EGFL7 loss correlates with increased VEGF-D expression, upregulating hippocampal adult neurogenesis and improving spatial learning and memory.

Neural stem cells reside in the subgranular zone, a specialized neurogenic niche of the hippocampus. Throughout adulthood, these cells give rise to neurons in the dentate gyrus, playing an important role in learning and memory. Given that these core cognitive processes are disrupted in numerous disease states, understanding the underlying mechanisms of neural stem cell proliferation in the subgranular zone is of direct practical interest. Here, we report that mature neurons, neural stem cells and neural precursor cells each secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this hippocampal niche. We further demonstrate that EGFL7 knock-out in a Nestin-CreERT2-based mouse model produces a pronounced upregulation of neurogenesis within the subgranular zone. RNA sequencing identified that the increased expression of the cytokine VEGF-D correlates significantly with the ablation of EGFL7. We substantiate this finding with intraventricular infusion of VEGF-D upregulating neurogenesis in vivo and further show that VEGF-D knock-out produces a downregulation of neurogenesis. Finally, behavioral studies in EGFL7 knock-out mice demonstrate greater maintenance of spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation. Taken together, our findings demonstrate that both EGFL7 and VEGF-D affect neurogenesis in the adult hippocampus, with the ablation of EGFL7 upregulating neurogenesis, increasing spatial learning and memory, and correlating with increased VEGF-D expression.

A new approach for clinical translation of infrared spectroscopy: exploitation of the signature of glioblastoma for general brain tumor recognition.

PURPOSE: Infrared (IR) spectroscopy has the potential for tumor delineation in neurosurgery. Previous research showed that IR spectra of brain tumors are generally characterized by reduced lipid-related and increased protein-related bands. Therefore, we propose the exploitation of these common spectral changes for brain tumor recognition. METHODS: Attenuated total reflection IR spectroscopy was performed on fresh specimens of 790 patients within minutes after resection. Using principal component analysis and linear discriminant analysis, a classification model was developed on a subset of glioblastoma (n = 135) and non-neoplastic brain (n = 27) specimens, and then applied to classify the IR spectra of several types of brain tumors. RESULTS: The model correctly classified 82% (517/628) of specimens as "tumor" or "non-tumor", respectively. While the sensitivity was limited for infiltrative glioma, this approach recognized GBM (86%), other types of primary brain tumors (92%) and brain metastases (92%) with high accuracy and all non-tumor samples were correctly identified. CONCLUSION: The concept of differentiation of brain tumors from non-tumor brain based on a common spectroscopic tumor signature will accelerate clinical translation of infrared spectroscopy and related technologies. The surgeon could use a single instrument to detect a variety of brain tumor types intraoperatively in future clinical settings. Our data suggests that this would be associated with some risk of missing infiltrative regions or tumors, but not with the risk of removing non-tumor brain.

Leptomeningeal Metastases in Melanoma Patients: An Update on and Future Perspectives for Diagnosis and Treatment.

Leptomeningeal disease (LMD) is a devastating complication of cancer with a particularly poor prognosis. Among solid tumours, malignant melanoma (MM) has one of the highest rates of metastasis to the leptomeninges, with approximately 10-15% of patients with advanced disease developing LMD. Tumour cells that metastasise to the brain have unique properties that allow them to cross the blood-brain barrier, evade the immune system, and survive in the brain microenvironment. Metastatic colonisation is achieved through dynamic communication between metastatic cells and the tumour microenvironment, resulting in a tumour-permissive milieu. Despite advances in treatment options, the incidence of LMD appears to be increasing and current treatment modalities have a limited impact on survival. This review provides an overview of the biology of LMD, diagnosis and current treatment approaches for MM patients with LMD, and an overview of ongoing clinical trials. Despite the still limited efficacy of current therapies, there is hope that emerging treatments will improve the outcomes for patients with LMD.

Modeling and simulation of diffusion and reaction processes during the staining of tissue sections on slides.

Histological slides are an important tool in the diagnosis of tumors as well as of other diseases that affect cell shapes and distributions. Until now, the research concerning an optimal staining time has been mainly done empirically. In experimental investigations, it is often not possible to stain an already-stained slide with another stain to receive further information. To overcome these challenges, in the present paper a continuum-based model was developed for conducting a virtual (re-)staining of a scanned histological slide. This model is capable of simulating the staining of cell nuclei with the dye hematoxylin (C.I. 75,290). The transport and binding of the dye are modeled (i) along with the resulting RGB intensities (ii). For (i), a coupled diffusion-reaction equation is used and for (ii) Beer-Lambert's law. For the spatial discretization an approach based on the finite element method (FEM) is used and for the time discretization a finite difference method (FDM). For the validation of the proposed model, frozen sections from human liver biopsies stained with hemalum were used. The staining times were varied so that the development of the staining intensity could be observed over time. The results show that the model is capable of predicting the staining process. The model can therefore be used to perform a virtual (re-)staining of a histological sample. This allows a change of the staining parameters without the need of acquiring an additional sample. The virtual standardization of the staining is the first step towards universal cross-site comparability of histological slides.

DMD genomic deletions characterize a subset of progressive/higher-grade meningiomas with poor outcome.

Progressive meningiomas that have failed surgery and radiation have a poor prognosis and no standard therapy. While meningiomas are more common in females overall, progressive meningiomas are enriched in males. We performed a comprehensive molecular characterization of 169 meningiomas from 53 patients with progressive/high-grade tumors, including matched primary and recurrent samples. Exome sequencing in an initial cohort (n = 24) detected frequent alterations in genes residing on the X chromosome, with somatic intragenic deletions of the dystrophin-encoding and muscular dystrophy-associated DMD gene as the most common alteration (n = 5, 20.8%), along with alterations of other known X-linked cancer-related genes KDM6A (n =2, 8.3%), DDX3X, RBM10 and STAG2 (n = 1, 4.1% each). DMD inactivation (by genomic deletion or loss of protein expression) was ultimately detected in 17/53 progressive meningioma patients (32%). Importantly, patients with tumors harboring DMD inactivation had a shorter overall survival (OS) than their wild-type counterparts [5.1 years (95% CI 1.3-9.0) vs. median not reached (95% CI 2.9-not reached, p = 0.006)]. Given the known poor prognostic association of TERT alterations in these tumors, we also assessed for these events, and found seven patients with TERT promoter mutations and three with TERT rearrangements in this cohort (n = 10, 18.8%), including a recurrent novel RETREG1-TERT rearrangement that was present in two patients. In a multivariate model, DMD inactivation (p = 0.033, HR = 2.6, 95% CI 1.0-6.6) and TERT alterations (p = 0.005, HR = 3.8, 95% CI 1.5-9.9) were mutually independent in predicting unfavorable outcomes. Thus, DMD alterations identify a subset of progressive/high-grade meningiomas with worse outcomes.

Correction to: DNA methylation-based reclassification of olfactory neuroblastoma.

In the original publication, the second name of the twentieth author was incorrect. It should read as 'Miguel Sáinz-Jaspeado'. The original publication of the article has been updated to reflect the change. This correction was authored by Ulrich Schüller on behalf of all authors of the original publication.

DNA methylation-based reclassification of olfactory neuroblastoma.

Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n = 66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed four subgroups among institutionally diagnosed ONB. The largest group (n = 42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n = 7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis, these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10, and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of tumors. Expression of cytokeratin, chromogranin A, the mutational status of IDH2 as well as DNA methylation patterns may greatly aid in the precise classification of ONB.

IDH1 mutation in human glioma induces chemical alterations that are amenable to optical Raman spectroscopy.

INTRODUCTION: Mutations in the isocytrate dehydrogenase 1 (IDH1) gene are early genetic events in glioma pathogenesis and cause profound metabolic changes. Because this genotype is found in virtually every tumor cell, therapies targeting mutant IDH1 protein are being developed. The intraoperative administration of those therapies would require fast technologies for the determination of IDH1 genotype. As of today, there is no such diagnostic test available. Recently, infrared spectroscopy was shown to bridge this gap. Here, we tested Raman spectroscopy for analysis of IDH1 genotype in glioma, which constitutes an alternative contact-free technique with the potential of being applicable in situ. METHODS: Human glioma samples (n = 36) were obtained during surgery and cryosections were prepared. IDH1 mutations were assessed using DNA sequencing and 100 Raman spectra were obtained for each sample. RESULTS: Analysis of Raman spectra revealed increased intensities in spectral bands related to DNA in IDH1 mutant glioma while bands assigned to molecular vibrations of lipids were significantly decreased. Moreover, intensities of Raman bands assigned to proteins differed in IDH1 mutant and IDH1 wild-type glioma, suggesting alterations in the protein profile. The selection of five bands (498, 826, 1003, 1174 and 1337 cm-1) allowed the classification of Raman spectra according to IDH1 genotype with a correct rate of 89%. CONCLUSION: Raman spectroscopy constitutes a simple, rapid and safe procedure for determination of the IDH1 mutation that shows great promise for clinically relevant in situ diagnostics.

Label-free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis.

The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti-Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two-photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG-active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co-localized with positive albumin immunohistochemistry and might indicate degeneration-associated vascular leakage. We present a label-free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label-free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo.

Prognostic impact of urokinase-type plasminogen activator system components in clear cell renal cell carcinoma patients without distant metastasis.

BACKGROUND: Members of the urokinase-type plasminogen activator (uPA) system including uPA, its receptor uPAR and the plasminogen activator inhibitor 1 (PAI-1) play an important role in tumour invasion and progression in a variety of tumour types. Since the majority of clear cell renal cell carcinoma (ccRCC) shows distant metastasis at time of diagnosis or later, the interplay of uPA, uPAR and PAI-1 might be of importance in this process determining the patients' outcome. METHODS: Corresponding pairs of malignant and non-malignant renal tissue specimens were obtained from 112 ccRCC patients without distant metastasis who underwent tumour nephrectomy. Tissue extracts prepared from fresh-frozen tissue samples by detergent extraction were used for the determination of antigen levels of uPA, uPAR and PAI-1 by ELISA. Antigen levels were normalised to protein concentrations and expressed as ng per mg of total protein. RESULTS: Antigen levels of uPA, uPAR, and PAI-1 correlated with each other in the malignant tissue specimens (rs=0.51-0.65; all P<0.001). Antigen levels of uPA system components were significantly higher in tissue extracts of non-organ confined tumours (pT3+4) compared to organ-confined tumours (pT1+2; all P<0.05). Significantly elevated levels of uPAR and PAI-1 were also observed in high grade ccRCC. When using median antigen levels as cut-off points, all three uPA system factors were significant predictors for disease-specific survival (DSS) in univariate Cox's regression analyses. High levels of uPA and uPAR remained independent predictors for DSS with HR=2.86 (95% CI 1.07-7.67, P=0.037) and HR=4.70 (95% CI 1.51-14.6, P=0.008), respectively, in multivariate Cox's regression analyses. A combination of high antigen levels of uPA and/or uPAR further improved the prediction of DSS in multivariate analysis (HR=14.5, 95% CI 1.88-111.1, P=0.010). Moreover, high uPA and/or uPAR levels defined a patient subgroup of high risk for tumour-related death in ccRCC patients with organ-confined disease (pT1+2) (HR=9.83, 95% CI 1.21-79.6, P=0.032). CONCLUSIONS: High levels of uPA and uPAR in tumour tissue extracts are associated with a significantly shorter DSS of ccRCC patients without distant metastases.

Vertebral body replacement with PEEK-cages after anterior corpectomy in multilevel cervical spinal stenosis: a clinical and radiological evaluation.

INTRODUCTION: A growing number of industrially manufactured implants have been developed in the last years for vertebral replacement in anterior cervical corpectomy and fusion (ACCF). Polyetheretherketone (PEEK)-cages are used in many centers, but outcome reports are scarce. This study assesses the clinical and radiological outcome after one- or two-level ACCF by the use of a PEEK-cage augmented by a plate-screw osteosynthesis. METHODS: A total of 21 patients received one-level (18 patients) or two-level (3 patients) ACCF by a PEEK-cage and plate-screw osteosynthesis for multilevel degenerative stenosis. The Visual Analogue Scale, Nurick Score, Neck Disability Index and European Myelopathy Score were used for clinical assessment. Radiological outcome-osseous fusion and loss of height-was evaluated by CT. RESULTS: The mean follow-up was 28 ± 12 months. In 19 patients, bony fusion was achieved after the primary operation. Graft failure that required surgical revision occurred in two patients. In these patients, osseous fusion was achieved after the second operation. Myelopathy improved significantly. The loss of height was 2.2 ± 2.3 and 5.3 ± 2.1 mm after one- and two-level ACCF, respectively. CONCLUSION: Anterior corpectomy and fusion by a PEEK-cage and plate-screw osteosynthesis resulted in clinical improvement in all patients. Bony fusion was achieved in all patients in the long run. PEEK cages are allegedly less rigid than other xenografts. Similar to those, however, their use bears the risk of early cage-dislocation and subsidence. A comparison of industrial xenografts and autologous bone implants is required to challenge the different fusion techniques.

PARK2 and PACRG are commonly downregulated in clear-cell renal cell carcinoma and are associated with aggressive disease and poor clinical outcome.

PARK2 is an E3 ligase, known to be involved in ubiquitination of several proteins and to play a role in neuronal protection. The gene PARK2 and its potentially co-regulated gene PACRG have been previously found to be deleted in clear-cell renal cell carcinomas (ccRCCs). The aim of our study was to evaluate the mRNA and protein expression of PARK2 and PACRG in a large cohort of ccRCC, and to investigate their association with outcome. The expression of both genes was measured by quantitative PCR in 94 primary ccRCCs and autologous nonmalignant kidney tissues. PACRG and PARK2 protein expression was determined immunohistochemically using tissue microarrays comprising 133 ccRCCs. The mRNA and protein expression of PARK2 and PACRG was significantly downregulated in ccRCCs compared with nonmalignant tissues. Low levels of PARK2 mRNA were associated with high-grade ccRCC and lymph node metastasis. Patients with low PARK2 mRNA levels showed a higher tumor-specific mortality rate and a shorter overall survival (OS) than those with high PARK2 expression. Patients without PACRG mRNA expression in the tumor had a shorter disease-free survival and OS than those with tumors expressing PACRG. In multivariate analyses, neither PARK2 nor PACRG expression were independent prognostic factors. The protein expression of PARK2 and PACRG was significantly downregulated in ccRCCs (82.8, and 96.9%, respectively), but no association with clinical outcome was noticed.

Personalized identification and characterization of genome-wide gene expression differences between patient-matched intracranial and extracranial melanoma metastasis pairs.

Melanoma is the most serious type of skin cancer that frequently spreads to other organs of the human body. Especially melanoma metastases to the brain (intracranial metastases) are hard to treat and a major cause of death of melanoma patients. Little is known about molecular alterations and altered mechanisms that distinguish intra- from extracranial melanoma metastases. So far, almost all existing studies compared intracranial metastases from one set of patients to extracranial metastases of an another set of melanoma patients. This neglects the important facts that each melanoma is highly individual and that intra- and extracranial melanoma metastases from the same patient are more similar to each other than to melanoma metastases from other patients in the same organ. To overcome this, we compared the gene expression profiles of 16 intracranial metastases to their corresponding 21 patient-matched extracranial metastases in a personalized way using a three-state Hidden Markov Model (HMM) to identify altered genes for each individual metastasis pair. This enabled three major findings by considering the predicted gene expression alterations across all patients: (i) most frequently altered pathways include cytokine-receptor interaction, calcium signaling, ECM-receptor interaction, cAMP signaling, Jak-STAT and PI3K/Akt signaling, (ii) immune-relevant signaling pathway genes were downregulated in intracranial metastases, and (iii) intracranial metastases were associated with a brain-like phenotype gene expression program. Further, the integration of all differentially expressed genes across the patient-matched melanoma metastasis pairs led to a set of 103 genes that were consistently down- or up-regulated in at least 11 of the 16 of the patients. This set of genes contained many genes involved in the regulation of immune responses, cell growth, cellular signaling and transport processes. An analysis of these genes in the TCGA melanoma cohort showed that the expression behavior of 11 genes was significantly associated with survival. Moreover, a comparison of the 103 genes to three closely related melanoma metastasis studies revealed a core set of eight genes that were consistently down- or upregulated in intra- compared to extracranial metastases in at least two of the three related studies (down: CILP, DPT, FGF7, LAMP3, MEOX2, TMEM119; up: GLDN, PMP2) including FGF7 that was also significantly associated with survival. Our findings contribute to a better characterization of genes and pathways that distinguish intra- from extracranial melanoma metastasis and provide important hints for future experimental studies to identify potential targets for new therapeutic approaches.

Network-based analysis of heterogeneous patient-matched brain and extracranial melanoma metastasis pairs reveals three homogeneous subgroups.

Melanoma, the deadliest form of skin cancer, can metastasize to different organs. Molecular differences between brain and extracranial melanoma metastases are poorly understood. Here, promoter methylation and gene expression of 11 heterogeneous patient-matched pairs of brain and extracranial metastases were analyzed using melanoma-specific gene regulatory networks learned from public transcriptome and methylome data followed by network-based impact propagation of patient-specific alterations. This innovative data analysis strategy allowed to predict potential impacts of patient-specific driver candidate genes on other genes and pathways. The patient-matched metastasis pairs clustered into three robust subgroups with specific downstream targets with known roles in cancer, including melanoma (SG1: RBM38, BCL11B, SG2: GATA3, FES, SG3: SLAMF6, PYCARD). Patient subgroups and ranking of target gene candidates were confirmed in a validation cohort. Summarizing, computational network-based impact analyses of heterogeneous metastasis pairs predicted individual regulatory differences in melanoma brain metastases, cumulating into three consistent subgroups with specific downstream target genes.

Prediction of WHO grade and methylation class of aggressive meningiomas: Extraction of diagnostic information from infrared spectroscopic data.

Background: Infrared (IR) spectroscopy allows intraoperative, optical brain tumor diagnosis. Here, we explored it as a translational technology for the identification of aggressive meningioma types according to both, the WHO CNS grading system and the methylation classes (MC). Methods: Frozen sections of 47 meningioma were examined by IR spectroscopic imaging and different classification approaches were compared to discern samples according to WHO grade or MC. Results: IR spectroscopic differences were more pronounced between WHO grade 2 and 3 than between MC intermediate and MC malignant, although similar spectral ranges were affected. Aggressive types of meningioma exhibited reduced bands of carbohydrates (at 1024 cm-1) and nucleic acids (at 1080 cm-1), along with increased bands of phospholipids (at 1240 and 1450 cm-1). While linear discriminant analysis was able to discern spectra of WHO grade 2 and 3 meningiomas (AUC 0.89), it failed for MC (AUC 0.66). However, neural network classifiers were effective for classification according to both WHO grade (AUC 0.91) and MC (AUC 0.83), resulting in the correct classification of 20/23 meningiomas of the test set. Conclusions: IR spectroscopy proved capable of extracting information about the malignancy of meningiomas, not only according to the WHO grade, but also for a diagnostic system based on molecular tumor characteristics. In future clinical use, physicians could assess the goodness of the classification by considering classification probabilities and cross-measurement validation. This might enhance the overall accuracy and clinical utility, reinforcing the potential of IR spectroscopy in advancing precision medicine for meningioma characterization.

Immunohistochemical analysis of prognostic protein markers for primary localized clear cell renal cell carcinoma.

Recurrence is not reliably predictable in localized clear cell renal cell carcinoma. Proteinmarkers could improve predictive accuracy. Tissue-microarrays from 132 patients with primary localized ccRCC were immunohistochemically analyzed for VHL, Ki67, p53, p21, survivin, and, for microvessel-density, UEA-1. Nuclear stainings of Ki67, p21, and survivin were significantly associated with disease-specific survival and increased predictive ability from 74% to 76%, 77%, and 78%, respectively in a multivariate model including T-stage and Fuhrman grade. A score-variable, combining Ki67-, p21-, and nS-staining identified a subset of patients with high risk of disease recurrence and increased predictive ability in the multivariate model to 84%.