This study was designed to investigate the feasibility of using Fas-associated phosphatase-1 (FAP-1), nuclear factor kappa B (NF-κB) and p53 as markers for chemo-radio sensitivity in oral squamous cell carcinoma (OSCC). FAP-1 plays a role as an anti-apoptotic factor through Fas-dependent apoptosis after chemo-radiotherapy. NF-κB and p53 might be involved in modulation of FAP-1 expression. FAP-1, NF-κB and p53 expression were immunohistochemically examined using biopsy specimens in 50 OSCC patients treated with chemotherapy and/or radiotherapy. FAP-1 was expressed in 52%, NF-κB in 52% and p53 in 46% of patients. There was no significant difference in FAP-1, p53 or NF-κB expression according to the clinicopathological features. No correlation was found among FAP-1, p53 or NF-κB expression. FAP-1-positive cases showed a poorer survival rate than FAP-1-negative cases ( P = 0.0409) and NF-κB-positive cases showed a poorer survival rate than NF-κB-negative cases ( P = 0.0018). Multivariate analysis showed that FAP-1 expression, NF-κB expression, clinical stage and age were significant independent variables for survival (clinical stage: P = 0.0016; age: P = 0.0016; NF-κB: P = 0.0314; FAP-1: P = 0.0366). These results suggest that FAP-1 and NF-κB might play a role as chemo-radioresistant factor during chemo-radiotherapy, and FAP-1 and NF-κB expression in OSCC would be feasible markers for chemo-radio sensitivity and prognosis.
The treatment of oral squamous cell carcinoma (OSCC), and other head and neck cancers, involves surgical resection, radiotherapy, chemotherapy or a combination of these treatment modalities. Complete surgical resection for OSCC can alter oral functions such as speech, swallowing, and occlusions, as well as the appearance of the mouth, despite recent advances in reconstructive techniques. Any functional treatment that preserves the anatomical structures would contribute greatly to the quality of life of patients. Chemotherapy and radiotherapy may thus play an important role in organ preservation and quality of life . The clinicopathological criteria for employing chemotherapy and radiotherapy without surgery and predicting the outcome for patients have not been established.
Induction of apoptosis after chemo-radiotherapy is correlated with tumour response. Fas (APO-1/CD95), a member of the tumour necrosis factor receptor family, is recognized as a major pathway for the induction of apoptosis . Concomitantly, death receptor signals are tightly regulated by anti-apoptotic factors to avoid inappropriate apoptosis. Fas-associated phosphatase-1 (FAP-1), a cytoplasmic tyrosine phosphatase, which consists of 2485 amino acid residues with a molecular mass of 275 kDa, binds the cytoplasmic tail of Fas and inhibits Fas-dependent apoptosis . Down-regulation of the FAP-1 expression induces enhancement of the Fas-signalling pathway . FAP-1 is widely expressed in normal human tissue, such as the renal tubules, skeletal muscle, pituitary gland, neurons, and bronchial epithelial cells, as well as in 78% of tumour cells, including breast, gastric, colon, and lung cancer cells, and in several types of sarcoma .
It has been reported that nuclear factor kappa B (NF-κB) and p53 are involved in the modulation of FAP-1 expression. The FAP-1 gene might be controlled by transcriptional regulatory elements, including NF-κB and p53, both of which are assumed to promote Fas-induced cell death by enhancing Fas expression . B ennett et al. found that p53 could regulate sensitivity to Fas-dependent apoptosis by allowing cytoplasmic Fas to redistribute to the cell surface . I vanov et al. reported that FAP-1 was associated with Fas trafficking to the cell surface . I rie et al. found that there was a potential binding site for p53 in the FAP-1 promoter, suggesting that activated p53 might suppress transcription of the FAP-1 gene . FAP-1 dephosphorylates tyrosine 275 in the carboxyl terminus of Fas in astrocytoma cells , and it has been suggested that FAP-1 phosphatase activity could be responsible for NF-κB activation . NF-κB-mediated expression of the FAP-1 protein might be a specific mechanism that could restrict Fas surface levels , and ionizing radiation has been shown to suppress the expression of FAP-1 mRNA via the activation of p53 .
The tumour suppressor activity of p53 is damaged in many types of human carcinoma because of mutational events or interactions with other proteins. The chemoresistance and radioresistance of carcinomas are dependent on the expression of the mutant p53 protein . p53 has several target genes in the Fas receptor-signalling pathway. NF-κB is an inducible transcription factor that mediates signal transduction between the cytoplasm and nucleus and is a member of a key transcriptional factor family that consists of five members in mammalian cells: RelA (p65), RelB, c-Rel, NF-κB1 (p50/p105) and NF-κB2 (p52/p100) . The best characterized variant is the combination of RelA (p65)–p50 . NF-κB is implicated in multiple physiological and pathological processes, including cell proliferation and differentiation, inflammatory and immune responses, cell survival and apoptosis, cellular stress reactions and tumorigenesis .
FAP-1 has been shown to play a role as an anti-apoptotic factor in the apoptotic process in several previous in vitro studies . The clinical influence of FAP-1 expression on patients with OSCC has been unclear. The authors considered that FAP-1 might serve as a marker of the efficacy of chemotherapy and/or radiotherapy because it has been suggested that FAP-1 expression confers chemo-radioresistance to malignant cells. The authors immunohistochemically examined the expression of FAP-1, NF-κB and p53 in biopsy samples of primary OSCC to evaluate the relation between these expressions and clinicopathological factors or clinical outcome.
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).
|Characteristics||Patients treated with chemo and/or RT|
|No. of patients (%)|
|Number of patients||50 (100)|
|Site of tumour|
|Maxillary sinus||1 (2.0)|
|Floor of the mouth||3 (6.0)|
|Buccal mucosa||5 (10.0)|
|Retromolar area||4 (8.0)|
|Submandibular region||1 (2.0)|
|Moderately differentiated||16 (32.0)|
|Poorly differentiated||3 (6.0)|
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.
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%.
Immunoreactivities of FAP-1, p53 and NF-κB p65
FAP-1-positive reactivity was seen as cytoplasmic staining ( Fig. 1 a and b). FAP-1 immunoreactivity was observed in 26 (52%) of the 50 samples. In 26 samples, 3 samples were graded as combined staining score ( P × I ) 3, 8 samples scored 4, 10 samples scored 6, 3 samples scored 8, 1 sample scored 9, and 1 sample scored 12. Of the positive patients, 14 patients died and 12 patients lived without OSCC (median follow-up term 44.3 months). Of the negative patients, 8 died and 16 lived without OSCC (median follow-up term 79.5 months). FAP-1 expression did not significantly correlate with tumour size, tumour differentiation or clinical stage ( Table 2 ).
|Patients treated with chemo and/or RT|
|FAP-1 expression||NF-κB expression||p53 expression|
|Negative||Positive||P -value||Negative||Positive||P -value||Negative||Positive||P -value|
|Site of tumour|
|Floor of the mouth||2||1||1||2||1||2|