Assessment of inverse correlation of p16 and pRb expression in carcinoma ex pleomorphic adenoma

Introduction

Several pathways need to be abrogated in the genesis of a malignant neoplasm. There is strong evidence that deregulation of one or more of the cell cycle checkpoints is among the most common abnormalities in human neoplasia. The protein p16 is a negative regulator of the cell cycle and is the product of the cyclin dependent kinase 2 (CDKN2) gene. Studies of the molecular genetics of oral cancer have shown that the CDKN2 gene was frequently inactivated by methylation or homozygous deletion [1].

The retinoblastoma protein (pRb) is one of the key cell-cycle regulating proteins and its inactivation leads to neoplastic transformation and carcinogenesis [2]. Phosphorylation of pRb is mediated by complexes comprised of a D-type cyclin and cyclin-dependent protein kinases (CDK4)/(CDK6). The activity of these kinases is in turn negatively regulated by cyclin kinase inhibitors including p16. The cascade composed of pRb, cyclin D1, CDK4/CDK6 and p16 plays a central role in cell cycle control. p16 controls cell proliferation through maintenance of a hypophosphorylated state of pRb [3]. It has been postulated that the p16-pRb pathway is regulated by a feedback loop involving pRb [4, 5]. In such a case, deregulated expression of pRb, by hyperphosphorylation, mutation, or association with viral oncoproteins, would permit high levels of p16.

Carcinoma ex pleomorphic adenoma (CPA) is considered to be a malignant transformation product of pre-existing pleomorphic adenoma [6]. Carcinoma ex pleomorphic adenoma is the most common malignant mixed tumour and has been estimated to account for 10% of all salivary gland malignancy [7]. The goals of the present study were to investigate the alterations and aberrations in the expression of p16 and pRb in carcinoma ex pleomorphic adenoma, and also to evaluate the inverse correlation between p16 and pRb expression in 27 cases of carcinoma ex pleomorphic adenoma.

Material and methods

Case selection



Twenty seven cases of CPA from July 2011 to July 2012 were studied (Table I). Normal tissue of the salivary gland surrounding the tumour was used as a control. The criteria used to identify CPA by Nagao et al. [8] were considered. Malignant changes in the pleomorphic adenoma include three types (World Health Organization, 2005): CPA, carcinosarcoma, and metastasizing pleomorphic adenoma. The inclusion criteria for CPA including malignant transformation in pleomorphic adenoma include poorly defined and/or infiltrative tumour margins, the presence of foci of haemorrhage, and necrosis. Benign and malignant elements are considered as well. Exclusion criteria include the other well-recognized salivary carcinomas and those of uncertain type included in the current WHO histological classification of tumours [9]. All microscopic slides were reviewed by two pathologists to confirm the diagnosis. The ethical approval was included (Ref: 5/2012).Immunohistochemistry



Serial 4-µm sections were cut from 27 specimens. Streptavidin-biotin-peroxidase method was used. Endogenous peroxidase was blocked by 3% hydrogen peroxidase for 5 minutes, followed by TBS (Tris-buffered saline) wash. Nonspecific immunoreactivity was blocked by incubation with normal goat serum for 20 minutes. A purified mouse anti-human monoclonal antibody p16 (Pharmingen, San Diego) was diluted to 5 µ/ml in 10 µ/ml tris buffer saline (TSA) for 1 hour at room temperature. A primary mouse anti-human retinoblastoma protein RB (Pharmingen, San Diego) was diluted to 5 µ/ml in 10 µ/ml tris buffer saline (TSA) containing 0.1% bovine serum albumin at room temperature. All sections were washed with TBS for 5 minutes. Sections were incubated with the biotinylated secondary antibody reagent for 30 minutes, followed by TBS wash for 5 minutes. Slides were incubated with streptavidin and horseradish peroxidase for 30 minutes, followed by TBS wash for 5 minutes, and incubated with a prepared chromogenic substrate solution (diaminobenzidine) for 15 minutes. Sections were counterstained with 0.25% methyl green in distilled water for 5 minutes. Sections were dehydrated and mounted in Depax. A positive control was included, squamous cell carcinoma. A negative control was included, substitution of the primary antibody with TBS. The assessment of p16 and pRb positive nuclei was as follows: negative (0) no expression of nuclear protein, (1) weak staining 0-25% of the total cells shows positive staining in the nucleus, (2) moderate staining > 25-75% of the total cells in the test area show positive nuclear staining, (3) strong staining > 75-100% cells show positive nuclear staining.



Statistical analysis



Cells of the carcinomatous component of the CPA were always scored. The statistical analysis included the use of descriptive statistics; frequencies proportion. Also, statistical analyses, including Wilcoxon’s nonparametric tests (ordinal data), were performed on the data. All statistical tests were two-sided and p-values less than 0.05 were considered to be statistically significant.

Results

p16 expression of the nuclear staining was studied in an adjacent area of CPA. p16 nuclear staining of duct cells showed strong positive nuclear staining in 23 (85%) cases out of 27, 3 (11.1%) with moderate staining, and 1 (3.7%) with weak staining. p16 nuclear staining of the acinar cells showed negative staining in 1 (3.7%) case out of 27, 11 (40.7%) had weak staining, and 15 (55.5%) had moderate staining.

p16 expressed negative nuclear staining in 22 (81.4%) cases out of 27, and 5 (18.5%) cases expressed moderate staining. There was a significant difference (Wilcoxon test, p value < 0.001) between p16 expression in the nucleus in the duct cells of normal tissue surrounding the tumour and the tumour area. 23 out of 27 cases had strong positive staining detected in the duct cells (normal tissue) and no strong staining was noted in all cases of carcinoma arising in pleomorphic adenoma.

The results of nuclear staining of duct cells, acinar cells, and stroma indicated that pRb was not expressed in any of the 27 cases (control group).

pRb showed negative staining in 5 cases (18.5%), 1 case had low staining (3.7%), 2 moderate staining (7.4%), and 19 cases strong staining (70.4%).

There was a significant difference (Wilcoxon test, p value < 0.001) between pRb expression in the nucleus in the duct cells of normal tissue surrounding the tumour and the tumour area. pRb was not expressed in any of the 27 cases in the control group surrounding CPA. Nineteen cases out of 27 cases had strong positive staining detected in carcinoma arising in pleomorphic adenoma.

We reclassified the nuclear staining in the carcinoma cases to study the relation between p16 and pRb. Low and negative staining were considered negative but moderate and strong nuclear staining were considered positive. Table II shows the inverse correlation between p16 (normal expression) and pRb (mutated) in 15 cases. Also 3 cases showed an inverse correlation between p16 (mutated) and pRb (normal expression). p16 and pRb (both proteins with normal expression) were identified in 3 cases. p16 and pRb (both proteins inactivated) were identified in 6 cases.

Discussion

No study has been published on the inverse correlation between p16 and pRb in carcinoma arising in pleomorphic adenoma.

Nielsen et al. [10] reported that p16 showed positive staining in the duct and acinar cells of salivary glands. Hu et al. [11] investigated the p16 protein expression and promoter methylation of the p16 gene in CPA and their roles in the malignant transformation of pleomorphic adenoma to CPA. They found no correlations between p16 protein expression and promoter methylation of the p16 gene in either benign or malignant components. Schache et al. [12] demonstrated the successful application of quantitative methylation-specific real-time polymerase chain reaction (qMSP) analysis to a large series of historical carcinoma ex pleomorphic adenoma samples and reported on a panel of tumour suppressor genes (TSGs) with significant differences in their methylation profiles between benign and malignant variants of pleomorphic salivary adenoma. They concluded that qMSP analysis could be developed as a useful clinical tool to differentiate between CPA and its benign precursor. Augello et al. [13] reported that p16(INK4A) promoter hypermethylation was found in 100% (5/5) of carcinomas including 4 cases of cystic adenocarcinomas and 1 case of CPA. Patel et al. [14] examined cyclin D1 and p16 expression in 43 parotid tumours (29 pleomorphic salivary adenoma and 14 carcinoma ex pleomorphic). They reported that cyclin D1 and p16 were both significantly more likely to be expressed in the neoplastic than in the normal epithelial and stromal components of pleomorphic adenoma and carcinoma ex pleomorphic adenoma (P < 0.001 and P < 0.005, respectively).

The inverse correlation between p16 and pRb in carcinoma arising in pleomorphic adenoma

Fang et al. [15] demonstrated pRb expression at higher levels in cell lines of human ovarian cancer lacking p16 than those with normal p16. They mentioned that if the function of one gene such as p16 or pRb was abrogated through deletion or mutation, the other gene would be overexpressed or unaffected. Therefore, they suggested that the growth inhibitory activity of this pathway would be maintained.

Yeager et al. [16] reported an inverse correlation between p16 and pRb in human urothelial cells. Ten samples with normal expression of pRb showed low or no detectable p16 levels, while 7 samples with known pRb alterations showed abundant p16.

Nagger et al. [17] showed that pRb and p16 expression exhibited a significant reciprocal correlation from primary oral and laryngeal squamous carcinoma specimens. Thirty-two tumours (91.4%) showed an inverse relationship, 31 cases had negative p16 and positive pRb staining, and 1 showed negative pRb and positive p16 staining. Three (8.6%) tumours were positive for both, and 1 tumour was negative for both proteins.

Sakaguchi et al. [18] also demonstrated an inverse relationship between p16 and pRb expression in non-small cell lung cancers. The expression of p16 and pRb protein was studied by immunochemistry in 61 cases. They found that 28 of 30 specimens did not stain for p16 but stained for pRb and 21 of 31 p16 positive specimens did not stain for pRb.

There are discrepancies in the results found in the literature because of different methodological problems. First, there may have been selection of patients and differences in treatment of different types of salivary gland tumours. Second, there are often differences in tissue processing from study to study, especially with regard to the type of antibody and the application of antigen retrieval. Also there are several steps in tissue processing that may influence staining patterns and intensity. These include type and duration of fixation, section thickness, antigen retrieval procedures, and type and concentrations of primary, second and third step antibodies. Third, and possibly most important, interpretation of staining and presentation of the results are not standardized, resulting in low intra-observer and inter-observer reproducibility. Different criteria (e.g. 0 = negative staining, 1 = low, 2 = moderate, 3 = strong; or 0-3 = negative and 4 = positive; or 0-2 = negative and 3-4 = positive; or negative and positive staining) have been used in the literature to assess the expression of p16 and pRb. Therefore, the use of one criterion such as negative or positive staining only for the assessment of staining will avoid any confusion in the interpretation of the results. There is therefore a clear need for a consensus on a protocol for scoring of immunohistochemical staining.

This study comprised 27 cases of CPA, which is much bigger than other published studies. The published literature on tumour markers in CPA is limited due to the fact that these tumours are rare. This study has shown an inverse correlation in 18 out of 27 cases of CPA, as mentioned above, so that if p16 or pRb is inactivated the other gene will show overexpression. The conclusion of this study is that the alteration of p16 and pRb expression has been detected in CPA cases. P16 and pRb might have a role in development of CPA.



The authors declare no conflict of interest.

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Address for correspondence



Bassel Tarakji

Department of Oral Maxillofacial Sciences

Al-Farabi Dental College, Riyadh, Saudi Arabia

fax: 00966-2324580

e-mail: denpol@yahoo.co.uk