The microenvironment in sporadic and neurofibromatosis type II-related vestibular schwannoma: the same tumor or different? A comparative imaging and neuropathology study.

OBJECTIVE: Inflammation and angiogenesis may play a role in the growth of sporadic and neurofibromatosis type 2 (NF2)-related vestibular schwannoma (VS). The similarities in microvascular and inflammatory microenvironment have not been investigated. The authors sought to compare the tumor microenvironment (TME) in sporadic and NF2-related VSs using a combined imaging and tissue analysis approach. METHODS: Diffusion MRI and high-temporal-resolution dynamic contrast-enhanced (DCE) MRI data sets were prospectively acquired in 20 NF2-related and 24 size-matched sporadic VSs. Diffusion metrics (mean diffusivity, fractional anisotropy) and DCE-MRI-derived microvascular biomarkers (transfer constant [Ktrans], fractional plasma volume, tissue extravascular-extracellular space [ve], longitudinal relaxation rate, tumoral blood flow) were compared across both VS groups, and regression analysis was used to evaluate the effect of tumor size, pretreatment tumor growth rate, and tumor NF2 status (sporadic vs NF2-related) on each imaging parameter. Tissues from 17 imaged sporadic VSs and a separate cohort of 12 NF2-related VSs were examined with immunohistochemistry markers for vessels (CD31), vessel permeability (fibrinogen), and macrophage density (Iba1). The expression of vascular endothelial growth factor (VEGF) and VEGF receptor 1 was evaluated using immunohistochemistry, Western blotting, and double immunofluorescence. RESULTS: Imaging data demonstrated that DCE-MRI-derived microvascular characteristics were similar in sporadic and NF2-related VSs. Ktrans (p < 0.001), ve (p ≤ 0.004), and tumoral free water content (p ≤ 0.003) increased with increasing tumor size and pretreatment tumor growth rate. Regression analysis demonstrated that with the exception of mean diffusivity (p < 0.001), NF2 status had no statistically significant effect on any of the imaging parameters or the observed relationship between the imaging parameters and tumor size (p > 0.05). Tissue analysis confirmed the imaging metrics among resected sporadic VSs and demonstrated that across all VSs studied, there was a close association between vascularity and Iba1+ macrophage density (r = 0.55, p = 0.002). VEGF was expressed by Iba1+ macrophages. CONCLUSIONS: The authors present the first in vivo comparative study of microvascular and inflammatory characteristics in sporadic and NF2-related VSs. The imaging and tissue analysis results indicate that inflammation is a key contributor to TME and should be viewed as a therapeutic target in both VS groups.

M-CSF and IL-34 expression as indicators for growth in sporadic vestibular schwannoma.

Macrophage colony stimulating factor and IL-34 are associated with clinical vestibular schwannoma progression. Investigating the biology behind vestibular schwannoma progression helps understanding tumor growth. Inflammation is important in the microenvironment of neoplasms. Macrophages are major players in the intratumoral infiltrate. These tumor-associated macrophages are known to stimulate angiogenesis and cell growth. M-CSF and IL-34 are cytokines that can regulate tumor-infiltrating macrophages. They are expressed by tumors and form potential targets for therapy. The goal of this study was to investigate these cytokines in vestibular schwannomas and to see if their expression is related to angiogenesis, macrophage numbers, cystic degeneration, and volumetric tumor progression. Immunohistochemical expression of M-CSF and IL-34 was analyzed in ten fast-growing vestibular schwannomas and in ten slow-growing vestibular schwannomas. Expression M-CSF and IL-34 were compared between fast- versus slow-growing and cystic versus non-cystic tumors. Data on macrophage numbers and microvessel density, known from earlier research, was also included. All tumors expressed M-CSF and its expression was higher in fast-growing tumors (p = 0.003) and in cystic tumors (p = 0.035). CD163 expression was higher in tumors with strong M-CSF expression (p = 0.003). All tumors expressed IL-34 as well, but no significant differences were found in relation to clinicopathological characteristics. This study demonstrated the expression of M-CSF and IL-34 in vestibular schwannomas. The results suggest that M-CSF is related to macrophage activity and tumor progression, making it a potential target for therapy. If a similar assumption can be made for IL-34 remains unclear.

The phosphorylation status of merlin in sporadic vestibular Schwannomas.

The events leading to Schwannomas development are still largely unknown. Some studies have demonstrated that merlin acts as a tumor suppressor by blocking Ras-mediated signaling. In this study, we analyze the clinical and biological behaviors of seven randomly selected sporadic vestibular Schwannomas removed from the patients. We find that merlin was commonly lost in these Schwannomas, due to loss of merlin expression or phosphorylation status of merlin expression. Heightened CDKs/cyclins signal transduction concomitant with loss of p27 was well correlated with loss of functional merlin in Schwannomas. More, we show that phosphorylated merlin Schwannomas exhibited increased Ras/Rac/PAK signal transduction. That was in agreement with the severe clinical behaviors, i.e., phosphorylation status of merlin increased tumor size in sporadic vestibular Schwannomas. These results led us to suggest that phosphorylated merlin, a kind of type of mutation merlin, is involved in tumorigenesis of sporadic vestibular Schwannomas.

Neurofibromatosis type 2: optic nerve sheath meningioma in one orbit, intramuscular schwannoma in the other.

A highly unusual patient with neurofibromatosis type 2 (NF2) presenting with simultaneous bilateral orbital tumors is described. A 12-year-old girl with a family history of NF2 was examined because of bilateral proptosis. Visual acuities were light perception RE and 20/40 LE. Magnetic resonance imaging studies showed bilateral cerebellopontine angle tumors, a tumor surrounding the right intraorbital optic nerve, and a large left lateral orbital mass mixed with the lateral rectus muscle. The histopathological diagnoses following incisional biopsies were right optic nerve sheath meningioma and left intramuscular schwannoma. The left-sided orbital schwannoma and the right-sided vestibular schwannoma were treated with fractionated stereotactic radiotherapy. This patient enlarges the spectrum of clinical presentations that can be encountered at young age in patients with NF2.

Immunohistochemical demonstration of vascular endothelial growth factor in vestibular schwannomas correlates to tumor growth rate.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is one of the most potent mediators of angiogenesis, which is a mandatory process during tumor growth. The present objectives were to determine expression of VEGF in vestibular schwannomas by immunohistochemistry and to examine a possible correlation with symptom duration, tumor size, or growth rate. STUDY DESIGN: Retrospective patient file review; immunohistochemistry and light microscopy of vestibular schwannomas removed by surgery. METHODS: Vestibular schwannomas from 18 patients were immunolabelled using a polyclonal antibody against VEGF, followed by light microscopy and blinded semiquantitation of VEGF expression. Fifteen patients had a well-defined tumor growth rate defined by repeated preoperative magnetic resonance imaging scans. RESULTS: All tumors showed expression of VEGF in the Schwann cell cytoplasm, with a more intense staining of the perinuclear region of some cells. The staining intensity varied from tumor to tumor, and semiquantitation revealed a significant correlation between VEGF expression and tumor growth rate, but not symptom duration or tumor size. CONCLUSION: VEGF is expressed in vestibular schwannomas and the level of expression correlates positively with tumor growth rate, but not with tumor size and symptom duration. We conclude that VEGF seems to be a factor involved in the growth of vestibular schwannomas.

Detection of antibody to sialyl-i, a possible antigen in patients with Meniere's disease.

An autoimmune hypothesis for the etiology of Meniere's disease has been proposed. In this study, we focused on gangliosides as potential antigens for autoantibodies in Meniere's disease patients. In an attempt to investigate ganglioside antigens which respond to the serum of patients with Meniere's disease, we analyzed gangliosides of human acoustic neurinomas, and used them as antigens to broadly explore gangliosides that react to serum. All the acoustic neurinoma samples used in the present study showed a similar ganglioside profile on TLC (thin-layer chromatography). For the microscale ganglioside analysis, a newly developed TLC blotting/secondary ion mass spectrometry (SIMS) system together with TLC immunostaining method was employed. Most of the ganglioside bands could be analyzed, and they were identified as GM3, GM2, SPG, GM1a, GD3, S-i (sialyl-i ganglioside) and GD1a. GD1a was the predominant ganglioside and many neolactoseries gangliosides were recognized by immunological analysis. Next, the immune reactivity of serum samples, from patients with Meniere's disease, with the acoustic neurinoma gangliosides was studied by TLC immunostaining. The result showed that five of 11 patients with Meniere's disease and one of eight normal subjects reacted with a specific band, which was identified as S-i by the TLC blotting/SIMS system. The findings of the present study indicate that S-i ganglioside is an autoantigen and possibly involved in the pathogenesis of Meniere's disease.

Transforming growth factor-beta1 expression in human acoustic neuroma.

HYPOTHESIS: The differing clinical behavior of acoustic neuroma (AN) may be explained by the presence of specific biological features involved in tumorigenesis and growth. BACKGROUND: Transforming growth factor (TGF) beta1 is known to participate in the regulation of peripheral nerve tumors, modulating cell proliferation and differentiation with mechanisms different from those of glial growth factors (GGF) and fibroblastic growth factors (FGF), which are responsible for Schwann cells' mitogen activity. METHODS: Surgically removed human AN specimens were fixed in formalin and embedded in paraffin for immunohistochemistry studies. Expression and localization of TGF-beta1 in different tumor regions were assessed after incubation of paraffin sections with a mouse monoclonal anti-TGF beta1 antibody (DBA, Milan, Italy). Clinically, the time elapsed between the beginning of symptomatology and AN size as shown by preoperative computed tomography, magnetic resonance imaging, or both was calculated as rough value of growth rate, which enabled slow-growing and fast-growing ANs to be distinguished. RESULTS: Eighty-four percent of AN specimens expressed TGF-beta1 positivity at the level of the cytoplasm of the Schwann cells. TGF-beta1 reactivity was also shown in the blood vessel walls (96.15%) and the tumor capsule (80.86%). TGF-beta1 reaction appeared higher in Antoni A regions than in Antoni B regions. No significant relationship was found between TGF-beta1 positivity and AN growth rate in the two groups. CONCLUSIONS: TGF-beta1 could participate in the biological behavior of AN, particularly as an important factor of tumor growth prediction by allowing rapidly progressive or potentially recurrent tumors to be differentiated from slow-growing tumors that are unlikely to recur. The clinical course of patients with AN is currently still of little help in predicting the rate of AN growth.

Rosenthal fibers and eosinophilic granular bodies in a classic acoustic schwannoma.

We describe unique features seen in a case of classic acoustic schwannoma. In the central portion of the tumor, abundant Rosenthal fibers and occasional eosinophilic granular bodies were present. Rosenthal fibers are homogeneous eosinophilic structures commonly seen in central nervous system lesions, such as pilocytic astrocytoma, or in the gliotic tissues adjacent to slowly growing neoplasms and some congenital malformations. Eosinophilic granular bodies are also structural markers of slow-growing, well-differentiated neuroglial neoplasms, such as pleomorphic xanthoastrocytoma, ganglion cell tumors, and pilocytic astrocytoma. To the best of our knowledge, however, these two structures have never before been described in schwannomas.

Immunohistochemical detection of p53 protein in tumours of the central nervous system.

The results of immunohistochemical detection of p53 protein in a large variety of CNS neoplasms with the polyclonal antiserum CM1 are presented. Immunoreactivity in at least several cells were regularly found in astrocytic neoplasms including glioblastomas; it was also frequent in medulloblastomas and oligodendrogliomas and could be found in more than 50% of acoustic schwannomas and esthesioneuroblastomas. Meningiomas also showed several immunoreactive cells in about 40% of the cases. Certain neuronal/neuronal-glial tumours (such as central neurocytomas, dysembryoplastic neuroepithelial tumours, gangliocytomas, dysplastic gangliocytomas of the cerebellum etc.) were consistently negative, whereas gangliogliomas (with the exception of the regularly positive desmoplastic gangliogliomas) very rarely displayed immunoreactive glial cells. Positive findings, however, seem to be more frequent in anaplastic gangliogliomas where they may be of prognostic significance. The same may be valid in ependymomas. The underlying mechanism for p53 immunopositivity, however, can be very heterogeneous and possibly even contrary (either accumulation of inactive mutant protein or detection of active wild-type protein), therefore isolated immunohistochemical findings call for very cautious interpretation.

Expression of steroid receptors in acoustic neuroma.

The expression of steroid receptors has been investigated in an attempt to clarify the role of steroid hormones in the proliferation and progression of acoustic neuromas. Specimens of tumours taken during translabyrinthine surgery were tested for cytosolic (c) and nuclear (n) steroid receptors. Oestrogen and progesterone receptor levels were evaluated by enzymatic immuno-assay, while androgen receptor binding levels were detected by dextran-coated charcoal method in a single-step determination. In some cases, the six point Scatchard analysis of cytosolic and nuclear androgen receptor was also performed. Threshold values were: 3 fmol/mg of proteins for cytosolic steroid receptors and 20 fmol/mg DNA for nuclear steroids, which corresponded to approximate median values of cytosolic and nuclear oestrogen and progesterone, respectively. Oestrogen and progesterone appeared to be localized more frequently in the nuclei rather than in the cytosol (70% oestrogen and progesterone positivity in the nuclei; 30% oestrogen, 40% progesterone positivity in the cytosol), while androgen receptors were preferentially localized in the cytosol (80% positivity in the cytosol; 40% positivity in the nuclei). A negative non-linear correlation between cytosolic oestrogen and cytosolic androgen receptors was found. There was a direct linear correlation between cytosolic oestrogen and nuclear oestrogen levels. A strict correlation between nuclear oestrogen and nuclear progesterone incidence was shown. Preliminary analysis of clinical data and biochemical parameters showed that cytosolic progesterone levels inversely correlated with tumour size.

Acoustic schwannoma and pregnancy: a DNA flow cytometric, steroid hormone receptor, and proliferation marker study.

For a long time, it has been speculated that pregnancy stimulates the growth of acoustic schwannomas. To test this hypothesis, immunohistochemical stains for estrogen receptor, progesterone receptor, and proliferating cell nuclear antigen (PCNA) were performed. Flow cytometric studies for DNA ploidy and S-phase fraction determinations were also performed. The study subjects included 6 female patients with unilateral acoustic tumors; at the time of tumor removal, 1 woman was pregnant and the other 5 women were 2 to 10 months postpartum. The age-sex-matched control group consisted of 6 men and 12 nonpregnant women, all with acoustic schwannomas similar in size to those of the study group. The study found no statistically significant association between the presence or quantity of estrogen or progesterone receptors and pregnancy, DNA ploidy, proliferation indices, or clinical data. Based on PCNA indices, large tumors tended to be less "biologically active" than small lesions (P < .01). The authors concluded that pregnancy does not significantly stimulate the cellular growth of acoustic schwannomas.

[Bone mineral density of the internal auditory meatus with acoustic neuroma].

The bone mineral density of the internal auditory meatus was investigated by means of quantitative computed tomography (QCT) in 12 patients with acoustic tumor. Investigated portions of the internal auditory meatus were the anterior and posterior porus and the anterior and posterior fundus. The bony vestibule and cortical bone of the mastoid cavity were also investigated. The bone density values (calcium carbonate equivalent value) for each portion were analyzed statistically. Another investigation by CT program "Profile" revealed changes in CT values along the posterior wall of the internal auditory meatus, and the results were compared with that of the QCT. The following results were obtained: 1) The highest calcium carbonate equivalent value was found in the posterior fundus, the lowest in the anterior porus. There were significant differences between the bilateral fundus and porus values. 2) There was a marked difference only in the anterior fundus of the abnormal side in comparison with the normal side, while differences in the other 5 portions were not statistically significant. 3) There were no significant differences in any of the portions between cases of superior vestibular nerve origin and those of inferior vestibular nerve origin. 4) In Profile, the CT values increased gradually from the porus to the fundus on both sides. A dip formation, indicating a lower CT value, was found between the porus and fundus in many cases. It was suggested that this dip formation was influenced by pneumatization of the posterior wall of the internal auditory meatus.

Raised levels of latent collagenase activating angiogenesis factor (ESAF) are present in actively growing human intracranial tumours.

Endothelial cell stimulating angiogenesis factor (ESAF) is a potent, low molecular mass mitogen, specific for endothelial cells. In common with various protein growth factors, it displays angiogenic activity in a variety of biological test systems. However, it differs from these other factors by virtue of its low molecular mass and its ability to activate latent matrix metalloproteinases in a dose dependent manner. This activity has been used to quantify the factor in both normal and diseased brain tissue. The concentration of ESAF determined in biopsies from different types of intracranial tumours varied: in some tumour types the level was close to that of control samples whereas in others it rose to levels comparable to those encountered in the pineal gland, the richest source of ESAF in mature mammals. Tumours considered to be benign contained significantly less ESAF than those neoplasms classified as being malignant (P = 0.025). There was also a correlation between the mitotic activity of tumour samples, as determined by conventional H & E histochemical staining and the ESAF concentration present. These findings agree with previous studies in which elevated ESAF levels have been found in tissue where proliferation of vascular elements has been observed.

Identification of perilymph proteins by two-dimensional gel electrophoresis.

Perilymph has a total protein component that is quantitatively distinct from serum and cerebrospinal fluid (CSF). The goal of this research was to determine if perilymph contains any qualitatively unique protein constituents that will distinguish it from serum or CSF. To test this hypothesis, matched sets of perilymph, serum, and CSF were obtained from 18 guinea pigs and seven human subjects. The purity of each sample was assured by measurement of the protein concentration of each sample and comparison of this parameter to known normal values for perilymph, serum, and CSF. Each sample was then subjected to two-dimensional gel electrophoresis, separating proteins by isoelectric point in the horizontal dimension and by relative molecular weight in the vertical dimension. All gels were processed under precisely identical physical conditions by use of a diamine silver stain. A small number of perilymph proteins not found in plasma were identified in both the guinea pig and the human specimens. The finding of unique perilymph proteins may permit the development of a sensitive marker that will aid in the diagnosis of perilymph fistula.

Oestrogen and progesterone receptors in acoustic neuroma.

Tissue samples from fourteen consecutive (8 male: 6 female) acoustic neuromas were assayed for hormone receptors using either a monoclonal antibody (MA), dextran coated charcoal (DCC) or isoelectric focusing (IEF) technique. In this series there were no unequivocally positive results, a finding at variance with previously published results.

Immunohistochemical localization of vimentin and S-100 antigen in small acoustic tumors and adjacent cochlear nerves.

To clarify the involvement of cochlear nerves in small acoustic tumors, we used immunoperoxidase techniques to determine the presence and distribution of vimentin and S-100 antigens in two acoustic tumor specimens and a transected vestibular nerve. Schwann cells and acoustic tumor cells failed to react positively with monoclonal antibody to vimentin. Reaction was observed in mesenchymal-appearing cells within both the normal nerve and the acoustic tumors, predominantly in association with blood vessels. Normal schwann cells and acoustic tumor cells reacted with polyclonal antibody to S-100 antigen with a similar, uniform distribution. Mesenchymal-appearing cells did not react with antibody to S-100. Immunostaining of a vestibular nerve from a Meniere's disease patient, used as a control, did not differ significantly from nerves adjacent to acoustic tumors. Because tumor cells and normal schwann cells stained similarly with antibody to S-100, it was not possible to establish with certainty if tumor cells invaded adjacent nerves.