PURPOSE: To evaluate ophthalmological and molecular findings in eight patients with a clinical diagnosis of neurofibromatosis type 2 (NF2). New pathological mutations are described and variability in the ophthalmic phenotype and NF2 allelic heterogeneity are discussed.
METHODS: Eye examination was performed in eight NF2 patients, and it included the measurement of the visual acuity, biomicroscopy, dilated fundus examination, color fundus photography, infrared photography, and spectral domain optical coherence tomography (SD-OCT). Molecular analysis was performed with whole-exome sequencing using DNA derived from peripheral blood mononuclear cells from each individual.
RESULTS: Ophthalmological features were present in all patients, ranging from subtle retinal alterations identified only using SD-OCT to severe ocular damage present at birth. Six mutations were observed: two patients with stop codon mutation as shown on table 1 and result section, three patients with frameshift mutation as shown on table 1 and result section. Three novel mutations were found among them.
CONCLUSIONS: It is a descriptive study of a rare disease, with poor previous literature. Clinical and genetic data are shown, reviving the need to further studies to clarify the genotype-phenotype correlations in NF2.

BACKGROUND: Owing to the aggressive nature of high-grade gliomas (HGGs), its early diagnosis holds the key to a favorable prognosis. Currently, tissue biopsy is the gold standard to verify HGG's initial diagnosis and can be challenging due to its invasive nature. In this study, our objective was a noninvasive panel for timely detection of HGG and its progression using cell-free circulating tumor DNA (cfTDNA).
MATERIALS AND METHODS: Twenty-seven patients with HGG were tested with a 50-gene tumor panel. cfTDNA isolated from serum was checked for single-nucleotide variations (SNVs) or copy number alterations using targeted next-generation sequencing, with further validation of results by checking respective formalin-fixed paraffin-embedded tumor tissues for the same genetic alterations.
RESULTS: About 88.8% of the patients were detected with HGG-associated cfTDNA. Around 25% patients were detected with one, 25% patients had three, 25% patients had four, and 12.5% patients each had five and six genetic alterations. About 12 of 50 genes were detected in the serum samples. The SNVs detected included TP53 in 87.5% of patients; PIK3CA and EGFR in 50% of patients; PTEN in 37.5%; KIT and VHL in each 25% of patients; and RB1, NF2, MET, ATRX, CDK2A, and CTNNB1 each in 8.3%-16.6%. On combining EGFR, KIT, PTEN, PIK3CA, TP53, and VHL genes (Govardhan Diagnostic Genetic Module for high-grade glioma), at least one of the genetic alterations was found in 100% of patients.
Conclusion: These findings illustrate that cfTDNA is easily demonstrable and can be used as a surrogate to tissue biopsy in brain tumor.

Inactivation of the tumor suppressor protein Merlin leads to the development of benign nervous system tumors in neurofibromatosis type2 (NF2). Documented causes of Merlin inactivation include deleterious mutations in the encoding neurofibromin2 gene (NF2) and aberrant Merlin phosphorylation leading to proteasomal degradation. Rare somatic NF2 mutations have also been detected in common human malignancies not associated with NF2, including colorectal and lung cancer. Furthermore, tumors without NF2 mutations and with unaltered NF2 transcript levels, but with low Merlin expression, have been reported. The present study demonstrated that NF2 is also regulated by microRNAs (miRNAs) through direct interaction with evolutionarily conserved miRNA response elements (MREs) within its 3'‑untranslated region (3'UTR). Dual‑Luciferase assays in human colorectal carcinoma (HCT116) and lung adenocarcinoma (A549) cells revealed downregulation of NF2 by miR‑92a‑3p via its wild‑type 3'UTR, but not NF2‑3'UTR with mutated miR‑92a‑3p MRE. HCT116 cells overexpressing miR‑92a‑3p exhibited significant downregulation of endogenous NF2 mRNA and protein levels, which was rescued by co‑transfection of a target protector oligonucleotide specific for the miR‑92a‑3p binding site within NF2‑3'UTR. miR‑92a‑3p overexpression in HCT116 and A549 cells promoted migration, proliferation and resistance to apoptosis, as well as altered F‑actin organization compared with controls. Knockdown of NF2 by siRNA phenocopied the oncogenic effects of miR‑92a overexpression on HCT116 and A549 cells. Collectively, the findings of the present study provide functional proof of the unappreciated role of miRNAs in NF2 regulation and tumor progression, leading to enhanced oncogenicity.

Ehm2 [also known as erythrocyte membrane protein band 4.1‑like protein 4B (EPB41L4B)] is a member of the NF2/ERM/4.1 superfamily. The overexpression of Ehm2 has been observed in metastatic cancer cells. Through alternative splicing, the Ehm2 gene produces two transcript variants that encode the two different isoforms, Ehm2/1 and Ehm2/2. The biological functions of these different Ehm2 transcript variants remain unclear. The present study aimed to determine the expression of the Ehm2 variants in lung adenocarcinoma and their involvement in the disease progression of the patients. The expression of Ehm2 transcript variants in human lung adenocarcinoma tissues was analyzed using immunohistochemistry and western blot analysis. Ehm2 variants were overexpressed or knocked down in A549 human lung adenocarcinoma cells. The consequent effects of the genetic modifications on the cellular functions of lung cancer cells were then examined using in vitro cell viability, invasion and migration assays. The expression of epithelial‑mesenchymal transition (EMT)‑related markers was evaluated by western blot analysis in the cell models. The association of Ehm2 variant expression with patient survival was analyzed using Kaplan‑Meier survival analysis. The expression of Ehm2/1 was significantly decreased in lung cancers compared with the paired normal lung tissues (P<0.05), while the Ehm2/2 protein levels were higher in the tumors than in the paired normal lung tissues, although this was not statistically significant. The overexpression of Ehm2/1 exerted inhibitory effects, while the knockdown of Ehm2/1 promoted the growth, invasion and migration of A549 cells in vitro. Ehm2/2 was expressed at low levels in the A549 cells and the enforced expression of Ehm2/2 significantly increased the invasiveness and migration of the A549 cells. Immunofluorescence staining revealed that Ehm2/1 was confined to the plasma membrane, while Ehm2/2 was observed at both the plasma membrane and cytoplasm. The overexpression of Ehm2/1 resulted in the upregulation of the epithelial marker, E‑cadherin, and in the decreased expression of the mesenchymal markers, N‑cadherin and Snail1, while the knockdown of Ehm2/1 and the enforced expression of Ehm2/2 had the opposite effects on the protein levels of EMT‑related markers. Kaplan‑Meier survival analysis revealed that higher Ehm2/1 transcript levels were associated with the longer survival of patients with lung adenocarcinoma, while the lower expression of Ehm2/2 exhibited a similar association with patient survival. Taken together, the two Ehm2 variants appear to be differentially expressed in lung adenocarcinoma. Ehm2/1 may function as a putative tumor suppressor in the disease progression of lung adenocarcinoma, while Ehm2/2 may have an opposite function.

BACKGROUND: Genomic profiling of colorectal cancer aims to identify actionable somatic mutations but can also discover incidental germline findings.
OBJECTIVE: The purpose of this study was to report the detection of pathogenic germline variants that confer heritable cancer predisposition.
DESIGN: This was a retrospective study.
SETTINGS: The study was conducted at a tertiary-referral institution.
PATIENTS: Between 2012 and 2015, 1000 patients with advanced cancer underwent targeted exome sequencing of a 202-gene panel. The subgroup of 151 patients with advanced colorectal cancer who underwent matched tumor-normal (blood) sequencing formed our study cohort.
INTERVENTIONS: Germline variants in 46 genes associated with hereditary cancer predisposition were classified according to a defined algorithm based on in silico predictions of pathogenicity. Patients with presumed pathogenic variants were examined for type of mutation, as well as clinical, pedigree, and clinical genetic testing data.
MAIN OUTCOME MEASURES: We measured detection of pathogenic germline variants.
RESULTS: A total of 1910 distinct germline variants were observed in 151 patients. After filtering, 15 pathogenic germline variants (9.9%) were found in 15 patients, arising from 9 genes of varying penetrance for colorectal cancer (APC (n = 2; 13%), ATM (n = 1; 6%), BRCA1 (n = 2; 13%), CDH1 (n = 2; 13%), CHEK2 (n = 4; 27%), MSH2 (n = 1; 7%), MSH6 (n = 1; 7%), NF2 (n = 1; 7%), and TP53 (n = 1; 7%)). Patients with pathogenic variants were diagnosed at a younger age than those without (median, 45 vs 52 y; p = 0.03). Of the 15 patients, 7 patients (46.7%) with variants in low/moderate- penetrant genes for colorectal cancer would likely have not been tested based on clinical and pedigree criteria, where 2 harbored clinically actionable variants (CDH1 and NF2, 28.5% of 7).
LIMITATIONS: This study was limited by its small sample size and advanced-stage patients.
CONCLUSIONS: Tumor-normal sequencing can incidentally discover clinically unsuspected germline variants that confer cancer predisposition in 9.9% of patients with advanced colorectal cancer. Precision medicine should integrate clinical cancer genetics to inform and interpret the actionability of germline variants and to provide follow-up care to mutation carriers. See Video Abstract at http://links.lww.com/DCR/A906.

Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. In this protocol, we describe how to inject schwannoma cells into the mouse brain cerebellopontine angle (CPA) region. We also describe how to apply state-of-the-art intravital imaging and hearing assessment techniques to study tumor growth and hearing loss. In addition, ataxia, angiogenesis, and tumor-stroma interaction assays can be applied, and the model can be used to test the efficacy of novel therapeutic approaches. By studying the disease from every angle, this model offers the potential to unravel the basic biological underpinnings of NF2 and to develop novel therapeutics to control this devastating disease. Our protocol can be adapted to study other diseases within the CPA, including meningiomas, lipomas, vascular malformations, hemangiomas, epidermoid cysts, cerebellar astrocytomas, and metastatic lesions. The entire surgical procedure takes ~45 min per mouse and allows for subsequent longitudinal imaging, as well as neurological and hearing assessment, for up to 2 months.

Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2-knockout human mesothelial cell line, MeT-5A (NF2-KO). In NF2-KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2-WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2-WT and NF2-KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c-Jun, and retinoblastoma (Rb) in NF2-KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2-KO clone. In addition, the disruption of FGFR2 in the NF2-KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c-Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2-negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2-positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.

Malignant mesothelioma (MM) constitutes a very aggressive tumor that is caused by asbestos exposure after long latency. The NF2 tumor suppressor gene is mutated in 40-50% of MM; moreover, one of its downstream signaling cascades, the Hippo signaling pathway, is also frequently inactivated in MM cells. Although the YAP transcriptional coactivator, which is regulated by the Hippo pathway, can function as a pro-oncogenic protein, the role of TAZ, a paralog of YAP, in MM cells has not yet been clarified. Here, we show that TAZ is expressed and underphosphorylated (activated) in the majority of MM cells compared to immortalized mesothelial cells. ShRNA-mediated TAZ knockdown highly suppressed cell proliferation, anchorage-independent growth, cell motility, and invasion in MM cells harboring activated TAZ. Conversely, transduction of an activated form of TAZ in immortalized mesothelial cells enhanced these in vitro phenotypes and conferred tumorigenicity in vivo. Microarray analysis determined that activated TAZ most significantly enhanced the transcription of genes related to "cytokine-cytokine receptor interaction." Among selected cytokines, we found that IL-1 signaling activation plays a major role in proliferation in TAZ-activated MM cells. Both IL1B knockdown and an IL-1 receptor antagonist significantly suppressed malignant phenotypes of immortalized mesothelial cells and MM cells with activated TAZ. Overall, these results indicate an oncogenic role for TAZ in MMs via transcriptional induction of distinct pro-oncogenic genes including cytokines. Among these, IL-1 signaling appears as one of the most important cascades, thus potentially serving as a target pathway in MM cells harboring Hippo pathway inactivation.

Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.

OBJECTIVES/HYPOTHESIS: Unilateral vestibular schwannoma (VS) occurs with a lifetime risk of around 1 in 1,000 and is due to inactivation of the NF2 gene, either somatically or from a constitutional mutation. It has been postulated that familial occurrence of unilateral VS occurs more frequently than by chance, but no causal mechanism has been confirmed.
STUDY DESIGN: Retrospective database analysis.
METHODS: The likelihood of chance occurrence of unilateral VS, or occurring in the context of neurofibromatosis type 2 (NF2), was assessed using national UK audit data and data from the national NF2 database. Families with familial unilateral VS (occurrence in first- and second-degree relatives) were assessed for constitutional NF2 and LZTR1 genetic variants, and where possible the tumor was also analyzed.
RESULTS: Approximately 1,000 cases of unilateral VS occurred annually in the United Kingdom between 2013 and 2016. Of these, 2.5 may be expected to have a first-degree relative who had previously developed a unilateral VS. The likelihood of this occurring in NF2 was considered to be as low as 0.05 annually. None of 28 families with familial unilateral VS had a constitutional NF2 intragenic variant, and in nine cases where the VS was analyzed, both mutational events in NF2 were identified and excluded from the germline. Only three variants of uncertain significance were found in LZTR1.
CONCLUSIONS: Familial occurrence of unilateral VS is very unlikely to be due to a constitutional NF2 or definitely pathogenic LZTR1 variant. The occurrence of unilateral VS in two or more first-degree relatives is likely due to chance. This phenomenon may well increase in clinical practice with increasing use of cranial magnetic resonance imaging in older patients.
LEVEL OF EVIDENCE: 2b Laryngoscope, 129:967-973, 2019.

BACKGROUND: The great majority of sporadic vestibular schwannomas (VSs) are due to the mutations of the NF2 gene encoding merlin. Sporadic VSs exhibit variable growth patterns and only a small fraction of the tumours are fast-growing; however, the underlying mechanisms remain undefined.
METHODS: DNA sequencing and dosage analysis were used to identify the NF2 mutation status in sporadic schwannomas. The expression and sub-cellular localization of merlin and p53-MDM2 were assessed by immunoblotting, qRT-PCR and immunofluorescence. In vitro and in vivo studies were performed to reveal the effects of Nutlin-3 (a MDM2 inhibitor) and/or MG-132(a proteasome inhibitor) on schwannomas. The proliferation of schwannoma cells was assessed by CCK-8 assay, EdU staining and Flow cytometry analysis.
FINDINGS: Double genetic hits of NF2 tended to occur in fast-growing tumours, characterized by the absence of merlin. The deregulation of p53-MDM2 was demonstrated to mediate merlin-deficient tumour growth, characterized by a nuclear accumulation of stabilized MDM2, contributing to a nuclear export of p53 for degradation. Nutlin-3 blocked the proliferation of schwannoma cells via a cooperative recovery of merlin and p53, accompanied by the shuttling of both proteins from the cytoplasm to the nucleus. We further demonstrated a difference in the sensitivity to Nutlin-3 between schwannoma cells with and without merlin expression. Nutlin-3 combined with MG-132 narrowed this between-group difference and triggered stronger inhibitory effects on the growth of schwannomas through coordinated reactivation of p53.
INTERPRETATION: These findings present treatment strategies directed on the pathogenesis of sporadic schwannomas. FUND: National Natural Science Foundation of China.

BACKGROUND: Although intracranial and spinal ependymomas are histopathologically similar, the molecular landscape is heterogeneous. An urgent need exists to identify differences in the genomic profiles to tailor treatment strategies. In the present study, we delineated differential gene expression patterns between intracranial and spinal ependymomas.
METHODS: We searched the Gene Expression Omnibus database using the term "ependymoma" and analyzed the raw gene expression profiles of 292 ependymomas (31 spinal and 261 intracranial). The gene expression data were analyzed to find differentially expressed genes (DEGs) between 2 regions. The fold change (FC) and false discovery rate (FDR) were used to assess DEGs after gene integration (|log
RESULTS: A total of 201 genes (105 upregulated and 96 downregulated) were significant DEGs in the data sets. The underexpression of NF2 in spinal ependymomas was statistically significant (FDR P = 7.91 × 10
CONCLUSIONS: The most substantial magnitude of DEGs in ependymoma might be HOX genes. However, whether the differential expression of these genes is the cause or consequence of the disease remains to be elucidated in a larger prospective study.

Schwannomatosis and neurofibromatosis type 2 (NF2) are both characterized by the development of multiple schwannomas but represent different genetic entities. Whereas NF2 is caused by mutations of the NF2 gene, schwannomatosis is associated with germline mutations of SMARCB1 or LZTR1. Here, we studied 15 sporadic patients with multiple non-intradermal schwannomas, but lacking vestibular schwannomas and ophthalmological abnormalities, who fulfilled the clinical diagnostic criteria for schwannomatosis. None of them harboured germline NF2 or SMARCB1 mutations as determined by the analysis of blood samples but seven had germline LZTR1 variants predicted to be pathogenic. At least two independent schwannomas from each patient were subjected to NF2 mutation testing. In five of the 15 patients, identical somatic NF2 mutations were identified (33%). If only those patients without germline LZTR1 variants are considered (n = 8), three of them (37.5%) had mosaic NF2 as concluded from identical NF2 mutations identified in independent schwannomas from the same patient. These findings imply that a sizeable proportion of patients who fulfil the diagnostic criteria for schwannomatosis, are actually examples of mosaic NF2. Hence, the molecular characterization of tumours in patients with a clinical diagnosis of schwannomatosis is very important. Remarkably, two of the patients with germline LZTR1 variants also had identical NF2 mutations in independent schwannomas from each patient which renders differential diagnosis of LZTR1-associated schwannomatosis versus mosaic NF2 in these patients very difficult.

OBJECTIVE: Vestibular schwannomas (VSs) can cause serious neurological defects including hearing loss and facial paralysis. The aim of this study is to identify whether Hippo signaling could be a potential targetable pathway for clinical treatment in VSs.
METHODS: Gene expression profiling was performed in 10 sporadic VSs and 4 normal nerves to identify aberrant genes expression of the Hippo pathway. Western blotting and immunohistochemical staining were used to examine the expression of Hippo core components in 20 VS samples. Neurofibromatosis type 2 (NF2) gene sequencing was also performed in all tumors using sanger sequencing. Verteporfin, inhibitor of yes-associated protein (YAP)-TEA domain family member, was used to assess the effect of proliferation inhibition in human primary VS cells and RT4-D6P2T cell line.
RESULTS: We found 51 differentially expressed genes of the Hippo pathway between VSs and healthy controls. Unsupervised analysis identified the 2 molecular variants that significantly related with distinct NF2 mutation status. The phosphorylation levels of large tumor suppressor 1 and YAP were significantly decreased in NF2-mutated VSs compared with wild-type VSs and normal nerves. Immunohistochemical staining showed that increased nuclear YAP expression in VSs was positively correlated with high Ki-67 index and low Merlin expression. Verteporfin reduced viability of primary VS cells and RT4-D6P2T cells.
CONCLUSIONS: Our findings implicate that deregulation of the Hippo pathway as a molecular mechanism of pathogenesis in human VSs, and suggest inhibition of this pathway as a potential treatment strategy.

Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype.

The NF2 gene encodes the tumor and metastasis suppressor protein Merlin. Merlin exerts its tumor suppressive role by inhibiting proliferation and inducing contact-growth inhibition and apoptosis. In the current investigation, we determined that loss of Merlin in breast cancer tissues is concordant with the loss of the inhibitory SMAD, SMAD7, of the TGF-β pathway. This was reflected as dysregulated activation of TGF-β signaling that co-operatively engaged with effectors of the Hippo pathway (YAP/TAZ/TEAD). As a consequence, the loss of Merlin in breast cancer resulted in a significant metabolic and bioenergetic adaptation of cells characterized by increased aerobic glycolysis and decreased oxygen consumption. Mechanistically, we determined that the co-operative activity of the Hippo and TGF-β transcription effectors caused upregulation of the long non-coding RNA Urothelial Cancer-Associated 1 (UCA1) that disengaged Merlin's check on STAT3 activity. The consequent upregulation of Hexokinase 2 (HK2) enabled a metabolic shift towards aerobic glycolysis. In fact, Merlin deficiency engendered cellular dependence on this metabolic adaptation, endorsing a critical role for Merlin in regulating cellular metabolism. This is the first report of Merlin functioning as a molecular restraint on cellular metabolism. Thus, breast cancer patients whose tumors demonstrate concordant loss of Merlin and SMAD7 may benefit from an approach of incorporating STAT3 inhibitors.

RATIONALE: Neurofibromatosis, including type 1 and type 2, is inherited dominant disease that causes serious consequences. The genetic mechanism of these diseases has been described, but germline mutation of checkpoint 2 kinase gene, together with other DNA repair related genes, has not been fully elucidated in the context of neurofibromatosis.
PATIENT CONCERNS: In this article, we reported identical germline mutation of CHEK2 gene (p.R180C) in a 7-year-old Tibetan boy with NF1, and in a 12-year-old Chinese girl with NF2.
DIAGNOSES: Neurofibromatosis 1 and 2 with CHECK2 gene germline mutation.
INTERVENTIONS: Both patients underwent operation to obtain tumor tissue, and peripheral blood of their family was tested.
OUTCOMES: Identical germline mutation of CHEK2 gene (p.R180C) was detected in both patients, and germline mutations of POLE, MUTYH and ATR were also detected.
LESSONS: This is the first article to describe CHEK2 mutation in both NF1 and NF2. This article highlights a possible role of CHEK2, in association with other germline genetic mutations, in tumorigenesis of NF1 and NF2.

INTRODUCTION: Surgery and radiation therapy are the standard treatment options for meningiomas, but these treatments are not always feasible. Expression profiling was performed to determine the presence of therapeutic actionable biomarkers for prioritization and selection of agents.
METHODS: Meningiomas (n = 115) were profiled using a variety of strategies including next-generation sequencing (592-gene panel: n = 14; 47-gene panel: n = 94), immunohistochemistry (n = 8-110), and fluorescent and chromogenic in situ hybridization (n = 5-70) to determine mutational and expression status.
RESULTS: The median age of patients in the cohort was 60 years, with a range spanning 6-90 years; 52% were female. The most frequently expressed protein markers were EGFR (93%; n = 44), followed by PTEN (77%; n = 110), BCRP (75%; n = 8), MRP1 (65%, n = 23), PGP (62%; n = 84), and MGMT (55%; n = 97). The most frequent mutation among all meningioma grades occurred in the NF2 gene at 85% (11/13). Recurring mutations in SMO and AKT1 were also occasionally detected. PD-L1 was expressed in 25% of grade III cases (2/8) but not in grade I or II tumors. PD-1 + T cells were present in 46% (24/52) of meningiomas. TOP2A and thymidylate synthase expression increased with grade (I = 5%, II = 22%, III = 62% and I = 5%, II = 23%, III = 47%, respectively), whereas progesterone receptor expression decreased with grade (I = 79%, II = 41%, III = 29%).
CONCLUSION: If predicated on tumor expression, our data suggest that therapeutics directed toward NF2 and TOP2A could be considered for most meningioma patients.

Neurofibromatosis type 2 (NF2) is associated with the development of several types of benign nervous system tumours, while malignancies are rare. We report a 22-year-old man who presented with retroperitoneal and spinal high-grade sarcomas with epithelial features. Samples showed a mixed epithelioid and spindled cell content with little associated matrix and inconclusive immunochemistry. Genetic analysis of a schwannoma and matched blood samples demonstrated a constitutional de novo substitution at the splice donor site of intron 8 of the NF2 gene and aa acquired large deletion of the entire NF2 gene as a second hit, with some loss of SMARCB1. The sarcoma also showed evidence of loss of SMARCB1 and NF2 with loss of INI1 staining. Unfortunately the mass was unresectable and the patient died 6 months after diagnosis. This malignancy was most consistent with SMARCB1-deficient epithelioid malignant peripheral nerve sheath tumour, although a significant differential was proximal-type epithelial sarcoma. Each differential has previously been reported only once with NF2. This demonstrates an extremely rare potential complication of the condition.

BACKGROUND/AIM: Epigenetic inactivation of tumor suppressor genes is an important molecular mechanism in the formation and development of human tumors. The purpose of our study was to evaluate the correlation between the methylation level of the secreted frizzled-related protein 1 (SFRP1) gene and the risk of renal cell carcinoma (RCC).
METHODS: The relevant literature was searched in detail in several electronic databases. The methodological heterogeneity was analyzed by meta-regression and subgroup analyses. The odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated to summarize the dichotomous outcomes of our meta-analysis.
RESULTS: The ten included articles contained 535 RCC samples and 475 normal controls. The results demonstrated that the methylation level of the SFRP1 promoter region was significantly correlated with an increased incidence of RCC (OR=13.72; 95% CI: 6.01-31.28; P=0.000). Furthermore, the eligible studies that had sufficient clinical data about the RCC cases were included in the analysis, and the results indicated that the frequency of SFRP1 promoter methylation was associated with a higher histological grade (P=0.000), tumor stage (P=0.033), tumor size (≥5 cm; P=0.029), and distant metastasis (P=0.047).
CONCLUSION: Our results indicate that the methylation level of the SFRP1 promoter region is increased in patients with RCC compared to normal controls and might be involved in the occurrence and development of RCC. Additional well-designed studies are needed to further verify our conclusions.

OBJECTIVES: Neurofibromatoses (type 1: NF1; type 2: NF2) are autosomal dominant tumor predisposition syndromes mostly caused by loss-of-function mutations in the tumor suppressor genes NF1 and NF2, respectively. Genotyping is important for correct diagnosis of these diseases. The authors aimed to characterize NF1 and NF2 variants in patients from Southern Brazil.
METHODS: Ninety-three unrelated probands with NF1 and 7 unrelated probands with NF2 features were recruited from an Oncogenetics center in Southern Brazil. Two next generation sequencing panels were customized to identify point mutations: NF1 (NF1, RNF135, and SUZ12 genes) and NF2 (NF2 and SMARCB1 genes). Large rearrangements were assessed by Multiplex Ligation-dependent Probe Amplification.
RESULTS: Sixty-eight heterozygous NF1 variants were identified in 75/93 probands (80%) and 3 heterozygous NF2 variants were identified in 3/7 probands (43%). In NF1, 59 (87%) variants were pathogenic (4 large rearrangements - 6%), 6 (9%) were likely pathogenic, 3 (4%) were variants of uncertain significance and 28 (41%) were novel. In NF2, all variants were pathogenic. No novel genotype-phenotype correlations were observed; however, previously described correlations were confirmed in our cohort.
CONCLUSION: The clinical and molecular characterization of neurofibromatoses in different populations is very important to provide further insights into the pathogenesis of these diseases.

Purpose Carboxyamidotriazole orotate (CTO) is a novel oral inhibitor of non-voltage-dependent calcium channels with modulatory effects in multiple cell-signaling pathways and synergistic effects with temozolomide (TMZ) in glioblastoma (GBM) models. We conducted a phase IB study combining CTO with two standard TMZ schedules in GBM. Methods In cohort 1, patients with recurrent anaplastic gliomas or GBM received escalating doses of CTO (219 to 812.5 mg/m

To explore the relationship between NF2 promoter gene mutation and the risk of medulloblastomas (MBs). We collected tissues from 16 MB patients and 7 age-matched non-MB controls. Gene sequencing, qPCR (real-time quantitative polymerase chain reaction), IHC (immunohistochemistry), and WB (Western blot) were used to analyze the changes in the NF2 gene sequence and expression between patients and controls. We found that NF2 promoter gene mutations occurred in MB patients. The NF2 mRNA expression was higher in the controls than in patients (p = 0.03 < 0.05); however, the results of IHC and WB demonstrated that the NF2 protein expression was significantly higher in patients than in the controls (IHC: p = 0.0001; WB: p = 0.01). There was no significant difference in the CRL4 mRNA and protein levels. In addition, NF2 protein was mainly expressed in the nucleus in MB patients, while the NF2 protein was mainly expressed in the cytoplasm in the controls. NF2 promoter mutations exist in MB patients. NF2 mRNA expression was higher in controls than patients; whereas NF2 protein level was higher in patients than in controls.

Epigenetic patterns on the level of DNA methylation have already been shown to separate clinically relevant subgroups of meningiomas. We here set out to identify potential prognostic implications of epigenetic modification on the level of histones with focus on H3K27 trimethylation (H3K27me3). H3K27me3 was assessed by immunohistochemistry on 232 meningiomas from 232 patients. In 194 cases, trimethylation was detected in tumor cells. In 25 cases, staining was limited to vessels while all tumor cells were negative. Finally, 13 cases yielded equivocal staining patterns. Reduced abundance of H3K27me3 in cases with staining limited to vessels was confirmed by mass spectrometry on a subset of cases. Lack of staining for H3K27me3 in all tumor cells was significantly associated with more rapid progression (p = 0.009). In line, H3K27me3-negative cases were associated with a DNA methylation pattern of the more aggressive types among the recently introduced DNA methylation groups. Also, NF2 and SUFU mutations were enriched among cases with complete lack of H3K27me3 staining in tumor cells (p < 0.0001 and p = 0.029, respectively). H3K27me3 staining pattern added significant prognostic insight into WHO grade II cases and in the compound subset of WHO grade I and II cases (p = 0.04 and p = 0.007, respectively). However, it did not further stratify within WHO grade III cases. Collectively, these data indicate that epigenetic modifications beyond DNA methylation are involved in the aggressiveness of meningioma. It also suggests that H3K27me3 immunohistochemistry might be a useful adjunct in meningioma diagnostics, particularly for cases with WHO grade II histology or at the borderline between WHO grade I and II.

BACKGROUND/AIM: Individuals with type 2 Neurofibromatosis are predisposed for the appearance of schwannomas. In the present study we analyzed the loss of heterozygosity and mutations in the NF2 gene in patients with sporadic Schwannoma without Neurofibromatosis type 2.
MATERIALS AND METHODS: We analyzed 39 patients with sporadic spinal schwannoma. We quantified the number of alleles by FISH and sequenced the NF2 gene.
RESULTS: We identified 16/39 patients with point mutations and/or LOHs in the tumor samples analyzed. The LOHs were found in 7/39 patients. Two homozygous mutations were detected in 4/39 tumors, and the presence of the mutation in heterozygosis was revealed in 3/39 patients. In two tumors, we detected the loss of one allele of the NF2 gene, with no mutation.
CONCLUSION: The genetic alterations observed in the NF2 gene indicated that spinal schwannomas are associated with genetic alterations also found in other schwannomas and type 2 Neurofibromatosis, which reinforces the etiological role of this gene.

The neurofibromatosis type 2 (

OBJECTIVE: To investigate 10 candidate single nucleotide polymorphisms (SNPs) in 5 genes (CASP8, XRCC1, WRN, NF2, and BRIP1) to confirm the association between the 5 genes and the meningioma risk in a Chinese population.
METHODS: We examined 10 candidate SNPs in 5 genes (CASP8, XRCC1, WRN, NF2, and BRIP1) to confirm the association between the 5 genes and the meningioma risk and tumor-related phenotype in 433 individuals, including 215 patients with meningioma and 218 controls.
RESULTS: The polymorphisms rs4968451T>G in BRIP1 were significantly associated with the risk of meningioma (TT vs. TG vs. GG additive, P = 0.005; TT+TG vs. GG dominant, P = 0.015; TT/GT+GG recessive, P = 0.034). The significant association was found only in females for BRIP1 rs4968451T>G (TT+TG vs. GG dominant, P = 0.001; TT/GT+GG recessive, P = 0.044). We observed no significant association between genotypes and the meningioma risk for the other 9 SNPs. Through genotype-phenotype analysis, the genotype of BRIP1 rs4968451T>G was also strongly associated with tumor-related phenotypes, including the tumor grade and tumor subtypes. BRIP1 rs4968451T>G was associated with markedly grade I meningioma risk (TT+TG vs. GG dominant, P = 0.008; TT/GT+GG recessive, P = 0.020). In addition, BRIP1 rs4968451T>G was associated with markedly meningothelial and transitional meningioma risk. Furthermore, the genotype of CAPS8, XRCC1, and NF2 was associated with different subtype of meningioma risk.
CONCLUSIONS: This study indicated a role for BRIP1 gene variations in meningioma and may be informative for future genetic or biological studies of meningioma. These findings will assist in further understanding the genetic cause for meningiomas and guide more effective biological interventions to facilitate meningiomas.

Cardiovascular disease remains the leading cause of death around the globe. Cardiac deterioration is associated with irreversible cardiomyocyte loss. Understanding how the cardiovascular system develops and the pathological processes of cardiac disease will contribute to finding novel and preventive therapeutic methods. The canonical Hippo tumor suppressor pathway in mammalian cells is primarily composed of the MST1/2-SAV1-LATS1/2-MOB1-YAP/TAZ cascade. Continuing research on this pathway has identified other factors like RASSF1A, Nf2, MAP4Ks, and NDR1/2, further enriching our knowledge of the Hippo-YAP pathway. YAP, the core effecter of the Hippo pathway, may accumulate in the nucleus and initiate transcriptional activity if the pathway is inhibited. The role of Hippo signaling has been widely investigated in organ development and cancers. A heart of normal size and function which is critical for survival could not be generated without the proper regulation of the Hippo tumor suppressor pathway. Recent research has demonstrated a novel role of Hippo signaling in cardiovascular disease in the context of development, hypertrophy, angiogenesis, regeneration, apoptosis, and autophagy. In this review, we summarize the current knowledge of how Hippo signaling modulates pathological processes in cardiovascular disease and discuss potential molecular therapeutic targets.

Schwannomas are benign nerve tumors that occur sporadically in the general population and in those with neurofibromatosis type 2 (NF2), a tumor predisposition genetic disorder. NF2-associated schwannomas and most sporadic schwannomas are caused by inactivating mutations in Schwann cells in the neurofibromatosis type 2 gene (NF2) that encodes the merlin tumor suppressor. Despite their benign nature, schwannomas and especially vestibular schwannomas cause considerable morbidity. The primary available therapies are surgery or radiosurgery which usually lead to loss of function of the compromised nerve. Thus, there is a need for effective chemotherapies. We established an untransformed merlin-deficient human Schwann cell line for use in drug discovery studies for NF2-associated schwannomas. We describe the generation of human Schwann cells (HSCs) with depletion of merlin and their application in high-throughput screening of chemical libraries to identify compounds that decrease their viability. This NF2-HSC model is amenable for use in independent labs and high-throughput screening (HTS) facilities.

OBJECTIVE: Onset of symptoms in severe sporadic neurofibromatosis type 2 (NF2) is typically within childhood; however, there is poor awareness of presenting features in young children, potentially resulting in delayed diagnosis and poorer outcome. We have reviewed presentation of sporadic paediatric NF2 to raise awareness of early features, highlighting those requiring further investigation.
DESIGN: Patients diagnosed with NF2 at age ≤16 and seen between 2012 and 2015 were notified via the British Paediatric Neurology Surveillance Unit or identified through the English NF2 service.
RESULTS: Epidemiological data estimate that 1 in 110 611 births are affected with childhood-onset NF2. Notes of 32 patients with sporadic NF2 were reviewed. Of those presenting under the age of 5, 89% (17/19) had ocular, 74% (14/19) dermatological and 58% (11/19) neurological signs; in 84% (16/19) features were multisystemic. Sixty-six per cent (21/32) had ≥1 atypical feature, including cerebellar hypoplasia in three cases (9%) and focal cortical dysplasia in five out of seven seizure-related presentations. Five cases presented with a sometimes transient or intermittent cranial nerve mononeuropathy. The mean delay to diagnosis was 3.16 years; in eight cases (25%) this exceeded 6 years. Most significant delay occurred in mononeuropathy, ophthalmological and/or seizure presentations, with a mean delay of 3, 4.5 and 6 years, respectively. Eighty-four per cent (27/32) of cases needed intervention in childhood.
CONCLUSIONS: All non-vestibular schwannoma NF2 presentations in childhood had significant diagnostic delay. We emphasise the importance of detailed assessment of skin and eyes in unusual presentations and propose an aide to prompt timely referral to specialist services.