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Introduction

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide (1). Despite diagnostic advances and improvements in treatment modalities, the 5-year survival rate of such patients remains poor (2). HNSCC represents a wide spectrum of neoplasms with different patterns of biological behaviour (3) therefore, the current tumor classification is limited in its prediction of prognosis. Consumption of alcohol or tobacco is the primary risk factor for cancer of the oral cavity, larynx, oropharynx and hypopharynx, and accounts for 75% of HNSCCs (4). HNSCC usually presents with symptoms from the primary site; sore throat, hoarseness, difficulty in breathing and swallowing or ear pain (5). Enlargement of a cervical lymph node as the first presenting feature is not uncommon, particularly within certain ‘silent’ sites, including the tongue base, supraglottis and nasopharynx (5). Systemic metastases are uncommon at first presentation of HNSCC (6). The type of treatment required depends on the location of the primary tumor, the stage of the disease and the expected oncological/functional outcomes (4).

Early stage HNSCC (stage I/II) is usually treated with single-modality therapy (surgery or radiotherapy) while the management of locally advanced disease (stage III/IV) generally requires various combinations of treatment modalities (surgery, radiotherapy, chemotherapy or cetuximab). The 5-year survival rate for all patients with HNSCC is 40–60% (7).

Nuclear receptors are transcription factors that modulate cell proliferation, apoptosis and migration (8). Recent data suggests that there is an association between the intranuclear overexpression of estrogen receptor (ER) β and higher survival rates in patients with lung (9,10), prostate (11), breast (12) and colon cancer (13). A number of studies have demonstrated that nuclear receptors are expressed in head and neck cancer (14–20). However, the clinical significance of sex hormone receptor expression in HNSCC remains unclear. The aim of the present study was to assess ERβ expression in HNSCC and investigate its association with clinical and histopathological factors, disease recurrence and patient survival. To the best of our knowledge this is the largest study examining the role of ERβ in HNSCC.

Patients and methods

Patients

A total of 174 patients with squamous cell carcinoma of oral cavity, larynx and hypopharynx were included in the present study. The patients all underwent primary surgical treatment between January 2000 and December 2006 at the Division of Head and Neck Surgery, Department of Surgical Oncology, University Hospital for Tumors (Zagreb, Croatia). The inclusion criteria for the current retrospective study were as follows: No prior treatment for head and neck cancer and a patient follow-up period of ≥5 years. Cases were classified based on the international tumor-node-metastasis (TNM) classification from 2002 (21). Patients with adverse histopathological features (extracapsular spread, multiple positive lymph nodes, stage pT3 or pT4) underwent postoperative (adjuvant) irradiation. Informed consent was obtained from each patient and the study was carried out with the approval of the Ethical Committee of the Clinical Hospital Center Sisters of Charity.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue samples from 174 primary head and neck squamous cell carcinomas were obtained from the Department of Pathology. Representative 3 µm sections of tumour tissue were cut, dewaxed in xylene and rehydrated in graded ethanol and destilled water. Antigen retrieval was performed in Tris/Ethylenediaminetetraacetic acid (EDTA), pH 9.0 buffer (Dako Target retrieval solution; catalogue number S2367; Dako, Glostrup, Denmark) for 1 h at 98°C in a water bath. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 min. Sections were washed with the buffer and subsequently incubated with mouse monoclonal anti-ERβ antibody (Product code: NCL-ER-β Clone EMR02, dilution, 1:40, Novocastra, Laboratories, Ltd., Newcastle upon Tyne, UK) for 45 min at room temperature. Bound primary antibody was detected using biotinylated anti-mouse secondary antibody (EnVision FLEX, High pH Kit, catalogue number 8010; Dako, ready for use) for 45 min and visualized with diaminobenzidine as chromogen on Autostainer Link 48 (Dako). Slides were counterstained with hematoxilyn, dehydrated, cleared and cover-slipped. A breast cancer tissue sample was used as a positive control.

Stained tissue sections were evaluated by an experienced pathologist. Nuclear staining was considered positive and >100 cells were counted. Separate scores were assigned to each sample according to the percentage of positive nuclei observed (0–100%). The following scoring system was used: 0, no staining of tumour cells (Fig. 1A); 1, positive staining <10% of tumour cells; 2, positive staining in 11–50% of tumour cells; 3, positive staining in >51% tumour cells (Fig. 1B). For the purpose of statistical analysis, cases with scores of 0 and 1 were considered negative and cases with scores of 2 or 3 were considered positive (14).

Figure 1. Immunohistochemical staining (hemotoxylin). (A) Negative reaction to ERβ in the nuclei of tumor cells (magnification, ×100) (B) Strong positive reactions in the nuclei of tumor cells (magnification, ×100).

Statistical analysis

Normality of data distribution was assessed with Smirnov-Kolmogorov test and due to the results obtained, appropriate non-parametric tests were used in analyses. Differences between ER groups were analyzed with the χ2 test (categorical values) and Mann-Whitney U test (quantitative values). Spearman correlation coefficients were used to analyze associations between total survival time and other clinical variables for each tumor location. Overall survival (OS) was calculated using the Kaplan-Meier method, while the log-rank test tested the differences between the actuarial curves. OS was calculated from the time of surgery to death resulting from all causes. IBM SPSS Statistics ver. 21 (Armonk, NY, USA) was used in all statistical tests. P<0.05 was considered to indicate a statistically significant difference.

Results

The present study included 165 male and 9 female patients with a median age of 60.8 years, (range, 39–79), of which 80 (46%) exhibited no evidence of cervical lymph node metastasis, while 94 (54%) had neck metastases. The clinicopathological characteristics of the study group are listed in Table I.

Table I. Clinical and histopathological characteristics of the cohort.

Table I.

Clinical and histopathological characteristics of the cohort.

Characteristic	No. patients	%
Age, years
Median	60.78
Range	39–79
Gender
Female	9	5.2
Male	165	94.8
Tumor site
Oral cavity	37	21.3
Oropharynx	33	18.9
Larynx	63	36.2
Hypopharynx	41	23.6
pT stage
T1	33	19
T2	80	46
T3	41	23.5
T4	20	11.5
pN stage
N0	80	46
N1	22	12.3
N2	64	36.8
N3	8	4.6
Differentiation
Well	19	10.9
Moderate	112	64.4
Poor	43	24.7
Adjuvant radiotherapy
No	43	24.7
Yes	131	75.3
Survival
AwD	70	40.2
StD	54	31.1
StC	50	28.7

[i] pT stage, pathological tumor stage; pN stage, pathological regional lymph nodes stage; AwD, alive without disease; StD, succumbed to disease; StC, succumbed to other causes.

Immunohistochemistry for ERβ indicated positive staining in 73 patients (42%). Distribution of ERβ status among different head and neck subsites is listed in Fig. 2. A higher proportion of the ERβ negative cases (>40%) were located in laryngeal primaries compared with other areas, whereas incidence of other sublocalization within ERβ positive cases were similar (Fig. 2). This association was statistically significant (P=0.04; Table II).

Figure 2. Distribution of ERβ status among head and neck subsites.

Table II. Comparison of the clinicopathological features with respect to ERβ status.

Table II.

Comparison of the clinicopathological features with respect to ERβ status.

Clinical parameter	ER β+ No. patients (%)	ER β- No. patients (%)	P-value
Age, years
≤61	38 (52.1)	53 (52.5)	0.956
>61	35 (47.9)	48 (47.5)
Gender
Male	69 (94.5)	96 (95.0)	0.876
Female	4 (5.5)	5 (5.0)
Tumor site
Oral cavity	21 (28.8)	16 (15.8)	0.040a
Oropharynx	17 (23.3)	16 (15.8)
Larynx	19 (26.0)	44 (43.6)
Hypopharynx	16 (21.9)	25 (24.8)
pT stage
T1/T2	40 (54.8)	73 (72.3)	0.448
T3/T4	33 (45.2)	28 (27.7)
pN stage
N0	31 (42.5)	49 (48.5)	0.429
N+	42 (57.5)	52 (51.5)
pTNM stage
Stage I/II	21 (28.8)	34 (33.7)	0.493
Stage III/IV	52 (71.2)	67 (66.3)
Differentiation
Well	7 (9.6)	12 (11.9)	0.261
Moderate	52 (71.2)	60 (59.4)
Poor	14 (19.2)	29 (28.7)

a P<0.05, indicates a statistically significant result. P-values measured by the χ² test. pT stage, pathological tumor stage; pN stage, pathological regional lymph nodes stage; pTNM stage, pathological tumor-node-metastasis stage.

The univariate correlations between clinicopathological features and ERβ status are presented in Table II. Positive ERβ status was not significantly correlated with any clinicopathohistological parameter other than the survival of patients with respect to the primary tumor site.

Patients with oropharyngeal cancer with specimens that demonstrated an ERβ positive intranuclear reaction had an OS rate of 35.3% at 5 years compared with 25% for patients with negative reaction, a statistically significant difference (log-rank test, P=0.045) (Fig. 3A). By contrast, there was no significant association between ERβ expression and other primary tumor subsites with respect to survival (Fig. 3B-D). By the end of the study, 54 (31.1%) patients had died of disease recurrence while 50 (28.7%) had died of other causes. Follow-up information was available for all patients and average follow-up time was 38.5 months (range, 3–60 months).

Figure 3. Kaplan-Meier survival estimates. Association between level of ERβ expression and primary tumor subsites with respect to survival in the (A) oropharynx, (B) oral cavity (C) hypopharynx and (D) larynx. *P<0.05.

Discussion

A previous report has suggested that ER-mediated signals and pathways serve a critical role in malignant tumor growth, progression and metastasis (8). The most comprehensive analysis was performed on patients with breast cancer where positive expression of ERβ was associated with increased response rates to ER antagonist therapy with tamoxifen (12,22). However, the role of ER signaling in other malignancies remains unclear. Similarily, the significance of ER expression in HNSCC and its biological role in cell invasion, proliferation, metastasis and survival is controversial.

In the head and neck region, ER expression was initially evaluated in laryngeal cancer. This type of cancer is 11 times more common in males than females, despite similar rates of alcohol abuse and smoking in both genders (15,23). Previous studies have demonstrated that 23–79% of laryngeal cancer cases express ERs (16–18,24), concluding that positive ER expression is gender independent with a positive prognostic influence on survival. However, other authors have not been able to identify ER expression in laryngeal cancer (25,26), therefore laryngeal carcinogenesis may be a hormonally independant process. Non-identification of ER in epithelial components of the normal larynges and laryngeal carcinomas has been confirmed (27).

A previous study examined the role of ERβ in laryngeal cancer and demonstrated that its expression was positively correlated with the maintenance of E-cadherin and β-catenin at cell junctions and negatively with increased TNM stage (19). This suggests that ERβ serves a protective role in the pathogenesis of this malignancy.

A study by Nozoe et al (28) demonstrated that positive expression of ERα in combination with negative expression of ERβ proved to be an unfavorable prognostic indicator with respect to the survival of patients with esophageal squamous cell carcinoma (SCC). Wang et al (29) have reported a correlation between positive ERβ expression and lower malignant potential in oesophageal SCC. However, Dong et al (30) reported that ERα and ERβ levels were inversely correlated and that downregulation of ERα and upregulation of ERβ expression may indicate poor prognosis in patients with oesophageal SCC.

Few studies have investigated the role of ER expression in HNSCC. Lukits et al (14) performed a study on 67 patients with carcinoma of the larynx, oral cavity and hypopharynx. ERα and ERβ expressions were identified in 50% of patients and had no significant influence on patient prognosis.

Egloff et al (20) demonstrated that treating HNSCC cells with estrogen and epidermal growth factor (EGF) significantly increased cell invasion compared to treatment alone, whereas inhibiting the two pathways reduced cell invasion. Futhermore, patients with high intranuclear ERα and tumor EGFR levels exhibited significantly reduced rates of progression-free survival; ERβ expression, however, did not significantly affect patient survival (20).

Ishida et al (31) examined 4 cell lines and 15 tumors from patients with oral SCC. High ERβ expression was observed in the tumor cells of human primary SCC tissues and various SCC cultured cell lines. Treatment with the ER antagonists tamoxifen and raloksifen resulted in SCC apoptotic cell death and Erβ knockdown by small interfering RNA inhibited SCC proliferation (31).

In the current study, 73 (42%) patients exhibited intranuclear ERβ expression, which is in accordance with prior reports (14). Distribution of ERβ status among different head and neck subsites demonstrated that almost half of ERβ negative cases were located in the larynx, while incidence of all tumor sublocalizations within ERβ positive cases was similar and comparable. Further studies are required to evaluate the effects of this aberrant distribution.

According to the results of the present study, positive expression of ERβ indicates improved survival in patients with oropharyngeal cancer. To the best of our knowledge, this is the first study suggesting that positive expression of ERβ may be an indicator of improved survival of patients with oropharyngeal primaries. However, this is only the case in patients with oropharyngeal cancer-in other subsites, there was no correlation between ERβ expression and prolonged survival.

In conclusion, given the positive results of antihormonal therapy administration in patients with breast cancer and similar effects observed in HNSCC cell lines, future studies are necessary in order to implement novel therapeutic strategies.

References