Schwannomas and Their Pathogenesis

Abstract

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

Introduction

Schwannomas are among the most common of the peripheral nerve sheath tumors, which also include neurofibromas, perineuriomas, granular cell tumors and malignant peripheral nerve sheath tumors. Although the major neoplastic component of neurofibromas is the Schwann cell 134, these tumors are both clinically and pathologically distinct from schwannomas and will not be discussed further. This article will briefly review the human pathology of schwannomas and discuss the clinical syndromes associated with a predisposition to schwannomas, including a review of the role of merlin, the protein encoded by the neurofibromatosis type 2 (NF2) gene and its downstream signaling pathways in tumor formation, and how our understanding of these has allowed the development of new therapeutic approaches to the treatment of schwannoma.

Schwannomas are benign, well‐circumscribed tumors usually attached to peripheral nerves, consisting of a clonal population of Schwann cells, which often undergo cystic and degenerative change. Most cases are sporadic; however, some are associated with NF2, schwannomatosis or Carney's complex (see below), and they may rarely occur following radiation 153, 178. Postradiation schwannomas, including acoustic schwannomas, appear with a latency of up to 50 years following treatment 147, 153, 156, 166, 204.

Pathology

Schwannomas are usually solitary tumors that often affect the small peripheral nerves in the head and neck regions, and the flexor surfaces of the extremities. Central lesions commonly arise from sensory nerve roots, and a common intracranial site is the vestibular branch of the eighth nerve, but they may also arise from the trigeminal nerve and, in the setting of NF2, other lower cranial nerves. Autopsy studies have suggested a frequency of sporadic central schwannoma of 4.5% in this elderly population 157, with over 85% being vestibular and the remainder spinal. However, population‐based studies of vestibular schwannoma suggest a much lower incidence of around 0.01%–0.1% 57, 110, 141, 181. Motor root and sympathetic chain involvement is uncommon, and involvement of the brain or cord parenchyma is rare. Schwannomas rarely affect the visceral organs and gastrointestinal tract, particularly the stomach. Many schwannomas are asymptomatic, but some result in pain or sensory disturbances, and “dumbbell” spinal tumors with an intradural component can cause cord compression. Deep‐seated tumors, such as retroperitoneal and mediastinal lesions, may grow to a considerable size before becoming symptomatic by local compression or bony erosion, such as the giant sacral schwannoma 30.

Schwannomas have a smooth nodular outline, sometimes with the nerve of origin visible (Figure 1A), and on cut surface have a tan or yellow color, often with areas of hemorrhage and cystic change. Microscopically, schwannomas are well circumscribed, with a surrounding capsule, and contain areas composed of fascicles of Schwann cells that have a spindle cell morphology (Antoni A pattern) (Figure 1B), which may either merge with or abruptly change to other more loosely textured and microcystic areas (Antoni B pattern) (Figure 1C). Schwannomas show areas of nuclear alignment or palisading, often forming parallel nuclear arrays or Verocay bodies (Figure 1D), although these structures are less common in vestibular schwannomas 154. Large tumors often show extensive degenerative change, with thick hyalinized, and often thrombosed and ectatic, blood vessels (Figure 1E); areas of hemorrhage; lipidization; calcification; and cystic change. Infarct‐like areas of necrosis may occasionally be present (Figure 1F), which presumably relate to the vascular changes 154. Tumors with extensive degenerative change, also known as “ancient schwannomas,” may show marked degenerative nuclear atypia, which should not be confused with malignant change. Mast cells are often seen but are more numerous in neurofibromas and malignant peripheral nerve sheath tumors 50. In some schwannomas, a chronic inflammatory cell infiltrate may be prominent, particularly within the gastrointestinal tract where there is characteristically a peripheral cuff of lymphoid cells (Figure 1G) 84. The significance of inflammation in most schwannomas is unclear, but in vestibular schwannomas, the presence of CD163‐positive macrophages has been correlated with vascular density and tumor growth 45, 46. Schwannomas may show areas with an epithelioid morphology (Figure 1H), and some subcutaneous examples are predominantly epithelioid 97, 168, but true epithelial metaplasia is rare 152. A rare variant of schwannoma contains areas of neuroblastoma‐like rosettes, but this should not be confused with malignant neuroectodermal tumors 27, 177. Schwannoma tumor cells show uniform strong nuclear and cytoplasmic S100 immunoreactivity and are enveloped in a pericellular basal lamina, containing laminin and collagen type 4 130. Schwannomas may also show immunoreactivity with antibodies to glial fibrillary acidic protein (GFAP) 96, 123, and some of these latter immunoreactive tumors also express cytokeratins 59, 69.

Figure 1.

A. Well‐circumscribed lumbar region schwannoma, with attached nerve root. Characteristic histological features include cellular Antoni A tissue (B), with less cellular areas of loosely textured microcystic Antoni B tissue (C), and parallel arrays of nuclei forming a Verocay body (D). Blood vessels often show hyaline degeneration of their walls and thrombosis (E), and occasional areas of necrosis may be seen (F). A prominent pericapscular lymphocytic infiltrate is common in gastrointestinal schwannomas (G). Epitheloid morphology is seen in some benign schwannomas (H).

Schwannomas are benign tumors, which in surgically accessible sites respond well to excision and, unlike neurofibromas, only very rarely undergo malignant transformation 44, which may occur in the setting of sporadic tumors 44, 122, 197, or in association with schwannomatosis 53, 68, neurofibromatosis 24, 53, 111 and following therapeutic irradiation 47, 116, 198. Malignant transformation within a schwannoma usually results in an epithelioid or primitive neuroectodermal morphology 122, 197, rather than the fibrosarcomatous pattern which is most often seen following transformation of neurofibromas.

Neurofibromas are usually easy to distinguish from schwannomas by their lack of a capsule, mixed population of cells (some of which are S100 immunoreactive), cells with wavy nuclei, absence of Antoni A and B patterns, and presence of axons, often with their myelin sheaths passing through the lesion. However, hybrid tumors can occur, with areas of both neurofibroma and schwannoma within the same tumor, and these appear to be more common in schwannomatosis and NF1 and 2 77. Hybrid tumors containing both perineurioma and schwannoma‐like areas have also been described, but these do not appear to be associated with familial tumor syndromes 82.

Central nerve root schwannomas sometimes may resemble meningiomas, particularly fibroblastic meningiomas. Helpful immunocytochemical findings include pericellular basement membrane deposition with antibodies to either type 4 collagen or laminin in schwannomas, and the presence of widespread membrane immunoreactivity for epithelial membrane antigen in meningiomas, although it should be noted that schwannomas can also express this antigen 194. Demonstration of basement membrane material can help in distinction from lumbar region ependymomas. Other soft‐tissue tumors, such as leiomyomas and palisaded myofibroblastomas, may show nuclear palisades, but are usually otherwise easy to distinguish from schwannomas on morphology and show different patterns of immunoreactivity.

There are a few morphologically distinct variants of schwannomas, in addition to the conventional schwannoma, which are discussed later.

Cellular schwannoma

Cellular schwannomas have a higher cellularity and mitotic rate than conventional schwannomas, and may be locally erosive but are benign tumors without metastatic potential. Cellular schwannomas commonly occur in the spinal and paraspinal regions, but about 10% are intracranial 32. Most cases are sporadic, and there is a female predominance 32, 191. The tumors are composed predominantly of cellular Antoni A‐type tissue (Figure 2A), but without well‐formed Verocay bodies and occasionally contain small foci of necrosis. Most tumors have fewer than four mitoses per 10 high power fields, but in some cases the mitotic rate may be higher 191. Capsular and perivascular lymphocytic infiltrates may be a prominent feature 154. Useful features to distinguish cellular schwannomas from malignant peripheral nerve sheath tumors are a relatively high cellularity for the mitotic rate, good circumscription, perivascular hyalinization, uniform diffuse S100 protein immunoreactivity (Figure 2B) and variable GFAP immunoreactivity (Figure 2C) 32, 81, 123, 190. They can be distinguished from other cellular soft‐tissue tumors, such as leiomyosarcomas and rhabdomyosarcomas by immunocytochemistry. Cellular schwannomas have a significant local recurrence rate of up to 40%, depending on the extent of resection and location 32, 191.

Figure 2.

Cellular schwannomas are predominantly composed of Antoni A tissue and may show prominent nucleoli and mitotic activity (A). Like classical schwannomas, they show widespread S100 (B) and sometimes strong glial fibrillary acidic protein (C) immunoreactivity.

Plexiform schwannoma

These are usually sporadic dermal tumors that have an intraneural nodular pattern of growth (Figure 3A,B). They show typical histological features of schwannomas, with predominantly Antoni A‐type tissue and sometimes Verocay body formation (Figure 3C). A minority of cases are associated with NF1 and 2 or schwannomatosis 23, 89, 90, 144, 185. Plexiform schwannomas tend to occur in a superficial location and in young adults, and most respond well to surgical excision. Deep‐seated tumors also occur, and may be cellular and show necrosis, but behave in a benign manner 1. There is an infiltrative sporadic subtype occurring in infants and children, which has a propensity to recur following surgery and may be mitotically active 196. However, they show uniform S100 immunoreactivity, and most show GFAP immunoreactivity. These infantile and childhood plexiform schwannoma do not have the capacity to metastasize, so these should not be confused with malignant peripheral nerve sheath tumors 90, 196.

Figure 3.

Plexiform schwannoma has a multinodular architecture seen macroscopically (A) and in histological sections (B). They are predominantly composed of Antoni A tissue and sometimes form Verocay bodies (C).

Melanotic schwannoma

These are uncommon pigmented Schwann cell tumors, which have a spindle cell and epithelioid cell morphology, often with intranuclear cytoplasmic pseudoinclusions (Figure 4A), and usually lacking Verocay bodies. Melanotic schwannomas are usually deep seated and commonly involve the spinal nerves, cranial nerves and sympathetic chain, but may occasionally involve the gastrointestinal tract, soft tissues, skin, liver and heart. A psammomatous subtype with calcospherites (which may not be very numerous), and often associated with cytoplasmic vacuolation of the tumor cells, sometimes resembling adipose tissue (Figure 4B). Approximately half of patients with psammomatous melanotic schwannomas have Carney's complex (see later), which rises to approximately 80% if the tumors are multiple 31. Tumors arising in association with Carney's complex tend to occur earlier than sporadic tumors (median age 22 years, compared with 33 years), and may show loss of tumor PRKAR1a protein expression by immunocytochemistry 31, 180. Melanotic schwannomas are S100 immunoreactive, but negative with antibodies to GFAP 31, 205. These tumors show ultrastructural evidence of melanosome formation 48, 186 and are immunoreactive for melanosome proteins, such as Melan‐A and HMB‐45 (Figure 4C). Pericellular basement membrane material is present, which may be demonstrated with reticulin staining, and is immunoreactive for type 4 collagen and laminin (Figure 4D). A significant proportion of melanotic schwannomas behave in a malignant fashion with metastatic spread, and particularly spinal tumors 42, 151, 186. However, distinguishing multiple tumors from metastatic lesions may be difficult. There are no clear‐cut histological criteria for malignancy, although features such as large nuclei, prominent nucleoli, mitoses and necrosis are common 23, 180. A recent review of melanotic schwannomas with clinical follow‐up found a rate of local recurrence of 35% and metastatic spread of 44%, and the authors have suggested that melanotic schwannomas should be reclassified as malignant tumors 180.

Figure 4.

Melanotic schwannoma has prominent melanin pigment and nucleoli, often with intranuclear pseudoinclusions (A), prominent cytoplasmic vacuolation and rare calcified psammoma bodies (inset) (B). These tumors are immunoreactive with antibodies to melanosome proteins such as HMB‐45 (C) and basement membrane proteins such as laminin (D).

Melanotic schwannomas need to be distinguished from primary melanocytic lesions, and the presence of basement membrane material, psammoma bodies or adipose‐like cells can be helpful. If necessary, mutational analysis for GNAQ codon 209 mutations can be performed; as if present, these mutations are highly specific for leptomeningeal melanocytic tumors 102. Pigmented neurofibromas lack psammoma bodies or adipose‐like cells, and show other histological features of neurofibromas (see earlier).

Genetic Syndromes Associated with Schwannomas

NF2

NF2 is a dominantly inherited syndrome, characterized by the formation of multiple schwannomas, and has an annual incidence of between 1 in 25 000 and 1 in 40 000 14, 56. Individuals with NF2 may have a number of tumors in addition to schwannomas, including meningioma in 50%–60% and ependymoma in about 6%, together with non‐neoplastic features such as posterior subcapsular cataracts, retinal hamartomas, epiretinal membranes, meningoangiomatosis and in around 2 out of 3 patients there is electrophysiological evidence of a polyneuropathy, which is usually axonal 54, 118, 132, 169. Almost all glial tumors occurring in NF2 patients are ependymomas, although rare tumors showing astrocytic features have been reported 93. Most individuals present with hearing loss and tinnitus because of the development of bilateral vestibular schwannomas; however, unilateral vestibular schwannomas with a number of other features may be sufficient for diagnosis 19, 55. Peripheral schwannomas occur in about 70% of NF2 patients 54. Diagnosis of NF2 can be made clinically using established diagnostic criteria 19, 51—see Table 1. In addition to routine clinical assessment and a detailed family history, patients should have thorough cutaneous examination, slit lamp ophthalmic assessment, craniospinal magnetic resonance imaging (MRI) and molecular analysis 51. It is good practice to use thin adjacent slices to diagnose vestibular schwannoma with MRI 167. Cases of mosaic NF2 can be confused with schwannomatosis 136, but patients fulfilling the Manchester criteria are unlikely to be misdiagnosed 19.

Table 1.

Manchester criteria for diagnosis of neurofibromatosis type 2 51

1. Bilateral vestibular schwannoma (VS) or family history of NF2 plus: Unilateral VS or two of meningioma, glioma, neurofibroma, schwannoma, posterior subcapsular lenticular opacities

2. Unilateral VS plus two of meningioma, glioma, neurofibroma, schwannoma, posterior subcapsular lenticular opacities

3. Two or more meningioma plus unilateral VS or two of glioma, neurofibroma, schwannoma, cataracts

NF2 = neurofibromatosis type 2.

Following the identification of the NF2 gene, and its product merlin (schwannomin) on chromosome 22 in 1993 150, 182, a better understanding of the disease pathogenesis has developed. A large number of mutations have been described in the NF2 gene 18. Up to half of patients have de novo mutations and a third of patients, particularly those with milder features or without a family history, have mosaicism 51, 58, 101, 126. The risk of offspring inheriting NF2 in mosaic patients is difficult to quantify, but may be considerably less than 50%. Genetic testing is available and ideally should include testing of tumor tissue, which has higher sensitivity, and is particularly useful in mosaic cases where mutations may be of low abundance in blood and can help to distinguish between mosaic NF2 and schwannomatosis. In patients with mosaicism, the disease may be localized to a segment or side of the body. Although there is some heterogeneity within families, in general, mutations resulting in nonsense or frameshift change result in a more severe phenotype than those with an in‐frame deletion. In particular, these patients have a younger age of onset, higher proportion of meningiomas and spinal tumors, and higher number of non‐eighth cranial nerve tumors 20, 160.

Schwannomas in patients with NF2 have a similar morphology to sporadic tumors, but multifocal nerve involvement and whorl formation are more frequent (Figure 5A) 154. In addition there may be patchy loss of SMARCB1 (INI‐1) immunoreactivity (Figure 5B) 133, and neurofilament‐positive nerve fibers sometimes traverse the tumor 189, features also seen in schwannomatosis, but are uncommon sporadic tumors. Rarely, meningioma “islets” may be seen in intracranial schwannomas from patients with NF2 112. Malignant transformation is rare 53, but may occur particularly after radiation 52.

Figure 5.

Whorl formation is seen more commonly in schwannomatosis and neurofibromatosis type 2 (NF2) (A). INI‐1 immunoreactivity often shows a mosaic pattern in familial schwannomas, including cases of NF2 (B). Alcian blue stain showing an area of myxoid change within a schwannoma from a patient with schwannomatosis (C).

Schwannomatosis

These patients present with multiple schwannomas, which in about a third of patients are localized to a limb or segment of the body 114 and the tumors are often painful. A family history can be demonstrated in only 15%–25% 136, so the majority of cases appear to be sporadic. Most schwannomas are found in the neck, trunk and extremities, with visceral lesions being infrequent. The vestibular nerves are not usually involved, but rare cases of schwannomatosis with unilateral VS have been described 136. Diagnostic criteria have been proposed 113 and subsequently modified to include molecular findings 136 (see Table 2). Helpful features to distinguish cases of schwannomatosis from cases of NF2 include the absence of ocular pathology, ependymomas and vestibular schwannomas by the age of 30. The prevalence of schwannomatosis is difficult to estimate as cases may be difficult to distinguish from mosaic forms of NF2 128, but it is probably similar to the prevalence of NF2 14, 113. The histology of the schwannomas is similar to that seen in sporadic tumors, although peritumoral edema, myxoid change (Figure 5C) and intraneural growth are more common 113. In addition, the tumors may show patchy loss of SMARCB1 (INI‐1) immunoreactivity 133 and the presence of intratumoral nerve fibers 189. Other types of tumor are not generally a feature of schwannomatosis, but rare cases associated with multiple meningiomas 16 or neurofibromas 146 have been described.

Table 2.

Diagnostic criteria for schwannomatosis 136

Molecular diagnosis

1. Two or more schwannomas or meningiomasa and genetic studies of at least two tumors showing loss of heterozygosity at chromosome 22 and NF2 mutations. The presence of a common SMARCB1 mutation defines SMARCB1‐associated schwannomatosis.

2. One schwannoma or meningiomaa and a germ‐line pathogenic SMARCB1 mutation

Clinical diagnosis

1. Two or more non‐intradermal schwannomas (one with pathological confirmation) and the absence of vestibular schwannoma on thin‐sliced MRIb

2. One schwannoma or meningiomaa and affected first‐degree relative

3. Possible diagnosis if two or more non‐intradermal schwannomas (without pathological confirmation) and chronic pain associated with tumors

Exclusion criteria: germ‐line pathogenic NF2 mutation, fulfill criteria for NF2, first‐degree relative with NF2, schwannomas in radiation field only

^aPathologically confirmed.

^bThese criteria may include some mosaic NF2 patients, and some schwannomatosis patients may have unilateral vestibular schwannomas or multiple meningiomas.

MRI = magnetic resonance imaging; NF2 = neurofibromatosis type 2.

Germ‐line mutations in SMARCB1 have been reported in a number of patients with schwannomatosis 86, 87, including cases of mosaicism 86. Germ‐line mutations in SMARCB1 have also been associated with other tumor types including multiple rhabdoid tumors 92 and multiple meningiomas 38, 73. Mutations in schwannomatosis appear more frequent toward the ends of the SMARCB1 gene, whereas they are more often centrally located in inherited predisposition to rhabdoid tumors 136. SMARCB1 protein forms part of an adenosine triphosphate (ATP)‐dependant chromatin remodeling complex, important in central nervous system (CNS) development 107, 117 and has tumor suppressor functions including induction of cell cycle arrest, associated with downregulation of cyclin D1 207 and upregulation of p16 26. As well as the SMARCB1 mutations, additional somatic mutations of the NF2 gene or loss of heterozygosity of 22q have been described in schwannomas associated with schwannomatosis 161, giving rise to a four‐hit hypothesis 136. Thus, schwannomas in schwannomatosis have merlin loss, like schwannomas in NF2 or spontaneous schwannomas, but the initial mutation is different. SMARCB1 mutations have been identified in only a minority of schwannomatosis patients, suggesting that other genetic loci are important. Recently, it has been shown that germ‐line loss of function mutations in LZTR1 are common in patients with schwannomatosis, who do not have SMARCB1 mutations, but tumors from these patients also show deletions of 22q and somatic mutations of the remaining NF2 allele 135. LZTR1 is a tumor suppressor gene that is lost in a subset of glioblastomas 63, and although it may have several functions, it may interact with proteins that regulate SMARCB1 expression 139, 143, 188.

Carney's complex

This condition comprises a combination of conjunctival and mucosal lentigines, myxomas (including cardiac), schwannomas, endocrine abnormalities, including acromegaly, Sertoli cell tumors and Cushing's syndrome 15, 145, 193. Diagnostic criteria have been proposed 176 (see Table 3), and the majority of patients have a family history 175, 187. Two genetic loci have been identified on chromosome arms 17p and 2p 99, 174, 187, but about two‐thirds of cases are caused by mutations in the protein kinase A subunit on 17p24, PRKAR1a gene. A large number of PRKAR1a mutations have been reported 25, 83, which have a high penetrance and occur with a higher frequency in familial, compared with sporadic, cases of Carney's complex 25. The schwannomas in Carney's complex commonly occur in the upper gastrointestinal tract and sympathetic chain, and are characterized by melanin pigmentation and the presence of psammoma bodies and they may show loss of PRKAR1a protein expression 180. As already discussed, significant proportion of melanotic schwannomas behave in a malignant manner and metastasize 42, 151, 180.

Table 3.

Diagnostic criteria for Carney's complex 176

Main criteria (two required or one plus a supplementary criterion)
1. Spotty skin pigmentation with a typical distribution (lips, conjunctiva and inner or outer canthi, vaginal and penile mucosa)
2. Myxoma (cutaneous and mucosal)a
3. Cardiac myxomaa
4. Breast myxomatosisa or fat‐suppressed magnetic resonance imaging findings suggestive of this diagnosis
5. Pigmented nodular adrenocortical diseasea or paradoxical‐positive response of urinary glucocorticosteroids to dexamethasone administration during Liddle's test
6. Acromegaly because of GH‐producing adenomaa
7. Large cell calcifying Sertoli cell tumorsa or characteristic calcification on testicular ultrasonography
8. Thyroid carcinomaa or multiple, hypoechoic nodules on thyroid ultrasonography in a young patient
9. Psammomatous melanotic schwannomaa
10. Blue nevus, epithelioid blue nevus (multiple)a
11. Breast ductal adenoma (multiple)a
12. Osteochondromyxomaa

^aPathologically confirmed.

Supplemental criteria:

1. Affected first‐degree relative.
2. Inactivating mutation of the PRKAR1A gene.

GH = growth hormone.

PRKAR1a appears to act as a tumor suppressor gene, and inactivation results in increased cyclic adenosine monophosphate (cAMP) activity 99, 100 and Rac 1 activation 115, leading to suppression of merlin activity (see later) and the development of Schwann cell tumors.

Merlin and disease models

Defects in the NF2 gene are found in all sporadic and NF2‐associated schwannomas, which include deletions, mutations, allelic losses and gene promoter hypermethylation 22, 67, 71, 74, 98, 158, 172. No other consistent genetic changes have been identified in schwannomas 13, 88. The NF2 gene product, merlin (also known as schwannomin), is structurally similar to the ezrin radixin moesin (ERM) proteins that are involved in linking the cytoskeleton to the membrane. The term merlin is derived from moesin–ezrin–radixin‐like protein 182 but has some differences from these three proteins; in particular, it lacks the classical actin‐binding domain at its carboxyl terminus. There are multiple isoforms of merlin caused by alternate splicing 65, 72, 76, 155, but the predominant forms have tumor suppressor activity 21, 165. Merlin loss is causal in the development in a number of tumors in addition to schwannomas, including mesotheliomas 28, 179, meningiomas 22 and ependymomas 22, 71, 142. Merlin is expressed in several tissues during development 3, 72, and, at later developmental stages, expression can be found in tissues where tumors develop 34, 40, 70, 72.

Although murine models of NF2 have been developed, NF2 knockout mice die when homozygous and, when heterozygous, develop a variety of tumors including sarcomas and malignant epithelial tumors that have a propensity to metastasize, but do not develop schwannomas 121. Conditional knockout mice develop schwannomas late in life but only rarely vestibular schwannomas 64. In addition, the pathologic features of tumors in mice show some differences with human nerve sheath tumors; they tend to be cellular with a fascicular or storiform pattern, Verocay body formation is uncommon, they are not encapsulated, and they infiltrate adjacent tissues and lack degenerative vascular changes such as hyalinized blood vessels, hemorrhage and thrombosis 171. However, murine models may be helpful in the evaluation of some therapeutic approaches 39, 195. Much work on the characterization of merlin has been carried out using in vitro cell models.

On a cellular level, merlin has been shown to be present in actin‐rich cellular protrusions 66, 155, sites of cell matrix and cell–cell contact 60, 104, 155, and is present within the nucleus 127. Merlin is phosphorylated at multiple sites; however, phosphorylation at the serine 518 residue, by Rac 1‐dependent p21‐activated kinase (PAK) and cAMP‐dependent protein kinase A 4, 162, results in a more closed conformation, which is associated with loss of activity 163. A second key site for phosphorylation is the serine 10 residue, which may occur as the result of either protein kinase A or Akt activity 105, 106, and may target merlin for degradation 106. Thus, merlin phosphorylation at these sites is a mechanism of protein loss and inactivation 106.

At the cell membrane, merlin interacts with integrins and focal adhesion proteins 60, 131, and is involved in inhibition of tyrosine kinase receptor expression and signaling (see later), particularly on cell–cell contact 7, 43, 104, 200. Additionally, merlin's association with adherens junction proteins is required for the formation of intercellular junctions and contact inhibition of growth 60, 103. Merlin negatively regulates CD44 interactions 17, which is dependent on the phosphorylation status of merlin 148. In merlin‐deficient tumors, CD44 expression is increased 164, but merlin is required for the cell contact growth inhibitory function of CD44 124.

Merlin shuttles to the nucleus in a cell cycle‐dependent manner 127, and within the nucleus merlin inhibits the E3 ubiquitin ligase CRL4–DCAF1 complex 108, a ubiqiuitin ligase that regulates and stimulates transcription of multiple genes including those of integrins and tyrosine kinase receptors. Mutations in the NF2 gene inhibit merlin's interaction with CRL4–DCAF1 108, and this function appears central to the growth inhibitory functions of merlin 41, 108. Merlin loss thus leads to increase integrin expression, resulting in increased cell spreading on the extracellular matrix in vitro and pseudomesaxon formation in vivo 184.

Merlin loss also leads to increased expression and signaling of growth factors, resulting in increased proliferation in vitro and in vivo 7, 79. Growth factor receptor activation, and merlin loss itself, leads to impaired adherens junctions and impaired contact inhibition of growth via the wnt/catenin signaling pathway 208. Growth factor receptor activation also leads to activation of the Ras/Mek/Erk and phosphatidylinositol 3‐kinase (PI3K) pathways. Additionally, merlin appears to be able to directly inhibit the mitogen‐activated protein kinase (MAPK) and PI3K via pathways downstream of the receptors 62, 109, 125, 149, and further downstream, merlin can also inhibit the mammalian target of rapamycin (mTOR) complex 91. In addition to affecting these mostly mitogenic and survival pathways, merlin uncouples Ras/Rac growth factor receptor interactions 125 and has a negative feedback loop with PAK 80, 162, so that loss of merlin leads to Rac activation 94, which in turn leads to sustained and nonlocalized GTPase activation 61, 129. This, together with the fact that merlin controls polar localization of ezrin 78, contributes to loss of schwannoma cell polarization and the inability to attach to an axon. Merlin's interaction with ezrin also seems to be relevant for Ras pathway activation downstream of tyrosine kinase receptors 125, 170. In addition to the tyrosine kinase receptor pathways, there is growing evidence that merlin may also act via the Hippo/Yap pathway 159, 199, 202, 203, 206, but this has not been investigated in any detail in relation to schwannoma 8.

Driven by the fact that growth factor receptors have been investigated intensively in oncology as potential therapeutic targets, growth factor receptors have been studied in some detail in schwannomas 7. In vitro studies suggest that merlin binds ErbB2 at the cell membrane 60, inhibiting both the Src‐binding of ErbB2 85 and the expression of ErbB2 and 3 2. Studies of epidermal growth factor receptor (EGFR) in human tumor samples are conflicting, with some studies showing no expression of protein 140, 192, and others finding overexpression of the protein and messenger RNA 49, 173. In human schwannomas, there is strong expression of ErbB2, 3 and 4 49, 75, 192, along with the agonist neuregulin 1 173. In addition ErbB2 is constitutively activated 29. Alterations and expression of ErbB2 and 3 may be directly related to merlin deficiency via alterations in receptor expression; merlin associates with EGFR on cell contact, limiting it to the cell membrane, preventing receptor internalization and Akt/MAPK signaling 43, 104 2, 104. Based on these data, there was a case series with the EGFR inhibitor, erlotinib, which did not show a significant effect on hearing or tumor size in patients with progressive vestibular schwannomas 137. A phase II trial with the ErbB2/EGFR inhibitor, lapatinib, showed that lapatinib carries minor toxicity in NF2 and a moderate effect in tumor volume in a subset of patients 95.

Overexpression of platelet‐derived growth factor receptor β (PDGFRβ) and insulin‐like growth factor receptor (IGF 1R) are also seen, and these seem to be functional in schwannomas 6, 10, 12, 62, 201, offering further potential sites for therapeutic drug targeting 6, 7, 9, 11. Overexpression of PDGFR may relate to the loss of merlin's role in receptor internalization and degradation 62. Schwannoma cells also overexpress insulin‐like growth factor, resulting in autocrine‐mediated cell growth 10, 35 and reduced to apoptosis 36, 37. Currently, phase 0 trials with PDGFR inhibitors are ongoing in the United Kingdom, looking at drug concentration and molecular effects in tumor and blood.

Vascular endothelial growth factor (VEGF) and its receptor expression are elevated in schwannomas, correlating with tumor growth and volume 33, 138, 183. In addition, Schwann cells express Axl a member of the TAM kinase family 8. In vitro and animal studies suggest that expression of these receptors is functional in schwannomas, and that specific inhibition may be an effective treatment approach 5, 39, 62. Limited studies on patients have shown some promising results with the VEGF inhibitor, bevacizumab, in vestibular schwannomas with improvements in hearing and reductions in tumor size 119, 120, 138. Currently, a phase II study is underway in the United States.

Summary

Schwannomas are relatively common benign tumors of peripheral nerves, which occur sporadically or in the context of several genetic tumor syndromes (see Figure 6). Although many peripheral tumors may be asymptomatic and most are treated easily by surgical resection, deep‐seated and multiple tumors (particularly in the context of a genetic syndrome) may be more difficult to treat. Recent advances in our understanding of the pathogenesis of these tumors have indicated that defects in merlin are responsible for both sporadic and genetically acquired schwannomas, and the mechanisms by which merlin loss triggers tumor development are being unraveled (see summary in Figure 7). Although merlin is involved in multiple pathways, and has roles in the nucleus and at the cell membrane, these diverse roles are interconnected. Loss of merlin in the nucleus leads to overexpression of membrane proteins, including growth factor receptors and integrins, and activation of growth factor receptors influences cell–cell contact inhibition. It should be borne in mind that some of the interactions between merlin and signaling pathways have for good practical reasons been described in cell lines, and may not always be translated exactly into human schwannomas. However, the recent rapid growth in our understanding of these pathways and their complex interactions has allowed the development of novel treatment strategies that may be applicable to both schwannomas and other merlin‐related tumors, including meningiomas and ependymomas.

Figure 6.

Diagram summarizing genetic pathways leading to schwannoma pathogenesis.

Figure 7.

Diagram illustrating key pathways dysregulated in merlin^−/− tumor cells. Merlin loss leads to increased growth factor expression and activation of the Ras and phosphatidylinositol 3‐kinase (PI3K) pathways. A central mechanism is the loss of merlin‐induced inhibition of the CRL4–DCAF1 complex within the nucleus, resulting in increased transcription of a number of genes, including integrins and growth factor receptors. Merlin also interacts with cell surface proteins, including CD44 and adhesion junction proteins, so that merlin deficiency leads to reduced contact‐dependant cell cycle arrest. EGF = epidermal growth factor; ErbB2 = epidermal growth factor receptor 2; Gas 6 = growth arrest specific 6; IGFR = insulin‐like growth factor receptor; NRG = neuregulin; PDGFR = platelet‐derived growth factor receptor; VEGFR = vascular endothelial growth factor receptor.