The physiological phosphorylation of tau is critically changed in fetal brains of individuals with Down syndrome.

AIMS: Down syndrome (DS) is a common cause of mental retardation accompanied by cognitive impairment. Comprehensive studies suggested a link between development and ageing, as nearly all individuals with DS develop Alzheimer disease (AD)-like pathology. However, there is still a paucity of data on tau in early DS to support this notion. METHODS: Using morphometric immunohistochemistry we compared tau phosphorylation in normal brains and in brains of individuals with DS from early development until early postnatal life. RESULTS: We observed in DS a critical loss of physiological phosphorylation of tau. Rhombencephalic structures showed prominent differences between controls and DS using antibodies AT8 (Ser-202/Thr-205) and AT180 (Thr-231). In contrast, in the subiculum only a small portion of controls deviated from DS using antibodies AT100 (Thr-212/Ser-214) and AT270 (Thr-181). With exception of the subiculum, phosphorylation-independent tau did not differ between groups, as confirmed by immunostaining for the HT-7 antibody (epitope between 159 and 163 of the human tau) as well. DISCUSSION: Our observations suggest functional tau disturbance in DS brains during development, rather than axonal loss. This supports the role of tau as a further important player in the pathophysiology of cognitive impairment in DS and related AD.

Dysregulation of the (immuno)proteasome pathway in malformations of cortical development.

BACKGROUND: The proteasome is a multisubunit enzyme complex involved in protein degradation, which is essential for many cellular processes. During inflammation, the constitutive subunits are replaced by their inducible counterparts, resulting in the formation of the immunoproteasome. METHODS: We investigated the expression pattern of constitutive (ß1, ß5) and immunoproteasome (ß1i, ß5i) subunits using immunohistochemistry in malformations of cortical development (MCD; focal cortical dysplasia (FCD) IIa and b, cortical tubers from patients with tuberous sclerosis complex (TSC), and mild MCD (mMCD)). Glial cells in culture were used to elucidate the mechanisms regulating immunoproteasome subunit expression. RESULTS: Increased expression was observed in both FCD II and TSC; ß1, ß1i, ß5, and ß5i were detected (within cytosol and nucleus) in dysmorphic neurons, balloon/giant cells, and reactive astrocytes. Glial and neuronal nuclear expression positively correlated with seizure frequency. Positive correlation was also observed between the glial expression of constitutive and immunoproteasome subunits and IL-1ß. Accordingly, the proteasome subunit expression was modulated by IL-1ß in human astrocytes in vitro. Expression of both constitutive and immunoproteasome subunits in FCD II-derived astroglial cultures was negatively regulated by treatment with the immunomodulatory drug rapamycin (inhibitor of the mammalian target of rapamycin (mTOR) pathway, which is activated in both TSC and FCD II). CONCLUSIONS: These observations support the dysregulation of the proteasome system in both FCD and TSC and provide new insights on the mechanism of regulation the (immuno)proteasome in astrocytes and the molecular links between inflammation, mTOR activation, and epilepsy.

Relative survival of patients with non-malignant central nervous system tumours: a descriptive study by the Austrian Brain Tumour Registry.

BACKGROUND: Unlike malignant primary central nervous system (CNS) tumours outcome data on non-malignant CNS tumours are scarce. For patients diagnosed from 1996 to 2002 5-year relative survival of only 85.0% has been reported. We investigated this rate in a contemporary patient cohort to update information on survival. METHODS: We followed a cohort of 3983 cases within the Austrian Brain Tumour Registry. All patients were newly diagnosed from 2005 to 2010 with a histologically confirmed non-malignant CNS tumour. Vital status, cause of death, and population life tables were obtained by 31 December 2011 to calculate relative survival. RESULTS: Overall 5-year relative survival was 96.1% (95% CI 95.1-97.1%), being significantly lower in tumours of borderline (90.2%, 87.2-92.7%) than benign behaviour (97.4%, 96.3-98.3%). Benign tumour survival ranged from 86.8 for neurofibroma to 99.7% for Schwannoma; for borderline tumours survival rates varied from 83.2 for haemangiopericytoma to 98.4% for myxopapillary ependymoma. Cause of death was directly attributed to the CNS tumour in 39.6%, followed by other cancer (20.4%) and cardiovascular disease (15.8%). CONCLUSION: The overall excess mortality in patients with non-malignant CNS tumours is 5.5%, indicating a significant improvement in survival over the last decade. Still, the remaining adverse impact on survival underpins the importance of systematic registration of these tumours.

Prognostic significance of Ki67 proliferation index, HIF1 alpha index and microvascular density in patients with non-small cell lung cancer brain metastases.

BACKGROUND: Survival upon diagnosis of brain metastases (BM) in patients with non-small cell lung cancer (NSCLC) is highly variable and established prognostic scores do not include tissue-based parameters. METHODS: Patients who underwent neurosurgical resection as first-line therapy for newly diagnosed NSCLC BM were included. Microvascular density (MVD), Ki67 tumor cell proliferation index and hypoxia-inducible factor 1 alpha (HIF-1 alpha) index were determined by immunohistochemistry. RESULTS: NSCLC BM specimens from 230 patients (151 male, 79 female; median age 56 years; 199 nonsquamous histology) and 53/230 (23.0%) matched primary tumor samples were available. Adjuvant whole-brain radiation therapy (WBRT) was given to 153/230 (66.5%) patients after neurosurgical resection. MVD and HIF-1 alpha indices were significantly higher in BM than in matched primary tumors. In patients treated with adjuvant WBRT, low BM HIF-1 alpha expression was associated with favorable overall survival (OS), while among patients not treated with adjuvant WBRT, BM HIF-1 alpha expression did not correlate with OS. Low diagnosis-specific graded prognostic assessment score (DS-GPA), low Ki67 index, high MVD, low HIF-1 alpha index and administration of adjuvant WBRT were independently associated with favorable OS. Incorporation of tissue-based parameters into the commonly used DS-GPA allowed refined discrimination of prognostic subgroups. CONCLUSION: Ki67 index, MVD and HIF-1 alpha index have promising prognostic value in BM and should be validated in further studies.

[Rapid frozen sections in neuropathology]. / Der Schnellschnitt in der Neuropathologie.

Neuropathological evaluation of frozen sections requires a) special expertise in neuropathological specimen assessment and neurooncology as well as b) a trustful and open communication culture with the neurosurgeons. In addition to frozen sections, cytological examinations of smear and touch preparations as supporting methods are available to reach a correct diagnosis: these additional methods should therefore be performed whenever possible. Besides evaluation of biopsy specimens, appraisal of resection specimens and resection margin controls are of high clinical relevance. In the case of diffusely infiltrating central nervous system (CNS) neoplasms, in particular gliomas, resection margin control is often not feasible in contrast to other types of solid tumor.

FISH-based detection of 1p 19q codeletion in oligodendroglial tumors: procedures and protocols for neuropathological practice - a publication under the auspices of the Research Committee of the European Confederation of Neuropathological Societies (Euro-CNS).

The codeletion of chromosomal arms 1p 19q is a characteristic and early genetic event in oligodendroglial tumors, that is associated with a better prognosis and enhanced response to therapy. Over the last years, the increasing clinical demand to determine the 1p 19q status has led to the implementation of its testing in many neuropathology laboratories. Several different methods for 1p 19q testing are available: PCR-based loss of heterozygosity analysis, multiplex ligation-dependent probe amplification, array comparative genomic hybridization, and fluorescence in situ hybridization (FISH). Herein, we focus and critically discuss the latter method because a detailed description of procedures and protocols for FISH-based 1p 19q testing in practice is lacking. We present a practical approach to the FISH-based assessment of the 1p 19q status in oligodendroglial tumors, from commonly used locus-specific probes and technical protocols to the neuropathological interpretation of results. Thereby, we aim to facilitate the implementation of FISH-based 1p 19q testing for clinical purposes in standard neuropathology laboratories without special focus on brain tumor research.

Tumor mutational burden and immune infiltrates in renal cell carcinoma and matched brain metastases.

BACKGROUND: Tumor mutational burden (TMB) and density of tumor-infiltrating lymphocytes (TIL) have been postulated as predictive biomarkers for immunotherapy. Therefore, we investigated the concordance of TMB and TIL of primary/extracranial renal cell carcinoma (RCC) specimens and matched brain metastases (BM). PATIENTS AND METHODS: Twenty specimens from 10 patients were retrieved from the Vienna Brain Metastasis Registry (6/10 primary tumor, 4/10 lung metastasis, 10/10 matched BM). TMB was assessed using the TruSight Oncology 500 gene panel with libraries sequenced on a NextSeq instrument. TIL subsets (CD3+, CD8+, CD45RO+, FOXP3+, PD-L1+) were investigated using immunohistochemistry (Ventana Benchmark Ultra system) and automated tissue analysis (Definiens software). RESULTS: No significant difference in TMB, CD3+, CD8+, CD45RO+, FOXP3+ or PD-L1+ expression was observed between extracranial and matched intracranial specimens (P > 0.05). Higher CD8+ TIL (P = 0.053) and CD45RO+ TIL (P = 0.030) densities in the primary tumor compared with the intracranial samples were observed in specimens collected after exposure to systemic treatment. Neither extracranial sample origin (lung metastasis versus primary RCC) nor extracranial disease status at BM diagnosis (progressive versus stable disease) were significantly associated with TMB or TIL densities in extracranial and intracranial samples (P > 0.05). No significant correlation was found between the median differences of TMB or TIL densities from extracranial to intracranial samples and BM-free survival. CONCLUSION: The comparable immunological microenvironment of extra- and intracranial tumor samples in our study underscores the immunological activation also in BM from RCC, and therefore, supports the development of immune modulatory treatments also in patients with brain metastatic RCC.

Neuropathological biomarker candidates in brain tumors: key issues for translational efficiency.

Brain tumors comprise a large spectrum of rare malignancies in children and adults that are often associated with severe neurological symptoms and fatal outcome. Neuropathological tumor typing provides both prognostic and predictive tissue information which is the basis for optimal postoperative patient management and therapy. Molecular biomarkers may extend and refine prognostic and predictive information in a brain tumor case, providing more individualized and optimized treatment options. In the recent past a few neuropathological brain tumor biomarkers have translated smoothly into clinical use whereas many candidates show protracted translation. We investigated the causes of protracted translation of candidate brain tumor biomarkers. Considering the research environment from personal, social and systemic perspectives we identified eight determinants of translational success: methodology, funding, statistics, organization, phases of research, cooperation, self-reflection, and scientific progeny. Smoothly translating biomarkers are associated with low degrees of translational complexity whereas biomarkers with protracted translation are associated with high degrees. Key issues for translational efficiency of neuropathological brain tumor biomarker research seem to be related to (i) the strict orientation to the mission of medical research, that is the improval of medical practice as primordial purpose of research, (ii) definition of research priorities according to clinical needs, and (iii) absorption of translational complexities by means of operatively beneficial standards. To this end, concrete actions should comprise adequate scientific education of young investigators, and shaping of integrative diagnostics and therapy research both on the local level and the level of influential international brain tumor research platforms.

Lesion-Specific Language Network Alterations in Temporal Lobe Epilepsy.

BACKGROUND AND PURPOSE: Temporal lobe epilepsy, structural or nonlesional, may negatively affect language function. However, little is known about the lesion-specific influence on language networks. We hypothesized that different epileptogenic lesions are related to distinct alterations in the functional language connectome detected by fMRI. MATERIALS AND METHODS: One hundred one patients with epilepsy due to mesiotemporal sclerosis (21 left, 22 right), low-grade mesiotemporal tumors (12 left), or nonlesional temporal lobe epilepsy (22 left, 24 right) and 22 healthy subjects performed 3T task-based language fMRI. Task-based activation maps (laterality indices) and functional connectivity analysis (global and connectivity strengths between language areas) were correlated with language scores. RESULTS: Laterality indices based on fMRI activation maps failed to discriminate among patient groups. Functional connectivity analysis revealed the most extended language network alterations in left mesiotemporal sclerosis (involving the left temporal pole, left inferior frontal gyrus, and bilateral premotor areas). The other patient groups showed less extended but also predominantly ipsilesional network changes compared with healthy controls. Left-to-right hippocampal connectivity strength correlated positively with naming function (P = .01), and connectivity strength between the left Wernicke area and the left hippocampus was linked to verbal fluency scores (P = .01) across all groups. CONCLUSIONS: Different pathologies underlying temporal lobe epilepsy are related to distinct alterations of the functional language connectome visualized by fMRI functional connectivity analysis. Network analysis allows new insights into language organization and provides possible imaging biomarkers for language function. These imaging findings emphasize the importance of a personalized treatment strategy in patients with epilepsy.

Applied Precision Cancer Medicine in Neuro-Oncology.

Brain tumours that are refractory to treatment have a poor prognosis and constitute a major challenge in offering effective treatment strategies. By targeting molecular alterations, precision cancer medicine may be a viable option for the treatment of brain tumours. In this retrospective analysis of our PCM platform, we describe the molecular profiling of primary brain tumours from 50 patients. Tumour samples of the patients were examined by a 161-gene next-generation sequencing panel, immunohistochemistry, and fluorescence in situ hybridization (FISH). We identified 103 molecular aberrations in 36 (72%) of the 50 patients. The predominant mutations were TP53 (14.6%), IDH1 (9.7%) and PIK3CA (6.8%). No mutations were detected in 14 (28%) of the 50 patients. IHC demonstrated frequent overexpression of EGFR and mTOR, in 38 (76%) and 35 (70%) patients, respectively. Overexpression of PDGFRa and PDGFRb were less common and detected in 16 and four patients, respectively. For 35 patients a targeted therapy was recommended. In our database, the majority of patients displayed mutations, against which targeted therapy could be offered. Based on our observations, PCM may be a feasible novel treatment approach in neuro-oncology.

RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference.

Heterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inherited cancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancer syndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes. MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/or gastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-based mutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection by the analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered by the presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based on direct cDNA sequencing of RT-PCR products, we investigated two families with children suspected to suffer from MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients of the first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family. Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL "hybrid" allele carrier, that RNA-based PMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogene coamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMR deficiency and in HNPCC patients with PMS2 defects.

Ki67 index in intracranial ependymoma: a promising histopathological candidate biomarker.

AIMS: The Ki67 tumour cell proliferation index is an independent prognostic factor in ependymoma patients. Essential prerequisites for validation of the Ki67 index as a histopathological biomarker are the reproducibility of this factor and its prognostic influence by different observers (proof of objective clinical and analytical performance). To this end, the aim was to analyse systematically inter- and intraobserver agreement and reproducibility of the prognostic impact of the Ki67 index in intracranial ependymoma. METHODS AND RESULTS: The study cohort contained 78 cases of intracranial ependymoma. In all cases, the Ki67 index was assessed by four experienced observers (EOs) and by four inexperienced observers (IOs) using the manual hot-spot method. There was considerable agreement on Ki67 index assessment. There was higher observer agreement among EOs compared with IOs. For each observer, survival analysis showed significant association of low Ki67 index with favourable patient outcome. CONCLUSIONS: Our data show that the Ki67 index in intracranial ependymoma is a reproducible and robust prognostic factor and can be considered a promising histopathological candidate biomarker. Attainment of biomarker status requires further translational studies in the context of prospective therapeutic trials.

Cerebral cavernous malformations: congruency of histopathological features with the current clinical definition.

AIM: Cerebral cavernous malformations (CCMs) are defined as a mulberry-like assembly of thin walled vascular sinusoids lined by a thin endothelium lacking smooth muscle and elastin, displaying no intervening brain parenchyma. In this study, we analyse the congruency of histopathological features with the current clinical definition on a large series of neuroradiologically verified CCMs. METHODS: 87 patients who received no primary treatment prior to surgery were included. Preoperative MRIs of all patients were reviewed. 12 histopathological parameters were assessed systematically, using haematoxylin-eosin, Prussian blue, elastica van Gieson and congo red for amyloid detection. RESULTS: 71/87 (81.6%) of the cases fulfilled the basic histological criteria of CCMs. However, the thickness of the vessel walls and the calibre of the malformed vessels were highly variable. 16/87 cases (18.4%) were histologically non-diagnostic. Non-diagnostic specimens were significantly associated with radiological signs of haemorrhage (p = 0.001). A few cases (4.6%) regionally contained capillary-like malformed vessels. Intervening brain parenchyma between malformed vessels throughout the lesion was seen in 50/71 (70.4%) diagnosable lesions. Haemosiderin deposits, gliosis, thrombosis, fibrotic changes, hyalinised vessel walls, calcification and cholesterol crystals were present in a considerable range. In addition, we found amyloid deposits in 14/87 (16.1%) specimens. CONCLUSION: Contrary to the current clinical definition, the absence of intervening brain parenchyma does not represent an essential histopathological criterion of CCMs in our series. Furthermore, the diameter of the vessel lumina and the thickness of vessel walls varied considerably. Based on these findings, adaptation of the current definition on the basis of interdisciplinary interaction needs to be considered.

Reliability and reproducibility of PCR-based testing of O6-methylguanine-DNA methyltransferase gene (MGMT) promoter methylation status in formalin-fixed and paraffin-embedded neurosurgical biopsy specimens.

OBJECTIVE: To analyze the reliability and reproducibility of PCR-based testing of O6-methylguanine-DNA methyltransferase gene (MGMT) promoter methylation status in formalin-fixed and paraffin-embedded (FFPE) neurosurgical biopsy specimens. MATERIALS: We used 6 FFPE neurosurgical temporal lobe specimens of children and young adults with drug-resistant epilepsy. Histopathologically, all specimens showed CNS tissue with gliosis but no tumor tissue. METHODS: For MGMT promoter methylation analysis, we used methylation specific PCR (MGMT MSP). In all 6 tissue specimens, 4 repetitive runs of MGMT MSP were performed. RESULTS: We obtained conclusive results only in 13/24 (54.2%) MGMT MSP analyses. In 5/13 (38.5%) successful MSP runs, the results indicated presence of a methylated MGMT promoter. In only 1/6 specimens, MSP yielded consistent results in all 4 repetitive runs. CONCLUSIONS: In our hands, MGMT MSP shows poor reliability and reproducibility of test results on FFPE neurosurgical tissue specimens. A more reliable method for diagnostic MGMT promoter methylation testing needs to be identified and validated by systematic testing of intra- and interlaboratory reliability and reproducibility. Alternatively, methods of tissue fixation that do not impair DNA quality but at the same time warrant high quality histopathology could facilitate molecular diagnostics.

Brain metastases as first manifestation of advanced cancer: exploratory analysis of 459 patients at a tertiary care center.

Symptomatic brain metastases (BM) are a frequent and late complication in cancer patients. However, a subgroup of cancer patients presents with BM as the first symptom of metastatic cancer. Here we aimed to analyze the clinical course and prognostic factors of this particular BM patient population. Patients presenting with newly diagnosed BM without a history of metastatic cancer were identified from the Vienna Brain Metastasis Registry. Clinical characteristics and overall survival were retrieved by chart review. 459/2419 (19.0%) BM patients presented with BM as first symptom of advanced cancer. In 374/459 (81.5%) patients, an extracranial primary tumor, most commonly lung cancer, could be identified within 3 months after BM diagnosis. In 85/459 (18.5%) patients no extracranial primary tumor could be identified despite comprehensive diagnostic workup within the first 3 months after diagnosis of BM. Survival of patients with identified extracranial tumor differed only numerically from patients with cancer of unknown primary (CUP), however patients receiving targeted therapy after molecular workup showed significantly enhanced survival (20 months vs. 7 months; p = 0.003; log rank test). The GPA score showed a statistically significant association with median overall survival times in the CUP BM patients (class I: 46 months; class II: 7 months; class III: 4 months; class IV: 2 months; p < 0.001; log rank test). The GPA score has a strong prognostic value in patients with CUP BM and may be useful for patient stratification in the clinical setting. Comprehensive diagnostic workup including advanced imaging techniques and molecular tissue analyses appears to benefit patients by directing specific molecular targeted therapies.

Combination of isocitrate dehydrogenase 1 (IDH1) mutation and podoplanin expression in brain tumors identifies patients at high or low risk of venous thromboembolism.

Essentials Risk stratification for venous thromboembolism (VTE) in patients with brain tumors is challenging. Patients with IDH1 wildtype and high podoplanin expression have a 6-month VTE risk of 18.2%. Patients with IDH1 mutation and no podoplanin expression have a 6-month VTE risk of 0%. IDH1 mutation and podoplanin overexpression in primary brain tumors appear to be exclusive. SUMMARY: Background Venous thromboembolism (VTE) is a frequent complication in primary brain tumor patients. Independent studies revealed that podoplanin expression in brain tumors is associated with increased VTE risk, whereas the isocitrate dehydrogenase 1 (IDH1) mutation is associated with very low VTE risk. Objectives To investigate the interrelation between intratumoral podoplanin expression and IDH1 mutation, and their mutual impact on VTE development. Patients/Methods In a prospective cohort study, intratumoral IDH1 R132H mutation and podoplanin were determined in brain tumor specimens (mainly glioma) by immunohistochemistry. The primary endpoint of the study was symptomatic VTE during a 2-year follow-up. Results All brain tumors that expressed podoplanin to a medium-high extent showed also an IDH1 wild-type status. A score based on IDH1 status and podoplanin expression levels allowed prediction of the risk of VTE. Patients with wild-type IDH1 brain tumors and high podoplanin expression had a significantly increased VTE risk compared with those with mutant IDH1 tumors and no podoplanin expression (6-month risk 18.2% vs. 0%). Conclusions IDH1 mutation and podoplanin overexpression seem to be exclusive. Although brain tumor patients with IDH1 mutation are at very low risk of VTE, the risk of VTE in patients with IDH1 wild-type tumors is strongly linked to podoplanin expression levels.

Histopathological prognostic factors in medulloblastoma: high expression of survivin is related to unfavourable outcome.

Standard postoperative treatment of medulloblastoma consists of craniospinal irradiation and chemotherapy. Currently, only clinical factors are used for therapy stratification. To optimise treatment and patient outcome, biological prognostic markers are needed. In the present study we tested the prognostic influence of four histopathological parameters considered in recent publications as prognostic factors in medulloblastoma. We analysed a series of 82 Austrian medulloblastoma patients who were treated according to the consecutive HIT protocols for medulloblastoma conducted by the German Society of Paediatric Haematology and Oncology. Histological subtype and immunohistochemical expression of erbB-2, TRKC, and survivin were determined on paraffin embedded tumour tissue and correlated with patient outcome. Statistical analysis showed a significant correlation of high expression levels of survivin with decreased survival. None of the other investigated histopathological factors correlated significantly with patient outcome. Our data indicate that high survivin expression is related to unfavourable clinical outcome in medulloblastoma patients.