Image cytometric analysis of p53 and mdm-2 expression in primary and recurrent mucoepidermoid carcinoma of parotid gland: immunohistochemical study

Malignant tumors of the major salivary glands are rare, representing 7% of all head and neck cancers. Benign and malignant parotid gland neoplasms account for approximately 90% of all major salivary tumors. They are characterized by a variety of histologic subtypes with different biologic and prognostic behavior [1].

Recent studies of the molecular biology of cancers have demonstrated that the loss of function of tumor suppressor genes may elicit tumorigenesis, and may be associated with the development and progression of many different cancer types. One of the best known tumor suppressor genes is the p53 gene [2]. This gene is located on human chromosome 17(p13) and encodes a nuclear phosphoprotein (53 Kd) that thought to regulate proliferation of normal cells [2]. Various point mutations of the p53 gene usually determine the transcription of a mutated protein, which is much more metabolically stable than the wild protein and accumulates in the nucleus. Consequently, normal cells with the wild p53 gene do not show p53 immunoreactivity, whereas tumors with the mutant form of the p53 gene may express high levels of p53 protein that are immunohistochemically detectable. The accumulation of the p53 oncoprotein clearly has been shown to correlate with shorter survival in patients with carcinomas of the breast, lung, stomach, colon as well as bladder [3].

The mdm-2 oncogene was first cloned as an amplified gene on a murine double-minute chromosome in the 3T3DM cell line, a spontaneously transformed derivative of BALByc 3T3 cells [4]. The gene encodes a 489 amino acid polypeptide that contains a p53 binding domain, an acidic region, and three putative zinc-binding motifs (one zinc-finger and one RING finger). Overexpression of the mdm-2 gene in NIH 3T3 cells increases the tumorigenic potential of these cells, thus establishing mdm-2 as an oncogene [4]. The mdm-2 gene can immortalize rat embryo fibroblasts and cooperate with the activated ras oncogene to transform these cells [5]. The mdm-2 gene is amplified or overexpressed in about 40-60% of human osteogenic sarcomas and about 30% of soft tissue sarcomas [4, 5], implicating its role in the development of these malignancies.

An important function of mdm-2 is to bind to the p53 tumor suppressor protein, inhibiting its ability to act as a transcription factor [6]. P53 also activates mdm-2 expression at the level of transcription suggesting that mdm-2 can function as a negative feedback regulator of p53 [7].

Several studies were carried out to investigate the role of aberrant expression of p53 tumor suppressor gene and mdm-2 overexpression in different benign and malignant salivary gland tumors [8, 9]. However, no definite data was available in the literature regarding the exact role of p53 and mdm-2 mutation upon the clinical coutcome of muco-epidermoid carcinoma as one of the common malignant salivary gland tumors and also the factors that might be related to the aggressiveness and recurrence of these tumors.

The aim of this study is to analyze immunocytochemically p53 aberrant expression and mdm-2 expression in primary and recurrent MEC of parotid gland and to ascertain if expression of these markers correlates with tumor behavior, clinical outcome, histological grade and local recurrence.

After follow-up period of all patients, 6 cases showed evidence of recurrence (30% of cases). The time elapsed between the surgical removal of the primary lesion and the recurrence was variable and ranged from 32-40 months.

This aim of the present study was to investigate the immunoexpression of p53 and mdm2 oncoproteins in primary and recurrent MEC as a trial to clarify the possible role of those proteins in the pathogenesis of this tumor and also to correlate the effect of their expression on the recurrence and clinical outcome of these tumors of salivary glands.

Earlier studies focused on the analysis of p53 gene in tumors of the salivary glands; however the connection between salivary gland carcinogenesis and the mdm2 oncoprotein was still elusive [11]. Some authors demonstrated that not only p53 gene is a potential target in neoplastic transformation, but also genes involved in the regulation of its function since mdm2 can potently regulate p53, and functions as an oncogene in the process of cell transformation [12]. For this reason co-expression of both p53 and mdm2 was investigated for their role in the biological behavior of MEC.

Interpretation of immunohistochemical positivity as mild, moderate and intense reaction is now considered as an obsolete method as it lacks the basic standardized parameters upon which the results are interpreted and also due to difficulty to avoid the subjectivity in estimating the degree of immunostaining. For this reason, a novel method of image analysis by which the results were automatically estimated as a surface area of immunopositivity is widely used [13].

Many authors considered both nuclear and cytoplasmic staining or exclusively cytoplasmic staining as positive results [14], and this was chosen for assessment of immunopositive reaction of mdm2 in this study, because a strong evidence is now available that mdm2 is an RNA binding protein that can shuttle between the nucleus and the cytoplasm, and both mdm2 and p53 proteins contain a nuclear-import and nuclear-export signals (NES) that enable them to be directed into the nucleus and out again towards the cytoplasm [14]. This NES of mdm2 is essential for p53 degradation by interacting with cytoplasmic proteasomes, where p53 is specifically degraded [15].

Immunohistochemical results of the present study revealed that 80% of primary lesions showed immunopositive expression of p53. These results were in accordance with that found by many authors who reported an immunopositive expression of aberrant p53 in glandular carcinomas including those of salivary glands and considered expression of mutant p53 as an early event in malignant transformation of these tissues [9, 16‐18]. Nordkvist et al [19] and Ohki et al [20] have detected p53 gene alterations in some MEC lesions. Although a small number of MEC cases were analyzed in these studies, p53 gene alterations were identified, suggesting a possible involvement of this gene in MEC pathogenesis. Matizonkas-Antontio et al [1] utilizing single-stranded conformational polymorphism (SSCP) analysis for p53 mutations in salivary gland tumors confirmed these data and reported that p53 mutations in exons 5 and 8 were most likely related to salivary gland neoplasms. They added that mutations were observed in 1 of 3 of MEC cases under study. On the other hand, Karja et al [18] did not observe any p53 mutations in these lesions suggesting variable results regarding p53 gene status in these lesions.

P53 immunopositivity of epidermoid cells observed in the present study might indicate the proliferative nature of these cells when compared to a more differentiated mucous-secreting cells. These results together with that of mean area fraction of immunopositivity seen by image analysis suggested a more expression of p53 in these aggressive cell population in MEC indicating a possible relationship between alteration of p53 function and malignant transformation of these cells [18]. This suggestion might explain the immunopositivity of p53 in all intermediate and high grade cases containing predominantly a more aggressive epidermoid and intermediate cells while only 3 out of 7 cases classified as grade (I) showed p53 immunopositivity due to presence of a more differentiated and less aggressive mucous-secreting cells in these lesions.

Immunopositivity of p53 in all recurrent cases noted in the present study strongly suggested the argument of Oreste Gallo et al [8] who stated that p53 expression tended to be higher in late stage cancers, correlated with clinicopathologic variables indicative of aggressiveness, such as regional and distant metastases; and provided prognostic information for disease free and overall survival probabilities. In fact, the above-mentioned trends found in p53-positive parotid gland cancers indicated that p53 gene expression does influence tumor behavior and strongly suggested that parotid gland carcinomas showing high p53 oncoprotein immunoreactivity are aggressive and have a poor prognosis as already was observed for other glandular carcinomas such as lung, breast, stomach, and colon cancer [3].

Mdm-2 immunohistochemical results of the present study revealed that 70% of MEC showed mdm-2 immunopositivity. These data agreed with those reported by Haitel et al [21] and Higashiyama et al [22] who reported an overexpression of mdm-2 oncoprotein in clear cell renal carcinoma and non-small cell lung cancer respectively.

However, de Araujo et al [9] found that the expression of mdm-2 in malignant tumors of minor salivary glands is not significantly high when compared to that in benign tumors.

On the other hand, only 2 out of the six recurrent cases showed mdm-2 immunopositivity. These data suggested that expression of mdm-2 is an early event in the carcinogenic pathway of MEC and hence the lowered percentage of mdm-2 immunopositivity is usually related to tumors of better prognosis and/or low recurrence rate [23].

The data of the present study also revealed that out of the 16 p53 immunopositive primary cases, 12 were mdm-2- positive while only 4 cases were p53 +ve/mdm-2 -ve. The high percentage of primary MEC that showed co-expression of both p53 and mdm-2 together with the linear correlation noted by regression analysis might indicate the possible interaction between these two proteins. One possible scenario of this interaction is that p53 aberrant expression as an early event in the carcinogenic cascade of MEC led to activation of mdm-2 feedback loop for proteasome degradation of activated p53 [14]. Despite the activation of mdm-2, degradation of p53 do not takes place. One explanation of this argument is that mdm-2 had two apparently opposite functions, a tumorigenic function and a growth arrest one. This dual role of mdm-2 could depend on the level of mdm-2 expressed in the cell and also the balance of positive and negative regulators of cell cycle which is critical for the control of cell proliferation [24].

Another explanation is that phosphorylation of the amino terminus of p53 does not affect its DNA binding ability, but does affect its affinity for mdm-2 and subsequent p53 degradation [25]. So, overexpression of p53 would increase the levels of mdm-2 by the feed back loop but without affecting the activity of phosphorylated p53 [25].

These explanations on the immunohistochemical level would focus on the role of these two proteins in the pathogenesis and clinical outcome of MEC, however, further studies utilizing much more advanced research tools such as in situ hybridization and SSCP analysis are highly recommended.

Based upon the results of the present study, it could be concluded that: