|Year : 2018 | Volume
| Issue : 2 | Page : 42-46
Differential expression of cell proliferation and apoptosis markers in squamous cell carcinoma of the tongue in young and old patients
Saede Atarbashi-Moghadam1, Dorsa Yousef Monji2, Mahshid Namdari3, Sepideh Mokhtari4
1 Department of Oral and Maxillofacial Pathology, Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Private Dental Practitioner, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
3 Department of Community Oral Health, Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Education Development Office, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
|Date of Web Publication||25-Apr-2019|
Dr. Sepideh Mokhtari
School of Dentistry, Tehran University of Medical Sciences, North Kargar Avenue, Tehran
Source of Support: None, Conflict of Interest: None
Introduction: Squamous cell carcinoma (SCC) is the most common malignant neoplasm of tongue. Some investigations show that tongue SCC (TSCC) in young patients has a more aggressive behavior. Tumor progression is believed to be influenced by tumor cell proliferation as well as anti-apoptotic activity. The present study was conducted to assess ki-67 and bcl-2 expression in TSCC between young and older patients. Materials and Methods: Thirty paraffin block sections of TSCC were stained with monoclonal antibodies against bcl-2 and Ki-67. Data were analyzed by Mann–Whitney and Spearman correlation coefficient test. Results: Samples from 19 men and 11 women, with a mean age of 56 years, were evaluated. The patients were divided into two groups, A (>45 years) and B (≤45 years). Clinical and microscopic data such as tumor size, grade, and muscle invasion were extracted. Bcl-2 expression was negative for all the samples except one. Ki-67 expression was assessed as a percentage of Ki-67-positive neoplastic cells and scored subsequently. There was a significant association between the expression of ki-67 with microscopic grade and age (P < 0.05). Conclusion: These findings suggest that the more aggressive behavior of TSCC in younger age may be related with ki-67 expression and may serve as a valuable prognostic factor.
Keywords: Bcl-2, cell proliferation, Ki-67, squamous cell carcinoma, tongue
|How to cite this article:|
Atarbashi-Moghadam S, Monji DY, Namdari M, Mokhtari S. Differential expression of cell proliferation and apoptosis markers in squamous cell carcinoma of the tongue in young and old patients. Indian J Oral Health Res 2018;4:42-6
|How to cite this URL:|
Atarbashi-Moghadam S, Monji DY, Namdari M, Mokhtari S. Differential expression of cell proliferation and apoptosis markers in squamous cell carcinoma of the tongue in young and old patients. Indian J Oral Health Res [serial online] 2018 [cited 2020 Jul 12];4:42-6. Available from: http://www.ijohr.org/text.asp?2018/4/2/42/257150
| Introduction|| |
Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the tongue. The susceptibility of the tongue for cancers was explained as it has a great muscular structure and rich lymphatic network. Therefore, it poorly saves itself from invasion and metastasis. This cancer accounts for 4%–13% of all cases of oral SCC and is infrequent in persons younger than 45 years., Some investigations show that tongue SCC (TSCC) in young patients has a more aggressive behavior,,,,,, while others claim that there is no relationship between age and aggressive behavior.,,,,
Apoptosis happens in healthy and pathologic tissues. The relation between apoptosis inhibition and tumorigenesis is well documented and suggests new therapeutic targets for molecular cancer treatment. Bcl-2 is involved in inhibiting the cell death rather than stimulating cell proliferation. Ki-67 is expressed in all phases of cell cycle except G0. Tumors with higher proliferation rate demonstrate more aggressive clinical manners. Here, we aimed to assess the expression of bcl-2 and ki-67 proteins in TSCC of young and adult patients.
| Materials and Methods|| |
Archived samples at the laboratory of Oral and Maxillofacial Pathology Department in Shahid Beheshti University of Medical Sciences and a private laboratory were examined and samples with TSCC diagnosis were selected. Clinical and demographic data such as age, gender, and tumor size (T) were extracted from the patients' records. Cases with sufficient data as well as corresponding paraffin blocks with complete fixation and adequate tissue were finally selected. In addition, microscopic grade, muscular, intravascular, and perineural invasion were recorded.
Staining procedure for bcl-2 and ki-67 was performed by Envision technique (horseradish peroxides-based two-step immunostaining method) on 4-μm thick sections. The tissue sections were deparaffinized in xylene and rehydrated in descending ethanol series. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide methanol solution. After antigen retrieval by protease enzyme, the sections were incubated with monoclonal mouse anti-bcl-2 (clone 124, diluted 1:80, Dako, Glostrup, Denmark) for 30 min. For immunostaining of Ki-67, epitopes were unmasked by microwave irradiation in 10 mM citrate buffer, pH = 6, and then MIB-1 (ready to use, Dako, Glostrup, Denmark) was used for 1 h. The reaction products were visualized with 3,3′-diaminobenzidine as the chromogen counterstained with Mayer's hematoxylin and mounted.
Only cytoplasmic staining of tumoral cells for bcl-2 and nuclear staining for ki-67 was considered positive. Ten fields were chosen for each section. The percentage of positive neoplastic cells was calculated (HPF) from a minimum of 1000 tumoral cells (Li) and then scored subsequently for bcl-2 (≥10%: score 1 and <10%: score 2) and for ki-67 (1%–25% positive cells: score 1, 26%–50% positive cells: score 2, 51%–75% positive cells: score 3, and >75% positive cells: score 4) according to the previous articles., In addition, ki-67 and bcl-2 expression were studied in adjacent epithelium when adequate epithelium was available. For the negative control, the primary antibody was replaced with phosphate buffer saline. As positive controls, tonsil and breast carcinoma sections were respectively used for bcl-2 and Ki-67 immunostaining. In addition, the bcl-2 slides had internal control (lymphocytes in the sections).
The data were analyzed by the (IBM) SPSS Statistics version 21. The correlations between the expression of ki-67 and clinicopathologic parameters (gender, age, muscle invasion, and grade) were evaluated by Mann–Whitney U-test. Spearman correlation coefficient was used for association of ki-67 expression and tumor size and adjacent epithelium. P < 0.05 was considered statistically significant.
| Results|| |
A total of 30 samples including 22 cases of well-differentiated SCC (Grade I) and eight cases of moderately differentiated SCC (Grade II) were analyzed. There were 19 men and 11 women with a mean age of 56 years. The patients were divided into two groups, A (>45 years) and B (≤45 years) according to previous articles.,
[Table 1] demonstrates the clinical and histopathologic characteristics of 30 specimens. Bcl-2 was negative for all the samples except one case with score 1 expression. However, the adjacent epithelium showed basal cell staining [Figure 1] and lymphocytes were also positive [Figure 2]. Ki-67 protein was expressed in all samples (100%). There were seven cases with score 1 [Figure 3], 12 cases with score 2, three cases with score 3, and eight cases with score 4 [Figure 4]. The ki-67 expression was significantly higher in Grade II group than in Grade I group (P < 0.001) [Table 2], and in Group B than Group A (P = 0.04) [Table 3]. There was no significant difference between ki-67 expression and gender (P = 0.58), tumor size (P = 0.86), and muscular invasion (P = 0.53). In addition, an association was not found between the expression of ki-67 in adjacent epithelium [Figure 5] and ki-67 expression in the tumor (P = 0.58).
|Figure 1: Cytoplasmic expression of bcl-2 protein in the basal layer of adjacent epithelium (×100)|
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|Figure 2: Bcl-2 expression is only seen in lymphocytes. No immunostaining is observed in neoplastic squamous cells (×100)|
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|Figure 3: Ki-67 expression in the peripheral cells of tumoral squamous islands with keratin pearl formation (×100)|
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|Figure 4: Intense immunostaining of ki-67 protein (score 4) is observed in almost all neoplastic cells (×400)|
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|Table 2: Correlation of ki-67 expression (score) with histopathologic grade (P <0.001)|
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|Figure 5: Nuclear expression of ki-67 in the basal and parabasal layer of epithelium (×100)|
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| Discussion|| |
The present study revealed that 96% of TSCCs were negative for bcl-2. However, the adjacent epithelium showed only basal cell staining. These results are consistent with the results of several studies conducted on bcl-2 expression.,,,, The information regarding the expression of this protein in oral squamous cell carcinoma (OSCC) is inconsistent. Lo Muzio et al. found that 83% of SCC samples were negative for this marker and there was no correlation between the expression of bcl-2 and clinicopathologic parameters. The bcl-2 expression was limited to peripheral cells of neoplastic islands and was not expressed in keratinized tumors. Sulkowska et al. mentioned that this marker was expressed in 27% of SCCs and was related to microscopic grade, higher mitotic index, and atypical mitosis. McAlinden et al. examined precancerous epithelium and found that only one case was positive for bcl-2 protein. This finding is in relative accordance to our findings regarding the adjacent tumoral epithelia. In the current study, the expression of bcl-2 was not seen in any other layers but the basal cells of the epithelium. In addition, Birchal et al. indicated that bcl-2 gene expression was only present in the basal layer of epithelium and is suppressed in SCC and its adjacent epithelium in comparison with normal and hyperplastic epithelium. They also found that apoptosis is not related to the degree of dysplasia or onset of SCC. In contrast to our findings, Jordan et al. showed that this marker is expressed in 60% of oral SCCs, especially in poorly differentiated tumors. They also found the expression of bcl-2 in basal and keratinocyte layers of dysplastic epithelium. Therefore, one possible cause for lack of bcl-2 expression in our study is that the majority of TSCCs in this study was well-differentiated. Yao et al. found an association between expression of this marker and histopathologic grades and mode of invasion. In contrast, Staibano et al. revealed that higher expression of bcl-2 was related to better prognosis and Garewal et al. found that well-differentiated tumors had higher bcl-2 expression. Ahmed showed that 80% of the samples were positive for bcl-2; however, the microscopic figures in their article revealed nuclear staining for this protein and indicating improper procedure and invalid results. This problem was also seen in Arya et al.'s study.
In the current study, all the samples were positive for ki-67, and there was a significant association between the expression of this protein with younger age group and microscopic grade. In contrast to our study, other research did not find any association between ki-67 overexpression and age., This difference can be attributed to the fact that there was an increasing number of Grade II TSCCs in younger age group of our study. Our results were consistent with other research in relation to the microscopic grade.,,,,, The current study failed to find an association between the expression of KI-67 in adjacent epithelium and ki-67 expression in the tumor. This result is consistent with some studies, however on the contrary with Gonzalez-Moles et al. investigation. They found a relationship between the expression of ki-67 in parabasal cells of the adjacent epithelia and tumor cells.
Montebugnoli et al. examined the distant mucosa (i.e., on the contralateral side of the tumor) of patients with OSCC and showed that the proliferation rate in this group was higher than healthy patients. Mallick et al. compared the expression rate of ki-67 in OSCC, verrucous carcinoma, and verrucous hyperplasia, and it was concluded that the ki-67 expression rate was much higher in the OSCC group. This could lead to useful information especially in challenging cases and in incisional biopsies. Matsuhira et al. examined this marker in adjacent dysplastic epithelia and found that Ki-67 is useful for the early diagnosis of OSCC.
In our study, there was no association between Ki-67 expression and tumor size. Some studies also did not find an association between these two parameters., However, Guimarães et al. merged four stage groups into two ([T1, T2] and [T3, T4]) and found a relationship between ki-67 expression and tumor size. In the current study, no relationship was identified between the expression of this marker and muscular invasion. Moreover, five cases with perineural invasion were observed but were not examined since they were low in number. The relationship with muscular invasion was not investigated the various articles. Yet, perineural and intravascular invasion was associated with a poor prognosis and regional metastases. Therefore, it was suggested that elective neck dissection should be recommended for Stage I/II TSCC patients with these microscopic features.
The majority of the research that has been done in this field considered ki-67 as an adjuvant diagnostic tool for predicting tumor behavior and possible response to chemo/radiotherapy. A meta-analysis study in the Asian population was performed, and it was concluded that ki-67 has a high probability of determining the prognosis of OSCC patients and their response to therapy. However, some studies have not found these associations.,, It seems that further investigations are necessary for invasive treatments.,,
The mean expression of this marker was 47.5% which was very similar to that of Benevenuto's et al. study. However, it is higher than some other research. The underlying reason is the fact that the cutoff levels for the high or low expression of this marker varies in the studies and different values as 15%, 20%, and 28% have been reported in the literature. Therefore, a comprehensive study with a large sample size and complete clinicopathological information is necessary. This would result in a more reasonable cutoff level.
| Conclusion|| |
According to the current findings, it seems that expression of bcl-2 has no role in the pathogenesis of TSCC. Moreover, overexpression of ki-67 is associated with high-grade tumors. In addition, the more aggressive behavior of TSCC in younger age may be related to ki-67 expression. Therefore, ki-67 staining is recommended as a routine procedure in incisional biopsies of OSCCs. It is suggested that further research with larger samples and complete clinical data should be done to gain a reliable cutoff level.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]