18 countries agreed in only 40% of cases. Highest discordance in microscopic diagnosis occurred between GG and DNT variants, when oligodendroglial-like cell patterns prevail, or ganglion cells were difficult to discriminate from pre-existing neurons. Suggesting new terminology or major histopathological criteria did not satisfactorily increase the yield of histopathology agreement in four consecutive trials. To this end, the Task Force applied the WHO 2016 strategy of integrating phenotype analysis with molecular-genetic data obtained from panel sequencing and 450k methylation arrays. This strategy was helpful to distinguish DNT from GG variants in all cases. The Task Force recommends, therefore, to further develop diagnostic panels for the integration of phenotype-genotype analysis in order to reliably classify the spectrum of LEAT, carefully characterize clinically meaningful entities and make better use of published literature.

BackgroundDisease relapse occurs in around 30% of children with medulloblastoma, and is almost universally fatal. We aimed to establish whether the clinical and molecular characteristics of the disease at diagnosis are associated with the nature of relapse and subsequent disease course, and whether these associations could inform clinical management.MethodsIn this multicentre cohort study we comprehensively surveyed the clinical features of medulloblastoma relapse (time to relapse, pattern of relapse, time from relapse to death, and overall outcome) in centrally reviewed patients who relapsed following standard upfront therapies, from 16 UK Children's Cancer and Leukaemia Group institutions and four collaborating centres. We compared these relapse-associated features with clinical and molecular features at diagnosis, including established and recently described molecular features, prognostic factors, and treatment at diagnosis and relapse.Findings247 patients (175 [71%] boys and 72 [29%] girls) with medulloblastoma relapse (median year of diagnosis 2000 [IQR 1995–2006]) were included in this study. 17 patients were later excluded from further analyses because they did not meet the age and treatment criteria for inclusion. Patients who received upfront craniospinal irradiation (irradiated group; 178 [72%] patients) had a more prolonged time to relapse compared with patients who did not receive upfront craniospinal irradiation (non-irradiated group; 52 [21%] patients; p<0·0001). In the non-irradiated group, craniospinal irradiation at relapse (hazard ratio [HR] 0·27, 95% CI 0·11–0·68) and desmoplastic/nodular histology (0·23, 0·07–0·77) were associated with prolonged time to death after relapse, MYC amplification was associated with a reduced overall survival (23·52, 4·85–114·05), and re-resection at relapse was associated with longer overall survival (0·17, 0·05–0·57). In the irradiated group, patients with MBGroup3 tumours relapsed significantly more quickly than did patients with MBGroup4 tumours (median 1·34 [0·99–1·89] years vs 2·04 [1·39–3·42 years; p=0·0043). Distant disease was prevalent in patients with MBGroup3 (23 [92%] of 25 patients) and MBGroup4 (56 [90%] of 62 patients) tumour relapses. Patients with distantly-relapsed MBGroup3 and MBGroup4 displayed both nodular and diffuse patterns of disease whereas isolated nodular relapses were rare in distantly-relapsed MBSHH (1 [8%] of 12 distantly-relapsed MBSHH were nodular alone compared with 26 [34%] of 77 distantly-relapsed MBGroup3 and MBGroup4). In MBGroup3 and MBGroup4, nodular disease was associated with a prolonged survival after relapse (HR 0·42, 0·21–0·81). Investigation of second-generation MBGroup3 and MBGroup4 molecular subtypes refined our understanding of heterogeneous relapse characteristics. Subtype VIII had prolonged time to relapse and subtype II had a rapid time from relapse to death. Subtypes II, III, and VIII developed a significantly higher incidence of distant disease at relapse whereas subtypes V and VII did not (equivalent rates to diagnosis).InterpretationThis study suggests that the nature and outcome of medulloblastoma relapse are biology and therapy-dependent, providing translational opportunities for improved disease management through biology-directed disease surveillance, post-relapse prognostication, and risk-stratified selection of second-line treatment strategies.FundingCancer Research UK, Action Medical Research, The Tom Grahame Trust, The JGW Patterson Foundation, Star for Harris, The Institute of Child Health - Newcastle University - Institute of Child Health High-Risk Childhood Brain Tumour Network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

For children with cancer, the clinical integration of precision medicine to enable predictive biomarker-based therapeutic stratification is urgently needed.We have developed a hybrid-capture next-generation sequencing (NGS) panel, specifically designed to detect genetic alterations in paediatric solid tumours, which gives reliable results from as little as 50 ng of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. In this study, we offered an NGS panel, with clinical reporting via a molecular tumour board for children with solid tumours. Furthermore, for a cohort of 12 patients, we used a circulating tumour DNA (ctDNA)-specific panel to sequence ctDNA from matched plasma samples and compared plasma and tumour findings.A total of 255 samples were submitted from 223 patients for the NGS panel. Using FFPE tissue, 82% of all submitted samples passed quality control for clinical reporting. At least one genetic alteration was detected in 70% of sequenced samples. The overall detection rate of clinically actionable alterations, defined by modified OncoKB criteria, for all sequenced samples was 51%. A total of 8 patients were sequenced at different stages of treatment. In 6 of these, there were differences in the genetic alterations detected between time points. Sequencing of matched ctDNA in a cohort of extracranial paediatric solid tumours also identified a high detection rate of somatic alterations in plasma.We demonstrate that tailored clinical molecular profiling of both tumour DNA and plasma-derived ctDNA is feasible for children with solid tumours. Furthermore, we show that a targeted NGS panel-based approach can identify actionable genetic alterations in a high proportion of patients.

Abstract Background Relapse occurs in 50% of pediatric ependymoma cases and has poor prognosis. Few studies have investigated the clinical progress of relapsed disease, and treatment lacks a standardized approach. Methods and materials We analyzed 302 pediatric ependymoma cases. Tumor, demographic, and treatment variables were investigated for association with relapse risk, time to recurrence, and survival after relapse. DNA methylation profiling was performed for 135/302 cases, and predominant subgroups were EPN_PFA ( n = 95) and EPN_RELA ( n = 24). Chromosome 1q status was ascertained for 185/302 cases by fluorescent in‐situ hybridization (FISH), multiplex ligation‐dependent probe amplification (MLPA), and DNA methylation profiles. Results Sixty‐two percent of cases relapsed, with a median of two recurrences with no difference between posterior fossa and supratentorial locations (66% vs 55% relapse rate). One hundred seventeen (38%) cases relapsed within two years and five (2%) beyond 10 years. The late relapses were clinically heterogeneous. Tumor grade and treatment affected risk and time to relapse variably across subgroups. After relapse, surgery and irradiation delayed disease progression with a minimal impact on survival across the entire cohort. In the EPN_PFA and EPN_RELA groups, 1q gain was independently associated with relapse risk (subhazard ratio [SHR] 4.307, P = 0.027 and SHR 1.982, P = 0.010, respectively) while EPN_PFA had increased relapse risk compared with EPN_RELA (SHR = 0.394, P = 0.018). Conclusions Recurrent pediatric ependymoma is an aggressive disease with poor outcomes, for which current treatments are inadequate. We report that chromosome 1q gain increases relapse risk in common molecular subgroups in children but a deeper understanding of the underlying biology at relapse and novel therapeutic approaches are urgently needed.

Less than 5% of medulloblastoma (MB) patients survive following failure of contemporary radiation-based therapies. Understanding the molecular drivers of medulloblastoma relapse (rMB) will be essential to improve outcomes. Initial genome-wide investigations have suggested significant genetic divergence of the relapsed disease.We undertook large-scale integrated characterization of the molecular features of rMB-molecular subgroup, novel subtypes, copy number variation (CNV), and driver gene mutation. 119 rMBs were assessed in comparison with their paired diagnostic samples (n = 107), alongside an independent reference cohort sampled at diagnosis (n = 282). rMB events were investigated for association with outcome post-relapse in clinically annotated patients (n = 54).Significant genetic evolution occurred over disease-course; 40% of putative rMB drivers emerged at relapse and differed significantly between molecular subgroups. Non-infant MBSHH displayed significantly more chromosomal CNVs at relapse (TP53 mutation-associated). Relapsed MBGroup4 demonstrated the greatest genetic divergence, enriched for targetable (eg, CDK amplifications) and novel (eg, USH2A mutations) events. Importantly, many hallmark features of MB were stable over time; novel subtypes (>90% of tumors) and established genetic drivers (eg, SHH/WNT/P53 mutations; 60% of rMB events) were maintained from diagnosis. Critically, acquired and maintained rMB events converged on targetable pathways which were significantly enriched at relapse (eg, DNA damage signaling) and specific events (eg, 3p loss) predicted survival post-relapse.rMB is characterised by the emergence of novel events and pathways, in concert with selective maintenance of established genetic drivers. Together, these define the actionable genetic landscape of rMB and provide a basis for improved clinical management and development of stratified therapeutics, across disease-course.

Abstract Background SIOP Ependymoma I was a non-randomised trial assessing event free and overall survival (EFS/OS) of non-metastatic intracranial ependymoma in children aged 3–21 years treated with a staged management strategy. A further aim was to assess the response rate (RR) of subtotally resected (STR) ependymoma to vincristine, etoposide, and cyclophosphamide (VEC). We report final results with 12-year follow-up and post hoc analyses of recently described biomarkers. Methods Seventy-four participants were eligible. Children with gross total resection (GTR) received radiotherapy, whilst those with STR received VEC before radiotherapy. DNA methylation, 1q, hTERT, ReLA, Tenascin-C, H3K27me3, and pAKT status were evaluated. Results Five- and ten-year EFS was 49.5% and 46.7%, OS was 69.3% and 60.5%. GTR was achieved in 33/74 (44.6%) and associated with improved EFS (P = .003, HR = 2.6, 95% confidence interval (CI) 1.4–5.1). Grade 3 tumours were associated with worse OS (P = .005, HR = 2.8, 95%CI 1.3–5.8). 1q gain and hTERT expression were associated with poorer EFS (P = .003, HR = 2.70, 95%CI 1.49–6.10 and P = .014, HR = 5.8, 95%CI 1.2–28) and H3K27me3 loss with worse OS (P = .003, HR = 4.6, 95%CI 1.5–13.2). Methylation profiles showed expected patterns. 12 participants with STR did not receive chemotherapy; a protocol violation. However, best chemotherapy RR was 65.5% (19/29, 95%CI 45.7–82.1), exceeding the prespecified 45%. Conclusions Participants with totally resected ependymoma had the best outcomes. RR of STR to VEC exceeded the pre-specified efficacy criterion. However, cases of inaccurate stratification highlighted the need for rapid central review. 1q gain, H3K27me3 loss, and hTERT expression were all associated with poorer survival outcomes.

Whole-genome sequencing (WGS) of cancers is becoming an accepted component of oncological care, and NHS England is currently rolling out WGS for all children with cancer. This approach was piloted during the 100,000 genomes (100 K) project. Here we share the experience of the East of England Genomic Medicine Centre (East-GMC), reporting the feasibility and clinical utility of centralised WGS for individual children locally.Non-consecutive children with solid tumours were recruited into the pilot 100 K project at our Genomic Medicine Centre. Variant catalogues were returned for local scrutiny and appraisal at dedicated genomic tumour advisory boards with an emphasis on a detailed exploration of potential clinical value.Thirty-six children, representing one-sixth of the national 100 K cohort, were recruited through our Genomic Medicine Centre. The diagnoses encompassed 23 different solid tumour types and WGS provided clinical utility, beyond standard-of-care assays, by refining (2/36) or changing (4/36) diagnoses, providing prognostic information (8/36), defining pathogenic germline mutations (1/36) or revealing novel therapeutic opportunities (8/36).Our findings demonstrate the feasibility and clinical value of centralised WGS for children with cancer. WGS offered additional clinical value, especially in diagnostic terms. However, our experience highlights the need for local expertise in scrutinising and clinically interpreting centrally derived variant calls for individual children.

Although rare, craniopharyngiomas constitute up to 80% of tumours in the hypothalamic–pituitary region in childhood. Despite being benign, the close proximity of these tumours to the visual pathways, hypothalamus, and pituitary gland means that both treatment of the tumour and the tumour itself can cause pronounced long-term neuroendocrine morbidity against a background of high overall survival. To date, the optimal management strategy for these tumours remains undefined, with practice varying between centres. In light of these discrepancies, as part of a national endeavour to create evidence-based and consensus-based guidance for the management of rare paediatric endocrine tumours in the UK, we aimed to develop guidelines, which are presented in this Review. These guidelines were developed under the auspices of the UK Children's Cancer and Leukaemia Group and the British Society for Paediatric Endocrinology and Diabetes, with the oversight and endorsement of the Royal College of Paediatrics and Child Health using Appraisal of Guidelines for Research & Evaluation II methodology to standardise care for children and young people with craniopharyngiomas.

The present study aims to describe the cognitive profile of children with medically refractory extratemporal epilepsies who undergo focal surgery and to identify determinants for preoperative and postoperative cognitive level.This is a retrospective cohort study. Children who underwent operations between 1997 and 2008 with a focal lesion in frontal, parietal, or occipital cortices and with a presurgical or postsurgical cognitive evaluation, were eligible for the study.Sixty-six children (53% male) with a mean age of 9.3 ± 8.8 years were enrolled. The overall full-scale IQ (FSIQ) at cognitive testing was 77.4 ± 44.4 before surgery. Children did not show any significant change in their FSIQ after surgery. Duration of presurgical epilepsy, age at epilepsy onset, etiology, and gender were found to be independently associated with lower FSIQ before surgery. Presurgical cognitive level was the only factor independently associated with postsurgical FSIQ. Overall, 51.5% of children who underwent surgery were seizure-free; however, the good postsurgical epilepsy control did not seem to influence the cognitive outcome.Children with extratemporal lobe epilepsy are below the normal cognitive level range. Intellectual abilities of children undergoing surgery are determined independently by presurgical factors and surgery does not seem to affect the cognitive level in the postsurgical period, even for those who become free from clinical seizures.

Parkinson's disease (PD) is a common neurodegenerative disorder of unknown etiology. The characteristic α-synuclein aggregation of PD is also a feature of Sanfilippo syndrome, a storage disorder caused by α-N-acetylglucosaminidase (NAGLU) gene mutations. We explored genetic links between these disorders and studied the pathology of Sanfilippo syndrome to investigate a common pathway toward α-synuclein aggregation.We typed the 2 single-nucleotide polymorphisms that tag the common haplotypes of NAGLU in 926 PD patients and 2308 controls and also stained cortical tissue from 2 cases of Sanfilippo A syndrome using the anti-α-synuclein antibody, Per7.Allelic analysis showed an association between rs2071046 and risk for PD (P 1.3 × 10(-3) ). Intracellular α-synuclein accumulation was observed in the cortical tissue of both Sanfilippo A syndrome cases.This study suggests a possible role of NAGLU in susceptibility to PD while extending evidence for α-synuclein aggregation in the brain in lysosomal storage disorders. Our findings support a mechanism involving lysosomal dysfunction more generally in the pathogenesis of PD.

Infantile-onset X-linked spinal muscular atrophy (SMAX2) is a rare lethal disorder linked to mutations in the UBA1 (previously UBE1) gene, encoding ubiquitin-activating enzyme 1 that has an important role in the ubiquitin–proteasome pathway. Published pathological reports are scarce. Here we report a male infant who presented from birth with predominantly truncal hypotonia following an antenatal history of reduced fetal movements. He had a myopathic face, profound weakness, multiple contractures and areflexia. Creatine kinase was moderately raised. Brain MRI showed non-specific symmetrical periventricular white matter changes. Neurophysiology revealed evidence of motor and sensory involvement and muscle biopsy showed marked inflammatory changes with subtle features suggestive of acute denervation. UBA1 sequencing revealed a novel hemizygous missense mutation (c.1670A>T; p.Glu557Val). He died from progressive respiratory failure at 4 months. On post mortem assessment, in addition to severe ventral motor neuron pathology, there was widespread involvement of the sensory system, as well as developmental and degenerative cerebellar abnormalities. In contrast to typical SMN1-associated SMA, the thalamus was unaffected. These findings indicate that SMAX2 is more accurately classified as a motor sensory neuronopathy rather than a pure anterior horn cell disorder. Ubiquitin–proteasome pathway defects may not only cause neurodegeneration but also affect normal neuronal development.

Objectives To study muscle biopsy tissue from patients with juvenile dermatomyositis (JDM) in order to test the reliability of a score tool designed to quantify the severity of histological abnormalities when applied to biceps humeri in addition to quadriceps femoris. Additionally, to evaluate whether elements of the tool correlate with clinical measures of disease severity. Methods 55 patients with JDM with muscle biopsy tissue and clinical data available were included. Biopsy samples (33 quadriceps, 22 biceps) were prepared and stained using standardised protocols. A Latin square design was used by the International Juvenile Dermatomyositis Biopsy Consensus Group to score cases using our previously published score tool. Reliability was assessed by intraclass correlation coefficient (ICC) and scorer agreement (α) by assessing variation in scorers’ ratings. Scores from the most reliable tool items correlated with clinical measures of disease activity at the time of biopsy. Results Inter- and intraobserver agreement was good or high for many tool items, including overall assessment of severity using a Visual Analogue Scale. The tool functioned equally well on biceps and quadriceps samples. A modified tool using the most reliable score items showed good correlation with measures of disease activity. Conclusions The JDM biopsy score tool has high inter- and intraobserver agreement and can be used on both biceps and quadriceps muscle tissue. Importantly, the modified tool correlates well with clinical measures of disease activity. We propose that standardised assessment of muscle biopsy tissue should be considered in diagnostic investigation and clinical trials in JDM.

Primary melanoma of the CNS in children is extremely rare, and usually linked to congenital melanocytic naevus syndrome, caused by mosaicism for oncogenic NRAS mutations. Outcome is fatal in all cases. Data from murine and in vitro studies suggest that MEK inhibition is a possible therapeutic option.Four children with NRAS-mutated CNS melanoma were treated with Trametinib on a compassionate basis.All four had an improvement in symptoms and objectively in signs. These varied from mild improvement for 1 month, to a sustained symptom-free period of 9 months in one case. In all cases there was eventual disease progression through treatment, followed by rapid death after discontinuation. There were no clinically-significant side effects.Trametinib is the first therapy to show any objective or measurable effect in NRAS-mutated primary CNS melanoma, with few side effects in this small series. The role of this therapy should be explored further in this rare paediatric tumour.

Recessive nebulin (NEB) mutations are a common cause of nemaline myopathy (NM), typically characterized by generalized weakness of early-onset and nemaline rods on muscle biopsy. Exceptional adult cases with additional cores and an isolated distal weakness have been reported. The large NEB gene with 183 exons has been an obstacle for the genetic work-up. Here we report a childhood-onset case with distal weakness and a core-rod myopathy, associated with recessive NEB mutations identified by next generation sequencing (NGS). This 6-year-old boy presented with a history of gross-motor difficulties following a normal early development. He had distal leg weakness with bilateral foot drop, as well as axial muscle weakness, scoliosis and spinal rigidity; additionally he required nocturnal respiratory support. Muscle magnetic resonance (MR) imaging showed distal involvement in the medial and anterior compartment of the lower leg. A muscle biopsy featured both rods and cores. Initial targeted testing identified a heterozygous Nebulin exon 55 deletion. Further analysis using NGS revealed a frameshifting 4 bp duplication, c.24372_24375dup (P.Val8126fs), on the opposite allele. This case illustrates that NEB mutations can cause childhood onset distal NM, with additional cores on muscle biopsy and proves the diagnostic utility of NGS for myopathies, particularly when large genes are implicated.

A diagnostic feature of focal cortical dysplasia (FCD) type II on magnetic resonance imaging (MRI) is increased subcortical white matter (WM) signal on T2 sequences corresponding to hypomyelination, the cause of which is unknown. We aimed to quantify WM pathology in FCD type II and any deficiency in the numbers and differentiation of oligodendroglial (OL) cell types within the dysplasia.In 19 cases we defined four regions of interests (ROIs): ROI1 = abnormal WM beneath dysplasia, ROI2 =dysplastic cortex, ROI3 = normal WM, and ROI4 = normal cortex. We quantified axonal and myelin density using immunohistochemistry for neurofilament, myelin basic protein and quantified mature OL with NogoA, cyclic nucleotide 3-phosphodiesterase (CNPase) and OL precursor cell (OPC) densities with platelet derived growth factor receptor (PDGFR)α, β and NG-2 in each region.We observed a significant reduction in myelin and axons in the WM beneath dysplasia relative to normal WM and there was a correlation between relative reduction of myelin and neurofilament in each case. OL and OPC were present in the WM beneath dysplasia and although present in lower numbers with most markers, were not significantly different from normal WM. Neurofilament and myelin labeling highlighted disorganized orientation of fibers in dysplastic cortex but there were no significant quantitative differences compared to normal cortex. Clinical correlations showed an association between the severity of reduction of myelin and axons in the WM of FCD and duration of epilepsy.These findings indicate a reduction of myelinated axons in the WM of FCD type II rather than dysmyelination as the primary pathologic process underlying WM abnormalities, possibly influenced by duration of seizures. The range of OPC to OL present in FCD type II does not implicate a primary failure of cell recruitment and differentiation of these cell types in this pathology.

PurposeMutations in POLG, the most common single-gene cause of inherited mitochondrial disease, are diagnostically challenging owing to clinical heterogeneity and overlap between syndromes. We aimed to improve the clinical recognition of POLG-related disorders in the pediatric population.MethodsWe performed a multinational, phenotype: genotype study using patients from three centers, two Norwegian and one from the United Kingdom. Patients with age at onset <12 years and confirmed pathogenic biallelic POLG mutations were considered eligible.ResultsA total of 27 patients were identified with a median age at onset of 11 months (range 0.6-80.4). The majority presented with global developmental delay (n=24/24, 100%), hypotonia (n=22/23, 96%) and faltering growth (n=24/27, 89%). Epilepsy was common, but notably absent in patients with the myocerebrohepatopathy spectrum phenotype. We identified two novel POLG gene mutations.ConclusionOur data suggest that POLG-related disease should be suspected in any child presenting with diffuse neurological symptoms. Full POLG sequencing is recommended since targeted screening may miss mutations. Finally, we simplify the classification of POLG-related disease in children using epilepsy as the crucial defining element; we show that Alpers and myocerebrohepatopathy spectrum follow different outcomes and that they manifest different degrees of respiratory chain dysfunction.

Routine childhood vaccination against measles, mumps and rubella has virtually abolished virus-related morbidity and mortality. Notwithstanding this, we describe here devastating neurological complications associated with the detection of live-attenuated mumps virus Jeryl Lynn (MuVJL5) in the brain of a child who had undergone successful allogeneic transplantation for severe combined immunodeficiency (SCID). This is the first confirmed report of MuVJL5 associated with chronic encephalitis and highlights the need to exclude immunodeficient individuals from immunisation with live-attenuated vaccines. The diagnosis was only possible by deep sequencing of the brain biopsy. Sequence comparison of the vaccine batch to the MuVJL5 isolated from brain identified biased hypermutation, particularly in the matrix gene, similar to those found in measles from cases of SSPE. The findings provide unique insights into the pathogenesis of paramyxovirus brain infections.

Juvenile idiopathic inflammatory myopathies have been recently reclassified into clinico-serological subgroups. Myopathological correlates of the subgroups are incompletely understood.We studied muscle biopsies from 101 children with clinically and serologically defined juvenile idiopathic inflammatory myopathies from the UK JDM Cohort and Biomarker Study by applying the international JDM score tool, myopathological review and C5b-9 complement analysis.Autoantibody data were available for 90/101 cases with 18/90 cases positive for anti-TIF1γ, 15/90 anti-NXP2, 11/90 anti-MDA5, 5/90 anti-Mi2 and 6/90 anti-PmScl. JDM biopsy severity scores were consistently low in the anti-MDA5 group, high in the anti-Mi2 group, and widely distributed in the other groups. Biopsies were classified histologically as perifascicular atrophy (22/101), macrophage-rich necrosis (6/101), scattered necrosis (2/101), clustered necrosis (2/101), inflammatory fibre invasion (2/101), chronic myopathic change (1/101), diffuse endomysial macrophage infiltrates (40/101) and minimal change (24/101). MDA5 cases segregated with the minimal change group and showed no capillary C5b-9-deposition. The Mi2 group displayed high severity scores and a tendency towards sarcolemmal complement deposition. NXP2 and TIF1γ groups showed a variety of pathologies with a high proportion of diffuse endomysial macrophage infiltrates and a high proportion of capillary C5b-9 deposition.We have shown that juvenile idiopathic inflammatory myopathies have a spectrum of histopathological phenotypes and show distinct complement attack complex deposition patterns. Both correlate in some cases with the serological subtypes. Most cases do not show typical histological features associated with dermatomyositis (e.g. perifascicular atrophy). In contrast, more than half show relatively mild histopathological changes.

In this study, we report three paediatric cases of Diffuse Glioneuronal Tumours with Oligodendroglioma-like features and Nuclear Clusters (DGONC).

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation.We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587.

Patients with presumed nonlesional focal epilepsy-based on either MRI or histopathologic findings-have a lower success rate of epilepsy surgery compared with lesional patients. In this study, we aimed to characterize a large group of patients with focal epilepsy who underwent epilepsy surgery despite a normal MRI and had no lesion on histopathology. Determinants of their postoperative seizure outcomes were further studied.We designed an observational multicenter cohort study of MRI-negative and histopathology-negative patients who were derived from the European Epilepsy Brain Bank and underwent epilepsy surgery between 2000 and 2012 in 34 epilepsy surgery centers within Europe. We collected data on clinical characteristics, presurgical assessment, including genetic testing, surgery characteristics, postoperative outcome, and treatment regimen.Of the 217 included patients, 40% were seizure-free (Engel I) 2 years after surgery and one-third of patients remained seizure-free after 5 years. Temporal lobe surgery (adjusted odds ratio [AOR]: 2.62; 95% CI 1.19-5.76), shorter epilepsy duration (AOR for duration: 0.94; 95% CI 0.89-0.99), and completely normal histopathologic findings-versus nonspecific reactive gliosis-(AOR: 4.69; 95% CI 1.79-11.27) were significantly associated with favorable seizure outcome at 2 years after surgery. Of patients who underwent invasive monitoring, only 35% reached seizure freedom at 2 years. Patients with parietal lobe resections had lowest seizure freedom rates (12.5%). Among temporal lobe surgery patients, there was a trend toward favorable outcome if hippocampectomy was part of the resection strategy (OR: 2.94; 95% CI 0.98-8.80). Genetic testing was only sporadically performed.This study shows that seizure freedom can be reached in 40% of nonlesional patients with both normal MRI and histopathology findings. In particular, nonlesional temporal lobe epilepsy should be regarded as a relatively favorable group, with almost half of patients achieving seizure freedom at 2 years after surgery-even more if the hippocampus is resected-compared with only 1 in 5 nonlesional patients who underwent extratemporal surgery. Patients with an electroclinically identified focus, who are nonlesional, will be a promising group for advanced molecular-genetic analysis of brain tissue specimens to identify new brain somatic epilepsy genes or epilepsy-associated molecular pathways.

Pituitary adenomas are rare in children and young people under the age of 19 (hereafter referred to as CYP) but they pose some different diagnostic and management challenges in this age group than in adults. These rare neoplasms can disrupt maturational, visual, intellectual and developmental processes and, in CYP, they tend to have more occult presentation, aggressive behaviour and are more likely to have a genetic basis than in adults. Through standardized AGREE II methodology, literature review and Delphi consensus, a multidisciplinary expert group developed 74 pragmatic management recommendations aimed at optimizing care for CYP in the first-ever comprehensive consensus guideline to cover the care of CYP with pituitary adenoma. Part 2 of this consensus guideline details 57 recommendations for paediatric patients with prolactinomas, Cushing disease, growth hormone excess causing gigantism and acromegaly, clinically non-functioning adenomas, and the rare TSHomas. Compared with adult patients with pituitary adenomas, we highlight that, in the CYP group, there is a greater proportion of functioning tumours, including macroprolactinomas, greater likelihood of underlying genetic disease, more corticotrophinomas in boys aged under 10 years than in girls and difficulty of peri-pubertal diagnosis of growth hormone excess. Collaboration with pituitary specialists caring for adult patients, as part of commissioned and centralized multidisciplinary teams, is key for optimizing management, transition and lifelong care and facilitates the collection of health-related quality of survival outcomes of novel medical, surgical and radiotherapeutic treatments, which are currently largely missing. This article is the second part of the first-ever comprehensive, two-part, consensus guideline to cover the care of children and young people under 19 years of age with pituitary adenoma. Part 2 details 57 recommendations for CYP with prolactinomas, Cushing disease, growth hormone excess, clinically non-functioning adenomas and TSHomas.

Background— GTP-cyclohydrolase 1 (GTP-CH1) catalyzes the first step for the de novo production of tetrahydrobiopterin (BH 4 ), a cofactor for nitric oxide synthase (NOS). The hyperphenylalaninemic mutant mouse (hph-1) displays a 90% reduction in GTP-CH1 activity. Reduced BH 4 decreases NOS activity and may lead to endothelial dysfunction, and there is increasing evidence that a dysfunction of the NOS pathway may be implicated in pulmonary hypertension. The aim of the study was to investigate whether reduced BH 4 in the hph-1 mouse results in a pulmonary hypertensive phenotype. Methods and Results— Morphological characterization of the heart, lung, and kidney and measurements of systemic and right ventricular blood pressures were performed in both hph-1 and wild-type mice. BH 4 and NO x levels were also measured. Hph-1 mice had significantly lower NO x and BH 4 levels, consistent with previous findings. Both morphological and in vivo data were indicative of a pulmonary but not systemic hypertensive phenotype. We observed increased right ventricle–left ventricle plus septum ratios attributable only to an increase in right ventricular mass, increased smooth muscle medial area in pulmonary resistance vessels, and significantly higher right ventricular pressures in vivo. There were no significant differences between left ventricular masses and systemic pressures, and there was no observed evidence of systemic hypertension in kidney sections between wild-type and hph-1. Conclusions— This study demonstrates that mice deficient in GTP-CH1/BH 4 display a pulmonary hypertensive but not systemic hypertensive phenotype.

We describe three unusual tumours characterized by a mixture of glial and neuronal differentiation, involvement of the posterior fossa and formation of rosettes. Mixed glial‐neuronal tumours of the posterior fossa are rare and poorly described neoplasms. However, several distinctive entities have appeared in the literature over recent years under a variety of different names. Our cases demonstrate the morphological features of the ‘rosette‐forming glioneuronal tumour of the fourth ventricle’, a recently identified tumour characterised by its unique location, neurocytic pseudo‐rosette formation and the presence of a low grade astrocytoma component. The long term prognosis of these tumours remains unclear. However, the clinical data available including the cases presented here, along with the histological features, suggest that these are low grade tumours with a good prognosis after surgical resection.

Developing sympathetic neurons depend on NGF for survival. When sympathetic neurons are deprived of NGF in vitro, a well documented series of events, including c-Jun N-terminal kinase (JNK) pathway activation, release of cytochrome c from the mitochondria, and caspase activation, culminates in the death of the neuron by apoptosis within 24-48 h. This process requires de novo gene expression, suggesting that increased expression of specific genes activates the cell death program. Using rat gene microarrays, we found that NGF withdrawal induces the expression of many genes, including mkp1, which encodes a MAPK phosphatase that can dephosphorylate JNKs. The increase in mkp1 mRNA level requires the MLK-JNK-c-Jun pathway, and we show that Mkp1 is an important regulator of JNK-dependent apoptosis in sympathetic neurons. In microinjection experiments, Mkp1 overexpression can inhibit JNK-mediated phosphorylation of c-Jun and protect sympathetic neurons from apoptosis, while Mkp1 knockdown accelerates NGF withdrawal-induced death. Accordingly, the number of superior cervical ganglion (SCG) neurons is reduced in mkp1-/- mice at P1 during the period of developmental sympathetic neuron death. We also show that c-Jun and ATF2 bind to two conserved ATF binding sites in the mkp1 promoter in vitro and in chromatin. Both of these ATF sites contribute to basal promoter activity and are required for mkp1 promoter induction after NGF withdrawal. These results demonstrate that Mkp1 is part of a negative feedback loop induced by the MLK-JNK-c-Jun signaling pathway that modulates JNK activity and the rate of neuronal death in rat sympathetic neurons following NGF withdrawal.

Epilepsy is relatively uncommon in patients with Neurofibromatosis Type 1 (NF1) and seizures are usually well controlled with antiepileptic treatment. However, pharmacoresistance has been reported in patients with NF1 and MRI evidence of malformations of cortical development or glioneuronal tumours. Available information on epilepsy surgery in NF1 is limited to a few patients with gliomas and glioneuronal tumours who underwent lesionectomies. We conducted a survey amongst 25 European epilepsy surgery centres to collect patients with NF1 who had undergone surgery for drug-resistant seizures and identified 12 patients from eight centres. MRI abnormalities were present in all patients but one. They were unilateral temporal in eight, bilateral temporal in one and multilobar or hemispheric in two. Seizures originated from the temporal lobe in ten patients, from the temporo-parieto-occipital region in one, and were bitemporal in one. One year after surgery eight patients were seizure free, one had worthwhile improvement and the remaining three had experienced no benefit. Postoperative outcome, available at 2 years in ten patients and at 5 years in three, remained stable in all but one whose seizures reappeared. Histology revealed dysembryoplastic neuroepithelial tumour (DNET) in five patients, hippocampal sclerosis in four, mixed pathology in one and polymicrogyria in one. No histological abnormality was observed in the remaining patient. Epilepsy surgery can be performed effectively in patients with NF1 provided a single and well-delimited epileptogenic zone is recognized. The high prevalence of DNETs in this series might suggest a non-fortuitous association with NF1.

Sudden infant death syndrome (SIDS) is a leading cause of postneonatal infant death in the developed world. The cause of SIDS is unknown but several hypotheses have been proposed, including the 'triple risk hypothesis', which predicts that foetal development of infants who subsequently succumb to SIDS is abnormal, leaving them unable to respond appropriately to stressors. Consistent with this hypothesis, a large number of studies have reported changes in the brain in SIDS. However, on nearly every subject, the reported findings vary widely between studies. Inconsistencies in the definitions of SIDS used and in control group selection are likely to underlie much of this variability. Therefore, in our analysis, we have included only those studies that met simple criteria for both the definition of SIDS and the control group. Of the 153 studies retrieved by our review of the literature, 42 (27%) met these criteria. Foremost among the findings reported by these studies are abnormalities of the brain stem, in particular brain stem gliosis and defects of neurotransmission in the medulla. However, these studies have not identified what could be considered in diagnostic terms a causative structural or biochemical abnormality for use in routine clinical practice. An assessment of changes in the architecture and composition of brain regions and changes in neurotransmission in multiple systems in a single, large cohort of well- and consistently characterized infants dying suddenly of a range of causes is needed before the inter-relation of these different features can be appreciated.

Ocular coloboma is a congenital defect resulting from failure of normal closure of the optic fissure during embryonic eye development. This birth defect causes childhood blindness worldwide, yet the genetic etiology is poorly understood. Here, we identified a novel homozygous mutation in the SALL2 gene in members of a consanguineous family affected with non-syndromic ocular coloboma variably affecting the iris and retina. This mutation, c.85G>T, introduces a premature termination codon (p.Glu29*) predicted to truncate the SALL2 protein so that it lacks three clusters of zinc-finger motifs that are essential for DNA-binding activity. This discovery identifies SALL2 as the third member of the Drosophila homeotic Spalt-like family of developmental transcription factor genes implicated in human disease. SALL2 is expressed in the developing human retina at the time of, and subsequent to, optic fissure closure. Analysis of Sall2-deficient mouse embryos revealed delayed apposition of the optic fissure margins and the persistence of an anterior retinal coloboma phenotype after birth. Sall2-deficient embryos displayed correct posterior closure toward the optic nerve head, and upon contact of the fissure margins, dissolution of the basal lamina occurred and PAX2, known to be critical for this process, was expressed normally. Anterior closure was disrupted with the fissure margins failing to meet, or in some cases misaligning leading to a retinal lesion. These observations demonstrate, for the first time, a role for SALL2 in eye morphogenesis and that loss of function of the gene causes ocular coloboma in humans and mice.

Pilocytic astrocytomas are slow-growing tumors that usually occur in the cerebellum or in the midline along the hypothalamic/optic pathways. The most common genetic alterations in pilocytic astrocytomas activate the ERK/MAPK signal transduction pathway, which is a major driver of proliferation but is also believed to induce senescence in these tumors. Here, we have conducted a detailed investigation of microRNA and gene expression, together with pathway analysis, to improve our understanding of the regulatory mechanisms in pilocytic astrocytomas. Pilocytic astrocytomas were found to have distinctive microRNA and gene expression profiles compared to normal brain tissue and a selection of other pediatric brain tumors. Several microRNAs found to be up-regulated in pilocytic astrocytomas are predicted to target the ERK/MAPK and NF-κB signaling pathways as well as genes involved in senescence-associated inflammation and cell cycle control. Furthermore, IGFBP7 and CEBPB, which are transcriptional inducers of the senescence-associated secretory phenotype (SASP), were also up-regulated together with the markers of senescence and inflammation, CDKN1A (p21), CDKN2A (p16) and IL1B. These findings provide further evidence of a senescent phenotype in pilocytic astrocytomas. In addition, they suggest that the ERK/MAPK pathway, which is considered the major driver of these tumors, is regulated not only by genetic aberrations but also by microRNAs.

Mutations in the nuclear gene POLG (encoding the catalytic subunit of DNA polymerase gamma) are an important cause of mitochondrial disease. The most common POLG mutation, A467T, appears to exhibit considerable phenotypic heterogeneity. The mechanism by which this single genetic defect results in such clinical diversity remains unclear. In this study we evaluate the clinical, neuropathological and mitochondrial genetic features of four unrelated patients with homozygous A467T mutations. One patient presented with the severe and lethal Alpers-Huttenlocher syndrome, which was confirmed on neuropathology, and was found to have a depletion of mitochondrial DNA (mtDNA). Of the remaining three patients, one presented with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), one with a phenotype in the Myoclonic Epilepsy, Myopathy and Sensory Ataxia (MEMSA) spectrum and one with Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO). All three had secondary accumulation of multiple mtDNA deletions. Complete sequence analysis of muscle mtDNA using the MitoChip resequencing chip in all four cases demonstrated significant variation in mtDNA, including a pathogenic MT-ND5 mutation in one patient. These data highlight the variable and overlapping clinical and neuropathological phenotypes and downstream molecular defects caused by the A467T mutation, which may result from factors such as the mtDNA genetic background, nuclear genetic modifiers and environmental stressors.

Focal cortical dysplasias (FCDs) are a range of malformations of cortical development each with specific histopathological features. Conventional radiological assessment of standard structural MRI is useful for the localization of lesions but is unable to accurately predict the histopathological features. Quantitative MRI offers the possibility to probe tissue biophysical properties in vivo and may bridge the gap between radiological assessment and ex-vivo histology. This review will cover histological, genetic and radiological features of FCD following the ILAE classification and will explain how quantitative voxel- and surface-based techniques can characterise these features. We will provide an overview of the quantitative MRI measures available, their link with biophysical properties and finally the potential application of quantitative MRI to the problem of FCD subtyping. Future research linking quantitative MRI to FCD histological properties should improve clinical protocols, allow better characterisation of lesions in vivo and tailored surgical planning to the individual.

To evaluate the relationship between expression of myxovirus-resistance protein A (MxA) protein on muscle biopsies by immunohistochemistry and disease activity in juvenile dermatomyositis (JDM) patients. Also, another aim was to investigate whether the expression of MxA is related with myositis-specific autoantibodies (MSA) status in JDM patients.103 patients (median aged 6.3, interquartile range 0.5-15.9) enrolled in the Juvenile Dermatomyositis Cohort and Biomarker Study (JDCBS). Muscle biopsies were stained with MxA and scored. Clinical data at initial presentation were collected and autoantibodies were analysed. Multiple linear regression analysis was performed to estimate the association between MxA expression on muscle fibres and muscle disease activity, and MSA status.Expression of MxA protein on JDM samples was identified in 61.2%. There was a significant association between MxA scores and Childhood Myositis Assessment Scale (CMAS) (P = 0.002), and Manual Muscle Testing of Eight Muscles (MMT8) (P = 0.026). CMAS and MMT8 scores were significantly lower in the group of patients with strong MxA expression. MxA scores differed according to MSA subgroups (P = 0.002). Patients with positive nuclear matrix protein 2 autoantibodies had strong MxA expression, whereas anti-melanoma differentiation-associated gene 5 positive patients had no or weak MxA expression.This study reveals the significant association between level of MxA expression on muscle fibres and clinical measures of muscular disease activity in JDM patients and MSA status. This confirms type I interferonopathies in muscle fibres of JDM patients which could help with improving treatment outcome in JDM patients and underscoring the distinct pathophysiological pathways in different MSA status.

Tissue invasion and infiltration by brain tumours poses a clinical challenge, with destruction of structures leading to morbidity. We assessed whether micro-CT could be used to map tumour invasion in adamantinomatous craniopharyngioma (ACP), and whether it could delineate ACPs and their intrinsic components from surrounding tissue.Three anonymised archival frozen ACP samples were fixed, iodinated and imaged using a micro-CT scanner prior to the use of standard histological processing and immunohistochemical techniques.We demonstrate that micro-CT imaging can non-destructively give detailed 3D structural information of tumours in volumes with isotropic voxel sizes of 4-6 microns, which can be correlated with traditional histology and immunohistochemistry.Such information complements classical histology by facilitating virtual slicing of the tissue in any plane and providing unique detail of the three dimensional relationships of tissue compartments.

Embryonal tumours of the central nervous system ( CNS ) present a significant clinical challenge. Many of these neoplasms affect young children, have a very high mortality and therapeutic strategies are often aggressive with poor long‐term outcomes. There is a great need to accurately diagnose embryonal tumours, predict their outcome and adapt therapy to the individual patient's risk. For the first time in 2016, the WHO classification took into account molecular characteristics for the diagnosis of CNS tumours. This integration of histological features with genetic information has significantly changed the diagnostic work‐up and reporting of tumours of the CNS . However, this remains challenging in embryonal tumours due to their previously unaccounted tumour heterogeneity. We describe the recent revisions made to the 4th edition of the WHO classification of CNS tumours and review the main changes, while highlighting some of the more common diagnostic testing strategies.

Neurometabolic disorders are markedly heterogeneous, both clinically and genetically, and are characterized by variable neurological dysfunction accompanied by suggestive neuroimaging or biochemical abnormalities. Despite early specialist input, delays in diagnosis and appropriate treatment initiation are common. Next-generation sequencing approaches still have limitations but are already enabling earlier and more efficient diagnoses in these patients. We designed a gene panel targeting 614 genes causing inborn errors of metabolism and tested its diagnostic efficacy in a paediatric cohort of 30 undiagnosed patients presenting with variable neurometabolic phenotypes. Genetic defects that could, at least partially, explain observed phenotypes were identified in 53% of cases. Where biochemical abnormalities pointing towards a particular gene defect were present, our panel identified diagnoses in 89% of patients. Phenotypes attributable to defects in more than one gene were seen in 13% of cases. The ability of in silico tools, including structure-guided prediction programmes to characterize novel missense variants were also interrogated. Our study expands the genetic, clinical and biochemical phenotypes of well-characterized (POMGNT1, TPP1) and recently identified disorders (PGAP2, ACSF3, SERAC1, AFG3L2, DPYS). Overall, our panel was accurate and efficient, demonstrating good potential for applying similar approaches to clinically and biochemically diverse neurometabolic disease cohorts.

Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors. Presently, MRI is the mainstay of disease diagnosis and surveillance. We identify clinically significant computational features from MRI and create a prognostic machine learning model.We isolated tumor volumes of T1-post-contrast (T1) and T2-weighted (T2) MRIs from 177 treatment-naïve DIPG patients from an international cohort for model training and testing. The Quantitative Image Feature Pipeline and PyRadiomics was used for feature extraction. Ten-fold cross-validation of least absolute shrinkage and selection operator Cox regression selected optimal features to predict overall survival in the training dataset and tested in the independent testing dataset. We analyzed model performance using clinical variables (age at diagnosis and sex) only, radiomics only, and radiomics plus clinical variables.All selected features were intensity and texture-based on the wavelet-filtered images (3 T1 gray-level co-occurrence matrix (GLCM) texture features, T2 GLCM texture feature, and T2 first-order mean). This multivariable Cox model demonstrated a concordance of 0.68 (95% CI: 0.61-0.74) in the training dataset, significantly outperforming the clinical-only model (C = 0.57 [95% CI: 0.49-0.64]). Adding clinical features to radiomics slightly improved performance (C = 0.70 [95% CI: 0.64-0.77]). The combined radiomics and clinical model was validated in the independent testing dataset (C = 0.59 [95% CI: 0.51-0.67], Noether's test P = .02).In this international study, we demonstrate the use of radiomic signatures to create a machine learning model for DIPG prognostication. Standardized, quantitative approaches that objectively measure DIPG changes, including computational MRI evaluation, could offer new approaches to assessing tumor phenotype and serve a future role for optimizing clinical trial eligibility and tumor surveillance.

We have sequenced and defined the expression during rat embryogenesis of the protocadherin fat, the murine homologue of a Drosophila tumour suppressor gene. As previously described for human fat, the sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the b-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. No labelling was seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat.

Disease-causing mutations affecting either one of the transcription factor genes, PITX2 or FOXC1, have been previously identified in patients with Axenfeld-Rieger syndrome (AR). We identified a family who segregate novel mutations in both PITX2 (p.Ser233Leu) and FOXC1 (c.609delC). The most severely affected individual, who presented with an atypical phenotype of corneal opacification, lens extrusion, persistent hyperplastic primary vitreous (PHPV), and subsequent bilateral retinal detachment, inherited mutations in both genes, whereas the single heterozygous mutations caused mild AR phenotypes. This is the first report of such digenic inheritance. By analyzing cognate targets of each gene, we showed that FOXC1 and PITX2 can independently regulate their own and each other's target gene promoters and do not show synergistic action in vitro. Mutation in either gene caused reduced transcriptional activation to different extents on the FOXO1 and PLOD1 promoters, whereas both mutations in combination showed the lowest level of activation. These data show how the compensatory activity of one factor, when the other is impaired, may lessen the phenotypic impact of developmental anomalies, yet reduced activity of both transcription factors increased disease severity. This suggests an under-reported mechanism for phenotypic variability whereby single mutations cause mild AR phenotypes, whereas digenic inheritance increases phenotypic severity. Hum Mutat 32:1144–1152, 2011. ©2011 Wiley-Liss, Inc.

Aim The aim of the study was to describe seizure outcome following surgery for focal extratemporal epilepsy and identify factors associated with prolonged postsurgical freedom from seizures. Method In this retrospective cohort study, children with drug‐resistant focal extratemporal epilepsy were treated surgically and followed up in a single tertiary care centre between 1997 and 2008. Results Eighty children were identified for inclusion in the study (42 males, 38 females; median age 9y 1mo, range 3mo–18y 7mo). The aetiology was identified as focal cortical dysplasia ( n =37), low‐grade tumour ( n =22), tuberous sclerosis ( n =9), or non‐specific ( n =12). Children were followed for a median of 3 years 1 month (range 8mo–10y 7mo) after surgery. Overall, at last follow‐up, 50% of the children had been completely seizure free since surgery (Engel class Ia); of these 40 individuals, 15 had discontinued all antiepileptic drugs. Several presurgical factors were associated with a favourable outcome. However, after controlling for confounding factors, aetiology appeared to be the only determinant of long‐term seizure outcome as non‐specific lesion pathology was associated with seizure recurrence (hazard ratio 10.43; 95% confidence interval 3.26–33.39). Interpretation In 50% of cases, children with surgically treated drug‐resistant extratemporal epilepsies have an excellent long‐term outcome. The aetiology of the epileptogenic lesion appears to be the only significant determinant of surgical outcome in this population of children. It is difficult to correctly identify non‐specific pathology on presurgical magnetic resonance imaging.

Abstract Brain tumors are thought to originate from stem/progenitor cell populations that acquire specific genetic mutations. Although current preclinical models have relevance to human pathogenesis, most do not recapitulate the histogenesis of the human disease. Recently, a large series of human gliomas and medulloblastomas were analyzed for genetic signatures of prognosis and therapeutic response. Using a mouse model system that generates three distinct types of intrinsic brain tumors, we correlated RNA and protein expression levels with human brain tumors. A combination of genetic mutations and cellular environment during tumor propagation defined the incidence and phenotype of intrinsic murine tumors. Importantly, in vitro passage of cancer stem cells uniformly promoted a glial expression profile in culture and in brain tumors. Gene expression profiling revealed that experimental gliomas corresponded to distinct subclasses of human glioblastoma, whereas experimental supratentorial primitive neuroectodermal tumors (sPNET) correspond to atypical teratoid/rhabdoid tumor (AT/RT), a rare childhood tumor. Cancer Res; 73(18); 5834–44. ©2013 AACR.