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Background Relapsed ependymoma has a dismal prognosis, and the role of chemotherapy at relapse remains unclear. This study prospectively evaluated the efficacy of intensive intravenous (IV) etoposide in patients less than 21 years of age with relapsed intracranial ependymoma (NCT00278252). Methods This was a single-arm, open-label, phase II trial using Gehan’s two-stage design. Patients received IV etoposide 100 mg/m2 on days 1-3, 8-10, and 15-17 of each 28-day cycle, up to maximum of 6 cycles. Primary outcome was radiological response after 3 cycles. Pharmacokinetic analysis was performed in 10 patients. Results Twenty-five patients were enrolled and included in the intention-to-treat (ITT) analysis. Three patients were excluded in per-protocol (PP) analysis. After 3 cycles of etoposide, 5 patients (ITT 20%/PP 23%) had a complete response (CR), partial response (PR), or objective response (OR). Nine patients (ITT 36%/PP 41%,) had a best overall response of CR, PR, or OR. 1-year PFS was 24% in ITT and 23% in PP populations. 1-year OS was 56% and 59%, 5-year OS was 20% and 18%, respectively, in ITT and PP populations. Toxicity was predominantly hematological, with 20/25 patients experiencing a grade 3 or higher hematological adverse event. Conclusions This study confirms the activity of IV etoposide against relapsed ependymoma, however, this is modest, not sustained, and similar to that with oral etoposide, albeit with increased toxicity. These results confirm the dismal prognosis of this disease, provide a rationale to include etoposide within drug combinations, and highlight the need to develop novel treatments for recurrent ependymoma.

Summary The role of the homeobox transcriptional repressor HESX1 in embryonic stem cells (ESCs) remains mostly unknown. Here, we show that Hesx1 is expressed in the preimplantation mouse embryo, where it is required during developmental diapause. Absence of Hesx1 leads to reduced expression of epiblast and primitive endoderm determinants and failure of diapaused embryos to resume embryonic development after implantation. Genetic deletion of Hesx1 impairs self-renewal and promotes differentiation toward epiblast by reducing the expression of pluripotency factors and decreasing the activity of LIF/STAT3 signaling. We reveal that Hesx1-deficient ESCs show elevated ERK pathway activation, resulting in accelerated differentiation toward primitive endoderm, which can be prevented by overexpression of Hesx1. Together, our data provide evidence for a novel role of Hesx1 in the control of self-renewal and maintenance of the undifferentiated state in ESCs and mouse embryos., Highlights • Hesx1-deficient embryos fail to reinitiate normal development after diapause • Hesx1 expression in ESCs is controlled by core pluripotency factors • Hesx1 regulates ESC differentiation toward epiblast and primitive endoderm • Hesx1 promotes LIF/STAT3 signaling and inhibits ERK signaling, Pozzi and colleagues have used Hesx1-defficient ESCs to reveal that Hesx1 expression is regulated by intrinsic and extrinsic pluripotency-stimulating signals. They show that Hesx1 promotes ESC self-renewal and inhibits ESC differentiation toward both the epiblast and primitive endoderm cell fates. They report the expression of Hesx1 in preimplantation embryos, where it is required to resume normal embryonic development following diapause.

Aims The TJBM Platform Study (https://www.birmingham.ac.uk/research/crctu/trials/brain-matrix) is a programme of work aimed at improving the knowledge of, and treatment for, glioma. The study will develop a backbone infrastructure for a molecular diagnostic pathway, particularly epigenetic classification and whole genome sequencing (WGS), and a data-repository of disease imaging, including real-time centralised Response Assessment in Neuro-Oncology (RANO) review, treatment and outcome data. Developed with the involvement of patients and relatives, patient reported outcome measures (PROMs), particularly quality of life assessment will be collected for patients. We present the feasibility data collected from the initial ten UK centres selected to participate in the TJBM study. This presents an opportunity to understand current local neuro-oncology practice, identify differences between services and chances to optimise these, bearing in mind the heterogeneity in our patient populations, staff and hospital facilities. Method Ten UK centres were selected to participate in the TJBM platform study, and each centre completed a multi-disciplinary feasibility questionnaire to facilitate participation and collaboration across the centres. Data were collected from hospital electronic MDT (Multi-Disciplinary Team) records, clinic letters, operative and imaging notes, MDT or personal experience. Continuous variables were reported using medians and ranges due to the non-normality of the data and categorical variables were reported as numbers and percentages. Tables and bar charts were generated to display relevant data. Analysis and plots were generated using Microsoft Excel sheets and SPSS (IBM) version 26. Results Work load Between 2016-2018 service provision redistribution reflects a trend towards higher volume centres. Overall, glioma workload within ten TJBM centres has remained stable (Figure 1). Imaging All TJBM centres have good access to imaging techniques and neuroradiology expertise, including relevant ‘advanced’ imaging. All have RANO capability, although not widely used clinically. Neurosurgery All centres have access to 5 ALA, perform awake craniotomy for language assessment and motor/sensory mapping are typically performed asleep, with subtle variation in techniques. Pathology Despite molecular analysis advances, current practice is limited to the evaluation of formalin embedded tissue by traditional morphology/ immuno-histochemical staining, with limited targeted testing of specific genetic changes (Figure 2). Clinical oncology Oncology treatments for glioma were as per NICE guidance with some minor local variation. The data has informed the development of the TJBM protocol, an overview of which will be presented (Figure 3). Conclusion Through systematic real-world data collection the TJBM platform study will provide a detailed understanding of practice within the UK, linked to molecular tumour genotype, treatment response outcome measures, and also regular quality of life assessments. Use of the platform infrastructure, will facilitate trials and add-on observational and biological studies to obtain rapid, efficient, and cost-effective data collection, and integrate findings with comprehensive molecular biological profiling and radiological features, thereby reducing the time and administrative burdens in trial delivery. This infrastructure will help establish a trial-competent network on which future research and collaborations can be based. Academic and industry partners will be able to use the TJBM platform through collaboration, overseen by a strong governance framework. This will maximise the opportunities and abilities to translate advances into trials and patient benefit.

Over the last decade, the development of mouse models, coupled with advances in profiling of human tumour samples, has greatly advanced our understanding of the biology of craniopharyngioma. These studies have revealed the critical pathways underlying tumourigenesis, thus revealing potential novel targeted therapies against these tumours. In addition, the murine models have provided unexpected insights into the role of SOX2 pituitary stem cells in tumour formation, which challenge more traditional theories, and suggest that cellular senescence is playing an instructive tumour-inducing role. Additionally, mouse models are ideal platforms to test novel agents against craniopharyngioma in preclinical trials. In this chapter, we will discuss these findings with specific focus on genetically engineered mouse models (GEMMS) and patient-derived xenograft (PDX) models of craniopharyngioma.

Senescent cells activate genetic programmes that irreversibly inhibit cellular proliferation, but also endow these cells with distinctive metabolic and signalling phenotypes. Although senescence has historically been considered a protective mechanism against tumourigenesis, the activities of senescent cells are increasingly being associated with age-related diseases, including cancer. An important feature of senescent cells is the secretion of a vast array of pro-inflammatory cytokines, chemokines, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Recent research has shown that SASP paracrine signalling can mediate several pro-tumourigenic effects, such as enhancing malignant phenotypes and promoting tumour initiation. In this review, we summarise the paracrine activities of senescent cells and their role in tumourigenesis through direct effects on growth and proliferation of tumour cells, tumour angiogenesis, invasion and metastasis, cellular reprogramming and emergence of tumour-initiating cells, and tumour interactions with the local immune environment. The evidence described here suggests cellular senescence acts as a double-edged sword in cancer pathogenesis, which demands further attention in order to support the use of senolytic or SASP-modulating compounds for cancer treatment.

OVERVIEW The TJBM Platform Study (https://www.birmingham.ac.uk/research/crctu/trials/brain-matrix) is a programme of work aimed at improving the knowledge of, and treatment for, glioma. We present the feasibility data collected from the initial ten UK centres. METHOD The UK TJBM centres completed a multi-disciplinary feasibility questionnaire to facilitate participation and collaboration across centres. Data were collected from hospital electronic board review records, clinic letters, operative and imaging notes, MDT or personal experience. RESULTS Work load: Between 2016-2018 service provision redistribution reflects a trend towards higher volume centres. Overall, glioma workload within ten TJBM centres has remained stable. Imaging: All TJBM centres have good access to imaging techniques and neuroradiology expertise, including relevant ‘advanced’ imaging. All have RANO capability, although not widely used clinically. Neurosurgery: All centres have access to 5 ALA, perform awake craniotomy for language assessment and motor/sensory mapping are typically performed asleep, with subtle variation in techniques. Pathology: Despite molecular analysis advances, current practice is limited to the evaluation of formalin embedded tissue by traditional morphology/ immunohistochemically staining, with limited targeted testing of specific genetic changes. Clinical oncology: Oncology treatments for glioma were as per NICE guidance with some minor local variation. A relative lack of linking treatments to detailed clinical, treatment, toxicity, and quality of life data making communication of significance of findings to patients challenging. CONCLUSION Through systematic real-world data collection the TJBM platform study will provide a detailed understanding of practice within the UK, linked to molecular tumour genotype, treatment response outcome measures, and regular quality of life assessments. This infrastructure will help establish a trial-competent network for future collaborative research. Academic and industry partners will be able to use the TJBM platform through collaboration, overseen by a strong governance framework. This will maximise the opportunities and abilities to translate advances into trials and patient benefit.

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.

Adamantinomatous craniopharyngioma (ACP) is the commonest tumor of the sellar region in childhood. Two genetically engineered mouse models have been developed and are giving valuable insights into ACP biology. These models have identified novel pathways activated in tumors, revealed an important function of paracrine signalling and extended conventional theories about the role of organ-specific stem cells in tumorigenesis. In this review, we summarize these mouse models, what has been learnt, their limitations and open questions for future research. We then discussed how these mouse models may be used to test novel therapeutics against potentially targetable pathways recently identified in human ACP.

In 2016 there were 5250 brain cancer deaths in the UK. Standard treatment is surgical resection followed by chemo-radiotherapy. In most cases of diffuse glioma, complete tumour resection is not feasible. Many chemotherapy drugs have untested penetration through the blood brain barrier, potentially leading to sub-therapeutic concentrations in the tumour. There is need to refine current treatment strategies in relation to the understanding of tumour biology, and rapidly introduce and evaluate novel therapeutic approaches and agents through delivering rigorous clinical trials. The TESSA JOWELL BRAIN MATRIX Study will evaluate the feasibility of delivering precision medicine for brain cancer patients within the NHS. A multicentre, platform feasibility study of 1200 patients with diffuse glioma will build on the 100,000 genome project to develop and evaluate an infrastructure to collect and integrate: 1) real time comprehensive integrated molecular analysis, including whole genome sequencing and epigenetic classification; 2) serial sampling and annotation of tumours; 3) collection of matched clinical data; 4) assessment of patient quality of life; 5) centralised radiological review and response assessment as per RANO criteria. Once developed this will allow rapid introduction of therapeutic trials to specific patient groups. Secondary objectives include: understanding the association between extent of resection and molecular stratification to refine the role of surgery; optimisation and harmonisation of protocols to best collect, manage and store tissue, clinical data, and radiological images in order to provide a resource for researchers, both within and outside of the study. Improve patient recruitment by identifying and removing recruitment barriers and improve the information and consent processes for patients. Promote the development of a national network with expertise in brain cancer. Enrolment of the first patient is expected in late 2019. For further information, please contact the Brain Matrix Trial Office BrainMatrix@trials.bham.ac.uk.

This manuscript describes the study of two cases of craniopharyngioma, which have been examined repeatedly over three separate centuries. This includes analysis by Josef Engel in 1839, who sought to uncover the physiological role of the pituitary gland; Jacob Erdheim in 1904, who initially described the disease we now call craniopharyngioma, and recent high resolution MRI and micro-CT imaging and attempted DNA analyses of the tumours. The cases highlight how, rightly or wrongly, our interpretation of data is shaped by the technologies, methodologies and prevailing theories of a given time.

In 2006 we reported the youngest case of a large clival chordoma, a 15-week old baby, the second case to present without skull base involvement and the fourth case of chordoma in a patient with tuberous sclerosis. This unusually rare case (surgically un-resect able) underwent endoscopic skull-base diagnostic biopsy and a novel chemotherapy regime that aimed to control his disease[i]. Initial tumour control was achieved with chemotherapy (Ifosfamide, doxorubicin with dexrazonane, intrathecal hydrocortisone, methotrexate, cytarabine). Carboplatin and etoposide were later given for a further year. Following this, Sirolimus and imatinib were used for another twelve months due to primary tumour regrowth and three new skull-vault lesions. Sirolimus alone was continued for an additional year, but stopped due to optic neuritis. Imatinib was given until further progression two years later, leading to a change to everolimus. Surgery for the ventral foramen magnum was performed a year later. The patient received further surgery and radiotherapy for tumour recurrence. Sadly the tumour metastasised and he succumbed at age 13. Chordomas are aggressive and recur frequently. Complete primary resection followed by radiotherapy/proton beam therapy offers the best chance of cure but is not an option in infants with giant lesions, as in our case. We inform on alternative targeted treatment strategies and review the literature on these rare lesions. [i] Kambogiorgas D, St George EJ, Chapman S, English M, Solanki G: Infantile Clivus chordoma without clivus involvement: Case report and review of the literature, Childs Nerv System (2006) 22:1369–1374

Sonic hedgehog (SHH) is an essential morphogenetic signal dictating cell fate decisions in several developing organs in mammals. In vitro data suggest that SHH is required to specify LHX3+/LHX4+ Rathke's pouch (RP) progenitor identity. However, in vivo studies have failed to reveal such a function, supporting instead, a critical role for SHH in promoting proliferation of these RP progenitors and for differentiation of pituitary cell types. Here, we have used a genetic approach to demonstrate that activation of the SHH pathway is necessary to induce LHX3+/LHX4+ RP identity in mouse embryos. First, we show that conditional deletion of Shh in the anterior hypothalamus results in a fully penetrant phenotype characterised by a complete arrest of RP development, with lack of Lhx3/Lhx4 expression in RP epithelium at 9.0 dpc (days post coitum) and total loss of pituitary tissue by 12.5 dpc. Conversely, over-activation of the SHH pathway by conditional deletion of Ptch1 in RP progenitors leads to severe hyperplasia and enlargement of the Sox2+ve stem cell compartment by the end of gestation.

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. Electronic supplementary material The online version of this article (10.1007/s00401-018-1830-2) contains supplementary material, which is available to authorized users.

Background and Purpose: Wilms tumor (WT) is the commonest pediatric renal malignancy. A significant minority of cases have mutations in known genes or recurrent copy number aberrations, but few of these have prognostic value. We have previously observed MYCN copy number gain in WT, an aberration associated with poor outcome in several other childhood cancers. We therefore set out to assess the prognostic significance of MYCN gain in a large WT series, and to explore other mechanisms by which the function of this gene may be dysregulated. Methods: MYCN copy number was measured in 293 tumours by Multiplex ligation-dependent probe amplification. Whole exome sequencing (Agilent SureSelect V4+UTR/Illumina HiSeq) was carried out on 51 WT tumour/germline pairs. Sanger sequencing was used to confirm variants of interest and to assess an independent panel of 168 WTs. 65 WTs were profiled on Affymetrix SNP arrays (250K Nsp or SNP 6.0), Illumina HumanMethylation450 arrays, and Illumina HumanHT-12 expression arrays, and additional selected tumors on Illumina HumanCytoSNP-12 arrays. Results: MYCN gain is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. A somatic p.P44L (c.131C>t) mutation was detected in 3/45 exomes that gave informative data at this position, and in 5/168 additional tumors screened for this variant, an overall frequency of 3.8%. MYCN overexpression in high risk tumors was associated with hypomethylation at specific loci. We observed parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumors of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolved over time. In a second bilateral case, we detected MYCN gain as a germline aberration. Conclusions: MYCN gain is associated with poor outcome and anaplastic histology in WT. MYCN also appears to be disrupted by other mechanisms in WT, including DNA hypomethylation and a recurrent potential gain of function mutation, P44L. The discovery of a germline aberration suggests that MYCN dysregulation may contribute to tumor predisposition. Citation Format: Richard D. Williams, Tasnim Chagtai, Marisa Alcaide-German, John Apps, Jenny Wegert, Sergey Popov, Gordan Vujanic, Harm van Tinteren, Marry M. van den Heuvel-Eibrink, Marcel Kool, David Gisselsson, Norbert Graf, Manfred Gessler, Kathy Pritchard-Jones. Multiple mechanisms of MYCN dysregulation in Wilms tumor. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-59.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00401-017-1773-z) contains supplementary material, which is available to authorized users.

BACKGROUND: Nephrogenic rests (NRs) are abnormally persistent foci of embryonal cells, thought to be the precursor lesion of Wilms tumors (WTs). To date, their presence has not been systematically examined in WTs treated with preoperative chemotherapy. METHODS: A systematic analysis of the data on NRs in WTs treated with preoperative chemotherapy obtained from the UK cohort of the International Society of Pediatric Oncology (SIOP) WT 2001 Trial. The study was based on central pathology review of full sets of slides from pathological specimens, with a median of 28 slides reviewed per case. RESULTS: NRs were identified in 40% of unilateral WTs, including 25% perilobar nephrogenic rest (PLNR), 9% intralobar nephrogenic rest (ILNR), 5% both PLNR and ILNR, and 1% nephroblastomatosis, and in 93% of cases with bilateral lesions. ILNRs were associated with stromal histology and a younger age at diagnosis and found frequently in patients with congenital anomalies associated with WT1 mutation. PLNRs were found frequently in patients with overgrowth syndromes. CONCLUSIONS: The prevalence of NRs in WTs after preoperative chemotherapy observed in SIOP UK WT 2001 Trial is similar to the previously published data on NRs not treated with preoperative chemotherapy. Their epidemiology supports at least two pathways to Wilms tumorigenesis.

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+ve 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+ve cells with sustained proliferative capacity and disrupted pituitary differentiation. Together, our data demonstrate a critical function of the MAPK pathway in controlling the balance between proliferation and differentiation of Sox2+ve cells and suggest that persistent proliferative capacity of Sox2+ve cells may underlie the pathogenesis of PCP.

Since the first identification of CTNNB1 mutations in adamantinomatous craniopharyngioma (ACP), much has been learned about the molecular pathways and processes that are disrupted in ACP pathogenesis. To date this understanding has not translated into tangible patient benefit. The recent development of novel techniques and a range of preclinical models now provides an opportunity to begin to support treatment decisions and develop new therapeutics based on molecular pathology. In this review the authors summarize many of the key findings and pathways implicated in ACP pathogenesis and discuss the challenges that need to be tackled to translate these basic science findings for the benefit of patients.

Childhood malignancies are relatively poorly studied in terms of tumour/host interaction. Using tissue arrays of childhood cancers, we analysed immunohistochemical staining for CD68, CD3 and FOXP3 to evaluate infiltration of myeloid cells, lymphocytes and regulatory T cells. Staining for phosphorylated STAT3 was performed in a subset. The majority of paediatric tumours demonstrated a marked infiltration of CD68+ myeloid cells but, with the exception of neuroblastoma, most showed only sparse infiltration of CD3+/ FOXP3- cells. There was evidence for activation of STAT3 in pPNET (50%), ependymoma (45%) and undifferentiated sarcoma (38%), but it was rarely activated in other tumours.

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. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0321-8) contains supplementary material, which is available to authorized users.

In 100 primary colorectal carcinomas, we demonstrate by array comparative genomic hybridization (aCGH) that 33% show DNA copy number (DCN) loss involving PARK2 , the gene encoding PARKIN, the E3 ubiquitin ligase whose deficiency is responsible for a form of autosomal recessive juvenile parkinsonism. PARK2 is located on chromosome 6 (at 6q25–27), a chromosome with one of the lowest overall frequencies of DNA copy number alterations recorded in colorectal cancers. The PARK2 deletions are mostly focal (31% ∼0.5 Mb on average), heterozygous, and show maximum incidence in exons 3 and 4. As PARK2 lies within FRA6E, a large common fragile site, it has been argued that the observed DCN losses in PARK2 in cancer may represent merely the result of enforced replication of locally vulnerable DNA. However, we show that deficiency in expression of PARK2 is significantly associated with adenomatous polyposis coli ( APC ) deficiency in human colorectal cancer. Evidence of some PARK2 mutations and promoter hypermethylation is described. PARK2 overexpression inhibits cell proliferation in vitro. Moreover, interbreeding of Park2 heterozygous knockout mice with Apc Min mice resulted in a dramatic acceleration of intestinal adenoma development and increased polyp multiplicity. We conclude that PARK2 is a tumor suppressor gene whose haploinsufficiency cooperates with mutant APC in colorectal carcinogenesis.

BACKGROUND: Adamantinomatous Craniopharyngioma (ACP) is associated with considerable short and long-term morbidity. There are currently no well-established directed therapies. Recent studies demonstrated pro-inflammatory characteristics in both the solid and cyst compartments, but no data have examined whether immunosuppressive mechanisms are active in these tumors. METHODS: This study utilized a variety of platforms to examine immunosuppressive markers in cyst fluid and associated solid tumor components of ACP. Analyte levels in ACP were compared with other common pediatric brain tumors and normal tissue in order to identify immunosuppressive factors that were overexpressed in ACP. Cyst fluid and cells contained therein were analyzed using multiplex cytokine analysis and flow cytometry, respectively. A large transcriptomic database of ACP and other pediatric tumors and normal brain was queried for immunosuppressive gene expression. Immunohistochemistry was used to further validate gene expression data. RESULTS: ACP demonstrated highly elevated levels of IDO-1 (FC 9.43 versus all other tumor/tissue types, p=1.4x10-28), confirmed by IHC staining for IDO-1 in the epithelial tumor compartment. Elevated levels of IL-10 were demonstrated in cyst fluid and solid tumor compartments. Myeloid cells from the ACP cyst compartment demonstrated low levels of CD64 and HLA-DR expression, combined with high levels of CD163 expression. This pattern is most consistent with an immunosuppressive, pro-tumor milieu. Both CD4 and CD8(+) T-cells demonstrated positive staining for PD-1, most consistent with an exhausted phenotype. Consistent with this, PD-L1 mRNA was elevated in ACP tumor samples versus many other tumor types. CONCLUSIONS: Pediatric ACP is characterized by immunosuppressive factors in both the solid and cyst fluid compartments. This finding must be further considered within the context of the pro-inflammatory characteristics that have been previously described. It further raises the prospect of clinical translation through the application of available immune-directed therapies, in particular those that elicit therapeutic benefit through reversal of immunosuppression.

Background Adamantinomatous craniopharyngiomas (ACP) are histologically benign but clinically aggressive tumours that lead to hypothalamic injury, visual impairment, and pituitary dysfunction. Histologically they consist of several compartments, and most cases have somatic CTNNB1 -activating mutations. Similar functional activation of CTNNB1 in the developing murine pituitary gland results in murine ACP, study of which has given insight into human ACP (eg, showing activation of paracrine sonic hedgehog [SHH] signalling). We aimed to explore the genetic and transcriptional landscape of human ACP and test the functional significance of SHH signalling in vivo. Methods RNA sequencing and targeted next generation DNA sequencing was done in ACP samples from local and national biobanks. Weighted gene correlation network analysis (WGCNA) was used to derive modules of coexpressed genes, and results were further refined with sequencing of laser capture microdissected tissue. A preclinical vehicle (n=11) controlled trial of the SHH pathway inhibitor GDC0449 (n=12) was performed in the embryonic mouse model of ACP. Findings Next generation sequencing of laser capture microdissected tissue confirmed heterozygous CTNNB1 mutation in all tumour compartments but not reactive tissue. WGCNA identified expression signatures of different tissue components (eg, tumour epithelium, reactive tissue, and immune infiltrate) and identified novel ACP genes (eg, BCL11B ). The tumour epithelium showed high expression of ameloblast transcription factors (eg, MSX2 ), and expression of enamel proteins ( ENAM , AMELX , AMBN ) correlated with the presence of wet keratin. RNA sequencing of laser capture microdissected tissue identified expression of SHH, transforming growth factor β1, bone morphogenic proteins, fibroblast growth factors, Wnt genes, and many cytokines and chemokines within tumour epithelial whorls and evidence of paracrine SHH signalling. Interim analysis of the GDC0449 trial suggests that inhibition of the SHH pathway might reduce survival in murine ACP (median survival 82 vs 233 days, p=0·1). Further studies showed that treatment with GDC0449 caused premature tumour induction, increased proliferation, and halved the doubling time of the solid tumour component as assessed by MRI. Interpretation ACPs have clonal CTNNB1 mutations and share a common embryological origin with teeth. They exhibit a complex transcriptional landscape, including paracrine interactions between different cellular compartments. Studies of ACP murine models suggest that, as in pancreatic, colorectal, and bladder cancer models, caution is needed when considering the use of SHH pathway inhibitors as a treatment for human ACP. Funding Cancer Research UK; Children with Cancer, Childhood Cancer and Leukaemia Group; Great Ormond Street Hospital Biomedical Research Centre.

A career path in academic paediatric medicine is an extremely rewarding one, and while not traditionally considered an academic specialty, it offers a wealth of exciting research opportunities. Developing academic paediatrics is becoming increasingly important, as recently reviewed in the Royal College of Paediatrics and Child Health (RCPCH) Turning the Tide report, and developing future leaders in academic paediatrics is a key goal of the academic training pathways. Strategies are being implemented to ensure that the enthusiasm of academic trainees is maintained, and their development into future leaders is secured.

We conducted RNA-sequencing analysis of Adamantinomatous Craniopharnygioma (ACP), including Laser capture micro-dissection (LCM) of individual cellular compartments, to characterise both disease heterogeneity and targetable deregulated molecular pathways. RNA sequencing was performed on 18 samples of ACP (17 pediatric, 1 adult) and six control samples (three fetal pituitaries, three non-functioning pituitary adenomas). LCM and RNA sequencing were performed on a subset of cases selecting nuclear β catenin accumulating clusters, and paired non-cluster tumour tissue (palisading epithelium). Mouse ACP models indicate that these clusters initiate tumorigenesis in a non-cell autonomous manner. Consensus clustering analysis revealed two subgroups of human ACP, one associated with a high CTNNB1 mutational frequency (>30%), a relatively high tumour content and fibrous reactive tissue (Group H) and another with a lower mutation frequency ( The meta-gene signature of Group H defined by non-negative matrix factorization included secreted factors, such as Sonic Hedgehog (SHH) and Fibroblast Growth Factors (FGFs) 3, 4 and 19, Keratins and Matrix Metalloproteinases and was enriched for genes related to odontogenesis. Weighted gene co-expression network analysis independently showed correlation of expression of such genes with mutational frequency. Glial markers and neural differentiation genes were up-regulated in Group L tumors. Differential expression showed up-regulation in ACPs of potential therapeutic targets including SHH (32x), EGFR (8.9x) and TNF (10x). Similarly, differential expression analysis of LCM dissected nuclear β catenin accumulating clusters identified up-regulation of SHH, FGFs and other secreted factors including as WNTs and BMPs. Analysis of the SHH pathway found that SHH is over expressed in clusters (9x) whilst its downstream targets Ptch1 (1.7x), Gli1 (2.1x), Gli3 (2.9x) are over expressed in palisading epithelium, indicating the presence of paracrine signaling. These findings were independently confirmed by in situ hybridisation in human samples and the mouse model. Clustering of murine expression data with those genes differentially expressed in the human clusters revealed murine and human clusters group together, suggesting they are similar structures functionally and biologically. Together, these results suggest that a subset of cells, carrying CTNNB1 mutations and showing nuclear β-catenin accumulation, express secreted pro-oncogenic factors and the response may be observed in neighboring cell types. Targeting such signalling offers an attractive therapeutic opportunity, for which the results suggest the mouse model is well placed for testing. The clinical significance of the two putative ACP subgroups remains to be determined. Citation Format: John R. Apps, Nital Jani, Gabriela Carreno, Jose M. Gonzalez-Meljem, Kyoko Tossell, Thomas J. Stone, Mark A. Ungless, Hywel J. Williams, Thomas S. Jacques, Juan-Pedro Martinez-Barbera. Expression analysis of adamantinomatous craniopharyngioma suggests two subtypes associated with CTNNB1 mutational frequency and highlights potential therapeutic targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1804.

A career path in academic paediatric medicine is an extremely rewarding one, and while not traditionally considered an academic specialty, it offers a wealth of exciting research opportunities. Developing academic paediatrics is becoming increasingly important, as recently reviewed in the Royal College of Paediatrics and Child Health (RCPCH) Turning the Tide report, and developing future leaders in academic paediatrics is a key goal of the academic training pathways. Strategies are being implemented to ensure that the enthusiasm of academic trainees is maintained, and their development into future leaders is secured.

Aims Youth stabbing in London is increasing and of national concern. There is little published data quantifying the number of paediatric stabbings attending Emergency Departments. A recent audit at one hospital highlighted the significant numbers within one area of London and a lack of engagement with the Police and Social Services. This study aimed to characterise attendances across all 32 Emergency Departments in London and audit existing referral rates to the Police and Social Services against new GMC guidance. This study also assessed the feasibility of a Pan-London audit of an acute paediatric presentation. Methods Hospitals were contacted, a trainee identified and patients less than 18 who had been stabbed, between 1 March 2007 and 31 March 2009, were identified locally by the most appropriate method using local codings. Self harm and accidental injury were excluded. A proforma was provided to record basic details of the event, the nature and management of any wound and the involvement of parents, police and social services. Results Of the 32 Hospitals, 25 hospitals have submitted results (further data accrual is continuing and will be presented). 276 cases were identified (aged 9–17 years), 109 of whom were less than 16 years. The majority were seen only by A+E staff and only a minority (29%) were admitted. Sadly three died in the departments. A knife was the commonest weapon and limbs the most common site of injury. Referral rates to Police were documented in only 26% of patients (39% if Conclusion A significant number of paediatric stabbings presented to and were discharged from Emergency Departments across London. Of those discharged, documentation regarding referral rates to Police and social services was poor, and documented referral rates low. This audit covered a period prior to the introduction of new GMC guidance and a repeat audit to assess subsequent referral rates is required. This audit also demonstrates it is possible (with a lot of effort!) to conduct a Pan-London audit of an acute paediatric presentation.

The Royal College of Paediatrics and Child Health’s report highlights challenges to paediatric academic medicine, including loss of senior academics as mentors and role models, and disillusionment of junior trainees poorly equipped to undertake fi rst-rank research. There is evidence for the former, but the latter is belied by a recent national meeting. The fi rst national meeting of paediatric academic trainees was held on April 18, at the Institute of Child Health, London, UK, and was attended by 65 enthusiastic trainees. We did a survey of trainees’ research experiences and future plans. The survey response rate was 74% (48/65). Repondents were from all levels including six academic foundation doctors, 16 academic clinical fellows, 12 PhD fellows, and six lecturers. We noted a strong commitment to academic carreers: 78% of current academics intend to continue research in their next position. In the longer term, 63% hope to work in a clinical academic position, and the remaining 37% plan to work in a clinical position with some teaching and research. This is a strong base from which paediatric academic leaders will surely emerge. However, there was less certainty regarding future academic training and senior jobs. Only 12% believe that there is a good chance of fi nding a clinical academic position within their specialty at their level of training. This reiterates concerns about the loss of senior academic posts raised by the report. It would be good to see more universities commit to supporting paediatric academic postitions in the future. Part-time working also raises specifi c concerns. In the Wellcome Trust 2009 career tracker survey, half of PhD awardees were women, and only 63% continued in academia after completing their award (vs 93% men). Two-thirds of our respondents were women, mirroring recruitment to the specialty, and the majority saw themselves working part-time in the future. However, 88% believed that they would be substantially or somewhat negatively viewed by funding bodies with regards to fl exible working. We believe that the National Institute for Health Researchinspired push to get academic centres certifi ed by the Athena Swan charter is important in supporting paediatric academic training. Rather than disaff ection, the meeting showed paediatric academic trainees engaging with issues that concern their future. The key question is how to support their development into long-term academics? Trainees have a valuable contribution to make to that debate.

Introduction Biomarkers are increasingly used to support risk stratification and clinical decisions in Paediatric Oncology. Implicit in the clonal evolutionary theory of cancer development is the evolution of cells derived from an initiating clone through natural selection acting upon intra-tumoural genetic heterogeneity. Wilms tumours frequently present as large macroscopically heterogeneous masses, and can be multifocal in origin. Several studies have indicated the presence of intra-tumoural genetic heterogeneity in a range of tumours. This study aimed to characterise intra-tumoural genetic heterogeneity within Wilms tumours. Methods Multiple snap frozen samples were taken from consecutive post chemotherapy Wilms tumour nephrectomy specimens at Great Ormond Street Hospital, May-October 2011. DNA was extracted from frozen specimens and analysed using the Illumina CytoSNP array for copy number variation. Results 23 samples were analysed from 10 separate tumours. 2 cases showed clear heterogeneity between samples. In one case gains of chromosome 8 and 13 were conserved across two tumour samples however gains of chromosomes 2 and 12 were present in one sample and of chromosome 7 in the other. In a separate case partial gain of 22q was present in both samples and 1q and partial 1p gain in only one sample. A third case showed localised heterogeneity with loss of 7p and gain of 7q present in two samples whilst gain of chromosome 8 was more apparent in only one sample. In three other cases, the changes observed were conserved between two or more samples (gain of 12, 13 and 20 in one case, gain of 12 in the second, and partial 9q21-22 deletions in the third). 4 tumours had no apparent copy number changes. Discussion 6 of 10 tumours showed copy number variation. In all of these cases certain copy number variations were conserved between multiple samples of an individual tumour, consistent with a clonal origin. In three cases there was intra-tumoural genetic heterogeneity. Further understanding such heterogeneity may aid in deciphering the genetic changes in the evolution of Wilms tumours and help identify driver mutations. In addition, as the use of biomarkers, such as 1q gain, enters clinical practice understanding such heterogeneity will be important for establishing appropriate sampling strategies.

Case scenarios ##### Case 1 A 3 month old infant, previously breast fed, presented with a urticarial rash, irritability, and vomiting shortly after introduction of cow’s milk formula. He was referred to a paediatric allergy clinic where skin prick testing and specific IgE testing were positive for cow’s milk protein. The mother did not wish to continue breast feeding. The formula was changed to an extensively hydrolysed protein feed, and the symptoms resolved rapidly. Re-challenge with cow’s milk was deferred until 12 months and proceeded uneventfully. ##### Case 2 An otherwise well and thriving 6 week old breast fed infant presented with frequent stools, irritability, and perianal redness. Because physical examination was otherwise unremarkable and stool virology and culture were negative, he was referred to the paediatric (gastroenterology) outpatients department. Dietary protein induced proctocolitis was suspected. Because cow’s milk is the most common allergen implicated in this condition, the mother was advised to stop taking all dairy products, but to continue breast feeding. The symptoms resolved within 72 hours but reappeared on challenge. The mother therefore continued to avoid dairy products while breast feeding (an extensively hydrolysed feed would be an alternative), and the infant was weaned at 6 months on to dairy free solids. Cow’s milk was reintroduced at 12 months, and the child’s original symptoms did not recur. The mother was referred for dietetic advice and prescribed calcium supplements. Cow’s milk (protein) allergy is an adverse immunological response to cow’s milk proteins seen mainly in the first few years of life. It can have diverse manifestations. It can be broadly divided into IgE (type I hypersensitivity) mediated disease and non-IgE (usually type IV hypersensitivity) mediated disease, sometimes referred to as cow’s milk (protein) intolerance. These differ in clinical presentation, diagnostic testing, and prognosis; for example, type I hypersensitivity classically presents early, with symptoms such as urticaria, …