Evaluation of squamous cell carcinoma antigen 1 expression in oral squamous cell carcinoma (tumor cells and peritumoral T-lymphocytes) and verrucous carcinoma and comparison with normal oral mucosa

Abstract Background: Squamous cell carcinoma antigen (SCCA) is used as a prognostic marker for recurrence of squamous cell carcinoma in various sites, including head and neck. Studies suggest that its high serum levels are correlated to some clinical features, such as nodal metastasis. However, it is still unknown if high SCCA in patients with SCCA tissue expression in tumor cells are related to peripheral T-lymphocytes. Therefore, we did this study to evaluate SCCA expression in squamous cell carcinoma and verrucous carcinoma and to compare it with normal oral mucosa, also investigating the correlation between serum-based and tissue-based antigen levels. Methodology: In this study, the immunohistochemistry (IHC) technique was used to determine the SCCA1 expression pattern in 81 specimens divided into 3 groups, including oral squamous cell carcinoma, verrucous carcinoma, and normal oral mucosa. Serum-based and tissue-based antigen levels of 20 oral squamous cell carcinoma cases were compared by the western blot assay. SCCA expression was also evaluated and compared in both tumor cells and peripheral T-lymphocytes by the immunofluorescence assay. Results: Our results showed that the SCCA levels in SCC specimens were significantly lower than in verrucous carcinoma and normal and hyperplastic oral mucosa specimens. We found no correlation between the IHC expression of SCCA and serum levels. SCCA was well expressed in both tumor cells and peripheral T-lymphocytes. Conclusion: Decreasing SCCA in SCC specimens suggested that SCC tumor cells may affect more than the serum levels of SCCA in some patients. In addition, expression of SCCA in peripheral T-lymphocytes showed that both tumor cells and T-lymphocytes may cause serum SCCA.


Introduction
Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck (excluding non-melanoma skin cancer). 1 Despite progress in research and therapy, its 5-year survival rate remained the same in the last 45 years 2 because of delayed clinical detection, poor prognosis, expensive therapeutic alternatives, and lack of specific biomarkers for the disease. 3 Biomarkers are biological molecules found in blood, other body fluids, or tissues that indicate normal biological processes, pathogenic processes, or a pharmacological response to a therapeutic intervention. 4 One of these markers, called squamous cell carcinoma antigen (SCCA), was found in human cervical squamous cell carcinoma tissue for the first time by Kato and Torigoe 5 (1977). Patients with squamous cell carcinoma of the cervix or other origins, such as nasopharynx and bronchus, head and neck, healthy volunteers and patients with different tumors showed high serum levels of SCCA. 6,7 SCCA belongs to the serine proteinase inhibitor (serpin) superfamily. 8 SCCA1 and SCCA2 are two nearly identical genes, responsible for SCCA expression. 9 SCCA mRNA can be detected in patients with OSCC and patients with non-SCC cancers, but it is relatively low in non-SCCs. 10 Regarding tumor aggression, high mRNA levels of SCCA are characteristic to less aggressive head and neck SCC tumors. 11 Studies showed that immunohistochemical concentration of SCCA in SCC tissues and in normal squamous epithelial was much higher than in non-SCC cancer tissues. 10 hematoxylin. Squamous carcinoma specimen of the cervix was applied using PBS (phosphate-buffered saline) as positive controls, while the primary antibody was omitted using PBS as negative controls.
All the slides were evaluated by two blinded oral pathologists using an Olympus BX51 microscope.
Diffuse cytoplasmic brown immunoreaction was with SCCA expression in random 10 high-power fields, measured as follows: Score 0: no expression; Score 1: less than 10% of tumor cells; Score 2: 10-50% of tumor cells; Score 3: more than 50% of tumor cells. 18 Western blotting A total of 20 plasmas with 20 formalin-fixed paraffin-embedded blocks were obtained from patients with OSCC (Table 1). Human plasma samples were first emptied from albumin and enriched with lowabundant proteins by trichloroacetic acid (TCA)/ acetone method as described elsewhere. 19 Then,

Inflammation of cells in connective tissue around
the tumor was evaluated as previously described. 20 Briefly, 4 µ sections were deparaffinized twice with xylol for 10 min and hydrated with water for 3 min.
Antigen retrieval was done using 2H2O/CaCL2 at 37°C, followed by RT for 20 min and 10 min respectively, and washed twice by PBS-T (0.05%). Membranous permeability was increased using Triton (0.5%), then

Immunohistochemistry
We observed SCCA immunostaining in the cytoplasm of typical epithelial, verrucous carcinoma, cancer cells and T-lymphocytes peripheral to cancer tissue ( Figure 1). Among 27 primary SCC specimens, 10 did not express SCCA (37%). Ten patients expressed SCCA in less than 10% of total cells (37%).
Only 7 out of 27 cases expressed SCCA in more than 10% of the total cancer cells (22.2%: 10-50% of cells or score 2 and 3.7%: more than 50% of cells or score 3) (Table 2). Sixteen verrucous carcinoma specimens had scores 2 and 3 (both 29.6%), and 10 cases had

Discussion
Our results revealed that SCCA1 expression was significantly higher in hyperplastic epithelium and verrucous carcinoma than in OSCC (p=0.001 and p=0.007, respectively). We also found that SCCA1 serum levels were not related to tissue expression (p=0.71 and p=0.83 in two separate analyses).
Based on the above, we designed an experiment to determine origin cells with SCCA1 protein and found that peripheral T-lymphocytes produce more SCCA1 than tumor cells, but the SCCA1 expression is the same in both.
A biomarker is a substance that can help oncologists in the diagnosis, prognosis, and follow-ups of diseases.     Table 4-Correlation between IHC analysis and western blot analysis From these findings, one can conclude that SCCA1 serum levels increase; however, from our results, tissue expression is not associated with serum levels.
While the cause of SCCA1 in patients with SCC remains controversial, previous studies suggested that SCC tumor cells increase SCCA in a patient's serum.
Therefore, tumor size could influence serum levels of

Conclusion
In conclusion, our findings suggested that serum levels of SCCA1 are not associated with tissue-based expression. Decreasing SCCA in SCC specimens showed that SCC tumor cells may affect more than serum levels of SCCA in some patients. Finally, immunofluorescence results showed that both tumor cells and peripheral T-lymphocytes can express and produce SCCA1, causing serum SCCA.