Neuronal differentiation associated with Gli3 expression predicts favorable outcome for patients with medulloblastoma.

Medulloblastoma (MB) is a malignant cerebellar tumor arising in children, and its ontogenesis is regulated by Sonic Hedgehog (Shh) signaling. No data are available regarding the correlation between expression of Gli3, a protein lying downstream of Shh, and neuronal differentiation of MB cells, or the prognostic significance of these features. We re-evaluated the histopathological features of surgical specimens of MB taken from 32 patients, and defined 15 of them as MB with neuronal differentiation (ND), three as MB with both glial and neuronal differentiation (GD), and 14 as differentiation-free (DF) MB. Gli3-immunoreactivity (IR) was evident as a clear circular stain outlining the nuclei of the tumor cells. The difference in the frequency of IR between the ND+GD (94.4%) and DF (0%) groups was significant (P < 0.001). The tumor cells with ND showed IR for both Gli3 and neuronal nuclei. Ultrastructurally, Gli3-IR was observed at the nuclear membrane. The overall survival and event-free survival rates of the patients in the ND group were significantly higher than those in the other groups. The expression profile of Gli3 is of considerable significance, and the association of ND with this feature may be prognostically favorable in patients with MB.

Analysis of nuclear nestin localization in cell lines derived from neurogenic tumors.

Nestin is a class VI intermediate filament protein expressed in the cytoplasm of stem and progenitor cells in the mammalian CNS during development. In adults, nestin is present only in a small subset of cells and tissues, including the subventricular zone of the adult mammalian brain, where neurogenesis occurs. Nestin expression has also been detected under such pathological conditions as ischemia, inflammation, and brain injury, as well as in various types of human solid tumors and their corresponding cell lines. Furthermore, nestin was recently found in the nuclei of glioblastoma, neuroblastoma, and angiosarcoma cells and it was proved to interact directly with the nuclear DNA in neuroblastoma cells. Here, we perform the first study of the intracellular distribution of nestin in cell lines derived from neurogenic tumors. Using immunodetection methods, we examined nestin expression in tumor-derived cell lines obtained from 11 patients with neuroblastoma, medulloblastoma, or glioblastoma multiforme. Besides its standard cytoplasmic localization, nestin was present in the nuclei of two neuroblastoma cell lines and one medulloblastoma cell line. Nestin was only present in the nuclei of cells with diffuse cytoplasmic staining for this protein, and the proportion of cells positive for nestin in nuclei, as well as the intensity of staining, varied. The presence of nestin in the nuclei was confirmed by both transmission electron microscopy and Western blotting. Our results indicate that the presence of nestin in the nuclei of tumor cells is not very rare, especially under in vitro conditions.

Nerve growth factor activation of the TrkA receptor induces cell death, by macropinocytosis, in medulloblastoma Daoy cells.

Ectopic expression of the TrkA receptor tyrosine kinase in tumors of the nervous system can mediate nerve growth factor (NGF)-dependent cell death by apoptosis and /or autophagy. Herein, we demonstrate that TrkA can also induce cell death in medulloblastoma Daoy cells by a caspase-independent mechanism that involves the hyperstimulation of macropinocytosis. Specifically, NGF-stimulates the uptake of AlexaFluor546-dextran into lysosome-associated membrane protein-1 positive vacuoles which fuse with microtubule associated protein light chain 3 (LC3) positive autophagosomes, to form large intracellular vacuoles (> 1 mum), which then fuse with lysotracker positive lysosomes. While LC3 cleavage and the appearance of LC3 positive vacuoles suggest the induction of autophagy, siRNA reduced expression of four proteins essential to autophagy (beclin-1, Atg5, LC3 and Atg9) neither blocks NGF-induced vacuole formation nor cell death. TrkA activated cell death does not require p38, JNK or Erk1/2 kinases but does require activation of class III PI-3 kinase and is blocked by the casein kinase 1 (CK1) inhibitor, D4476. This inhibitor does not interfere with TrkA activation but does block NGF-dependent AlexaFluor546-dextran uptake and CK1 dependent phosphorylation of beta-catenin. Collectively, these data demonstrate that TrkA stimulates cell death by a novel mechanism involving CK1-dependent hyperstimulation of macropinocytosis.

Medulloblastoma demonstrating multipotent differentiation: case report.

We report a 6-year-old boy who presented with a medulloblastoma demonstrating classic, myoblastic, neuronal, glial, and melanotic differentiation and manifesting as severe morning headache. Magnetic resonance imaging revealed a mass lesion with cystic components in the cerebellar vermis. He underwent suboccipital craniotomy and total resection of the tumor. The specimen consisted of three morphologically distinct components. The first component consisted of densely packed cells with round-to-oval highly hyperchromatic nuclei surrounded by scanty cytoplasm. Immunohistochemical staining revealed diffuse expression of neurofilament protein and focal expression of desmin and myoglobin. The second component consisted of long spindle-shaped cells with elongated nuclei and eosinophilic cytoplasm. Immunohistochemical staining revealed diffuse expression of neurofilament protein, desmin, and myoglobin. The third component consisted of cells with small, densely hyperchromatic nuclei and scanty cytoplasm in a fine fibrillary background. Mature ganglion cells and melanotic tumor cells were also observed. Immunohistochemical staining revealed diffuse expression of synaptophysin and neurofilament protein, and focal expression of glial fibrillary acidic protein, S-100 protein, desmin, and myoglobin. The diagnosis was medulloblastoma with myoblastic, neuronal, astrocytic, and melanotic differentiation. Medulloblastoma demonstrating multipotent differentiation is rare, but the features observed in this case support the idea that medulloblastoma originates from multipotent stem cells.

Evaluation of poly (glycerol-adipate) nanoparticle uptake in an in vitro 3-D brain tumor co-culture model.

Despite the inherent problems associated with in vivo animal models of tumor growth and metastases, many of the current in vitro brain tumor models also do not accurately mimic tumor-host brain interactions. Therefore, there is a need to develop such co-culture models to study tumor biology and, importantly, the efficacy of drug delivery systems targeting the brain. So far, few investigations of this nature have been published. In this paper we describe the development of a new model system and its application to drug delivery assessment. For our new model, a co-culture of DAOY cell brain tumor aggregates and organo-typic brain slices was developed. Initially, the DAOY aggregates attached to cerebellum slices and invaded as a unit. Single cells in the periphery of the aggregate detached from the DAOY aggregates and gradually replaced normal brain cells. This invasive behavior of DAOY cells toward organotypic cerebellum slices shows a similar pattern to that seen in vivo. After validation of the co-culture model using transmission electron microscopy, nanoparticle (NP) uptake was then evaluated. Confocal micrographs illustrated that DAOY cells in this co-culture model took up most of the NPs, but few NPs were distributed into brain cells. This finding corresponded with results of NP uptake in DAOY and brain aggregates reported elsewhere.

Intracellular distribution of beta-catenin in human medulloblastoma cell lines with different degree of neuronal differentiation.

Gene mutations impairing the functions of the WNT signaling transduction pathway have been found in approximately 15% of human sporadic medulloblastomas. To understand the functional role of the WNT pathway in medulloblastoma, we have investigated the intracellular distribution of beta-catenin in a series of 17 human medulloblastomas to correlate such expression with neuronal differentiation and in cultured cell models following functional silencing of the APC gene by small-interference RNA (siRNA). Transient siRNA transfection resulted in a 50% reduction of the APC gene product levels in both DAOY and D283MED cell lines. In the former, less-differentiated cell line, beta-catenin levels remained unchanged or were slightly reduced, but beta-catenin translocated in the nucleus following APC gene siRNA silencing. In contrast, in the more differentiated D283MED cells, beta-catenin levels increased about twofold while mainly maintaining the cytoplasmic and cell membrane localization. Cytoplasmic/nuclear localization of beta-catenin was present in 12 of 17 cases of medulloblastoma with a prevalent distribution in the classic, 6/7 cases, and large cell/anaplastic variant, 4/4 cases. The nodular/desmoplastic lesions showed strongly positivity in the cell membrane mainly of intranodular cells with advanced neuronal differentiation. These observations support an important functional role of WNT/beta-catenin pathway in neuronal differentiation in medulloblastoma.

Morphophenotype of medulloblastoma in children and adults. The size of nuclei.

OBJECTIVE: Uniform cells with round, regular nuclei characterize the typical histologic aspect of medulloblastoma. Enlargement of nuclei distinguishes the large-cell medulloblastoma variant and is associated with a poor prognosis in pediatric medulloblastomas. The aim of the present study was to compare the size of nuclei between pediatric and adult medulloblastomas by a morphometric analysis. MATERIAL AND METHODS: In 79 neurosurgical specimens of cerebellar medulloblastomas, the maximum nuclear diameter of the largest nuclei was measured. Measurements were performed with a digital-image analysis system. The measure of the maximum diameter was chosen in order to reduce the split cell error. RESULTS: The difference between the mean values in children and adults was statistically significant (p = 0,001). The distribution of maximum values measured in each case had two distinct peaks in the two age groups, in 3.5% of adult cases and in more than 30% of pediatric cases the maximum nuclear size was superior to 12 microm. CONCLUSIONS: The present results show that nuclei of tumor cells in pediatric medulloblastomas are larger than those in adult medulloblastomas and confirm that the phenotype of medulloblastoma is different in the two age groups. Distinct genetic events can, thus, underlie medulloblastoma in childhood and adult age, the prognostic role of genetic variables can differ by age.

Differentiation of classic medulloblastoma into metastatic large cell medulloblastoma with focal rhabdoid differentiation in the absence of posterior fossa recurrence.

A case of classic medulloblastoma that metastasized, despite the absence of local recurrence, to extraneural sites 7 years after treatment is reported. The metastases were, in contrast to the primary tumor, of large cell type and displayed abortive myogenic and, in one site, also rhabdoid differentiation. The primary tumor expressed microtubule-associated protein 1B and neuron-specific nuclear protein (NeuN), and was desmin negative. The metastases were also positive for microtubule-associated protein 1B and NeuN, although the expression of the latter marker was weak and/or focal in two of four metastases and absent in the rhabdoid metastasis. They were, in contrast with the primary tumor, all strongly positive for desmin. The hSNF5/INI1 was expressed in the nuclei of all cells of the primary tumor and the metastases, including the one with rhabdoid differentiation. Two metastases were studied by cytogenetics. The composite karyotype of a large cell metastasis was 45~46,XY,add(1)(p36.1),t(2;8)(p21;q24.1),add(3)(q25),t(9;15)(q22;q13),add(12)(p11.2), +1approximately2mar,inc[cp12]/46,XY[12], while the rhabdoid metastasis contained additional changes including monosomy 22. These findings indicate that some rhabdoid (atypical teratoid/rhabdoid) tumors of the cerebellum and medulloblastoma may be histogenetically related.

Wet SEM: a novel method for rapid diagnosis of brain tumors.

The authors present the application of wet SEM for histopathological assessment, a technology for imaging fully hydrated samples at atmospheric pressure in a scanning electron microscope (SEM). Both transmission and scanning electron microscopy techniques usually require long and complex sample preparation of the tissues. In marked contrast, a rapid preparation of tissues is described for evaluation by SEM imaging. The wet SEM technology successfully demonstrated both histological and ultrastructural features of several CNS tumors: Rosette formation and intracytoplasmic lumens were observed in ependymoma; numerous fibrillary processes in fibrillary astrocytoma; and focal rosette formation with no intracytoplasmic lumens in medulloblastoma. Application of this method simultaneously with frozen section may improve rapid intraoperative diagnosis of these intracranial tumors.

Testing the "Go or Grow" hypothesis in human medulloblastoma cell lines in two and three dimensions.

OBJECTIVE: The "Go or Grow" hypothesis proposes that cell division and cell migration are temporally exclusive events and that tumor cells defer cell division to migrate. The purpose of this study was to assess the Go or Grow hypothesis using medulloblastoma cell lines in directional migration and invasion assays in monolayer and three-dimensional cultures. METHODS: Time-lapse videomicroscopy was used to continually monitor the directional migration, invasion, and mitosis of individual cells. The mitotic activity observed by time-lapse videomicroscopy was compared with staining for the proliferating cell nuclear antigen Ki-67. RESULTS: A positive correlation exists between the migratory/invasive and mitotic activities of the four medulloblastoma cell lines studied. Within individual cell lines, however, migration and invasion distances are not influenced by the number of cell divisions. Time-lapse videomicroscopy and Ki-67 staining revealed similar trends in mitotic activity between migrating and nonmigrating cells within cell lines. Analysis of cell velocities before, after, and between cell divisions revealed an increase in cell velocity after cell divisions. CONCLUSION: In the models studied, four medulloblastoma cell lines do not defer cell proliferation for migration across an uncoated surface or invasion of a Type I collagen matrix, contrary to the Go or Grow hypothesis. Migrating and invading cells continue to proliferate and migrate/invade a cell line-dependent distance irrespective of the number of divisions that take place. These findings emphasize the need to evaluate the effect of future therapies on both biological events and, if possible, to identify intracellular signaling proteins that negatively regulate medulloblastoma migration/invasion and proliferation.

Mouse embryos cloned from brain tumors.

Cancer cells escape from growth control by accumulating genetic and epigenetic alterations. In rare instances, epigenetic changes alone are oncogenic. Furthermore, agents that modify DNA methylation or chromatin structure can restore a normal phenotype to cells harboring oncogenic mutations. However, it is unclear to what extent epigenetic reprogramming can reverse oncogenesis. Using somatic nuclear transfer, we show that medulloblastomas arising in Ptc1+/- mice can direct preimplantation development. Additionally, blastocysts derived from medulloblastoma nuclei form postimplantation embryos with typical cell layers. Thus, tumor cells can be epigenetically reprogrammed into normal cell types. This approach could lead to a general strategy for assessing genetic and epigenetic contributions to tumorigenesis.

Ultrastructural spectrum of medulloblastoma with immunocytochemical correlations.

Electron microscopy was used to examine 72 cases of medulloblastoma to better characterize the ultrastructural spectrum of this tumor. Twenty-four cases showed prominent neural differentiation. Twenty-three cases showed minimal (21) or no (2) recognizable neural differentiation, and the remainder of the cases (25) showed intermediate differentiation. All 42 cases tested stained for neuron-specific enolase, 28 for synaptophysin, and 12 for neurofilament protein. All cases showed strong reactivity for glial fibrillary acidic protein (GFAP) within reactive astrocytes. Three cases showed reactivity for GFAP within tumor cells. Medulloblastoma exhibits a broad spectrum of neural differentiation, with nearly all cases showing at least some degree of this change, and it universally exhibits participation of reactive astrocytes which can create a potential for diagnostic confusion.

Ultrastructural changes in medullomyoblastoma. Similarities with foetal rhabdomyoma.

The light and electronmicroscopic changes are described in two cases of medullomyoblastoma, and compared with the changes seen in a case of foetal rhabdomyoma. The medullomyoblastomas in two children aged 8 and 5 years, consisted predominantly of classical type of medulloblastoma cells, along with few to many 'strap cells' or 'myoid cells' which, on closer examination, showed clear cross striations, consistent with muscle fibres or myofibrils. The primitive myoid cells were similar to those encountered in larger numbers in a post-auricular rhabdomyoma, possibly of foetal origin in a 40 day old infant. The four pathogenetic mechanisms i.e. (i) an embryonal stage of myofibrillar differentiation; (ii) a malformative factor; (iii) a teratoid factor on account of the presence of mesenchyme derived striated muscle tissue in the obviously predominant ectodermal medulloblastoma; and (iv) metaplasia of the vascular smooth muscle cells in the medullomyoblastoma, are discussed.

Activation of neurotrophin-3 receptor TrkC induces apoptosis in medulloblastomas.

Elevated expression of the neurotrophin-3 (NT-3) receptor TrkC by childhood medulloblastomas is associated with favorable clinical outcome. Here, we provide evidence that TrkC is more than simply a passive marker of prognosis. We demonstrate that: (a) medulloblastomas undergo apoptosis in vitro when grown in the presence of NT-3; (b) overexpression of TrkC inhibits the growth of intracerebral xenografts of a medulloblastoma cell line in nude mice; and (c) trkC expression by individual tumor cells is highly correlated with apoptosis within primary medulloblastoma biopsy specimens. TrkC-mediated NT-3 signaling promotes apoptosis by activating multiple parallel signaling pathways and by inducing immediate-early gene expression of both c-jun and c-fos. Considered collectively, these results support the conclusion that the biological actions of TrkC activation affect medulloblastoma outcome by inhibiting tumor growth through the promotion of apoptosis.

Medullomyoblastoma: a histological, immunohistochemical, ultrastructural and molecular genetic study.

Medullomyoblastoma is a rare variant of medulloblastoma containing myoblastic elements. A 9-year-old boy developed a cerebellar syndrome and signs of increased intracranial pressure, the cause of which was a tumor of the cerebellar vermis measuring 7 x 4.5 x 4.5 cm. Morphologically the tumor largely consisted of a medulloblastoma component but displayed glial, myoblastic and ganglionic differentiation on light microscopic, immunohistochemical and ultrastructural examination. The non-enhancing rim of the tumor on magnetic resonance imaging showed extensive ganglionic differentiation. The tumor did not express bcl-2, c-myc, or c-erb-B2 oncoproteins and was negative for the p53 gene product. On molecular genetic studies, the tumor did not show allelic loss on chromosome loci, frequently altered in medulloblastomas, such as 17p, 1q and 9q.

Effects of basic fibroblast growth factor on the differentiation, growth, and viability of a new human medulloblastoma cell line (UM-MB1).

We presently report the effects of human recombinant basic fibroblast growth factor (bFGF) on a newly established medulloblastoma cell line, UM-MB1. This predominantly adherent cell line has a mean doubling time of 39.3 hours and was found, by karyotypic analysis, to be near triploid. UM-MB1 consists of undifferentiated cells expressing markers of neuronal lineage such as the three neurofilament subunits as well as neuron-specific enolase, synaptophysin, and microtubule-associated proteins 1 and 5. In contrast, no immunoreactivity for glial fibrillary acidic protein was evident. When exposed to nanomolar amounts of bFGF, UM-MB1 cells began to extend neurite-like processes with arborizations and growth-cone-like structures. In addition, UM-MB1 cells treated with bFGF exhibited ultrastructural alterations that reflect their enhanced differentiation as well as the increased expression of at least one of the neurofilament subunits as evidenced both immunocytochemically and on Western blots. Furthermore, bFGF significantly decreased UM-MB1 cell growth as well as induced their death. UM-MB1 cells treated with bFGF for several days displayed DNA cleavage, nuclear shrinkage, and chromatin condensation while retaining their cytoplasmic and mitochondrial membrane integrity, all early indices of apoptosis. After this, cell death was evident with the concomitant appearance of the classical apoptotic bodies. By flow cytometry, bFGF was found to increase the proportion of cells in G1 before inducing their death by apoptosis. In conclusion, UM-MB1 cells can, when appropriately stimulated, be induced to differentiate along their neuronal lineage pathway. Their differentiation induced by bFGF, although incomplete, appears to promote or inhibit the expression of apoptotic effectors or suppressors in these cells, respectively, so to induce their death by an apoptotic-like mechanism.

NeuN: a useful neuronal marker for diagnostic histopathology.

The monoclonal antibody A60 specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates. In this study we demonstrate the potential use of NeuN as a diagnostic neuronal marker using a wide range of formalin-fixed, paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system. After microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya, and some proximal neuronal processes, whereas more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina, and in sympathetic chain ganglia. We examined nine gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma, and one dysplastic cerebellar gangliocytoma. The neuronal component of all of these lesions showed marked immunoreactivity for NeuN. In addition, NeuN immunoreactivity was focally seen in one of seven medulloblastomas with prominent neuronal differentiation. There was no staining of non-neuronal structures. The results indicate that NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues, and may be useful in diagnostic histopathology.

Medullocytoma (lipidized medulloblastoma). A cerebellar neoplasm of adults with favorable prognosis.

This study describes three cases of neuroectodermal cerebellar neoplasms occurring in adults, characterized by a monomorphic population of round cells with scanty cytoplasm and focal areas of lipid accumulation. Astrocytic and neuronal differentiation was confirmed in these cells by glial fibrillary acidic protein and synaptophysin immunoreactivity. Electron microscopy performed in two cases showed neuritic processes, synapses, and dense-core granules. Patients included two men and one woman, and the age at diagnosis was 36, 37, and 57 years, respectively. Two patients refused any postoperative treatment. One of these had two surgically removed recurrences after 10 and 11 years and died postoperatively from intracranial hemorrhage. The second had two recurrences after 10 and 15 years and is alive and in good health at the last follow-up. The third patient received postoperative radiotherapy and is alive and well after 2 years. Review of the literature revealed seven cases of cerebellar neoplasms with histological features similar to those observed in our series. These lesions have been considered a variant of medulloblastomas. The age of patients ranged from 42 to 77 years (mean age, 51 years); four were women, 3 men. Follow-up information available in two cases indicates a 5-year survival with surgery alone. These data indicate that these cerebellar neuroectodermal neoplasms have morphologically unique features and indolent biologic behavior that distinguish them from the highly aggressive medulloblastoma; the term medullocytoma for this form is suggested.

Lipomatous medulloblastoma in adults. A distinct clinicopathological entity.

We report on three patients who presented with a cerebellar medulloblastoma at age 48, 53, and 59 years. Histopathology showed typical features of medulloblastoma, in one case with marked neuronal differentiation. In addition, all neoplasms contained focal accumulations of mature fat cells. Immunoreactivity of adipocytes for S-100 protein, neuron-specific enolase, synaptophysin, microtubule-associated protein-2, and glial fibrillary acidic protein and the lack of immunoreactivity to type IV collagen suggest lipomatous differentiation of neoplastic primitive neuroectodermal cells rather than an admixture of mesenchymal elements. Mitotic activity was low and the growth faction, as determined by the MIB-1 labeling index, was less than 5%. All patients are alive with a recurrence-free interval ranging from 3.5 to 12 years. These three patients and five similar previously reported cases all fit into the concept of the lipomatous medulloblastoma as a new clinicopathological entity characterized by (a) typical features of a cerebellar medulloblastoma with advanced neuronal differentiation, (b) areas of lipomatous differentiation, (c) low proliferative potential, (d) manifestation in adults (mean age, 50 years), and (e) apparent favorable clinical prognosis.