Expression of transforming growth factor-beta receptor type 1 and type 2 in human sporadic vestibular Schwannoma.

Expression of the transforming growth factor-beta (TGF-beta) protein family in the peripheral nervous system is well established, but the role of their cognate receptors TGF-beta receptor type 1 (R1) and type 2 (R2) has been less well studied. TGF-beta plays an essential role in Schwann cell proliferation and differentiation, and is involved in neurotrophic effects of several neurotrophic substances. TGF-beta is also expressed in benign peripheral nervous system tumors such as vestibular schwannomas. In the present study, we aimed to detect TGF-beta R1 and R2 in a total of 40 sporadic vestibular schwannomas using immunohistochemistry, and correlated the findings to essential clinicopathologic data. TGF-beta, TGF-beta R1, and TGF-beta R2 mRNA was further analyzed by RT-PCR in six vestibular schwannomas. TGF-beta R1 immunoexpression was found in about 95% of the tumors. TGF-beta R1 was equally present in Antoni A and Antoni B areas of the tumors. TGF-beta R2 was found immunohistochemically in 77%. In addition, all tumors showed strong expression of TGF-beta. No correlation between TGF-beta R1 or R2 expression and clinicopathologic parameters such as age, sex, clinical symptoms, growth pattern, and proliferation acitivity as measured by Ki-67 (MIB-1) staining was found. Moreover, all schwannomas studied contained TGF-beta, TGF-beta R1, and TGF-beta R2 mRNA. Therefore, the TGF-beta/TGF-beta R1 and -R2 system is present in human schwannomas, but its biologic role for tumor development and growth remains unclear.

The neurotrophin receptor TrkB is colocalized to mitochondrial membranes.

The transmembrane tyrosine-specific protein kinase TrkB has been shown to serve as a receptor for the neurotrophic factors BDNF and NT-4. Neurotrophin binding to TrkB isoformes mediates many intracellular signaling pathways, including calcium signalling. Two truncated isoforms of the receptor, lacking the tyrosine kinase activity, signal through a yet unknown pathway. Specific signals modulate the surface expression of TrkB, which is localized in considerable amounts in intracellular pools. These intracellular pools has not been specified so far. We therefore investigated the intracellular distribution of TrkB by colocalisation studies. In contrast to the unspecific neurotrophin receptor NGFRp75, TrkB immunohistochemistry showed a staining pattern very similar to mitochondrial stainings in adult human skeletal muscle fibers. Immunofluorescence techniques revealed in different types of permeabilized cells that TrkB is bound to mitochondrial membranes. This observation was confirmed on isolated astrocyte mitoplasts. Colocalisation of the TrkB ligand NT-4 and the specific mitochondrial marker cytochrome c oxidase was also observed. Western blot analysis of isolated mitochondria from rat brain and skeletal muscle verified that a truncated isoform of TrkB is present in both, brain and muscle mitochondria, and full-length TrkB is additionally present in brain mitochondria. Our results imply that neurotrophins can be stored in mitochondria and possibly act as signalling molecules on mitochondria.

Analysis of a single nucleotide polymorphism in codon 388 of the FGFR4 gene in malignant gliomas.

The FGFR4 codon 388 polymorphism (Arg(388), Arg/Gly(388) or Gly(388)) was determined in glioblastoma multiforme (GBM), anaplastic astrocytomas (AA), diffuse astrocytomas (DA), and control muscles. Arg(388) was rare in AA, GBM, muscles, and was absent in DA. The Arg/Gly(388) and the Gly(388) frequency was equal among GBM and controls. FGFR4 expression was not related to codon 388 in GBM, and no survival differences between Arg/Gly(388) and Gly(388) tumors were found. U87 cells (Arg/Gly(388)) did not show higher invasion than U138 cells (Gly(388)). This suggests that the FGFR4 codon 388 status does not play a major role in malignant gliomas.

Different activation of mitogen-activated protein kinase and Akt signaling is associated with aggressive phenotype of human meningiomas.

PURPOSE: Activation of intracellular signaling cascades has been implicated in the growth control of benign meningiomas, but their role for meningioma progression and outcome is unknown. Here we determined the expression and function of proteins involved in mitogen-activated protein kinase (MAPK) and phosphinositol-3 kinase (PI3K)/Akt signaling in benign, atypical, and malignant meningiomas and studied their association with clinicopathologic data including meningioma recurrence. EXPERIMENTAL DESIGN: Expression of various MAPK and PI3K signaling proteins was determined in 70 primary meningiomas and, if present, in recurrent tumors by immunohistochemistry and Western blotting. The expression patterns in primary and recurrent tumors were related to clinical data. The effect of MAPK and PI3K pathway inhibition on cell proliferation and apoptosis was determined using a primary malignant meningioma cell culture. RESULTS: Atypical and malignant meningiomas showed higher levels of phospho-Akt compared with benign tumors, and their proliferation could be inhibited by PI3K blocking using wortmannin. PI3K inhibition did not induce apoptosis in malignant meningioma cells. In contrast, expression of phospho-Raf and phospho-MAPK was decreased in aggressive meningiomas compared with benign tumors, but MAPK inhibition by PD98059 resulted in tumor cell apoptosis and decreased proliferation. Reduced MAPK activation was associated with meningioma recurrence, and PI3K activation was associated with poor preclinical condition and brain invasion of malignant meningiomas. CONCLUSIONS: Both MAPK and PI3K/Akt pathways are activated at different levels in benign and malignant meningiomas. Activation of PI3K/Akt signaling contributes to the aggressive behavior of malignant meningiomas, whereas MAPK activation is involved in both proliferation and apoptosis of malignant meningiomas.

Expression and function of somatostatin receptors in peripheral nerve sheath tumors.

Although somatostatin receptors have been detected in many normal and neoplastic tissues, little is known of their expression and function in peripheral nerve tumors. In the present study, we examined the expression of all 5 somatostatin receptor subtypes (sst1-5) in 3 normal peripheral nerves, 3 traumatic neuromas, 27 schwannomas, 18 neurofibromas, and 177 malignant peripheral nerve sheath tumors (MPNSTs) by immunohistochemistry as well as by Western blot and reverse transcriptase-polymerase chain reaction investigations in 2 normal peripheral nerves, one neurofibroma, 5 schwannomas, and 5 MPNSTs. Immunoreactive somatostatin receptors were not detectable in normal peripheral nerve and in nonneoplastic Schwann cell proliferations. In contrast, sst2A mRNA and protein was present in 89% of schwannomas. This receptor subtype was less frequently detected in neurofibromas (22%) and MPNSTs (15%). Interestingly, sst4 was seen in 32% of MPNSTs and was almost exclusively expressed in this malignant tumor type. In support of a role in Schwann cell tumor growth control by somatostatin was the observation of induced internalization of sst2A and inhibition of cell proliferation in an NF1-associated MPNST cell line. Moreover, administration of an sst2A-selective agonist resulted in induction of MPNST cell apoptosis. We conclude that peripheral nerve sheath tumors often express at least one functional somatostatin receptor. Furthermore, our findings suggest a potential clinical role for somatostatin receptor agonists in tumor imaging and/or treatment of schwannomas and MPNSTs.

Gastric adenocarcinoma with leptomeningeal carcinomatosis as the presenting manifestation: an autopsy case report.

Leptomeningeal carcinomatosis is a clinically important and severe complication in patients with cancer. Leptomeningeal involvement as a secondary event in gastric carcinoma is rarely reported and usually occurs late in advanced disease. Herein, we report a case of leptomeningeal carcinomatosis as the initial manifestation of a previously asymptomatic gastric adenocarcinoma. The clinical features and the appropriate diagnostic procedures are discussed.

Differential expression of sst1, sst2A, and sst3 somatostatin receptor proteins in low-grade and high-grade astrocytomas.

We have previously reported that sst2A somatostatin receptors are frequently overexpressed in human meningiomas. Initial clinical observations suggest that somatostatin analogues may also be of value for imaging and treatment of other human intracranial tumors, including astrocytomas. However, contradictory results have been reported regarding the expression of somatostatin receptors in low-grade and high-grade astrocytomas. Therefore, we determined the precise pattern of somatostatin receptor protein expression in 8 diffuse astrocytoma (DA), 10 anaplastic astrocytomas (AA), and 32 glioblastoma multiforme (GBM) using immunohistochemistry and Western blot analysis. sst1 and sst2A somatostatin receptors were not present in DA and only occasionally detected in AA. In GBM, sst1 was present in 66%, and sst2A was found in 44% of the tumors. sst3 receptors were present in 38% of DA, 40% of AA, and 84% of GBM. Thus, loss of differentiation was significantly associated with increased expression of sst1, sst2A, and sst3 somatostatin receptors. In contrast, sst4 and sst5 receptors were found in 80% and 25% of all cases, respectively, in a manner independent of histological grade. No significant correlation was found between somatostatin receptor expression and the proliferation rate of the tumors as determined by MIB-I immunostaining. Furthermore, the presence or absence of the 5 somatostatin receptor subtypes did not significantly influence survival time in 14 GBM patients.

June 2004: a male in his late 60s with recurrent extracerebral tumor.

June 2004: Over the past year, this man in late-60s had complained about progressive weakness of concentration and memory disturbances, associated with word finding difficulties. MRI examination revealed an extra-axial, parasagittal tumor 3 cm in diameter located in the left frontoparietal region. Five years ago, a meningioma in the same region, with radiographic appearance comparable to the present tumor had been totally removed. The histological picture of the current tumor was dominated by sheets of large rounded pleomorphic tumor cells with abundant eosinophilic cytoplasm and eccentric nuclei (rhabdoid cells). Cytoplasmic inclusions were frequent; occasionally,multinucleatedtumorcellswereseen. Mitoticfigures were absent and the MIB was 3%. Meningothelial lobules were scarce, and regions with fibroblastic appearance were absent. There were no psammoma bodies, necrosis or brain invasion. Moderate immunoreactivity for EMA was found. Additionally, strong cytoplasmic immunoreaction for vimentin within the rhabdoid cells was observed. Review of the previous material showed small islets of rhabdoid cells. Rhabdoid meningioma is an uncommon meningioma variant. It has been suggested that rhabdoid meningiomas are highly aggressive tumors (WHO grade III)and that the rhabdoid phenotype represents a marker of malignant transformation in meningiomas. Histologically, rhabdoid meningiomas usually exhibit signs of anaplasia, a high mitotic activity, and a markedly increased MIB-1 labeling index. Extracranial metastases may occur in the course of the disease. However, not all rhabdoid tumors appear to have anaplastic features (as this case illustrates). Another interesting feature of rhabdoid meningiomas is that in a significant number of cases, the rhabdoid cells appear only at the time of recurrence. Alternatively, as seen in this case, the rhabdoid cells may be already present in the primary meningioma, but not as the predominating histological feature.

Changes of retinal glutamate transporter GLT-1 mRNA levels following optic nerve damage.

PURPOSE: Under physiological conditions, levels of the excitatory neurotransmitter glutamate within the retina are regulated by retinal glutamate transporters to prevent toxic accumulation. Alterations in this glutamate buffering have been implicated in retinal ganglion cell (RGC) death. We quantified the changes in the level of glutamate transporter mRNA in a model of acute rat optic nerve injury. METHODS: Optic nerve damage was induced in one eye of 25 adult Wistar rats by partial optic nerve crush (ONC). Total mRNA levels of the retinal glutamate transporter GLT-1 (EAAT-2) were determined by quantitative real-time PCR. GLT-1 mRNA levels were measured 1, 3, 7, 14, and 28 days following optic nerve injury. Additionally, control values were obtained from the retinas of five control rats (sham-crush). RESULTS: In the very early phase (1 day post-ONC), a 3.9 fold increase in levels of GLT-1 mRNA was observed in the ONC retinae compared with control eyes. This was followed by a rapid decrease towards control levels at day 3 post-ONC. GLT-1 mRNA levels remained up to 14 days post-crush. However, in the late phase post-ONC (day 28), the level of GLT-1 mRNA increased again, but still remained not significant to control levels. CONCLUSIONS: Changes in GLT-1 mRNA expression following axonal trauma of RGCs can lead to an imbalance of glutamate homeostasis. This may cause local accumulation of toxic concentrations of the neurotransmitter glutamate and further irreversible excitotoxic damage of RGCs.

Prognostic relevance of MAPK expression in glioblastoma multiforme.

The objective of this study was to determine the immunoexpression pattern of the mitogen-activated protein kinase (MAPK), and related signalling proteins [protein kinase C (PKC), phospholipase Cgamma (PLCgamma)], in glioblastoma multi-forme, and to investigate their prognostic value. Paraffin-embedded biopsy samples from 26 patients [13 patients with long-term survival (LTS; N=13; median 28 months, range 13-76 months), and, for comparison, 13 patients with short-term survival (STS; N=13; median 7 months, range 1-12 months)] were investigated for the immunoexpression of MAPK, the activated pMAPK, PKC, PLCgamma, EGFR, and PTEN. Additionally, the MIB-1 proliferation index was determined. The immunoexpression pattern were related to clinical data, including analysis of their prognostic value using the Cox-proportional hazard model. No significant differences were found between STS and LTS in terms of age, Karnofsky performance status, and treatment. Whereas EGFR expression did not differ between STS and LTS and does not influence survival, expression of MAPK and activated pMAPK was significantly correlated with survival time. The percentage of pMAPK expressing cells correlated strongly with the percentage of MIB-1 positive cells. Furthermore, survival in patients with tumors expressing PKC or PLCgamma was significantly shorter. No differences were found for PTEN expression. Our findings indicate that the MAPK pathway is correlated with proliferation in gliomas, and that patient subgroups exist, in which expression of MAPK-related signalling proteins (PKC, PLCgamma) is associated with poorer prognosis. These patient subgroups may benefit from additional chemotherapeutic agents which specifically inhibit these signalling proteins.

Distribution of p53 alterations in a case of gliomatosis cerebri.

Gliomatosis cerebri (GC) is a rare neuroepithelial tumor characterized by diffuse infiltration of large parts of the brain. The origin of GC is unknown, and the molecular alterations underlying this tumor have not been determined. Because mutations in the p53 tumor-suppressor gene are frequent in common gliomas, we investigated the distribution of p53 alterations by immunohistochemistry and direct sequencing in a GC case with a disease involving both hemispheres and the basal ganglia. Nuclear accumulation of p53 protein was detected in a single region with features of a high-grade glioma. In the remaining 10 regions, corresponding to low-grade gliomas, no p53 accumulation was seen. In 1 low-grade tumor sample, a pathogenic splice site mutation was detected. These findings suggest that p53 alterations occur in GC, but are no prerequisite of malignant progression. The distribution of p53 alterations demonstrates the existence of topographically different clones in 1 patient.

Immunohistochemical and molecular analysis of p53, RB, and PTEN in malignant peripheral nerve sheath tumors.

The molecular basis of both sporadic and neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs) is yet largely undetermined. Therefore, we analyzed a series of 12 MPNSTs - including two cases which arose in the setting of NF1 - for molecular alterations in the p53, retinoblastoma ( Rb), and PTEN tumor suppressor genes. Furthermore, the immunohistochemical expression of p53, RB, and PTEN protein was examined in these tumors. One mutation (8%), an A to T transversion leading to an amino acid exchange, was found in exon 5 of the p53 gene in a sporadic MPNST. In two other sporadic tumors (20%), loss of heterozygosity (LOH) of the p53 gene occurred. Nuclear overexpression of p53 protein was observed in ten tumors (83%). Loss of RB protein expression was seen in two MPNSTs (17%), and LOH of the Rb gene was detected in four tumors (44%), including the two NF1-associated MPNSTs, one of them showing concomitant loss of RB protein expression. No mutation in the PTEN gene was detected, and cytoplasmic immunoreactivity for the PTEN protein was maintained in eight MPNSTs (67%). We suggest that alterations in the p53 and RB pathway, both are essential in controlling the cell-cycle progression, are critical points in the tumorigenesis of sporadic and NF1-associated MPNSTs, whereas the PTEN gene seems to play no significant role in this process.

Single-cell analysis of mtDNA deletion levels in sporadic amyotrophic lateral sclerosis.

One possible cause for the neuronal loss in sporadic amyotrophic lateral sclerosis (S-ALS) is an increase of free radicals, which may produce oxidative damage to susceptible biomolecules, which, in turn, can damage the mitochondrial DNA (mtDNA). Following laser microdissection of single motor neurons from paraffin-embedded autopsy tissue, we analyzed the presence of a common mtDNA deletion, the 5 kb common deletion (CD). Spinal cord neurons showed slightly higher CD detection rate in patients than controls (94% vs 75%). No significant differences were found between patients and controls for neurons derived from other motor or non-motor regions. A PCR assay of serial DNA dilutions (10-fold) showed no CD level differences between motor neurons in S-ALS and controls. These data suggest that neuronal death in S-ALS is not associated with significant accumulation of mtDNA deletions.

Region-specific analysis of mitochondrial DNA deletions in neurodegenerative disorders in humans.

Mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) aberrations has been implicated in the neuronal death in neurodegenerative disorders. Significant neuronal damage can occur if the percentage of mtDNA mutations may reach a critical threshold. mtDNA mutations also accumulate during normal aging. Here we quantified the 5 kB common mtDNA deletion (CD) using real-time PCR in various brain regions from neurodegenerative disorders and controls. We confirmed previous results that the CD levels increase with age, reaching highest levels in the basal ganglia. High CD levels were also found in affected regions in frontotemporal dementia, Parkinson's disease, and dementia with Lewy bodies, but not in Alzheimer's disease. This suggests that mtDNA damage may occur in a region-specific distribution in neurodegenerative disorders.

Reduced PTEN expression in gastric cancer and in the gastric mucosa of gastric cancer relatives.

OBJECTIVES: Gastric cancer is a leading cause of cancer-related deaths worldwide, and the genetic and molecular alterations underlying its pathogenesis are largely unknown. PTEN, a tyrosine phosphatase, is frequently mutated in brain and breast cancers but not in gastric cancers. In order to assess the role of PTEN in gastric carcinogenesis, we analysed the expression of PTEN in human gastric cancer and in the gastric mucosa of cancer relatives. METHODS: Gastric cancer tissues were obtained from 26 patients with gastric cancer undergoing upper-gastrointestinal endoscopy or resection for gastric cancer. Furthermore, 21 first-degree relatives of gastric cancer patients and 12 healthy individuals agreed to undergo upper-gastrointestinal endoscopy. In all cases, biopsies were taken from the antrum and corpus. PTEN expression was assessed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. RESULTS: PTEN expression was reduced or absent in 21/26 gastric cancers compared with the matched non-malignant gastric biopsy (P < 0.001). Furthermore, PTEN expression was reduced significantly in the antrum biopsy of first-degree relatives compared with healthy controls (P < 0.05). CONCLUSIONS: Reduced expression of PTEN in gastric cancer points to another mechanism apart from PTEN mutation that may be involved in the pathogenesis of gastric cancer. The reduction of PTEN expression was also observed in first-degree relatives.

Single-cell analysis of mtDNA in amyotrophic lateral sclerosis: towards the characterization of individual neurons in neurodegenerative disorders.

Laser microdissection offers the separate analysis of neuronal cells within the central nervous system in certain neurodegenerative diseases. We have established the amplification of a common deletion of mitochondrial DNA (mtDNA) on the basis of single microdissected neurons. Using brain and spinal cord tissue from patients suffering from amyotrophic lateral sclerosis (ALS) and healthy controls, we detected the 5 kB common deletion of mtDNA in motor neurons from ALS and control cases. The deletion was also present in non-motor regions from diseased patients and controls, suggesting that the presence of the mtDNA deletion is not associated with the neuronal death in specific areas of the central nervous system in ALS.

Expression pattern of apoptotic markers in vestibular schwannomas.

The Fas-Fas-L system plays a major role in the regulation of apoptosis and hence in growth in benign and malignant human tumors. As the factors regulating cell death in benign schwannomas are not well understood, we investigated the immunoexpression of the Fas-Fas-L system, as well as that of the anti-apoptotic factor Bcl-2 and the pro-apoptotic factor Bax in 14 sporadic vestibular schwannomas, and related the findings to the MIB-1 labeling index as a marker for cell proliferation. Whereas cytoplasmic Fas expression was seen in only one tumor (7%), Fas-L was found in the nuclei of 12 schwannomas (86%). Bcl-2 expression was found in the cytoplasm of 9 tumors (64%), and Bax was found in 10 out of 14 schwannomas (71%). No significant correlations between different labeling indices were observed. However, schwannomas expressing Bax tended to show a higher proliferation rate as revealed by the MIB-1 LI, suggesting a balance between cell proliferation and cell death. Our study further showed that Fas-L is present in most vestibular schwannomas; however, due to the lack of Fas expression, apoptosis in vestibular schwannomas does not seem to be mediated via the Fas-Fas-L system.

Primary primitive neuroectodermal tumor of the spinal cord: case report and review of the literature.

We present the clinical, radiological, and pathological features of a primary primitive neuroectodermal tumor (PNET) that occurred in the thoracic spinal cord of a 69-year-old man. Magnetic resonance imaging (MRI) demonstrated on T1-weighted images a 2x1x5 cm isointense intraspinal mass with homogeneous contrast enhancement extending from the C7 to the Th3 level. There was no clinical or radiological evidence for the existence of an intracranial tumor. Histological examination revealed a small round cell tumor with rosette formation and immunohistochemical characteristics of a PNET. The patient is the oldest among the 20 cases with this rare spinal cord neoplasm reported so far in the literature; the previously published cases are briefly reviewed.

ADAM 12: a putative marker of oligodendrogliomas?

ADAM 12 (meltrin alpha) belongs to a large family of molecules, consisting of members with both disintegrin and metalloproteinase properties. ADAMs have been implicated in several cell physiological processes including cell adhesion, cell fusion, proteolysis and signalling. ADAM 12 is widely expressed, including skeletal muscle, testis, bone, intestine, heart and kidney. In addition, a variety of tumours show elevated expression of ADAM12; among them being breast-, colon-, gastric- and lung-carcinoma. As to the brain, ADAM 12 has been shown previously to be expressed in rat and human oligodendrocytes. However, little is known about the expression of this protease in brain tumours. This study demonstrates the presence of ADAM 12 in non-neoplastic oligodendroglial cells of normal human brain as well as in neoplastic oligodendroglia and minigemistocytes arising from four pure oligodendrogliomas and three mixed oligoastrocytomas. Double stainings revealed a notable preference of ADAM 12 for the oligodendroglial over astroglial components. The results of immunohistochemistry are in accordance with the results obtained from the RT-PCR, which further demonstrated a mild difference concerning the mRNA concentration of ADAM 12 between similar grades of eight astrocytomas and eight oligodendrogliomas (namely four astrocytomas grade II versus four oligodendrogliomas grade II and four astrocytomas grade III versus four oligodendrogliomas grade III). Both cellular immunostaining for ADAM 12 and ADAM 12 mRNA content decrease with higher histologic grade of the tumour. Surprisingly, the latter parameter (ADAM12 mRNA) showed a significant opposite correlation to the degree of histologic tumour malignancy. From our data showing that ADAM 12 is highly expressed in, but not restricted to, oligodendrogliomas, we conclude that ADAM 12 immunohistochemistry may be a helpful tool in the diagnosis of brain tumours.