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Background (1) Description of clinical and cranial MRI features in the original Pontine Autosomal Dominant Microangiopathy with Leukoencephalopathy (PADMAL) family and correlation with the segregation analysis of the causative collagen 4A1 gene (COL4A1) variant. (2) Sequence analysis of the COL4A1 miRNA-binding site containing the causative variant in two independent cross-sectional samples of sporadic stroke patients. Patients and methods Sanger sequencing of the COL4A1 miRNA-binding site in the PADMAL family and 874 sporadic stroke patients. Results PADMAL shows adult-onset usually between 30 and 50 years of age with initial brainstem-related symptoms most commonly dysarthria, with progression to dementia and tetraparesis. Radiologically pontine lacunes are followed by supratentorial white matter involvement. Radiological onset may precede clinical symptoms. We found no variants in the COL4A1 miRNA-binding site of sporadic stroke patients. Conclusion Our results allow an early diagnosis of PADMAL based on cranial MRI, clinical signs, and confirmatory sequencing of the COL4A1 miRNA-29-binding site. COL4A1 miRNA-29-binding site variants do not contribute to a sizeable proportion of sporadic stroke.

Background Plexiform neurofibromas can transform into atypical neurofibromas (ANF) and then further progress to aggressive malignant peripheral nerve sheath tumors (MPNST). ANF have been described to harbor distinct histological features and frequent loss of CDKN2A/B. However, histological evaluation may be rater-dependent, and detailed knowledge about the molecular mechanisms of malignant transformation is scarce. In general, malignant transformation can be accompanied by significant epigenetic changes, and global DNA methylation profiling is able to differentiate relevant tumor subgroups. Therefore, epigenetic profiling might provide a valuable tool to distinguish and characterize ANF with differing extent of histopathological atypia from neurofibromas and MPNST. Methods We investigated 40 tumors histologically diagnosed as ANF and compared their global methylation profile to other peripheral nerve sheath tumors. Results Unsupervised class discovery and t-SNE analysis indicated that 36/40 ANF cluster with benign peripheral nerve sheath tumors with clear separation from MPNST. 21 ANF formed a molecularly distinct cluster in proximity to schwannomas. Tumors in this cluster had a frequent heterozygous or homozygous loss of CDKN2A/B and significantly more lymphocyte infiltration than MPNST, schwannomas, and NF. Few ANF clustered closely with neurofibromas, schwannomas, or MPNST, raising the question, whether diagnosis based on histological features alone might pose a risk to both over- and underestimate the aggressiveness of these lesions. Conclusions Our data suggest that ANF with varying histological morphology show distinct epigenetic similarities and cluster in proximity to benign peripheral nerve sheath tumor entities. Future investigations should pay special respect to correlating this methylation pattern to clinical outcomes.

BACKGROUND: Medulloblastoma is the most common malignant paediatric brain tumour, and cerebrospinal fluid (CSF) dissemination (M1 stage) is a high-risk prognostic factor. Criteria for CSF evaluation and for differentiating M0 from M1 stage are not clearly defined, and the prognostic significance of M1 stage in this context is unknown.PATIENTS AND METHODS: CSF investigations from 405 patients with medulloblastoma of the prospective multicenter trial HIT-2000 (HIirnTumor-2000) were reviewed. Data from 213 patients aged ≥4 years were related to 5-year progression-free (5y-PFS) and overall survival.RESULTS: Patients with cytological tumour dissemination only (M1 stage only) aged ≥4 years (n = 18) and patients with radiologically detected metastases (M2/3, n = 85) showed a worse 5y-PFS than M0 patients (n = 110) without signs of metastatic disease (5y-PFS 61.1% and 59.6% vs 80.7%; p < 0.02 and p < 0.01, log rank). Patients with positive samples drawn early after surgery who turned negative within 14 days postoperatively (n = 9) and patients with atypical cells (n = 6) showed a 5y-PFS similar to M0 patients. No tumour cells were detected in samples containing CONCLUSION: Based on our results, we suggest that CSF medulloblastoma staging should be performed 14 days postoperatively by lumbar puncture, and specimens should contain at least 10 nucleated cells. Cytological tumour dissemination alone (M1 stage only) appears a high-risk prognostic factor associated with an outcome comparable to M2/M3 stage. Tumour cell clusters seem to have a greater impact on prognosis than single tumour cells. This should be validated further.

ZusammenfassungIn den letzten Jahren wurden biologische Subgruppen des Ependymoms (EPN) identifiziert und in die aktuelle WHO-Klassifikation eingeführt, allerdings haben diese derzeit noch keinen Einfluss auf die Therapiestratifizierung. Jedoch finden sich immer mehr Hinweise, dass das klinische Verhalten abhängig von der Tumorbiologie ist: PF-EPN-A mit einem Zugewinn des Chromosom 1q beispielsweise sind mit einem hohen Risiko für Rezidive assoziiert, während das Überleben bei ST-EPN-YAP1 bei>90% liegt. Die insgesamt noch nicht zufriedenstellende Prognose zeigt, dass die Therapiestrategien weiter verbessert werden müssen. Das Ausmaß der Tumorresektion ist der wichtigste klinische Prognosefaktor, weshalb die Evaluation einer erneuten OP bei einem postoperativen Resttumor höchste Priorität hat. Darüber hinaus wird die Wirksamkeit der Lokalbestrahlung als bewiesen angesehen und derzeit im Alter>1 Jahr empfohlen. Der Stellenwert der Chemotherapie ist weiterhin unklar. Ein Ziel der europäischen klinischen Studie SIOP Ependymoma II ist, die Wirksamkeit verschiedener Chemotherapien zu evaluieren, so dass deutsche Patienten im Rahmen dieser Studie behandelt werden sollten, um den Kenntnisstand voranzutreiben. Neue prognostische biologische Parameter werden im Rahmen des Begleitprojekts BIOMECA validiert. Die Erkenntnisse dieser Begleitstudie sollen auch genutzt werden, um zielgerichtete Therapien sog. Targeted Therapies zu entwickeln. Für Patienten, die nicht im Rahmen der interventionellen Strata der Studie behandelt werden können, stellt die HIT-MED Guidance (aktuelle Version 5.2) eine Handlungsempfehlung dar. Der vorliegende Artikel gibt einen Überblick über Empfehlungen zu Diagnostik und Therapie für Kinder- und Jugendliche mit intrakraniellem Ependymom, sowie zur Behandlung im Rahmen der SIOP Ependymoma II Studie.

Purpose Pituitary adenoma (PA) constitutes the third most common intracranial neoplasm. The mostly benign endocrine lesions express no hormone (null cell PA) or the pituitary hormone(s) of the cell lineage of origin. In 0.5–1.5% of surgical specimens and in up to 10% of autopsy cases, two or three seemingly separate PA may coincide. These multiple adenomas may express different hormones, but whether or not expression of lineage-restricted transcription factors and molecular features are distinct within multiple lesions remains unknown. Methods Searching the data bank of the German Pituitary Tumor Registry 12 double pituitary adenomas with diverse lineage were identified among 3654 adenomas and 6 hypophyseal carcinomas diagnosed between 2012 and 2020. The double adenomas were investigated immunohistochemically for expression of hormones and lineage markers. In addition, chromosomal gains and losses as well as global DNA methylation profiles were assessed, whenever sufficient material was available (n = 8 PA). Results In accordance with the literature, combinations of GH/prolactin/TSH–FSH/LH adenoma (4/12), GH/prolactin/TSH–ACTH adenoma (3/12), and ACTH–FSH/LH adenoma (3/12) were observed. Further, two out of 12 cases showed a combination of a GH/prolactin/TSH adenoma with a null-cell adenoma. Different expression pattern of hormones were confirmed by different expression of transcription factors in 11/12 patients. Finally, multiple lesions that were molecularly analysed in 4 patients displayed distinct copy number changes and global methylation pattern. Conclusion Our data confirm and extend the knowledge on multiple PA and suggest that such lesions may origin from distinct cell types.

Pituitary neuroendocrine tumour (PitNET)/adenoma classification is based on cell lineage and requires immunopositivity for adenohypophysial hormones and/or transcription factors (TFs) SF1, TPIT, or PIT1. PitNET/adenomas lacking lineage affiliation are termed "null cell" tumours (NCTs). NCT diagnosis may be afflicted by methodological limitations and inconsistent diagnostic approaches. Previous studies have questioned the existence of true NCTs. In this study, we explore the epigenomic identities of PitNET/adenomas lacking clear TF-immunopositivity.74 hormone-negative PitNET/adenomas were immunostained and scored for SF1, TPIT, and PIT1 expression. All tumours were classified as gonadotroph, corticotroph, PIT1-positive or "null cell". NCTs were subjected to global DNA methylation analysis. Epigenomic profiles of NCTs were compared to reference tumours using UMAP plotting and methylation-based classification.TF-immunostaining revealed definite lineage identity in 59 of 74 (79.7%) hormone-negative PitNET/adenomas. Of the remaining 15 NCTs, 13 demonstrated minimal and inconclusive nuclear SF1- or TPIT-expression (5 and 8, respectively). Two NCTs were entirely immunonegative. UMAP plotting and methylation-based classification demonstrated that the epigenomes of NCTs with minimal SF1- or TPIT-expression, were adequately affiliated with gonadotroph or corticotroph lineages, respectively. The two immunonegative NCTs were located near the corticotroph PitNET/adenomas via UMAP, while the methylation classifier could not match these two cases to predefined tumour classes.Epigenomic analyses substantiate lineage identification based on minimal TF-immunopositivity in PitNET/adenomas. This strategy dramatically decreases the incidence of NCTs and further challenges the legitimacy of NCTs as a distinct PitNET/adenoma subtype. Our study may be useful for guiding diagnostic efforts and future considerations of PitNET/adenoma classification.

Purpose The identification of prognostically and therapeutically relevant molecular markers is fundamental to the further development of personalised therapies in brain tumours. Current therapeutic options for the treatment of gliomas rely mainly on surgical resection and the inhibition of tumour cell proliferation by irradiation and chemotherapy. Glioma stem cells are a subpopulation of proliferating tumour cells that have self-renewal capacity and can give rise to heterogeneous cells that comprise the tumour and are thought to play a role in the resistance of gliomas to therapy. The aim of this study was to evaluate the expression of markers of glioma stem cells and differentiated glial cells in proliferating glioma cells in comparison to the overall expression of the respective markers in the tumour tissue. Methods Tissue microarrays were assembled from specimen of pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, and anaplastic ependymoma. These were immunohistochemically double stained with antibodies against the proliferation-associated antigen Ki67 and marker proteins for glioma stem cells (CD133, Nestin, Musashi, CD15, CD44), and differentiated glioma cells (GFAP, MAP2c). Results The expression of both glial and glioma stem cell markers differs between proliferating and non-proliferating glioma cells. Furthermore, the proliferating cells in the different glial tumour entities show a different expression profile. Conclusion Further analysis of marker expression in proliferating glioma cells and correlation with clinical outcome and susceptibility to irradiation and chemotherapy might help establish new biomarkers and therapies for glioma.

Astrocytes are increasingly being recognized as contributors to physiological brain function and behavior. Astrocytes engage in glia-synaptic interactions through peripheral astrocyte processes, thus modulating synaptic signaling, for example, by handling glutamate removal from the synaptic cleft and (re)provision to axonal terminals. Peripheral astrocyte processes are ultrafine membrane protrusions rich in the membrane-to-actin cytoskeleton linker Ezrin, an essential component of in vitro filopodia formation and in vivo peripheral astrocyte process motility. Consequently, it has been postulated that Ezrin significantly contributes to neurodevelopment as well as astrocyte functions within the adult brain. However, while Ezrin has been studied in vitro within cultured primary astrocytes, in vivo studies on the role of Ezrin in astrocytes remain to be conducted and consequences of its depletion to be studied. Here, we investigated consequences of Ezrin deletion in the mouse brain starting from early neuronal specification. While Ezrin knockout did not impact prenatal cerebral cortex development, behavioral phenotyping depicted reduced exploratory behavior. Starting with postnatal appearance of glia cells, Ezrin was verified to remain predominantly expressed in astrocytes. Proteome analysis of Ezrin deficient astrocytes revealed alterations in glutamate and ion homeostasis, metabolism and cell morphology - important processes for synaptic signal transmission. Notably, Ezrin deletion in astrocytes provoked (GFAP) glial fibrillary acidic protein upregulation - a marker of astrocyte activation and reactive astrogliosis. However, this spontaneous, reactive astrogliosis exhibited proteome changes distinct from ischemic-induced reactive astrogliosis. Moreover, in experimental ischemic stroke, Ezrin knockout mice displayed reduced infarct volume, indicating a protective effect of the Ezrin deletion-induced changes and astrogliosis.

We studied small vessel disease (SVD) pathology in Familial Alzheimer's disease (FAD) subjects carrying the presenilin 1 (PSEN1) p.Glu280Ala mutation in comparison to those with sporadic Alzheimer's disease (SAD) as a positive control for Alzheimer's pathology and Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) bearing different NOTCH3 mutations, as positive controls for SVD pathology. Upon magnetic resonance imaging (MRI) in life, some FAD showed mild white matter hyperintensities and no further radiologic evidence of SVD. In post-mortem studies, total SVD pathology in cortical areas and basal ganglia was similar in PSEN1 FAD and CADASIL subjects, except for the feature of arteriosclerosis which was higher in CADASIL subjects than in PSEN1 FAD subjects. Further only a few SAD subjects showed a similar degree of SVD pathology as observed in CADASIL. Furthermore, we found significantly enlarged perivascular spaces in vessels devoid of cerebral amyloid angiopathy in FAD compared with SAD and CADASIL subjects. As expected, there was greater fibrinogen-positive perivascular reactivity in CADASIL but similar reactivity in PSEN1 FAD and SAD groups. Fibrinogen immunoreactivity correlated with onset age in the PSEN1 FAD cases, suggesting increased vascular permeability may contribute to cognitive decline. Additionally, we found reduced perivascular expression of PDGFRβ AQP4 in microvessels with enlarged PVS in PSEN1 FAD cases. We demonstrate that there is Aβ-independent SVD pathology in PSEN1 FAD, that was marginally lower than that in CADASIL subjects although not evident by MRI. These observations suggest presence of covert SVD even in PSEN1, contributing to disease progression. As is the case in SAD, these consequences may be preventable by early recognition and actively controlling vascular disease risk, even in familial forms of dementia.

Background/aim Neurofibromatosis type 1 (NF) is an autosomal dominant hereditary disease. The cardinal clinical findings include characteristic skeletal alterations. Difficulties in diagnosis and therapy can arise if an individual has further illnesses. Case report This is a case report of a 16-year-old patient affected by NF1. She also suffered from Alagille syndrome and the consequences of fetal alcohol exposure. The patient's facial phenotype showed findings that could be assigned to one or more of the known diseases. The patient was referred for treating a cherubism-like recurrent central giant cell granuloma (CGCG) of the jaw. The patient developed bilateral, multilocular non-ossifying fibromas (NOF) of the long bones of the lower extremity. Treatment of the skeletal lesions consisted of local curettage. While NOF regressed after surgery, the CGCG of the jaw remained largely unchanged. Extensive genetic tests confirmed a previously unknown germline mutation in the JAG1 gene, the germline mutation of the NF1 gene, and the somatic mutation in the NF1 gene in the diffuse plexiform neurofibroma, but not in the CGCG. Conclusion Assigning facial findings to a defined syndrome is ambiguous in many cases and especially difficult in patients who have multiple diseases that can affect the facial phenotype. Surgical therapy should be adapted to the individual findings.

Purpose To analyze the co-expression of the intermediate filaments GFAP and cytokeratin in 326 pituitary adenomas with regard to the distribution pattern, the subtype of the adenoma and clinical prognostic data. Methods Tissue from 326 pituitary adenomas and 13 normal anterior pituitaries collected in the Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, between 2006 and 2009 was investigated by immunohistochemistry, immunofluorescence and electron microscopy. Results Co-expression of intermediate filaments GFAP and cytokeratin was associated with hormone expression in 62/278 cases (22%), but only found in 2/48 (4%) of null cell adenomas (p Conclusions Cellular co-expression of the intermediate filaments GFAP and cytokeratin in pituitary adenomas and the pituitary was demonstrated and shown to be associated with hormone expression and low recurrence rate. The results are discussed with regard to the biology of folliculostellate cells, neural transformation and tumor stem cells. This study may complement the understanding of pituitary adenoma biology.

Background Neurofibromatosis type 1 (NF1) is an autosomal dominant hereditary disease with complete penetrance and a very variable phenotype. Recent research has shown that postzygotic NF1 gene mutations occur to a far greater extent than previously thought. The phenotype of affected individuals reflects the time of somatic mutation and the phenotype is correspondingly diverse. This report describes histological and genetic findings in a case of mosaic NF1, the clinical control of which documents almost stationary skin findings over a period of 9 years. Case report The 55-year-old female first presented for advice on a strip of nodular skin tumours of the calf skin. She had no hallmarks of NF1. It was only 9 years later that she had the skin tumours removed, all of which were partially diffuse and partially plexiform neurofibroma. The genetic examination showed an atypical large deletion of the NF1 gene in the skin tumours, but not in overlying skin or blood. Conclusion Segmental NF1 is a distinct type of mosaic/somatic NF1 mutation. The phenotype of diffuse and plexiform skin neurofibromas can resemble cutaneous neurofibroma. Surgical therapy for segmental neurofibromatosis does not differ from the concepts for treating nerve sheath tumours in NF1 patients with a germline NF1 mutation.

OBJECTIVETo shed light on the role of relaxin in cerebral cavernous malformations (CCMs) in adults and children, the authors investigated endothelial cell (EC) expression of relaxin 1, 2, and 3; vascular endothelial growth factor receptor–1 and –2 (VEGFR-1 and -2); Ki-67; vascular geometry; and hemorrhage, as well as the clinical presentation of 32 patients with surgically resected lesions.METHODSParaffin-embedded sections of 32 CCMs and 5 normal nonvascular lesion control (NVLC) brain tissue samples were immunohistochemically stained with antibodies to relaxin 1, 2, and 3; angiogenesis growth factor receptors Flt-1 (VEGFR-1) and Flk-1 (VEGFR-2); and proliferation marker Ki-67. For morphometric analysis, Elastica van Gieson stain was used, and for hemorrhage demonstration, Turnbull stain was used. Data from the pediatric and adult CCMs were compared with each other and with those obtained from the NVLCs. Statistical analyses were performed with Fisher’s exact test, the chi-square test, the phi correlation coefficient, and the Student t-test. A p value < 0.05 was considered significant.RESULTSPediatric and adult cavernoma vessels did not significantly differ in diameter. Hemorrhage was observed in CCMs but not in NVLC samples (p < 0.05). There was no difference in expression of Ki-67, VEGFR-1 and -2, and relaxin 1, 2, and 3 in the ECs of pediatric and adult CCMs. The ECs of CCMs were largely negative for relaxin 3 compared to NVLCs (p < 0.05), whereas CCMs, compared to control brain tissue samples, more frequently expressed Flt-1 and relaxin 2 (p < 0.05). Ki-67 was not expressed in the NVLCs, but the difference was not statistically significant. Relaxin 1 and 2 expression and increased expression of VEGFR-1 were associated with a supra- versus infratentorial location (p < 0.05).CONCLUSIONSRelaxin 1 and 2 and VEGFR-1 play a role in supratentorial cavernomas. Relaxin 3 may play a physiological role in normal brain vasculature. Relaxin 1 and 3 are also found in normal cerebral vasculature. Relaxin 1, 2, and 3 are associated with increased VEGFR-1 expression.

The receptor tyrosine kinase Axl is described to promote migration, metastasis and resistance against molecular targeting, radiotherapy, and chemotherapy in various tumor entities, including head and neck squamous cell carcinoma (HNSCC). Since clinical data on Axl and its ligand Gas6 in HNSCC are sparse, we assessed the association of Axl and Gas6 expression with patient survival in a single center retrospective cohort in a tissue microarray format. Expression was evaluated manually using an established algorithm and correlated with clinicopathological parameters and patient survival. A number of 362 samples yielded interpretable staining, which did not correlate with T- and N-stage. Protein expression levels were not associated with the survival of patients with p16-positive oropharyngeal SCC. In HPV-negative tumors, Axl expression did not impact patients treated with primary or adjuvant radio(chemo)therapy, but was significantly associated with inferior overall and recurrence-free survival in patients treated with surgery alone. Gas6 was a positive predictor of survival in patients whose treatment included radiotherapy. Associations remained significant in multivariable analysis. Our data question a meaningful contribution of the Axl/Gas6 pathway to radio-resistance in HNSCC and instead suggest that strong Axl expression identifies tumors requiring adjuvant radio(chemo)therapy after surgery.

To characterize the growth pattern and antigen profile of peripheral nerve sheaths tumors (PNST) in a large series of tumors obtained from patients with neurofibromatosis type 1 (NF1).Tissue micro-array technique was applied to study 520 PNSTs of 385 patients with NF1 by immunohistochemistry for human epidermal growth factor receptors erb-b2 receptor tyrosine kinase 2 (ERBB2) and ERBB3, CD44, neuroregulin (NRG1) and proliferation index by Ki-67. PNSTs were classified as cutaneous neurofibroma (CNF) in 114 cases, diffuse neurofibroma (DNF) in 109, diffuse plexiform neurofibroma (DPNF) in 108, plexiform neurofibroma (PNF) in 110, and malignant PNST (MPNST) in 22.The Ki-67 proliferation index was significantly higher in MPNST than in benign PNST (p0.001). ERBB2 expression was significantly lower in PNST with diffuse growth than in PNF and MPNST (p0.001). ERBB3 expression was also higher in PNF and MPNST (both p0.001) than in diffuse PNST. NRG1 expression was significantly higher in PNF than in non-encapsulated benign PNST or MPNST (both p0.001). Co-expression of ERBB2, ERBB3 and ligand NRG1 was rare, mainly observed in PNST with a plexiform component (in four PNFs, nine DPNFs, one CNF, and two MPNSTs). Expression of CD44 in contrast was significantly stronger in diffusely growing PNST than in PNF (p0.001).Growth factor receptors ERBB2 and ERBB3 were significantly up-regulated in PNF and MPNST. The antigen expression pattern of DPNF resembled that of benign PNST with diffuse growth pattern rather than that of encapsulated PNF. Differentiating PNST may be important for the assessment of neurofibroma progression, and for the expected impact of drugs currently used for tumor reduction.

Background/Aim: The aim of this study was to present the long-term course of a patient with nevus sebaceous syndrome (NSS). Recent genetic studies place the syndrome in the emerging group of so-called RASopathies. The focus of the report is on surgical treatment and morphological and genetic findings of the face and oral cavity. Case Report: A female patient was treated for congenital alterations of facial skin and oral mucosa. The oral lesions were removed repeatedly. Eruption of teeth on the lesion sites was made easier by the measures taken. However, after repeated ablation of the affected gingiva, the periodontal papillomatous epithelium re-differentiated into the same reddish, conspicuous, hyperplastic epithelium. The teeth in the affected region showed noticeable changes in position, surface, and shape. A HRAS mutation was detected only in the regions of altered oral epithelia and not in adjacent soft tissues. Conclusion: Reports on NSS rarely address oral manifestations. The recorded alterations of oral soft and hard tissues in NSS indicate a topographical relationship between the development of oral mucosa and teeth as well as the long-lasting impact of a sporadic mutation on organ development at this site.