Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 57  |  Issue : 4  |  Page : 553--557

Association of Ki-67 antigen and p53 protein at invasive tumor front of oral squamous cell carcinoma


Ruchita Verma1, Anil Singh2, Rohit Jaiswal2, Akhilesh Chandra3, Renuka Verma4, Jalaj Tak1,  
1 Departments of Oral and Maxillofacial Pathology, Shree Bankey Bihari Dental College and Research Centre, Ghaziabad, Uttar Pradesh, India
2 Sardar Patel Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India
3 Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
4 Career Post Graduate Institute of Dental Sciences and Hospital, Lucknow, Uttar Pradesh, India

Correspondence Address:
Akhilesh Chandra
2/29 Rashmi Khand, Sharda Nagar, Lucknow - 226 002, Uttar Pradesh
India

Abstract

Aim: Studies reveal that invasive tumor front may be the best field for quantification of proliferative and apoptotic markers. The current study assessed the expression of Ki-67 antigen and p53 protein at invasive tumor front of oral squamous cell carcinoma (OSCC) and correlated the immunostaining with the histologic grades of malignancy. Materials and Methods: A total of 80 slides sample was prepared for the study, one each from ten normal oral mucosa cases and two each from 30 OSCC cases. The OSCC patients were biopsied along invasive tumor front, and samples were immunohistochemically analyzed for the expression of both Ki-67 antigen and p53 protein. Results: Of the total 30 OSCC cases, the expression of p53 was found positive in 20 cases (66.6%), while Ki-67 expression was found positive in 21 cases (70%). All the cases in the control group were negative. A higher p53 and Ki-67 expression were seen in OSCC group as compared to normal mucosa. On comparing the control group with various grades of OSCC a statistically significant result was obtained. Conclusion: Higher and statistically significant expression was noted for both p53 and Ki-67 antigen. The results emphasize the potential of Ki-67 and p53 as biomarkers of carcinogenesis in OSCC. Ki-67 expression was comparatively higher when compared with p53 except in poorly differentiated squamous cell carcinoma and signifies actively proliferating malignant cells at invasive tumor front.



How to cite this article:
Verma R, Singh A, Jaiswal R, Chandra A, Verma R, Tak J. Association of Ki-67 antigen and p53 protein at invasive tumor front of oral squamous cell carcinoma .Indian J Pathol Microbiol 2014;57:553-557


How to cite this URL:
Verma R, Singh A, Jaiswal R, Chandra A, Verma R, Tak J. Association of Ki-67 antigen and p53 protein at invasive tumor front of oral squamous cell carcinoma . Indian J Pathol Microbiol [serial online] 2014 [cited 2021 Nov 28 ];57:553-557
Available from: https://www.ijpmonline.org/text.asp?2014/57/4/553/142660


Full Text

 INTRODUCTION



World Health Organization predicts a continuing worldwide increase in a number of patients with oral cancer, the dominant being squamous cell carcinoma (SCC). [1] The development of oral squamous cell carcinoma (OSCC) is a multistep process requiring the accumulation of multiple genetic alterations, influenced by the patient's genetic predisposition as well as by environmental influences including tobacco, alcohol, chronic inflammation, and viral infection. [2]

Tumorigenic genetic alterations consist of two major types: Tumor suppressor genes (TSGs), which promote tumor development when inactivated and oncogenes, which promote tumor development when activated. TSGs can be inactivated through genetic events such as mutation, loss of heterozygosity, deletion, and epigenetic modifications like DNA methylation or chromatin remodeling. [2] The TSG p53 is located on the short arm of chromosome 17 and encodes a protein of 393 amino acids. Point mutations in p53 are important in the development of malignancy and are frequently observed in cancer of various tissue, including OSCC. [3]

Cell proliferation is a vital biological process to all living organisms. Abnormal cell proliferation appears to be a precursor and a possible predictor of tumorigenesis. [4] Ki-67 gene located on chromosome 10 (10q25) encodes a protein which is expressed in nucleolus in G1, S, G2, and M phases but not in G0 (arrest phase). [4] Ki-67 is a nuclear antigen which is present in the perichromosomal region. [5]

Studies reveal that tumor invasive front (TIF) may be the best field for growth fraction estimation. [4] The TIF has the deepest and most aggressive cells. [6] The molecular events at invasive tumor include increased angiogenesis, aberrant expression of adhesion-related molecules, overproduction of extracellular matrix degrading enzymes, and increased expression of proliferation-related molecules. [4]

Therefore, this study was planned to analyze the expression of p53 protein and Ki-67 antigen at invasive tumor front of OSCC and to correlate the immunostaining with the histologic grade of malignancy.

 MATERIALS AND METHODS



This quantitative retrospective study was carried out at Department of Oral Pathology and Microbiology, Sardar Patel PG Institute of Dental and Medical Sciences, Lucknow, India after the approval from the Ethical Committee of the Institute.

The study comprised of two groups. Group I contained 10 cases as control group with normal oral mucosa. Group II consisted of 30 cases as study group, in which biopsy samples of invasive tumor front of OSCC were included. The samples for study group were collected from the formalin-fixed and paraffin-embedded blocks of histologically proven OSCCs from the archives of Department of Oral Pathology.

From the paraffin-embedded blocks, one section of 5 μm thickness and two sections of 3 μm thickness were taken. 5 μm thick section was stained with hematoxylin and eosin for the diagnosis of the lesion; whereas the 3 μm thick sections were taken on poly-L-lysine-coated glass slides for immunohistochemical staining of p53 protein and Ki-67 antigen, respectively.

Heat-mediated antigen retrieval was performed using citrate buffer in the pressure cooker. [7],[8] Slides were then incubated in 3% hydrogen peroxide to block endogenous peroxidase activity. [8],[9] Ready-to-use mouse IgG-1 anti p53 (DO-7) monoclonal antibody (BioGenex, Milmont drive, Fremont, California- 94538 USA) and mouse IgG anti Ki-67 (BGX-297) monoclonal antibody (BioGenex, Milmont drive, Fremont, California- 94538, USA) were used as primary antibodiesand were incubated for 60 min, followed by super enhancer for 25 min. [8],[10] For secondary antibody application, slides were incubated with polymer-horseradish-peroxidase reagent (antimouse and antirabbit IgG labeled with enzyme polymer in phosphate-buffered saline) for 30 min. [8],[11] 3,3- diaminobenzidene (DAB) (DAB tetrahydrochloride) solution was used as the chromogen and counterstaining was done with Mayer's hematoxylin. [3],[8]

When a distinct brown staining was confined to the nuclei, p53 expression was considered positive. p53 exhibited a granular or reticular nuclear staining. [3] Similarly, tumor cells were considered positive for Ki-67 antigen if there was intranuclear light brown granular staining. [4],[12]

Counting procedure

Primarily, the hematoxylin and eosin stained tissue sections were examined to confirm the histological diagnosis at low-power magnification (10). Then, immunohistochemically stained sections were first analyzed for the expression of p53 and Ki-67 at low power (10). The selected fields were used for the quantitative assessment of the p53 and Ki-67 positive cells at high power. At least 600 tumor cells along the invasive tumor front were observed and counted in three different histological fields using a magnification of 40. An eyepiece grid was used to prevent the overlapping of fields.

The numbers of positively stained and nonstained cells were recorded separately. p53 and Ki-67 overexpression were considered positive for those cases which demonstrated >10% positively-stained nuclei of tumor cells. Negative result was defined as absence of stained nuclei or immunoexpression under 10% of tumor cells. [9] The indices were calculated as the percentage of positively stained cells among the total number of cells were counted. The results were tabulated and subjected to statistical analysis including Student's t-test, Chi-square test and Mann-Whitney U-test.

 STATISTICS AND RESULTS



In the study groups, out of the 30 cases, 17 cases were well differentiated squamous cell carcinoma (WDSCC), 10 cases were moderately differentiated squamous cell carcinoma (MDSCC), and 3 cases were poorly differentiated squamous cell carcinoma (PDSCC).

Of the total 30 OSCC cases, the expression of p53 was found positive in 20 cases (66.6%) and negative in 10 cases (33.3%). Although the expression of Ki-67 was found positive in 21 cases (70%) and negative in 9 cases (30%) out of total 30 OSCC cases [Table 1]. All the cases in the control group for both p53 and Ki-67 were negative, showing immunoexpression in <10% cells [Table 1]. U-test revealed a significantly higher (P < 0.001) p53 and Ki-67 expression in various grades of OSCC group as compared to the control group [Table 2] and [Table 3].{Table 1}{Table 2}{Table 3}

The mutual comparison of p53 and Ki-67 expression in different groups (control group and various grades of OSCC) is summarized in [Table 4]. It shows that the Ki-67 expressions in all groups were comparatively higher when compared to p53 expressions, except in PDSCC. On comparing the expressions of both in each group separately, U-test revealed significantly higher (P < 0.05) expression of Ki-67 in control and WDSCC group than p53. However, the expressions of both did not differ (P > 0.05) in MDSCC and PDSCC [Table 4].{Table 4}

 DISCUSSION



The abrogation of p53 functions through the mutation of its gene (TP53); the loss of heterozygosity of TP53; or interaction with viral proteins is one of the most common molecular alterations in SCC of the head and neck. The involvement of p53 in apoptosis and cell-cycle control makes it a plausible biomarker of prognosis. [13] Ki-67 can be detected in phases G1, S, G2 and M of the cell-cycle, but not on G0 phase, exclusively in the nuclei of cycling cells. This defined period of nuclear expression makes the Ki-67 protein a reliable marker of proliferating cells. [14]

The histologic features of OSCC may differ widely from area to area within the same tumor, and it is believed that the most useful prognostic information can be deduced from the invasive front of the tumors, where the deepest and presumably most aggressive cells reside. [3]

In the present study, p53 expression was detected in 66.7% cases of OSCC along the invasive tumor front [Figure 1] and 33.3% cases were p53 negative. The mean p53 index in OSCC in our study was 61.95 ± 24.17. Xie et al. in their research work evaluated p53 expression in tongue carcinoma and found that 61% cases expressed detectable levels of p53. [15] Lazarus et al. stated that incidence of p53 mutations have been observed in around 63% of OSCC. [16] {Figure 1}

On the contrary, other workers found the lower frequency of p53 immunoexpression, that is 46% by Shiraki et al. [17] and 43% by Siegelmann-Danieli et al. [18] Wong et al. suggested that p53 has been shown to interact with the oncogenic protein E6 of the human papilloma virus, which results in the rapid degradation of the p53 protein by the ubiquitin-mediated proteolysis system. [19]

p53 expression increased linearly from the control group through various grades of OSCC in the present study. In pairwise comparison, a significant difference was obtained between normal mucosa against WDSCC, MDSCC, and PDSCC (P < 0.001). The results were in accordance with Kannan et al. [20] and Xu et al. [21] who showed a significant difference between the expression pattern of normal mucosa and OSCC.

However, nonsignificant result was obtained on comparing various grades of OSCC (P > 0.001) in the present study. Similar studies were reported by Lal et al. found that p53 was an important prognostic factor and was independent of histologic grade. [22] Siegelmann-Danieli et al. also stated that p53 status was not associated with tumor progression, as reflected by similar mutation rates in tumors of different stages, nodal involvement, and histology grade. [18]

On the contrary, Jain et al. observed that the percentage of positive cases and p53 positivity showed a corresponding increase in values with increasing grade of OSCC. A significant difference in p53 positivity was noted between cases of WDSCC and MDSCC and between WDSCC and PDSCC. [5] The reason to this may be that different antigen retrieval techniques may be responsible for obscuring any correlation by creating false positive cases. They also said that in advanced disease, several genetic changes may mask the true influence of p53. [22]

All cases of normal oral mucosa (control group) assessed in the present study were p53 negative [Figure 2] as they had <10% positively stained tumor cells (cut-off in the present study). The mean expression of p53 in normal mucosa was 0.22 ± 0.048. The expression was restricted to few basal cells. Nylander et al. stated that due to its short half-life, around 20 min, the p53 protein is hard to detect in normal tissue. However, if occasional positive cells can be detected in normal tissue, they are located in the basal layer of the epithelium. [23] {Figure 2}

The study showed that, Ki-67 expression was positive in 70% cases [Figure 3] and negative in 30% cases of OSCC. Mean Ki-67 index in OSCC was 72.86 ± 18.51. Rodrigues et al. reported a high expression of Ki-67 in 80.8% cases of OSCC. Ki-67 expression was evident in 73.3% of the patients with metastatic lymph nodes and in 75% of high-grade tumors. [9] However, Kurokawa et al. reported Ki-67 mean score in 32.8% cases in OSCC. [3]{Figure 3}

Comparing the mean Ki-67 expression between study group and control group significantly (P < 0.001) different and higher Ki-67 expression was seen in the study group when compared to control the group. The Ki-67 expression was linearly increased from normal mucosa through various grades of OSCC. Pairwise comparison showed a significant difference between normal mucosa against WDSCC, MDSCC, and PDSCC (P < 0.001). Similar results have been reported by Kannan et al. [20] and Xu et al. [21] who showed a significant difference between the expression pattern of Ki-67 in normal mucosa and OSCC.

However, Ki-67 expression did not show statistically significant difference in various histological grade of malignancy (P > 0.05) in this study. Our results were in accordance with Torres-Rendon et al. [8] who showed that there was no correlation between the level of tumor differentiation and labeling index of Ki-67 protein.

In the present study, Ki-67 expression was seen along the parabasal and sometimes basal layer of normal oral mucosa, predominantly being restricted parabasally [Figure 4]. However, all cases were negative as they had <10% positive Ki-67 cells. The mean labeling index of Ki-67 in normal mucosa was 2.07 ± 0.56. Torres-Rendon et al. [8] also reported the similar observations. The reason for positive staining in parabasal compartment could be due to that in normal oral mucosa, most cells are in the G0 phase (not in the cycle) with a lesser number in a licensed G0-G1 transition. Only a few cells are actually in the cell-cycle (demonstrated by Ki-67 expression). [8] {Figure 4}

Mean Ki-67 expression in all groups was comparatively higher as compared to p53 expression, except PDSCC. Similar results had been observed by Rodrigues et al. who reported 61.5% expression for p53 and 80.8% expression for Ki-67. They concluded that in cases of higher histological grade and advanced disease, such difference failed to achieve statistical significance. [9]

According to Kannan et al., Ki-67 exhibited a more pronounced association with tumor progression. The variation in expression may be of prognostic importance for the early detection of premalignant lesions. [20] Higher Ki-67 labeling values may indicate biologic malignancy with higher proliferative activity. [3]

 CONCLUSION



On the basis of the present study, we concluded that abnormal p53 protein and Ki-67 antigen are detected in 66.6% and 70% cases, respectively, at invasive tumor front of OSCC. Their expressions were found increased linearly, from normal mucosa through various grades of OSCC. The results emphasize the potential of Ki-67 and p53 as biomarkers of carcinogenesis in OSCC. Higher expression of Ki-67 as compared to p53 signifies actively proliferating malignant cells at this site and may help in the prognosis of tumor.

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