NF2-associated Vestibular Schwannomas Research Articles

EXTH-03. A VEGF+ SUBPOPULATION OF SCHWANN CELLS DRIVES VESTIBULAR SCHWANNOMA TUMORIGENESIS

Abstract INTRODUCTION Up to 95% of vestibular schwannomas (VS) are sporadic, however their molecular underpinnings remain obscure. OBJECTIVE To perform transcriptomic profiling of VS and investigate novel tumorigenic mechanisms. METHODS We analyzed 19 VS samples (7 sporadic, 12 NF2), and performed single-cell RNA sequencing (scRNAseq, n = 4) or single-nucleus (snRNAseq, n = 15), assays for transposase-accessible chromatin (snATACseq, n = 15) and bulk DNA methylation (n = 14). CellChat was used to predict cell-cell interactions. We characterized transcription factor binding using ChIPseq. We validated our key findings at the mRNA and protein levels using RNAscope (n = 5) and multiplex immunocytochemistry (mIHC; n = 5). Mechanistic studies were conducted using NF2-null Periostin-Cre+ mice versus their Cre- control littermates. RESULTS We profiled the VS transcriptome and identified markers for Schwann cells, macrophages, T cells and endothelial cells. We identified eight Schwann cell subclusters which were enriched for known markers for myelinating, immature, repair and non-myelinating subtypes. We also unexpectedly identified a subset of VS Schwann cells with high expression of VEFGA, and these cells were enriched for signaling via laminin, NCAM, NRXN and NEGR pathways. Using RNAscope and mIHC, we verified VEGFA expression in S100B+ Schwann cells. snRNAseq and snATACseq demonstrated increased TEAD1 expression in Schwann cells, as well as increased chromatin accessibility for TEAD family transcription factor motifs. We used ChIPseq to confirm increased TEAD1 binding at the promoters of 714 genes including VEGFA. NF2-null Periostin-Cre+ mice developed spontaneous schwannomas within their dorsal root ganglia which showed increased vegfa expression, and this was partly reversible using a novel pharmacological TEAD inhibitor in-vivo. CONCLUSIONS We performed detailed epigenetic and transcriptomic profiling of sporadic and NF2-associated VS and uncovered TEAD1-mediated overexpression of VEGFA in a subset of VS Schwann cells. TEAD inhibition may constitute a novel therapeutic target for VS.

Advances in Targeted Therapy for Neurofibromatosis Type 2 (NF2)-Associated Vestibular Schwannomas.

Neurofibromatosis 2 (NF2) is an autosomal-dominant genetic disorder characterized by bilateral vestibular schwannomas (VS), meningiomas, ependymomas, spinal and peripheral schwannomas, optic gliomas, and juvenile cataracts. Ongoing studies provide new insight into the role of the NF2 gene and merlin in VS tumorigenesis. As NF2 tumor biology becomes increasingly understood, therapeutics targeting specific molecular pathways have been developed and evaluated in preclinical and clinical studies. NF2-associated VS are a source of significant morbidity with current treatments including surgery, radiation, and observation. Currently, there are no FDA-approved medical therapies for VS, and the development of selective therapeutics is a high priority. This manuscript reviews NF2 tumor biology and current therapeutics undergoing investigation for treatment of patients with VS.

Qu-Du-San-Jie decoction induces growth inhibition and vascular normalization in NF2-associated vestibular schwannoma.

Background: Neurofibromatosis type 2 (NF2) is a rare genetic syndrome that predisposes individuals to develop bilateral vestibular schwannomas (VSs) causing a high risk of life-threatening neurological complications. Traditional treatment options for NF2-associated VS usually cause neurological damage, and to date, there are no FDA-approved pharmacotherapies for NF2. The aim of this study was to evaluate the antitumor efficacy of Qu-Du-San-Jie (QDSJ) decoction, a traditional Chinese medicine formula, on NF2-associated VS and to investigate the potential underlying mechanisms. Methods: Ultra high-performance liquid chromatography-mass spectroscopy (UHPLC-MS) analysis was performed to identify the components of QDSJ and their targets. To determine the relationships between the putative targets of QDSJ and the differential genes of NF2-associated VS, the drug-disease crossover genes were screened using the UHPLC-MS data combined with our previous gene expression profiling data. The differentially expressed genes were imported into the STRING database to generate a PPI network. Differentially expressed gene targets and pathways were identified using GO and KEGG pathway enrichment analyses. The in vitro and in vivo drug efficacy of QDSJ decoction was tested using a patient-derived schwannoma cell line and a patient-derived xenograft mouse model, respectively. H&E staining, immunochemistry, and immunofluorescence staining were used to evaluate the cell proliferation and tumor vessels. Results: A total of 133 compounds were identified in QDSJ decoction using UHPLC-MS analysis. Network pharmacology showed that the regulation of necroptosis, apoptosis, cell cycle, angiogenesis, adherens junction, and neuroactive ligand-receptor interaction could be associated with the efficacy of QDSJ in treating NF2-associated VS. Treatment with QDSJ induced necrotic cell death and apoptosis of schwannoma cells in vitro and suppressed the tumor growth in vivo. Histopathological analysis revealed areas of cell necrosis and enlarged tumor blood vessels in the QDSJ-treated tumors. The numbers of cells positive for Cyclin D1 and Ki-67 were significantly reduced in QDSJ-treated tumors compared to control tumors. Immunofluorescence staining of CD31 and αSMA showed a decreased number and density of tumor vessels and normalized vessel structure in QDSJ-treated tumors. Conclusion: Our study demonstrates that QDSJ decoction shows significant antitumor activity against NF2-associated schwannoma and is a possible candidate for future clinical trials.

Novel patient-derived xenograft and cell line models for therapeutic screening in NF2-associated schwannoma.

Treatment of schwannomas in patients with neurofibromatosis type 2 (NF2) is extremely unsatisfactory, and innovative therapeutic approaches are urgently needed. However, the lack of clinically relevant NF2-associated schwannoma models has severely hampered drug discovery in this rare disease. Here we report the first establishment and characterization of patient-derived xenograft (PDX) and cell line models of NF2-associated schwannoma, which recapitulates the morphological and histopathological features of patient tumors, retain patient NF2 mutations, and maintain gene expression profiles resembling patient tumor profiles with the preservation of multiple key signaling pathways commonly dysregulated in human schwannomas. Using gene expression profiling, we identified elevated PI3K/AKT/mTOR networks in human NF2-associated vestibular schwannomas. Using high-throughput screening of 157 inhibitors targeting the PI3K/AKT/mTOR pathways in vitro, we identified a dozen inhibitors (such as BEZ235, LY2090314, and AZD8055) with significant growth-suppressive effects. Interestingly, we observed that three cell lines displayed differential therapeutic responses to PI3K/AKT/mTOR inhibitors. Furthermore, we demonstrated that two orally bioavailable inhibitors, AZD8055 and PQR309, suppressed NF2-associated schwannoma growth both in vitro and in vivo. In conclusion, our novel patient-derived models of NF2-associated schwannoma closely mimic the phenotypes and genotypes of patient tumors, making them reliable preclinical tools for testing novel personalized therapies. © 2022 The Pathological Society of Great Britain and Ireland.

Stereotactic radiosurgery for vestibular schwannomas in neurofibromatosis type 2 patients: a systematic review and meta-analysis.

Neurofibromatosis type 2 (NF2) is characterized by often bilateral vestibular schwannomas (VS) that result in progressive hearing loss and compression of nearby brainstem structures causing cranial nerve palsies. Treatment of these tumors remains challenging, as both surgical removal and expectant management can result in symptom progression. Stereotactic radiosurgery (SRS) has been investigated for the management of NF2-associated VS; however, the role, promises, and pitfalls of this treatment modality remain unclear. Ovid MEDLINE, EMBASE, Web of Science, and Cochrane Reviews were searched for studies assessing SRS outcome in NF2-associated VS only. Primary endpoints included tumor control, serviceable hearing, presence of tinnitus, and cranial nerve V and VII symptoms. A total of 16 studies (589 patients harboring 750 tumors) were analyzed. Clinical tumor control was achieved in 88% of cases (95% CI 80-95%); salvage surgery was needed in 8% (95% CI 4-13%) of cases. Treatment resulted in a worsening of pre-treatment serviceable hearing (OR = 0.26, p < 0.01), increased facial nerve (OR = 1.62, p < 0.01) and trigeminal nerve (OR = 1.42, p = 0.07) impairment. The incidence of vestibular symptoms and hydrocephalus were not consistently reported and thus could not be assessed. The treatment of NF2-associated VS continues to pose a challenge, as current SRS regimens result in impaired hearing and worse cranial nerve comorbidities, despite achieving high tumor control. It remains unclear if these findings have to be regarded as treatment complications or, rather, continued disease progression.

Risk Stratification for Immediate Postoperative Hearing Loss by Preoperative BAER (Brainstem Auditory Evoked Response) and Audiometry in NF2-Associated Vestibular Schwannomas.

Simple SummaryHearing preservation is one of the major goals in the surgical treatment of Neurofibromatosis Type 2 (NF2) associated vestibular schwannomas (VS) and hearing diagnostics are important monitoring parameters and intraoperative tools to pursue this goal. Our monocentric retrospective analysis aimed to predict postoperative hearing deterioration or loss based on preoperative audiometry and neurophysiological (brainstem auditory evoked potentials, BAEP) findings. In this respect and according to our data evaluation in 100 NF2-associated VS of 72 patients both parameters seem to be prognostic markers, particularly BAEP findings. Large discrepancies between both audiometry and BAEPs which were identified in twenty-one cases of our cohort appear to be at high risk of significant postoperative hearing worsening.Both brainstem auditory evoked potentials (BAEP) and audiometry play a crucial role in neuro-oncological treatment decisions in Neurofibromatosis Type 2 associated (NF2) vestibular schwannoma (VS) as hearing preservation is the major goal. In this study, we investigated the risk of immediate postoperative hearing deterioration (>15 dB and/or 15% loss in pure-tone average [PTA]/ speech discrimination score [SDS] in a cohort of 100 operated VS (ears) in 72 NF2 patients by retrospective analysis of pre- and postoperative hearing data (PTA, SDS, American Association of Otolaryngology–Head and Neck Surgery [AAO-HNS], and brainstem auditory evoked potential [BAEP] class) taking into account relevant influencing factors, particularly preoperative audiometry and BAEP status and the extent of resection. Immediately after surgery, the hearing was preserved in 73% of ears and approximately ~60% of ears kept their hearing classes. Preoperative BAEP (p = 0.015) and resection amount (p = 0.048) significantly influenced postoperative hearing outcome. The prediction model for postoperative hearing deterioration/loss between preoperative BAEP and AAO-HNS class showed increased risk by increasing BAEP class. Twenty-one tumors/ears were identified with large BAEP and AAO-HNS class discrepancies (≥2 points) and were associated with a high (48–100%) risk of deafness after surgery in ears with preoperative available hearing. Overall, the results were heterogeneous but the better both BAEP and audiometry class before surgery, the higher the chance of hearing maintenance afterwards. Large resection amounts (e.g., 100% risk in near-total resections) exhibit a significant (p < 0.05) higher risk compared to smaller amounts (e.g., 10/20% in laser-coagulated/partially resected tumors). Our results emphasized the indispensable role of both hearing monitoring in form of audiometry and neurophysiology (BAEP) in the pre-and perioperative monitoring of NF2-associated VS. Both BAEP and audiometry are good prognostic markers for the postoperative hearing outcome. The extent of resection should be strictly guided by and adjusted to the intraoperative neurophysiological monitoring.

LGG-42. BEVACIZUMAB-ASSOCIATED SECONDARY AMENORRHEA AND PREMATURE OVARIAN FAILURE IN ADOLESCENT FEMALE PATIENTS WITH LOW-GRADE CNS DISEASE

Compelling body of evidence exists to use bevacizumab, a humanized monoclonal anti-VEGF antibody, in selected paediatric patients with low-grade CNS tumours. Common toxicities of bevacizumab, hypertension, proteinuria, epistaxis, mucosal perforation, decreased wound healing are well reported. However, the effect of bevacizumab on female ovarian function and long-term fertility is still being documented. Current evidence for bevacizumab-associated decline in ovarian function is largely from breast and colon cancer cohorts where exposure to multimodal chemotherapy confounds causative relationships. Fertility counseling and oocyte cryopreservation is currently offered as standard of care to post-pubertal females at high risk of infertility due to high-dose radiation and chemotherapy. Adolescent females with low-grade CNS tumours on bevacizumab represent a unique population which could potentially be at high risk for infertility. We report 2 cases of adolescent girls treated with bevacizumab as a single agent and in combination with vinblastine for NF2-associated vestibular schwannomas and brainstem glioma respectively. Both patients were post-pubertal with established menstrual cycles and normal baseline FSH/LH/oestradiol/AMH values prior to commencement of therapy. They became amenorrhoeic shortly after starting of therapy with levels of FSH/LH/oestradiol suggestive of premature ovarian failure. One patient has remained asymptomatic, whereas the other has developed profound post-menopausal symptoms interfering with quality of life which necessitated commencement of hormone-replacement therapy. Appropriate pretreatment fertility investigation and consultation should be offered to all post-pubertal females starting on bevacizumab. Further research into the long-term effects of gonadal toxicity in both females and males with drugs inhibiting angiogenesis is needed.

New developments in neurofibromatosis type 2 and vestibular schwannoma.

Neurofibromatosis type 2 (NF2) is a rare autosomal dominant disorder characterized by the development of multiple nervous system tumors due to mutation in the NF2 tumor suppressor gene. The hallmark feature of the NF2 syndrome is the development of bilateral vestibular schwannomas (VS). Although there is nearly 100% penetrance by 60 years of age, some patients suffer from a severe form of the disease and develop multiple tumors at an early age, while others are asymptomatic until later in life. Management options for VS include surgery, stereotactic radiation, and observation with serial imaging; however, currently, there are no FDA-approved pharmacotherapies for NF2 or VS. Recent advancements in the molecular biology underlying NF2 have led to a better understanding of the etiology and pathogenesis of VS. These novel signaling pathways may be used to identify targeted therapies for these tumors. This review discusses the clinical features and treatment options for sporadic- and NF2-associated VS, the diagnostic and screening criteria, completed and ongoing clinical trials, quality of life metrics, and opportunities for future research.

NCOG-12. STEREOTACTIC RADIOSURGERY FOR VESTIBULAR SCHWANNOMAS IN NEUROFIBROMATOSIS TYPE 2 PATIENTS: A SYSTEMATIC REVIEW AND META-ANALYSIS

Abstract BACKGROUND One of the hallmarks of neurofibromatosis type 2 (NF2) is the presence of bilateral vestibular schwannomas (VSs) that result in progressive hearing loss and compression of nearby brainstem structures causing cranial nerve palsies. Treatment of these tumors remains challenging, as both surgical removal and expectant management can result in symptom progression. Stereotactic radiosurgery (SRS) has recently been investigated for the management of NF2-associated VSs; however, the role, promises, and pitfalls of this treatment modality remain unclear. METHODS Ovid MEDLINE, EMBASE, Web of Science, and Cochrane Reviews were searched for studies assessing SRS outcome in NF2-associated VSs only. Primary endpoints included tumor control, serviceable hearing, presence of tinnitus, and cranial nerve V and VII symptoms. RESULTS A total of 16 studies (589 patients harboring 750 tumors) was included in this analysis. Clinical tumor control was achieved in 88% of cases (95% CI: 80%-95%); salvage surgery was needed in 8% (95% CI: 4%-13%) of cases. Treatment resulted in a worsening of pre-treatment serviceable hearing (OR of 0.26, p &amp;lt; 0.01), in an increase of facial nerve impairment (OR of 1.62, p &amp;lt; 0.01), and showed a trend for increase trigeminal nerve impairment as well (OR of 1.42, p = 0.07). The incidence of vestibular symptoms and hydrocephalus requiring shunting could not be assessed since these measures were not reported consistently. CONCLUSIONS The treatment of NF2-associated VS continues to pose a challenge, as current SRS regimens result in impaired hearing and worse cranial nerve comorbidities, despite achieving high tumor control. It remains unclear if these findings have to be intended as treatment complications or, rather, continuous disease progression despite tumor control.

Gene Expression, Network Analysis, and Drug Discovery of Neurofibromatosis Type 2-Associated Vestibular Schwannomas Based on Bioinformatics Analysis.

Neurofibromatosis Type 2- (NF2-) associated vestibular schwannomas (VSs) are histologically benign tumors. This study aimed to determine disease-related genes, pathways, and potential therapeutic drugs associated with NF2-VSs using the bioinformatics method. Microarray data of GSE108524 were downloaded from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) were screened using GEO2R. The functional enrichment and pathway enrichment of DEGs were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes Genomes (KEGG). Furthermore, the STRING and Cytoscape were used to analyze the protein-protein interaction (PPI) network of all differentially expressed genes and identify hub genes. Finally, the enriched gene sets belonging to the identified pathways were queried against the Drug-Gene Interaction database to find drug candidates for topical use in NF2-associated VSs. A total of 542 DEGs were identified, including 13 upregulated and 329 downregulated genes, which were mainly enriched in terms of focal adhesion, PI3K-Akt signaling pathway, ECM-receptor interaction, Toll-like receptor signaling pathway, Rap1 signaling pathway, and regulation of actin cytoskeleton. 28 hub genes were identified based on the subset of PPI network, and 31 drugs were selected based on the Drug-Gene Interaction database. Drug discovery using bioinformatics methods facilitates the identification of existing or potential therapeutic drugs to improve NF2 treatment.

Managing NF2-associated vestibular schwannomas in children and young adults: review of an institutional series regarding effects of surgery and bevacizumab on growth rates, tumor volume, and hearing quality

We reviewed our experience in managing of NF2-associated vestibular schwannoma (VS) in children and young adults regarding the effect of surgery and postoperative bevacizumab treatment. A total of 579 volumetric and hearing data sets were analyzed. The effect of surgery on tumor volume and growth rate was investigated in 46 tumors and on hearing function in 39 tumors. Long-term hearing follow-up behavior was compared with 20 non-operated ears in additional 15 patients. Sixteen operated VS were treated with bevacizumab. Mutation analysis of the NF2 gene was performed in 25 patients. Surgery significantly slowed down VS growth rate. Factors associated with a higher growth rate were increasing patient age, tumor volume, and constitutional truncating mutations. Immediately after surgery, functional hearing was maintained in 82% of ears. Deterioration of hearing was associated with initial hearing quality, larger tumor volumes, and larger resection amounts. Average hearing scores were initially better in the group of non-operated VS. Over time, hearing scores in both groups worsened with a similar dynamic. During bevacizumab treatment of residual tumors, four different patterns of growth were observed. Decompression of the internal auditory canal with various degrees of tumor resection decreases the postoperative tumor growth rates. Carefully tailored BAEP-guided surgery does not cause additional hearing deterioration. Secondary bevacizumab treatment showed heterogenous effects both regarding tumor size and hearing preservation. It seems that postoperative tumor residuals, that grow slower, behave differently to bevacizumab than reported for not-operated faster growing VS.

Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma

Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.

Bevacizumab for NF2-associated vestibular schwannomas of childhood and adolescence.

Seventeen children at six institutions with neurofibromatosis type 2 (NF2)-related vestibular schwannomas received bevacizumab. Eight of the 13 patients with initial hearing loss (61%) showed objective hearing improvement within six months of treatment. No patients showed hearing deterioration during therapy; however, only two patients showed objective radiological response. Seven of eight patients had tumor progression or worsening hearing loss upon cessation of treatment. Bevacizumab was well tolerated with no patients discontinuing therapy. Bevacizumab appears to postpone hearing loss in childhood NF2-associated vestibular schwannomas, but responses are not durable, suggesting that either longer maintenance therapy or new strategies are required.

Contribution of mTOR and PTEN to Radioresistance in Sporadic and NF2-Associated Vestibular Schwannomas: A Microarray and Pathway Analysis.

The use of radiation treatment has increased for both sporadic and neurofibromatosis type 2 (NF2)-associated vestibular schwannoma (VS). However, there are a subset of radioresistant tumors and systemic treatments that are seldom used in these patients. We investigated molecular alterations after radiation in three NF2-associated and five sporadically operated recurrent VS after primary irradiation. We compared these findings with 49 non-irradiated (36 sporadic and 13 NF2-associated) VS through gene-expression profiling and pathway analysis. Furthermore, we stained the key molecules of the distinct pathway by immunohistochemistry. A total of 195 differentially expressed genes in sporadic and NF2-related comparisons showed significant differences based on the criteria of p value < 0.05 and a two-fold change. These genes were involved in pathways that are known to be altered upon irradiation (e.g., mammalian target of rapamycin (mTOR), phosphatase and tensin homolog (PTEN) and vascular endothelial growth factor (VEGF) signaling). We observed a combined downregulation of PTEN signaling and an upregulation of mTOR signaling in progressive NF2-associated VS after irradiation. Immunostainings with mTOR and PTEN antibodies confirmed the respective molecular alterations. Taken together, mTOR inhibition might be a promising therapeutic strategy in NF2-associated VS progress after irradiation.

Bevacizumab Therapy of Neurofibromatosis Type 2 Associated Vestibular Schwannoma in Japanese Patients.

We conducted a feasibility study to investigate the therapeutic effect of bevacizumab on vestibular schwannomas (VS) associated with neurofibromatosis type 2 (NF2) in a sample of Japanese patients. Ten NF2 patients were selected between 2013 and 2018: nine women and one man, with ages ranging from 12 to 45 years (mean: 29.4). Bevacizumab was administered intravenously in 5 mg/kg doses four times, with an inter-dose interval of 2 weeks. Seventeen tumors were followed for 3–72 months (mean: 39). A reduction from baseline tumor volume of at least 20% was considered a therapeutic radiologic response. Maximum reduction in tumor volume was identified in the 3rd month in 11 tumors, and in the 6th month in three tumors. Three tumors did not show any response to bevacizumab. A radiologic response was detected in seven tumors (41%). There was a significantly lower tumor volume mean in the 3rd month in comparison to the baseline for the entire sample. Tumors in patients aged 25 and above showed a significant reduction in volume in the 3rd month and significantly lower tumor-volume-to-baseline ratio than younger patients in both the 3rd and 6th months. The interaction between ‘time’ and ‘age group’ factors significantly affected the therapeutic outcome of bevacizumab on tumor volume. This study investigated the therapeutic effects of bevacizumab on NF2-associated vestibular schwannomas in Japanese patients. Bevacizumab appears to be a useful therapeutic choice in NF2 cases to control the growth of VS. Therefore, a randomised control trial to prove this assumption is necessary.

Management of NF2-associated vestibular schwannomas in children and young adults: influence of surgery and clinical factors on tumor volume and growth rate.

The authors' aim was to evaluate the tumor volume and growth rate of neurofibromatosis type 2 (NF2)-associated vestibular schwannomas (VSs) and the clinical factors or type of mutations before and after surgery in children and adults younger than 25 years at the time of diagnosis. A total of 579 volumetric measurements were performed in 46 operated tumors in 28 NF2 patients, using thin-slice (< 3 mm) T1-weighted contrast-enhanced MRI. The follow-up period ranged from 21 to 167 months (mean 75 months). Growth rate was calculated using a multilinear regression model. Mutation analysis of the NF2 gene was performed in 25 patients. Surgery significantly (p = 0.013) slowed the VS growth rate from 0.69 ± 1.30 cm3/yr to 0.23 ± 0.42 cm3/yr. Factors significantly associated with a higher growth rate of VSs were increasing patient age (p < 0.0005), tumor volume (p = 0.006), tumor size (p = 0.001), and constitutional truncating mutations in the NF2 gene (p = 0.018). VS growth rates tended to be higher in patients with spinal ependymomas and in right-sided tumors and lower in the presence of peripheral schwannomas; however, no statistical significance was achieved. Decompression of the internal auditory canal with various degrees of tumor resection decreases the postoperative tumor growth rate in children and young adults with NF2-associated VS. Patients with potential risk factors for accelerated growth (e.g., large volume, truncating mutations) and with increasing age should be monitored more closely before and after surgery.

A mosaic pattern of INI1/SMARCB1 protein expression distinguishes Schwannomatosis and NF2-associated peripheral schwannomas from solitary peripheral schwannomas and NF2-associated vestibular schwannomas.

The INI1/SMARCB1 gene protein product has been implicated in the direct pathogenesis of schwannomas from patients with one form of schwannomatosis [SWNTS1; MIM # 162091] showing a mosaic pattern of loss of protein expression by immunohistochemistry [93% in familial vs. 55% in sporadic cases]. To verify whether such INI1/SMARCB1 mosaic pattern could be extended to all schwannomas arising in the sporadic and familial schwannomatoses [i.e. to SMARCB1-related (SWNTS1) or LZTR1-related (SWNTS2) schwannomatosis or to SMARCB1/LZTR1-negative schwannomatosis] and whether it could be involved in classical NF2 or solitary peripheral schwannomas METHODS: We blindly analysed schwannoma samples obtained from a total of 22 patients including (a) 2 patients (2 males; aged 38 and 55years) affected by non-familial SMARCB1-associated schwannomatosis (SWTNS1); (b) 1 patient (1 female; aged 33years) affected by familial schwannomatosis (SWTNS1/ SMARCB1 germ line mutations); (c) 5 patients (3 males, 2 females; aged 33 to 35years) affected by non-familial (sporadic) LZTR1-associated schwannomatosis (SWNTS2); (d) 3 patients (3 males; aged 35 to 47years) affected by familial schwannomatosis (SWTNS2/ LZTR1 germ line mutations); (e) 2 patients (1 male, 1 female; aged 63 and 49years, respectively) affected by non-familial schwannomatosis (SWTNS, negative for SMARCB1, LZTR1 and NF2 gene mutations); (f) 4 patients (3 males, 1 females; aged 15 to 24years) affected by classical NF2 (NF2: harbouring NF2 germ line mutations; and (g) 5 patients (3 males, 2 females; aged 33 to 68years) who had solitary schwannomas. [follow-up=15-30years; negative for constitutional/somatic mutation analysis for the SMARCB1, LZTR1 and NF2 genes] were (blindly) analyzed. The INI1/SMARCB1 immunostaining pattern was regarded as (1) diffuse positive nuclear staining [= retained expression] or (2) mosaic pattern [mixed positive/negative nuclei = loss of expression in a subset of tumour cells]. All solitary peripheral schwannomas and NF2-associated vestibular schwannomas showed diffuse nuclear INI1/SMARCB1 staining in 97-100% of neoplastic cells; schwannomas obtained from all cases of non-familial and familial schwannomatosis and NF2-associated non-vestibular schwannomas showed a mosaic pattern ranging from 10 to 70% of INI1/SMARCB1-positive expression. We did not record a complete lack of nuclear staining. The present data suggests that (a) mosaic loss of immunohistochemical INI1/SMARCB1 expression, despite the interlesional variability, is a reliable marker of schwannomatosis regardless of the involved gene and it might help in the differential diagnosis of schwannomatosis vs. solitary schwannomas and (b) INI1/SMARCB1 expression is not useful in the differential with mosaic NF2, since NF2-associated peripheral schwannomas show the same immunohistochemical pattern.

A Lack of Association between Human Herpes Viridiae (HHV) and Vestibular Schwannoma: Analysis of 121 Cases

Objectives: Vestibular schwannoma (VS) is a benign tumor thatarises from the Schwann cells of the vestibular nerve. Human herpes virus (HHV) 1 and 2 have been found within the parenchyma of virtually all the organs of the body and the dorsal root ganglia of the spinal cord in humans. HHV has been shown to replicate within Schwann cells, and infection by HHV has been shown to mimic VS on magnetic resonance imaging. Others hypothesized that the likely cause of VS is HHV infection and that prior exposure to HHV-3 is a potential risk factor in VS development. Our objective wasto assess for the presence of HHV viral DNA using immunohistochemical techniques in surgically excised vestibular schwannoma samples. Methods: Tissue microarrays (TMAs) representing sporadic and NF2-associated vestibular schwannomas from 121 patients, as well as both positive and negative controls, were studied. TMA sections were immunostained-using antibodies directed against HHV-1, HHV-2, HHV-3, HHV-5, and HHV-8. Results: There was no immunohistochemically detectable HHV (HHV-1, HHV-2, HHV-3, HHV-5, HHV-8) in any of the 121 cases of sporadic or NF2 cases analyzed. Conclusions: These data offer no support for an association between HHV and the development of sporadic or NF2 associated VS in humans.

Assessment of antiangiogenic effect of imatinib mesylate on vestibular schwannoma tumors using in vivo corneal angiogenesis assay

Angiogenesis and the platelet-derived growth factor (PDGF) pathway are active in the pathogenesis of vestibular schwannomas (VSs). The purpose of this study was to test whether imatinib mesylate (Gleevec), a PDGF receptor (PDGFR) blocker, reduces angiogenic capacity in sporadic VS and in VS associated with neurofibromatosis Type 2 (NF2) using a corneal angiogenesis assay. From 121 VS tissue samples stored in the tumor bank at the Marmara University Institute of Neurological Sciences, 10 samples (6 from sporadic cases, 4 from NF2-associated cases) were selected at random for use in this study. Expression of PDGF-A and PDGF-B and their receptors was evaluated in sporadic and NF2-associated VS as well as in glioblastoma (GBM) and normal brain tissue by means of immunohistochemistry and Western blot analysis. Corneal angiogenesis assay was then used to evaluate the angiogenic capacity of tissue specimens from sporadic and NF2-associated VS with and without imatinib treatment as well as positive and negative controls (GBM and normal brain tissue). The angiogenic potential of the sporadic and NF2-associated VS tumor tissue differed significantly from that of the positive and negative control tissues (p <0.05). Furthermore, NF2-associated VS showed significantly lower angiogenic potential than sporadic VS (p <0.05). Imatinib treatment significantly reduced the angiogenic potential in both the sporadic VS and the NF2-associated VS groups. The level of PDGF-A and PDGFR-α as well as PDGF-B and PDGFR-β expression in sporadic VS and NF2-associated VS also differed significantly (p <0.05) from the levels in controls. Additionally the level of PDGFR-β was significantly higher in sporadic VS than in NF2-associated VS (p <0.05). The findings of this study indicate that NF2-associated VS has significantly more angiogenic potential than sporadic VS and normal brain tissue. Additionally, imatinib reduces the angiogenic activity of both sporadic and NF2-associated VS. The authors conclude that imatinib may be a potential treatment for VS, especially for NF2-associated lesions that cannot be cured with resection or radiosurgery.

Cyclooxygenase-2 Supports Tumor Proliferation in Vestibular Schwannomas

Recent studies have shown that cyclooxygenase-2 (COX-2) plays an important role in tumor growth and neovascularization. However, COX-2 expression in vestibular schwannomas (VSs) has not been investigated. To analyze the pattern of COX-2 expression in sporadic and neurofibromatosis type 2 (NF2)-associated VSs and its relationship with tumor proliferation and microvessel density. Fifteen sporadic and 15 NF2-associated VSs were examined for COX-2 expression, microvessel density, and proliferation rate by immunohistochemical methods. Immunohistochemical scores were used to interpret the extent and intensity of COX-2 staining. Microvessel density (MVD) was determined using von Willebrand factor (vWf). Proliferation rate was quantified using Ki-67. The relationship among COX-2 expression, MVD, and proliferation rate was statistically analyzed. COX-2 expression was detected in 29 (96.67%) of 30 VSs, with no significant difference between sporadic and NF2-associated VSs (P = .722). In 6 (20%) VSs, COX-2 expression was graded as strong, in 12 (40%) as moderate, and in 11 (36.7%) as weak. VSs with high proliferation showed significantly higher COX-2 expression (P = .015) than VSs with low proliferation. COX-2 expression and MVD did not show specific biological correlations (P = .035). Our data demonstrate that COX-2 is expressed in VSs. High COX-2 expression in VSs with high proliferation rates suggests that the COX-2 pathway may be involved in the development and growth of VSs.