Indian Journal of Oral Health and Research

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 4  |  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

Correspondence Address:
Dr. Sepideh Mokhtari
School of Dentistry, Tehran University of Medical Sciences, North Kargar Avenue, Tehran
Iran

Abstract

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.



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;4:42-46


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 2019 May 19 ];4:42-46
Available from: http://www.ijohr.org/text.asp?2018/4/2/42/257150


Full Text



 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.[1] This cancer accounts for 4%–13% of all cases of oral SCC and is infrequent in persons younger than 45 years.,[2] Some investigations show that tongue SCC (TSCC) in young patients has a more aggressive behavior[3],[4],[5],[6],[7],[8],[9] while others claim that there is no relationship between age and aggressive behavior.[10],[11],[12],[13],[14]

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.[15] Ki-67 is expressed in all phases of cell cycle except G0.[16] Tumors with higher proliferation rate demonstrate more aggressive clinical manners.[17] 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.[16],[18] 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.[19],[20]

[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).{Table 1}{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Table 2}{Table 3}{Figure 5}

 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.[21],[22],[23],[24],[25] The information regarding the expression of this protein in oral squamous cell carcinoma (OSCC) is inconsistent. Lo Muzio et al.[21] 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.[22] 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.[23] 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.[24] 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.[25] In contrast to our findings, Jordan et al.[26] 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.[27] found an association between expression of this marker and histopathologic grades and mode of invasion. In contrast, Staibano et al.[28] revealed that higher expression of bcl-2 was related to better prognosis and Garewal et al.[29] found that well-differentiated tumors had higher bcl-2 expression. Ahmed[30] 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.[31]

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.[2],[17] 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.[2],[16],[17],[32],[33],[34] 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[19],[35] however on the contrary with Gonzalez-Moles et al.[16] investigation. They found a relationship between the expression of ki-67 in parabasal cells of the adjacent epithelia and tumor cells.

Montebugnoli et al.[35] 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.[36] 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.[37] 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.[16],[38] However, Guimarães et al.[39] 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.[40]

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.[41] However, some studies have not found these associations.[16],[42],[43] It seems that further investigations are necessary for invasive treatments.[44],[45],[46]

The mean expression of this marker was 47.5% which was very similar to that of Benevenuto's et al. study.[2] 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%,[47] 20%,[44] and 28%[39] 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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Akbari ME, Atarbashi Moghadam S, Atarbashi Moghadam F, Bastani Z. Malignant tumors of tongue in Iranian population. Iran J Cancer Prev 2016;9:e4467.
2Benevenuto TG, Nonaka CF, Pinto LP, de Souza LB. Immunohistochemical comparative analysis of cell proliferation and angiogenic index in squamous cell carcinomas of the tongue between young and older patients. Appl Immunohistochem Mol Morphol 2012;20:291-7.
3Popovtzer A, Shpitzer T, Bahar G, Marshak G, Ulanovski D, Feinmesser R, et al. Squamous cell carcinoma of the oral tongue in young patients. Laryngoscope 2004;114:915-7.
4Soudry E, Preis M, Hod R, Hamzany Y, Hadar T, Bahar G, et al. Squamous cell carcinoma of the oral tongue in patients younger than 30 years: Clinicopathologic features and outcome. Clin Otolaryngol 2010;35:307-12.
5Sarkaria JN, Harari PM. Oral tongue cancer in young adults less than 40 years of age: Rationale for aggressive therapy. Head Neck 1994;16:107-11.
6Vargas H, Pitman KT, Johnson JT, Galati LT. More aggressive behavior of squamous cell carcinoma of the anterior tongue in young women. Laryngoscope 2000;110:1623-6.
7Siriwardena BS, Tilakaratne A, Amaratunga EA, Tilakaratne WM. Demographic, aetiological and survival differences of oral squamous cell carcinoma in the young and the old in Sri Lanka. Oral Oncol 2006;42:831-6.
8Vered M, Dobriyan A, Dayan D, Yahalom R, Talmi YP, Bedrin L, et al. Tumor-host histopathologic variables, stromal myofibroblasts and risk score, are significantly associated with recurrent disease in tongue cancer. Cancer Sci 2010;101:274-80.
9Garavello W, Spreafico R, Gaini RM. Oral tongue cancer in young patients: A matched analysis. Oral Oncol 2007;43:894-7.
10Annertz K, Anderson H, Biörklund A, Möller T, Kantola S, Mork J, et al. Incidence and survival of squamous cell carcinoma of the tongue in scandinavia, with special reference to young adults. Int J Cancer 2002;101:95-9.
11Sasaki T, Moles DR, Imai Y, Speight PM. Clinico-pathological features of squamous cell carcinoma of the oral cavity in patients and lt; 40 years of age. J Oral Pathol Med 2005;34:129-33.
12Udeabor SE, Rana M, Wegener G, Gellrich NC, Eckardt AM. Squamous cell carcinoma of the oral cavity and the oropharynx in patients less than 40 years of age: A 20-year analysis. Head Neck Oncol 2012;4:28.
13Pytynia KB, Grant JR, Etzel CJ, Roberts D, Wei Q, Sturgis EM, et al. Matched analysis of survival in patients with squamous cell carcinoma of the head and neck diagnosed before and after 40 years of age. Arch Otolaryngol Head Neck Surg 2004;130:869-73.
14Pitman KT, Johnson JT, Wagner RL, Myers EN. Cancer of the tongue in patients less than forty. Head Neck 2000;22:297-302.
15Atarbashi S, Elahi M, Khani M, Rakhshan V. Immunohistochemical analysis of B-cell lymphoma-2 in pleomorphic adenoma and mucoepidermoid carcinoma. Dent Res J (Isfahan) 2014;11:257-63.
16Gonzalez-Moles MA, Ruiz-Avila I, Gil-Montoya JA, Esteban F, Bravo M. Analysis of ki-67 expression in oral squamous cell carcinoma: Why ki-67 is not a prognostic indicator. Oral Oncol 2010;46:525-30.
17Yu YH, Morales J, Feng L, Lee JJ, El-Naggar AK, Vigneswaran N, et al. CD147 and ki-67 overexpression confers poor prognosis in squamous cell carcinoma of oral tongue: A tissue microarray study. Oral Surg Oral Med Oral Pathol Oral Radiol 2015;119:553-65.
18Moreno-Galindo C, Hermsen M, García-Pedrero JM, Fresno MF, Suárez C, Rodrigo JP, et al. P27 and BCL2 expression predicts response to chemotherapy in head and neck squamous cell carcinomas. Oral Oncol 2014;50:128-34.
19Bascones-Martínez A, Rodríguez-Gutierrez C, Rodríguez-Gómez E, Gil-Montoya JA, Gómez-Font R, González-Moles MÁ, et al. Evaluation of p53, caspase-3, bcl-2, and ki-67 markers in oral squamous cell carcinoma and premalignant epithelium in a sample from Alava Province (Spain). Med Oral Patol Oral Cir Bucal 2013;18:e846-50.
20Knopf A, Lempart J, Bas M, Slotta-Huspenina J, Mansour N, Fritsche MK, et al. Oncogenes and tumor suppressor genes in squamous cell carcinoma of the tongue in young patients. Oncotarget 2015;6:3443-51.
21Lo Muzio L, Mignogna MD, Pannone G, Rubini C, Grassi R, Nocini PF, et al. Expression of bcl-2 in oral squamous cell carcinoma: An immunohistochemical study of 90 cases with clinico-pathological correlations. Oncol Rep 2003;10:285-91.
22Sulkowska M, Famulski W, Sulkowski S, Reszeć J, Koda M, Baltaziak M, et al. Correlation between bcl-2 protein expression and some clinicopathological features of oral squamous cell carcinoma. Pol J Pathol 2003;54:49-52.
23McAlinden RL, Maxwell P, Napier S, Hamilton P, Cowan CG, Lundy FT, et al. Bcl-2 expression in sequential biopsies of potentially malignant oral mucosal lesions assessed by immunocytochemistry. Oral Dis 2000;6:318-26.
24Birchall MA, Schock E, Harmon BV, Gobé G. Apoptosis, mitosis, PCNA and bcl-2 in normal, leukoplakic and malignant epithelia of the human oral cavity: Prospective, in vivo study. Oral Oncol 1997;33:419-25.
25Birchall MA, Winterford CM, Gob G, Harmon BV. Apoptosis and mitosis in oral and oropharyngeal epithelium; evidence for a topographical switch in premalignant lesion. Cell Proliferation, 1996;29 (8):447-456.
26Jordan RC, Catzavelos GC, Barrett AW, Speight PM. Differential expression of bcl-2 and bax in squamous cell carcinomas of the oral cavity. Eur J Cancer B Oral Oncol 1996;32B: 394-400.
27Yao L, Iwai M, Furuta I. Correlations of bcl-2 and p53 expression with the clinicopathological features in tongue squamous cell carcinomas. Oral Oncol 1999;35:56-62.
28Staibano S, Mignogna MD, Lo Muzio L, Di Alberti L, Di Natale E, Lucariello A, et al. Overexpression of cyclin-D1, bcl-2, and bax proteins, proliferating cell nuclear antigen (PCNA), and DNA-ploidy in squamous cell carcinoma of the oral cavity. Hum Pathol 1998;29:1189-94.
29Garewal J, Garewal R, Sircar K. Expression of bcl-2 and MIB-1 markers in oral squamous cell carcinoma (OSCC) – A comparative study. J Clin Diagn Res 2014;8:QC01-4.
30Ahmed MM. Expression profile of apoptotic mediators and proliferative markers in oral squamous cell carcinoma. J Egypt Natl Canc Inst 2009;21:85-92.
31Arya V, Singh S, Daniel MJ. Clinicopathological correlation of bcl-2 oncoprotein expression in oral precancer and cancer. J Oral Biol Craniofac Res 2016;6:18-23.
32van Monsjou HS, Wreesmann VB, van den Brekel MW, Balm AJ. Head and neck squamous cell carcinoma in young patients. Oral Oncol 2013;49:1097-102.
33Olimid DA, Simionescu CE, Mărgăritescu C, Florescu A. Immunoexpression of ki67 and cyclin D1 in oral squamous carcinomas. Rom J Morphol Embryol 2012;53:795-8.
34Dragomir LP, Simionescu C, Mărgăritescu C, Stepan A, Dragomir IM, Popescu MR, et al. P53, p16 and ki67 immunoexpression in oral squamous carcinomas. Rom J Morphol Embryol 2012;53:89-93.
35Montebugnoli L, Gissi DB, Badiali G, Marchetti C, Cervellati F, Farnedi A, et al. Ki-67 from clinically and histologically “normal” distant mucosa as prognostic marker in early-stage (T1-T2N0) oral squamous cell carcinoma: A prospective study. J Oral Maxillofac Surg 2011;69:2579-84.
36Mallick S, Breta M, Gupta SD, Dinda AK, Mohanty BK, Singh MK, et al. Angiogenesis, proliferative activity and DNA ploidy in oral verrucous carcinoma: A Comparative study including verrucous hyperplasia and squamous cell carcinoma. Pathol Oncol Res 2015;21:1249-57.
37Matsuhira A, Noguchi S, Sato K, Tanaka Y, Yamamoto G, Mishima K, et al. Cytokeratin 13, cytokeratin 17, ki-67 and p53 expression in upper layers of epithelial dysplasia surrounding tongue squamous cell carcinoma. Bull Tokyo Dent Coll 2015;56:223-31.
38Diniz MG, Silva Jde F, de Souza FT, Pereira NB, Gomes CC, Gomez RS, et al. Association between cell cycle gene transcription and tumor size in oral squamous cell carcinoma. Tumour Biol 2015;36:9717-22.
39Guimarães EP, de Carli ML, Sperandio FF, Hanemann JA, Pereira AA. Cyclin D1 and ki-67 expression correlates to tumor staging in tongue squamous cell carcinoma. Med Oral Patol Oral Cir Bucal 2015;20:e657-63.
40Matsushita Y, Yanamoto S, Takahashi H, Yamada S, Naruse T, Sakamoto Y, et al. Aclinicopathological study of perineural invasion and vascular invasion in oral tongue squamous cell carcinoma. Int J Oral Maxillofac Surg 2015;44:543-8.
41Xie S, Liu Y, Qiao X, Hua RX, Wang K, Shan XF, et al. What is the prognostic significance of ki-67 positivity in oral squamous cell carcinoma? J Cancer 2016;7:758-67.
42Perisanidis C, Perisanidis B, Wrba F, Brandstetter A, El Gazzar S, Papadogeorgakis N, et al. Evaluation of immunohistochemical expression of p53, p21, p27, cyclin D1, and ki67 in oral and oropharyngeal squamous cell carcinoma. J Oral Pathol Med 2012;41:40-6.
43Hwa JS, Kwon OJ, Park JJ, Woo SH, Kim JP, Ko GH, et al. The prognostic value of immunohistochemical markers for oral tongue squamous cell carcinoma. Eur Arch Otorhinolaryngol 2015;272:2953-9.
44Freudlsperger C, Freier K, Hoffmann J, Engel M. Ki-67 expression predicts radiosensitivity in oral squamous cell carcinoma. Int J Oral Maxillofac Surg 2012;41:965-9.
45Myoung H, Kim MJ, Lee JH, Ok YJ, Paeng JY, Yun PY, et al. Correlation of proliferative markers (Ki-67 and PCNA) with survival and lymph node metastasis in oral squamous cell carcinoma: A clinical and histopathological analysis of 113 patients. Int J Oral Maxillofac Surg 2006;35:1005-10.
46Bôas DS, Takiya CM, Coelho-Sampaio TL, Monção-Ribeiro LC, Ramos EA, Cabral MG, et al. Immunohistochemical detection of ki-67 is not associated with tumor-infiltrating macrophages and cyclooxygenase-2 in oral squamous cell carcinoma. J Oral Pathol Med 2010;39:565-70.
47Freudlsperger C, Rohleder SE, Reinert S, Hoffmann J. Predictive value of high ki-67 expression in stage I oral squamous cell carcinoma specimens after primary surgery. Head Neck 2011;33:668-72.