Materials and methods

50 patients with a histopathological diagnosis of squamous cell carcinoma in biopsy specimens were evaluated. The patients had refused surgical treatment and were treated with chemotherapy and/or radiotherapy at the Department of Oral and Maxillofacial Surgery, Shimane University Hospital, from 1990 to 2002. Patients who received palliative chemotherapy or radiotherapy due to disseminated disease, other serious illness, or a poor general condition were included, but patients who died within 6 months were excluded from the study. No patient had received treatment at the time of the diagnosis. The primary tumour was clinically staged according to the TNM classification defined by the 2002 UICC .

The grade of tumour differentiation was determined according to the WHO criteria for histological typing of oral and oropharyngeal tumours . Clinicopathological data including age, gender, smoking and alcohol intake history, site of tumour, tumour differentiation and tumour stage are summarized in Table 1 . Chemotherapy protocols were based on 5-fluorouracil, platinum, or taxane, combined with or without other anticancer agents. Radiotherapy was carried out as external, intracavitary, or interstitial irradiation, or a combination of these irradiations. The average irradiation dose was 65.1 Gy (range 38–91 Gy).

All biopsy samples were fixed with 10% neutral buffered formalin and embedded in paraffin. 3 μm thick sections were stained immunohistochemically with the FAP-1, p53 or NF-κB p65 antibody. The sections were deparaffinized in xylene and dehydrated in ethanol, and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in absolute methanol for 20 min. The sections were treated with normal rabbit serum for 15 min followed by overnight incubation at 4 °C with primary anti-FAP-1 polyclonal antibody (1:250 dilution, sc-1138; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p53 monoclonal antibody (1:200 dilution, DO-7; Dako Corporation, Carpinteria, CA, USA), or anti-NF-κB p65 subunit monoclonal antibody (1:100 dilution, sc-8008; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were incubated at room temperature with biotinylated anti-goat IgG rabbit antibody for anti-FAP-1 antibody, or biotinylated anti-mouse IgG rabbit antibody for anti-p53 or anti-NF-κB p65 antibody for 10 min. They were incubated with streptavidin peroxidase reagents (Nichirei, Tokyo, Japan) for 5 min, visualized with diaminobenzidine solution (Nichirei, Tokyo, Japan) for 3 min, resulting in a brown precipitate, and counterstained with Mayer’s haematoxylin. As a negative control, phosphate buffered saline was used in place of the primary antibodies. Positive controls were OSCC slides known to have positive immunohistochemical staining for FAP-1, p53 or NF-κB p65.

Two of the authors without knowledge of any clinicopathological parameters examined the immunohistochemical stains. The immunohistochemical stains for FAP-1 and NF-κB p65 were analyzed semi-quantitatively using the R emmele score , which took into account the percentage of positive cells and the intensity of staining. The scores of the percentage of positive cells ( P ) were classified as follows: 0 no positive cells; 1 <10% positive cells; 2 11–50% positive cells; 3 51–80% positive cells; and 4 >81% positive cells. The scores for the intensity of staining ( I ) were classified as follows: 0 no staining; 1 weak staining; 2 moderate staining; and 3 strong staining. The combined staining scores ( S ) were designated as P × I for each section.

The immunohistochemical staining of FAP-1 was conducted in the cytoplasm, with a staining score of 0–2 considered negative and a score of 3–12 considered positive. The immunohistochemical staining of NF-κB was conducted in the nucleus and/or cytoplasm using NF-κB p65 , with staining scores of 0–3 being considered negative and nuclear staining or staining scores of 4–12 being considered positive .

The immunohistochemical staining for p53 was also evaluated semi-quantitatively according to the presence or absence of nuclear staining. Staining of p53 was considered positive if >10% of malignant cells showed nuclear staining. This cutoff point for p53 was selected based on previous studies .

The Mann–Whitney U -test was used to compare FAP-1, NF-κB and p53 expression with clinicopathological factors. The relationship between FAP-1, NF-κB and p53 expression was examined using Fisher’s exact test. Overall survival rates were estimated by the Kaplan–Meier method and compared using a log-rank test. To evaluate the effects of the expressions on the clinicopathological variables and the patients’ prognoses, multivariate analysis using the Cox stepwise proportional-hazards model was performed. Age, gender, smoking, alcohol, tumour size, tumour stage, tumour differentiation, FAP-1 expression, NF-κB expression and p53 expression were included in variables. A hazard ratio of 1 was considered to indicate equivalence for the different levels of a factor. All statistical analyses were performed using Statview ^® version 5.0 for Macintosh (SAS Institute Inc., Cary, NC, USA) and a P -value < 0.05 was considered statistically significant.

Results

The subjects consisted of 35 male and 15 female patients, with a mean age of 68.5 years (range 43–89 years). The patients’ characteristics are summarized in Table 1 . Over the median follow-up term of 61.2 months (range 7–179 months), 16 of 50 patients died of OSCC and 6 patients died of other diseases. The overall survival rate at 5 years was 61%.