Introduction Head and neck squamous cell carcinoma constitutes the sixth most common cancer in the world. Male to female incidence rates are greater than 2: 1. The five year survival rate for patients with this disease has not improved appreciably since the early 1980s and remains at approximately 52%. A common problem occurring in cases of head and neck cancer is the recurrence of the disease either in the location of primary surgical resection (although the surgical resection margins were histopathologically free of cancer cells) or in a new place in upper respiratory tract. The risk of a local recurrence of the disease is stili high, estimated at about 10-30%. Molecular biology achievements enabled to determine mechanisms responsible for tumor escape from host immunological surveillance, which are controlled by the regulation of apoptosis through the expression of various factors on cells surface. Tumor associated antigen RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) having gene at 8q23, is a type II membrane protein, expressed in various human cancer cells. It has been reported to be expressed with high frequency in breast, lung, larynx and pharynx, gallbladder, ovarian, uterine, bile duet cancer cells, in Reed Sternberg cells and erythroid progenitor cells. The RCAS1 protein acts as a ligand for a putative receptor present in various h ; uman cells including normal peripheral lymphocytes such as T, B, and NK (natural killer) cells. RCAS1 inhibits growth of receptor expressing cells in vitro and in vivo and induces apoptotic cell death through activation of caspase 3 and caspase 8 and collapse of mitochondria! transmembrane potential. Thus, it facilitates escape from host immune surveillance by inducing apoptosis of activated CD3+ and NK cells and enables the disease to spread. High RCAS1 expression in many examined cancer was connected with high TIL apoptosis rate, and was related to poor prognosis in these cases. The aim of his work was to determine RCAS1 expression in pharyngeal and laryngeal cancers and non-cancerous tissue samples (histological elear surgical margin, chronic inflammation), analyze the expression with respect to the clinicopathological features of the tumor (TNM, grading) in order to evaluate its possible prognostic value in cancer. Materials and Methods All patients with pharyngeal and laryngeal cancer in this study had undergone surgery between January 2002 and December 2003 at the Department of Otolaryngology and Head and Neck Surgery of Jagiellonian University in Krakow. In all cases patient's agreement was received. The approval for the research program was obtained from the Ethical Committee of the Jagiellonian University in Krakow: KBET/379/13/2002. The patients ; were randomly selected and observed within 18 months after the surgical treatment. A total of 120 tissue samples was obtained during surgery. RCAS1 was estimated in 51 tissue samples derived from laryngeal and pharyngeal squamous cell carcinoma, 34 elear surgical margins, 8 nasal polyps, and 27 healthy mucous membrane specimens. Surgically removed materiał was evaluated to determine histological type and metastases of the lymph nodes using histological methods in Pathology Department of Jagiellonian University. In 27 cases of laryngeal and pharyngeal cancer two samples were collected, one derived from the cancer tissue and one from its elear surgical margin. The elear surgical margin was defined as the lcm2 area of tumor adjacent tissue macroscopically and histological free of neoplasmatfo texture. Directly after the surgical procedure the pathologist examined and collected tissue samples in - 90°C. Using Westem blot method RCAS1 protein and β-Actin control protein were analyzed. For the identification RCAS 1 anti-RCAS 1 mouse Mo-22-1-1 monoelonal antibody was used. Results RCAS1 was determined as a 32kDa band while β-Actin control protein was identified as a 42 kDa band. The presence of RCAS 1 protein was identified in all cancerous tissue samples examined. The protein was also revealed in histological elear surgical margin and chronic inflammation of muco ; us membrane of upper respiratory tract (nasal polyps). RCAS1 expression was not observed in healthy mucous membrane of upper respiratory tract derived from patients with not neoplasmatic disease. No difference was found in the expression of β-Actin control protein between these tissue samples. • RCAS1 relative amount was observed to be statistically significantly higher in pharyngeal and laryngeal cancer than in chronic inflammation of upper respiratory tract (nasal polyps). • Higher average relative amount of RCAS1 protein was observed in tissue samples derived from patients with the presence of lymph node metastases in comparison to samples derived from patients without the presence of lymph node metastasis. • Among the group of patients with the presence of lymph node metastases the level of RCAS I expression was statistically significantly higher in patients with those, in which the neoplasmatic infiltration spread extracapsulary in comparison to the patients without extracapsular transition. • The analysis of RCAS1 average relative amount with respect to the tumor grading revealed statistically significantly higher value in cancers of high grade (G3) in comparison to G2 and Gł. • The RCAS1 expression level was found to be higher in cancers infiltrating pharynx than in laryngeal cancers. In 27 cases of pharyngeal and laryngeal cancers two samples wer ; e collected, one derived from the cancer tissue and one from its elear surgical margin. The RCAS1 average relative amount in cancer samples was statistically significantly higher than in elear surgical margin specimens. It was noticed, however that in single cases the RCAS1 average relative amount was higher in elear surgical margin samples than in cancer samples. Thus, two RCAS1 values were compared, elear surgical margin average relative amount to the cancer average relative amount, obtaining tumor environmental indicator (TEI) value. lt was analyzed with respect to the tumor size, Iocal Iymph node metastases presence (according to the TNM classification), tumor grading, tumor localization, cancer recurrence and prognosis. • Statistically significantly higher TEI value was observed in cases of the largest tumor size (T3). • TEI value was compared with the tumor grading and was found to be higher in the cases of high grade squamous cell carcinoma than in the cases of moderately differentiated cancer cells. • The analysis of TEI value and tumor Iocalization revealed the highest TEI values in cases with tumors affecting both pharynx and larynx, where the tumor range transgressed anatomical barriers. • High TEI value was related to poor prognosis. High TEI value was identified in four cases involving patients who, in spite of histopatologically cancer fre ; e surgical margins, died of cancer within a year following radical surgical treatment. • In 13 cases of patients with high TEI values the recurrence of the disease was confirmed within a year following surgical treatment. Conelusions RCAS1 protein expression is an unfavorable prognostic marker in pharyngeal and laryngeal cancer. Its expression is associated with other well known negative prognostic factors. High RCAS1 expression in elear surgical margin of pharyngeal and laryngeal cancer is connected with higher risk of !ocal cancer recurrence and dissemination of the tumor. We have indicated that comparison of RCAS1 in elear surgical margin and tumor cells might thus have both diagnostic and treatment consequences. RCAS1 expression could be used to predict the possibility of cancer recurrence. Molecular diagnosis of elear surgical margin might thus have a decisive significance for subsequent therapy and survival.