Effects of ErbB2 signaling on the response of vestibular schwannoma cells to gamma-irradiation

Objective: For vestibular schwannomas (VSs) that require treatment, options are limited to microsurgery or irradiation (IR). Development of alternative therapies that augment or replace microsurgery or IR would benefit patients not suitable for current therapies. This study explored the ability of ErbB2 inhibitors to modulate the effects of IR on VS cells. Study Design: Prospective study using primary cultures derived from human VSs. Methods: Primary cultures of VS cells were derived from acutely resected tumors. Cultures received single escalating doses (15–40 Gy) of γ‐irradiation from a ¹³⁷Cs γ‐irradiation source. Cell proliferation was determined by BrdU uptake and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Trastuzumab (Herceptin) and PD158780 were independently used to inhibit ErbB2 signaling while neuregulin‐1β (NRG‐1) was used to activate ErbB2. Results: IR induces VS cell cycle arrest and apoptosis in doses greater than 20 Gy, demonstrating that VS cells are relatively radioresistant. This radioresistance likely arises from their low proliferative capacity as a sublethal dose of IR (10 Gy) strongly induces deoxyribonucleic acid (DNA) damage evidenced by histone H2AX phosphorylation. Inhibition of ErbB2, which decreases VS cell proliferation, protects VS cells from radiation‐induced apoptosis, while NRG‐1, an ErbB2 ligand and VS cell mitogen, increases radiation‐induced VS cell apoptosis. Conclusions: Compared with many neoplastic conditions, VS cells are relatively radioresistant. The radio‐protective effect of ErbB2 inhibitors implies that the sensitivity of VS cells to IR depends on their proliferative capacity. These results hold important implications for current and future treatment strategies.     

ErbB and Nrg: potential molecular targets for vestibular schwannoma pharmacotherapy.

OBJECTIVE: Identify molecular targets for development of tumor-specific pharmacotherapeutics aimed at treating vestibular schwannomas (VSs). Activated epidermal growth factor receptor B (ErbB) 2 and ErbB3 are abundantly expressed in VS. ErbB2 signaling is essential for Schwann cell differentiation, survival, and proliferation. VS arise after loss of functional merlin, a putative tumor suppressor. Merlin internalizes ErbB2 receptors in rodent Schwann cells.Unregulated ErbB signaling may contribute to VS tumorigenesis. STUDY DESIGN: Molecular analyses, retrospective clinical correlation. SETTING: Tertiary referral center. PATIENTS: Thirty-eight specimens from patients operated for sporadic (n=21) and neurofibromatosis (NF) 2-related (n=17) VS. INTERVENTION(S): VS analyses via real-time polymerase chain reaction, immunohistochemistry, and correlation with patient clinical data. MAIN OUTCOME MEASURE(S): ErbB signaling molecule expression, tumor size, age, and NF2 status. RESULTS: VS upregulated epidermal growth factor (EGF) receptor in 68% (62% sporadic and 75% NF2-associated VS) and ErbB2 in 84% (76% sporadic and 94% NF2-related VS). ErbB3 was upregulated in 34%, and ErbB4 is downregulated in NF2-related VS. Of EGF receptor (EGFR) ligands, EGF was upregulated in all NF2-related VS, but none of the sporadic VS (p<0.01), and transforming growth factor alpha and beta-cellulin showed upregulation in 67% of NF2-related VS but not sporadic VS (p=0.02 and p=0.01, respectively). Neuregulin (Nrg) was upregulated in 86% of sporadic VS versus 19% of NF2-related VS (p<0.01). EGFR expression levels correlated directly with VS tumor size and inversely with patient age, whereas Nrg expression correlated directly with age (p=0.0005). EGF expression predicts NF2 status, whereas Nrg predicts non-NF2 status (p<0.01). CONCLUSION: These findings implicate the ErbB pathway in VS growth and as potential molecular targets for VS pharmacotherapy.     

Merlin蛋白在听神经瘤组织中的表达与分布

目的明确听神经瘤中merlin蛋白的表达及其在细胞内的分布。方法收集临床诊断并且经过术后病理证实的听神经瘤组织石蜡包埋标本54例,用免疫组织化学方法分析merlin蛋白在肿瘤组织中的表达,取三叉神经痛和晚期梅尼埃病患者接受手术治疗术中切除的神经组织作为对照组。用Western blot方法测试肿瘤组织中merlin蛋白的电泳迁移行为。用图像分析方法计算每例患者的阳性表达率,将merlin蛋白表达阳性率与患者年龄、性别、肿瘤生长指数、肿瘤直径和临床分期进行比较。结果merlin蛋白在听神经瘤组织中有不同程度的表达,阳性率为0～87．5％,平均(46．66±5．75)％,与肿瘤生长指数之间无明显相关性,与患者年龄、性别、肿瘤直径和临床分期无明显相关性。merlin细胞内分布部位有一定差异,以细胞核内、核周分布为主,也有部分分布于细胞质中。Western blot显示在听神经瘤组织中merlin蛋白位于65 000和125 000。结论在听神经瘤组织中存在merlin蛋白的表达,其在细胞内分布部位有一定变异。听神经瘤组织中merlin蛋白可能同时与其他蛋白以复合物的形式存在。     

Primary cultures of human vestibular schwannoma: selective growth of schwannoma cells.

OBJECTIVE: To establish primary vestibular schwannoma (VS) cultures that selectively favor the growth of schwannoma cells. BACKGROUND: The lack of a suitable in vitro model of human VS cells has directly limited the progress of research on tumorigenesis and therapy. The problems of establishing pure VS culture include control of fibroblast proliferation. Current efforts to extend VS cell life span using viral oncogenes, by conferring the ability to proliferate in vitro, will yield cells intrinsically different from in vivo VS tumors. Much more desirable is the ability to culture VS cells without cellular transformation. METHODS: Tumor specimens from 17 patients were processed for cell culture and grown at 37 degrees C with 5% CO2 and 100% humidity. Key modifications limiting fibroblast proliferation included using selective medium without L-valine, supplemented by Nu-Serum for at least a week; the use of cytosine arabinoside to kill contaminating fibroblasts; and using the Dulbecco modified medium, supplemented with brain-derived neurotrophic factor and 10% fetal calf serum after the initial serum-free period. RESULTS: Twelve of 17 VS were successfully cultured. The presence of schwannoma cells and the absence of fibroblasts were confirmed immunohistochemically using S100 and CD90 markers, respectively. Scanning and transmission electron microscopy demonstrated typical spindle-shaped cells and the presence of "fibrous long-spacing collagen." CONCLUSION: We describe a method for obtaining short-term, essentially fibroblast-free, primary VS cultures. Such pure VS cultures, retaining in vivo characteristics, are extremely useful as an in vitro model to study the pathobiology of schwannoma cells.     

The natural history of vestibular schwannoma.

OBJECTIVE: The incidence of vestibular schwannomas (VSs) approaches 20 per million/yr. As treatment may depend on tumor growth, there is a demand of a treatment strategy based on hard data on the growth pattern of these tumors. This article reports growth data registered prospectively in 552 patients. STUDY DESIGN: Of the 1,818 consecutive patients, diagnosed with VS during the period from 1975 to 2005, 729 patients were allocated to observation by repetitive magnetic resonance imaging. At least two scans had been performed in 552 patients at the time of data analysis. Two hundred thirty patients had a tumor confined to the internal acoustic meatus, whereas 322 patients had a tumor with an extrameatal extension. Growth to extrameatal extension was the definition for growth in intrameatal tumors, whereas a largest diameter change of more than 2 mm was the criteria for growth/shrinkage of extrameatal tumors. The mean observation time was 3.6 years (range, 1-15 yr). RESULTS: Seventeen percent of the intrameatal tumors grew, whereas significantly more of the extrameatal tumors displayed growth during the period (28.9%). Growth occurred within the first 5 years after diagnosis. No correlation could be demonstrated between tumor growth rate, sex, or age. CONCLUSION: VS growth occurs within the first 5 years after diagnosis in a limited number of tumors, primarily in tumors with an extrameatal extension. We found no relation between tumor growth and sex or age. These findings justify primary observation of small tumors. A treatment strategy is proposed for this disease, focusing on the patient group allocated to observation.     

Cochlear implantation in patients with neurofibromatosis type 2 and bilateral vestibular schwannoma.

OBJECTIVE: To investigate the results of cochlear implantation in patients with neurofibromatosis Type 2 (NF2) and bilateral vestibular schwannoma. STUDY DESIGN: Retrospective case review. SETTING: Three academic tertiary referral centers. PATIENTS: Seven patients with NF2 and bilateral vestibular schwannoma who lost hearing in at least one ear after treatment of their tumor (surgery or radiation therapy). INTERVENTION: Cochlear implantation after treatment of their vestibular schwannoma. MAIN OUTCOME MEASURE: Postimplantation audiometric scores (pure-tone average thresholds, consonant-nucleus-consonant (CNC) words/phonemes, Central Institute for the Deaf (CID) sentences, Hearing in Noise Test (HINT) quiet/noise, and Monosyllable, Trochee, Spondee (MTS) recognition/category tests), patient satisfaction, and device use patterns. RESULTS: The average age at implantation was 40 years (range, 16-57 yr). Follow-up ranged from 6 to 88 months after implantation. Three patients were implanted with residual useful hearing in the contralateral ear, whereas four patients had no hearing in the contralateral ear. Hearing loss was due to surgical excision of tumor (n=5) or gamma-knife radiotherapy (n=2). Postactivation pure-tone average thresholds in the implanted ear ranged from 30 to 55 dB (average, 32.5 dB), although speech reception testing varied considerably among subjects. Despite this variability, all patients continue to use the device on a daily basis. CONCLUSION: In selected cases of deafness in patients with NF2 where there has been anatomic preservation of the auditory nerve after acoustic neuroma resection or radiation therapy, cochlear implantation may offer some improvement in communication skills, including the possibility of open-set speech communication in some patients. These results compare favorably to the auditory brainstem implant offering an alternative for hearing rehabilitation in patients with NF2.     

The learning curve in vestibular schwannoma surgery.

OBJECTIVE: This study aimed to examine the effect of surgical team experience on facial nerve function and complication rate in vestibular schwannoma surgery. STUDY DESIGN: The study design was a retrospective analysis of a case series. SETTING: The study was conducted at a tertiary referral center. PATIENTS: One hundred sixty consecutive patients undergoing vestibular schwannoma excision participated. INTERVENTION: Surgical excision of vestibular schwannoma via a translabyrinthine, middle cranial fossa, suboccipital, or combined approach was performed. MAIN OUTCOME MEASURES: Facial nerve function (House-Brackmann score) and complication rates including cerebrospinal fluid leak and meningitis compared by groups of 20 patients were measured. RESULTS: There was a statistically significant improvement in the number of patients achieving a House-Brackmann grade I result between the first 20 patients (35% House-Brackmann grade 1) and the ensuing 7 groups of 20 patients (74% House-Brackmann grade 1) by chi2 analysis. When considering House grades I and II together, there was no statistically significant difference in facial nerve function in the first 20 patients (80%) compared to the last 7 groups of 20 patients (88%) by Tukey's pairwise comparisons (p = 0.245). Mean tumor size was not significantly different in the groups studied (p = 0.54). The total cost of patient care declined over the study period; however, the wide case-to-case variance made it so that this trend was not statistically significant (p = 0.448). CONCLUSIONS: A learning curve of 20 patients was demonstrated by this study to have been necessary for attaining acceptable standards in the surgical removal of vestibular schwannomas by a new surgical team. The findings of this study may have implications for patient care and surgeon training.     

Co-existing cholesteatoma and vestibular schwannoma

A 69-year-old man presented with a cholesteatoma in the right mastoid process and a vestibular schwannoma at the left internal acoustic meatus. Cholesteatoma co-existing with a vestibular schwannoma has not been documented previously in the contemporary literature. The clinical dilemma in the management of his progressive bilateral hearing loss is discussed. He presented with dizziness and bilateral hearing loss worse on the right side. Pressure over the mastoid process elicited vertigo and nystagmus. He had no history of previous operation or infection in the ear canal. Audiograms confirmed high-tone hearing loss. Radiological investigations revealed a symptomatic cholesteatoma on the right side and an incidental vestibular schwannoma on the left. We have elected to manage both lesions conservatively. Bilateral cholesteatoma and bilateral vestibular schwannomas have been previously reported. Co-existing lesions, as in our patient have, however, not been reported previously. The management options of his hearing loss are discussed.     

Clinical picture of vestibular schwannoma

PURPOSE: To characterize the clinical picture with vestibular schwannoma, we retrieved 122 patients with vestibular schwannoma from the vestibular unit of the Helsinki University Central Hospital. PROCEDURES: The patients filled out a questionnaire concerning their symptoms, earlier diseases, accidents, and the use of tobacco and alcohol. This information was then completed with results of audiometric, neurotologic and imaging studies. The data were prospectively stored to the database of neurotologic expert system called ONE. RESULTS: The average tumor size was 21 mm. Most of the patients had hearing loss (94%) and tinnitus (83%) but only half of them (49%) had vertigo attacks. The most common initial symptom was hearing loss combined with tinnitus (34%, n=44). Vertigo only was the initial symptom for 12 patients. The mean duration of a vertigo attack was from 5 min to 4 h and the intensity of the attack was regularly mild (37%) or moderate (32%). The vertigo of the vestibular schwannoma patients differed from vertigo in other diseases by the absence (63%) or low intensity (18%) of nausea. Spontaneous nystagmus was present in 56 patients (46%), and caloric asymmetry (>25%) was observed in 66% (n=69). Caloric asymmetry increased with tumor size. The prevalence of headache was 18% (n=21). Sudden slips seldom (7%) occurred. CONCLUSION: Hearing loss and tinnitus are the main symptoms of vestibular schwannoma. Only half of patients have vertigo. Screening to detect vestibular schwannoma is more yielding in sudden deafness patients rather than in patients with Meniere's disease.     

Management of intrameatal vestibular schwannoma

The growth of purely intrameatal vestibular schwannoma (VS) was investigated, in the period 1973-96 in a series of 40 patients with 40 unilateral VS. In the present study, the material was analysed and updated. By the end of the observation period (mean 3.6 years), 27 tumours (67.5%) revealed growth and 13 tumours (32%) had no measurable growth. Four growth patterns were observed: (A) 15 tumours (37.5%) exhibited constant growth; (B) 13 tumours (32.5%) had no measurable growth; (C) 8 tumours (20%) revealed growth subsequent to a no-growth period; and (D) 4 tumours (10%) showed different growth patterns during the observation period. The annual diameter growth rate ranged between 00 mm/year and 6.5 mm/year and the mean diameter growth per year was 3.2 mm. The findings of the present study, especially those for group B (the non-growing tumours) and C (tumour growth subsequent to a silent period) bring into question the reliability of the results achieved by radiosurgery, as without any intervention it may be that no tumour growth occurs.     

Neurotrophic factor expression in vestibular schwannoma. An overview

The vestibular schwannoma is a benign, slow-growing neoplasm that originates from the neurolemmal sheath of the vestibular branch of the VIIIth cranial nerve. This tumor entity accounts for 6 % of all intracranial tumors and the annual incidence of newly diagnosed vestibular schwannoma is reported as 13 per million. The molecular pathogenesis of both sporadic vestibular schwannoma and those occurring in neurofibromatosis type II appears to be associated with an aberration of a tumor suppressor gene on chromosome 22q12. The biological background for the various growth patterns of vestibular schwannoma is, however, largely unknown. This differing clinical and biological behaviour of vestibular schwannoma may be explained by the presence of neurotrophic factors. The results of recent immunohistochemical studies demonstrate the co-expression of transforming growth factor (TGF)-beta 1 and glial cell line-derived neurotrophic factor (GDNF) in vestibular schwannoma and suggest a trophic synergism of both neurotrophic factors in this tumor. Moreover, expression of numerous different neurotrophic factors has been shown in studies of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), neuregulin (NRG) and erythropoietin (EPO) indicating a biological role in development, maintainance or growth of vestibular schwannoma. In this article, we summarize the findings on neurotrophic factor expression and discuss their characteristics and biological role in vestibular schwannoma.     

Auditory pathway function after vestibular schwannoma surgery.

We studied seven patients before and after vestibular schwannoma surgery. Four patients became unilaterally profoundly deaf and three patients preserved their hearing. Cortical responses were recorded with a 122-channel whole-scalp SQUID neuromagnetometer using tone-burst stimuli to the healthy ear. Brainstem auditory evoked potentials (BAEPs) were measured using alternating clicks. Ten healthy volunteers served as a control group. In patients, preoperative cortical response latencies and strengths did not differ significantly from those of controls. However, 6 months after the operation the latency was, on average, 7 ms longer than preoperatively over both hemispheres. BAEPs were in the normal range both before and after the operation. These results suggest that unilateral lesion in peripheral auditory pathways also affects cortical reactivity to stimuli presented to the non-affected ear, possibly reflecting altered binaural interaction in the auditory pathways.