Context.—The significance of promoter methylation of the p16 gene and intracellular localization of p16 protein in the carcinogenesis of salivary carcinoma ex pleomorphic adenoma (Ca-ex-PA) is not clear. The correlation of the promoter methylation of the p16 gene and the expression and localization of p16 protein in Ca-ex-PA need to be further clarified.

Objective.—To investigate the p16 protein expression and promoter methylation of p16 gene in Ca-ex-PA and their roles in the malignant transformation of pleomorphic adenoma to Ca-ex-PA.

Design.—The p16 protein expression and promoter methylation of the p16 gene were determined in both benign and malignant components of 50 primary salivary Ca-ex-PA tissues by immunohistochemistry and methylation-specific polymerase chain reaction. Expression of p16 protein and promoter methylation of the p16 gene between the benign and the malignant components was compared statistically.

Results.—The tumor cells in the malignant components showed significantly higher p16 protein expression in the cytoplasm and lower expression in the nuclei than those in the benign components. Promoter methylation frequency of the p16 gene in the malignant components (36%) was significantly higher than that in the benign components (16%). There were no correlations between p16 protein expression and promoter methylation of the p16 gene in either benign or malignant components.

Conclusions.—Overexpression of p16 protein in the cytoplasm and decreased expression of p16 protein in the nucleus may play important roles in the evolution of pleomorphic adenoma to Ca-ex-PA. Promoter methylation of the p16 gene may be correlated with the malignant transformation of pleomorphic adenoma.

Pleomorphic adenoma (PA) is the most common neoplasm of salivary gland origin. Although this lesion is generally considered benign, approximately 6.2% of PAs become malignant,1 evolving into carcinoma ex PA (Ca-ex-PA). However, the molecular mechanisms involved in the malignant transformation of PA are still uncertain.

The p16 protein is encoded by the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, which is located at chromosome region 9p21. The p16 protein is located in the cell nucleus and acts as a tumor suppressor protein by binding to cyclin-dependent kinases 4 and 6, blocking their activity and leading to cell cycle arrest. The inactivation of the p16 gene is recognized as a crucial event in the development of several types of human tumors.26 Different mechanisms have been reported for inactivation of the p16 gene, including gene mutations, deletions, and promoter methylation. Promoter methylation of the p16 gene has been detected in a high percentage of various tumors, such as gastric carcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, and salivary adenoid cystic carcinoma.710 

There have been several studies of p16 protein expression in salivary gland tumors, and most data on Ca-ex-PA are restricted to a few individual case reports,11,12 except 1 study published by Patel et al13 that included 14 cases of Ca-ex-PAs. The studies of promoter methylation of the p16 gene in Ca-ex-PA are very limited, and the findings of the studies seemed conflicting.14,15 However, none of these studies focused on the correlation between p16 protein expression and promoter methylation of the p16 gene in Ca-ex-PA. With this background, in the current study we evaluated the p16 protein expression and promoter methylation status of the p16 gene in both benign and malignant components of 50 primary salivary Ca-ex-PA tissues by immunohistochemistry and methylation-specific polymerase chain reaction (PCR). We also analyzed the correlations between the molecular alterations of the p16 gene and clinicopathologic characteristics of these tumors to determine the role of the p16 gene in the malignant transformation of salivary PA and progression of Ca-ex-PA.

Tissue Samples

Samples of salivary Ca-ex-PA were obtained from 149 unrelated patients who were diagnosed between 1985 and 2006 in the Department of Oral Pathology at Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Tumor samples were fixed in formalin and embedded in paraffin. Tissue sections (4 µm) were stained with hematoxylin–eosin and reviewed by 2 pathologists (Y.-H.H. and J.L.). We selected 50 samples of Ca-ex-PA in which the conspicuous benign PA components and carcinoma components were simultaneously presented. The clinical and pathologic data were collected for all 50 patients. The invasiveness and histologic subtype of the tumors were determined according to the 2005 World Health Organization Pathology and Genetics of Head and Neck Tumours.16 The malignant components were subclassified as grade 1, 2, or 3 based on nuclear pleomorphism, mitotic activity, and necrosis (adenocarcinoma not otherwise specified [ANOS] and myoepithelial carcinoma) or 2005 World Health Organization Pathology and Genetics of Head and Neck Tumours (mucoepidermoid carcinoma).16 In addition, 1 case of subtype of epithelial-myoepithelial carcinoma and 1 case of undifferentiated carcinoma were subclassified as grade 1 and grade 3 respectively. Tumor stage was determined according to American Joint Committee on Cancer cancer staging criteria.17 

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissues were cut into 4-µm sections and then were deparaffinized in graded alcohol. Antigen retrieval was accomplished by microwave irradiation (in citrate buffer, pH 6.0) for 20 minutes. Endogenous peroxidase activity was blocked with 3% H2O2 for 20 minutes. The slides were incubated overnight at 4°C with monoclonal mouse anti-human p16 antibody (Neomarkers, Fremont, California) at 1∶50 dilution, followed by incubation with secondary peroxidase-labeled polymer conjugated to goat anti-mouse immunoglobulin antibody (Dako, Carpinteria) at room temperature for 30 minutes. Expression of p16 was detected with the diaminobenzidine chromogen system (Dako, Carpinteria). The nuclei were counterstained with hematoxylin. Negative controls, with omission of the first antibody, were run simultaneously. Sections of colon carcinoma were used as positive controls for p16 expression.

Evaluation of Immunohistochemistry

Because there was no significant difference in the intensity of reactivity of tumor cells among all the cases, a scoring system based on the percentage of immunopositive cells was used to assess p16 protein expression. The extent of nuclear and cytoplasmic p16 expression in both benign and malignant components was visually estimated at whole tissue sections. Nuclear/cytoplasmic p16 expression was interpreted as N-0/C-0 (no positive reaction in nucleus/cytoplasm), N-1/C-1 (1%–30% of cells were positive in nuclei/cytoplasms), or N-2/C-2 (>30% of cells were positive in nuclei/cytoplasms).

Microdissection and DNA Extraction

Tissue sections (10 µm) were stained with hematoxylin and eosin. Areas containing benign PA cells and carcinoma cells were dissected under a stereomicroscope and then digested in 300 µL of digestion buffer (500 mM Tris, 20 mM ethylenediaminetetraacetic acid, 10 mM NaCl [pH 9.0], 1% sodium dodecyl sulfate, and 0.5 mg/mL of proteinase K) at 42°C for 36 hours. The digested products were purified twice using phenol and chloroform. DNA was precipitated with ethanol and resuspended in double-distilled water.

Methylation-Specific PCR

The methylation status of the p16 gene promoter was determined by methylation-specific PCR analysis. Genomic DNA (2 µg), with 1 µg of salmon sperm DNA as a carrier and in a total volume of 50 µL, was denatured using NaOH (final concentration, 0.2 M) for 10 minutes at 37°C; 30 µL of 10 mM hydroquinone (Sigma Chemical Company, St. Louis, Missouri) and 520 µL of 3 M sodium bisulfite (Sigma) at pH 5.0, both freshly prepared, were added and mixed. The samples were then incubated at 50°C for 16 hours.

Modified DNA was purified using the Wizard DNA purification resin (Promega, Madison, Wisconsin) according to the manufacturer's instructions and then eluted into 50 µL of water. The reaction was stopped with use of NaOH (final concentration, 0.3 M) for 10 minutes at room temperature, followed by ethanol precipitation. The DNA was resuspended in double-distilled water and was either used immediately or stored at −20°C.

Bisulfite-modified DNA was then amplified by methylation-specific PCR using methylated-specific primers for the p16 gene (forward 5′-TTATTAGAGGGTGGGGTGGATTGT-3′, reverse 5′-CAACCCCAAACCACAACCATAA-3′) and annealing at 64°C, producing a 150-bp fragment, and using unmethylated-specific primers for the p16 gene (forward 5′-TTATTAGAGGGTGGGGTGGATTGT-3′, reverse 5′-CAACCCCAAACCACAACCATAA-3′) and annealing at 60°C, producing a 151-bp fragment. The PCR mixture contained 10× PCR buffer (0.166 M NH4SO4, 0.67 M Tris [pH 8.8], 67 mM MgCl2, 100 mM β-mercaptoethanol, 67 µM ethylenediaminetetraacetic acid, and 9% dimethyl sulfoxide), HotStar Taq DNA polymerase (0.625 unit; Qiagen, Valencia, California), dimethyl sulfoxide (0.5 µL), dATP, dGTP, dCTP, and dTTP (each 1.25 mM), primers (75 ng per reaction), and bisulfite-modified DNA (50–100 ng) in a final volume of 12.5 µL. Water was substituted for DNA as a negative control. The DNA from the ACC-2 cell line treated with SssI methylase (New England Biolabs, Beverly, Massachusetts) was used as a positive control. The PCR conditions were set as follows: 95°C for 15 minutes, followed by 40 cycles at 94°C for 30 seconds, at the annealing temperature for 45 seconds, and at 72°C for 1 minute, with a final 5-minute extension at 72°C. The PCR products were subjected to electrophoresis on 2% agarose gels, stained with ethidium bromide, and directly visualized under ultraviolet illumination.

Statistical Analysis

The differences in p16 protein expression and promoter methylation of the p16 gene between benign and malignant components and the correlations between p16 protein expression, promoter methylation of the p16 gene, and clinicopathologic characteristics were analyzed statistically. Nonparametric statistical analysis and χ2 test (or Fisher's exact test) were performed for categoric data, using the SAS program (version 6.12, SAS Institute, Cary, North Carolina). All P values were 2-sided, and statistical significance was set at P ≤ .05.

Clinical and Pathologic Characteristics

The clinical and pathologic findings of the 50 patients with primary salivary Ca-ex-PA are summarized in Table 1. Fourteen patients were women and 36 were men, with a median age of 55 years (range, 34–78 years). Forty-two tumors (84%) originated from the major salivary glands and 8 (16%) from the minor salivary glands. Histologically, 14 tumors were classified as grade 1, 28 as grade 2, and 8 as grade 3. Five patients (10%) had TNM stage I disease, 23 (46%) had stage II, 19 (38%) had stage III, and 3 (6%) had stage IV. The subtypes of the malignant components were 20 cases of myoepithelial carcinoma (Figure 1, A), 25 cases of ANOS (Figure 1, B), 3 cases of mucoepidermoid carcinoma (Figure 1, C and D), 1 case of epithelial-myoepithelial carcinoma, and 1 case of undifferentiated carcinoma. Twenty-five tumors were invasive Ca-ex-PAs, 6 were minimally invasive Ca-ex-PAs, and 19 were noninvasive Ca-ex-PAs.

Figure 1

Hematoxylin–eosin photomicrographs of the different histologic subtypes of carcinoma ex pleomorphic adenoma. A, Myoepithelial carcinoma. B, Adenocarcinoma not otherwise specified. C. Mucoepidermoid carcinoma. D, Mucous cells from Figure C are shown in high-power field (original magnifications ×200 [A and B], ×100 [C], and ×400 [D]).

Figure 1

Hematoxylin–eosin photomicrographs of the different histologic subtypes of carcinoma ex pleomorphic adenoma. A, Myoepithelial carcinoma. B, Adenocarcinoma not otherwise specified. C. Mucoepidermoid carcinoma. D, Mucous cells from Figure C are shown in high-power field (original magnifications ×200 [A and B], ×100 [C], and ×400 [D]).

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Table 1

Summary of Clinical and Pathologic Findings and Nuclear and Cytoplasmic Expression of p16 Protein of 50 Cases of Ca-ex-PAa

Summary of Clinical and Pathologic Findings and Nuclear and Cytoplasmic Expression of p16 Protein of 50 Cases of Ca-ex-PAa
Summary of Clinical and Pathologic Findings and Nuclear and Cytoplasmic Expression of p16 Protein of 50 Cases of Ca-ex-PAa

p16 Protein Expression in Ca-ex-PA

In Ca-ex-PA tissues, p16 protein expression was highly heterogeneous. The detailed results are shown in Table 1. We found that malignant components showed significantly lower p16 expression in the nuclei than benign components (P < .001) and significantly higher expression in the cytoplasm than benign components (P < .001) (Table 2). There was significant difference in nuclear (P < .001) and cytoplasmic (P  =  .047) p16 expression between benign and malignant glandular components, and there was significant difference in nuclear (P  =  .02) and cytoplasmic (P  =  .007) p16 expression between benign and malignant myoepithelial components (Table 2). In the benign components, there was no significant difference in nuclear (P  =  .77) and cytoplasmic (P  =  .45) p16 expression between the ductal cells and the myoepithelial cells (Figure 2, A; Table 2). Similarly, there was no significant difference in nuclear (P  =  .16) and cytoplasmic (P  =  .31) p16 expression between ANOS components (Figure 2, B) and myoepithelial carcinoma components (Figure 2, C; Table 3).

Figure 2

Immunoreactivity of p16 in the benign and malignant components of carcinoma ex pleomorphic adenoma. There was positive reaction of p16 protein in benign ductal cells and myoepithelial cells, and expression primarily occurred in tumor cell nuclei (A). There was strong positive reaction in the cytoplasms of many adenocarcinoma not otherwise specified cells (B) and myoepithelial carcinoma cells (C) (original magnifications ×400).

Figure 2

Immunoreactivity of p16 in the benign and malignant components of carcinoma ex pleomorphic adenoma. There was positive reaction of p16 protein in benign ductal cells and myoepithelial cells, and expression primarily occurred in tumor cell nuclei (A). There was strong positive reaction in the cytoplasms of many adenocarcinoma not otherwise specified cells (B) and myoepithelial carcinoma cells (C) (original magnifications ×400).

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Table 2

Comparisons of p16 Protein Expression and Promoter Methylation of the p16 Gene Between Benign and Malignant Components of Carcinoma Ex Pleomorphic Adenoma

Comparisons of p16 Protein Expression and Promoter Methylation of the p16 Gene Between Benign and Malignant Components of Carcinoma Ex Pleomorphic Adenoma
Comparisons of p16 Protein Expression and Promoter Methylation of the p16 Gene Between Benign and Malignant Components of Carcinoma Ex Pleomorphic Adenoma
Table 3

Analysis of the Relationship Between the p16 Protein Expression and Promoter Methylation of the p16 Gene of Malignant Components and the Clinicopathologic Features of Carcinoma Ex Pleomorphic Adenoma

Analysis of the Relationship Between the p16 Protein Expression and Promoter Methylation of the p16 Gene of Malignant Components and the Clinicopathologic Features of Carcinoma Ex Pleomorphic Adenoma
Analysis of the Relationship Between the p16 Protein Expression and Promoter Methylation of the p16 Gene of Malignant Components and the Clinicopathologic Features of Carcinoma Ex Pleomorphic Adenoma

Promoter Methylation of the p16 Gene in Ca-ex-PA and Its Correlation With p16 Protein Expression

Promoter methylation frequency of the p16 gene in benign and malignant components of Ca-ex-PA was 8% (4 of 50) and 36% (18 of 50) respectively (Figure 3, A and B). In all 4 cases in which p16 gene promoter was methylated in the benign components, p16 gene promoter was also methylated in the corresponding malignant components. There was significant difference in the prevalence of the p16 gene promoter methylation between benign and malignant components (P  =  .001; Table 2).

Figure 3

Analysis by methylation-specific polymerase chain reaction on the p16 gene in the benign and malignant components of carcinoma ex pleomorphic adenoma. In the benign components, the methylation bands were present in cases 19 and 23 (A). In the malignant components, the methylation bands were present in cases 23, 25, 26, and 28 (B). Abbreviations: +, positive control; CM, negative control for methylation; CU, negative control for unmethylation; M, methylated DNA; U, unmethylated DNA.

Figure 3

Analysis by methylation-specific polymerase chain reaction on the p16 gene in the benign and malignant components of carcinoma ex pleomorphic adenoma. In the benign components, the methylation bands were present in cases 19 and 23 (A). In the malignant components, the methylation bands were present in cases 23, 25, 26, and 28 (B). Abbreviations: +, positive control; CM, negative control for methylation; CU, negative control for unmethylation; M, methylated DNA; U, unmethylated DNA.

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No associations were found between cytoplasmic (P  =  .57) or nuclear (P  =  .18) p16 expression and promoter methylation status of the p16 gene in benign components, and no associations were found between cytoplasmic (P  =  .79) or nuclear (P  =  .32) p16 expression and promoter methylation status of the p16 gene in malignant components (Table 4).

Table 4

Correlations Between Promoter Methylation of the p16 Gene and Cytoplasmic or Nuclear p16 Protein Expression in Benign and Malignant Components of Carcinoma Ex Pleomorphic Adenoma

Correlations Between Promoter Methylation of the p16 Gene and Cytoplasmic or Nuclear p16 Protein Expression in Benign and Malignant Components of Carcinoma Ex Pleomorphic Adenoma
Correlations Between Promoter Methylation of the p16 Gene and Cytoplasmic or Nuclear p16 Protein Expression in Benign and Malignant Components of Carcinoma Ex Pleomorphic Adenoma

Correlations Between p16 Protein Expression or Promoter Methylation of the p16 Gene and Clinicopathologic Characteristics

No correlations were found between nuclear p16 protein expression and patients' sex (P  =  .40), age (P  =  .51), tumor location (P  =  .33), TNM stage (P  =  .56), grade (P  =  .75), histologic subtype (ANOS or myoepithelial carcinoma; P  =  .16) or invasiveness (P  =  .59), and no correlations were found between cytoplasmic p16 protein expression and patients' sex (P  =  .57), age (P  =  .70), tumor location (P  =  .50), TNM stage (P  =  .82), grade (P  =  .12), histologic subtype (ANOS or myoepithelial carcinoma; P  =  .31) or invasiveness (P  =  .81). Furthermore, no associations were found between promoter methylation of the p16 gene and patients' sex (P  =  .98), age (P  =  .26), tumor location (P  =  .69), TNM stage (P  =  .96), grade (P > .99), histologic subtype (ANOS or myoepithelial carcinoma; P > .99) or invasiveness (P  =  .08) (Table 3).

In an attempt to clarify the role of the p16 gene in Ca-ex-PA, we analyzed the p16 protein expression and the promoter methylation status of the p16 gene in both benign and malignant components of 50 cases of Ca-ex-PA. Ca-ex-PA is considered a lesion derived from preexisting or recurrent PA. All of the 50 cases selected in our study had conspicuous benign and malignant components simultaneously in the same tumor tissue. Therefore, the paired comparison of molecular alterations of these 2 distinct components in the same sample might provide important information on the role of the p16 gene in Ca-ex-PA.

Nuclear p16 protein plays an important role as a direct inhibitor of cyclin-dependent kinase to exert its oncosuppressor activity. Decrease or loss of p16 protein expression in the nuclei of tumor cells has been reported in many kinds of human carcinomas.5,1820 Similar to those previous findings, our results showed that the tumor cells in malignant components of Ca-ex-PA showed significantly lower p16 expression in the nuclei than that in the benign components. With regard to cytoplasmic p16 accumulation, it was initially considered as nonspecific expression by some investigators. However, Evangelou et al21 detected simultaneous nuclear and cytoplasmic p16 staining of tumor cell in non–small cell lung carcinomas and then observed granular immunoreactivity in the cytoplasm near the rough endoplasmic reticulum by immunoelectron microscopy. Furthermore, Di Vinci et al22 reported that the p16 protein was localized almost exclusively in nuclei in breast fibroadenoma and nontumoral epithelia, whereas in breast carcinoma, the staining was presented in both nuclei and cytoplasms or only in cytoplasms. Zhao et al23 found that most colon carcinomas showed more cytoplasmic p16 expression than the adenomas or the adjacent normal mucosa, whereas most normal epithelia presented more p16 expression in the nucleus than their adjacent adenomas and carcinomas. These findings indicated that specific p16 expression not only could occur in cytoplasms of tumor cells, but also might be related to carcinogenesis. In the current study we also found that carcinoma cells showed significantly more cytoplasmic expression of p16 than did benign tumor cells. In addition, there was no significant difference in p16 expression between the glandular epithelial cells and the myoepithelial cells in either benign or malignant components. Our results suggest that overexpression of p16 in the cytoplasm as well as decreased p16 expression in the nucleus may be important in the evolution of salivary PA to Ca-ex-PA whenever epithelial components or myoepithelial components undergo malignant transformation. Contrary to our results, Patel et al13 reported there was no difference in p16 protein expression of tumor cells between the benign and the malignant components of 14 cases of Ca-ex-PAs. However, their study did not distinguish the localization of p16 expression between cytoplasm and nucleus. So we propose that when evaluating the immunohistochemical result of p16, expression in both nucleus and cytoplasm should be analyzed.

The mechanisms leading to the cytoplasmic p16 accumulation remain unclear. Neither mutation nor loss of heterozygosity of the p16 gene was detected in 3 breast cancer cell lines with cytoplasmic p16 expression.24 In the present study, we found that cytoplasmic p16 protein expression was not correlated with promoter methylation of the p16 gene. Therefore, we suppose that cytoplasmic p16 protein expression may be induced not only by alterations of p16 on the gene level, but also by other mechanisms, such as posttranslational modification of p16 protein, cytoplasmic accumulation of unknown protein that binds to p16 protein, or even more complex pathways. For example, Zhao et al23 suggested that cytoplasmic localization of p16 might be due to its binding with cyclin-dependent kinase 4, forming a larger molecule that is therefore difficult to pass through the nuclear membrane.

Early studies have suggested that cytoplasmic p16 expression was also related to tumor progression. Emig et al24 detected that cytoplasmic p16 expression was associated with poor differentiation in breast cancer. Arifin et al25 found that cytoplasmic p16 expression was associated with poor prognosis in high-grade astrocytoma. Unlike these previous reports, there was no correlation between cytoplasmic p16 expression and clinicopathologic factors of Ca-ex-PA in this study, suggesting that the cytoplasmic p16 expression may be significant in the malignant transformation of PA, but not in the progression of Ca-ex-PA. In addition, in the current study, there was no association between nuclear p16 expression and any clinicopathologic factor of Ca-ex-PA.

Promoter methylation of the p16 gene occurs frequently in cancer tissue and is considered a crucial event in carcinogenesis. In terms of Ca-ex-PA, only 2 English papers have been published.14,15 Suzuki and Fujioka14 reported that no case with promoter methylation of the p16 gene was detected among the 4 cases studied, whereas Weber et al15 determined promoter methylation of the p16 gene in the 1 case of Ca-ex-PA studied. The conflicting findings may be attributed to the very small sample size in their studies. Our results suggest that promoter methylation of the p16 gene was a more frequent event in the malignant components of Ca-ex-PA than in the benign components. Howerer, correlation between promoter methylation of the p16 gene and any clinicopathologic variable failed to be statistically significant.

The p16 gene is silenced by promoter methylation epigenetically in many human tumors.6,2629 We examined whether promoter methylation of the p16 gene was associated with reduced nuclear p16 protein expression in Ca-ex-PA. However, our study did not show a significant association between promoter methylation of the p16 gene and decreased nuclear p16 protein expression. The possible explanation of the absence of their correlation is that in Ca-ex-PA p16 expression is also regulated by other mechanisms, for example genetic changes, such as gene mutations and deletions. In another study,14 the investigators detected homozygous deletions of the p16 gene in 1 of 4 Ca-ex-PAs. Extensive research is needed to further clarify the relationship between the genetic and epigenetic alterations and p16 expression changes in Ca-ex-PA.

In conclusion, we suggest that overexpression of p16 protein in the cytoplasm and decreased expression of p16 protein in the nucleus may play important roles in the evolution of PA to Ca-ex-PA. Promoter methylation of the p16 gene may be correlated with the malignant transformation of PA.

This work was supported by the Natural Science Fund of China (30872905 and 81072211), the Science and Technology Commission of Shanghai (08DZ2271100), and the Shanghai Leading Academic Discipline Project (S30206).

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Author notes

From the Department of Oral Pathology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, China.

The authors have no relevant financial interest in the products or companies described in this article.