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Tumor Suppressor Mutations and Growth Factor Signaling in the Pathogenesis of NF1-Associated Peripheral Nerve Sheath Tumors. I. The Role of Tumor Suppressor Mutations

Posted on: Wednesday, 17 November 2004, 03:00 CST

Abstract. Patients with neurofibromatosis type 1 (NF1), a common autosomal dominant tumor predisposition syndrome, develop benign cutaneous, intraneural, and plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs), an aggressive form of Schwann cell neoplasm that frequently arises from plexiform neurofibromas. Impressive advances have been made in defining the molecular mechanisms responsible for neurofibroma and MPNST tumorigenesis, including the identification of key tumor suppressor gene mutations, an improved understanding of the functions of these tumor suppressors, and the production of transgenic mouse models in which tumor suppressor gene mutations predispose animals to the development of neurofibromas and MPNSTs. It has also become apparent that dysregulated growth factor signaling cooperates with tumor suppressor mutations to promote neurofibroma and MPNST tumorigenesis. In Part I of this two-part review, we consider findings demonstrating that Schwann cells are the primary neoplastic cell type in neurofibromas and MPNSTs and that specific tumor suppressor gene mutations promote the development of these tumors. In Part II, which will be published in a later issue, we will review evidence indicating that inappropriate growth factor signaling contributes to this process by stimulating the proliferation, survival, and migration of Schwann cells whose regulatory mechanisms have been crippled by a loss of tumor suppressor function.

Key Words: Growth factor; Neurofibromatosis; Schwann cell; Tumorigenesis; Tumor progression; Tumor suppressor.

INTRODUCTION

Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor predisposition syndrome that occurs in approximately 1 in 3,500 newborn infants worldwide (1-3). The manifestations of NF1 are protean and include pigmentary lesions (caf-au-lait macules, axillary freckling, and Lisch nodules), the development of several tumor types (optic gliomas, pheochromocytomas, and juvenile chronic myeloid leukemia), bony dysplasias, and learning disabilities. The hallmark of NF1, however, is the occurrence of multiple neurofibromas. Neurofibromas are benign peripheral nerve sheath tumors (PNSTs) that present as fleshy nodules in skin (dermal neurofibromas), circumscribed masses in nerves (intraneural or nodular neurofibromas), or diffuse lesions spreading through multiple fascicles of large nerves or nerve plexuses (plexiform neurofibromas). These neoplasms, particularly the plexiform variants, can cause significant dysfunction in NF1 patients, producing disfigurement, pain, and neurologic deficits (4). Plexiform neurofibromas may also become quite large, sometimes involving entire body segments or limbs. These large plexiform neurofibromas develop an extensive vascular network and elaborate factors that stimulate the growth of adjacent bone and soft tissue, further impairing the function of the affected limb or body segment.

Plexiform and, less commonly, intraneural neurofibromas may also undergo transformation to malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy developing in NF1 patients. Overall, NF1 patients have a 10% lifetime risk of developing MPNSTs and NF1 patients with symptomatic plexiform neurofibromas may have a risk as high as 30% (5). The prognosis for patients with MPNSTs is poor, with overall 5- and 10-year survival rates of 34% and 23%, respectively (6, 7). Surgery, ideally resulting in complete tumor resection, is the primary treatment for MPNSTs (8). Radiotherapy can help control local disease and delay recurrence, but has little effect on overall survival (5). Chemotherapy has, at best, a modest effect on the survival of patients with MPNSTs (8).

Since neurofibromas and MPNSTs produce considerable morbidity and mortality in NF1 patients, there is great interest in elucidating the molecular mechanisms responsible for the development of these tumors. Over the past two decades, significant advances have been made in our understanding of the role tumor suppressor gene mutations play in the pathogenesis of neurofibromas and MPNSTs. However, it is widely held that tumor suppressor mutations alone are insufficient for the formation of PNSTs and that these mutations must cooperate with dysregulated growth factor signaling to promote the proliferation, survival, and migration of neoplastic Schwann cells. At present, our understanding of the role of dysregulated growth factor signaling in neurofibroma and MPNST pathogenesis is much more limited than our knowledge of the role tumor suppressor mutations play in their development. This is unfortunate, given that growth factor receptor inhibitors such as Herceptin (trastuzumab; an anti-erbB2 antibody useful in treating patients with c-neu overexpressing breast cancers) and Gleevec (STI-571, imatinib; used to treat chronic myeloid leukemia and gastrointestinal stromal tumors) have proven quite effective in treating other tumor types.

Our goals in the first part of this two-pail review are to i) examine evidence that Schwann cells are the primary neoplastic cell type in neurofibromas and MPNSTs and, ii) consider findings in human neoplasms and animal models that indicate that specific tumor suppressor mutations occur in neoplastic Schwann cells and promote neurofibroma and MPNST tumorigenesis. The second part of this review, which will be published in a subsequent issue, will examine evidence implicating specific growth factors and growth factor receptors in the pathogenesis of neurofibromas and MPNSTs, focusing particularly on findings that shed light on the action of each growth factor on neoplastic Schwann cells. We will also suggest some future lines of investigation that will likely be important for the development of new treatments for neurofibromas and MPNSTs.

EVIDENCE THAT SCHWANN CELLS ARE THE PRIMARY NEOPLASTIC CELL TYPE IN NF1-ASSOCIATED PERIPHERAL NERVE SHEATH TUMORS

Neurofibromas are composed of multiple cell types including Schwann cells, mast cells, perineurial cells, fibroblasts, and endothelial cells (see Fig. 1 for a comparison of the histology of normal nerve and the three major tumor types that develop in peripheral nerve). The histogenesis of neurofibromas has long been controversial, in large part because of the significant cellular heterogeneity observed in these neoplasms. However, it is now generally accepted that Schwann cells represent the primary neoplastic cell type in neurofibromas. Forty to 80% of the cells in neurofibromas are Schwann cells (9) and these glia demonstrate several functional abnormalities. Schwann cells isolated from neurofibromas, unlike normal Schwann cells or neurofibroma-derived fibroblasts, are invasive (10, 11) and promote angiogenesis (10). Neurofibroma-derived Schwann cells do not form colonies in soft agar (a measure of tumorigenicity and anchorage-independent growth) or masses when grafted subcutaneously into immunodeficient (nude) mice. Unlike normal Schwann cells, however, Schwann cells isolated from neurofibromas do survive and show infiltrative growth when xenografted into the sciatic nerve of nude mice (12). Cytogenetic abnormalities, when identified, are found in the Schwann cell component of neurofibromas (13). MPNSTs derived from plexiform neurofibromas (as well as sporadically occurring MPNSTs) frequently demonstrate histologic features associated with Schwann cells, including immunoreactivity for the Schwann cell marker S-100β, expression of long-spacing fibrous collagen (Luse bodies), and partial investment of tumor cells by basement membrane (14). Considered together with studies of tumor suppressor gene mutations (see below), these findings argue that Schwann cells are the primary neoplastic cell type in neurofibromas and MPNSTs.

Although the central role Schwann cells play in peripheral nerve sheath tumorigenesis is becoming increasingly clear, the origin of these tumor cells remains poorly understood. Given their morphologic and immunohistochemical features, it is tempting to speculate that these tumor elements are derived from mature Schwann cells or from immature Schwann cell-like elements persistently present in the peripheral nervous system. Alternatively, neoplastic Schwann cells in neurofibromas and MPNSTs may arise from multipotent neural crest stem cells, which have been identified in mammalian fetal peripheral nerve (15); this latter possibility is potentially consistent with the observation that some MPNSTs demonstrate local divergent differentiation (e.g. the presence of a rhabdomyosarcomatous component in the MPNST variant known as a malignant Triton tumor). At present, our ability to distinguish between these possibilities is hampered by our limited understanding of normal Schwann cell development and a paucity of phenotypic markers that can be used to define neurofibroma- and MPNST-associated Schwann cells.

TUMOR SUPPRESSOR MUTATIONS IN PERIPHERAL NERVE SHEATH TUMORS

Mutations of the NF1 Locus in Neoplastic Schwann Cells: A Key Early Step in the Pathogenesis of Neurofibromas and MPNSTs Resulting in Increased Ras Activity

NF1 patients carry a constitutional mutation in the NF1 locus, a tumor suppressor gene that is located on the long ar\m of chromosome 17 (17q11.2). This mutation may be inherited from a parent, but the NF1 locus has a relatively high mutation rate and up to 50% of NF1 cases represent new mutations (4). In keeping with Knudson's "two- hit" hypothesis (9), it is believed that neoplasms develop in NF1 patients when a somatic mutation disrupts the remaining functional copy of the NF1 allele. Consistent with this postulate, loss of heterozygosity (LOH) of the NF1 locus is evident in both neurofibromas and MPNSTs (2, 5, 9, 16-19), occurring in Schwann cells, but not fibroblasts, isolated from these neoplasms (13, 20- 22). Fluorescent in situ hybridisation (FISH) analyses of plexiform neurofibromas and MPNSTs confirm that NF1 deletions are found in S100β-immunoreactive Schwann cells in vivo as well (23). The observation that biallelic inactivation of NF1 occurs in Schwann cells in both neurofibromas and MPNSTs suggests that mutation of the remaining functional copy of the NF1 gene is a key early step in the development of these neoplasms.

Fig. 1. Comparison of the histology of normal human peripheral nerve, schwannomas, neurofibromas, and malignant peripheral nerve sheath tumors (MPNSTs). A: Sensory nerve adjacent to a thoracic dorsal root ganglion showing an orderly arrangement of Schwann cells associated with and myelinating axons. B: Schwannoma arising in a thoracic spinal nerve root from a 22-year-old woman. Shown is an area of densely packed neoplastic Schwann cells (Antoni A pattern); unlike neurofibromas, schwannomas are almost completely composed of Schwann cells and compress, rather than infiltrate, the nerve in which they develop. C: Neurofibroma developing in a lumbar spinal nerve root from a 56-year-old woman with NF1. The infiltrative nature of this neoplasm is demonstrated by the presence of entrapped myelinated axons (indicated by the arrow; note the "neurokeratin" artifact [herringbone pattern] produced by formalin fixation of the myelin sheath). D: MPNST resected from a 56-year-old man with NF1. Note the numerous mitotic figures evident in this tumor (arrows). Scale bar for A-D: 50 m.

NF1 mutations have important functional consequences in Schwann cells. Neurofibroma-derived Schwann cells lacking expression of neurofibromin, the product of the NF1 locus, have a growth advantage over neurofibromin-positive Schwann cells in vitro and in vivo (12). Schwann cells derived from Nf1 null mice (see below) also show increased chemotactic and chemokinetic migration relative to normal Schwann cells (24), consistent with the infiltrative behavior of neurofibromas and MPNSTs. As our understanding of the functions of neurofibromin has grown, the mechanisms underlying these altered Schwann cell responses have become increasingly clear. Neurofibromin is a large (~220 to 250 kD) protein composed of multiple functional domains, including a GTPase activating protein (GAP)-related domain. Neurofibromin is thought to act, at least in part, by negatively regulating the activity of Ras proteins, a family of small G- proteins that act as a key regulators of mitogenesis and other cellular responses (19). Ras proteins are essentially binary switches which get turned "on" when they bind GTP. Neurofibromin turns this switch "off" by using its GAP-related domain to stimulate an intrinsic GTPase activity in Ras proteins, thereby accelerating the cleavage of GTP to GDP. Consistent with this action, loss of NF1 function is associated with increased levels of activated (GTP- associated) Ras proteins in neurofibroma (22) and MPNST (25, 26) cells.

The Increase in Ras Activity Resulting from Neurofibromin Loss Activates Key Signaling Cascades Mediating Mitogenesis, Migration, and Transformation

Three "classical" neurofibromin-regulatable Ras proteins (H-, N- , and K-Ras) are known. All three of these proteins are expressed in wild-type and Nf1 null (-/-) mouse Schwann cells (24) and likely contribute to neoplasia. H-Ras proteins appear to play a particularly important role because farnesyltransferase inhibitors, which inhibit the activation of H-Ras while sparing N- and K-Ras, interfere with the abnormal proliferation of Nf1 -/Schwann cells (27). There is little evidence to indicate that activating Ras mutations occur in neurofibromas and MPNSTs. H-, N-, and K-Ras are instead likely activated when growth factors, such as those that will be discussed in Part II of this review, bind to and activate integral membrane tyrosine kinase receptors expressed by neoplastic Schwann cells. Growth factor-activated Ras proteins in turn stimulate the activation of at least two mitogen-activated protein (MAP) kinase cascades (Fig. 2). The first of these cascades, the Ras- Raf-MEK1/2-ERK1/2 pathway, plays an essential and well-established role in promoting mitogenesis and the transformation of several cell types, including Schwann cells (28). Ras also activates Rac and Cdc42, two members of the Rho family of small G-proteins, leading to the sequential activation of p21-activated kinase 1 (Pak1), MAPK/ ERK kinase kinase (MEKK), stress-activated protein kinase (SEK), and Jun N-terminal kinase (JNK; also known as stress-activated protein kinase or SAPK). JNK regulates the action of Jun and multiple other transcription factors that are essential for the control of mitogenesis and survival in nonneoplastic cells (29). Activation of JNK is required for Ras-induced transformation in several cell types (29) and for in vivo oncogenesis in at least some settings (30); although potentially important in the pathogenesis of neurofibromas and MPNSTs, the precise role JNK plays in these neoplasms remains to be defined.

One protein in the Rac signaling cascade, Pak1, is additionally notable for its ability to interact with other signaling cascades. Pak1 is clearly an essential mediator of Ras action in neoplastic Schwann cells as dominant negative Pak1 mutants block Ras-mediated transformation of T antigen-immortalized rat Schwann cells and inhibit the ability of human ST88-14 MPNST cells to form colonies in soft agar and tumors in nude mice (28). Although Pak1 activates JNK. in these cells, its promotion of Ras-mediated transformation is dependent on "cross-talk" with the Ras-Raf-MEK1/2-ERK1/2 pathway rather than activation of JNK (28); this reinforcement of ERK signaling is thought to promote a persistent activation of ERK that is necessary for transformation. Pak1 also forms part of a "feedforward" signaling loop (Fig. 2) in which merlin, a tumor suppressor produced by the neurofibromatosis type 2 (NF2) locus, inhibits Pak1 action (31), and activated Pak1, in turn, inhibits merlin (32). The regulatory interactions of Pak1 and merlin are likely disrupted in at least a subset of MPNSTs, as deletions of the region of chromosome 22 encoding merlin (band 22q12.2) were detected in 30% (33) and 45% (34) of the MPNSTs studied in two series. Consistent with the hypothesis that NF2 loss contributes to the pathogenesis of human MPNSTs, mice with Schwann cell specific ablation of Nf2 develop MPNSTs at a low frequency (35) and animals lacking both Nf2 and p53 develop multiple MPNST-like neoplasms (36).

In contrast to its effects on proliferation, expression of an H- Ras dominant negative mutant (which inhibits the action of H-, N- and K-Ras) in Nf1 -/- mouse Schwann cells does not reduce the enhanced migration evident in these glia (24). Based on this observation, it was concluded that classical Ras proteins make little contribution to enhanced migration in neoplastic Schwann cells and that other neurofibromin-regulated proteins must mediate these responses. Consistent with this hypothesis, neurofibromin also stimulates the GTPase activity of nonclassical Ras proteins such as R-Ras and TC21/R-Ras2 (37), two small G-proteins that have been implicated in the control of cell motility in other systems (38). Although the contribution R-Ras makes to the increased mobility of Nf1 -/- Schwann cells remains poorly understood, TC21/R-Ras2 is essential for increased migration in Schwann cells lacking neurofibromin, acting through a signaling pathway that includes phosphatidylinositol 3-kinase to mediate this effect (Fig. 2). Nf1 - /- Schwann cells also secrete an as yet unidentified factor that enhances the chemotaxis of neoplastic Schwann cells and do so at levels greater than either wild-type or Nf1 haploinsufficient (+/-) Schwann cells (24). Loss of neurofibromin expression therefore enhances the migration of neoplastic Schwann cells both by activating a TC21/R-Ras2-dependent signaling cascade that promotes migration and by enhancing the secretion of factors that act in an autocrine or paracrine fashion to promote Schwann cell migration.

At present, the functions of other neurofibromin domains are poorly understood. Neurofibromin has been shown to regulate cyclic AMP (cAMP)-dependent signaling pathways (39), but the significance that loss of this activity has for Schwann cell neoplasia is unclear.

Fig. 2. Ras-dependent signaling pathways affected by neurofibromin loss in neoplastic Schwann cells. Growth factors that may activate these signaling pathways include the molecules that will be discussed in Part II of this review. The cross-activation of ERK 1/2 by Pak1 is indicated by a dashed arrow as Pak1 is also capable of phosphorylating Raf and MEK 1/2 and the level at which Pak1 cross-talks with Ras-Raf-MEK-ERK cascade is not yet clear. We would also emphasize that it is likely that other, as yet unidentified, molecules participate in some of these signaling cascades. Abbreviations: Receptor TKs, receptor tyrosine kinuses; PI3K, phosphatidylinositol 3-kinase; MEK, mitogen-activated kinase (MEK)/extracellular signal-regulated kinase (ERK) kinase; MEKK, MAPK/ ERK kinase kinase; SEK, stress-activated protein kinase; JNK, Jun N- terminal kinase.

Mutations of p53 and Cell Cycle Regulatory Pathway Genes Accumulate as Neurofibromas Progress to Become MPNSTs

As NF1 deletio\ns occur in both neurofibromas and MPNSTs, it is evident that NF1 mutations alone are not sufficient for MPNST pathogenesis and that additional mutations are required for progression from neurofibroma to MPNST. In at least some MPNSTs, these additional mutations include loss-of-function mutations of the p53 tumor suppressor, a molecule controlling cell cycle progression and cell death that is mutated in more than half of human cancers (40). Deletions and other mutations of the p53 locus are common in MPNSTs, being found in 29% to 75% of the tumors studied in three series (41-43). Nuclear accumulation of p53 protein, a finding that can be associated with ineffective p53 action occurring secondary to mutations in p53 functional domains (particularly the DNA binding domain), has been identified in MPNSTs in some studies (44-46); others, however, have found a poor correlation between p53 immunoreactivity and mutations of this tumor suppressor (43).

Mutations of genes that regulate cell cycle progression also accumulate in MPNSTs. The INK4A (also known as CDKN2A/p16) gene is located on the short arm of chromosome 9 (9p21), a region that is deleted or otherwise altered in up to 75% of MPNSTs (47, 48). This locus encodes both p16^sup INK4A^, a protein that arrests cells in the G1 phase of the cell cycle by inhibiting cyclin-dependent kinases 4 (CDK4) and 6 (CDK6), and p19^sup ARF^, a polypeptide that binds to Mdm2 and prevents it from degrading p53. Immunoreactivity for p16^sup INK4A^ is evident in virtually all neurofibromas (49). In contrast, the p16^sup INK4A^ antigen is commonly undetectable in MPNSTs and deletions of the INK4A gene were identified in 50% of the MPNSTs studied in two series (49, 50). The expression of p27^sup kipl^, a cyclin-dependent kinase inhibitor that controls G1/S progression, is also altered in MPNSTs. Nuclear p27^sup kipl^ immunorcactivity is prominent in neurofibromas, while this protein accumulates in the cytoplasm of MPNST cells (46). It has been suggested that this alteration results from epigenetic phenomena rather than mutations of the p27^sup kipl^ gene. The Retinoblastoma (Rb) protein, a tumor suppressor that impedes cell cycle progression and becomes inactivated when phosphorylated by CDK4/6 kinases, may contribute to peripheral nerve sheath tumorigenesis in some cases. Rb protein is undetectable in a small subset of MPNSTs, with LOH of the Rb locus evident in some of these same tumors (45). It remains to be determined whether Rb mutations are more common than indicated by initial studies and at what stage they contribute to peripheral nerve sheath tumorigenesis.

Neurofibromas and MPNSTs Developing in Transgenic Mice with Null Mutations of the Nf1 and p53 Tumor Suppressor Genes

Shortly after the identification of the NF1 locus, several laboratories began to develop transgenic mice with null mutations of the murine Nf1 gene for the purpose of gaining further insight into neurofibroma and MPNST pathogenesis. Mice homozygous for a germline Nf1 "knock-out" mutation (Nf1 -/- mice) die in utero secondary to cardiac malformations (double outlet right ventricle and endocardial cushion defects) (51-53) and, in some cases, exencephaly (54). Mice heterozygous for the Nf1 mutation (Nf1 +/- mice) are viable, fertile and, like human NF1 patients, develop pheochromocytomas and myeloid leukemias (52). They do not, however, develop neurofibromas, suggesting that the "second hit" of the Nf1 locus in murine Schwann cells is rate-limiting, occurring infrequently relative to the occurrence of Nf1 mutations in murine adrenal medulla and bone marrow. To overcome this difficulty, Nf1 -/- mouse embryos have been fused with wild-type embryos to produce chimeric mice in which only a portion of their somatic cells are Nf1 -/-. These chimeric mice, unlike animals with germline Nf1 mutations, develop multiple tumors resembling human neurofibromas in association with dorsal root ganglia, nerve trunks within limbs or, less commonly, other sites such as the trigeminal nerve; Nf1 -/- chimeric mice do not develop dermal neurofibromas (55). In tumors arising in chimeric mice, the overwhelming majority of the tumor cells are derived from the Nf1 -/ - embryonic stem cells, indicating that there is minimal recruitment of wild-type cells into these neoplasms.

Although experiments with Nf1 -/- chimeric mice demonstrated that a loss of neurofibromin predisposes mice to the formation of lesions histologically similar to human neurofibromas, they did not establish the identity of the cell type key to tumor initiation. In an elegant set of experiments, Parada et al addressed this issue by producing mice with a Schwann cell-specific ablation of the Nf1 locus (56). Mice with Nf1 -/- Schwann cells developed microscopic hyperplastic lesions in cranial nerves, but not the other histologic features of neurofibromas. However, when mice with Nf1 -/- Schwann cells and Nf1 haploinsufficiency (Nf1 +/-) in all other cell types were produced, these animals developed neurofibroma-like neoplasms within cranial nerves and spinal cord nerve roots composed of mixtures of the same cell types evident in human neurofibromas. These findings indicate that neurofibroma tumorigenesis requires both a complete loss of neurofibromin in Schwann cells and Nf1 haploin-sufficiency in other, as yet unidentified, cellular elements (56). As a corollary, it is evident that interactions between Nf1 -/ - Schwann cells and other Nf1 +/- cell types are required for neurofibroma formation.

In addition to the models described above, two laboratories have generated transgenic mice that carry cis linked null mutations of the Nf1 and p53 loci, rendering them haploinsufficient for both genes (55, 57). In addition to neoplasms previously observed in mice with knockouts of the Nf1 and p53 genes alone (lymphomas, osteosarcomas, and hemangiosarcomas), Nf11p53 haploinsufficient mice develop soft tissue sarcomas, the majority of which have the histologic and immunohistochemical characteristics of human MPNSTs and malignant Triton tumors (MTTs). Consistent with the hypothesis that NF1 and p53 mutations cooperate to promote the progression of neurofibromas to MPNSTs in humans, the MPNST-like tumors developing in mice with cis linked Nf1 and p53 null mutations demonstrate LOH of the remaining functional alleles of the Nf1 and p53 genes. To further investigate the origin of the soft tissue sarcomas developing in Nf1/p53 haploinsufficient mice, Vogel et al established permanent cell lines from approximately 70 of these neoplasms and examined their expression of mRNAs encoding neural crest, Schwann cell, and myogenic markers. These cell lines expressed, to varying degrees, all three sets of markers, leading these investigators to propose that the MPNST- and MTT-like neoplasms developing in mice carrying cis linked Nf1 and p53 null mutations are derived from a multipotent neural crest stem cell (57).

Fig. 3. The development of neurofibromas and their subsequent progression to become malignant peripheral nerve sheath tumors (MPNSTs) is associated with a series of tumor suppressor gene mutations. The process is initiated when a Schwann cell or Schwann cell precursor (NF1 +/- Schwann cell) in an NF1 patient undergoes loss of the remaining functional NF1 allele. A process involving as yet undefined interactions with other haploinsufficient cell types in the nerve then leads to the formation of a neurofibroma. The subsequent loss or abnormal function of other tumor suppressor genes including p53, INK4A, and p27^sup kip^ results in the malignant transformation of NF1 -/- Schwann cells in the neurofibroma and the development of an MPNST.

The transgenic neurofibromas and MPNSTs described above were among a group of genetically engineered murine (GEM) neoplasms whose histologic, immunohistochemical, and ultrastructural features were recently reviewed and compared to their human counterparts by a panel of pathologists with expertise in neuropathology and soft tissue neoplasms (58). Based on these comparisons, a grading scheme was proposed in which GEM neoplasms were classified as Grade I (tumors with low cellularity, bland/uniform cytology, and no mitoses or necrosis), II (tumors with increased cellularity, nuclear atypia and some mitotic activity), or III (tumors with marked cellularity, atypia, high mitotic activity, and areas of hemorrhage or necrosis). This group also specified that GEM PNSTs should be designated, based on their cellular composition, as GEM schwannomas, GEM neurofibromas, GEM perineuriomas, or GEM PNSTs (tumors arising in peripheral nerves with some immunohistochemical or ultrastructural evidence of schwannian differentiation that do not fit into the preceding categories). This group concluded that the pathology of the mouse neurofibromas and MPNSTs described above was highly similar to that of their human counterparts and classified them as GEM Grade I neurofibromas and GEM Grade III PNSTs, respectively; the term "malignant" was not applied to the MPNST-like neoplasms arising in transgenic mice as their clinical behavior has not yet been adequately defined.

SOME UNRESOLVED QUESTIONS AND CONCLUSIONS

Over the last 15 years, a coherent picture of the genetic abnormalities responsible for the pathogenesis of NF1-associated neurofibromas and MPNSTs has begun to emerge. There is now strong evidence that Schwann cells in NF1 patients are the primary neoplastic cell type in neurofibromas. The Schwann cells in these tumors, which are already haploinsufficient for NF1, become neoplastic when they lose the function of their remaining NF1 allele, an event that results in the activation of several Ras- dependent signaling pathways that regulate mitogenesis and migration and promote transformation. These Schwann cell changes are not themselves sufficient for the development of neurofibromas; NF1 -/- Schwann cells must interact with other NF1 +/-cell types (mast cells, perineurial cells, fibroblasts and/or Schwann cells) to form a neurofibroma. Abnormalities of additional tumor suppressor genes accumulate in MPNSTs, some of which (p53, INK4A, p27^sup kipl^) have been identified. Considered together, the studies cited above support a multistage model of tumor progression in which mutation of both Schwann cell NF1 alleles leads to the development of a neurofibroma and the subsequent accumulation of mutations or other abnormalities involving additional tumor suppressor genes (i.e. p53, INK4A, and p27^sup kipl^) is necessary for progression to MPNST (Fig. 3).

Although the evidence cited above is compelling, we must emphasize that this story is incomplete. It is likely that several other tumor suppressors and oncogenes relevant to peripheral nerve sheath tumorigenesis remain to be identified. Cytogenetic analyses of MPNSTs have shown that these neoplasms frequently have near- triploid or hypodiploid karyotypes and show losses (1p, 9p, 11, 12p, 14q, 17q, 18, 22q, X, and Y) and gains (chromosome 7) in several chromosomal regions (47, 59); some of these alterations, as well as other abnormalities, have also been identified using comparative genomic hybridization (60-65). Although some of the regions of chromosomal loss contain known tumor suppressors (e.g. the INK4A locus in 9p, the NF1 gene in 17q, and the NF2 gene in 22q), others do not and at least one chromosomal region, 1p, has been suggested to contain an unidentified tumor suppressor gene that contributes to MPNST pathogenesis (66). In addition, the chromosomal rearrangements, gains, and losses observed in MPNSTs are highly variable and differ between individual tumors (13), suggesting that there may be more than one pathway that can lead to the development and progression of these neoplasms. In support of this latter hypothesis, some human MPNST cell lines have been found to express functional p53 (67); a subset of these lines express activated Notch receptors, suggesting the existence of an alternative, Notch- dependent, pathway for progression from neurofibroma to MPNST. Identifying other oncogenic molecules and tumor suppressors contributing to peripheral nerve sheath pathogenesis and deciphering alternative neoplastic pathways will be highly important if new, more effective therapies for neurofibromas and MPNSTs are to be developed.

ACKNOWLEDGMENTS

We thank Drs. Kevin A. Roth (Dept. of Pathology, UAB), Bruce R. Korf (Dept. of Genetics, UAB) and Robert E. Schmidt (Dept. of Pathology, Washington University School of Medicine) for helpful comments on this manuscript.

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STEVEN L. CARROLL, MD, PHD AND MARK S. STONECYPHER, BS

From Division of Neuropathology, Department of Pathology, The University of Alabama School of Medicine, Birmingham, Alabama.

Correspondence to: Steven L. Carroll, MD, PhD, The University of Alabama at Birmingham, Division of Neuropathology, Department of Pathology, 1720 Seventh Avenue South, SC843, Birmingham, AL 35294- 0017. E-mail: carroll@path.uab.edu

Supported by National Institute of Neurological Disorders and Stroke Grant R01 NS048353.

Copyright American Association of Neuropathologists, Inc. Nov 2004

Source: Journal of Neuropathology and Experimental Neurology

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