Clinicopathological significance of androgen receptor, HER2, Ki-67 and EGFR expressions in salivary duct carcinoma.

BACKGROUND: Salivary duct carcinoma (SDC) is a highly aggressive disease which often metastasizes to distant sites, and there is no established standard therapy for this systemic disease. Given that SDC is biologically similar to breast and prostate cancer, anti-androgenic receptor (AR) and anti-human epidermal growth factor receptor 2 (HER2) therapies have the potential to exert effects, not only on patients with breast and prostate cancer but also on those with SDC. METHODS: The expression levels of HER2, epidermal growth factor receptor (EGFR), Ki-67, and AR were assessed in 32 patients with SDC, and their correlations with overall survival (OS) and disease-free survival (DFS) were analyzed retrospectively. SDC was classified into five subtypes using a method similar to that used for breast cancer. RESULTS: Anti-AR, HER2, and EGFR were positive in 23 (71.9 %), 14 (43.8 %), and 26 (81.3 %) cases, respectively. One or more of these 3 factors were positive in 30 (93.8 %) cases. The Ki-67 labeling index was greater than 15 % in all cases. While molecular status did not correlate with OS, EGFR and AR positivity were significantly associated with DFS in univariate analysis. Multivariate analysis revealed that EGFR was the only independent predictor of DFS. CONCLUSIONS: The statuses of some molecules are useful to predict DFS in patients with SDC. Ki-67 overexpression suggests that cytotoxic agents are effective for SDC. Since the majority of SDCs express AR, HER2, and/or EGFR, assessing and targeting these molecules are promising strategies to improve the prognosis of unresectable, metastatic or recurrent SDC, and a classification system according to the molecular expression status may be useful to select appropriate therapy.

Differential expression of topoisomerase IIalpha protein in salivary gland carcinomas: histogenetic and prognostic implications.

BACKGROUND: Salivary gland carcinomas are relatively uncommon heterogeneous malignancies characterized by locoregional invasion and distant metastasis. Topoisomerase IIalpha (topoIIalpha), located at chromosome 17q21-22, is considered a major mediator of cell proliferation and DNA replication. The purpose of this study was to evaluate the expression of topoIIalpha in various types of salivary gland tumors and its biological significance. METHODS: The protein expression of topoIIalpha was evaluated immunohistochemically in formalin-fixed, paraffin-embedded tissue from 54 salivary gland carcinomas and 20 benign tumors (10 pleomorphic adenomas and 10 Warthin's tumors). The primary salivary gland carcinoma specimens consisted of 17 adenoid cystic carcinomas, 7 adenocarcinomas not otherwise specified, 7 mucoepidermoid carcinomas, 6 salivary duct carcinomas, 3 acinic cell carcinomas, 3 carcinomas ex pleomorphic adenomas, 3 epithelial-myoepithelial carcinomas, 2 carcinosarcomas, 2 lymphoepithelial carcinomas, 2 myoepithelial carcinomas, 1 oncocytic carcinoma, and 1 squamous cell carcinoma. The associations between clinicopathological factors and outcome were analyzed. RESULTS: Of the 54 primary salivary gland carcinomas, 38 (70%) showed positive expression (> or = 10%) of topoIIalpha protein, and 16 carcinomas (30%) and all benign tumors were negative (p < 0.001). Expression of topoIIalpha was more frequently observed in salivary duct carcinoma, carcinoma ex pleomorphic adenoma, adenocarcinoma, and adenoid cystic carcinoma, solid type, and it was associated with advanced stage and shortened survival. CONCLUSION: The results of the present study suggest that topoIIalpha expression is associated with histologically aggressive subtypes and shortened survival. Furthermore, it may provide useful prognostic information and suggests the potential efficacy of topoIIalpha-targeting therapy in patients with salivary gland carcinoma.

[Treatment results of chemotherapy with S-1 for head and neck cancer].

A study of chemotherapy treatment with S-1 in patients with head and neck cancer was conducted in 20 patients with residual tumor after primary chemoradiotherapy, or recurrent tumors. Treatment courses consisted of oral administration of S-1 at a dose of 80 to 120 mg/day depending on the body surface area for 14 consecutive day followed by a 7- day rest period. The response rate in patients with residual tumors after primary chemoradiotherapy was 55.6%(5/9). In patients with recurrent tumors, the response rate was 0%(0/11), however, median survival time was 534.5 days. Therefore, S-1 seemed to be useful for the treatment of patients with head and neck cancer from the point of QOL.

Effects of small interfering RNA targeting thymidylate synthase on survival of ACC3 cells from salivary adenoid cystic carcinoma.

BACKGROUND: Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer and high expression of TS has been associated with poor prognosis or refractory disease in several cancers including colorectal and head and neck cancer. Although TS is known to regulate cell cycles and transcription factors, its potency as a therapeutic target has not been fully explored in adenoid cystic carcinoma (ACC). METHODS: An ACC cell line (ACC3) was transfected with siRNA targeting the TS gene and inhibition of cell growth and induction of apoptosis-associated molecules were evaluated in vitro. In addition, the in vivo effect of TS siRNA on tumor progression was assessed using a xenograft model. RESULTS: Our results demonstrated that ACC3 cells showed significantly higher TS expression than non-cancer cell lines and the induction of TS siRNA led to inhibition of cell proliferation. The effect was associated with an increase in p53, p21, and active caspase-3 and S-phase accumulation. We also found up-regulation of spermidine/spermine N1-acetyltransferase (SSAT), a polyamine metabolic enzyme. Furthermore, treatment with TS siRNA delivered by atelocollagen showed a significant cytostatic effect through the induction of apoptosis in a xenograft model. CONCLUSION: TS may be an important therapeutic target and siRNA targeting TS may be of potential therapeutic value in ACC.

Implications of methylation patterns of cancer genes in salivary gland tumors.

PURPOSE: We investigated the methylation status and protein expression of four tumor suppressor genes to determine their role in salivary gland tumorigenesis. EXPERIMENTAL DESIGN: We performed methylation-specific PCR and protein analyses of 29 normal salivary glands, 23 benign, and 79 malignant salivary gland neoplasms to determine the pattern and potential diagnostic and/or biological role of the RASSF1, RARbeta2, DAPK, and MGMT tumor suppressor gene methylation in these tumors. RESULTS: No methylation was detected in the normal tissues. Methylation occurred in 9 of 23 (39.1%) benign tumors; 3 (25.0%) pleomorphic adenomas and 6 (66.7%) Warthin's tumors at the MGMT, DAPK, or RASSF1 genes. Methylation occurred in 33 of 79 (41.8%) malignant tumors; 8 (30.8%) adenoid cystic carcinomas, 6 (33.3%) mucoepidermoid carcinomas, 6 (42.9%) acinic cell carcinomas, and 13 (62.0%) salivary duct carcinomas. RASSF1 and RARbeta2 represented 75.8% of methylation events occurring most frequently in salivary duct and acinic cell carcinomas. Overall, we found no significant correlation between protein expression and methylation status of individual genes, but observed low or absent protein expression in several methylated tumors. Significant correlations were found between methylation and aggressive malignant phenotypes (P = 0.0004) and age (P = 0.05). CONCLUSIONS: (a) Benign and malignant salivary tumors differed in the frequency and pattern of gene methylation; (b) high-grade carcinomas were significantly methylated compared with low-grade phenotypes; (c) RASSF1 and RARbeta2 were highly methylated in malignant tumors and can be targeted for therapy; and (d) methylation pattern may serve as a diagnostic and biological marker in assessing these tumors.

Localization of dopamine receptor subtypes in the rat spiral ganglion.

Although dopaminergic neurons are thought to exist in the lateral olivocochlear efferent system and modulate the afferent nerve activity, the distribution of dopamine (DA) receptor subtypes is still obscure. In the present study, we investigated the localization of five subtypes of DA receptor (D1-5) by immunocytochemical analysis and the gene expression of D1-5 using RT-PCR procedure in the rat cochlea. Most, but not all, spiral ganglion neurons were immunolabeled with all the anti-DA receptor subunit antibodies and faint punctuate immunoreactivities were observed in inner hair cell regions. Gene expression for all receptors was detected. These results suggest that all DA receptor subtypes are present in spiral ganglion cells, and potentially regulate afferent neurotransmission.

Differential expression of p63 isotypes (DeltaN and TA) in salivary gland neoplasms: biological and diagnostic implications.

To determine the association between the expression of p63 gene isoforms (TA and DeltaN) and salivary gland tumorigenesis, we performed reverse transcription-polymerase chain reaction analysis of these markers in 71 benign and malignant salivary gland neoplasms. The results were correlated with the expression of Notch ligand JAG1 gene and the clinicopathologic features and the full-length p63 protein expression by immunohistochemistry. Both p63 isoforms were either negative or weakly expressed in normal salivary gland tissues. TAp63 was highly expressed in most benign tumors and was either negative or weakly positive in most carcinomas. Conversely, DeltaNp63 was negative or faintly positive in most benign neoplasms and was highly expressed in adenoid cystic, mucoepidermoid, and myoepithelial carcinomas. Immunohistochemical analysis using anti-full-length p63 protein showed ubiquitous nuclear staining in basal and myoepithelial cells in both benign and malignant neoplasms. JAG1 was expressed in most benign and malignant tumors and did not correlate with p63 isoforms expression. We conclude that (1) p63 isoforms are differentially expressed in most benign and malignant tumors and may play distinct biological roles in certain salivary gland neoplasms; (2) p63 immunostaining do not correlate with the isoforms expression; and (3) isoform-specific antibodies are required for better cellular localization and biological correlations.

Differential methylation status of tumor-associated genes in head and neck squamous carcinoma: incidence and potential implications.

PURPOSE: Promoter hypermethylation is one of the major mechanisms in the transcriptional inactivation of certain carcinoma-associated genes. Concurrent methylation analysis of multiple, functionally distinct genes may provide important information on their differential alterations and potential association in head and neck squamous carcinogenesis. EXPERIMENTAL DESIGN: Methylation-specific PCR analysis of the CpG islands of 8 cancer-related genes was performed on 19 cell lines and 32 primary head and neck squamous cell carcinoma (HNSC) specimens with matched histologically normal mucosa and 6 dysplastic lesions. The methylation status and histological features of the specimens were investigated. RESULTS: In histologically normal squamous mucosa, no to low-level methylation (0-22%) was noted in some specimens at all genes except RARbeta2 (50%). Considerable variation in the incidence of methylation of these genes within and between cell lines and tumor specimens was noted. The highest incidences of methylation in the cell lines and primary tumors were noted in RARbeta2 (53%), MGMT (37%), p16 (33%), and DAP-K (25%); low incidence of methylations were noted in E-cadherin (2%), p73 (2%) RASSF1A (10%), and p14 (20%) genes. The incidences of methylation of each gene were almost similar between the HNSC cell lines and primary cancer specimens, although methylation of RASSF1A was observed in cell line (26%), but not in dysplasia and primary tumor. RARbeta, p16, and MGMT genes showed the highest incidences of methylation in premalignant and invasive carcinomas. CONCLUSIONS: Methylation of p16, RARbeta, and MGMT may constitute early events in HNSC tumorigenesis. The infrequent methylation at certain genes suggests a minimal role for this feature in their functional assessment in HNSC. The variability within and between cell lines and tumor specimens supports a heterogeneous and dynamic state of methylation in genes associated with HNSC tumorigenesis.

Inflammatory pseudotumour (plasma cell granuloma) arising in the maxillary sinus.

CONCLUSIONS: Inflammatory pseudotumours in the maxillary sinus may present as malignant tumours and manifest locally aggressive features characteristic of such tumours. Despite their locally destructive features, they pursue a benign course after local excision. OBJECTIVE: Inflammatory pseudotumour (plasma cell granuloma) is an uncommon non-neoplastic lesion comprising a proliferation of spindle myofibroblasts and chronic inflammatory cells. Despite its benign histopathological nature, it may exhibit aggressive behaviour that is yet to be characterized in the head and neck area. MATERIAL AND METHODS: We present the cases of two adult patients with inflammatory pseudotumour arising from the maxillary sinus. Immunohistochemistry and polymerase chain reaction for immunoglobulin from tissue sections were performed to confirm the polyclonality of the infiltrating plasma cells. RESULTS: CT and MRI disclosed expansive soft masses eroding surrounding soft and bony tissues. Histopathologically, the lesions were unencapsulated and composed of numerous plasma cells, histiocytes and spindle cells with minimal nuclear pleomorphism.

Gene expression screening of salivary gland neoplasms: molecular markers of potential histogenetic and clinical significance.

Salivary gland neoplasms comprise phenotypically and biologically diverse lesions of uncertain histogenesis. The molecular events associated with their development and clinicopathological heterogeneity remain unknown. To reveal these events, we performed microarray expression analysis using a nylon-filter membrane platform on 18 primary lesions representing the most common benign and malignant types. Our study identified a small set of genes that are differentially altered between normal salivary gland tissues and benign and malignant tumors. Of the 5000 genes arrayed, 136 genes were differentially expressed by normal tissue, benign tumors, and various malignant neoplasms. Hierarchical clustering analysis differentiated between adenoid cystic carcinomas (ACCs) and other malignant subtypes. Non-ACC specimens manifested overlapping patterns of gene expression within and between tumors. Most of the differentially expressed genes share functional similarities with members of the adhesion, proliferation, and signal transduction pathways. Our study identified: 1) a set of genes that differentiate normal tissue from tumor specimens, 2) genes that differentiate pleomorphic adenoma and ACCs from other malignant salivary gland neoplasms, and 3) different patterns of expression between ACCs arising from major and minor salivary gland sites. The differentially expressed genes provide new information on potential genetic events of biological significance in future studies of salivary gland tumorigenesis.

Phase I/II study of docetaxel, cisplatin and S-1 in locally advanced, recurrent and metastatic head and neck squamous cell carcinoma.

The purpose of this study was to evaluate the maximum tolerated dose, dose-limiting toxicities and preliminary efficacy of chemotherapy with cisplatin, docetaxel and S-1 (TPS) to treat advanced head and neck squamous cell cancer. S-1 was administered orally twice daily on days 1-14 and docetaxel and cisplatin were injected intravenously on day 8, with one course lasting 4 weeks. The recommended dose obtained from a phase I study was set at docetaxel 60 mg/m(2), cisplatin 60 mg/m(2) and S-1 80 mg/m(2)/day. The phase II study revealed that the overall response rate was 81%, comprising 95% in untreated patients with localized advanced cancer and no distant metastases, 50% in untreated patients with distant metastases and 33% in previously treated patients with recurrence. The overall survival rate of untreated patients with localized advanced cancer and no distant metastases was 95% at 1 year and 64.33% at 2 years. In terms of grade 3 or higher hematotoxicity, neutropenia occurred in 100%, thrombocytotopenia in 4% and anemia in 4%. Febrile neutropenia occurred in 46%, with the rate rising to 57% in elderly patients ≥66 years. Grade 3 or higher non-hematotoxicity consisted of loss of appetite in 8%, diarrhea in 8%, hyponatremia in 13% and hypokalemia in 13%. This TPS therapy may be recommended for use as induction chemotherapy. For patients ≤65 years, the appropriate dose was docetaxel 60 mg/m(2), cisplatin 60 mg/m(2) and S-1 80 mg/m(2), whereas for those ≥66 years, it was docetaxel 60 mg/m(2), cisplatin 60 mg/m(2) and S-1 60 mg/m(2).

Inflammatory pseudotumor of the parapharyngeal space: a case report.

Inflammatory pseudotumor is an idiopathic granuloma characterized by infiltrative proliferation of inflammatory cells and myofibroblastic cells, as well as locally aggressive features, clinically and radiologically mimicking a neoplastic process. The occurrence of inflammatory pseudotumor in the head and neck area is uncommon, especially in the parapharyngeal space. The case of a 54-year-old female with inflammatory pseudotumor of the parapharyngeal space is presented. The patient initially complained of hoarseness, dysarthria, aspiration, and hearing impairment. MRI disclosed an expansive soft mass in the parapharyngeal space encompassing the carotid arteries. Histopathologically, the lesions were composed of numerous plasma cells, lymphocytes, histiocytes, and spindle myofibroblastic cells, showing perineural infiltration of inflammatory cells. The patients' symptoms, including conductive hearing loss, improved dramatically with reduction in lesion size after corticosteroid treatment.

Autologous serum eardrops therapy with a chitin membrane for closing tympanic membrane perforations.

OBJECTIVE: Office treatment for chronic tympanic membrane (TM) perforations has limitations, and alternative methods to myringoplasty are sometimes needed. Serum lacks antigenicity and contains a large variety of growth factors known to modulate proliferation of various tissues to promote wound healing effects. Our purpose was to evaluate the feasibility of autologous serum eardrops therapy with a chitin membrane for closing TM perforations. INTERVENTION: In the outpatient clinic, the perforation margin was cauterized with silver nitrate, and the perforation was covered with a chitin membrane. Patients were instructed to apply autologous serum eardrops daily. Patients were examined every 2 weeks, and the procedure was repeated. RESULTS: We treated 19 sequential patients with chronic TM perforation in 1 ear between October 2005 and September 2007. Closure of the TM was achieved in 11 (58%) of 19 ears, and reduction of the perforation size was observed in 2 ears (11%). Closure rates for small, medium, and large perforations were 57 (8 of 14), 0 (0 of 1), and 75% (3 of 4), respectively. Closure rates for perforations attributable to intratympanic dexamethasone treatment, after myringoplasty and chronic otitis media were 67 (2 of 3), 67 (2 of 3), and 54% (7 of 13), respectively. Time for closure took from 15 to 175 days, with an average of 68 days (5.9 clinic visits). During autologous serum eardrop therapy with a chitin membrane, no remarkable side effects in the treated ears were observed. Measurement of the concentration of the epidermal growth factor, transforming growth factor beta1, fibronectin, and interleukin 6 in the serum showed no decrease in 14 days, suggesting activity remained stable in that period. CONCLUSION: Autologous serum eardrops therapy with a chitin membrane, which requires no surgical intervention, was found to be a promising office-based technique for the closure of chronic TM perforations because of its ease, safeness, and feasibility. However, additional studies are needed to independently analyze the specific benefits of the serum drops and the chitin membrane.

Developmental expression of the outer hair cell motor prestin in the mouse.

The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (C (v)), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, C (v) was about 70% of the mature level. C (v) saturated at P18 when hearing shows full maturation. On the other hand, C (lin), which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor's operating voltage midpoint, V (pkcm), tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, V (pkcm) reached a steady-state level near -67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate V (pkcm), may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.

Simvastatin inactivates beta1-integrin and extracellular signal-related kinase signaling and inhibits cell proliferation in head and neck squamous cell carcinoma cells.

The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, also called statins, are commonly used as lipid-lowering drugs that inhibit cholesterol biosynthesis. An anticancer effect, as a pleiotropic function of certain statins, has been hypothesized. In the present study, we investigated the effect of simvastatin, one of the natural statins, on cell proliferation, cell cycle, invasive activity, and molecular expressions associated with cell-extracellular matrix adhesion, signal transduction, and DNA synthesis in Tu167 and JMAR cells from head and neck squamous cell carcinoma. The addition of simvastatin resulted in a dose-dependent inhibition of cell growth and migration into the extracellular matrix. Considerable morphological changes occurred after treatment with simvastatin, demonstrating loss of cell adhesion and disruption of actin filaments in cytoplasm. The inhibitory effect of simvastatin on cell proliferation seemed to be associated with cell cycle arrest and increased expression of p21, p27, and activated caspase-3. The expression of beta1-integrin, a counter adhesion for the extracellular matrix, phosphorylated FAK, and phosphorylated ERK was decreased by treatment with simvastatin. The proapoptotic effect of simvastatin was inhibited by treatment with mevalonate. cDNA microarray assay demonstrated that molecular changes resulting from treatment with simvastatin included the up-regulation of cell cycle regulators and apoptosis-inducing factors and the down-regulation of integrin-associated molecules and cell proliferation markers. Of down-regulated genes induced by simvastatin treatment, a significant depletion of thymidylate synthase was confirmed using western blot analysis. These results imply that simvastatin has the potential to be effective for the prevention of the growth and metastasis of cancer cells.

Salivary gland carcinoma treated with concomitant chemoradiation with intraarterial cisplatin and docetaxel.

Malignant neoplasms of the salivary gland are uncommon entities in which surgical resection of the primary lesion has been accepted as a standard therapeutic option. The efficacy of radiation and systemic chemotherapy has been limited for patients with recurrent, metastatic, or unresectable disease because of unfavorable response rates and the short duration of the response. We treated one patient with recurrent adenoid cystic carcinoma arising from the sublingual gland and one patient with primary adenocarcinoma arising from the parotid gland with transfemoral intraarterial chemotherapy, based on full-dose cisplatin and docetaxel and concurrent external-beam radiotherapy. The doses of cisplatin and docetaxel in the two patients were 80-100 mg/m2 and 10-15 mg/m2, respectively. Docetaxel was infused first, followed by cisplatin. Both patients obtained complete responses. Although complications such as mucositis, anorexia, neutropenia, and ischemic colitis were observed, they were well tolerated and manageable. The concomitant chemoradiotherapy of cisplatin and docetaxel seemed to be a practicable option for patients with recurrent and unresectable salivary gland carcinomas.

Promoter methylation and protein expression of the E-cadherin gene in the clinicopathologic assessment of adenoid cystic carcinoma.

Adenoid cystic carcinoma, a relatively uncommon tumor of salivary glands, is characterized by a prolonged clinical course and a fatal outcome. The molecular events underlying their progression are unknown. In this study, we examined the methylation status of E-cadherin gene and its protein expression in 23 cases of adenoid cystic carcinoma and correlated the results with the clinicopathologic factors to determine its role in these tumors. We also analyzed the effect of 5-azacytidine on the re-expression in a methylated cell line of adenoid cystic carcinoma for this gene. In our study, E-cadherin immunoreactivity, although heterogeneous, showed a progressive reduction with high histological grade and in metastatic and recurrent lesions. Promoter methylation was detected in 16 of 23 cases (70%), but there was no correlation with the histological grade or patient prognosis. Microdissection of immuno-negative cells in heterogeneous tumors showed positive methlyation. In the cell line from salivary adenoid cystic carcinoma with methylated E-cadherin, 5-azacytidine restored the E-cadherin expression. Our results indicate that: (1) E-cadherin gene promoter is frequently methylated in adenoid cystic carcinoma, leading to reduced E-cadherin expression, (2) variable E-cadherin expression might result from the intratumoral heterogeneity, and (3) increased extent of methylated areas may be associated with progression and advancement of the disease.

Expression of p16 protein and hypermethylation status of its promoter gene in adenoid cystic carcinoma of the head and neck.

Reduced expression of the p16 gene product (protein), an inhibitor of cyclin-D-dependent protein kinase which regulates cell cycle at the G1/S boundary, is implicated in tumor progression in various neoplasms. Hypermethylation of the p16 promoter gene has recently been suggested to be one of the reasons for the reduced protein expression. To explore the role of p16 in the biological behavior of adenoid cystic carcinomas (ACC), we investigated the immunohistochemical expression of p16 protein in 38 ACC tumors (32 primary, 3 recurrent, and 3 metastatic tumors) and the methylation status of its promoter gene. We also examined their relationships to the histological grade of malignancy. Positive reaction of p16 protein was demonstrated in the nuclei of luminar cuboidal cells in areas with tubular patterns. The reactions were reduced in the areas with solid or large cribriform patterns. The levels of p16 expression correlated with the histological grade of malignancy. Recurrent or metastatic tumors did not differ with respect to histological grades from the original tumor except for 1 case, in which p16 expression was reduced compared to the primary tumor. Methylation-specific PCR demonstrated the hypermethylation status of the p16 promoter gene in 4 of 22 primary tumors (21%), all of which showed negative or low expression of the p16 protein. The study indicated that p16 expression was reduced in ACC cases of higher histological grade of malignancy and that hypermethylation of its promoter gene may be involved in its process in some cases.