Int. J. Morphol., 26(3):567-572, 2008.

Inmunohistochemical Expression of PCNA, p53 and bcl-2 in Pleomorphic Adenomas

Expresión Inmunohistoquímica de PCNA, p53 y bcl-2 en Adenomas Pleomórficos

*Manuel Antonio Gordón-Núñez; **Gustavo Pina Godoy; ***Rosilene Calazans Soares; ****Lélia Batista de Souza; ****Roseana de Almeida Freitas &****Lélia María Guedes Queiroz

* Dr. M.D., Ph.D. in Oral Pathology. Post Doctor Student of the IFARHU -SENACYT Program - Panamá/UFRN, Natal/RN, Brazil.
** Prof. Dr., M.D., Ph.D. of Oral Pathology / UEPB / Campiña Grande, PB, Brazil.
*** Prof., Dr., M.D., Ph.D. Morphology Department of Sergipe Federal University, Sergipe, S, Brazil.
**** Prof., Dr., M.D., Ph.D. Post Graduation Program in Oral Pathology, UFRN, RN, Brazil.

Dirección para correspondencia

SUMMARY: The aim of the study was to determine the immunohistochemical expression of the PCNA, p53 and bcl-2 proteins in pleomorphic adenomas.onineteen specimens of pleomorphic adenomas were selected for analysis by the streptavidin-biotin-peroxidase method with antibodies againstp53, PCNA and bcl-2 proteins. It was observedweak labeling for p53 in 12 cases (63.1%) andforPCNA in 8 (42.1%). With respect to the bcl-2 labeling index,ono expression of this protein was detected in 12 cases, corresponding to 63.1% of the sample. Based on these findings, it was concluded that p53 and PCNA can favor the proliferative activity of pleomorphic adenomas, whereas bcl-2 probably doesonot effectively particípate in the pathogenesis of this tumor.

KEY WORDS: Pleomorphic adenoma; Salivary gland tumour; p53; PCNA; bcl-2.

RESUMEN: El objetivo del estudio fue determinar la expresión inmmunohistoquímica de las proteínas PCNA, p53 y bcl-2 en adenomas pleomórficos. Fueron seleccionados 19 especímenes de adenomas pleomórficos para análisis a través del método de la estreptavidina-biotina-peroxidasa con anticuerpos contra las proteínas p53, PCNA y bcl-2. Fue observada leve marcación para p53 en 12 casos (63,1%) y para PCNA en 8 (42,1%). Con relación al índice de marcación para bcl-2,ono fue detectada en 12 casos (63,1%) expresión de esta proteína. En base a los resultados, se concluyó que las proteínas p53 y PCNA pueden favorecer la actividad proliferativa de adenomas pleomórficos, y por otro lado, la bcl-2 probablementeono participaría efectivamente de la patogenia de este tumor.

PALABRAS CLAVE: Adenoma Pleomórfico; Tumor de Glándula Salival; p53; PCNA; bcl-2.

INTRODUCTION

Pleomorphic adenoma, also known as a benign mixed tumor, is a benignoneoplasm of the salivary gland which shows a remarkable degree of morphological diversity (Jaeger et al, 1997). It is the most common tumor of the salivary gland, accounting for approximately 40 to 70% of all salivary gland tumors (Matturri et al, 1996; Jorge et al, 2002; Neville et al, 2002).

Most pleomorphic adenomas occur in the superficial lobe of the parotid gland; however, when they affect the minor salivary glands, the most frequent site of involvement is the región of the hard palate, followed by the upper lip and buccal mucosa (Neville et al.). Clinically, pleomorphic adenoma presents as a slow growing mass, firm on palpation, which is usually encapsulated and may ulcerate in some situations. It affects any age but is more frequent in adults aged 30 to 50 years, with a slight predominance among females (Félix et al, 1999; Louro et al, 2002). Histologically, the tumor is characterized by histomorphologic heterogeneity consisting of an epithelial component fhat forms ducts and cystic structures as well as cell clusters arranged in islands oronests, whereas the stroma can be mucoid, myxoid or chondroid depending on the product derived from the altered metabolism of epithelial cells (Matturri et al).

Recurrence of pleomorphic adenomas is observed in 10 to 12% of cases and malignant transformation isonot rare (Matturri et al). A well-established fact in the literature is that carcinomatous foci occasionally develop in pleomorphic adenomas and that the risk of malignant transformation increases with the duration of these lesions (Ohtake et al, 2002; Marioni et al, 2003).

The expression of genes related to cell proliferation and oncogenesis seems to be associated with the prognosis of some oral tumors. Mutation in the tumor suppressor gene p53 is the most common genetic modification found in malignant oral tumors. Additionally, numerous studies involving proliferating cell nuclear antigen (PCNA) and the bcl-2 protein have been conducted to determine the processes related to cell proliferation and, consequently, the susceptibility of some tumors to malignant transformation (Ohtake et al; Rosa et al, 1997; Yanez et al, 1999; Alves et al, 2002).

The objective of the present study was to determine the immunohistochemical expression of PCNA, p53 and bcl-2 in pleomorphic adenomas in order to contribute to the understanding of cell proliferation and the possible malignant transformation of this tumor.

MATERIAL AND METHOD

Nineteen representative pleomorphic adenoma specimens were selected for immunohistochemical analysis from the files of the Service of Pathologic Anatomy, Discipline of Oral Pathology, Department of Dentistry, Federal University of Rio Grande doonorte. The material from the files was cut into 5-µm thick slides and stained with hematoxylin-eosin for morphologic analysis by light microscopy.

For immunohistochemistry, 3-µm thick slides were obtained from each specimen and mounted on glass slides with organosilane (3-aminopropyltriethoxy-silane, Sigma Chemical Co., St. Louis, MO, USA). The streptavidin-biotin-peroxidase method (SABC - streptavidin-biotin complex) with antibodies against proliferation (p53 and PCNA) and anti-apoptosis (bcl-2) proteins was used. The sections were deparaffinized, submitted to antigen retrieval and incubated with the anti-p53, anti-PCNA and anti-bcl-2 primary antibodies as shown in Table I. For inactivation of endogenous peroxidase, the sections were immersed twice in hydrogen peroxide (10 volumes) for 5 min each. Between steps, the samples were washed with Tris-HCl, pH 7.4. The clones, dilutions and incubation times used are Usted in Table I. The reaction was developed with 0.03% diaminobenzidine (Sigma) as chromogen, and the material was counterstained with Mayer's hematoxylin.

The slides submitted to immunohistochemistry were analyzed regarding the presence or absence of labeling of the antigens studied.

For quantitative analysis of p53, PCNA and bcl-2 positive cells, the cells were counted under aonikon® light microscope using a 0.25-mm² WeibelonGW2® grid at a final magnification of lOOOx by two independent examiners. The mean obtained was then used to calcúlate the labeling index based on the ratio of the number of immunopositive cells per 1000 randomly counted cells per case studied, multiplied by 100 to express the index in percentage. The labeling intensity wasonot considered in this analysis.

Since the quantitative data showedonoonormal distribution, the Kruskal-Wallis test was used for comparison at the 5% level of significance. Spearman's correlation coefficient at a level of significance of 5% was applied to determine a possible correlation between the three markers. The data were analyzed using the SPSS 13.0 software for Windows. For qualitative analysis which permitted comparison of the present data with those reported in the literature, the absolute labeling indices were transformed into the following scores according to the criteria of Al ves et al.: (-) negative, ≡ 5%; (+) weak, ≤ 5 and ≤ 25%; (++) moderate, >25 and ≤ 50%; (+++) strong, >50%. These data were analyzed using the chi-square test for proportions at the 5% level of significance.

RESULTS

The Kruskal-Wallis test revealed a significant difference between markers (p = 0.0044). Application of Dunn's múltiple comparisons test showed that this difference was significantly only between p53 and bcl-2 (Table II).

The results of Spearman's correlation analysis between the absolute labeling indices obtained for the three markers are shown in Table III. A significant positive correlation was observed between p53 and PCNA and between PCNA and bcl-2, which was higher for p53 x PCNA. No significant correlation was observed between p53 and bcl-2.

Table IV shows the labeling indices obtained for p53, PCNA and bcl-2 in the pleomorphic adenomas studied. Taking into account the percent distribution of the labeling scores obtained for p53, the chi-square test for proportions revealed a significant difference, with weak labeling predominating in 12 cases, correspondmg to 63.1% of the sample (p <0.00001) (Fig. 1). A predominance of weakly labeled cases was also observed for PCNA, although this difference wasonot significant (p = 0.246). Weak labeling was observed in 8 cases, correspondmg to 42.1% of all cases evaluated (Fig. 2). However, some cases shown occasional strong expression of PCNA. A significant difference was observed in the percent distribution of bcl-2 labeling scores (p < 0.0001), with a predominance of bcl-2-negative cases, correspondmg to 63.1% of the sample (12 cases). It was observed a weak expression of bcl-2 in 7 cases (Fig. 3). Taking into account the distribution of each score between markers, the proportion test revealed a significant difference for theonegative score, which was observed for bcl-2 in most cases (p = 0.0136). With respect to the distribution of the other scores,ono significant difference was observed between markers (p = 0.0712, p = 0.152 and p = 0.319, respectively).

DISCUSSION

Pleomorphic adenoma is the most frequent salivary gland tumor, whose clinical and microscopic characteristics have been well established in the literature. However, the pathogenesis of this tumor is still unknown, a fact that encouraged the present immunohistochemical study of PCNA and bcl-2 in order to determine the role of these proteins in cell proliferation and even to establish a possible relationship of p53 expression with the malignant transformation of pleomorphic adenoma. PCNA is a nuclear protein that plays an important role in DNA synthesis, with a significant participation in cell replication (Zhu et al, 1997). In the present study, most cases showing a predominantly weak labeling for PCNA. This result agrees with the studies of Matturri et al. and Alves et al., who reported a predominance of weak to modérate labeling of this protein in pleomorphic adenomas.

Strong labeling of PCNA was observed in four of the present cases, indicating a greater proliferative activity of these tumors and suggesting a tendency toward recurrence and possible susceptibility of these lesions to malignant transformation, in agreement with Matturri et al. Additionally, Ohtaké et al. observed high positivity for PCNA, especially in atypical cells, and suggested a potential of malignant transformation for pleomorphic adenomas. However, other investigators such as Zhu et al. have reported that the proliferative activity of pleomorphic adenomas isonot significant and have associated their high rate of recurrence with inadequate surgical procedures. The predominance of cases showing weak orono labeling in the present study suggests a less aggressive behavior and possibly lower tendency toward malignant transformation for most pleomorphic adenomas in this sample.

p53 is a tumor suppressor gene located on the short arm of chromosome 17 and is the most commonly mutated gene in tumors (Ohki et al, 2001). Many studies have correlated the expression ofonormal and mutantp53 with the differentiation, aggressiveness and prognosis of salivary gland tumors, but the results are highly controversial. In the present study, most cases showed weak labeling for p53, in agreement with the findings of Marioni et al, Lazzaro & Cleveland (2000) and Weber et al. (2002). However different results have been reported by Rosa et al. and Alves et al, who observedono expression of p53 in any case, emphasizing that this protein isonot implicated in the pathogenesis of pleomorphic adenomas. In contrast, most cases were positive for the p53 protein in the studies of Ohki et al. and Ohtake et al. indicating, according to these authors, a tendency toward malignant transformation of pleomorphic adenomas. This statement is also supported by Li et al. (1997) who observed that the expression of p53 was frequently associated with deletion of the p53 gene detected by fluorescence in situ hybridization test (FISH), favoring a possible installation of carcinoma ex-pleomorphic adenoma.

Based on the present findings, especially the significant correlation between the expression of PCNA and p53, we may infer that this correlation it could be associated with a more evident proliferative profile in some cases of pleomorphic adenoma.

The bcl-2 protein is responsible for the inhibition of apoptosis and is therefore important for greater cell survival. According to Yanez et al, bcl-2 plays an important role in the development of pleomorphic adenomas since all cases analyzed by the authors were positive forthis protein. Aoki et al. (2004) also detected ahigh percentage of bcl-2-positive cases, with immunoreactivity being observed in 33 (94.3%) of 35 cases analyzed. The present findings are in contrast with these results since most cases wereonegative for bcl-2 protein, suggesting that this protein isonot involved in the pathogenesis of pleomorphic adenoma. In addition, a significant correlation was observed between bcl-2 and PCNA, Le., in most cases in which PCNA was expressed bcl-2 was also identified. These findings demónstrate a concomitant proliferative and apoptotic activity of pleomorphic adenoma cells, a profile expected for benignoneoplasm.

We concluded that the correlation of p53 and PCNA expression can favor the proliferative activity of pleomorphic adenomas and that bcl-2 didonot effectively particípate in the pathogenesis of this tumor in the cases studied here. Considering that the incomplete surgical removal is told in the literature as main cause of recurrence for the pleomorphic adenomas, thefindings of this study in relation to the highest cellular proliferative activity evidenced by the labeling for PCNA and p53 can to suggest that in some cases of pleomorphic adenoma with this profile, the recurrence risk becomes relatively higher. However, more detailed studies areonecessary to confirm this assumption.

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Correspondence to:

Dr. Manuel Antonio Gordón-Nuñez.
Avenida Senador Salgado Filho, 1787
Lagoaonova.
CEP:59056-000
onatal-RN
Brasil

Telephone/fax number: (55)(84) 215-4238

Email: gordonnunez28@yahoo.com

Received: 18-02-2008 Accepted: 22-06-2008