Cutting Edge: Codeletion of the Ras GTPase-Activating Proteins (RasGAPs) Neurofibromin 1 and p120 RasGAP in T Cells Results in the Development of T Cell Acute Lymphoblastic Leukemia.

Ras GTPase-activating proteins (RasGAPs) inhibit signal transduction initiated through the Ras small GTP-binding protein. However, which members of the RasGAP family act as negative regulators of T cell responses is not completely understood. In this study, we investigated potential roles for the RasGAPs RASA1 and neurofibromin 1 (NF1) in T cells through the generation and analysis of T cell-specific RASA1 and NF1 double-deficient mice. In contrast to mice lacking either RasGAP alone in T cells, double-deficient mice developed T cell acute lymphoblastic leukemia/lymphoma, which originated at an early point in T cell development and was dependent on activating mutations in the Notch1 gene. These findings highlight RASA1 and NF1 as cotumor suppressors in the T cell lineage.

Antibodies to neurofascin exacerbate adoptive transfer experimental autoimmune neuritis.

Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy are autoimmune disorders of the peripheral nervous system in which autoantibodies are implicated in the disease pathogenesis. Recent work has focused on the nodal regions of the myelinated axon as potential autoantibody targets. Here we screened patient sera for autoantibodies to neurofascin and assessed the pathophysiological relevance of anti-neurofascin antibodies in vivo. Levels of anti-neurofascin antibodies were higher in sera from patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy when compared with those of controls. Anti-neurofascin antibodies exacerbated and prolonged adoptive transfer experimental autoimmune neuritis and caused conduction defects when injected intraneurally.

Neurofibromin specific antibody differentiates malignant peripheral nerve sheath tumors (MPNST) from other spindle cell neoplasms.

Malignant peripheral nerve sheath tumors (MPNST) derive from the Schwann cell or perineurial cell lineage and occur either sporadically or in association with the tumor syndrome neurofibromatosis type 1 (NF1). MPNST often pose a diagnostic challenge due to their frequent lack of pathognomonic morphological or immunohistochemical features. Mutations in the NF1 tumor suppressor gene are found in all NF1-associated and many sporadic MPNST. The presence of NF1 mutation may have the potential to differentiate MPNST from several morphologically similar neoplasms; however, mutation detection is hampered by the size of the gene and the lack of mutational hot spots. Here we describe a newly developed monoclonal antibody binding to the C-terminus of neurofibromin (clone NFC) which was selected for optimal performance in routinely processed formalin-fixed and paraffin-embedded tissue. NFC immunohistochemistry revealed loss of neurofibromin in 22/25 (88 %) of NF1-associated and 26/61 (43 %) of sporadic MPNST. There was a strong association of neurofibromin loss with deletions affecting the NF1 gene (P < 0.01). In a series of 256 soft tissue tumors of different histotypes NFC staining showed loss of neurofibromin in 2/8 myxofibrosarcomas, 2/12 (16 %) pleomorphic liposarcomas, 1/16 (6 %) leiomyosarcomas, and 4/28 (14 %) unclassified undifferentiated pleomorphic sarcomas. However, loss of neurofibromin was not observed in 22 synovial sarcomas, 27 schwannomas, 23 solitary fibrous tumors, 14 low-grade fibromyxoid sarcomas, 50 dedifferentiated liposarcomas, 27 myxoid liposarcomas, 13 angiosarcomas, 9 extraskeletal myxoid chondrosarcomas, and 7 epitheloid sarcomas. Immunohistochemistry using antibody NFC may substantially facilitate sarcoma research and diagnostics.

K-ras is critical for modulating multiple c-kit-mediated cellular functions in wild-type and Nf1+/- mast cells.

p21(ras) (Ras) proteins and GTPase-activating proteins (GAPs) tightly modulate extracellular growth factor signals and control multiple cellular functions. The specific function of each Ras isoform (H, N, and K) in regulating distinct effector pathways, and the role of each GAP in negatively modulating the activity of each Ras isoform in myeloid cells and, particularly, mast cells is incompletely understood. In this study, we use murine models of K-ras- and Nf1-deficient mice to examine the role of K-ras in modulating mast cell functions and to identify the role of neurofibromin as a GAP for K-ras in this lineage. We find that K-ras is required for c-kit-mediated mast cell proliferation, survival, migration, and degranulation in vitro and in vivo. Furthermore, the hyperactivation of these cellular functions in Nf1(+/-) mast cells is decreased in a K-ras gene dose-dependent fashion in cells containing mutations in both loci. These findings identify K-ras as a key effector in multiple mast cell functions and identify neurofibromin as a GAP for K-ras in mast cells.

Molecular analysis of astrocytomas presenting after age 10 in individuals with NF1.

BACKGROUND: Fifteen to 20% of children with neurofibromatosis type 1 (NF1) develop low-grade astrocytomas. Although brain tumors are less common in teenagers and adults with NF1, recent studies have suggested that patients with NF1 are at a significantly increased risk of developing astrocytomas. OBJECTIVE: S: To investigate the genetic basis for astrocytoma development in patients with NF1 beyond the first decade of life. METHODS: The authors performed molecular genetic analyses of 10 NF1-associated astrocytomas representing all World Health Organization (WHO) malignancy grades using fluorescence in situ hybridization, loss of heterozygosity, immunohistochemistry, and direct sequencing. RESULTS: Later-onset NF1-associated astrocytomas, unlike histologically identical sporadic astrocytomas, exhibit NF1 inactivation, supporting a direct association with NF1 rather than a chance occurrence. Furthermore, some of these astrocytomas have homozygous NF1 deletion. In addition, genetic changes observed in high-grade sporadic astrocytomas, including TP53 mutation and CDKN2A/p16 deletion, are also seen in NF1-associated high-grade astrocytomas. CONCLUSIONS: Neurofibromatosis type 1-associated astrocytomas occurring in patients older than 10 years exhibit genetic changes observed in sporadic high-grade astrocytomas. Patients with neurofibromatosis type 1 and germline NF1 deletions may be at risk for developing late-onset astrocytomas.

Lymphoproliferative defects in mice lacking the expression of neurofibromin: functional and biochemical consequences of Nf1 deficiency in T-cell development and function.

Ras plays an essential role in lymphocyte development and function. However, in vivo consequence(s) of regulation of Ras activity by guanosine triphosphatase (GTPase)-activating proteins (GAPs) on lymphocyte development and function are not known. In this study we demonstrate that neurofibromin, the protein encoded by the NF1 tumor suppressor gene functions as a GAP for Ras in T cells. Loss of Nf1 in T cells results in enhanced Ras activation, which is associated with thymic and splenic hyperplasia, and an increase in the absolute number of immature and mature T-cell subsets compared with control mice. Interestingly, in spite of a profound T-cell expansion and higher thymidine incorporation in unstimulated Nf1-deficient T cells, T-cell receptor and interleukin-2 receptor-mediated proliferation of thymocytes and mature T cells was substantially reduced compared with control mice. Collectively, these results identify neurofibromin as a GAP for Ras in T cells for maintaining immune homeostasis in vivo.

Immunoexpression of neurofibromin, S-100 protein, and leu-7 and mutation analysis of the NF1 gene at codon 1423 in osteofibrous dysplasia.

The NF1 (neurofibromatosis type 1, or von Recklinghausen disease) gene, is a tumor-suppressor gene, and its product, neurofibromin, down-regulates ras protein by its guanosine triphosphatase-activating protein (GAP)-related domain. Osteofibrous dysplasia (OFD) is characterized by fibroblast-like spindle cells and osseous tissue and is generally seen in the tibia or fibula during childhood. The precise nature of OFD remains controversial. Cosegregations of OFD and NF1 have been reported, and it has been surmised that OFD is associated with the NF1 gene. We studied the expressions of NF1 gene product (neurofibromin) and so-called Schwann cell markers (S-100 protein, Leu-7) in 17 cases of OFD immunohistochemically. Ten cases of fibrous dysplasia (FD) were also used for the purpose of comparison. Five OFD and 7 FD cases were analyzed for NF1 gene mutation at codon 1423, which is a GAP-related domain, by single-strand conformation polymorphism. Fibroblast-like cells of OFD showed the expression of neurofibromin (5 of 17), S-100 protein (9 of 17), and Leu-7 (5 of 17), and those of FD did not show these expressions, with the exception of 1 case that showed Leu-7 expression. Regarding the OFD cases, significant correspondence was found between cases showing expression of neurofibromin and S-100 protein, between cases showing expression of neurofibromin and Leu-7, and between cases showing expression of S-100 protein and Leu-7 (P < .01). NF1 gene mutation at codon 1423 was not detected in either the OFD (0 of 5) or FD (0 of 7) cases. These results seem to suggest the possible involvement of neurofibromin in the development of OFD, which is associated with the expression of Schwann cell markers (S-100 protein and Leu-7). Furthermore, NF1 gene mutation at codon 1423 did not seem to be related to OFD.