Background
Salivary gland carcinomas are uncommon neoplasms, accounting for approximately 5% of those arising in the head and neck region [
1]. These tumors are characterized by widely varied histological features with heterogeneous and unpredictable clinical behaviors. In general, these carcinomas may be categorized into low grade and high grade malignancies based on their origin from either the terminal (intercalated) or the excretory ductal epithelium, respectively [
2]. Biological and clinical classifications have been dependent on this histogenetic classification, but they are occasionally diverse even within those categories [
3]. Patients with higher histological grades appear to be more susceptible to locoregional recurrence and distant metastasis. However, effective treatment for unresectable tumors has not been established [
4]. Although chemotherapy remains the main treatment modality for patients with advanced disease, the efficacy of systemic chemotherapy appears to be limited or controversial in high-grade salivary gland carcinomas.
Topoisomerase IIα (topoIIα), a nuclear enzyme, has a key role in DNA replication. Consistent with its major role in DNA replication, overexpression of topoIIα has been detected in cells with high proliferation activity [
5,
6]. The gene encoding topoIIα is located at chromosome 17q21-22 and encodes for a 170 kDa protein. Interestingly, this gene is also in close proximity to the HER2 oncogene, which encodes for a transmembrane tyrosine kinase receptor protein and has been shown to be frequently coamplified in several cancers, including breast cancer [
7]. Overexpression of HER2 has been reported in salivary gland carcinomas of excretory cell origin, including salivary duct and squamous cell carcinomas, but not in those of intercalated cell origin, including adenoid cystic, acinic cell, and adenocarcinomas, and it is associated with poor clinical prognosis in patients with salivary duct carcinoma [
8]. On the other hand, little information is available about the expression of topoIIα protein in salivary gland tumors.
TopoIIα is a known cellular target for anticancer agents such as anthracyclines. Anthracyclines inhibit the activity of topoIIα by stabilizing DNA cleavage, thus exerting anticancer effects [
6]. Previous clinical investigations have shown that amplification and overexpression of topoIIα are associated with good response to anthracycline-based chemotherapy in several cancers, such as breast cancer [
9‐
11]. Anthracyclines as a single agent or in combination therapy have shown certain effects in mucoepidermoid carcinomas and high-grade adenocarcinomas, whereas they likely have a limited effect in adenoid cystic carcinomas [
4]. These observations suggest that identifying biological markers that can predict sensitivity to drugs may also allow stratification of patients for treatment. The purpose of the present study was to evaluate topoIIα expression levels in different histological types of salivary gland tumors and to estimate its potential use as a prognostic marker or clinical target in the treatment of patients with salivary gland cancers.
Methods
Tissue samples from surgical cases
Tumor specimens from 54 patients with primary salivary gland carcinomas who underwent surgery or biopsy between 1996 and 2007 were retrieved from the Department of Pathology, Hirosaki University, and associated hospitals. Tumor specimens consisted of 17 adenoid cystic carcinomas, 7 mucoepidermoid carcinomas, 7 adenocarcinomas not otherwise specified, 6 salivary duct carcinomas, 3 carcinomas ex pleomorphic adenomas, 3 acinic cell carcinomas, 3 epithelial-myoepithelial carcinomas, 2 myoepithelial carcinomas, 2 lymphoepithelial carcinomas, and 2 carcinosarcomas. There was only one sample of squamous cell carcinoma and one of oncocytic carcinoma. To compare the expression status of benign tumors, 10 pleomorphic adenomas and 10 Warthin's tumors were also selected (Table
1). All samples were fixed in a 10% formaldehyde solution and embedded in paraffin. Diagnosis and histological classification were based on the World Health Organization Classification (version 2005) [
12]. Informed consents from patients were obtained for the use of resected tumor specimens. The study design and procedure involving the human tissue sampling collection were approved by the ethical board of Hirosaki University.
Table 1
TopoIIa expression status in different histologic types
Benign tumor
| | | | | |
Pleomorphic adenoma | 10 | 0 | 0 | 0 | 10 |
Warthin's tumor | 10 | 0 | 0 | 0 | 10 |
Malignant tumor
| | | | | |
Adenoid cystic carcinoma | 5 | 7 | 3 | 2 | 17 |
Adenocarcinoma, | 2 | 0 | 2 | 3 | 7 |
not otherwise specified | | | | | |
Mucoepidermoid carcinoma | 3 | 2 | 2 | 0 | 7 |
Salivary duct carcinoma | 0 | 1 | 0 | 5 | 6 |
Acinic cell carcinoma | 2 | 0 | 0 | 1 | 3 |
Carcinoma ex pleomorphc adenoma | 0 | 0 | 0 | 3 | 3 |
Epithelial-myoepithelial carcinoma | 2 | 1 | 0 | 0 | 3 |
Carcinosarcoma | 0 | 0 | 1 | 1 | 2 |
Lymphoepithelial carcinoma | 1 | 0 | 1 | 0 | 2 |
Myoepithelial carcinoma | 0 | 1 | 0 | 1 | 2 |
Oncocytic carcinoma | 1 | 0 | 0 | 0 | 1 |
Squamous cell carcinoma | 0 | 0 | 0 | 1 | 1 |
Immunohistochemistry
For immunohistochemistry, 4-μm-thick sections were deparaffinized in xylene and rinsed in ethanol. For antigen retrieval, the sections were heated for 15 min at 121°C in Target Retrieval Solution (DakoCytomation, Glostrup, Denmark) by autoclaving. Endogenous peroxidase was blocked by incubation in methanol with 0.3% H2O2 for 15 min, followed by rinsing in PBS containing 0.1% Tween 20. Immunohistochemical staining was performed with the EnVision™+ System Kit (DakoCytomation) according to the manufacturer's protocol. After treatment with blocking solution, the slides were incubated with the monoclonal primary antibody against topoisomerase IIα (clone Ki-S1; DakoCytomation; dilution 1:100) overnight at 4°C. The slides were then incubated at room temperature for 30 min with a dextran polymer conjugated with horseradish peroxidase enzyme and secondary anti-mouse antibody. The reaction products were detected with 3,3'-diaminobenzidine tetrahydrochloride as the chromogen, and the slides were counterstained with hematoxylin. The number of positive cells in 1000 tumor cells within 4–6 microscopic fields at ×200 magnification was counted and semiquantitatively graded as follows: 0 (negative, 0% to 9%), 1+ (focal, 10% to 24%), 2+ (moderate, 25% to 49%), and 3+ (diffuse, ≥50%). Quantitation of positive cells was conducted by 3 independent observers (S.M., H.M. and H.K.), and inter-observer concordance was over 95%.
Statistical analysis
Statistical analysis was performed using Fisher's exact test to evaluate significant differences between pairs of findings. The Kaplan-Meier method and the log-rank test were used for univariate survival analysis. For multivariate survival analysis, each clinicopathologic parameter was analyzed using a Cox proportional hazards regression model. Differences with a P < 0.05 were considered statistically significant. All statistical examinations were performed using SPSS 16.0 software package (SPSS Japan, Inc., Tokyo, Japan).
Discussion
The present results show that topoIIα is differentially expressed in salivary gland carcinomas and is associated with aggressive histologic type, advanced disease, and poor clinical outcome. To the best of our knowledge, this is the first study to correlate clinical and prognostic features with topoIIα expression in various types of salivary gland tumors. In a multifactorial analysis, topoIIα expression was more significantly correlated with overall survival than clinical stage (Table
2). Several studies have indicated that high expression of topoIIα is considered a feature of enhanced cellular proliferation [
5,
6]. It has been reported that overexpression of topoIIα is associated with poorer survival rates in several malignancies, including head and neck squamous cell carcinoma, glioblastoma, and breast cancer [
13‐
15]. The present findings also support an association between topoIIα expression and aggressive histological subtypes (salivary duct carcinoma, carcinoma ex pleomorphic adenoma, adenocarcinoma, high-grade adenoid cystic carcinoma, and carcinosarcoma) [
16]. Of 9 cases with salivary duct carcinoma and carcinoma ex pleomorphic adenoma, most (8 cases) showed diffuse and strong nuclear expressions of topoIIα; 2 cases of carcinosarcoma had high expression of topoIIα. Carcinosarcoma, which is composed of both malignant epithelial and malignant mesenchymal components, occasionally occurs from pleomorphic adenoma [
17]. Paradoxically, there was no topoIIα expression in benign tumors. In this context, overexpression of topoIIα may be a key event in the malignant transformation of pleomorphic adenoma.
Currently, standard chemotherapy regimens for salivary gland carcinomas have not been established [
4]. TopoIIα is not only known to be a prognostic marker, but it is an important target for topoisomerase II poisons, such as anthracycline and epipodophyllotoxin, in several human cancers. These agents exert an anticancer effect by stabilizing the cleavage of double-stranded DNA. Previous investigations have shown that the sensitivity of topoIIα inhibitors, such as anthracyclines or epipodophyllotoxin, is dependent on increased topoIIα expression levels [
18,
19]. Coincidentally, the gene encoding topoIIα is located at chromosome 17q21-22, which is contiguous with the region encoding HER2. In the field of salivary gland carcinomas, a previous study has shown that overexpression of HER2 is more common in salivary gland carcinomas of excretory cell origin, such as salivary duct carcinoma, mucoepidermoid carcinoma, and squamous cell carcinoma [
8]. Nguyen
et al. [
20] also reported a potential association between HER2 overexpression and histological aggressiveness in mucoepidermoid carcinoma. The present data demonstrated that the expression status of topoIIα might be identical to that of HER2, showing common expression in cases of
de novo salivary duct carcinoma and in malignant components from carcinoma ex pleomorphic adenoma. The molecules associated with amplification of chromosome 17q21-22 may have important roles as diagnostic and therapeutic targets in salivary gland carcinomas. This hypothesis is underscored by a recent phase II trial of herceptin in salivary gland cancers overexpressing HER2. A single dose of herceptin (trastuzumab), a monoclonal antibody directed against HER2 protein, contributed to stabilization of disease in several cases of salivary duct carcinoma [
4,
8]. Recently, Prat
et al. [
21] reported a metastatic salivary duct carcinoma in which complete response was obtained by the combination of paclitaxel, carboplatin, and herceptin. These observations imply that the therapeutic approaches targeting chromosome17q21-22 region encoding topoisomerase IIα and HER2 proteins may benefit patients with high-grade salivary gland carcinomas.
Adenoid cystic carcinoma is the subtype that has been most widely investigated in various types of salivary gland carcinomas. It has been well known that its clinical behavior is dependent on histological grade, with poorer prognosis in the solid type than in the tubular or cribriform type, and the propensity for perineural invasion is histologically characterized [
3]. We previously reported that low expressions of p16 and E-cadherin were associated with poor prognosis and aggressive histological type in adenoid cystic carcinoma [
22,
23]. In the present study, high expressions of topoIIα were observed in tumors predominantly composed of solid areas, though the difference was not statistically significant due to the small number of cases. Other major proliferation or anti-apoptotic markers, such as Ki-67, bcl-2, and cyclin D1, were frequently expressed in adenoid cystic carcinoma, but they had less of an impact on the discrimination of histological grade of adenoid cystic carcinoma [
24‐
26]. In this context, topoIIα may be an excellent marker to evaluate biological and histological aggressiveness in adenoid cystic carcinoma, although further evaluation in a large subset of tumors is needed.
The present study showed a significant association between topoIIα expression and clinical staging, suggesting that overexpression of topoIIα was associated with rapid growth of cancer cells and susceptibility to locoregional or distant metastasis. High expression of topoIIα is considered to be a general feature of highly proliferating cells with rapid cell cycle acceleration [
5,
6]. Our observations may be supported by a previous investigation in oral squamous cell carcinoma, which showed that topoIIα expression was more significant in cases with lymph node metastasis than those without lymph node metastasis [
27]. Thus, topoIIα may be a valuable marker for evaluating the proliferative activity of salivary gland tumor cells.
Conclusion
In conclusion, the present study suggests that topoIIα expression is an independent prognostic factor for salivary gland carcinoma, and assessment of topoIIα expression may be valuable for predicting clinical aggressiveness. While certain factors, such as clinical stage and histopathological grade, play a pivotal role in the management of patients with salivary gland carcinoma, high expression of topoIIα may warrant an intensive modality of therapy that includes adjuvant chemotherapy targeting topoIIα protein and radiation. Further investigations are needed to clarify the significance of topoIIα as a practical therapeutic target.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SM and TS coordinated the study, performed immunohistochemical and statistical analyses, and drafted the manuscript. HK and HM performed histological evaluation of immunostaining. SK and TN contributed to conception and design of the study and interpretation of the data. HS participated in the design and coordination of the study and helped draft the manuscript. All authors have read and approved the final manuscript.