Bcl-2 and LMP1 expression in nasopharyngeal carcinomas

Original Contributions Bcl-2 and LMP1 Expression in Nasopharyngeal Carcinomas Sarp Sarac, MD,* M. Umut Akyol, MD,* Bilge Kanbur, MD,† Aylar Poyraz, MD,‡ Gu¨len Akyol, MD,‡ Taner Yilmaz, MD,* and Arzu Sungur, MD§ In vitro studies of B-cells immortalized by Epstein-Barr Virus (EBV) have shown that B-cell leukemia/lymphoma-2 (bcl-2) protein expression is upregulated by the EBV-latency–associated antigen, latent membrane protein 1 (LMP1). The same phenomenon has also been observed in epithelial cells in vitro. However, such correlation between these 2 markers has not been shown in nasopharyngeal carcinoma (NPC), in which EBV infection is a well-known etiologic factor. This retrospective study examined 35 cases of undifferentiated NPC to answer the question of whether LMP1 and/or bcl-2 presence can be used as biomarkers at treatment response, as well as to see the relationship between bcl-2 and LMP in NPC. Of the 35 patients, 26 (74.3%) were bcl-2, and 10 (28.6%) were LMP1-positive. Although a significant correlation was observed between bcl-2 and LMP1 staining (P ⫽ .003), it appeared that bcl-2 expression could be independent of LMP1. Statistical analysis showed that cervical lymph node metastasis (P ⫽ .04) and cranial nerve involvement (P ⫽ .03) are the only variables that significantly affect patient survival. At this time, bcl-2 and LMP1 presence are not significant indicators of outcome; however, although they are not directly related to survival, expression of both bcl-2 and LMP1 was strongly correlated with cervical lymph node metastasis, which is a potent predictor of patient survival. (Am J Otolaryngol 2001;22:377-382. Copyright © 2001 by W.B. Saunders Company)

Epstein-Barr virus (EBV) is a herpes virus associated with a number of malignant diseases, such as Burkitt’s lymphoma, nasopharyngeal carcinoma, (NPC) and Hodgkin’s disease.1,2 Infection of normal B-lymphocytes with EBV leads to the establishment of lymphoblastoid cell lines (LCLs) with infinite proliferative potential. This transformation is closely associated with the expression of 6 Epstein-Barr nuclear antigens (EBNAs 1-6), 2 latent membrane proteins (LMPs 1-3), and 2 small, nonpolyadenylated RNAs, which are constitutively expressed from the viral ge-

From the *Hacettepe University Medical School, Department of Otolaryngology; †Otolaryngologist; ‡Gazi University Medical School, Department of Pathology; and the §Hacettepe University Medical School, Department of Pathology, Ankara, Turkey. Address reprint requests to M. Umut Akyol, MD, Iran Cad. 47/10, GOP, Ankara, 06700 Turkey. Copyright © 2001 by W.B. Saunders Company 0196-0709/01/2206-0001$35.00/0 doi:10.1053/ajot.2001.28071

nome in LCLs.3 EBNA 2, EBNA-3A, EBNA-3C and LMP1 are particularly essential for this process.4-6 Although successful transformation of B-cells requires a coordinated expression of these genes, many of the transformation-associated properties of EBV can be reproduced in various cell systems by expression of just 1 viral gene, whose product is a 386-amino acid integral membrane protein, LMP1.7 The immortalizing effect of LMP1 on B-lymphocytes has been shown to be mediated by the B-cell leukemia/lymphoma-2 (bcl-2) gene.8 The product of this oncogene, bcl-2 protein, is an inner mitochondrial membrane protein whose overexpression blocks the programmed apoptotic cell death.9 Induction of bcl-2 expression by LMP has been shown in cases of Burkitt’s lymphoma,10 non-Hodgkin’s lymphoma,11 and acquired immunodeficiency syndrome–related primary brain lymphomas.12 Although some studies state that upregulation of bcl-2 by LMP1 is a B-cell-specific response,7 cooperative interaction of these 2 proteins has also

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been observed in the growth transformation of human epithelial cells.13 Still, a previous study on NPC has failed to show any correlation between bcl-2 and LMP1.14 This study was designed to investigate the possible prognostic values of bcl-2 and LMP1 in NPC and their correlation. MATERIALS AND METHODS Patients The study sample consisted of 35 patients with undifferentiated NPC who were diagnosed and treated at Hacettepe University Medical School Hospital, Ankara, Turkey, between 1979 and 1993. Patient charts were examined for gender; age; primary tumor, regional lymph nodes, and distant metastasis (TNM) status; stage; cranial nerve involvement; cervical lymph node metastases (histopathologic); distant metastases; disease recurrences; and survival periods. Patients were included only if they had posttreatment follow-up of at least 3 years, or died of disease within this period. Plain radiographs, computed tomography, and magnetic resonance imaging (after 1992) were used to determine soft tissue and bone involvement. All patients were treated primarily with radiotherapy, with surgery limited to neck dissections in which radiotherapy has controlled the primary tumor but failed in the neck.

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ered saline. Peroxidase activity was detected with 3,3⬘diaminobenzidine tetrahydrochloride and hydrogen peroxide. The sections were counterstained with Mayer’s hematoxylin. Appropriate positive and negative controls were prepared in the same fashion. Both cytoplasmic and membranous staining pattern was considered positive for LMP and bcl-2. The frequency of positive cells was evaluated semiquantitatively and determined as the ratio of positive-stained tumor cells to overall tumor cell population. They were graded from 0 to ⫹3 as follows: 0 ⫽ no tumor cells, ⫹1 ⫽ less than 25% tumor cells, ⫹2 ⫽ 25% to 50% tumor cells, and ⫹3 ⫽ greater than 50% tumor cells. The intensity of staining was not graded, and any expression of LMP1 and bcl-2 were classified as positive.

Statistical Analysis Statistical analysis was performed by using the SPSS for Windows, version 7.5.1, computer program (SPSS Inc, Chicago, IL). Bivariate correlation analysis was used to define any correlation between bcl-2 and LMP1. The Mann Whitney U test was used to find any significant difference between bcl-2 and LMP1 staining in patients with and without cervical lymph node metastasis. Univariate survival analysis with log rank test was run on the clinical, histopathologic, and immunohistochemical data to pinpoint which factors affected overall survival. Cox regression analysis with forward selection was used to define the factors affecting the survival independently. A significance level of .05 was used for all statistical tests.

Histopathology

RESULTS

For conventional histopathology, 6-␮m-thick paraffin sections that were stained with hemotoxylin and eosin were reexamined.

Patients

Immunohistochemical Analysis The 6-␮m-thick sections were deparaffinized in xylene and rehydrated through graded alcohol row. Ten minutes of microwave pretreatment for antigen retrieval in citrate buffer was performed for bcl-2 detection. The samples were incubated in 0.3% hydrogen peroxide for 15 minutes and then with normal goat serum for 10 minutes. Immunostaining was performed with prediluted monoclonal mouse antibodies to c-LMP1 (DAKO, Glostrup, Denmark) and to bcl-2 protein (BioGenex Laboratories, San Ramon, CA). Further steps involved the use of a multilink streptavidin-biotin peroxidase kit (BioGenex Laboratories, San Ramon, CA), in which the samples were first incubated with biotinylated link for 30 minutes and then for another 30 minutes with a labeling reagent that peroxidase-conjugated streptavidin. These steps were undertaken at room temperature and were followed by washing in phosphate-buff-

Twenty-six (74.3%) of the patients were men and 9 (25.7%) were women. Patient age ranged from 10 to 71 years, with a median of 35 years. All individuals had undifferentiated NPC. One patient had stage I (2.9%), 3 had stage II (8.6%), 6 had stage III (17.1%), and 25 had stage IV (71.4%) disease. Cervical lymph node metastasis was present in 20 (57.1%), and 11 (31.4%) patients had paralysis of 1 or more cranial nerves at the time of diagnosis. The most frequently affected cranial nerves were abducens and glossopharyngeus, seen in 5 (14.3%) patients each. Hypoglossal and accessory nerve involvement was seen in 3 (8.6%), and trigeminal and vagus nerve in 2 (5.7%) patients each. One (2.9%) patient had facial nerve involvement. Locoregional recurrence was diagnosed in 19 (54.3%), and 1 or more distant metastases were found in 11

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(31.4%) patients. Most frequent sites of distant metastases were bone (5, 14.3%); lung (4, 11.4%); liver (3, 8.6%); and brain (3, 8.6%). The follow-up interval ranged from 24 to 208 months, with a median of 66 months. A total of 14 (40%) patients died of their disease. Histological Findings Pathologic slides of the cases were reevaluated, and all diagnoses were confirmed as undifferentiated carcinoma on histopathologic examination. Immunohistochemical Analysis Twenty-six cases (74.3%) were stained with anti– bcl-2. Although a cytoplasmic staining pattern was noticed, the dominant pattern was membranous. Positive cells were high in frequency in most of the stained cases (Table 1), and their intensity varied from case to case. LMP1 presence was detected less often than bcl-2 positivity. Only 10 (28.6%) cases were found to be positive, and the staining pattern was membranous (Fig 1). The intensity of staining was not as strong as bcl-2 staining, and the positive cells were evenly distributed in most of the cases (Table 1). The distribution of LMP1 expression in bcl-2 positive and negative patients is given in Table 2. All of the 10 LMP1-positive patients were also positive for bcl-2. On the other hand, there were 16 bcl-2-positive patients who were LMP1 negative. Statistical Analysis A significant positive correlation was observed between bcl-2 and LMP1 (Pearson correlation coefficient⫽0.48, P ⫽ .003). Mann Whitney U tests showed higher staining pos-

TABLE 1. Distribution of bcl-2 and LMP1 Staining Among Patients Bcl-2 Grade 0 1⫹ 2⫹ 3⫹

Patients 9 5 6 15

Fig 1. Positive membranous staining of LMP1 in neoplastic cells. (Immunoperoxidase stain; original magnification ⴛ 460.)

itivity for both bcl-2 (Z ⫽ ⫺3.06, P ⫽ .003) and LMP1 (Z ⫽ ⫺2.37, P ⫽ .05) in patients with cervical lymph node metastasis when compared with those without metastasis. Kaplan-Meier analysis showed that cervical lymph node metastasis (Fig 2) and cranial nerve involvement (Fig 3) were the only variables that significantly affected patient survival, with P values of .04 and .01, respectively. Mean survival time was 110 months for patients with neck metastasis and 151 months for those without this type of involvement. Survival was 66 months for patients with craTABLE 2. Distribution of LMP1 Expression in bcl-2-Negative and Positive Patients

LMP1

Percent (%) 25.7 14.3 17.1 42.9

Patients 25 4 4 2

LMP1

Percent (%) 71.4 11.4 11.4 5.8

Bcl-2

Negative

Positive

Total

Negative Positive Total

9 16 25

0 10 10

9 26 35

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Fig 2. Cumulative survival of the patients with node negative (solid line) versus node positive (dotted line) necks.

nial nerve involvement and 138 months in cases where cranial nerves were unaffected. Bcl-2 expression (P ⫽ .25), LMP1 expression (P ⫽ .08), sex (P ⫽ .60), tumor stage (P ⫽ .41), none stage (P ⫽ .06), and metastasis stage (P ⫽ .47) were not correlated with overall patient survival. Multivariate analysis was performed with the Cox regression analysis with forward

Fig 3. Cumulative survival of the patients with and without cranial nerve involvement.

selection. All the clinical, histologic, and immunohistochemical data were entered, and the factors independently affecting the survival were found to be cervical lymph node metastasis (␹2 ⫽ 4.94, P ⫽ .02) and cranial nerve involvement (␹2 ⫽ 6.24, P ⫽ .012), with hazard ratios of 2.71 and 0.26 consecutively for 95% confidence interval.

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DISCUSSION The results concerning the prognostic significance of bcl-2 expression in head and neck cancers are conflicting. The overexpression of bcl-2 was shown to be associated with poor treatment outcome and shorter overall survival in early-stage squamous cell carcinoma of head and neck (SCCHN) in 2 different studies.15,16 Theoretically, the bcl-2 gene should result in treatment resistance and poor prognosis via 2 mechanisms: (1) Prevention of spontaneous apoptosis, resulting in more rapid accumulation of tumor cells; and (2) blockage of treatment-related apoptosis, regardless of the type of therapy used.15 The major drawback of these studies, however, is that the patients included have tumors in various sites of the head and neck, and, thus, do not constitute homogeneous groups. On the other hand, a better clinical outcome in patients with bcl-2 overexpression was observed in NPC.17 The investigators concluded that this better clinical outcome in bcl-2 positive NPC cases might depend on the histologic type of tumor because of the significantly higher bcl-2 expression in undifferentiated NPC. Based on this observation, we picked up only undifferentiated NPC patients for our study to create a more homogeneous group. Our data failed to show any correlation between bcl-2 and LMP1 expression and survival. At this time, bcl-2 and LMP1 presence are not significant indicators of outcome, and the more standard clinical indicators of disease severity, cervical lymph node metastasis, and cranial nerve involvement are still to be used for prognosticating patients. Another interesting finding is the positivity rate of bcl-2 expression in different epithelial malignancies. Positivity rates between 18% and 36% have been reported in early SCCHN15,16, a rate comparable with a 27% rate in lung cancer.18 In contrast, bcl-2 positivity ranging between 76% and 80% has been reported in NPCs.14,17,19 These findings are consistent with our 74.3% bcl-2 positivity rate. This higher rate of bcl-2 expression in NPC when compared with other epithelial, non-EBV-related malignancies indicate that induction of bcl-2 gene expression seems to be part of the survival strategy of the virus independently of the host cell infected, leading to prolonged cell survival.

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This bcl-2 induction might be mediated by LMP1 as seen in many lymphoid malignancies.10-12 The data on LMP1 expression in epithelial cells are limited. High-level expression of the LMP1 gene in a human epithelial cell line (RHEK-1) was shown to induce apoptosis.20 The same investigators showed in another study that LMP1-induced apoptosis could be specifically blocked with the ectopic expression of bcl-2 and coexpression of LMP1. Bcl-2 in RHEK-1 cells enabled the cells to grow under low-serum conditions and to form colonies in semisoft agar medium.13 These results suggest that these 2 proteins may play important complementary roles in the process of EBV-associated epithelial cell transformation. Our data showed a significant positive correlation between bcl-2 and LMP1. All the 10 LMP1-positive patients were also positive for bcl-2. However, all bcl-2-positive cases were not also positive for LMP1, and there are 16 bcl-2-positive patients who are LMP1 negative. So it is difficult to conclude whether this phenomenon really indicates an upregulation of bcl-2 by LMP1 or a coexpression of these genes. It appeared that bcl-2 expression could be independent of LMP1, so it is possible that LMP1 is not critical for bcl-2 expression in this tumor type. This observation suggests that bcl-2 expression may be independent of LMP1, which would provide a testable hypothesis for further in vitro studies. Nevertheless, although they are not directly related to survival, both bcl-2 and LMP1 were strongly correlated with cervical lymph node metastasis, which is a powerful predictor of patient survival. It seems that, although our data reject the hypothesis that bcl-2 and LMP1 are direct prognostic indicators in NPC, studies involving much larger series are needed to define the role of bcl-2 and LMP1 as single entities and as an interactive pair. REFERENCES 1. Miller G: Epstein-Barr virus. Biology, pathogenesis, and medical aspects, in Fieldsand BN, Knipe DM (eds): Fields Virology (ed 2). New York, NY, Raven Press, 1990, pp 1921-1958 2. Young LS, Rowe M: Epstein-Barr virus, lymphomas and Hodgkin’s disease. Semin Cancer Biol 3:273-284, 1992 3. Kieff E, Leibowitz D: Epstein-Barr virus and its replication, in Fieldsand, BN, Knipe DM (eds): Fields Virology (ed 2). New York, NY, Raven Press, 1990, pp 18891920 4. Hammerschmidt W, Sugden B: Genetic analysis im-

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