A macroprolactinoma becoming resistant to cabergoline and developing atypical pathology

Pituitary adenomas are a common intracranial neoplasm, usually demonstrating a benign phenotype. They can be classified according to pathological, radiological or clinical behaviour as typical, atypical or carcinomas, invasive or noninvasive, and aggressive or nonaggressive. Prolactinomas account for 40-60% of all pituitary adenomas, with dopamine agonists representing the first-line treatment and surgery/radiotherapy reserved for drug intolerance/resistance or in neuro-ophthalmological emergencies. We present the case of a 62-year-old man with an apparently indolent prolactin-secreting macroadenoma managed with partial resection and initially showing a biochemical response to cabergoline. Five years later, the tumour became resistant to cabergoline, despite a substantial increase in dosage, showing rapid growth and causing worsening of vision. The patient then underwent two further transsphenoidal operations and continued on high-dose cabergoline; despite these interventions, the tumour continued enlarging and prolactin increased to 107 269 U/L. Histology of the third surgical specimen demonstrated features of aggressive behaviour (atypical adenoma with a high cell proliferation index) not present in the tumour removed at the first operation. Subsequently, he was referred for radiotherapy aiming to control tumour growth

Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations

Accumulation of the DNA/RNA binding protein fused in sarcoma (FUS) as inclusions in neurons and glia is the pathological hallmark of amyotrophic lateral sclerosis patients with mutations in FUS (ALS-FUS) as well as in several subtypes of frontotemporal lobar degeneration (FTLD-FUS), which are not associated with FUS mutations. Despite some overlap in the phenotype and neuropathology of FTLD-FUS and ALS-FUS, significant differences of potential pathomechanistic relevance were recently identified in the protein composition of inclusions in these conditions. While ALS-FUS showed only accumulation of FUS, inclusions in FTLD-FUS revealed co-accumulation of all members of the FET protein family, that include FUS, Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 15 (TAF15) suggesting a more complex disturbance of transportin-mediated nuclear import of proteins in FTLD-FUS compared to ALS-FUS. To gain more insight into the mechanisms of inclusion body formation, we investigated the role of Transportin 1 (Trn1) as well as 13 additional cargo proteins of Transportin in the spectrum of FUS-opathies by immunohistochemistry and biochemically. FUS-positive inclusions in six ALS-FUS cases including four different mutations did not label for Trn1. In sharp contrast, the FET-positive pathology in all FTLD-FUS subtypes was also strongly labeled for Trn1 and often associated with a reduction in the normal nuclear staining of Trn1 in inclusion bearing cells, while no biochemical changes of Trn1 were detectable in FTLD-FUS. Notably, despite the dramatic changes in the subcellular distribution of Trn1 in FTLD-FUS, alterations of its cargo proteins were restricted to FET proteins and no changes in the normal physiological staining of 13 additional Trn1 targets, such as hnRNPA1, PAPBN1 and Sam68, were observed in FTLD-FUS. These data imply a specific dysfunction in the interaction between Trn1 and FET proteins in the inclusion body formation in FTLD-FUS. Moreover, the absence of Trn1 in ALS-FUS provides further evidence that ALS-FUS and FTLD-FUS have different underlying pathomechanism

Pathogenesis of FUS-associated ALS and FTD: insights from rodent models

Disruptions to genes linked to RNA processing and homeostasis are implicated in the pathogenesis of two pathologically related but clinically heterogeneous neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in the Fused-in-Sarcoma (FUS) gene encoding a 526 amino-acid RNA-binding protein are found in a small subset of ALS cases, but FUS mutations do not appear to be a direct cause of FTD. Structural and functional similarities between FUS and another ALS-related RNA-binding protein, TDP-43, highlight the potential importance of aberrant RNA processing in ALS/FTD, and this pathway is now a major focus of interest. Recently, several research groups have reported transgenic vertebrate models of FUSopathy, with varying results. Here, we discuss the evidence for FUS pathogenicity in ALS/FTD, review the experimental approaches used and phenotypic features of FUS rodent models reported to date, and outline their contribution to our understanding of pathogenic mechanisms. Further refinement of vertebrate models will likely aid our understanding of the role of FUS in both diseases

Study protocol for putting the ‘Person’ in the PiCTuRE: an exploratory sequential mixed methods-based design, exploring how precision medicine is implemented and experienced by people living with a primary tumour of the craniospinal axis

Introduction: Primary tumours of the brain and spine are rare, heterogeneous, and frequently associated with significant morbidity and mortality. Advances in precision oncology and personalised medicine offer the potential to accelerate diagnosis, improve clinical outcomes, and yield critical insights into the molecular biology of these cancers of unmet need. Despite this, patient engagement in this area remains limited. Well-organised neuro-oncological biorepositories—those that are clinically integrated, fully consented, and derived from routine care—are limited and fragmented, which impedes progress. Therefore, it is crucial to examine the barriers to tissue donation and data integration within the NHS by analysing patients’ lived experiences. The PiCTuRE (Personalised Consent in Tissue donation for neuroscience Research, lived Experiences) study aims to develop a digital platform that provides customised, individualised, and interactive support to assist patients in their decision-making regarding tissue donation for research and participation in related clinical trials. Methods & Analysis: PiCTuRE is a multistage, mixed-methods, exploratory sequential investigation aimed at understanding the lived experiences of individuals donating tissue for research. It consists of three phases: Phase 1 involves an online survey to collect lived experience data, followed by semi-structured interviews to further explore individual perspectives. Thematic analysis will be performed to identify key themes. In Phase 2, patient-reported experience data will be gathered through co-design and statistically analysed to validate content for the development of the digital platform. Phase 3 will refine this intervention through iterative cycles of Phases 1 and 2, in collaboration with patients with lived experience of brain or spine tumours, to prepare it for integration into routine clinical practice. Ethics and Dissemination: Ethical approval has been obtained via the Medical Sciences Interdivisional Research Ethics Committee (MS IDREC), University of Oxford (R79248/RE001). Findings will be disseminated via podium presentations, public patient initiatives in partnership with charities, in peer-reviewed publications and via social media. Trial Registration Number: ISRCTN12601034

FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations

Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing's sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing's sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing's sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing's sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma patholog

A comprehensive assessment of benign genetic variability for neurodegenerative disorders

Over the last few years, as more and more sequencing studies have been performed, it has become apparent that the identification of pathogenic mutations is, more often than not, a complex issue. Here, with a focus on neurodegenerative diseases, we have performed a survey of coding genetic variability that is unlikely to be pathogenic. We have performed whole-exome sequencing in 478 samples derived from several brain banks in the United Kingdom and the United States of America. Samples were included when subjects were, at death, over 60 years of age, had no signs of neurological disease and were subjected to a neuropathological examination, which revealed no evidence of neurodegeneration. This information will be valuable to studies of genetic variability as a causal factor for neurodegenerative syndromes. We envisage it will be particularly relevant for diagnostic laboratories as a filter step to the results being produced by either genome-wide or gene-panel sequencing. We have made this data publicly available at www.alzforum.org/exomes/hex

Functional expression of calcium-permeable Canonical Transient Receptor Potential 4-containing channels promotes migration of medulloblastoma cells

Aberrant intracellular Ca(2+) signalling contributes to the formation and progression of a range of distinct pathologies including cancers. Rises in intracellular Ca(2+) concentration occur in response to Ca(2+) influx through plasma membrane channels and Ca(2+) release from intracellular Ca(2+) stores, which can be mobilised in response to activation of cell surface receptors. OGR1 (Ovarian cancer G protein coupled Receptor 1, aka GPR68) is a proton-sensing Gq -coupled receptor that is most highly expressed in cerebellum. Medulloblastoma (MB) is the most common paediatric brain tumour that arises from cerebellar precursor cells. We find that nine distinct human MB samples all express OGR1. In both normal granule cells and the transformed human cerebellar granule cell line DAOY, OGR1 promotes expression of the proton-potentiated member of the Canonical Transient Receptor Potential (TRPC) channel family, TRPC4. Consistent with a role for TRPC4 in MB, we find that all MB samples also express TRPC4. In DAOY cells, activation of TRPC4-containing channels resulted in large Ca(2+) influx and enhanced migration, while in normal cerebellar granule (precursor) cells and MB cells not derived from granule precursors, only small levels of Ca(2+) influx and no enhanced migration was observed. Our results suggest that OGR1-dependent increases in TRPC4 expression may favour formation of highly Ca(2+) -permeable TRPC4-containing channels that promote transformed granule cell migration. Increased motility of cancer cells is a prerequisite for cancer invasion and metastasis, and our findings may point towards a key role for TRPC4 in progression of certain types of MB. This article is protected by copyright. All rights reserved

Rapid intraoperative molecular genetic classification of gliomas using Raman spectroscopy

Background - The molecular genetic classification of gliomas, particularly the identification of isocitrate dehydrogenase (IDH) mutations, is critical for clinical and surgical decision-making. Raman spectroscopy probes the unique molecular vibrations of a sample to accurately characterize its molecular composition. No sample processing is required allowing for rapid analysis of tissue. The aim of this study was to evaluate the ability of Raman spectroscopy to rapidly identify the common molecular genetic subtypes of diffuse glioma in the neurosurgical setting using fresh biopsy tissue. In addition, classification models were built using cryosections, formalin-fixed paraffin-embedded (FFPE) sections and LN-18 (IDH-mutated and wild-type parental cell) glioma cell lines.Methods - Fresh tissue, straight from neurosurgical theatres, underwent Raman analysis and classification into astrocytoma, IDH-wild-type; astrocytoma, IDH-mutant; or oligodendroglioma. The genetic subtype was confirmed on a parallel section using immunohistochemistry and targeted genetic sequencing.Results - Fresh tissue samples from 62 patients were collected (36 astrocytoma, IDH-wild-type; 21 astrocytoma, IDH-mutated; 5 oligodendroglioma). A principal component analysis fed linear discriminant analysis classification model demonstrated 79%–94% sensitivity and 90%–100% specificity for predicting the 3 glioma genetic subtypes. For the prediction of IDH mutation alone, the model gave 91% sensitivity and 95% specificity. Seventynine cryosections, 120 FFPE samples, and LN18 cells were also successfully classified. Meantime for Raman data collection was 9.5 min in the fresh tissue samples, with the process from intraoperative biopsy to genetic classification taking under 15 min.Conclusion - These data demonstrate that Raman spectroscopy can be used for the rapid, intraoperative, classification of gliomas into common genetic subtypes

Targeting NKG2D ligands in glioblastoma with a bispecific T-cell engager is augmented with conventional therapy and enhances oncolytic virotherapy of glioma stem-like cells

Background: Glioblastoma (GBM) almost invariably becomes resistant towards conventional treatment of radiotherapy and temozolomide (TMZ) chemotherapy, partly due to subpopulations of intrinsically resistant glioma stem-like cells (GSC). The oncolytic herpes simplex virus-1 G207 is a promising approach for GBM virotherapy although its efficacy in patients with GBM is often limited. Natural killer group 2 member D ligands (NKG2DLs) are minimally expressed by healthy cells but are upregulated by the DNA damage response (DDR) and in malignant cells with chronic DDR signaling, resulting in innate immune activation. Methods: We have designed a bispecific T-cell engager (BiTE) capable of cross-linking CD3 on T cells with NKG2DL-expressing GBM cells. We then engineered the G207 virus to express the NKG2D BiTE and secrete it from infected cells. The efficacy of the free BiTE and BiTE delivered by G207 was evaluated in combination with conventional therapies in GBM cells and against patient-derived GSCs in the context of T-cell activation and target cell viability. Results: NKG2D BiTE-mediated cross-linking of GBM cells and T cells causes antigen-independent T-cell activation, pro-inflammatory cytokine release, and tumor cell death, thereby combining direct viral oncolysis with BiTE-mediated cytotoxicity. Surface NKG2DL expression was further elevated on GBM cells following pretreatment with sublethal doses of TMZ and radiation to induce the DDR, increasing sensitivity towards G207-NKG2D BiTE and achieving synergistic cytotoxicity. We also demonstrate a novel strategy for targeting GSCs that are non-permissive to G207 infection but remain sensitive to NKG2D BiTE. Conclusions: We propose a potential model for targeting GSCs in heterogeneous tumors, whereby differentiated GBM cells infected with G207-NKG2D BiTE produce NKG2D BiTE locally, directing T-cell cytotoxicity towards the GSC subpopulations in the tumor microenvironment