Universal absence of merlin, but not other ERM family members, in schwannomas.

NF2 (neurofibromatosis 2, encoding the merlin protein) gene mutations and chromosome 22q loss have been demonstrated in the majority of sporadic and NF2-associated schwannomas, but many schwannomas fail to demonstrate genetic evidence of biallelic NF2 gene inactivation. In addition, the role of the merlin-related ERM family members (ezrin, radixin, and moesin) remains unclear in these tumors. We therefore studied expression of NF2-encoded merlin as well as ezrin, radixin, and moesin in 22 vestibular and peripheral schwannomas that had been evaluated for NF2 mutations and chromosome 22q loss. Western blotting and immunohistochemistry with antibodies directed against the amino and carboxy termini of merlin demonstrated loss of merlin expression in all studied schwannomas, including 12 tumors lacking genetic evidence of biallelic NF2 gene inactivation. Western blotting with antibodies directed against ezrin, radixin, and moesin, however, showed expression of these proteins in all schwannomas. In addition, immunohistochemistry with an antibody to moesin revealed widespread expression in tumor and endothelial cells. These data indicate that the specific loss of merlin is universal to schwannomas and is not linked to loss of ezrin, radixin, or moesin expression.

Gene therapy for metastatic brain tumors by vaccination with granulocyte-macrophage colony-stimulating factor-transduced tumor cells.

We have developed an ex vivo gene therapy paradigm for the treatment of brain tumors using granulocyte-macrophage colony-stimulating factor (GM-CSF). Murine B16 melanoma cells were infected with MFG recombinant retrovirus containing the mouse GM-CSF cDNA. Subcutaneous vaccination of syngeneic mice with irradiated GM-CSF-secreting B16 melanoma cells was capable of completely protecting animals against subsequent intracranial B16 tumor inoculation, with up to 5 x 10(3) cells. Histologic evaluation revealed the presence of neutrophils, eosinophils, and lymphocytes, including CD4+, CD8+, and CD45R+ cells, in the intracerebral inoculation site, peaking 4 days after intracranial inoculation. In contrast, nonvaccinated animals or animals vaccinated with irradiated, nontransduced B16 cells succumbed to intracranial tumor within 3 weeks after inoculation. Treatment of established intracranial B16 melanoma tumors with subcutaneous injection of irradiated GM-CSF-secreting B16 cells significantly delayed death, as compared to injection of irradiated nontransduced B16 cells or no treatment. In addition, treatment of established intracerebral GL261 gliomas by vaccination with irradiated GM-CSF-secreting B16 cells mixed with irradiated, transduced, or nontransduced GL261 cells also extended survival. These B16/GL261 co-vaccinations also improved outcome and, in some cases, induced immunological memory that protected survivors from subsequent intracranial challenge with GL261 tumor cells. These findings indicate that peripheral vaccination with irradiated tumor cells in the presence of GM-CSF-producing cells can initiate a potent antitumor immune response against intracranial neoplasms.

Expression of NF2-encoded merlin and related ERM family proteins in the human central nervous system.

Germline mutations of the neurofibromatosis 2 (NF2) gene are associated with an increased incidence of gliomas and glial harmartomas, suggesting a role for the NF2-encoded protein, merlin, in glial growth control. Using monoclonal and polyclonal anti-merlin antibodies for Western blotting and immunohistochemistry, we evaluated the cellular pattern of merlin expression in the normal human central nervous system (CNS), reactive gliosis; and NF2-associated glial hamartomas. In the normal CNS, merlin is widely expressed in coarse cytoplasmic granules in both glia and neurons, with less pronounced expression in other cells. Merlin is also expressed in reactive astrocytes and in the astrocytes of NF2-associated glial hamartomas. In reactive astrocytes, however, merlin is also present at the cell membrane and in cellular processes, suggesting redistribution of the protein in activated cells. Merlin is structurally related to ezrin, radixin and moesin, which are also expressed in the CNS, as demonstrated by Western blotting. The pattern of merlin expression, however, is distinct from that of ezrin, which has been previously described, and that of moesin, in which immunohistochemistry with an anti-moesin antibody showed expression in endothelial cells, glia and neurons in a membranous or diffuse cytoplasmic pattern. These findings imply that merlin has widespread and specific functions in the human central nervous system.

Refined deletion mapping of the chromosome 19q glioma tumor suppressor gene to the D19S412-STD interval.

Allelic loss of chromsome 19q occurs frequently in malignant gliomas, suggesting the presence of a chromosome 19q glioma tumor suppressor gene. Deletion mapping studies have delineated a 3.5 Mb candidate region between D19S219 and HRC. Cloned sequences from the proximal 425 kb of this interval, however, have not shown tumor-specific alterations. To refine the location of the tumor suppressor gene further, we conducted loss of heterozygosity studies on 191 malignant gliomas using nine PCR-based polymorphisms. These included the previously identified and physically mapped markers D19S219, DM, D19S112, HRC and the recently physically mapped polymorphisms at D19S412, STD, D19S596 and GYS. In addition, we isolated a novel microsatellite polymorphism that maps 400 kb telomeric to D19S112. Oligodendroglial tumors showed frequent loss of heterozygosity in all grades, and typically displayed allelic loss at all studied markers. Astrocytomas, however, showed frequent loss primarily in anaplastic astrocytomas and displayed deletion breakpoints within the candidate region. Deletion mapping revealed a minimal region of overlap between D19S412 and STD, a distance of 900 kb. These data suggest that the D19S412-STD interval represents the most likely location for a chromsome 19q glioma tumor suppressor gene involved in astrocytoma, and perhaps oligodendroglioma, tumorigenesis.