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Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer regions in squamous cell carcinoma: relationships with tumor stage and grade, and apoptosis
Int. J. Oral Maxillofac. Surg. 2005; 34: 900–906 doi:10.1016/j.ijom.2005.03.006, available online at http://www.sciencedirect.com
Research Paper Head and Neck Oncology
Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer regions in squamous cell carcinoma: relationships with tumor stage and grade, and apoptosis
N. Guler1, S. Uckan2, I. Celik3, Y. Oznurlu3, D. Uckan4 1 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University of Yeditepe, Istanbul, Turkey; 2Department of Oral and Maxillofacial Surgery, University of Bas¸kent, Faculty of Dentistry, Ankara, Turkey; 3 Department of Histology, Faculty of Veterinary, University of Selc¸uk, Konya, Turkey; 4Department of Pediatric Hematology, Faculty of Medicine, University of Hacettepe, Ankara, Turkey
N. Guler, S. Uckan, I. Celik,Y. Oznurlu, D. Uckan: Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer regions in squamous cell carcinoma: relationships with tumor stage and grade, and apoptosis. Int. J. Oral Maxillofac. Surg. 2005; 34: 900–906. # 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. The aim of this study was to investigate whether levels of Fas and Fas-ligand (Fas-L) expression in oral and oropharyngeal squamous cell carcinomas (SCCs) are associated with tumor stage and grade, and to assess whether parameters related to argyrophilic nucleolar organizer regions (AgNORs) correlate with apoptosis in SCC cells and adjacent normal epithelium. Fifty-two specimens taken from the tumoral and neighboring normal tissues of 26 patients with oral and oropharyngeal SCC were analyzed for Fas/Fas-L expression, and 24 specimens from 12 patients for AgNOR parameters. Seventeen (65%) of the tumors were Fas and/or Fas-L-positive (by immunohistochemistry). A significant positive correlation was found between Fas/FasL expression and clinical tumor stage (P < 0.01). Mean AgNOR number per nucleus, AgNOR size and the percentage area of each nucleus occupied by AgNORs (percent of nuclear area) were significantly increased in the SCC cells (4.49 1.28, 4.48 1.42, 5.56 1.22, respectively) when compared with the control neighboring squamous epithelial cells (2.58 0.61, 1.64 0.59 and 4.35 0.62%, respectively) (P < 0.01). A significant positive correlation was found between the AgNOR parameters and Fas/ Fas-L expression as apoptotic markers in the tumoral cells of SCC (P < 0.05). There was also a significant positive correlation between the AgNOR parameters and the grading of tumors (P < 0.05). In conclusion, AgNOR count was a strong proliferation marker in patients with SCC, and Fas and Fas-L staining was useful in tumor grading.
0901-5027/080900+07 $30.00/0
Accepted for publication 15 March 2005 Available online 19 May 2005
# 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer The process of apoptosis, or programmed cell death, was first described by KERR et al. in 197218. Apoptosis is an active bioenergysaving cell-elimination mechanism by which aged, injured or sub-lethally damaged cells are destroyed, and their valuable caloric contents re-used by macrophages or adjacent cells by which they are phagocytosed18,41. This process is believed to be involved in numerous physiological and pathologic events, including the destruction of cells during embryogenesis, hormone-dependent involution of tissues in adults, elimination of cells in proliferating cell populations, cell death in tumors, death of immune cells, pathologic atrophy of hormone-dependent tissues, cell injury in certain viral diseases, and in the killing of cancer cells by chemotherapeutic drugs2,18,22,36,39. The cell-surface protein known as Fas/ CD95 (Fas) is expressed on activated lymphocytes. Cross-linking of Fas with its ligand, Fas L/CD95L (Fas-L), causes transudation of the apoptotic signal from the cell surface into the cytoplasm, thereby triggering cell death. Fas and Fas-L are typical members of the tumor necrosis factor (TNF) receptor and TNF ligand family, respectively, and they play pivotal roles in the regulation of apoptotic processes such as activation-induced cell death, Tcell-induced cytotoxicity, immune privilege, and tumor surveillance38. Fas-L induces suicidal death (apoptosis) of accumulated cytotoxic T-cells by binding to Fas expressed on the surface of these cells. Cancer cells that arise from several mutations of normal ancestor cells come under host immune attack as the body attempts to eliminate them26. However, most tumors escape this attack by imitating the strategies of the cells in the immuneprivileged sites through overexpressing Fas-L or down-regulating Fas5,13. This mechanism of immune escape through altered expression of Fas and Fas-L has been reported in squamous cell carcinoma (SCC) of the head and neck by several investigators1,11,16,21,25,33,37. Nucleolar organizer regions (NORs) are loops of DNA that contain genes for ribosomal RNA. These regions are transcribed by RNA polymerase I, and are of vital importance for protein synthesis8. NORs that are stained by silver are called AgNORs, and proteins associated with these are called AgNOR proteins12,31. AgNOR staining is a histochemical technique that provides valuable information about cellular status. In interphasic nuclei, AgNOR staining usually appears as dark granules within the nucleoli. The intensity of staining depends on the transcriptional
activity of the cells23,34. Recent studies suggest that the number of silver-stained proteins in cell nucleoli is related to cell proliferation29,40. More numerous and often atypical AgNORs have been described in malignant neoplasm than in benign tumors and normal tissues9. The main aim of this study was to investigate whether levels of Fas and Fas-L expression in oral and oropharyngeal SCCs are correlated with tumor stage and grade. Another goal was to assess whether AgNOR-related parameters— AgNOR number per nucleus, AgNOR size and the percentage area of each nucleus occupied by AgNORs (percent of nuclear area)—correlate with levels of apoptosis in SCC cells and adjacent normal epithelium. Material and methods
The study was performed on specimens of tumoral and adjacent normal tissue, 52 specimens taken from 26 patients (8 females and 18 males; mean age, 61.4 9.5 years; age range, 41–77 years), with oral and oropharyngeal SCCs. The stage of SCC, the grade of tumor, and levels of Fas and Fas-L expression were recorded for each tumor. Twenty-four of the specimens (12 tumoral and corresponding 12 adjacent normal tissues) were randomly selected and assessed for AgNORs. TNM (tumor size, lymph node metastasis, distant metastasis) and clinical stage of oral cavity cancers were classified according to the guidelines from the American Joint Committee on Cancer. The patients did not receive radiation therapy and/or chemotherapy prior to surgery. They did not show any distant metastases. The adjacent normal tissues were taken from the surgical margins with non-tumoral tissue following confirmation that the tissue was not tumoral using frozen sections. The grade of each tumor was also assessed (well differentiated, moderately differentiated or poorly differentiated) using the criteria described by CAWSON et al.4 Each patient had undergone standard treatment appropriate to the stage of the SCC. At the time of surgery, part of the resected SCC and a sample of adjacent normal tissue were immediately placed in 10% buffered neutral saline (10 mM, pH 7.4) and then embedded in paraffin for Fas and Fas-L staining (and AgNOR staining in 12 cases). The remaining portion of each SCC specimen and a sample of adjacent normal tissue were fixed in 10% formaldehyde and embedded in a paraffin block using routine histological technique. Seven-micrometer-thick sections were cut from 26 blocks containing the tumor spe-
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cimens and 26 blocks of corresponding adjacent normal tissue and were stained with hematoxylin and eosin. The tumor tissues were examined by a pathologist who confirmed the diagnosis of SCC in each case. Immunohistochemical methods
Fas and Fas-L staining
Four-micrometer-thick sections were cut from each of the 52 specimens designated for immunohistochemistry, and these were mounted on slides coated with poly-Llysine. The sections were de-paraffinized in xylene and then rehydrated through a series of baths containing decreasing concentrations of ethanol in de-ionized water. They were then treated with citrate buffer (pH 6.6) at 95 8C for 5 min to retrieve the antigens (Fas and Fas-L surface molecules). The sections were then brought to room temperature (20 8C), rinsed three times in de-ionized distilled water, and endogenous peroxidase activity was stopped by 20 min of immersion in 0.3% H2O2 in methanol. After three rinses in cold (4 8C) phosphate-buffered saline (10 mM, pH 7.4), the sections were incubated for 30 min with primary polyclonal antibody, either a rabbit polyclonal antihuman Fasspecific IgG (C-20, Sc-715, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a rabbit polyclonal antihuman Fas-L-specific IgG (N-20, Sc-834, Santa Cruz Biotechnology). After this, each section was rinsed three times in cold phosphate-buffered saline and then stained with secondary monoclonal antibody using the Santa Cruz Staining System (Sc-2053, goat antirabbit IgG-horse radish peroxidase conjugate, Santa Cruz Biotechnology). The manufacturer’s recommended dilutions and incubation periods for the primary and secondary monoclonal antibody were carefully followed. Peroxidase staining was demonstrated using the method of KOLB19 JØRNSEN et al. The cell nuclei were stained for 10 min with 1% methyl green prepared in 0.1 M acetate buffer (pH 4.2). Each slide was examined under the light microscope at high magnification (1000), and cells with brownish-black reaction products in the cell membrane and cytoplasm were considered positive for Fas or Fas-L, depending on which primary antibody had been applied. In all specimens, rabbit nonimmune serum purchased from Santa Cruz was used as a control. For each of the 52 specimens, intensity and extent of Fas and Fas-L expression were evaluated by a comprehensive scoring formula originally designed by BUSCH et al.3 The extent of
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staining in the samples was scored as: 1 = less than one-third, 2 = one-third to two-thirds, and 3 = more than two-thirds of the tumor cells stained positively. The results obtained with the two scales were multiplied against each other, yielding a single scale with steps of 1–4, 6 and 9, in which 1–4 were considered to be weak staining and 6 and 9 were considered to be strong staining. Staining intensity was scored as follows: 1 = weak expression, 2 = moderate expression, and 3 = strong expression. Staining the AgNORs
The paraffin-embedded tissues from 12 patients (12 tumoral tissue and 12 neighboring normal tissue samples) were stained for AgNORs. Four-micrometerthick sections were de-paraffinized and rehydrated as described above, and then stained with a specially prepared solution that consisted of one volume 2% gelatin in 1% aqueous formic acid, and two volumes 50% silver nitrate. Sections were immersed in this solution in the dark for 10–12 min at 37 8C, and then washed three times with distilled deionized water. Each section was then dehydrated and mounted using routine methods20. The sections were examined under high magnification (100), and AgNORs were identified as silver-stained (black) dots. Three AgNOR-related parameters were recorded for each case: AgNOR number per nucleus, AgNOR size and the percentage area of each nucleus occupied by AgNORs (percent nuclear area). For each lesion, 100 nuclei in 10 different areas of the tumor were analyzed in interphase using a 100 oil-immersion objective. The threshold for AgNOR dots was selected for individual cases, after enhancing the contrast up to a point at which the AgNORs dots were easily identified as black points within the nuclei. The relative AgNOR area (percentage of total nuclear area occupied by AgNORs) was determined as representative of the AgNOR quantity. Where two or more dots joined in clusters these were counted as a single AgNOR20. Statistical analysis
Clinical parameters, histological parameters, Fas and Fas-L expression, and the findings for the three AgNOR parameters were compared using the Spearman rank correlation coefficient and x2 testing, as appropriate. Two-tailed independent Student’s t-test was used to compare data between groups, whereas one-
way analysis of variance test was used to make comparisons within groups. The level of significance was set at P < 0.05. Results
Table 1 lists the characteristics of patients in relation to presence/absence of Fas and/ or Fas-L expression. Eight of 26 SCCs (31%) were in the oral region and 18 (69%) were oropharyngeal. Nine (53%) of the 17 Fas- or Fas-L-positive tumors showed strong expression of both antigens; 5 (29%) were positive for Fas only, and 3 (18%) were positive for Fas-L only. Twelve (86%) of the 14 tumors that were positive for Fas showed strong staining. The usual pattern of positive staining of Fas-L was similar to that of staining for Fas. The Fas-L staining varied in intensity, with tumor areas ranging from weak to strong intensity. Ten (83%) of the 12 tumors that were positive for Fas-L showed strong staining. Of the 26 specimens of normal tissue adjacent to the SCCs, seven (27%) showed both Fas and Fas-L expression. The remaining 19 specimens did not stain for either antigen. Two of the seven cases positive for Fas
and Fas-L showed weak staining for both antigens, two cases showed strong Fas staining, while three cases showed weak Fas staining. Eight (30.8%) patients exhibited primary metastasis to regional lymph nodes and accounted for three (37.5%) of the eight T3 tumors, and five (62.5%) of the seven T4 tumors. The frequency of primary metastasis to regional nodes in the T4 group was significantly higher than that in the T3 group (P < 0.01). There was no correlation between presence of Fas and/ or Fas-L expression and primary lymph node metastasis. There was a significant positive correlation between Fas and FasL expression and tumor stage (r = 0.62, P < 0.01 and r = 0.64, P < 0.01, respectively), but neither Fas nor Fas-L expression was correlated with clinical tumor size (T classification), lymph node metastasis (N classification) (Table 2). Of the 14 Fas-positive tumors eight were well, five were moderately and one was poorly differentiated, whereas four out of 12 Fas-L-positive tumors were well, five moderately and three poorly differentiated. Thus, Fas expression was more frequent in well-differentiated SCCs, and
Table 1. Clinical data for the 26 oral and oropharyngeal SCC cases in relation to presence of Fas and/or Fas-L staining Fas and/or Fas-L staining Age (years, mean SD) Sex Female Male
Positive (n = 17, 65%)
Negative (n = 9, 35%)
61.1 10.7
60.8 16.9
5 12
3 6
Tumor size T1 T2 T3 T4
1 (5.9%) 5 (29.4%) 6 (35.3%) 5 (29.4%)
4 1 2 2
Cervical nodal involvement N0 N1 N2 N3
10 (59%) 3 (17.6%) 4 (23.4%) –
8 (88.9%) 1 (11.1%) – –
Clinical stage I II III IV
1 (5.9%) 4 (23.5%) 7 (41.2%) 5 (29.4%)
4 1 2 2
Fas/Fas-L expression None Weak Moderate Strong
4 (23.5%) 4 (23.5%) 9 (53%)
– –
Differentiation grade Well differentiated Moderately differentiated Poorly differentiated
8 (47.1%) 6 (35.3%) 3 (17.6%)
8 (88.9%) 1 (11.1%) –
(44.5%) (11.1%) (22.2%) (22.2%)
(44.5%) (11.1%) (22.2%) (22.2%)
9 (100%)
Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer Table 2. Clinical features of the study population in relation to expression of Fas, Fas-L, and both antigens Fas and Fas-L positive (n = 9)
Fas positive (n = 5)
Fas-L positive (n = 3)
– 2 (22.2%) 3 (33.3%) 4 (44.5%)
1 (20%) 2 (40%) 2 (40%) –
– 1 (33.3%) 1 (33.3%) 1 (33.3%)
4 (80%) 1 (20%)
– 1 (33.3%) 2 (66.7%) –
– 2 (22.2%) 3 (33.3%) 4 (44.5%)
1 (20%) 1 (20%) 3 (60%) –
– 1 (33.3%) 1 (33.3%) 1 (33.3%)
– 9
2 (40%) 3 (60%)
2 (66.7%) 1 (33.3%)
Tumor size T1 T2 T3 T4
Cervical nodal involvement N0 6 (66.7%) N1 1 (11.1%) N2 2 (22.2%) N3 Clinical Stage I II III IV Staining Weak Strong
Fas-L expression was more frequent in moderately and poorly differentiated tumors (P < 0.05). Mean AgNOR number per nucleus and mean AgNOR size in tumoral and adjacent normal tissues are shown in Table 3. There were significant differences in the mean AgNOR number, size and nuclear area ratio between the control and SCC groups (P < 0.01) (Figs. 1 and 2). The relationships between the mean AgNOR number per nucleus and mean individual AgNOR size values and the percentage area of each nucleus occupied by AgNORs (percent of nuclear area) are also shown in Table 3 for tumoral and adjacent normal tissues in 12 oral and oropharygeal SCCs. The clinical
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stage, degree of differentiation and Fas and/or Fas-L expression for the 12 AgNOR-stained specimens are given in Table 4, the most common parameters being Stage III (50%), moderate differentiation (50%), and both Fas and Fas-L staining (50%). A significant positive correlation was found between the AgNOR parameters and Fas and Fas-L expression in the tumoral cells of SCC (r = 0.65 and 0.66, P < 0.05), and also between the AgNOR parameters and the grading of tumors (r = 0.61, P < 0.05). Discussion
The expression of Fas-L may play an important role in establishing immunologically privileged environments that allow tumors to escape the host’s immune surveillance11,14,35; that is, the relative predominance of the striking power of lymphocytes and opposing striking power
Fig. 1. AgNORs in the nuclei of normal cells. The number and size of the silver-stained regions are relatively low compared to SCC (see Fig. 2; 1700).
Table 3. Comparison of AgNOR parameters between normal and tumoral tissue
AgNOR number
AgNOR size
The percentage area of each nucleus occupied by AgNORs (percent of nuclear area)
Patients (n = 12)
Normal tissue
Tumoral tissue
Normal tissue
Tumoral tissue
Normal tissue
Tumoral tissue
1 2 3 4 5 6 7 8 9 10 11 12
1.60 0.52 3.70 0.68 3.40 0.52 3,10 0,32 2.30 0.82 3.10 0.88 2.30 0.82 2.10 0.74 2.30 0.82 2.30 0.82 2.40 0.97 2.30 0.82
4.10 1.10* 5.0 1.25* 6.70 1.06* 6.20 0.63* 2.82 1.17 3.80 1.03 4.40 1.17* 3.70 2.11* 6.10 0.99* 4.60 1.84* 3.10 0.99 3.30 0.68
1.38 0.22 1.62 0.33 2.94 1.06 1.40 0.46 1.26 0.71 1.51 0.44 1.26 0.71 2.73 1.04 1.26 0.71 1.26 0.71 1.83 0.91 1.26 0.71
2.76 0.44* 3.93 0.47* 5.09 1.19* 6.61 1.21* 3.54 1.97* 4.76 1.74* 6.07 1.06* 4.90 2.46* 3.12 0.74* 6.60 1.36* 2.63 0.93 3.75 1.69*
3.42 0.25 3.60 0.19 4.84 0.87 3.54 0.00 4.67 1.30 3.78 0.79 4.67 1.30 5.37 0.90 4.67 1.30 4.67 1.30 4.31 0.79 4.67 1.30
3.42 0.37 3.66 0.25 6.61 1.14* 6.08 1.18* 5.07 1.15 6.56 1.71* 5.31 0.62* 7.14 1.61* 5.19 0.83 6.73 0.69* 4.60 0.87 6.31 1.44
Mean P
2.58 0.61
4.49 1.28 <0.001*
1.64 0.59
4.48 1.42 <0.001*
4.35 0.62
5.56 1.22 <0.001*
*
P < 0.05.
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Fig. 2. AgNORs in the nuclei of SCC cells. The AgNORs are larger and more numerous than in normal cells (see Fig. 1, 1700).
Table 4. Clinical stage, degree of differentiation, and expression of Fas and/or Fas-L in AgNOR-stained tumoral cells Specimen (n = 12) 1 2 3 4 5 6 7 8 9 10 11 12
Clinical stage
Degree of differentiation
Stage III Stage II Stage III Stage IV Stage III Stage IV Stage III Stage III Stage III Stage II Stage II Stage I
Moderate Moderate Moderate Poor Moderate Well Poor Well Moderate Moderate Well Well
of tumor cells affects the biological behavior of a tumor, and Fas-system-mediated apoptosis plays a role in this mechanism27. In the present study, Fas and Fas-L expression was detected in oral and oropharyngeal SCCs. A significant correlation was found between tumor staging and Fas/FasL expression frequency. The number of patients examined was not large (n = 26), but 65% of the SCC specimens stained strongly positive for Fas or Fas-L. In the specimens from Stages III and IV patients, a strong staining reaction for Fas-L was observed, while Fas staining was observed for the lower stages. This result is in agreement with MURAKI et al.25 who showed a high incidence of Fas antigen expression in the lower stage SCCs that may act as a controlling factor by promoting apoptosis in the slower growth of Faspositive SCC. T cells in the tumor microenvironment of head and neck cancer have showed evidence of apoptosis as well as decreased expression of signaling molecules. Immunohistochemical studies have shown that Fas antigen is widely distributed in normal and pathological epithelia, including oral mucosa24,42. While in normal oral mucosa
Fas and/or Fas-L staining Fas and/or Fas and/or Fas and/or Fas and/or Fas and/or Negative Fas-L Fas and/or Negative Fas-L Fas Fas
Fas-L Fas-L Fas-L Fas-L Fas-L Fas-L
Fas-L is primarily detected in cells of the basal layer32, Fas antigen is mainly located in the epithelial cell cytoplasm of human oral SCCs24. Fas expression has been reported to be down-regulated in invasive oral SCCs whereas Fas-L expression showed up-regulation5,16. Fas-L-expressing tumor cells may up-regulate Fas-L mRNA levels by direct contact with Fas-bearing cells from the antigenic host organism, thus taking advantage of Fas-L expression as a factor of tissue invasion10. It has been reported that poor expression of Fas and positive Fas-L staining in oral SCC correlates negatively with degree of differentiation and apoptosis32. Furthermore, it has been shown that Fas antigen expression is higher in lower stage than in advanced stage SCC25. In this study, Fas-L was found to be highly expressed in poorly differentiated tumors whereas in well differentiated tumors expression was considerably lower. Faspositive cells were found in only one poorly differentiated tumor. This may reflect the potential of tumor cells to undergo spontaneous apoptotic cell death17. Also in this study, 13 (50%) of 26 tumors had poor or no Fas expression,
which suggests that this may be a common mechanism for tumors to escape from the Fas-mediated host immune surveillance. In addition, the absence of Fas in Fas-Lpositive tumors precluded autocrine Fas-L attack for oral and oropharygeal SCC. This study confirms that the frequency of Fas-L expression is highest in T3 SCCs followed by T4 SCCs suggesting that FasL expression may be associated with tumor progression and poor prognosis. Certain normal tissues constitutively coexpressing Fas and Fas-L are characterized by apoptotic cell turnover that functions in the control of tissue homeostasis1–6. There is an increased awareness of the importance of apoptosis in carcinogenesis. SCC is the most common malignancy of head and neck regions. It is important that these lesions be diagnosed early and accurately. Determination of the state of nuclear DNA and proliferation markers is important in oral and oropharyngeal carcinomas presenting with Fas and Fas-L coexpression. It remains unclear as to whether Fas and Fas-L are associated with tumor growth. The results of this study show the usefulness of the staining of Fas and Fas-L antigens in tumor grading, and also might suggest a role for these antigens in carcinogenesis. A series of studies indicate that the quantity of protein AgNOR is related to the rapidity of cell proliferation, and a relationship between AgNOR counts and the prognosis of malignant tumors has been suggested6,30. Several studies have shown variations in the number, shape and volume of AgNORs of normal mucosa cells and malignant cells9,30. The data available indicate that cancer cells have a greater amount of interphase AgNORs than benign or normal cells, and therefore a malignant lesion may frequently be distinguished from a benign lesion of the same tissue by interphase AgNOR quantitative evaluation. Since the tumor mass growth rate is one of the most important factors influencing clinical outcome, it follows that the AgNOR value is a valuable parameter for providing information about the progression of disease6. AgNOR number may allow more precise tumor staging if it is shown that it is a marker for progression rather than malignant potential15. It is also likely that the mean number of AgNORs is related to the number of cells in S phase and with ploidy. Thus, AgNOR counts may assist in the grading of neoplasm7. In the ¨ Z et al.28, the present study, like that of O number of NOR dots increased with increasing grade of SCC while the area of the NOR decreased and dots became more irregular with increasing grade. There
Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer were significant differences in the mean AgNOR counts, area and nuclear area ratio between the control and SCC groups (P < 0.05). Significant positive correlation was found between the AgNOR parameters and Fas/Fas-L expression in the tumoral cells of SCC. In conclusion, in this study a significant positive correlation was found between Fas/Fas-L expression and tumor stage; and AgNOR enumeration appeared to be a useful tool in distinguishing between normal epithelium and SCC. In addition, AgNOR count was found to be a strong proliferation marker, when compared with the presence of Fas and Fas-L antigen in patients with SCC. Acknowledgements. We thank Dr Kayhan Oztuok for his kind assistance. This study was supported by Grant No. SB-99-117 from the Scientific Research Council of Selc¸ule University.
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12.
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14.
15.
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24. Murakami Y, Yoshioka C, Fukuda J, Haneji T, Kobayashi N. Immunohistochemical detection of Fas antigen in oral epithelia. J Oral Pathol Med 1997: 26: 57–62. 25. Muraki Y, Tateishi A, Seta C, Fukuda J, Haneji T, Oya R, Kobayashi N. Fas antigen expression and outcome of oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2000: 29: 360–365. 26. Nagata S. Apoptosis by death factor. Cell 1997: 88: 355–365. 27. Nagata S, Golstein P. The Fas death factor. Science 1995: 267: 1449–1456. ¨ z F, O ¨ z S, Uraz S. Nucleolar organizer 28. O regions in squamous cell carcinoma of the larynx. Turkish Arch Otolaryngol 1997: 35: 71–74. 29. Pession A, Farabegoli F, Trere D, Novello F, Montanaro L, Sperti S, Rambelli F, Derenzini M. The AgNOR proteins transcription and duplication of ribosomal genes in mammalian cell nucleoli. Chromosoma 1991: 100: 242–250. 30. Piffko J, Bankfalvi A, Ofner D, Bryne M, Rasch D, Joos U, Bocker W, Schmid KW. Prognostic value of histobiological factors (malignancy grading and AgNOR content) assessment at the invasive tumour front of oral squamous cell carcinomas. Br J Cancer 1997: 75: 1543–1546. 31. Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet J-J. Improvement in the staining and in the visualization of the argyrophylic proteins of the nucleolar organizer region at the optical level. Histochem J 1986: 18: 5–14. 32. Ravi D, Ramadas K, Mathew BS, Nalinakumari KR, Nair MK, Pillai MR. De novo programmed cell death in oral cancer. Histopathology 1999: 34: 241– 249. 33. Reichert TE, Strauss L, Wagner EM, Gooding W, Whiteside TL. Signaling abnormalities, apoptosis, and reduced proliferation of circulating and tumorinfiltrating lymphocytes in patients with oral carcinoma. Clin Cancer Res 2002: 8: 3137–3145. 34. Schmiady H, Mu¨nke M, Sperling K. Ag staining of nucleolus organizer regions on human prematurely condensed chromosomes from cells with different ribosomal RNA gene activity. Exp Cell Res 1979: 121: 425–428. 35. Schneider P, Bodmer JL, Holler N, Mattmann C, Scuderi P, Terskikh A, Peitsch MC, Tschopp J. Characterization of Fas (Apo-1, CD95)- Fas ligand interaction. J Biol Chem 1997: 272: 18827–18833. 36. Sealer J, Kerr JFR, Bishop CJ. Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol Ann 1982: 17: 229– 259. 37. Seta C, Fujita M, Muraki Y, Fukuda J, Kobayashi N, Haneji T. Fas expres-
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sion and Fas monoclonal antibodyinduced apoptosis in a human squamous cell carcinoma cell line, SCC-25. J Oral Pathol Med 2000: 29: 271–278. 38. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993: 75: 1169–1178. 39. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995: 267: 1456–1461.
40. Trere D, Pession A, Derenzini M. The silver-stained proteins of interphasic nucleolar organizer regions as a parameter of cell duplication rate. Exp Cell Res 1989: 184: 131–137. 41. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980: 68: 251–300. 42. Yoshioka C, Murakami Y, Fukuda J, Haneji T, Kobayashi N. Identification of the Fas antigen in human gingival. J Dent Res 1996: 75: 1353–1357.
Address: Nurhan Guler ¨ niversitesi Yeditepe U Dis¸ Hekimlig˘i Faku¨ltesi Ag˘ız-Dis¸-C¸ene Hastalıkları ve Cerrahisi AD Bagdat cad. No. 238 Goztepe Istanbul Turkey Tel: +90 216 3636044 Fax: +90 216 3636211. E-mail: [email protected]
Research Paper Head and Neck Oncology
Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer regions in squamous cell carcinoma: relationships with tumor stage and grade, and apoptosis
N. Guler1, S. Uckan2, I. Celik3, Y. Oznurlu3, D. Uckan4 1 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University of Yeditepe, Istanbul, Turkey; 2Department of Oral and Maxillofacial Surgery, University of Bas¸kent, Faculty of Dentistry, Ankara, Turkey; 3 Department of Histology, Faculty of Veterinary, University of Selc¸uk, Konya, Turkey; 4Department of Pediatric Hematology, Faculty of Medicine, University of Hacettepe, Ankara, Turkey
N. Guler, S. Uckan, I. Celik,Y. Oznurlu, D. Uckan: Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer regions in squamous cell carcinoma: relationships with tumor stage and grade, and apoptosis. Int. J. Oral Maxillofac. Surg. 2005; 34: 900–906. # 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. The aim of this study was to investigate whether levels of Fas and Fas-ligand (Fas-L) expression in oral and oropharyngeal squamous cell carcinomas (SCCs) are associated with tumor stage and grade, and to assess whether parameters related to argyrophilic nucleolar organizer regions (AgNORs) correlate with apoptosis in SCC cells and adjacent normal epithelium. Fifty-two specimens taken from the tumoral and neighboring normal tissues of 26 patients with oral and oropharyngeal SCC were analyzed for Fas/Fas-L expression, and 24 specimens from 12 patients for AgNOR parameters. Seventeen (65%) of the tumors were Fas and/or Fas-L-positive (by immunohistochemistry). A significant positive correlation was found between Fas/FasL expression and clinical tumor stage (P < 0.01). Mean AgNOR number per nucleus, AgNOR size and the percentage area of each nucleus occupied by AgNORs (percent of nuclear area) were significantly increased in the SCC cells (4.49 1.28, 4.48 1.42, 5.56 1.22, respectively) when compared with the control neighboring squamous epithelial cells (2.58 0.61, 1.64 0.59 and 4.35 0.62%, respectively) (P < 0.01). A significant positive correlation was found between the AgNOR parameters and Fas/ Fas-L expression as apoptotic markers in the tumoral cells of SCC (P < 0.05). There was also a significant positive correlation between the AgNOR parameters and the grading of tumors (P < 0.05). In conclusion, AgNOR count was a strong proliferation marker in patients with SCC, and Fas and Fas-L staining was useful in tumor grading.
0901-5027/080900+07 $30.00/0
Accepted for publication 15 March 2005 Available online 19 May 2005
# 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer The process of apoptosis, or programmed cell death, was first described by KERR et al. in 197218. Apoptosis is an active bioenergysaving cell-elimination mechanism by which aged, injured or sub-lethally damaged cells are destroyed, and their valuable caloric contents re-used by macrophages or adjacent cells by which they are phagocytosed18,41. This process is believed to be involved in numerous physiological and pathologic events, including the destruction of cells during embryogenesis, hormone-dependent involution of tissues in adults, elimination of cells in proliferating cell populations, cell death in tumors, death of immune cells, pathologic atrophy of hormone-dependent tissues, cell injury in certain viral diseases, and in the killing of cancer cells by chemotherapeutic drugs2,18,22,36,39. The cell-surface protein known as Fas/ CD95 (Fas) is expressed on activated lymphocytes. Cross-linking of Fas with its ligand, Fas L/CD95L (Fas-L), causes transudation of the apoptotic signal from the cell surface into the cytoplasm, thereby triggering cell death. Fas and Fas-L are typical members of the tumor necrosis factor (TNF) receptor and TNF ligand family, respectively, and they play pivotal roles in the regulation of apoptotic processes such as activation-induced cell death, Tcell-induced cytotoxicity, immune privilege, and tumor surveillance38. Fas-L induces suicidal death (apoptosis) of accumulated cytotoxic T-cells by binding to Fas expressed on the surface of these cells. Cancer cells that arise from several mutations of normal ancestor cells come under host immune attack as the body attempts to eliminate them26. However, most tumors escape this attack by imitating the strategies of the cells in the immuneprivileged sites through overexpressing Fas-L or down-regulating Fas5,13. This mechanism of immune escape through altered expression of Fas and Fas-L has been reported in squamous cell carcinoma (SCC) of the head and neck by several investigators1,11,16,21,25,33,37. Nucleolar organizer regions (NORs) are loops of DNA that contain genes for ribosomal RNA. These regions are transcribed by RNA polymerase I, and are of vital importance for protein synthesis8. NORs that are stained by silver are called AgNORs, and proteins associated with these are called AgNOR proteins12,31. AgNOR staining is a histochemical technique that provides valuable information about cellular status. In interphasic nuclei, AgNOR staining usually appears as dark granules within the nucleoli. The intensity of staining depends on the transcriptional
activity of the cells23,34. Recent studies suggest that the number of silver-stained proteins in cell nucleoli is related to cell proliferation29,40. More numerous and often atypical AgNORs have been described in malignant neoplasm than in benign tumors and normal tissues9. The main aim of this study was to investigate whether levels of Fas and Fas-L expression in oral and oropharyngeal SCCs are correlated with tumor stage and grade. Another goal was to assess whether AgNOR-related parameters— AgNOR number per nucleus, AgNOR size and the percentage area of each nucleus occupied by AgNORs (percent of nuclear area)—correlate with levels of apoptosis in SCC cells and adjacent normal epithelium. Material and methods
The study was performed on specimens of tumoral and adjacent normal tissue, 52 specimens taken from 26 patients (8 females and 18 males; mean age, 61.4 9.5 years; age range, 41–77 years), with oral and oropharyngeal SCCs. The stage of SCC, the grade of tumor, and levels of Fas and Fas-L expression were recorded for each tumor. Twenty-four of the specimens (12 tumoral and corresponding 12 adjacent normal tissues) were randomly selected and assessed for AgNORs. TNM (tumor size, lymph node metastasis, distant metastasis) and clinical stage of oral cavity cancers were classified according to the guidelines from the American Joint Committee on Cancer. The patients did not receive radiation therapy and/or chemotherapy prior to surgery. They did not show any distant metastases. The adjacent normal tissues were taken from the surgical margins with non-tumoral tissue following confirmation that the tissue was not tumoral using frozen sections. The grade of each tumor was also assessed (well differentiated, moderately differentiated or poorly differentiated) using the criteria described by CAWSON et al.4 Each patient had undergone standard treatment appropriate to the stage of the SCC. At the time of surgery, part of the resected SCC and a sample of adjacent normal tissue were immediately placed in 10% buffered neutral saline (10 mM, pH 7.4) and then embedded in paraffin for Fas and Fas-L staining (and AgNOR staining in 12 cases). The remaining portion of each SCC specimen and a sample of adjacent normal tissue were fixed in 10% formaldehyde and embedded in a paraffin block using routine histological technique. Seven-micrometer-thick sections were cut from 26 blocks containing the tumor spe-
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cimens and 26 blocks of corresponding adjacent normal tissue and were stained with hematoxylin and eosin. The tumor tissues were examined by a pathologist who confirmed the diagnosis of SCC in each case. Immunohistochemical methods
Fas and Fas-L staining
Four-micrometer-thick sections were cut from each of the 52 specimens designated for immunohistochemistry, and these were mounted on slides coated with poly-Llysine. The sections were de-paraffinized in xylene and then rehydrated through a series of baths containing decreasing concentrations of ethanol in de-ionized water. They were then treated with citrate buffer (pH 6.6) at 95 8C for 5 min to retrieve the antigens (Fas and Fas-L surface molecules). The sections were then brought to room temperature (20 8C), rinsed three times in de-ionized distilled water, and endogenous peroxidase activity was stopped by 20 min of immersion in 0.3% H2O2 in methanol. After three rinses in cold (4 8C) phosphate-buffered saline (10 mM, pH 7.4), the sections were incubated for 30 min with primary polyclonal antibody, either a rabbit polyclonal antihuman Fasspecific IgG (C-20, Sc-715, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a rabbit polyclonal antihuman Fas-L-specific IgG (N-20, Sc-834, Santa Cruz Biotechnology). After this, each section was rinsed three times in cold phosphate-buffered saline and then stained with secondary monoclonal antibody using the Santa Cruz Staining System (Sc-2053, goat antirabbit IgG-horse radish peroxidase conjugate, Santa Cruz Biotechnology). The manufacturer’s recommended dilutions and incubation periods for the primary and secondary monoclonal antibody were carefully followed. Peroxidase staining was demonstrated using the method of KOLB19 JØRNSEN et al. The cell nuclei were stained for 10 min with 1% methyl green prepared in 0.1 M acetate buffer (pH 4.2). Each slide was examined under the light microscope at high magnification (1000), and cells with brownish-black reaction products in the cell membrane and cytoplasm were considered positive for Fas or Fas-L, depending on which primary antibody had been applied. In all specimens, rabbit nonimmune serum purchased from Santa Cruz was used as a control. For each of the 52 specimens, intensity and extent of Fas and Fas-L expression were evaluated by a comprehensive scoring formula originally designed by BUSCH et al.3 The extent of
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staining in the samples was scored as: 1 = less than one-third, 2 = one-third to two-thirds, and 3 = more than two-thirds of the tumor cells stained positively. The results obtained with the two scales were multiplied against each other, yielding a single scale with steps of 1–4, 6 and 9, in which 1–4 were considered to be weak staining and 6 and 9 were considered to be strong staining. Staining intensity was scored as follows: 1 = weak expression, 2 = moderate expression, and 3 = strong expression. Staining the AgNORs
The paraffin-embedded tissues from 12 patients (12 tumoral tissue and 12 neighboring normal tissue samples) were stained for AgNORs. Four-micrometerthick sections were de-paraffinized and rehydrated as described above, and then stained with a specially prepared solution that consisted of one volume 2% gelatin in 1% aqueous formic acid, and two volumes 50% silver nitrate. Sections were immersed in this solution in the dark for 10–12 min at 37 8C, and then washed three times with distilled deionized water. Each section was then dehydrated and mounted using routine methods20. The sections were examined under high magnification (100), and AgNORs were identified as silver-stained (black) dots. Three AgNOR-related parameters were recorded for each case: AgNOR number per nucleus, AgNOR size and the percentage area of each nucleus occupied by AgNORs (percent nuclear area). For each lesion, 100 nuclei in 10 different areas of the tumor were analyzed in interphase using a 100 oil-immersion objective. The threshold for AgNOR dots was selected for individual cases, after enhancing the contrast up to a point at which the AgNORs dots were easily identified as black points within the nuclei. The relative AgNOR area (percentage of total nuclear area occupied by AgNORs) was determined as representative of the AgNOR quantity. Where two or more dots joined in clusters these were counted as a single AgNOR20. Statistical analysis
Clinical parameters, histological parameters, Fas and Fas-L expression, and the findings for the three AgNOR parameters were compared using the Spearman rank correlation coefficient and x2 testing, as appropriate. Two-tailed independent Student’s t-test was used to compare data between groups, whereas one-
way analysis of variance test was used to make comparisons within groups. The level of significance was set at P < 0.05. Results
Table 1 lists the characteristics of patients in relation to presence/absence of Fas and/ or Fas-L expression. Eight of 26 SCCs (31%) were in the oral region and 18 (69%) were oropharyngeal. Nine (53%) of the 17 Fas- or Fas-L-positive tumors showed strong expression of both antigens; 5 (29%) were positive for Fas only, and 3 (18%) were positive for Fas-L only. Twelve (86%) of the 14 tumors that were positive for Fas showed strong staining. The usual pattern of positive staining of Fas-L was similar to that of staining for Fas. The Fas-L staining varied in intensity, with tumor areas ranging from weak to strong intensity. Ten (83%) of the 12 tumors that were positive for Fas-L showed strong staining. Of the 26 specimens of normal tissue adjacent to the SCCs, seven (27%) showed both Fas and Fas-L expression. The remaining 19 specimens did not stain for either antigen. Two of the seven cases positive for Fas
and Fas-L showed weak staining for both antigens, two cases showed strong Fas staining, while three cases showed weak Fas staining. Eight (30.8%) patients exhibited primary metastasis to regional lymph nodes and accounted for three (37.5%) of the eight T3 tumors, and five (62.5%) of the seven T4 tumors. The frequency of primary metastasis to regional nodes in the T4 group was significantly higher than that in the T3 group (P < 0.01). There was no correlation between presence of Fas and/ or Fas-L expression and primary lymph node metastasis. There was a significant positive correlation between Fas and FasL expression and tumor stage (r = 0.62, P < 0.01 and r = 0.64, P < 0.01, respectively), but neither Fas nor Fas-L expression was correlated with clinical tumor size (T classification), lymph node metastasis (N classification) (Table 2). Of the 14 Fas-positive tumors eight were well, five were moderately and one was poorly differentiated, whereas four out of 12 Fas-L-positive tumors were well, five moderately and three poorly differentiated. Thus, Fas expression was more frequent in well-differentiated SCCs, and
Table 1. Clinical data for the 26 oral and oropharyngeal SCC cases in relation to presence of Fas and/or Fas-L staining Fas and/or Fas-L staining Age (years, mean SD) Sex Female Male
Positive (n = 17, 65%)
Negative (n = 9, 35%)
61.1 10.7
60.8 16.9
5 12
3 6
Tumor size T1 T2 T3 T4
1 (5.9%) 5 (29.4%) 6 (35.3%) 5 (29.4%)
4 1 2 2
Cervical nodal involvement N0 N1 N2 N3
10 (59%) 3 (17.6%) 4 (23.4%) –
8 (88.9%) 1 (11.1%) – –
Clinical stage I II III IV
1 (5.9%) 4 (23.5%) 7 (41.2%) 5 (29.4%)
4 1 2 2
Fas/Fas-L expression None Weak Moderate Strong
4 (23.5%) 4 (23.5%) 9 (53%)
– –
Differentiation grade Well differentiated Moderately differentiated Poorly differentiated
8 (47.1%) 6 (35.3%) 3 (17.6%)
8 (88.9%) 1 (11.1%) –
(44.5%) (11.1%) (22.2%) (22.2%)
(44.5%) (11.1%) (22.2%) (22.2%)
9 (100%)
Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer Table 2. Clinical features of the study population in relation to expression of Fas, Fas-L, and both antigens Fas and Fas-L positive (n = 9)
Fas positive (n = 5)
Fas-L positive (n = 3)
– 2 (22.2%) 3 (33.3%) 4 (44.5%)
1 (20%) 2 (40%) 2 (40%) –
– 1 (33.3%) 1 (33.3%) 1 (33.3%)
4 (80%) 1 (20%)
– 1 (33.3%) 2 (66.7%) –
– 2 (22.2%) 3 (33.3%) 4 (44.5%)
1 (20%) 1 (20%) 3 (60%) –
– 1 (33.3%) 1 (33.3%) 1 (33.3%)
– 9
2 (40%) 3 (60%)
2 (66.7%) 1 (33.3%)
Tumor size T1 T2 T3 T4
Cervical nodal involvement N0 6 (66.7%) N1 1 (11.1%) N2 2 (22.2%) N3 Clinical Stage I II III IV Staining Weak Strong
Fas-L expression was more frequent in moderately and poorly differentiated tumors (P < 0.05). Mean AgNOR number per nucleus and mean AgNOR size in tumoral and adjacent normal tissues are shown in Table 3. There were significant differences in the mean AgNOR number, size and nuclear area ratio between the control and SCC groups (P < 0.01) (Figs. 1 and 2). The relationships between the mean AgNOR number per nucleus and mean individual AgNOR size values and the percentage area of each nucleus occupied by AgNORs (percent of nuclear area) are also shown in Table 3 for tumoral and adjacent normal tissues in 12 oral and oropharygeal SCCs. The clinical
903
stage, degree of differentiation and Fas and/or Fas-L expression for the 12 AgNOR-stained specimens are given in Table 4, the most common parameters being Stage III (50%), moderate differentiation (50%), and both Fas and Fas-L staining (50%). A significant positive correlation was found between the AgNOR parameters and Fas and Fas-L expression in the tumoral cells of SCC (r = 0.65 and 0.66, P < 0.05), and also between the AgNOR parameters and the grading of tumors (r = 0.61, P < 0.05). Discussion
The expression of Fas-L may play an important role in establishing immunologically privileged environments that allow tumors to escape the host’s immune surveillance11,14,35; that is, the relative predominance of the striking power of lymphocytes and opposing striking power
Fig. 1. AgNORs in the nuclei of normal cells. The number and size of the silver-stained regions are relatively low compared to SCC (see Fig. 2; 1700).
Table 3. Comparison of AgNOR parameters between normal and tumoral tissue
AgNOR number
AgNOR size
The percentage area of each nucleus occupied by AgNORs (percent of nuclear area)
Patients (n = 12)
Normal tissue
Tumoral tissue
Normal tissue
Tumoral tissue
Normal tissue
Tumoral tissue
1 2 3 4 5 6 7 8 9 10 11 12
1.60 0.52 3.70 0.68 3.40 0.52 3,10 0,32 2.30 0.82 3.10 0.88 2.30 0.82 2.10 0.74 2.30 0.82 2.30 0.82 2.40 0.97 2.30 0.82
4.10 1.10* 5.0 1.25* 6.70 1.06* 6.20 0.63* 2.82 1.17 3.80 1.03 4.40 1.17* 3.70 2.11* 6.10 0.99* 4.60 1.84* 3.10 0.99 3.30 0.68
1.38 0.22 1.62 0.33 2.94 1.06 1.40 0.46 1.26 0.71 1.51 0.44 1.26 0.71 2.73 1.04 1.26 0.71 1.26 0.71 1.83 0.91 1.26 0.71
2.76 0.44* 3.93 0.47* 5.09 1.19* 6.61 1.21* 3.54 1.97* 4.76 1.74* 6.07 1.06* 4.90 2.46* 3.12 0.74* 6.60 1.36* 2.63 0.93 3.75 1.69*
3.42 0.25 3.60 0.19 4.84 0.87 3.54 0.00 4.67 1.30 3.78 0.79 4.67 1.30 5.37 0.90 4.67 1.30 4.67 1.30 4.31 0.79 4.67 1.30
3.42 0.37 3.66 0.25 6.61 1.14* 6.08 1.18* 5.07 1.15 6.56 1.71* 5.31 0.62* 7.14 1.61* 5.19 0.83 6.73 0.69* 4.60 0.87 6.31 1.44
Mean P
2.58 0.61
4.49 1.28 <0.001*
1.64 0.59
4.48 1.42 <0.001*
4.35 0.62
5.56 1.22 <0.001*
*
P < 0.05.
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Fig. 2. AgNORs in the nuclei of SCC cells. The AgNORs are larger and more numerous than in normal cells (see Fig. 1, 1700).
Table 4. Clinical stage, degree of differentiation, and expression of Fas and/or Fas-L in AgNOR-stained tumoral cells Specimen (n = 12) 1 2 3 4 5 6 7 8 9 10 11 12
Clinical stage
Degree of differentiation
Stage III Stage II Stage III Stage IV Stage III Stage IV Stage III Stage III Stage III Stage II Stage II Stage I
Moderate Moderate Moderate Poor Moderate Well Poor Well Moderate Moderate Well Well
of tumor cells affects the biological behavior of a tumor, and Fas-system-mediated apoptosis plays a role in this mechanism27. In the present study, Fas and Fas-L expression was detected in oral and oropharyngeal SCCs. A significant correlation was found between tumor staging and Fas/FasL expression frequency. The number of patients examined was not large (n = 26), but 65% of the SCC specimens stained strongly positive for Fas or Fas-L. In the specimens from Stages III and IV patients, a strong staining reaction for Fas-L was observed, while Fas staining was observed for the lower stages. This result is in agreement with MURAKI et al.25 who showed a high incidence of Fas antigen expression in the lower stage SCCs that may act as a controlling factor by promoting apoptosis in the slower growth of Faspositive SCC. T cells in the tumor microenvironment of head and neck cancer have showed evidence of apoptosis as well as decreased expression of signaling molecules. Immunohistochemical studies have shown that Fas antigen is widely distributed in normal and pathological epithelia, including oral mucosa24,42. While in normal oral mucosa
Fas and/or Fas-L staining Fas and/or Fas and/or Fas and/or Fas and/or Fas and/or Negative Fas-L Fas and/or Negative Fas-L Fas Fas
Fas-L Fas-L Fas-L Fas-L Fas-L Fas-L
Fas-L is primarily detected in cells of the basal layer32, Fas antigen is mainly located in the epithelial cell cytoplasm of human oral SCCs24. Fas expression has been reported to be down-regulated in invasive oral SCCs whereas Fas-L expression showed up-regulation5,16. Fas-L-expressing tumor cells may up-regulate Fas-L mRNA levels by direct contact with Fas-bearing cells from the antigenic host organism, thus taking advantage of Fas-L expression as a factor of tissue invasion10. It has been reported that poor expression of Fas and positive Fas-L staining in oral SCC correlates negatively with degree of differentiation and apoptosis32. Furthermore, it has been shown that Fas antigen expression is higher in lower stage than in advanced stage SCC25. In this study, Fas-L was found to be highly expressed in poorly differentiated tumors whereas in well differentiated tumors expression was considerably lower. Faspositive cells were found in only one poorly differentiated tumor. This may reflect the potential of tumor cells to undergo spontaneous apoptotic cell death17. Also in this study, 13 (50%) of 26 tumors had poor or no Fas expression,
which suggests that this may be a common mechanism for tumors to escape from the Fas-mediated host immune surveillance. In addition, the absence of Fas in Fas-Lpositive tumors precluded autocrine Fas-L attack for oral and oropharygeal SCC. This study confirms that the frequency of Fas-L expression is highest in T3 SCCs followed by T4 SCCs suggesting that FasL expression may be associated with tumor progression and poor prognosis. Certain normal tissues constitutively coexpressing Fas and Fas-L are characterized by apoptotic cell turnover that functions in the control of tissue homeostasis1–6. There is an increased awareness of the importance of apoptosis in carcinogenesis. SCC is the most common malignancy of head and neck regions. It is important that these lesions be diagnosed early and accurately. Determination of the state of nuclear DNA and proliferation markers is important in oral and oropharyngeal carcinomas presenting with Fas and Fas-L coexpression. It remains unclear as to whether Fas and Fas-L are associated with tumor growth. The results of this study show the usefulness of the staining of Fas and Fas-L antigens in tumor grading, and also might suggest a role for these antigens in carcinogenesis. A series of studies indicate that the quantity of protein AgNOR is related to the rapidity of cell proliferation, and a relationship between AgNOR counts and the prognosis of malignant tumors has been suggested6,30. Several studies have shown variations in the number, shape and volume of AgNORs of normal mucosa cells and malignant cells9,30. The data available indicate that cancer cells have a greater amount of interphase AgNORs than benign or normal cells, and therefore a malignant lesion may frequently be distinguished from a benign lesion of the same tissue by interphase AgNOR quantitative evaluation. Since the tumor mass growth rate is one of the most important factors influencing clinical outcome, it follows that the AgNOR value is a valuable parameter for providing information about the progression of disease6. AgNOR number may allow more precise tumor staging if it is shown that it is a marker for progression rather than malignant potential15. It is also likely that the mean number of AgNORs is related to the number of cells in S phase and with ploidy. Thus, AgNOR counts may assist in the grading of neoplasm7. In the ¨ Z et al.28, the present study, like that of O number of NOR dots increased with increasing grade of SCC while the area of the NOR decreased and dots became more irregular with increasing grade. There
Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer were significant differences in the mean AgNOR counts, area and nuclear area ratio between the control and SCC groups (P < 0.05). Significant positive correlation was found between the AgNOR parameters and Fas/Fas-L expression in the tumoral cells of SCC. In conclusion, in this study a significant positive correlation was found between Fas/Fas-L expression and tumor stage; and AgNOR enumeration appeared to be a useful tool in distinguishing between normal epithelium and SCC. In addition, AgNOR count was found to be a strong proliferation marker, when compared with the presence of Fas and Fas-L antigen in patients with SCC. Acknowledgements. We thank Dr Kayhan Oztuok for his kind assistance. This study was supported by Grant No. SB-99-117 from the Scientific Research Council of Selc¸ule University.
11.
12.
13.
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