Updated diagnostic criteria and nomenclature for neurofibromatosis type 2 and schwannomatosis: An international consensus recommendation.

PURPOSE: Neurofibromatosis type 2 (NF2) and schwannomatosis (SWN) are genetically distinct tumor predisposition syndromes with overlapping phenotypes. We sought to update the diagnostic criteria for NF2 and SWN by incorporating recent advances in genetics, ophthalmology, neuropathology, and neuroimaging. METHODS: We used a multistep process, beginning with a Delphi method involving global disease experts and subsequently involving non-neurofibromatosis clinical experts, patients, and foundations/patient advocacy groups. RESULTS: We reached consensus on the minimal clinical and genetic criteria for diagnosing NF2 and SWN. These criteria incorporate mosaic forms of these conditions. In addition, we recommend updated nomenclature for these disorders to emphasize their phenotypic overlap and to minimize misdiagnosis with neurofibromatosis type 1. CONCLUSION: The updated criteria for NF2 and SWN incorporate clinical features and genetic testing, with a focus on using molecular data to differentiate the 2 conditions. It is likely that continued refinement of these new criteria will be necessary as investigators study the diagnostic properties of the revised criteria and identify new genes associated with SWN. In the revised nomenclature, the term "neurofibromatosis 2" has been retired to improve diagnostic specificity.

miR-497 and 219 in blood aid meningioma classification.

INTRODUCTION: The current WHO classification and methylation status help predict meningioma recurrence and prognosis. However, up to date, there is no circulating biomarker showing clinical value in meningioma diagnosis or classification. Circulating miRNAs showed the potential to be used as cancer biomarkers in various tumours. This research evaluated specific miRNAs, miR-497 and miR-219, as convenient and efficient predictors of meningioma grades. METHODS: We studied serum and exosomal levels of miR-497 in 74 meningioma samples (WHO grade I = 25, WHO grade II = 25, and WHO grade III = 24) and 53 healthy controls. The serum level of miR-219 was studied in 56 meningioma samples WHO grade I = 22, WHO grade II = 14, and WHO grade III = 20). We used qPCR for miRNA quantification. We also tested two different normalisers, endogenous and external, and evaluated their impact on the diagnostic value of miR-497. RESULTS: The serum and exosomal levels of miR-497 distinguished meningioma from the control samples. Moreover, miR-497 was a suitable identifier for meningioma grade. When we combined miR-497 and miR-219, the efficacy of the combined biomarker was higher than miR-497 or miR-219 when used individually in meningioma classification. Both miR-497 and miR-219 showed a noticeable change with the methylation class of meningioma. CONCLUSION: This study shows that serum miR-497 is an effective and easy-to-measure biomarker for meningioma diagnosis and classification. Moreover, when we combined miR-497 and miR-219, the combined biomarker showed enhanced accuracy in meningioma classification. Furthermore, this is the first study to evaluate the correlation between serum circulating miRNA and the methylation status in meningioma.

Molecular and translational advances in meningiomas.

Meningiomas are the most common primary intracranial neoplasm. The current World Health Organization (WHO) classification categorizes meningiomas based on histopathological features, but emerging molecular data demonstrate the importance of genomic and epigenomic factors in the clinical behavior of these tumors. Treatment options for symptomatic meningiomas are limited to surgical resection where possible and adjuvant radiation therapy for tumors with concerning histopathological features or recurrent disease. At present, alternative adjuvant treatment options are not available in part due to limited historical biological analysis and clinical trial investigation on meningiomas. With advances in molecular and genomic techniques in the last decade, we have witnessed a surge of interest in understanding the genomic and epigenomic landscape of meningiomas. The field is now at the stage to adopt this molecular knowledge to refine meningioma classification and introduce molecular algorithms that can guide prediction and therapeutics for this tumor type. Animal models that recapitulate meningiomas faithfully are in critical need to test new therapeutics to facilitate rapid-cycle translation to clinical trials. Here we review the most up-to-date knowledge of molecular alterations that provide insight into meningioma behavior and are ready for application to clinical trial investigation, and highlight the landscape of available preclinical models in meningiomas.

Actin-rich protrusions and nonlocalized GTPase activation in Merlin-deficient schwannomas.

Schwannomas lack both alleles for the tumor suppressor Merlin, a cytoskeleton-membrane linker. Previous results showed increased cell spreading of schwannoma cells, but little is known about the underlying mechanisms. Electron microscopy reveals that schwannoma cells not only show more lamellipodia/ruffles but also multiple filopodia. We show that Cdc42, important in filopodia formation, is activated. Both Rac1 and Cdc42 are found all around the cell periphery and in colocalization with their effector phospho-p21 activated kinase in human schwannoma cells. We therefore claim that Rac1 and Cdc42 are activated in a nonlocalized manner, which explains the disperse distribution of lamellipodia/ruffles and filopodia. Using live cell imaging, we further demonstrate continuous remodeling of the many actin-rich protrusions in schwannoma cells. The underlying cytoskeleton of these structures is thin and extensively branched. The actin-related protein 2/3 complex, a major regulator of actin branching, is enriched in the many lamellipodia and ruffles of human primary schwannoma cells. We suggest that the Merlin deficiency in human primary schwannoma cells leads to a random, nonlocalized activation of Rac1 and Cdc42, inducing many actin-rich protrusion zones, not only at the leading edge but also all around the cell periphery. Their nondirectional occurrence together with the continuous and highly dynamic actin remodeling results in the dedifferentiation of these tumor cells.

The p53/mouse double minute 2 homolog complex deregulation in merlin-deficient tumours.

Deficiency of the tumour suppressor merlin leads to the development of schwannomas, meningiomas and ependymomas occurring spontaneously or as a part of the hereditary disease Neurofibromatosis type 2 (NF2). Merlin loss is also found in a proportion of other cancers like mesothelioma, melanoma, breast cancer and glioblastoma. The tumour suppressor/transcription factor p53 regulates proliferation, survival and differentiation and its deficiency plays a role in the development of many tumours. 53 can be negatively regulated by FAK, PI3K/AKT and MDM2 and possibly positively regulated by merlin in different cell lines. In this study we investigated the role of p53 in merlin-deficient tumours. Using our in vitro model of primary human schwannoma cells we have previously demonstrated that FAK is overexpressed/activated and localises into the nucleus of schwannoma cells increasing proliferation. AKT is strongly activated via platelet-derived growth factor (PDGF) - and insulin-like growth factor 1 (IGF1) - receptors increasing survival. Here we investigated p53 regulation and its role in proliferation and survival of human primary schwannoma cells using western blotting, immunocytochemistry, immunohistochemistry and proliferation, survival and transcription factor assays. In human primary schwannoma cells p53 was found to be downregulated while MDM2 was upregulated leading to increased cell proliferation and survival. p53 is regulated by merlin involving FAK, AKT and MDM2. Merlin reintroduction into schwannoma cells increased p53 levels and activity, and treatment with Nutlin-3, a drug which increases p53 stability by disrupting the p53/MDM2 complex, decreased tumour growth and reduced cell survival. These findings are important to dissect the mechanisms responsible for the development of merlin-deficient tumours and to identify new therapeutic targets. We suggest that Nutlin-3, possibly in combination with FAK or PI3K inhibitors, can be employed as a novel treatment for schwannoma and other merlin-deficient tumours.

Schwannomas and their pathogenesis.

Schwannomas may occur spontaneously, or in the context of a familial tumor syndrome such as neurofibromatosis type 2 (NF2), schwannomatosis and Carney's complex. Schwannomas have a variety of morphological appearances, but they behave as World Health Organization (WHO) grade I tumors, and only very rarely undergo malignant transformation. Central to the pathogenesis of these tumors is loss of function of merlin, either by direct genetic change involving the NF2 gene on chromosome 22 or secondarily to merlin inactivation. The genetic pathways and morphological features of schwannomas associated with different genetic syndromes will be discussed. Merlin has multiple functions, including within the nucleus and at the cell membrane, and this review summarizes our current understanding of the mechanisms by which merlin loss is involved in schwannoma pathogenesis, highlighting potential areas for therapeutic intervention.

Nilotinib alone or in combination with selumetinib is a drug candidate for neurofibromatosis type 2.

Loss of the tumor suppressor merlin is a cause of frequent tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas, which occur spontaneously or as part of neurofibromatosis type 2 (NF2). Because there is medical need for drug therapies for these tumors, our aim is to find therapeutic targets. We have studied the pathobiology of schwannomas, because they are the most common merlin-deficient tumors and are a model for all merlin-deficient tumors. With use of a human schwannoma in vitro model, we previously described strong overexpression/activation of platelet-derived growth factor receptor-ß (PDGFR-ß) leading to strong, long-lasting activation of extracellular-signal-regulated kinase (ERK1/2) and AKT and increased schwannoma growth, which we successfully inhibited using the PDGFR/Raf inhibitor sorafenib. However, the benign character of schwannomas may require long-term treatment; thus, drug tolerability is an issue. With the use of Western blotting, proliferation assays, viability assays, and a primary human schwannoma cell in vitro model, we tested the PDGFR/c-KIT inhibitors imatinib (Glivec(;) Novartis) and nilotinib (Tasigna(;) Novartis). Imatinib and nilotinib inhibited PDGF-DD-mediated ERK1/2 activation, basal and PDGF-DD-mediated activation of PDGFR-ß and AKT, and schwannoma proliferation. Nilotinib is more potent than imatinib, exerting its maximal inhibitory effect at concentrations lower than steady-state trough plasma levels. In addition, nilotinib combined with the MEK1/2 inhibitor selumetinib (AZD6244) at low concentrations displayed stronger efficiency toward tumor growth inhibition, compared with nilotinib alone. We suggest that therapy with nilotinib or combinational therapy that  simultaneously inhibits PDGFR and the downstream Raf/MEK1/2/ERK1/2 pathway could represent an effective treatment for schwannomas and other merlin-deficient tumors.

Merlin-deficient human tumors show loss of contact inhibition and activation of Wnt/ß-catenin signaling linked to the PDGFR/Src and Rac/PAK pathways.

Neurofibromatosis type 2 (NF2) is an inherited predisposition cancer syndrome characterized by the development of multiple benign tumors in the nervous system including schwannomas, meningiomas, and ependymomas. Using a disease model comprising primary human schwannoma cells, we previously demonstrated that adherens junctions (AJs) are impaired in schwannoma cells because of a ubiquitous, upregulated Rac activity. However, the mechanism by which loss of contact inhibition leads to proliferation remains obscure in merlin-deficient tumors. In this study, we show that proliferative Wnt/ß-catenin signaling is elevated as active ß-catenin (dephosphorylated at serine 37 and threoine 41) localizes to the nucleus and the Wnt targets genes c-myc and cyclin D1 are upregulated in confluent human schwannoma cells. We demonstrate that Rac effector p21-activated kinase 2 (PAK2) is essential for the activation of Wnt/ß-catenin signaling because depletion of PAK2 suppressed active ß-catenin, c-myc, and cyclin D1. Most importantly, the link between the loss of the AJ complex and the increased proliferation in human schwannoma cells is connected by Src and platelet-derived growth factor receptor-induced tyrosine 654 phosphorylation on ß-catenin and associated with degradation of N-cadherin. We also demonstrate that active merlin maintains ß-catenin and N-cadherin complex at the plasma membrane through direct regulation. Finally, we demonstrate that phosphorylation of tyrosine 654 is critical for the increased proliferation in human schwannoma cells because overexpression of a Y654F mutant ß-catenin reduces hyperproliferation of schwannoma cells. We suggest a model that these pathways are coordinated and relevant for proliferation in merlin-deficient tumors.

Quinidine impairs proliferation of neurofibromatosis type 2-deficient human malignant mesothelioma cells.

BACKGROUND: Human malignant mesotheliomas (HMMs) are aggressive tumors that arise from the mesothelium. They respond poorly to conventional tumor treatment and outcome is often fatal. Inactivating mutations of the neurofibromatosis type 2 (NF2) tumor suppressor gene merlin have been described in nearly 60% of primary malignant mesothelioma and in approximately 20% of the mesothelioma cell lines. Studies regarding human NF2 schwannoma cells revealed a higher proliferation and a larger noninactivating K(+) outward current compared with controls. The enhanced proliferation of merlin-deficient NF2 schwannoma cells could be reduced in the presence of quinidine, a K(+) channel blocker, whereas the proliferation of normal Schwann cells is not affected. The current study was undertaken to evaluate the effect of quinidine on the proliferation of HMM cell lines in relation to their NF2 status. METHODS: Proliferation analyses using bromodeoxyuridine incorporation was performed by immunocytochemical staining and fluorescence assisted cell sorting. The patch-clamp technique was applied for electrophysiologic characterization of the HMM cell lines. The cytochrome P450 2D6 locus, known to be mutated at high frequencies in NF2 patients and to be specifically inhibited by quinidine, was screened for mutations by cycle sequencing. RESULTS: Quinidine selectively reduces the proliferation of merlin-deficient HMM cell lines by causing a G(0)/G(1) arrest, whereas the proliferation rates of merlin-expressing HMM cell lines remain unchanged. The effect of quinidine on the proliferation of HMM cell lines appears to be correlated with the NF2 gene status but not with the K(+) outward current. No relation to cytochrome P450 2D6 mutations was detected. CONCLUSIONS: Quinidine or quinidine analogs are of potential therapeutic interest for the subset of merlin-deficient mesothelioma tumors.

A novel nonsense mutation in the ABC1 gene causes a severe syringomyelia-like phenotype of Tangier disease.

Tangier disease is a rare autosomal recessive disorder caused by mutations in the recently identified ATP-binding cassette transporter 1 gene (ABC1). A typical clinical manifestation of Tangier disease is peripheral neuropathy. Former studies differentiated between two manifestations: the more frequent mono- or polyneuropathic form and a syringomyelia-like type. It is unknown whether specific mutations in the ABC1 gene or a particular genetic background are responsible for either of these forms. A family is presented comprising a case with a severe syringomyelia-like phenotype of Tangier disease and absence of cardiovascular disease. Sequencing analysis of the ABC1 gene was performed. A new homozygous C-->T transition in exon 18 was found in the index patient. This mutation results in a stop codon at position 909 (R909X) leading to premature termination of translation. Her clinically asymptomatic daughters, her sister and one of her nieces were heterozygous. Sural nerve biopsies were studied in the index patient at the age of 45 and 54 years; both revealed a severe neuropathy, characterized by a subtotal and finally complete loss of nerve fibres. The entire loss of Schwann cells resulted in an extraordinary form of endoneurial sclerosis. Only rare capillaries, lipid-laden macrophages and fibroblasts had survived in the endoneurium. This case appears to be unique in respect to the underlying novel mutation in the ABC1 gene and its association with complete endoneurial sclerosis of all fascicles in the sural nerve and absence of cardiovascular disease.