Expression Pattern of Apoptotic Markers in Vestibular Schwannomas

PATHOLOGY

Original Paper

RESEARCH AND PRACTICE © Urban & Fischer Verlag http://www.urbanfischer.de/journals/prp

Expression Pattern of Apoptotic Markers in Vestibular Schwannomas Christian Mawrin1,2, Elmar Kirches1, Knut Dietzmann1, Albert Roessner3, Carsten Boltze3 Departments of 1Neuropathology, 2Psychiatry, and 3Pathology, Otto-von-Guericke-University, Magdeburg, Germany

Summary 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. Key words: Bcl-2 – Bax – Fas – Fas-L – MIB – Vestibular – Schwannoma

Introduction It is now well accepted that clonal expansion and tumor growth are the result of the deregulation of intrinsic proPathol. Res. Pract. 198: 813–819 (2002)

liferation and cell death (apoptosis). Failure of apoptotic cell death may result in survival of transformed cells that are prone to undergo further genetic damages and thus play an important role in tumorigenesis [22, 29]. The Fas-Fas ligand (Fas-L) system is regarded as a major pathway for the induction of apoptosis in cells and tissues [26, 29]. The Fas (F7-associated surface protein) antigen, also referred to as apoptosis-inducing protein 1 (APO-1) or cluster of differentiation-antigen 95 (CD95), is a 40–50 kDa type I transmembrane receptor which belongs to the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor family. Fas functions as a mediator of apoptosis, and loss of the Fas protein has been reported to induce resistance to apoptosis; however, apoptotic resistance has also been reported to occur in some Fas-expressing malignant cells [32]. Fas ligand (Fas-L) is a key molecule in normal immune development, homeostasis, modulation and cell function, inducing apoptosis by binding to its receptor Fas. Fas-L is a type II integral membrane protein that is homologous to TNF. It is cleaved by a metalloproteinase to produce an active, soluble molecule [8]. Tumor cells can escape from Fas-mediated apoptosis by the presence of inhibitory mechanisms, including the production of soluble Fas (sFas) [5], production of decoy receptor for Fas-L [27], or lack of cell surface Fas expression [24]. Another escaping mechanism is the

Address for correspondence: Carsten Boltze, Department of Pathology, Otto-von-Guericke University, Leipziger Strasse 44, D-39120 Magdeburg, Germany. Phone: +49-391-671 7871, Fax: +49-391-671 5818. E-mail: [email protected] 0344-0338/02/198/12-813 $15.00/0

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overexpression of inhibitory proteins in signal transduction pathways, such as Fas-associated phosphatase 1 (FAP-1) [30], and members of the Bcl-2 family [11]. Bcl-2 is an inner membrane protein present in mitochondria and rough endoplasmic reticulum, but its mechanisms of action are not well understood [10]. Although Bcl-2 overexpression was initially found in follicular lymphomas harboring a t(14;18) translocation that activates the Bcl-2 gene, this protein is now known to be widely expressed in non-lymphoid cells and various tumors, including tumors of the nervous system, despite the absence of the t(14;18) translocation [9, 20, 23]. Bax is another member of the Bcl-2 family; however, in contrast to Bcl-2, it has an apoptosis-stimulating function [2]. The pro-apoptotic action of Bax depends on the formation of Bax homodimers on the outer mitochondrial membrane [31]. The formation of Bcl-2-Bax heterodimers is at least partly responsible for the antiapoptotic effects of Bcl-2 [25]. Schwannomas are benign tumors arising predominantly from cranial and spinal nerves and, less frequently, from peripheral nerves. They account for approximately 8% of all primary intracranial tumors, and they usually occur as solitary and sporadic lesions [3]. Furthermore, they can occur in the setting of neurofibromatosis [35]. Although malignant transformation of schwannomas is very rare [34], there is a significant number of cases showing recurrences irrespective of the method of treatment [4, 12, 13]. However, the role of disregulated apoptosis for the growth of sporadic schwannomas is not well investigated. The function of the Fas-Fas-L system, in particular, has not been determined in these tumors so far. Therefore, in the present study, we investigated the immunoexpression of Fas and its ligand, as well as the expression of Bcl-2 and Bax in sporadic vestibular schwannomas, and related the findings to the proliferation rate of the tumors.

using 0.01 M sodium acetat buffer (pH 6.0) for 3 × 10 min. Endogeneous peroxidase activity was blocked by incubation (30 min) in 0.3% H2O2 in methanol. The sections were gently rinsed with TBSbuffer and then incubated with bovine serum albumin for 30 minutes to reduce non-specific antibody binding. Sections were incubated with monoclonal antibodies against MIB-1 (clone Ki-S5; dilution 1:50), Bcl-2 (clone 124, 1:100), CD95/Fas (clone APO-1, 1:10; all from DAKO, Hamburg, Germany), and polyclonal antibodies against Bax (DAKO, Hamburg, Germany; 1:100) and Fas-L (Santa Cruz, CA; 1:100) for 60 min at 37 °C in a humified chamber. Negative controls included omission of the primary antibody and its substitution by an irrelevant mouse monoclonal antibody. The signal was detected using the streptavidin-biotin-peroxidase complex method (LSAB) according to the manufacturer’s recommendation (DAKO, Hamburg, Germany). DAB (3,3′diaminobenzidine hydrochloride containing 0.08% hydrogen peroxide) was used as a chromogen to visualize peroxidase activity. Finally, the sections were counterstained with hematoxylin. The immunoreaction of the antibodies was evaluated using two different approaches. Immunoreaction localized within the cytoplasm (Fas, Bcl-2, Bax) was graded semiquantitatively as follows: – no immunoreaction, + weak, ++ moderate, +++ strong. In addition, a labeling index (LI [%]) was calculated for Bcl-2 and Bax as the percentage of stained cytoplasms after counting 10 high power fields (HPF; ×400). At least 1,000 cells were counted. For all antibodies demonstrating immunoreaction restricted to the tumor cell nuclei (MIB-1, Fas-L), a LI was calculated by determining the number of immunopositive nuclei among 100 tumor cell nuclei in the same manner as described above. Only a distinct nuclear immunoreaction was jugded as positive for MIB-1 and Fas-L. To avoid inappropriate counting, no tumor areas with lymphocytic infiltrates were evaluated, because lymphocytes were usually positive for the apoptotic markers.

Materials and Methods We investigated a total of 14 sporadic vestibular schwannomas, which were selected from the files of the Department of Neuropathology, Otto-von-GuerickeUniversity Magdeburg, Germany. The diagnoses were based on clinical information, review of hematoxylin and eosin-stained tissue sections, and previous immunohistochemical investigations (i.e. S-100 protein expression). None of the patients examined had bilateral familial vestibular schwannomas or other clinical stigmata suggestive of the presence of neurofibromatosis. The tumor specimens were routinely formalin-fixed and paraffin-embedded. For immunohistochemistry, 4-µm-thick sections were deparaffinized with xylene for 15 min and dehydrated through a series of graded alcohols. Sections were pretreated in a microwave oven

Statistical analysis Correlations between different LI´s were determined by calculating the Pearson correlation coefficient, as well as the Spearman rank order correlation coefficient; pvalues below 0.05 were considered significant. Differences in the labeling indices determined, in the context of the presence or absence of an apoptotic marker were assessed using the nonparametric Mann-Whitney U-test. All analyses were carried out using the SPSS for Windows software (SPSS, Chicago, Ill., Version 10.0.5).

Results The clinicopathologic data of all 14 patients examined in this study are summarized in Table 1. In all schwan-

13.9 15.4 28.5 – 28.1 17.4 10.4 31.8 – 24.1 19.6 38.8 24.3 36.2 – + – – – – – – – – – – – – 9.2 17.8 3.2 – 12.5 17.0 – – 13.8 – 7.3 23.6 6.8 2.7 ++ +++ + – + +++ – – + – + + + + F = female, M = male; R = right, L = left; C = cytoplasm, N = nuclei; LI = labeling index

10.7 – 3.8 4.3 12.7 – – 6.7 – – 10.7 10.6 – 12.6 +++ + + ++ +++ – – ++ – – +++ + – +++ 6.8 3.7 3.8 2.0 6.3 4.5 3.3 6.2 7.3 3.3 4.0 3.9 5.9 3.5 deafness deafness sudden deafness deafness deafness deafness deafness deafness deafness deafness deafness deafness tinnitus, sudden deafness deafness R R R L L L R L R R R L R L 56 58 52 52 59 59 53 60 73 59 63 56 54 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14

F M F M M F F F M F F M M M

Bcl-2 (C) MIB-1 (N) LI (%) Clinical Symptoms Localization Sex Age (years) Case No.

Table 1. Clinical and immunohistochemical data of patients with vestibular schwannomas.

Bcl-2 (N) LI (%)

Bax (C)

Bax (N) LI (%)

Fas (C)

Fas-L (N) LI (%)

Apoptotic Markers in Schwannomas · 815

nomas, strong expression of S-100 protein within the cytoplasm of the neoplastic schwann cells was seen (data not shown). The mean proliferation index of the tumors as jugded by MIB-1 immunostaining was 4.6% ± 0.4%. There were no marked differences in the number of stained nuclei between different tumor areas; the MIB-1 LI´s ranged from 2.0% to 7.3%. Weak cytoplasmic immunoexpression of Fas (+) was observed only in one patient (7%). In addition, immunoexpression was restricted to small regions within this vestibular schwannoma (Figs. 1E–H). In contrast, most tumors showed strong nuclear immunoreaction for Fas-L (12/14, 86%). Nuclear Fas-L expression was observed in highly cellular Antoni A-regions, as well as in the Antoni B-regions with low cellularity (Figs. 1I, 1K). The mean Fas-L LI was 20.6% ± 3.2%. Variable numbers of lymphocytes also expressed Fas-L. Anti-apoptotic Bcl-2 was expressed in 9 schwannomas (64%). Immunoreaction was found in the cytoplasm and tumor cell nuclei. Among the tumors with Bcl-2 expression, strong immunoreaction (+++) was found in 4 tumors (44%), moderate immunoreaction (++) in 2 (22%), and weak expression (+) in 3 tumors (33%) (Figs. 1A, 1B). In the cellular Antoni-A regions, the expression of Bcl-2 tends to be stronger than in the Antoni-B regions. Sometimes, endothelial cells and lymphocytes were also stained. The mean nuclear Bcl-2 LI was 5.2% ± 1.4%. Pro-apoptotic Bax expression was observed in the cytoplasm of schwannoma cells and within the tumor cell nuclei (Figs. 1C, 1D). Cytoplasmic immunoexpression of Bax was found in 10 schwannomas (71%). Compared to Bcl-2, the cytoplasmic immunoreaction of Bax was more diffuse. Strong cytoplasmic immunoexpression of Bax (+++) was seen in 2 cases (20%); moderate (++) immunoexpression was found only in one case (10%), whereas most schwannomas (7 cases; 70%) exhibited a weak (+) expression of Bax protein within the cytoplasm. Cytoplasmic immunoreaction was always stronger in the Antoni-A areas than in the Antoni-B areas (Fig. 1D). The mean labeling index for the nuclear staining of Bax in these 10 schwannomas was determined as 8.1% ± 2.1%. Nuclear Bax expression did not differ between Antoni-A and Antoni-B areas. Statistical analysis revealed no significant correlations between the different labeling indices. However, there was a slight tendency towards a positive correlation between Fas L LI and Bcl-2 LI (r = 0.503, p = 0.067; Pearson´s correlation coefficient). It is interesting to note that all schwannomas expressing Bcl-2 were negative for Fas, whereas the single Fas-positive tumor (case no. 2) contained only small amounts of Bcl2. To define an association between the expression of cell death markers and cell proliferation in vestibular

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schwannomas, we compared the MIB-1 labeling indices as a marker for the proliferation rate of tumors expressing apoptotic proteins (Bcl-2 LI, Bax LI) using the nonparametric Mann-Whitney U test. We found no significant association between the expression of Bcl-2 and MIB-1 LI (p = 0.947, NS). However, schwannomas expressing pro-apoptotic Bax tended to have a higher MIB-1 LI than tumors lacking Bax (Fig. 2). However, this association was not statistically significant (p = 0.066), but this may, at least to some extent, be attributed to the limited number of cases studied.

Discussion The aim of our study was to ascertain whether proteins of the apoptotic pathway via the Fas-Fas-L system are expressed in benign vestibular schwannomas. We found that nearly no Fas was immunohistochemically detectable in these tumors, whereas Fas-L was present in most vestibular schwannomas. Our findings suggest that the Fas-Fas-L system does not play an important role in the induction of cell death in vestibular schwannomas. However, the occurrence of apoptosis in benign schwannomas has already been reported. Apoptotic figures were found to be related to high proliferation rates as determined by MIB-1 immunostaining [1], and were observed after radiotherapy for recurrent schwannoma [7, 13]. High apoptotic rates have also been described in schwannomas of the dog [16]. Schwannoma cell culture studies have shown that apoptosis can be induced by transforming growth factor beta 1 (TGF-β1), and is at least in part mediated via the mitogen-activated protein kinase and phosphatidylinositol-3-kinase/Akt-(PI3/Akt) pathway [17, 18]. Apoptosis via PI3/Akt can be induced by the tumor suppressor PTEN, and we recently found that the expression of PTEN is maintained in most vestibular schwannomas [19]. Interestingly, a recent study analyzing cDNA microarrays found a significant downregulation of the apoptosis-related LUCA-15 gene in vestibular schwannomas [33]. In contrast to benign schwannomas, in malignant soft tissue tumors, such as malignant schwannomas, rhabdomyosarcomas, and Ewing´s sarcoma, Fas-L was found to be present in a high percentage of tumors [14, 21]. However, although the biologic significance of the Fas-Fas-L system has been demonstrated in Ewing’s sarcoma by the simultaneous presence of Fas and Fas-L [21], no data are currently available for malig-

nant soft tissue tumors originating from neoplastic schwann cells, i.e. malignant peripheral nerve sheath tumors (MPNSTs). In our study, we found that a high percentage of schwannomas expressed Bcl-2, which can act as an antiapoptotic factor by inhibiting Fas-mediated apoptosis [11]. As previous studies have also demonstrated a widespread expression of Bcl-2 in schwannomas [9, 20, 23], Fas-Bcl-2-interaction may significantly contribute to the lack of Fas-expression. In sympathetic neurons, high levels of anti-apoptotic Bcl-2 were reported, but apoptosis was not mediated via Fas signaling [28]. Therefore, our finding of high Bcl-2 expression but low Fas levels suggests that apoptosis in schwannomas is predominantly induced via the so called “intrinsic” pathway by releasing mitochondrial cytochrome c and, subsequently, by opening the transition pore with caspase activation and ultimate cell death [15]. In contrast, the “extrinsic” pathway, which involves death receptors of the TNF superfamily and activation of the Fas-Fas-L system, is less likely to operate in schwannoma cell death. However, it has been shown that in non-glial brain tumors, despite the presence of Fas-Fas-L proteins, correlation with Bcl-2 expression was missing, suggesting that protection from Fas-mediated cell death may involve other mechanisms [6]. Finally, our finding that a high number of tumors expressed both Bcl-2 and Bax can be considered a hint for

Fig. 2. Relation between the presence (+) or absence (–) of cytoplasmic Bax staining and MIB-1 labeling index in vestibular schwannomas. The difference is not statistically significant (Mann-Whitney U-test: p = 0.066).

b Fig. 1. Examples of immunohistochemical staining of Bcl-2 (A, B), Bax (C, D), Fas (E–H) and Fas-L (I, K). Most cases were immunonegative for Fas (E, F); only in case No. 2 (G, H) was Fas detectable focally. (A, C, E, G, I: magnification ×100; B, D, F, H, K: magnification ×400).

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the proposed formation of heterodimers between both proteins [25], inhibiting apoptosis in these cases. It has already been reported that schwannomas with high numbers of apototic figures simultaneously show increased cell proliferation with an increased number of MIB-1 positive tumor cells [1]. Therefore, our finding that increased MIB-1 labeling indices are seen in tumors expressing pro-apoptotic factor Bax suggests that the balance between cell proliferation and cell death is maintained in benign vestibular schwannomas, which may, at least partly, be responsible for the slow growth of these tumors. Acknowlegdements. The technical assistance of S. Hartmann and T. Fuchs is gratefully acknowlegded.

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