Alex Virasami

Astrovirus VA1/HMO-C: An Increasingly Recognized Neurotropic Pathogen in Immunocompromised Patients

Brain biopsy from a child with unknown cause of encephalopathy was deep-sequenced. Astrovirus VA1/HMO-C was identified, highly divergent from human astroviruses and 95% identical to astrovirus previously associated with encephalitis. Findings suggest astrovirus VA1/HMO-C is an under-recognized cause of viral encephalitis.

Human Coronavirus OC43 Associated with Fatal Encephalitis.

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.

Stem cell senescence drives age-attenuated induction of pituitary tumours in mouse models of paediatric craniopharyngioma

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.Senescent cells can promote tumour progression through the activation of a senescenceassociated secretory phenotype (SASP). Here, the authors show that SASP activation is associated with non-cell autonomous cell transformation and tumour initiation in an in vivo model of adamantinomatous craniopharyngioma.

Imaging Invasion: Micro-CT imaging of adamantinomatous craniopharyngioma highlights cell type specific spatial relationships of tissue invasion

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.

Molecular Analyses Reveal Inflammatory Mediators in the Solid Component and Cyst Fluid of Human Adamantinomatous Craniopharyngioma.

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.

MAPK pathway control of stem cell proliferation and differentiation in the embryonic pituitary provides insights into the pathogenesis of papillary craniopharyngioma

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. Highlighted Article: Constitutive activation of the MAPK/ERK pathway causes pituitary hyperplasia, abnormal morphogenesis, and abnormal endocrine cell specification due to the sustained proliferation of the Sox2+ stem cell compartment.

Vps33b is crucial for structural and functional hepatocyte polarity

Graphical abstract

Preclinical transgenic and patient-derived xenograft models recapitulate the radiological features of human adamantinomatous craniopharyngioma

To assess the clinical relevance of transgenic and patient‐derived xenograft models of adamantinomatous craniopharyngioma (ACP) using serial magnetic resonance imaging (MRI) and high resolution post‐mortem microcomputed tomography (μ‐CT), with correlation with histology and human ACP imaging. The growth patterns and radiological features of tumors arising in Hesx1Cre/+;Ctnnb1lox(ex3)/+ transgenic mice, and of patient‐derived ACP xenografts implanted in the cerebral cortex, were monitored longitudinally in vivo with anatomical and functional MRI, and by ex vivo μ‐CT at study end. Pathological correlates with hematoxylin and eosin stained sections were investigated. Early enlargement and heterogeneity of Hesx1Cre/+;Ctnnb1lox(ex3)/+ mouse pituitaries was evident at initial imaging at 8 weeks, which was followed by enlargement of a solid tumor, and development of cysts and hemorrhage. Tumors demonstrated MRI features that recapitulated those of human ACP, specifically, T1‐weighted signal enhancement in the solid tumor component following Gd‐DTPA administration, and in some animals, hyperintense cysts on FLAIR and T1‐weighted images. Ex vivo μ‐CT correlated with MRI findings and identified smaller cysts, which were confirmed by histology. Characteristic histological features, including wet keratin and calcification, were visible on μ‐CT and verified by histological sections of patient‐derived ACP xenografts. The Hesx1Cre/+;Ctnnb1lox(ex3)/+ transgenic mouse model and cerebral patient‐derived ACP xenografts recapitulate a number of the key radiological features of the human disease and provide promising foundations for in vivo trials of novel therapeutics for the treatment of these tumors.

Comprehensive molecular characterisation of epilepsy-associated glioneuronal tumours

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.

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target

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.