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Invasiveness in pituitary adenomas has been defined and investigated from multiple perspectives, with varying results when its predictive value is considered. A systematic literature review, following PRISMA guidelines, was performed, searching PubMed and Scopus databases with terms that included molecular markers, histological, radiological, anatomical and surgical data on invasiveness of pituitary adenomas. The results showed that differing views are still present for anatomical aspects of the sellar region that are relevant to the concept of invasiveness; radiological and histological diagnoses are still limited, but might improve in the future, especially if they are related to surgical findings, which have become more accurate thanks to the introduction of the endoscope. The aim is to achieve a correct distinction between truly invasive pituitary adenomas from those that, in contrast, present with extension in the parasellar area through natural pathways. At present, diagnosis of invasiveness should be based on a comprehensive analysis of radiological, intra-operative and histological findings.

Cerebral organoids (cORGs) obtained from induced pluripotent stem cells (iPSCs) have become significant instruments for investigating human neurophysiology, with the possibility of simulating diseases and enhancing drug discovery. The current approaches require a strict process of manual inclusion in animal-derived matrix Matrigel® and are challenged by unpredictability, operators’ skill and expertise, elevated costs, and restricted scalability, impeding their extensive applicability and translational potential. In this study, we present a novel method to generate brain organoids that address these limitations. Our approach does not require a manual, operator-dependent embedding. Instead, it employs a chemically defined hydrogel in which the Matrigel® is diluted in a solution enriched with sodium alginate (SA) and sodium carboxymethylcellulose (CMC) and used as a bioink to print neural embryoid bodies (nEBs). Immunohistochemical, immunofluorescence, and gene expression analyses confirmed that SA-CMC-Matrigel® hydrogel can sustain the generation of iPSC-derived cortical cORGs as the conventional Matrigel®-based approach does. By day 40 of differentiation, hydrogel-based 3D-bioprinted cORGs showed heterogeneous and consistent masses, with a cytoarchitecture resembling an early-stage developmental fetal brain composed of neural progenitor cells PAX6+/Ki67+ organized into tubular structures, and densely packed cell somas with extensive neurites SYP+, suggestive of cortical tissue-like neuronal layer formation.

Microglia contribute to development, homeostasis, and immunity of the CNS. Like other tissue-resident macrophage populations, microglia express the surface receptor triggering receptor expressed on myeloid cells 2 (TREM2), which binds polyanions, such as dextran sulphate and bacterial LPS, and activates downstream signaling cascades through the adapter DAP12. Individuals homozygous for inactivating mutations in TREM2 exhibit demyelination of subcortical white matter and a lethal early onset dementia known as Nasu-Hakola disease. How TREM2 deficiency mediates demyelination and disease is unknown. Here, we addressed the basis for this genetic association using Trem2(-/-) mice. In WT mice, microglia expanded in the corpus callosum with age, whereas aged Trem2(-/-) mice had fewer microglia with an abnormal morphology. In the cuprizone model of oligodendrocyte degeneration and demyelination, Trem2(-/-) microglia failed to amplify transcripts indicative of activation, phagocytosis, and lipid catabolism in response to myelin damage. As a result, Trem2(-/-) mice exhibited impaired myelin debris clearance, axonal dystrophy, oligodendrocyte reduction, and persistent demyelination after prolonged cuprizone treatment. Moreover, myelin-associated lipids robustly triggered TREM2 signaling in vitro, suggesting that TREM2 may directly sense lipid components exposed during myelin damage. We conclude that TREM2 is required for promoting microglial expansion during aging and microglial response to insults of the white matter.

Early B-cell factor-1 (EBF1) is a transcription factor with an important role in cell lineage specification and commitment during the early stage of cell maturation. Originally described during B-cell maturation, EBF1 was subsequently identified as a crucial molecule for proper cell fate commitment of mesenchymal stem cells into adipocytes, osteoblasts and muscle cells. In vessels, EBF1 expression and function have never been documented. Our data indicate that EBF1 is highly expressed in peri-endothelial cells in both tumor vessels and in physiological conditions. Immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and fluorescence-activated cell sorting (FACS) analysis suggest that EBF1-expressing peri-endothelial cells represent bona fide pericytes and selectively express well-recognized markers employed in the identification of the pericyte phenotype (SMA, PDGFRβ, CD146, NG2). This observation was also confirmed in vitro in human placenta-derived pericytes and in human brain vascular pericytes (HBVP). Of note, in accord with the key role of EBF1 in the cell lineage commitment of mesenchymal stem cells, EBF1-silenced HBVP cells showed a significant reduction in PDGFRβ and CD146, but not CD90, a marker mostly associated with a prominent mesenchymal phenotype. Moreover, the expression levels of VEGF, angiopoietin-1, NG2 and TGF-β, cytokines produced by pericytes during angiogenesis and linked to their differentiation and activation, were also significantly reduced. Overall, the data suggest a functional role of EBF1 in the cell fate commitment toward the pericyte phenotype.

Oligodendroglioma is defined by IDH mutation and 1p/19q codeletion. The latter is mutually exclusive to ATRX immunohistochemical loss and has been recently associated with the loss of H3K27me3 immunostaining. We aimed to assess the diagnostic and prognostic value of H3K27me3 immuno-expression in diffuse gliomas with oligodendroglial or mixed oligoastrocytic morphology. H3K27me3 immunostaining was performed in 69 diffuse gliomas with oligodendroglial (n = 62) or oligoastrocytic (n = 7) morphology. The integration with routinely assessed IDH mutations, ATRX immunostaining, and 1p/19q codeletion classified these cases as 60 oligodendroglial and 9 astrocytic. H3K27me3 was lost in 58/60 oligodendrogliomas with retained (n = 47) or non-conclusive (n = 11) ATRX staining, 3/6 IDH-mutant astrocytomas with ATRX loss, and 3/3 IDH-wt astrocytomas. H3K27me3 was retained in 2/60 oligodendrogliomas with retained ATRX, and in 3/6 IDH-mutant astrocytomas, two of which had lost and one retained ATRX. The combination of H3K27me3 and ATRX immunostainings with IDH mutational status correctly classified 55/69 (80%) cases. In IDH-mutant gliomas, ATRX loss indicates astrocytic phenotype, while ATRX retention and H3K27me3 loss identify oligodendroglial phenotype. Only 14 (20%) IDH-mutant cases with retained ATRX and H3K27me3 or inconclusive ATRX immunostaining would have requested 1p/19q codeletion testing to be classified. Furthermore, H3K27me3 retention was associated with significantly shorter relapse-free survival (P < 0.0001), independently from IDH mutation or 1p/19q codeletion (P < 0.005). Our data suggest that adding H3K27me3 immunostaining to the diagnostic workflow of diffuse gliomas with oligodendroglial or mixed morphology is useful for drastically reducing the number of cases requiring 1p/19q codeletion testing and providing relevant prognostic information.

Glioblastomas (GBM) can be classified into three major transcriptional subgroups (proneural, mesenchymal, classical), underlying different molecular alterations, prognosis, and response to therapy. However, transcriptional analysis is not routinely feasible and assessment of a simplified method for glioblastoma subclassification is required. We propose an integrated molecular and immunohistochemical approach aimed at identifying GBM subtypes in routine paraffin-embedded material. RNA-sequencing analysis was performed on representative samples (n = 51) by means of a “glioblastoma transcriptional subtypes (GliTS) redux” custom gene signature including a restricted number (n = 90) of upregulated genes validated on the TCGA dataset. With this dataset, immunohistochemical profiles, based on expression of a restricted panel of gene classifiers, were integrated by a machine-learning approach to generate a GliTS based on protein quantification that allowed an efficient GliTS assignment when applied to an extended cohort (n = 197). GliTS redux maintained high levels of correspondence with the original GliTS classification using the TCGA dataset. The machine-learning approach designed an immunohistochemical (IHC)-based classification, whose concordance was 79.5% with the transcriptional- based classification, and reached 90% for the mesenchymal subgroup. Distribution and survival of GliTS were in line with reported data, with the mesenchymal subgroup given the worst prognosis. Notably, the algorithm allowed the identification of cases with comparable probability to be assigned to different GliTS, thus falling within overlapping regions and reflecting an extreme heterogeneous phenotype that mirrors the underlying genetic and biological tumor heterogeneity. Indeed, while mesenchymal and classical subgroups were well segregated, the proneural types frequently showed a mixed proneural/classical phenotype, predicted as proneural by the algorithm, but with comparable probability of being assigned to the classical subtype. These cases, characterized by concomitant high expression of EGFR and proneural biomarkers, showed lower survival. Collectively, these data indicate that a restricted panel of highly sensitive immunohistochemical markers can efficiently predict GliTS with high accuracy and significant association with different clinical outcomes. The authors developed a novel simplified assay for glioblastoma transcriptional classification on formalin-fixed-paraffin-embedded tissue samples. On such dataset, immunohistochemical profiles, based on expression of a restricted panel of gene classifiers, were integrated by machine learning approach to generate a glioblastoma transcriptional signature based on protein quantification that allowed to efficiently assign transcriptional subgroups to an extended cohort. Correlations with both histopathological features and clinical outcome have been also performed.

Somatic mutations activating MAPK/PI3K signalling play a pivotal role in both tumours and brain developmental disorders. We developed a zebrafish model of brain tumour based on somatic expression of oncogenes that activate MAPK/PI3K signalling in neural progenitor cells. HRASV12 was the most effective in inducing both heterotopia and invasive tumours. Tumours, but not heterotopias, require persistent activation of phospho‑(p)ERK and express a gene signature similar to the mesenchymal glioblastoma subtype, with a strong YAP component. Application of a 8-gene signature to human brain tumours establishes that YAP activation distinguishes between mesenchymal glioblastoma and low grade glioma in a wide TCGA sample set including gliomas and glioblastomas (GBMs). This suggests that the activation of YAP may be an important event in brain tumour development, promoting malignant versus benign brain lesions. Indeed, co-expression of dominant active YAP (YAPS5A) and HRASV12 abolishes the development of heterotopias and leads to the sole development of aggressive tumours. Thus, we have developed a model proving that neurodevelopmental disorders and brain tumours may originate from the same somatic mutations activating oncogenes and established that YAP activation is a hallmark of malignant brain tumours.

Abstract A 54-yr-old man presented with the radiological recurrence of a glioblastoma at the level of the left anterior cingulate gyrus, 46 mo after first surgery, which had been complicated by bone flap infection. Due to the relatively small recurrence, the long survival, and the good neurological status, surgery was warranted. A new, high-definition (4 K) and 3-dimensional exoscope (ORBEYE; Sony Olympus Medical Solutions Inc, Tokyo, Japan) was used during the surgical approach and throughout tumor removal, which was aided by five-aminolevulinic acid (5-ALA) derived fluorescence. The postoperative course was characterized by supplementary motor area syndrome, which quickly improved, leading to a discharge home 1 wk after surgery. Histological examination confirmed a wild-type (WT) IDH1/2, MGMT (DNA repair enzyme O6-methylguanine-DNA methyltransferase) methylated glioblastoma with a proliferative index focally as high as 20%. He is now being considered for a second-line treatment. As recently reported for spinal surgery,1 we believe this technology has significant potential for its small dimension (which can provide optimal positioning even in ergonomically challenging positions), ease of movement, and image quality, including 5-ALA fluorescence. The patient's consent was obtained for publication.

Background To perform genetic screening for ARMC5 gene germline pathogenic variants in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH). Subjects and Methods In a group of 10 PBMAH patients, we performed complete sequencing of the coding region of the ARMC5 gene and MLPA analysis for large deletion detection. In subjects with the ARMC5 variant, we searched ARMC5 gene somatic variants on tumor samples. Results Among 10 PBMAH patients, we identified four ARMC5 germline variants (40%). One variant, c:174dupC p.Glu59Argfs*44, was already known; one variant p.Gly323Asp, was already reported and classified as likely disease‐causing VUS (class 3–4); two variants p.Leu596Arg and p.Arg811Pro, were never reported before. For p.Gly323Asp and p.Arg811Pro, we identified second deleterious variants at the somatic level, enforcing the possible pathogenic effect of germline variants. Conclusions Our results underscore the importance of performing genetic testing also in sporadic PBMAH patients and broaden the spectrum of molecular variants involved in PBMAH syndrome. Among 10 PBMAH patients, we identified four ARMC5 germline variants (40%). One variant, 174dupC p.Glu59Argfs*44, was already known; one variant p.Gly323Asp, was already reported and classified as likely disease‐causing VUS (class 3–4); two variants p.Leu596Arg and p.Arg811Pro, were never reported before. For p.Gly323Asp and p.Arg811Pro, we identified a second deleterious mutation at the somatic level, enforcing the possible pathogenic effect of germline variants.