Table of Contents

    Topic review

    Neurofibromatosis and Schwannomatosis

    Subjects: Neurosciences
    View times: 9
    Submitted by: Ryota Tamura


    Neurofibromatosis (NF) is a neurocutaneous syndrome characterized by the development of tumors of the central or peripheral nervous system including the brain, spinal cord, organs, skin, and bones. There are three types of NF: NF1 accounting for 96% of all cases, NF2 in 3%, and schwannomatosis (SWN) in <1%. The NF1 gene is located on chromosome 17q11.2, which encodes for a tumor suppressor protein, neurofibromin, that functions as a negative regulator of Ras/MAPK and PI3K/mTOR signaling pathways. The NF2 gene is identified on chromosome 22q12, which encodes for merlin, a tumor suppressor protein related to ezrin-radixin-moesin that modulates the activity of PI3K/AKT, Raf/MEK/ERK, and mTOR signaling pathways. In contrast, molecular insights on the different forms of SWN remain unclear. Inactivating mutations in the tumor suppressor genes SMARCB1 and LZTR1 are considered responsible for a majority of cases.

    1. Introduction

    Neurofibromatosis (NF) is a genetic disorder that causes multiple tumors on nerve tissues, including brain, spinal cord, and peripheral nerves [1][2][3]. There are three types in NF: NF1, NF2, and schwannomatosis (SWN) [4]. NF1 is the most prevalent, accounting for 96% of all cases and characterized by neurofibromas (peripheral nerve tumors) that induce skin changes and bone deformation. Characterized by tumors originating from Schwann cells, NF2 and SWN are rare compared to NF1, occurring in 3% and <1%, respectively. NF2 typically causes hearing loss and vestibular dysfunction [5][6]. Whereas, SWN causes intense pain [5][6].
    Different mutations result in the three types of NF. Currently, there is no way to prevent or cure these diseases. In this review, we discuss the clinical, genetic, and molecular characteristics of NF and the current molecular targeted therapies, and review the recent clinical trials for the patients with NF.

    2. Neurofibromatosis Type 1

    2.1. Clinical Characteristics

    NF1, which is known as von Recklinghausen’s disease, causes various manifestations such as multiple flat, light-brown patches of skin pigment (café-au-lait spots), skinfold freckling, visible neurofibromas under the skin, and small nodules of the iris (Lisch nodules) (Table 1) [7][8][9][10]. NF1 occurs in 1 in 3000–4000 people worldwide. Although NF1 is inherited via autosomal dominance, 50% of detected mutations are de novo [7][8][9][10][11]. The condition typically progresses over time since childhood. It has been shown that NF1 patients have decreased life expectancy of 15 years compared to the general population [12]. Malignant tumors and vascular disease have been significantly associated with the death of NF1 patients aged <40 years [13].
    Table 1. Diagnostic criteria of neurofibromatosis type 1.
    A: The Diagnostic Criteria for NF1 Are Met in an Individual Who Does Not Have a Parent Diagnosed with NF1 if Two or More of the Following Are Present:
    At least six café-au-lait macules (>5 mm diameter in prepubertal individuals and >15 mm in postpubertal individuals)
    Freckling in axillary or inguinal regions #1
    Optic glioma
    At least two Lisch nodules identified by slit lamp examination or two or more choroidal abnormalities—defined as bright, patchy nodules imaged by optical coherence tomography/near-infrared reflectance imaging
    At least two neurofibromas of any type, or one plexiform neurofibroma
    A distinctive osseous lesion such as sphenoid dysplasia, #2 anterolateral bowing of the tibia, or pseudarthrosis of a long bone
    A heterozygous pathogenic NF1 variant with a variant allele fraction of 50% in apparently normal tissue such as white blood cells
    B: A child of a parent who meets the diagnostic criteria specified in A merits a diagnosis of NF1 if one or more of the criteria in A are present
    #1 If only café-au-lait macules and freckling are present, the diagnosis is most likely NF1 but exceptionally the person might have another diagnosis such as Legius syndrome. At least one of the two pigmentary findings (café-au-lait macules or freckling) should be bilateral. #2 Sphenoid wing dysplasia is not a separate criterion in case of an ipsilateral orbital plexiform neurofibroma.



    2.2. Genetic and Molecular Characteristics

    The NF1 gene is located on 17q11.2 of chromosome 17. The point mutations are responsible for 90% of NF1 patients. A single exon or whole NF1 gene deletion is associated with the remaining 5–7% [14][15]. NF1 codes for neurofibromin, which is a Ras-GTPase-activating protein (Ras-GAP) [15]. Neurofibromin protein is produced in nerve cells, oligodendrocytes, and Schwann cells. NF1 gene deficiency leads to Ras hyperactivation, leading to the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways [15] (Figure 1). ERK activates SYN1, modulating GABA release. Ras-GTP also activates Rac1 and Cdc42 pathways, leading to overactivation of PAK1 [16][17]. Nonfunctional neurofibromin protein influences the growth of neurofibromas along the nerves of the whole body. However, it currently remains unclear how NF1 gene mutations cause café-au-lait spots and learning disabilities.
    Figure 1. Molecular pathogenesis of NF1. NF1 codes for neurofibromin, which is a Ras-GTPase-activating protein (Ras-GAP). NF1 gene deficiency leads to Ras hyperactivation, which causes the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways. ERK activates SYN1 modulating GABA release. Ras-GTP also activates Rac1 and Cdc42 pathways, leading to overactivation of PAK1.
    Several thousand pathogenic NF1 variants have been identified in NF1 patients [18]. Some patients who have a higher incidence of intellectual disability, developmental delay, dysmorphic facial features, and earlier appearance of cutaneous neurofibromas tend to have malignant peripheral nerve sheath tumors (MPNSTs) [19][20][21]. The c.2970–2972 delAAT (p.M992del) mutation is associated with a mild phenotype [22]. A severe phenotype including plexiform neurofibromas, spinal neurofibromas, optic glioma, skeletal dysplasia, and malignant transformation is associated with missense variants in codons 844 to 848 [23]; p.Met1149, p.Arg1276, or p.Lys1423 missense variants with a Noonan syndrome-like phenotype [24]; and p.Arg1276 and p.Met1149 with spinal neurofibromas and café-au-lait macules, respectively [14].
    Furthermore, NF1 can be associated with specific genetic lesions. NF1 gene product acts as a negative regulator of the product of RAS genes which are activated in myelodysplastic syndromes and acute myeloid leukemia through point mutations [25].
    Genetic testing can be performed to confirm the diagnosis and to assist the direct screening of family members [26]. However, a negative test does not completely exclude the diagnosis as it may also represent mosaicism for a pathogenic variant. NF1 is noted for the considerable inter and intrafamilial variation observed in the clinical phenotype, even in patients who share the same germline mutation. This variability poses disease prediction and management problems. Allelic heterogeneity may be a possible cause of the multiple phenotypes in NF1 [27]. Meanwhile, a positive NF1 mutation does not predict the severity of the disease. In general, targeted testing is performed to detect the mutation rather than comprehensively analyze the mutation of the entire gene. The development of next-generation sequencing technologies allows for rapid diagnosis of NF1 [28]. Amniocentesis or chorionic villus sampling can be performed to obtain a sample for genotyping the fetus if the precise mutation of an affected NF1 family member is detected [28].

    2.3. Therapeutic Strategies

    Surgery is the principal mode of treatment for neurofibromas, but comes with a high recurrence rate after partial removal of large plexiform neurofibromas. In the case of NF1-related tumors, there is no consensus with regard to the treatment strategy due to the multiple pathways involved in the growth of NF1-related tumors. Targeted therapy can show a great impact [17][29]. Anti-Ras therapies are ideal because Ras-GTP is upregulated in neurofibromas. Agents targeting Ras signaling and other pathways (tipifarnib, pirfenidone, sirolimus, pegylated interferon alfa-2b, and imatinib) have been used for plexiform neurofibromas in phase II clinical trials. Targeting downstream effectors of the Ras signaling pathway, such as agents inhibiting MEK and PI3K, and the pharmacological inhibition of kit activity and α4β1 adhesion are considered promising therapeutic strategies. A new clinical trial suggests that the MEK inhibitor selumetinib induces partial responses in children with NF1 who have inoperable plexiform neurofibromas [30]. In April 2020, the US Food and Drug Administration approved selumetinib (KOSELUGO, AstraZeneca) for pediatric NF1 patients aged at least 2 years who have symptomatic, inoperable plexiform neurofibromas [31]. Rapamycin is an inhibitor of the mTOR pathway, but induces AKT activation, thus demonstrating both therapeutic potential and limits [17][29].
    Low grade optic gliomas are the most common central nervous system tumors in NF1 patients [32], with pilocytic astrocytoma known as its indolent subtype. Regimens involving carboplatin and vincristine are the most frequently used chemotherapy for optic nerve glioma [33].

    2.4. Ongoing Clinical Trials

    Table 2 shows ongoing phase I/II clinical trials for NF1 patients using various molecular targeted agents such as selumetinib and mirdametinib (MEK inhibitor), binimetinib and trametinib (MEK1/2 inhibitor), and cabozantinib (VEGFR2 inhibitor). The primary outcome is volumetric response, toxicity, and event-free survival.
    Table 2. Ongoing clinical trials for the patients with NF1.
    ID Initiation Date Phase Nation N Disease Treatment Primary Outcome
    NCT04495127 8, 2020 1 Japan 12 NF1 Selumetinib Toxicity
    NCT01968590 8, 2017 2 USA 320 NF1 Cholecalciferol Bone mineral density
    NCT03962543 9, 2019 2 USA 100 NF1
    Plexiform Neurofibroma
    Mirdametinib (PD-0325901) oral capsule Complete or partial response rate compared to baseline.
    NCT03231306 11, 2017 2 USA 40 NF1
    Plexiform Neurofibroma
    Binimetinib Change from Baseline Target Tumor Volume at 12 months
    NCT02839720 4, 2017 2 USA 24 Cutaneous Neurofibroma
    Optic Nerve Glioma
    Selumetinib Change in the size
    NCT02407405 1, 2016 2 USA 60 NF1
    Plexiform Neurofibromas
    Selumetinib Determine objective response rate
    NCT04461886 7, 2020 3 Japan 100 NF NPC-12G gel Discontinuation rate associated with adverse events
    NCT03871257 10, 2019 3 USA 290 Low Grade Glioma NF1
    Visual Pathway Glioma
    Selumetinib Sulfate Vincristine Sulfate
    Event-free survival
    NCT02101736 6, 2014 2 USA 48 NF1
    Neurofibromatosis Plexiform Neurofibromas
    Cabozantinib The change in tumor size based on radiographic assessment
    NCT03326388 9, 2019 1/2 USA 30 NF1
    Plexiform Neurofibroma
    Optic Nerve Glioma
    Selumetinib To evaluate the Maximum Tolerated Dose
    Objective response rate
    NCT03741101 6, 2019 2 Sweden 15 NF1
    Plexiform Neurofibromas
    Trametinib Remission of tumor volume ≥20%
    NCT02728388 8, 2016 2 USA 30 NF1 aminolevulinic acid Time to disease progression
    NCT04435665 8, 2020 2 USA 48 NF1
    Cutaneous Neurofibroma
    NFX-179 Gel Phospho-erk (p-ERK) levels of Target cNF Tumors
    NCT02390752 4, 2015 1/2 USA 81 Neurofibroma, Plexiform PLX3397 Toxicity
    Objective response rate
    NCT03688568 9, 2018 2 USA 20 Neurofibroma, Plexiform Imatinib Mesylate Quantitative Functional Airway Response
    NCT03433183 10, 2019 2 USA 21 Malignant Peripheral Nerve Sheath Tumors
    Selumetinib Sirolimus Clinical benefit rate of selumetinib in combination with sirolimus
    NCT04085159 9, 2019 1/2 China 100 Neurofibromatosis Schwannomatosis Antigen-specific T cells CART/CTL and DCvac Percentage of adverse effects
    NF1, neurofibromatosis type 1.

    2.5. Animal Models

    The development of genetically engineered mouse models of NF1-related tumors has promoted preclinical trials of targeted agents [34][35][36][37]. Initial studies focused on a targeted mutation of the Nf1 gene. Traditional NF1+/- mice were generated in which one allele of the murine Nf1 gene is inactivated by the insertion of a neomycin cassette [38][39][40]. Second-generation models included NF1-/- chimeric mice and NF1 exon-specific knockout mice [41]. Tissue-specific NF1 inactivation can be accomplished by the Cre/LoxP technology, in which LoxP recombinatorial sequences are inserted into noncoding regions of the NF1 gene [40], thus providing important insights into the function of neurofibromin in specific cell types.
    Several NF1-associated high-grade glioma models have been established by coupling complete NF1 gene inactivation with loss of other tumor suppressor genes (p53, PTEN), for example, strategies targeting genes such as CRISPR/Cas9 [42], standard and conditional knockout mice [43][44], and viral gene silencing [45]. NF1 optic glioma requires a combination of a germline inactivating NF1 gene mutation and somatic NF1 loss in neuroglial progenitor cells [46].
    Recently, NF1 porcine models have been established [47][48]. The anatomical, biochemical, and cellular components of porcine nerves are comparable to humans. This innovative model may recapitulate the wide spectrum of the phenotypic and pathological changes associated with NF1, accelerating NF1 research and therapies.

    3. Neurofibromatosis Type 2

    3.1. Clinical Characteristics

    NF2 is an autosomal dominantly inherited syndrome that predisposes individuals to multiple nervous tumors. A de novo mutation may take place after fertilization, resulting in a mosaic expression [49][50]. Diagnosis is based on clinical and neuroimaging studies (Table 3). Two large population-based studies reported that this condition occurs in 1 in 25,000 people [51]. The actuarial survival after diagnosis is 15 years, with an average age at death of 36 years [52] and a 10-year survival rate of 67% [53]. NF2 patients uniformly develop schwannomas on the bilateral vestibular portion of the eighth cranial nerve and on other cranial nerves, spinal roots, or peripheral nerves [54]. In addition, NF2 patients often develop multiple meningiomas and ependymomas at an early age.
    Table 3. Diagnostic criteria of neurofibromatosis type 2.
    Bilateral vestibular schwannomas or
    First-degree relative with neurofibromatosis type 2 plus
    1. Unilateral vestibular schwannomas or
    2. Any two of the following: Meningioma, glioma, schwannoma, or juvenile PLO
    PLO, posterior lenticular opacities.
    NF2 patients often experience hearing loss, balance problems, flesh colored skin flaps, and muscle wasting. Some develop mononeuropathy, often involving the facial nerve. Severe polyneuropathy is noted in 3–5% of adult NF2 patients [55]. Visual impairment is likely due to cataracts, optic nerve meningiomas, and retinal hamartomas [56][57][58]. Approximately 70% of NF2 patients have cutaneous manifestations: only 10% have more than 10 skin tumors [54]. Plaque-like lesions may be more pigmented than the surrounding skin with increased hair. Subcutaneous nodules are identified along the peripheral nerve. Intracutaneous schwannomas, which are similar to those observed in NF1 patients, are occasionally seen.

    3.2. Genetic and Molecular Characteristics

    NF2 is caused by a defect in the gene that normally produces merlin, located at 22q12.2 of chromosome 22, which regulates multiple proliferative signaling pathways. At the membrane, merlin blocks signaling caused by integrins and tyrosine receptor kinases. Merlin can also inhibit downstream signalings, including the p21-activated kinase signaling, Ras/Raf/MEK/ERK, FAK/Src, PI3K/AKT, Rac/PAK/JNK, mTORC1, and Wnt/β-catenin pathways (Figure 2). In the nucleus, merlin suppresses the E3 ubiquitin ligase CRL4DCAF1, which also regulates the expression of integrins and tyrosine receptor kinases. The Hippo signaling pathway regulates tissue homeostasis. Merlin is implicated as one of the upstream regulators of the Hippo signaling pathway [49][50][59][60][61].
    Figure 2. Molecular pathogenesis of NF2. NF2 gene encodes merlin. Merlin regulates multiple proliferative signaling pathways. At the membrane, merlin blocks signaling caused by integrins and tyrosine receptor kinases. Merlin can also inhibit downstream signalings, including the p21-activated kinase signaling, Ras/Raf/MEK/ERK, FAK/Src, PI3K/AKT, Rac/PAK/JNK, mTORC1, and Wnt/β-catenin pathways. Downstream signaling of NF2 includes VEGF-A.
    A de novo mutation results in a mosaic expression. Somatic mosaicism may prevent the molecular diagnosis unless tumor tissue is analyzed [62]. The growth of schwannomas requires inactivation of both NF2 alleles. The “second hit” occurs through loss of the entire NF2 gene and most of chromosome 22. Various types of mutations, such as protein-truncating alterations (frameshift deletions/insertions and nonsense mutations), splice-site mutations, missense mutations, are identified. Truncating mutations (nonsense and frameshifts) are the most frequent germline event and cause the most severe disease. The presence of a truncated protein is associated with younger age at diagnosis and a higher prevalence of meningiomas, spinal tumors, and cranial nerve tumors other than VIII [62]. Deletions in the NH2-terminal domain of merlin proteins are associated with early tumor onset and disease progression [62]. The most common alterations are splice-site mutations or nonsense mutations in exons 1–8. Missense or in-frame deletions have been associated with milder clinical courses. A positional effect with mutations in the latter parts of the gene (exons 14 and 15) is associated with milder disease and fewer meningiomas [63]. Alterations in the conserved N-terminal FERM domain and truncating mutations are typically associated with the Wishart phenotype including younger age at diagnosis, a higher incidence of meningiomas, ophthalmologic and cutaneous lesions, and poor outcomes. Missense and splice-site mutations, particularly in the 3′ end of the gene, are associated with the Gardner phenotype and a better prognosis with fewer meningiomas [64].
    The role of mutation screening for NF2 in all patients with a unilateral vestibular schwannoma is less certain. Although these patients show an increased risk for the development of NF2, routine screening for germline mutations is not recommended except in patients younger than 30 years [65][66], but can undergo prenatal diagnosis and pre-implantation genetic diagnosis.

    3.3. Therapeutic Strategies

    To date, there is no established effective treatment for NF2 patients because tumors are highly likely to regrow after surgical resection [67]. Treatment is generally indicated when the patient has risk of brainstem compression, deterioration of hearing, and/or facial nerve dysfunction. Vestibular schwannomas may involve facial nerve fibers, possibly posing a significant risk of damage to the facial nerve during surgery [68]. While the use of stereotactic radiosurgery has recently become an effective management modality for NF2 schwannomas, it is not advocated for multiple or large tumors [69]. Vestibular dysfunction and trigeminal neuropathy have been reported after radiosurgery [70]. Malignant transformation associated with radiosurgery is evidently uncommon [71][72]. Furthermore, surgical resection may be more difficult following stereotactic radiosurgery [69][73].
    Vascular endothelial growth factor (VEGF)-A is an important factor for the growth of schwannoma that mainly depends on VEGF-A/VEGF receptor (VEGFR) pathway (not other factors such as estrogen and progesterone) [74][75]. Tumor shrinkage and hearing improvement are identified after administration of bevacizumab (a monoclonal antibody against VEGF-A) in >50% of progressive vestibular schwannomas in NF2 patients. Stable hearing is retained in the majority of the patients [76][77][78][79][80][81][82][83][84]. Bevacizumab is recently considered as a first-line medical therapy for rapidly growing vestibular schwannomas [85]. In a recent meta-analysis of eight observational studies involving 161 patients with NF2-associated vestibular schwannomas, the best response to bevacizumab was partial regression in 41%, no change in 47%, and progression in 7% of the patients [86]. The median treatment duration was 16 months. Hearing improved in 20%, remained stable in 69%, and worsened in 6 percent. The incidence of serious toxicity was 17%, with amenorrhea (70%), proteinuria (43%), and hypertension (33%). Although the dose and schedule of bevacizumab has not been standardized, a proposed regimen is 5–7.5 mg/kg every 2–3 weeks for at least 6 months, followed by maintenance therapy at 2.5–5 mg/kg every 4 weeks [85]. A higher dose regimen (10 mg/kg every 2 weeks for 6 months) offered no clear advantage compared with lower-dose regimens [87], and furthermore higher dose may increase the risk of renal impairment.
    Contrarily, problems arise after administration of bevacizumab such as the need for frequent administration, occurrence of hypertension and thrombosis, and apparent drug resistance. Tumor growth rebound following bevacizumab treatment discontinuation was pointed out [88]. Recently, a clinical trial using VEGFR1/2 peptide vaccine was also conducted in patients with progressive NF2-derived schwannomas, showing hearing improvement and tumor volume reduction [89].
    The NF2 gene product involves multiple molecular pathways in cell growth. Some studies reported on the efficacy of everolimus, an oral inhibitor of the mTORC1, for progressive vestibular schwannomas in NF2 patients [90][91]. Lapatinib showed objective activity in 4 out of 17 patients with NF2-related progressive vestibular schwannoma in a phase II trial [92], whereas erlotinib was not effective in a retrospective series with 11 NF2 patients [93]. Mirdametinib (PD-0325901) is an orally delivered inhibitor of the dual specificity kinases (MEK1 and MEK2), which demonstrated tumor shrinkage and sustained inhibition of pERK. Furthermore, the recent study demonstrated that hypoxia was significantly associated with shorter progression-free survival in NF2 schwannomas [94]. HIF-1-targeted therapy might be considered for some NF2 schwannomas that are difficult to treat by surgical resection and stereotactic radiosurgery [94].
    For patients with severe hearing impairment, strategies using cochlear or brainstem implants may offer some benefit [95][96]. Pilot studies have demonstrated the feasibility of novel psychosocial interventions delivered to deaf individuals with NF2 via teleconferencing with captioning technology [97].
    NF2 patients tend to develop meningiomas at an earlier age than those with sporadic meningiomas [98]. The meningiomas seen in NF2 patients are more frequently atypical or anaplastic compared with sporadic tumors [99]. Although radiation therapy has been performed in those patients, long-term follow-up is lacking. Targeted therapies are under investigation [100]. Lapatinib showed some activity in a small number of patients with NF2-associated progressive meningiomas [101]. There is little evidence that bevacizumab has activity in NF2-related meningiomas [102]. These molecular studies led to clinical trials using mTORC1 inhibitor everolimus, a rapamycin analog, for NF2 and sporadic meningiomas. Alternate treatment options for NF2 tumors include inhibitors of the epidermal growth factor receptor, an inhibitor of platelet-derived growth factor, and an inhibitor of histone deacetylase [100].

    3.4. Ongoing Clinical Trials

    Table 4 shows ongoing phase II clinical trials of NF2 patients using various molecular targeted agents besides bevacizumab: icotinib (EGFR inhibitor), axitinib (VEGFR inhibitor), everolimus (mTOR1 inhibitor), crizotinib (c-Met and ALK inhibitor), vistusertib (dual mTORC1/2 inhibitor), brigatinib (ALK and EGFR inhibitor), or selumetinib (MEK inhibitor). Primary outcome is volumetric response or hearing response. Recently, the first phase III randomized clinical trial using bevacizumab was conducted in Japan [103].
    Table 4. Ongoing clinical trials for the patients with NF2.
    ID Initiation Date Phase Nation N Disease Treatment Primary Outcome
    NCT02934256 7, 2016 2 China 20 NF2 Icotinib Change from Baseline in volume of tumor
    NCT02129647 4, 2014 2 USA 12 NF2
    Progressive VS
    Axitinib volumetric response rates
    NCT01345136 7, 2015 2 USA 4 NF2 RAD001, everolimus Vestibular schwannoma volume
    NCT01767792 5, 2013 2 USA 22 NF2
    Progressive VS
    Bevacizumab Hearing
    NCT04283669 2, 2020 2 USA 19 NF2
    Progressive VS
    Crizotinib Volumetric response rate
    NCT02831257 8, 2016 2 USA 18 NF2
    AZD2014 Volumetric response rate
    NCT04374305 6, 2020 2 USA 80 NF2
    Vestibular Schwannoma
    Non-vestibular Schwannoma
    Brigatinib Volumetric response rate
    NCT03095248 5, 2017 2 USA 34 NF2
    Vestibular Schwannoma
    Selumetinib Hearing response
    Volumetric response rate
    NCT03079999 6, 2018 2 USA 300 NF2
    Vestibular schwannoma
    Aspirin Progression-free survival
    NF2, neurofibromatosis type 2.

    3.5. Animal Models

    In the Nf2 mutant allele, the 3′ part of exon 2 up to the 5′ part of intron 3 has been replaced by a selection marker [104]. Two different mutant Nf2 alleles were generated [105]. One mutant allele, Nf2KO3, was generated by an insertional mutation in exon 3. The other mutant allele, Nf2Δ2, carried an in-frame deletion of exon 2. Tissue-specific Nf2 inactivation can be accomplished by the Cre/LoxP technology. LoxP recombinatorial sequences are inserted into noncoding regions flanking exon 2 of the Nf2 gene to produce phenotypically normal Nf2 flox mice. Tissue-specific inactivation is mediated by the expression of the bacteriophage Cre recombinase from a tissue-specific promoter or by direct injection of adenoviral Cre [106].
    Recently, Chen et al. described the injection of schwannoma cells into the mouse brain cerebellopontine angle region and the application intravital imaging and hearing assessment techniques to study tumor growth and hearing loss. In addition, ataxia, angiogenesis, and tumor–stroma interaction assays could be shown [107].
    To date, models of NF2-associated ependymoma remain yet to be generated whereas genetically engineered mouse strain of meningiomas have been. Somatic Nf2 loss after subarachnoid or subdural viral injection of Cre recombinase into newborn Nf2 flox/flox conditional knockout mice result in meningioma [106]. More aggressive tumors develop when NF2 loss is coupled with the loss of other tumor suppressor genes (Ink4a) [108]. It has been challenging to maintain NF-associated tumors as patient-derived xenografts. To date, successful orthotopic transplant models have only been developed for NF2-associated meningioma [109].

    4. Schwannomatosis

    4.1. Clinical Characteristics

    SWN is the rarest form of NF characterized by multiple schwannomas in the absence of bilateral vestibular schwannomas inherited via autosomal dominance in 15–20% [2][110] (Table 5). Patients with SWN have a median tumor count of 4, and a median whole body tumor volume of 39 mL [111]. The incidence of SWN have ranged from 1 in 40,000 to 1 in 1.7 million people. The median age at diagnosis is approximately 40 years [112], with chronic pain, numbness, tingling, and weakness appearing in early adulthood. Chronic pain is localized or diffused and often does not correlate with the location of schwannomas. Total tumor burden, size, and location do not correlate with pain-related morbidity [113]. While life expectancy of patients with SWN is normal [110][112][113], more data are needed to understand the risk of MPNST and other malignancies in patients with SWN [112][114]. Patients with SWN do not have learning disabilities [113][115]. The incidence of meningioma is 5% [116].
    Table 5. Diagnostic criteria of schwannomatosis.
    Definite Schwannomatosis
    A. Age >30 years and two or more schwannomas (not intradermal), at least one with histologic confirmation with no evidence of vestibular tumor on brain MRI scan and no known NF mutation
    B. Vestibular schwannoma (pathologically confirmed) plus first-degree relative who meets the criteria of schwannomatosis
    Possible schwannomatosis
    A. Age <30 years plus two or more schwannomas (not intradermal), at least one with histologic confirmation with no evidence of vestibular tumor on brain MRI scan and no known NF mutation
    B. Age >45 years plus two or more schwannomas (not dermal), at least one with histologic confirmation and no symptoms of 8th nerve dysfunction and NF type 2
    C. Evidence of a non-vestibular schwannoma and first-degree relative meeting criteria for definite schwannomatosis
    MRI, magnetic resonance imaging; NF, neurofibromatosis.

    4.2. Genetic and Molecular Characteristics

    Mutations in SMARCB1 and LZTR1 genes cause SWN. Causative inactivating germline mutations in the tumor suppressor genes SMARCB1 and LZTR1 are present in approximately 85% of families with SWN and up to 40% of sporadic cases. Mutations in the SMARCB1 or LZTR1 gene alone are not sufficient to trigger SWN and require additional somatic mutations. Genetic testing is available for both SMARCB1 and LZTR1 [117].
    In SMARCB1 (INI1) mutation-positive schwannomas, there can be additional genetic alterations, including loss of one copy of chromosome 22 and inactivating mutations in the NF2 gene [118][119][120][121][122][123]. This suggests a four-hit, three-step model of tumorigenesis in considerable SMARCB1-associated SWN patients. The mutated germline SMARCB1 gene copy is retained in the tumor (hit 1), whereas chromosome 22, or at least a segment of chromosome 22 containing the wildtype SMARCB1 gene copy and a wildtype copy of the NF2 gene is lost (hits 2 and 3), followed by a mutation in the remaining wildtype NF2 gene copy (hit 4) [119][121][124] (Figure 3). Germline mutations in SMARCB1 can cause an inherited predisposition to atypical teratoid/rhabdoid tumors (ATRT) of the central nervous system [125]. Reports of families with both SWN and rhabdoid tumor phenotypes are rare [114][126][127]. Mosaic loss of immunohistochemical expression of SMARCB1/INI1 is a reliable marker of SWN and can assist in distinguishing SWN from an isolated schwannoma [128]. The presence of abundant myxoid stroma or a hybrid tumor may be also associated with an underlying syndromic diagnosis [124].
    Figure 3. Molecular pathogenesis of SWN.
    Mutations in another tumor suppressor gene, LZTR1, may explain the predisposition to SWN in patients without mutations in SMARCB1 [129][130]. LZTR1 is located on chromosome 22q11.21, centromeric to both SMARCB1 (22q11.23) and NF2 (22q12.2) [129][131]. A larger study on SMARCB1 and NF2 mutation-negative patients revealed LZTR1 mutations in 6 of 16 patients with familial SWN (38%), 11 of 49 sporadic patients (22%), and 2 of 39 patients with unilateral vestibular schwannoma. Somatic LZTR1 mutations have also been found in several other cancers [129][132].
    Although biallelic mutations of SMARCB1 or LZTR1 have been detected in the patients with SWN, the classical two-hit model of tumorigenesis is insufficient to account for schwannoma growth, since NF2 is frequently inactivated in these tumors. Tumorigenesis in SWN involves the mutation of at least two different tumor suppressor genes, an occurrence frequently mediated by loss of heterozygosity of large parts of chromosome 22q harboring not only SMARCB1 and LZTR1 but also NF2.

    4.3. Therapeutic Strategies

    To date, there are no established medical therapies that target the SWN, and the use of gabapentin or pregabalin and short-acting opioids and/or nonsteroidal anti-inflammatories has been reportedly successful in reducing pain in patients with SWN. Additional agents, including tricyclic antidepressants such as amitriptyline, serotonin-norepinephrine reuptake inhibitors such as duloxetine, or antiepileptics such as topiramate or carbamazepine may be used as adjuncts or independently. Surgical resection of painful schwannomas may be considered if pain is not successfully controlled. Other surgical indications include spinal cord compression or impingement of other organs.
    Both SMARCB1 and LZTR1 interact with histone deacetylase 4 [133][134] which indicates therapeutic implications in SWN. Histone deacetylase inhibitors are currently under development as antitumor drugs [133][134].

    4.4. Ongoing Clinical Trials

    Table 6 shows ongoing clinical trials for SWN patients. Tanezumab is a monoclonal antibody against nerve growth factor as a treatment for pain, which is undergoing Phase II clinical trials for the treatment of various pain entities in SWN patients. Primary outcome is pain relief.
    Table 6. Ongoing clinical trials for the patients with schwannomatosis.
    ID Initiation Date Phase Nation N Disease Treatment Primary Outcome
    NCT04163419 4, 2020 2 USA 46 Schwannomatosis Tanezumab Change in pain level
    NCT04085159 9, 2019 1/2 China 100 Neurofibromatosis Schwannomatosis Antigen-specific T cells CART/CTL and DCvac Percentage of adverse effects

    The entry is from 10.3390/ijms22115850


    1. Coy, S.; Rashid, R.; Stemmer-Rachamimov, A.; Santagata, S. An update on the CNS manifestations of neurofibromatosis type 2. Acta Neuropathol. 2020, 139, 643–665.
    2. Evans, D.G.; Bowers, N.L.; Tobi, S.; Hartley, C.; Wallace, A.J.; King, A.T.; Lloyd, S.K.W.; Rutherford, S.A.; Hammerbeck-Ward, C.; Pathmanaban, O.N.; et al. Schwannomatosis: A genetic and epidemiological study. J. Neurol. Neurosurg. Psychiatry 2018, 89, 1215.
    3. Nix, J.S.; Blakeley, J.; Rodriguez, F.J. An update on the central nervous system manifestations of neurofibromatosis type 1. Acta Neuropathol. 2020, 139, 625–641.
    4. Kresak, J.L. Neurofibromatosis: A Review of NF1, NF2, and Schwannomatosis. J. Pediatr. Genet. 2016, 5, 98–104.
    5. Blakeley, J.O.; Plotkin, S.R. Therapeutic advances for the tumors associated with neurofibromatosis type 1, type 2, and schwannomatosis. Neuro. Oncol. 2016, 18, 624–638.
    6. Campian, J.; Gutmann, D.H. CNS Tumors in Neurofibromatosis. J. Clin. Oncol. 2017, 35, 2378–2385.
    7. Evans, D.G.; Howard, E.; Giblin, C.; Clancy, T.; Spencer, H.; Huson, S.M.; Lallo, F. Birth incidence and prevalence of tumor-prone syndromes: Estimates from a UK family genetic register service. Am. J. Med. Genet. A 2010, 152A, 327.
    8. Kallionpää, R.A.; Uusitalo, E.; Leppävirta, J.; Pöyhönen, M.; Peltonen, S.; Peltonen, J. Prevalence of neurofibromatosis type 1 in the Finnish population. Genet. Med. 2018, 20, 1082.
    9. Ruggieri, M.; Huson, S.M. The clinical and diagnostic implications of mosaicism in the neurofibromatoses. Neurology 2001, 56, 1433.
    10. Legius, E.; Messiaen, L.; Wolkenstein, P.; Pancza, P.; Avery, R.A.; Berman, Y.; Blakeley, J.; Babovic-Vuksanovic, D.; Cunha, K.S.; Ferner, R.; et al. Revised diagnostic criteria for neurofibromatosis type 1 and Legius syndrome: An international consensus recommendation. Genet. Med. 2021, 19.
    11. Stephens, K.; Kayes, L.; Riccardi, V.M.; Rising, M.; Sybert, V.P.; Pagon, R.A. Preferential mutation of the neurofibromatosis type 1 gene in paternally derived chromosomes. Hum. Genet. 1992, 88, 279.
    12. Rasmussen, S.A.; Yang, Q.; Friedman, J.M. Mortality in neurofibromatosis 1: An analysis using U.S. death certificates. Am. J. Hum. Genet. 2001, 68, 1110–1118.
    13. Landry, J.P.; Schertz, K.L.; Chiang, Y.J.; Bhalla, A.D.; Yi, M.; Keung, E.Z.; Scally, C.P.; Feig, B.W.; Hunt, K.K.; Roland, C.L.; et al. Comparison of Cancer Prevalence in Patients With Neurofibromatosis Type 1 at an Academic Cancer Center vs in the General Population From 1985 to 2020. JAMA Netw. Open. 2021, 4, e210945.
    14. Pasmant, E.; Sabbagh, A.; Hanna, N.; Masliah-Planchon, J.; Jolly, E.; Goussard, P.; Ballerini, P.; Cartault, F.; Barbarot, S.; Landman-Parker, J.; et al. SPRED1 germline mutations caused a neurofibromatosis type 1 overlapping phenotype. J. Med. Genet. 2009, 46, 425.
    15. Scheffzek, K.; Ahmadian, M.R.; Wiesmüller, L.; Kabsch, W.; Stege, P.; Schmitz, F.; Wittinghofer, A. Structural analysis of the GAP-related domain from neurofibromin and its implications. EMBO J. 1998, 17, 4313–4327.
    16. Peltonen, S.; Kallionpää, R.A.; Peltonen, J. Neurofibromatosis type 1 (NF1) gene: Beyond café au lait spots and dermal neurofibromas. Exp. Dermatol. 2017, 26, 645–648.
    17. Trovó-Marqui, A.B.; Tajara, E.H. Neurofibromin: A general outlook. Clin. Genet. 2006, 70, 1–13.
    18. Messiaen, L.; Wimmer, K. NF1 mutational spectrum. Monogr. Hum. Genet. 2008, 16, 63.
    19. Kluwe, L.; Friedrich, R.E.; Peiper, M.; Friedman, J.; Mautner, V.F. Constitutional NF1 mutations in neurofibromatosis 1 patients with malignant peripheral nerve sheath tumors. Hum. Mutat. 2003, 22, 420.
    20. Farid, M.; Demicco, E.G.; Garcia, R.; Ahn, L.; Merola, P.R.; Cioffi, A.; Maki, R.G. Malignant peripheral nerve sheath tumors. Oncologist 2014, 19, 193–201.
    21. Upadhyaya, M.; Spurlock, G.; Majounie, E.; Griffiths, S.; Forrester, N.; Baser, M.; Huson, S.M.; Gareth, E.D.; Ferner, R. The heterogeneous nature of germline mutations in NF1 patients with malignant peripheral serve sheath tumours (MPNSTs). Hum. Mutat. 2006, 27, 716.
    22. Koczkowska, M.; Callens, T.; Gomes, A.; Sharp, A.; Chen, Y.; Hicks, A.D.; Aylsworth, A.S.; Azizi, A.A.; Basel, D.G.; Bellus, G.; et al. Expanding the clinical phenotype of individuals with a 3-bp in-frame deletion of the NF1 gene (c.2970_2972del): An update of genotype-phenotype correlation. Genet. Med. 2019, 21, 867.
    23. Koczkowska, M.; Chen, Y.; Callens, T.; Gomes, A.; Sharp, A.; Johnson, S.; Hsiao, M.C.; Chen, Z.; Balasubramanian, M.; Barnett, C.P.; et al. Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844-848. Am. J. Hum. Genet. 2018, 102, 69.
    24. Koczkowska, M.; Callens, T.; Chen, Y.; Gomes, A.; Hicks, A.D.; Sharp, A.; Johns, E.; Uhas, K.A.; Armstrong, L.; Bosanko, K.A.; et al. Clinical spectrum of individuals with pathogenic NF1 missense variants affecting p.Met1149, p.Arg1276, and p.Lys1423: Genotype-phenotype study in neurofibromatosis type 1. Hum. Mutat. 2020, 41, 299.
    25. Quesnel, B.; Preudhomme, C.; Vanrumbeke, M.; Vachee, A.; Lai, J.L.; Fenaux, P. Absence of rearrangement of the neurofibromatosis 1 (NF1) gene in myelodysplastic syndromes and acute myeloid leukemia. Leukemia 1994, 8, 878–880.
    26. Messiaen, L.M.; Callens, T.; Mortier, G.; Beysen, D.; Vandenbroucke, I.; Roy, N.V.; Paepe, A.D. Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum. Mutat. 2000, 15, 541.
    27. Castle, B.; Baser, M.E.; Huson, S.M.; Cooper, D.N.; Upadhyaya, M. Evaluation of genotype-phenotype correlations in neurofibromatosis type 1. J. Med. Genet. 2003, 40, e109.
    28. Nardecchia, E.; Perfetti, L.; Castiglioni, M.; Di Natale, D.; Imperatori, A.; Rotolo, N. Bullous lung disease and neurofibromatosis type-1. Monaldi Arch. Chest Dis. 2012, 77, 105.
    29. Katz, D.; Lazar, A.; Lev, D. Malignant peripheral nerve sheath tumour (MPNST): The clinical implications of cellular signalling pathways. Expert Rev. Mol. Med. 2009, 11, e30.
    30. Gross, A.M.; Wolters, P.L.; Dombi, E.; Baldwin, A.; Whitcomb, P.; Fisher, M.J.; Weiss, B.; Kim, A.; Bornhorst, M.; Shah, A.C.; et al. Selumetinib in Children with Inoperable Plexiform Neurofibromas. N. Engl. J. Med. 2020, 382, 1430–1442.
    31. Mukhopadhyay, S.; Maitra, A.; Choudhury, S. Selumetinib: The first ever approved drug for neurofibromatosis-1 related inoperable plexiform neurofibroma. Curr. Med. Res. Opin. 2021, 8, 1.
    32. DeBella, K.; Szudek, J.; Friedman, J.M. Use of the national institutes of health criteria for diagnosis of neurofibromatosis 1 in children. Pediatrics 2000, 105, 608–614.
    33. Kaufman, L.M.; Doroftei, O. Optic glioma warranting treatment in children. Eye 2006, 20, 1149–1164.
    34. Brannan, C.I.; Perkins, A.S.; Vogel, K.S.; Ratner, N.; Nordlund, M.L.; Reid, S.W.; Buchberg, A.M.; Jenkins, N.A.; Parada, L.F.; Copeland, N.G. Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. Genes Dev. 1994, 8, 1019–1029.
    35. Jacks, T.; Shih, T.S.; Schmitt, E.M.; Bronson, R.T.; Bernards, A.; Weinberg, R.A. Tumor predisposition in mice heterozygous for a targeted mutation in NF1. Nat. Genet. 1994, 7, 353–361.
    36. Lakkis, M.M.; Epstein, J.A. Neurofibromin modulation of ras activity is required for normal endocardial-mesenchymal transformation in the developing heart. Development 1998, 125, 4359–4367.
    37. Lakkis, M.M.; Golden, J.A.; O’Shea, S.; Epstein, J.A. Neurofibromin deficiency in mice causes exencephaly and is a modifier for Splotch neural tube defects. Dev. Biol. 1999, 212, 80–92.
    38. Bajenaru, M.L.; Donahoe, J.; Corral, T.; Keilly, K.M.; Brophy, S.; Pellicer, A.; Gutmann, D.H. Neurofibromatosis 1 (NF1) heterozygosity results in a cell—Autonomous growth advantage for astrocytes. Glia 2001, 33, 314–323.
    39. Gutmann, D.H.; Loehr, A.; Zhang, Y.; Kim, J.; Henkemeyer, M.; Cashen, A. Haploinsufficiency for the neurofibromatosis 1 (NF1) tumor suppressor results in increased astrocyte proliferation. Oncogene 1999, 18, 4450–4459.
    40. Zhu, Y.; Romero, M.I.; Ghosh, P.; Ye, Z.; Charnay, P.; Rushing, E.J.; Marth, J.D.; Parada, L.F. Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Genes Dev. 2001, 15, 859–876.
    41. Costa, R.M.; Yang, T.; Huynh, D.P.; Pulst, S.M.; Viskochil, D.H.; Silva, A.J.; Brannan, C.I. Learning deficits, but normal development and tumor predisposition, in mice lacking exon 23a of Nf1. Nat. Genet. 2001, 27, 399–405.
    42. Zuckermann, M.; Hovestadt, V.; Knobbe-Thomsen, C.B.; Zapatka, M.; Northcott, P.A.; Schramm, K.; Belic, J.; Jones, D.T.; Tschida, B.; Moriarity, B.; et al. Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling. Nat. Commun. 2015, 6, 7391.
    43. Kwon, C.H.; Zhao, D.; Chen, J.; Alcantara, S.; Li, Y.; Burns, D.K.; Mason, R.P.; Lee, E.Y.; Wu, H.; Parada, L.F. Pten haploinsufficiency accelerates formation of high-grade astrocytomas. Cancer Res. 2008, 68, 3286–3294.
    44. Reilly, K.M.; Loisel, D.A.; Bronson, R.T.; McLaughlin, M.E.; Jacks, T. Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects. Nat. Genet. 2000, 26, 109–113.
    45. Marumoto, T.; Tashiro, A.; Friedmann-Morvinski, D.; Scadeng, M.; Soda, Y.; Gage, F.H.; Verma, I.M. Development of a novel mouse glioma model using lentiviral vectors. Nat. Med. 2009, 15, 110–116.
    46. Bajenaru, M.L.; Hernandez, M.R.; Perry, A.; Zhu, Y.; Parada, L.F.; Garbow, J.R.; Gutmann, D.H. Optic nerve glioma in mice requires astrocyte Nf1 gene inactivation and Nf1 brain heterozygosity. Cancer Res. 2003, 63, 8573–8577.
    47. Isakson, S.H.; Rizzardi, A.E.; Coutts, A.W.; Carlson, D.F.; Kirstein, M.N.; Fisher, J.; Vitte, J.; Williams, K.B.; Pluhar, G.E.; Dahiya, S.; et al. Genetically engineered minipigs model the major clinical features of human neurofibromatosis type 1. Commun Biol. 2018, 1, 158.
    48. White, K.A.; Swier, V.J.; Cain, J.T.; Kohlmeyer, J.L.; Meyerholz, D.K.; Tanas, M.R.; Uthoff, J.; Hammond, E.; Li, H.; Rohret, F.A.; et al. A porcine model of neurofibromatosis type 1 that mimics the human disease. JCI Insight. 2018, 3, e120402.
    49. Evans, D.G.; Hartley, C.L.; Smith, P.T.; King, A.T.; Bowers, N.L.; Tobi, S.; Wallace, A.J.; Perry, M.; Anup, R.; Lloyd, S.K.W.; et al. Incidence of mosaicism in 1055 de novo NF2 cases: Much higher than previous estimates with high utility of next-generation sequencing. Genet. Med. 2020, 22, 53.
    50. Evans, D.G.; Ramsden, R.T.; Shenton, A.; Gokhale, C.; Bowers, N.L.; Huson, S.M.; Pichert, G.; Wallace, A. Mosaicism in neurofibromatosis type 2: An update of risk based on uni/bilaterality of vestibular schwannoma at presentation and sensitive mutation analysis including multiple ligation-dependent probe amplification. J. Med. Genet. 2007, 44, 424.
    51. Evans, D.G.; Moran, A.; King, A.; Saeed, S.; Gurusinghe, N.; Ramsden, R. Incidence of vestibular schwannoma and neurofibromatosis 2 in the North West of England over a 10-year period: Higher incidence than previously thought. Otol. Neurotol. 2005, 26, 93.
    52. Godel, T.; Bäumer, P.; Farschtschi, S.; Gugel, I.; Kronlage, M.; Hofstadler, B.; Heiland, S.; Gelderblom, M.; Bendszus, M.; Mautner, V.F. Peripheral nervous system alterations in infant and adult neurofibromatosis type 2. Neurology 2019, 93, e590.
    53. Picry, A.; Bonne, N.X.; Ding, J.; Aboukais, R.; Lejeune, J.P.; Baroncini, M.; Dubrulle, F.; Vincent, C. Long-term growth rate of vestibular schwannoma in neurofibromatosis 2: A volumetric consideration. Laryngoscope 2016, 126, 2358–2362.
    54. Evans, D.G. Neurofibromatosis type 2 (NF2): A clinical and molecular review. Orphanet J. Rare Dis. 2009, 4, 16.
    55. Sperfeld, A.D.; Hein, C.; Schröder, J.M.; Ludolph, A.C.; Hanemann, C.O. Occurrence and characterization of peripheral nerve involvement in neurofibromatosis type 2. Brain 2002, 125, 996.
    56. Gaudioso, C.; Listernick, R.; Fisher, M.J.; Campen, C.J.; Paz, A.; Gutmann, D.H. Neurofibromatosis 2 in children presenting during the first decade of life. Neurology 2019, 93, e964.
    57. McLaughlin, M.E.; Pepin, S.M.; Maccollin, M.; Choopong, P.; Lessell, S. Ocular pathologic findings of neurofibromatosis type 2. Arch. Ophthalmol. 2007, 125, 389.
    58. Painter, S.L.; Sipkova, Z.; Emmanouil, B.; Halliday, D.; Parry, A.; Elston, J.S. Neurofibromatosis Type 2-Related Eye Disease Correlated with Genetic Severity Type. J. Neuroophthalmol. 2019, 39, 44.
    59. Castellanos, E.; Plana, A.; Carrato, C.; Carrió, M.; Rosas, I.; Amilibia, E.; Roca-Ribas, F.; Hostalot, C.; Castillo, A.; Ros, A.; et al. Early Genetic Diagnosis of Neurofibromatosis Type 2 from Skin Plaque Plexiform Schwannomas in Childhood. JAMA Dermatol. 2018, 154, 341.
    60. Kluwe, L.; Mautner, V.; Heinrich, B.; Dezube, R.; Jacoby, L.B.; Friedrich, R.E.; MacCollin, M. Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas. J. Med. Genet. 2003, 40, 109.
    61. Moyhuddin, A.; Baser, M.E.; Watson, C.; Purcell, S.; Ramsden, R.T.; Heiberg, A.; Wallace, A.J.; Evans, D.G. Somatic mosaicism in neurofibromatosis 2: Prevalence and risk of disease transmission to offspring. J. Med. Genet. 2003, 40, 459.
    62. Selvanathan, S.K.; Shenton, A.; Ferner, R.; Wallace, A.J.; Huson, S.M.; Ramsden, R.T.; Evans, D.G. Further genotype—Phenotype correlations in neurofibromatosis 2. Clin. Genet. 2010, 77, 163.
    63. Smith, M.J.; Higgs, J.E.; Bowers, N.L.; Halliday, D.; Paterson, J.; Gillespie, J.; Huson, S.M.; Freeman, S.R.; Lloyd, S.; Rutherford, S.A.; et al. Cranial meningiomas in 411 neurofibromatosis type 2 (NF2) patients with proven gene mutations: Clear positional effect of mutations, but absence of female severity effect on age at onset. J. Med. Genet. 2011, 48, 261.
    64. Hexter, A.; Jones, A.; Joe, H.; Heap, L.; Smith, M.J.; Wallace, A.J.; Halliday, D.; Parry, A.; Taylor, A.; Raymond, L.; et al. Clinical and molecular predictors of mortality in neurofibromatosis 2: A UK national analysis of 1192 patients. J. Med. Genet. 2015, 52, 699.
    65. Evans, D.G.; Ramsden, R.T.; Gokhale, C.; Bowers, N.; Huson, S.M.; Wallace, A. Should NF2 mutation screening be undertaken in patients with an apparently isolated vestibular schwannoma? Clin. Genet. 2007, 71, 354.
    66. Evans, D.G.; Wallace, A.J.; Hartley, C.; Freeman, S.R.; Lloyd, S.K.; Thomas, O.; Axon, P.; Hammerbeck-Ward, C.L.; Pathmanaban, O.; Rutherford, S.A.; et al. Familial unilateral vestibular schwannoma is rarely caused by inherited variants in the NF2 gene. Laryngoscope 2019, 129, 967.
    67. Evans, D.G.; Baser, M.E.; O’Reilly, B.; Rowe, J.; Gleeson, M.; Saeed, S.; King, A.; Huson, S.M.; Kerr, R.; Thomas, N.; et al. Management of the patient and family with neurofibromatosis 2: A consensus conference statement. Br. J. Neurosurg. 2005, 19, 5–12.
    68. Dewan, R.; Pemov, A.; Kim, H.J.; Morgan, K.L.; Vasquez, R.A.; Chittiboina, P.; Wang, X.; Chandrasekharappa, S.C.; Ray-Chaudhury, A.; Butman, J.A.; et al. Evidence of polyclonality in neurofibromatosis type 2-associated multilobulated vestibular schwannomas. Neuro. Oncol. 2015, 17, 566.
    69. Mathieu, D.; Kondziolka, D.; Flickinger, J.C.; Niranjan, A.; Williamson, R.; Martin, J.J.; Lunsford, L.D. Stereotactic radiosurgery for vestibular schwannomas in patients with neurofibromatosis type 2: An analysis of tumor control, complications, and hearing preservation rates. Neurosurgery 2007, 60, 460.
    70. Sharma, M.S.; Singh, R.; Kale, S.S.; Agrawal, D.; Sharma, B.S.; Mahapatra, A.K. Tumor control and hearing preservation after Gamma Knife radiosurgery for vestibular schwannomas in neurofibromatosis type 2. J. Neurooncol. 2010, 98, 265–270.
    71. McClelland, S., 3rd; Gerbi, B.J.; Cho, K.H.; Hall, W.A. The treatment of a large acoustic tumor with fractionated stereotactic radiotherapy. J. Robot. Surg. 2007, 1, 227–230.
    72. Seferis, C.; Torrens, M.; Paraskevopoulou, C.; Psichidis, G. Malignant transformation in vestibular schwannoma: Report of a single case, literature search, and debate. J. Neurosurg. 2014, 121, 160–166.
    73. Balasubramaniam, A.; Shannon, P.; Hodaie, M.; Laperriere, N.; Michaels, H.; Guha, A. Glioblastoma multiforme after stereotactic radiotherapy for acoustic neuroma: Case report and review of the literature. Neuro Oncol. 2007, 9, 447.
    74. Cayé-Thomasen, P. VEGF and VEGF receptor-1 concentration in vestibular schwannoma homogenates correlates to tumor growth rate. Otol. Neurotol. 2005, 26, 98–101.
    75. Komotar, R.J.; Starke, R.M.; Sisti, M.B.; Connolly, E.S. The role of bevacizumab in hearing preservation and tumor volume control in patients with vestibular schwannomas. Neurosurgery 2009, 65, N12.
    76. Alanin, M.C.; Klausen, C.; Caye-Thomasen, P.; Thomsen, C.; Fugleholm, K.; Poulsgaard, L.; Lassen, U.; Mau-Sorensen, M.; Hofland, K.F. The effect of bevacizumab on vestibular schwannoma tumour size and hearing in patients with neurofibromatosis type 2. Eur. Arch. Otorhinolaryngol. 2015, 272, 3627–3633.
    77. Blakeley, J.; Schreck, K.C.; Evans, D.G.; Korf, B.R.; Zagzag, D.; Karajannis, M.A.; Bergner, A.L.; Belzberg, A.J. Clinical response to bevacizumab in schwannomatosis. Neurology 2014, 83, 1986–1987.
    78. Eminowicz, G.K.; Raman, R.; Conibear, J.; Plowman, P.N. Bevacizumab treatment for vestibular schwannomas in neurofibromatosis type two: Report of two cases, including responses after prior gamma knife and vascular endothelial growth factor inhibition therapy. J. Laryngol. Otol. 2012, 126, 79–82.
    79. Liu, P.; Yao, Q.; Li, N.A.; Liu, Y.; Wang, Y.; Li, M.; Li, Z.; Li, J.; Li, G. Low-dose bevacizumab induces radiographic regression of vestibular schwannomas in neurofibromatosis type 2: A case report and literature review. Oncol. Lett. 2016, 11, 2981–2986.
    80. Mautner, V.F.; Nguyen, R.; Kutta, H.; Fuensterer, C.; Bokemeyer, C.; Hagel, C.; Friedrich, R.E.; Panse, J. Bevacizumab induces regression of vestibular schwannomas in patients with neurofibromatosis type 2. Neuro. Oncol. 2010, 12, 14–18.
    81. Plotkin, S.R.; Stemmer-Rachamimov, A.O.; Barker, F.G., 2nd; Halpin, C.; Padera, T.P.; Tyrrell, A.; Sorensen, A.G.; Jain, R.K.; di Tomaso, E. Hearing improvement after bevacizumab in patients with neurofibromatosis type 2. N. Engl. J. Med. 2009, 361, 358–367.
    82. Plotkin, S.R.; Merker, V.L.; Halpin, C.; Jennings, D.; McKenna, M.J.; Harris, G.J.; Barker, F.G., 2nd. Bevacizumab for progressive vestibular schwannoma in neurofibromatosis type 2: A retrospective review of 31 patients. Otol. Neurotol. 2012, 33, 1046–1052.
    83. Subbiah, V.; Slopis, J.; Hong, D.S.; Ketonen, L.M.; Hamilton, J.; McCutcheon, I.E.; Kurzrock, R. Treatment of patients with advanced neurofibromatosis type 2 with novel molecularly targeted therapies: From bench to bedside. J. Clin. Oncol. 2012, 30, 64–68.
    84. Versleijen, M.W.; Verbist, B.M.; Mulder, J.J.; de Geus-Oei, L.F.; van Herpen, C.M. Avastin scintigraphy in surveillance of bevacizumab treatment in a patient with neurofibromatosis type 2: A case report. Clin. Nucl. Med. 2014, 39, 277–280.
    85. Morris, K.A.; Golding, J.F.; Axon, P.R.; Afridi, S.; Blesing, C.; Ferner, R.E.; Halliday, D.; Jena, R.; Pretorius, P.M. Bevacizumab in Neurofibromatosis type 2 (NF2) related vestibular schwannomas: A nationally coordinated approach to delivery and prospective evaluation. Neuro-Oncol. Pract. 2016, 3, 281–289.
    86. Lu, V.M.; Ravindran, K.; Graffeo, C.S.; Perry, A.; Van Gompel, J.J.; Daniels, D.J.; Link, M.J. Efficacy and safety of bevacizumab for vestibular schwannoma in neurofibromatosis type 2: A systematic review and meta-analysis of treatment outcomes. J. Neurooncol. 2019, 144, 239.
    87. Plotkin, S.R.; Duda, D.G.; Muzikansky, A.; Allen, J.; Blakeley, J.; Rosser, T.; Campian, J.L.; Clapp, D.W.; Fisher, M.J.; Tonsgard, J.; et al. Multicenter, Prospective, Phase II and Biomarker Study of High-Dose Bevacizumab as Induction Therapy in Patients with Neurofibromatosis Type 2 and Progressive Vestibular Schwannoma. J. Clin. Oncol. 2019, 37, 3446.
    88. Ouerdani, A.; Goutagny, S.; Kalamarides, M.; Trocóniz, I.F.; Ribba, B. Mechanism-based modeling of the clinical effects of bevacizumab and everolimus on vestibular schwannomas of patients with neurofibromatosis type 2. Cancer Chemother. Pharmacol. 2016, 77, 1263–1273.
    89. Tamura, R.; Fujioka, M.; Morimoto, Y.; Ohara, K.; Kosugi, K.; Oishi, Y.; Sato, M.; Ueda, R.; Fujiwara, H.; Hikichi, T.; et al. A VEGF receptor vaccine demonstrates preliminary efficacy in neurofibromatosis type 2. Nat. Commun. 2020, 11, 2028.
    90. Goutagny, S.; Raymond, E.; Esposito-Farese, M.; Trunet, S.; Mawrin, C.; Bernardeschi, D.; Larroque, B.; Sterkers, O.; Giovannini, M.; Kalamarides, M. Phase II study of mTORC1 inhibition by everolimus in neurofibromatosis type 2 patients with growing vestibular schwannomas. J. Neurooncol. 2015, 122, 313.
    91. Karajannis, M.A.; Legault, G.; Hagiwara, M.; Giancotti, F.G.; Filatov, A.; Derman, A.; Hochman, T.; Goldberg, J.D.; Vega, E.; Wisoff, J.H.; et al. Phase II study of everolimus in children and adults with neurofibromatosis type 2 and progressive vestibular schwannomas. Neuro. Oncol. 2014, 16, 292.
    92. Karajannis, M.A.; Legault, G.; Hagiwara, M.; Ballas, M.S.; Brown, K.; Nusbaum, A.O.; Hochman, T.; Goldberg, J.D.; Koch, K.M.; Golfinos, J.G.; et al. Phase II trial of lapatinib in adult and pediatric patients with neurofibromatosis type 2 and progressive vestibular schwannomas. Neuro. Oncol. 2012, 14, 1163.
    93. Plotkin, S.R.; Halpin, C.; McKenna, M.J.; Loeffler, J.S.; Batchelor, T.T.; Barker, F.G., 2nd. Erlotinib for progressive vestibular schwannoma in neurofibromatosis 2 patients. Otol. Neurotol. 2010, 31, 1135.
    94. Tamura, R.; Morimoto, Y.; Sato, M.; Kuranari, Y.; Oishi, Y.; Kosugi, K.; Yoshida, K.; Toda, M. Difference in the hypoxic immunosuppressive microenvironment of patients with neurofibromatosis type 2 schwannomas and sporadic schwannomas. J. Neurooncol. 2020, 146, 265–273.
    95. Deep, N.L.; Patel, E.J.; Shapiro, W.H.; Waltzman, S.B.; Jethanamest, D.; McMenomey, S.O.; Roland, J.T., Jr.; Friedmann, D.R. Cochlear Implant Outcomes in Neurofibromatosis Type 2: Implications for Management. Otol. Neurotol. 2021, 42, 540–548.
    96. Neff, B.A.; Wiet, R.M.; Lasak, J.M.; Cohen, N.L.; Pillsbury, H.C.; Ramsden, R.T.; Welling, D.B. Cochlear implantation in the neurofibromatosis type 2 patient: Long-term follow-up. Laryngoscope 2007, 117, 1069.
    97. Funes, C.J.; Mace, R.A.; Macklin, E.A.; Plotkin, S.R.; Jordan, J.T.; Vranceanu, A.M. First report of quality of life in adults with neurofibromatosis 2 who are deafened or have significant hearing loss: Results of a live-video randomized control trial. J. Neurooncol. 2019, 143, 505.
    98. Evans, D.G.; Watson, C.; King, A.; Wallace, A.J.; Baser, M.E. Multiple meningiomas: Differential involvement of the NF2 gene in children and adults. J. Med. Genet. 2005, 42, 45.
    99. Larson, J.J.; van Loveren, H.R.; Balko, M.G.; Tew, J.M., Jr. Evidence of meningioma infiltration into cranial nerves: Clinical implications for cavernous sinus meningiomas. J. Neurosurg. 1995, 83, 596.
    100. Wentworth, S.; Pinn, M.; Bourland, J.D.; Deguzman, A.F.; Ekstrand, K.; Ellis, T.L.; Glazier, S.S.; McMullen, K.P.; Munley, M.; Stieber, V.W.; et al. Clinical experience with radiation therapy in the management of neurofibromatosis-associated central nervous system tumors. Int. J. Radiat. Oncol. Biol. Phys. 2009, 73, 208.
    101. Osorio, D.S.; Hu, J.; Mitchell, C.; Allen, J.C.; Stanek, J.; Hagiwara, M.; Karajannis, M.A. Effect of lapatinib on meningioma growth in adults with neurofibromatosis type 2. J. Neurooncol. 2018, 139, 749.
    102. Nunes, F.P.; Merker, V.L.; Jennings, D.; Caruso, P.A.; di Tomaso, E.; Muzikansky, A.; Barker, F.G., 2nd; Stemmer-Rachamimov, A.; Plotkin, S.R. Bevacizumab treatment for meningiomas in NF2: A retrospective analysis of 15 patients. PLoS ONE 2013, 8, e59941.
    103. Fujii, M.; Ichikawa, M.; Iwatate, K.; Bakhit, M.; Yamada, M.; Kuromi, Y.; Sato, T.; Sakuma, J.; Saito, K. Bevacizumab Therapy of Neurofibromatosis Type 2 Associated Vestibular Schwannoma in Japanese Patients. Neurol. Med. Chir. 2020, 60, 75–82.
    104. McClatchey, A.I.; Saotome, I.; Ramesh, V.; Gusella, J.F.; Jacks, T. The NF2 tumor suppressor gene product is essential for extraembryonic development immediately prior to gastrulation. Genes Dev. 1997, 11, 1253–1265.
    105. Giovannini, M.; Robanus-Maandag, E.; Niwa-Kawakita, M.; van der Valk, M.; Woodruff, J.M.; Goutebroze, L.; Merel, P.; Berns, A.; Thomas, G. Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein. Genes Dev. 1999, 13, 978–986.
    106. Kalamarides, M.; Niwa-Kawakita, M.; Leblois, H.; Abramowski, V.; Perricaudet, M.; Janin, A.; Thomas, G.; Gutmann, D.H.; Giovanni, M. Nf2 gene inactivation in arachnoidal cells is rate-limiting for meningioma development in the mouse. Genes Dev. 2002, 16, 1060–1065.
    107. Chen, J.; Landegger, L.D.; Sun, Y.; Ren, J.; Maimon, N.; Wu, L.; Ng, M.R.; Chen, J.W.; Zhang, N.; Zhao, Y.; et al. A cerebellopontine angle mouse model for the investigation of tumor biology, hearing, and neurological function in NF2-related vestibular schwannoma. Nat. Protoc. 2019, 14, 541–555.
    108. Kalamarides, M.; Stemmer-Rachamimov, A.O.; Takahashi, M.; Han, Z.Y.; Chareyre, F.; Niwa-Kawakita, M.; Black, P.M.; Carroll, R.S.; Giovannini, M. Natural history of meningioma development in mice reveals: A synergy of Nf2 and p16(Ink4a) mutations. Brain Pathol. 2008, 18, 62–70.
    109. Burns, S.S.; Chang, L.S. Generation of noninvasive, quantifiable, orthotopic animal models for NF2-associated schwannoma and meningioma. Methods Mol. Biol. 2016, 1427, 59–72.
    110. MacCollin, M.; Woodfin, W.; Kronn, D.; Short, M.P. Schwannomatosis: A clinical and pathologic study. Neurology 1996, 46, 1072.
    111. Plotkin, S.R.; Bredella, M.A.; Cai, W.; Kassarjian, A.; Harris, G.J.; Esparza, S.; Merker, V.L.; Munn, L.L.; Muzikansky, A.; Askenazi, M.; et al. Quantitative assessment of whole-body tumor burden in adult patients with neurofibromatosis. PLoS ONE 2012, 7, e35711.
    112. Merker, V.L.; Esparza, S.; Smith, M.J.; Stemmer-Rachamimov, A.; Plotkin, S.R. Clinical features of schwannomatosis: A retrospective analysis of 87 patients. Oncologist 2012, 17, 1317.
    113. MacCollin, M.; Chiocca, E.A.; Evans, D.G.; Friedman, J.M.; Horvitz, R.; Jaramillo, D.; Lev, M.; Mautner, V.F.; Niimura, M.; Plotkin, S.R.; et al. Diagnostic criteria for schwannomatosis. Neurology 2005, 64, 1838.
    114. Carter, J.M.; O’Hara, C.; Dundas, G.; Gilchrist, D.; Collins, M.S.; Eaton, K.; Judkins, A.R.; Biegel, J.A.; Folpe, A.L. Epithelioid malignant peripheral nerve sheath tumor arising in a schwannoma, in a patient with “neuroblastoma-like” schwannomatosis and a novel germline SMARCB1 mutation. Am. J. Surg. Pathol. 2012, 36, 154.
    115. Jacoby, L.B.; MacCollin, M.; Parry, D.M.; Kluwe, L.; Lynch, J.; Jones, D.; Gusella, J.F. Allelic expression of the NF2 gene in neurofibromatosis 2 and schwannomatosis. Neurogenetics 1999, 2, 101.
    116. Van den Munckhof, P.; Christiaans, I.; Kenter, S.B.; Baas, F.; Hulsebos, T.J. Germline SMARCB1 mutation predisposes to multiple meningiomas and schwannomas with preferential location of cranial meningiomas at the falx cerebri. Neurogenetics 2012, 13, 1.
    117. Hadfield, K.D.; Newman, W.G.; Bowers, N.L.; Wallace, A.; Bolger, C.; Colley, A.; McCann, E.; Trump, D.; Prescott, T.; Evans, D.G. Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis. J. Med. Genet. 2008, 45, 332.
    118. Boyd, C.; Smith, M.J.; Kluwe, L.; Balogh, A.; Maccollin, M.; Plotkin, S.R. Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis. Clin. Genet. 2008, 74, 358.
    119. Hadfield, K.D.; Smith, M.J.; Urquhart, J.E.; Wallace, A.J.; Bowers, N.L.; King, A.T.; Rutherford, S.A.; Trump, D.; Newman, W.G.; Evans, D.G. Rates of loss of heterozygosity and mitotic recombination in NF2 schwannomas, sporadic vestibular schwannomas and schwannomatosis schwannomas. Oncogene 2010, 29, 6216.
    120. Hulsebos, T.J.; Kenter, S.B.; Jakobs, M.E.; Baas, F.; Chong, B.; Delatycki, M.B. SMARCB1/INI1 maternal germ line mosaicism in schwannomatosis. Clin. Genet. 2010, 77, 86.
    121. Sestini, R.; Bacci, C.; Provenzano, A.; Genuardi, M.; Papi, L. Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associated schwannomas. Hum. Mutat. 2008, 29, 227.
    122. Rousseau, G.; Noguchi, T.; Bourdon, V.; Sobol, H.; Olschwang, S. SMARCB1/INI1 germline mutations contribute to 10% of sporadic schwannomatosis. BMC Neurol. 2011, 11, 9.
    123. Smith, M.J.; Wallace, A.J.; Bowers, N.L.; Rustad, C.F.; Woods, C.G.; Leschziner, G.D.; Ferner, R.E.; Evans, D.G. Frequency of SMARCB1 mutations in familial and sporadic schwannomatosis. Neurogenetics 2012, 13, 141.
    124. Plotkin, S.R.; Blakeley, J.O.; Evans, D.G.; Hanemann, C.O.; Hulsebos, T.J.; Hunter-Schaedle, K.; Kalpana, G.V.; Korf, B.; Messiaen, L.; Papi, L.; et al. Update from the 2011 International Schwannomatosis Workshop: From genetics to diagnostic criteria. Am. J. Med. Genet. A 2013, 161A, 405.
    125. Versteege, I.; Sévenet, N.; Lange, J.; Rousseau-Merck, M.F.; Ambros, P.; Handgretinger, R.; Aurias, A.; Delattre, O. Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 1998, 394, 203.
    126. Eaton, K.W.; Tooke, L.S.; Wainwright, L.M.; Judkins, A.R.; Biegel, J.A. Spectrum of SMARCB1/INI1 mutations in familial and sporadic rhabdoid tumors. Pediatr. Blood Cancer 2011, 56, 7.
    127. Swensen, J.J.; Keyser, J.; Coffin, C.M.; Biegel, J.A.; Viskochil, D.H.; Williams, M.S. Familial occurrence of schwannomas and malignant rhabdoid tumour associated with a duplication in SMARCB1. J. Med. Genet. 2009, 46, 68.
    128. Caltabiano, R.; Magro, G.; Polizzi, A.; Praticò, A.D.; Ortensi, A.; D’Orazi, V.; Panunzi, A.; Milone, P.; Maiolino, L.; Nicita, F.; et al. A mosaic pattern of INI1/SMARCB1 protein expression distinguishes Schwannomatosis and NF2-associated peripheral schwannomas from solitary peripheral schwannomas and NF2-associated vestibular schwannomas. Childs Nerv. Syst. 2017, 33, 933.
    129. Piotrowski, A.; Xie, J.; Liu, Y.F.; Poplawski, A.B.; Gomes, A.R.; Madanecki, P.; Fu, C.; Crowley, M.R.; Crossman, D.K.; Armstrong, L.; et al. Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas. Nat. Genet. 2014, 46, 182.
    130. Smith, M.J.; Isidor, B.; Beetz, C.; Williams, S.G.; Bhaskar, S.S.; Richer, W.; O’Sullivan, J.; Anderson, B.; Daly, S.B.; Urquhart, J.E.; et al. Mutations in LZTR1 add to the complex heterogeneity of schwannomatosis. Neurology 2015, 84, 141.
    131. Frattini, V.; Trifonov, V.; Chan, J.M.; Castano, A.; Lia, M.; Abate, F.; Keir, S.T.; Ji, A.X.; Zoppoli, P.; Niola, F.; et al. The integrated landscape of driver genomic alterations in glioblastoma. Nat. Genet. 2013, 5, 1141.
    132. Forbes, S.A.; Bindal, N.; Bamford, S.; Cole, C.; Kok, C.Y.; Beare, D.; Jia, M.; Shepherd, R.; Leung, K.; Menzies, A.; et al. COSMIC: Mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011, 39, D945.
    133. Algar, E.M.; Muscat, A.; Dagar, V.; Rickert, C.; Chow, C.W.; Biegel, J.A.; Ekert, P.G.; Saffery, R.; Craig, J.; Johnstone, R.W.; et al. Imprinted CDKN1C is a tumor suppressor in rhabdoid tumor and activated by restoration of SMARCB1 and histone deacetylase inhibitors. PLoS ONE 2009, 4, e4482.
    134. Emanuele, M.J.; Elia, A.E.; Xu, Q.; Thoma, C.R.; Izhar, L.; Leng, Y.; Guo, A.; Chen, Y.N.; Rush, J.; Hsu, P.W.; et al. Global identification of modular cullin-RING ligase substrates. Cell 2011, 147, 459.
    1. Please check and comment entries here.