Medulloblastoma in adults: evaluation of the Dutch society for neuro-oncology treatment protocol.

PURPOSE: Medulloblastoma is a rare tumor in adults. The objective of this nationwide, multicenter study was to evaluate the toxicity and efficacy of the Dutch treatment protocol for adult medulloblastoma patients. METHODS: Adult medulloblastoma patients diagnosed between 2010 and 2018 were identified in the Dutch rare tumors registry or nationwide pathology database. Patients with intention to treat according to the national treatment protocol were included. Risk stratification was performed based on residual disease, histological subtype and extent of disease. All patients received postoperative radiotherapy [craniospinal axis 36 Gy/fossa posterior boost 19.8 Gy (14.4 Gy in case of metastases)]. High-risk patients received additional neoadjuvant (carboplatin-etoposide), concomitant (vincristine) and adjuvant chemotherapy (carboplatin-vincristine-cyclophosphamide) as far as feasible by toxicity. Methylation profiling, and additional next-generation sequencing in case of SHH-activated medulloblastomas, were performed. RESULTS: Forty-seven medulloblastoma patients were identified, of whom 32 were treated according to the protocol. Clinical information and tumor material was available for 28 and 20 patients, respectively. The histological variants were mainly classic (43%) and desmoplastic medulloblastoma (36%). Sixteen patients (57%) were considered standard-risk and 60% were SHH-activated medulloblastomas. Considerable treatment reductions and delays in treatment occurred due to especially hematological and neurotoxicity. Only one high-risk patient could complete all chemotherapy courses. 5-years progression-free survival (PFS) and overall survival (OS) for standard-risk patients appeared worse than for high-risk patients (PFS 69% vs. 90%, OS 81% vs. 90% respectively), although this wasn't statistically significant. CONCLUSION: Combined chemo-radiotherapy is a toxic regimen for adult medulloblastoma patients that may result in improved survival.

Development of a microfluidic collection system to measure electrolyte variations in sweat during exercise.

A wide variety of electrochemical sweat sensors are recently being developed for real-time monitoring of biomarkers. However, from a physiological perspective, little is known about how sweat biomarkers change over time. This paper presents a method to collect and analyze sweat to identify inter and intraindividual variations of electrolytes during exercise. A new microfluidic sweat collection system is developed which consists of a patch covering the collection surface and a sequence of reservoirs. Na+, Cl- and K+ are measured with ion chromatography afterwards. The measurements show that with the new collector, variations in these ion concentrations can be measured reliably over time.

Expression-based intrinsic glioma subtypes are prognostic in low-grade gliomas of the EORTC22033-26033 clinical trial.

INTRODUCTION: The European Organisation for Research and Treatment of Cancer (EORTC) 22033-26033 clinical trial (NCT00182819) investigated whether initial temozolomide (TMZ) chemotherapy confers survival advantage compared with radiotherapy (RT) in low-grade glioma (LGG) patients. In this study, we performed gene expression profiling on tissues from this trial to identify markers associated with progression-free survival (PFS) and treatment response. METHODS: Gene expression profiling, performed on 195 samples, was used to assign tumours to one of six intrinsic glioma subtypes (IGSs; molecularly similar tumours as previously defined using unsupervised expression analysis) and to determine the composition of immune infiltrate. DNA copy number changes were determined using OncoScan arrays. RESULTS: We confirm that IGSs are prognostic in the EORTC22033-26033 clinical trial. Specific genetic changes segregate in distinct IGSs: most samples assigned to IGS-9 have IDH-mutations and 1p19q codeletion, samples assigned to IGS-17 have IDH-mutations without 1p19q codeletion and samples assigned to other intrinsic subtypes often are IDH-wildtype. A trend towards benefit from RT was observed for samples assigned to IGS-9 (hazard ratio [HR] for TMZ is 1.90, P = 0.065) but not for samples assigned to IGS-17 (HR 0.87, P = 0.62). We did not identify genes significantly associated with PFS within intrinsic subtypes, although follow-up time is limited. We also show that LGGs and glioblastomas differ in their immune infiltrate, which suggests that LGGs are less amenable to checkpoint inhibitor-type immune therapies. Gene expression analysis also allows identification of relatively rare subtypes. Indeed, one patient with a pilocytic astrocytoma was identified. CONCLUSION: IGSs are prognostic for PFS in EORTC22033-26033 clinical trial samples.

An improved system approach towards future cochlear implants.

Cochlear implants (CIs) have been used for many years to restore hearing for deaf patients. Unfortunately, today's CIs are still bulky devices and uncomfortable to wear. In this paper we present three innovations that ultimately should pave the way to a fully implantable bionic ear. First a microfabrication process used to fabricate the polymer metal microelectrode array for auditory nerve stimulation is discussed. Subsequently, a compact biphasic programmable stimulator chip to be used along with this electrode array is presented. By using a double loop feedback circuit topology, the circuit provides a precise stimulation current while requiring only little voltage headroom. The resulting low power consumption and reduced chip area allow for integration of the electronic circuitry onto the electrode array. Finally, as reliability and data transmission rate are two of the most critical issues in CI devices, we propose a software method to improve both data rate and reliability of transmitting digital data from the external part of the CI to the internal part with negligible power consumption.

Gene expression profiles of gliomas in formalin-fixed paraffin-embedded material.

BACKGROUND: We have recently demonstrated that expression profiling is a more accurate and objective method to classify gliomas than histology. Similar to most expression profiling studies, our experiments were performed using fresh frozen (FF) glioma samples whereas most archival samples are fixed in formalin and embedded in paraffin (FFPE). Identification of the same, expression-based intrinsic subtypes in FFPE-stored samples would enable validation of the prognostic value of these subtypes on these archival samples. In this study, we have therefore determined whether the intrinsic subtypes identified using FF material can be reproduced in FFPE-stored samples. METHODS: We have performed expression profiling on 55 paired FF-FFPE glioma samples using HU133 plus 2.0 arrays (FF) and Exon 1.0 ST arrays (FFPE). The median time in paraffin of the FFPE samples was 14.1 years (range 6.6-26.4 years). RESULTS: In general, the correlation between FF and FFPE expression in a single sample was poor. We then selected the most variable probe sets per gene (n=17,583), and of these, the 5000 most variable probe sets on FFPE expression profiles. This unsupervised selection resulted in a better concordance (R(2)=0.54) between expression of FF and FFPE samples. Importantly, this probe set selection resulted in a correct assignment of 87% of FFPE samples into one of seven intrinsic subtypes identified using FF samples. Assignment to the same molecular cluster as the paired FF tissue was not correlated to time in paraffin. CONCLUSION: We are the first to examine a large cohort of paired FF and FFPE samples. We show that expression data from FFPE material can be used to assign samples to intrinsic molecular subtypes identified using FF material. This assignment allows the use of archival material, including material derived from large-randomised clinical trials, to determine the predictive and/or prognostic value of 'intrinsic glioma subtypes' on Exon arrays. This would enable clinicians to provide patients with an objective and accurate diagnosis and prognosis, and a personalised treatment strategy.

The impact of morphology on light transport in cancellous bone.

In recent years, optical techniques based on diffusion approximation have demonstrated their ability to gain rich spectral information about bone. However, these methods normally assume homogeneity, while cancellous bone and marrow form a highly heterogeneous two-phase medium. This paper studies the limitations of this assumption, and quantifies the role of microstructure on long-range transport properties. The propagation of light pulses through trabecular bone is calculated by Monte Carlo simulation of the scattering and absorption in reconstructions of bone samples obtained from x-ray micro tomographic scans. The time-resolved responses are then fitted with the analytical response of a homogeneous material to obtain the apparent transport properties. These properties are used to test different homogenization equations that have been postulated in the past for heterogeneous tissues and to check their accuracy. The results show that nonlinearity and crosstalk between absorption and scattering are statistically significant, although their impact is relatively small. More importantly, we found that the weight of the components is not only affected by their volume fractions, but need to be corrected by other morphologic measures like trabecular spacing or connectivity density. These deviations from the homogeneous assumption are stronger for scattering than for absorption. In conclusion, the average optical properties of cancellous bone are strongly determined by its microstructure, meaning that optical techniques are a valid method for tissue evaluation, but careful consideration of structure-related perturbation sources is required.

The CASPR2 cell adhesion molecule functions as a tumor suppressor gene in glioma.

Genomic translocations have been implicated in cancer. In this study, we performed a screen for genetic translocations in gliomas based on exon-level expression profiles. We identified a translocation in the contactin-associated protein-like 2 (CASPR2) gene, encoding a cell adhesion molecule. CASPR2 mRNA was fused to an expressed sequence tag that likely is part of the nuclear receptor coactivator 1 gene. Despite high mRNA expression levels, no CASPR2 fusion protein was detected. In a set of 25 glioblastomas and 22 oligodendrogliomas, mutation analysis identified two additional samples with genetic alterations in the CASPR2 gene and all three identified genetic alterations are likely to reduce CASPR2 protein expression levels. Methylation of the CASPR2 gene was also observed in gliomas and glioma cell lines. CASPR2-overexpressing cells showed decreased proliferation rates, likely because of an increase in apoptosis. Moreover, high CASPR2 mRNA expression level is positively correlated with survival and is an independent prognostic factor. These results indicate that CASPR2 acts as a tumor suppressor gene in glioma.

A case of pilomyxoid astrocytoma presenting with CSF rhinorrhoea in a 15-year-old.

We report a pilomyxoid astrocytoma (PMA) presenting with CSF rhinorrhoea in a 15-year-old. This uncommon, recently described entity typically presents in infancy with focal neurological or endocrine symptoms, has distinctive histologic features and displays a more aggressive behaviour than pilocytic astrocytoma (PA) with which it was previously classified.

Echinoderm microtubule-associated protein like protein 4, a member of the echinoderm microtubule-associated protein family, stabilizes microtubules.

Echinoderm microtubule-associated protein (EMAP) is the major microtubule binding protein in dividing sea urchin (Strongylocentrotus purpuratus) eggs. Echinoderm microtubule-associated protein like protein 4 (Eml4, restrictedly overexpressed proliferation-associated protein 120 kDa (Ropp120)) is one of the five mammalian EMAP homologues, the cellular function of which remains to be elucidated. In our first set of experiments we determined the spatio-temporal expression pattern of Eml4 in mouse brain. Our results demonstrate that Eml4 is a highly developmentally regulated gene with high expression levels in the developing nervous system of E11 embryos declining to low levels in adult. Spatially, Eml4 expression becomes restricted to the olfactory bulb, hippocampus and cerebellum. Transient transfection of a fusion construct of full-length mouse Eml4 with green fluorescent protein (GFP-Eml4) into Cos7 and HeLa cells resulted in colocalization of GFP-Eml4 with microtubules. This colocalization was observed both with microtubules of non-dividing cells and with the mitotic spindle of dividing cells. In addition, transient overexpression of GFP-Eml4 in Cos7 cells resulted in microtubules that were resistant to nocodazole treatment suggesting that Eml4 stabilizes microtubules. A consequence of microtubule stabilization is a net reduction in the amount of free tubulin. Microtubule stabilizing proteins therefore are expected to indirectly decrease the microtubule growth rate. Indeed, transient transfection of GFP-Eml4 resulted in a marked decrease in the microtubule growth rate, which is in line with our hypothesis that Eml4 functions as a microtubule stabilizing protein. In summary, our results suggest that Eml4 is a developmentally regulated protein that colocalizes with and stabilizes microtubules.

A case of raised intracranial pressure due to multiple lumbar nerve root melanotic schwannomas.

We report a patient with multiple cranial nerve and lumbar spine nerve root melanotic schwannomas, a rare variant of schwannoma, with raised intracranial pressure. Possible pathophysiology of the raised intracranial pressure by decreased CSF resorption due to blocked spinal lymphatics and other mechanisms is discussed.

Eml5, a novel WD40 domain protein expressed in rat brain.

We have isolated a novel transcript with homology to the major microtubule-associated protein in dividing sea urchin embryos, EMAP. The protein has a predicted MW of approximately 180 kDa and we have named it Eml5 (EMAP-like protein 5). Eml5 contains 11 putative WD40 domains and 3 hydrophobic stretches of 43 aa, HELP domains, which have been suggested to be involved in microtubule binding. Eml5 appears to consist of two tandem repeats of the complete EMAP protein separated by a putative dimerization domain. Eml5 mRNA and protein is expressed at high levels in the hippocampus, cerebellum and olfactory bulb, as determined by in situ hybridization and immunocytochemistry. Eml5 transcripts can be detected in fore- and hindbrain structures from embryonic day 13 onwards. Because other EMAP-like proteins are involved in regulating microtubule dynamics, it is likely that Eml5 plays a role in the regulation of cytoskeletal rearrangements during neuronal development and in adult brain

Ntab, a novel non-coding RNA abundantly expressed in rat brain.

We have identified a novel transcript that is abundantly and specifically expressed in both the adult and developing rat CNS. Within the full-length cDNA sequence we were unable to identify a clear open reading frame. Moreover, we were unable to detect any protein product derived from the full-length cDNA sequence using an in vitro translation assay. Therefore, we suggest this gene is one of a growing number of non-coding mRNA-like RNA transcripts that exert their cellular functions directly as an RNA. We have named this novel gene Ntab for non-coding transcript abundantly expressed in brain (accession number AY035551). In addition, in some regions of the brain we find evidence for RNA accumulation in cellular processes at some distance from the soma. These findings suggest that Ntab is actively transported and may function within cellular processes. Since Ntab is a targeted non-coding RNA, such cellular functions could include the targeting and/or regulation of localised translation of other mRNA species.

Subfield-specific immediate early gene expression associated with hippocampal long-term potentiation in vivo.

It is not known whether NMDA receptor-dependent long-term potentiation (LTP) is mediated by similar molecular mechanisms in different hippocampal areas. To address this question we have investigated changes in immediate early gene and protein expression in two hippocampal subfields following the induction of LTP in vivo and in vitro. In granule cells of the dentate gyrus, LTP induced in vivo by tetanic stimulation of the perforant path was followed by strong induction of the immediate early genes (IEGs) Zif268, Arc and Homer. The increase in Zif268 mRNA was accompanied by an increase in protein expression. In contrast, we were unable to detect modulation of the IEGs Zif268, Arc, Homer and HB-GAM following induction of LTP by high-frequency stimulation of the commissural projection to CA1 pyramidal cells in vivo. In this pathway, we also failed to detect modulation of Zif268 protein levels. Zif268, Arc and Homer can be modulated in CA1 pyramidal cells approximately twofold after electroshock-induced maximal seizure, which demonstrates potential responsiveness to electrical stimuli. When LTP was induced in vitro neither CA1 pyramidal cells nor granule cells showed an increase in Zif268, Arc or Homer mRNA. However, in the slice preparation, granule cells have a different transcriptional state as basal IEG levels are elevated. These results establish the existence of subfield-specific transcriptional responses to LTP-inducing stimulation in the hippocampus of the intact animal, and demonstrate that in area CA1-enhanced transcription of Zif268, Arc and Homer is not required for the induction of late LTP.

A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories.

The induction of long-term potentiation (LTP) in the dentate gyrus of the hippocampus is associated with a rapid and robust transcription of the immediate early gene Zif268. We used a mutant mouse with a targeted disruption of Zif268 to ask whether this gene, which encodes a zinc finger transcription factor, is required for the maintenance of late LTP and for the expression of long-term memory. We show that whereas mutant mice exhibit early LTP in the dentate gyrus, late LTP is absent when measured 24 and 48 hours after tetanus in the freely moving animal. In both spatial and non-spatial learning tasks, short-term memory remained intact, whereas performance was impaired in tests requiring long-term memory. Thus, Zif268 is essential for the transition from short- to long-term synaptic plasticity and for the expression of long-term memories.

Seizure-induced gene expression in area CA1 of the mouse hippocampus.

Synaptic plasticity in the hippocampus requires activity-dependent gene expression. We have therefore profiled gene expression in area CA1 following the induction of an electroshock-evoked maximal seizure. Using cDNA microarrays, the differential expression of approximately 9000 cDNAs was examined. In situ hybridization on 14 transcripts that showed strongest modulation in the microarray screen (1.8-2-fold) confirmed the differential expression of a single gene that encodes for the nuclear hormone receptor NGFI-B (Nur77, N10). Although this gene is only modestly up-regulated (approximately 2-fold) in area CA1, in situ hybridization revealed that maximal seizures induce a marked (approximately 12-fold) up-regulation of NGFI-B in the dentate gyrus. These data support the notion [French et al. (2001) Eur. J. Neurosci., 13, 968-976] that CA1 pyramidal neurons are more refractory than granule cells of the dentate gyrus with respect to activity-dependent gene transcription. Furthermore, our results argue against a large cohort of activity-dependent genes in area CA1.

Molecular mechanisms of long-term potentiation in the insular cortex in vivo.

We have investigated molecular mechanisms of synaptic plasticity in the pathway between two forebrain structures important for taste learning, the basolateral amygdala (BLA) and the insular cortex. We report here that in vivo long-term potentiation (LTP) induced by BLA stimulation requires functional NMDA receptors and is modulated by muscarinic acetylcholine receptors. In addition, LTP results in the activation of cortical extracellular regulated kinase 1/2 (ERK1/2) and is blocked by inhibitors of ERK1/2 activation. Previous findings demonstrated the involvement of the same molecular mechanisms in the same cortical area during novel taste learning. The results demonstrate that both synaptic and behavioral plasticity share common molecular mechanisms in the insular cortex.

Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism.

Previous studies have revealed an adenosine 3',5'-cyclic monophosphate (cAMP)-independent activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by the tyrosine kinase inhibitor genistein. To further explore its mechanism of action, we have reconstituted genistein activation of CFTR in excised inside-out membrane patches. In the presence or absence of ATP, genistein appeared unable to open silent CFTR Cl- channels. However, on CFTR prephosphorylation by cAMP-dependent protein kinase (cAK), genistein enhanced CFTR activity by twofold, resulting from a prolonged burst duration. Genistein could also hyperactivate partially phosphorylated CFTR in the absence of cAK and therefore is different from 5'-adenylylimidodiphosphate, which required fully phosphorylated CFTR. Phosphatase-resistant thiophosphorylation likewise primed the CFTR Cl- channel for hyperactivation by genistein in the absence of cAK. Replacement of ATP by GTP as a hydrolyzable nucleotide triphosphate for CFTR did not impair the ability of genistein to activate thiophosphorylated CFTR, despite the fact that GTP is a poor substrate for tyrosine kinases. These findings argue against a role of protein phosphatases or tyrosine kinases but suggest a more direct interaction of genistein with CFTR, possibly at the level of the second nucleotide-binding domain.

Cystic fibrosis transmembrane conductance regulator mediates the cyclic adenosine monophosphate-induced fluid secretion but not the inhibition of resorption in mouse gallbladder epithelium.

We have studied the physiological role of the cystic fibrosis (CF) gene product (cystic fibrosis transmembrane conductance regulator [CFTR]) in gallbladder epithelium using a knockout mouse model for CF. We found that normal mouse gallbladder epithelium expresses functional CFTR as shown by reverse-transcription polymerase chain reaction (RT-PCR) analysis and Ussing chamber experiments. Gallbladders from Cftr -/- mice were structurally intact as shown by microscopic and physiological parameters but lacked the cyclic adenosine monophosphate (cAMP)-induced chloride current observed in normal gallbladders. In fluid transport measurements, normal and Cftr -/- gallbladders were equally active in basal resorption. The addition of forskolin, which activates CFTR anion channel activity through the cAMP system, resulted in net fluid secretion in normal gallbladders. In contrast, Cftr -/- gallbladders were unable to secrete fluid while a complete inhibition of resorption by forskolin was observed. We conclude that, in normal mouse gallbladder epithelium, cAMP-induced fluid secretion involves simultaneous inhibition of apical sodium chloride resorption and activation of CFTR. Our data support the hypothesis that gallbladder disease in CF is at least in part caused by a deficient secretory response to the endogenous cAMP-linked hormones VIP and secretin.

CFTR expression and mucin secretion in cultured mouse gallbladder epithelial cells.

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in humans is frequently associated with progressive liver disease, which appears to result from obstruction of biliary ducts with mucous material. CFTR in the liver is expressed in the biliary epithelium. With the use of a mouse model for cystic fibrosis (CF) we have studied the relationship between CFTR expression and glycoprotein secretion in primary culture of mouse gallbladder epithelial cells (MGBC) MGBC in culture maintain a well-differentiated phenotype as shown by microscopy. The cells produce CFTR mRNA to levels comparable to the intact tissue. With patch-clamp analysis we could frequently observe a linear protein kinase A-regulated Cl- channel that shows all the major characteristics of human CFTR, although its conductance is lower (5 pS compared with 8 pS). MGBC in culture produce and secrete high molecular weight glycoproteins (HMG) in a time-dependent and temperature-sensitive manner. Secretion of HMG was not stimulated significantly by either adenosine 3',5'-cyclic monophosphate (cAMP), Ca2+, or protein kinase C agonists in this system. High concentrations (3 mM) of extracellular ATP stimulated secretion threefold, but low concentrations (0.3 mM) had no effect. Approximately one-third of the HMG produced and secreted consisted of mucin. Cultured MGBC from CFTR-deficient mice produced and secreted mucin to a similar extent as normal cells. We conclude that cultured mouse gallbladder cells are a convenient model to study both CFTR function and mucin secretion. In this system, we found no evidence for a direct link between mucin secretion and CFTR activity, as has been suggested for other cell types.

A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo.

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.