ⅠINTRODUCTION
Squamous cell carcinoma(OSCC) represents about 3% of human cancers and over 90% of malignant tumors at oral cavity with new cases being diagnosed worldwide over 350,000 each year.1) Malignant tumors of the head and neck including oral cancers tend to spread to adjacent lymph nodes as progressive metastatic tumor. Distant metastases are relatively few. The prognostic evaluation of OSCC is determined by histological and immunohistochemical findings, including tumor size, lymph node involvement and histological grade. The presence of metastasis to cervical lymph nodes acts as an important factor for evaluating the extent of the disease and determining the prognosis.
The proliferating tendency of the cancer cells is believed to have prognostic significance.2) Moreover, uncontrolled cell proliferation is considered one of the most important biological mechanisms associated with oncogenesis.3) The ratio of cells growing within the cycle can be easily identified by Ki-67 or MIB-1 antibodies, identifying antigen expression on phases G1, S, G2 and M of the cell cycle.4, 5) In fact, several studies using antibodies that recognize the Ki-67 nuclear antigen have provided a reliable method to evaluate tumor growth rate.6) Ki-67 is considered a useful tool in determining the aggressiveness of malignant neoplasms. Many studies support that the expression of Ki-67 is a reliable prognostic factor in head and neck tumors.7)
p53 is a nuclear phosphoprotein inhibiting the cell cycle, eventually leading to cell apoptosis. It ultimately functions as a tumor suppressor gene. R. Todd et al. reported that there was a correlation between p53 expression and clinicopathologic parameters in head and neck cancer.8) The expression of P53 protein were detected in approximately 50-60% of the cases of oral cavity or tongue carcinoma, which may be explained by a dramatic increase in P53 expression or possibility of random mutation.9,10)
There are many researches about the correlation of p53 or Ki-67 with oral squamous cell carcinoma. However, few studies have investigated on the histopathological differences between primary squamous cell carcinoma in oral cavity (POSCC) and metastatic squamous cell carcinoma in lymph nodes (MOSCC). In the present study, we have evaluated the immunohistochemical expression of Ki-67 and p53 in both POSCC and paired MOSCC.
ⅡMATERIALS and METHODS
Patients and tissue sample
This study was conducted with 10 patients, confirmed with the lymph node metastasis, who underwent surgical treatment including neck dissection after the diagnosis of OSCC by tissue biopsy at Department of Oral & Maxillofacial Surgery, Kyungpook National University Hospital from Jan. 2010 to Jun. 2015. Paraffin-embedded tissue samples from 10 POSCC and paired 10 MOSCC cases were obtained from the Department of Pathology at Kyungpook National University Hospital. These 20 specimens were possible to be re-examined with favorable storage condition of paraffin-embedded tissue blocks. Informed consent was obtained from all patients before surgical treatment. This study was approved by the Institutional Review Board of Kyungpook National University Hospital (KNUH_2015-01030).
Immunohistochemistry
Paraffin-embedded tissue blocks were obtained from 10 POSCC specimens and paired 10 MOSCC specimens by being soaked in xylene solution for 30 minutes. Paraffin was then eliminated and samples were hydrated in 100%, 90%, 70% ethyl alcohol sequentially as preparation for immunohistochemical staing. Stainings for ki-67 and p53 was performed using microwave oven method. Slides were put in 10mM citrate buffer (pH 6.0), heated at 95℃ for 10 minutes The slides were then placed at room temperature for 20 minutes, cleansed with buffered saline (TBS, 50Mm, pH 7.4) followed by application of 0.3% hydrogen peroxidase. In order to prevent unspecific antigen binding in tissue, specimens were reacted in normal horse serum for 30 minutes and placed in 4℃ water overnight for anti-ki-67(MIB-1 antigen was diluted in 1:60, anti-P53 antigen was diluted in 1:100) primary antibody attachment. The specimens were cleansed with TBS for 3 times. They were reacted with secondary antibody (Vector Elite kit, Vector Laboratorieds, USA) for 30 minutes, followed by ABC (avidin-biotin conjugate) reagent reaction for 45 minutes at room temperature. After cleansing in TBS and Tris-HCl (pH 7.6) buffer, color development was processed with diaminobenzidine tetrahydrochloride (DAB, Sigma Chemicals, USA). The slides were cleansed with water, counterstained with Mayer's hematoxylin for 20 seconds, and then mounted for complete preparation.
Expression of Ki-67 and p53 was evaluated independently by two investigators who were not informed of the clinical data of the patients. The staining reaction was determined by counting a total of 1000 cancer cells in each sample (10 random areas, 100 cells / 1 area, 400 magnification) and by accessing the percentage of labeled cells. The immunostaining for Ki-67 and p53 was evaluated as 0 (no positive cells), weak (1~25% positive cells), moderate (26-50% positive cells) and strong (>50% positive cells).11)
Statistical analysis
All of the statistical analyses were performed using the IBM SPSS(IBM Corporation, NY, USA). Correlation between expression of Ki-67 or P53 in POSCC or MOSCC were analyzed by Mann-Whitney test. The relationship between the expression of Ki-67 or p53 and the degree of differentiation in OSCC was evaluated using the Spearman’s rank correlation coefficient. The values were considered statistically significant when the p value was lower than 0.05.
ⅢRESULTS
1Clinicopathological characteristics
Clinicopathologic information of the 10 patients (20 specimens) was summarized in Table 1. The age of patients ranged from 49 to 77 years, with mean age 61.1±8.1. 8 male and 2 female patients participated. The histological differentiation of POSCC resulted in 5 well-differentiated and 8 moderately differentiated specimens. The histological differentiation of MOSCC showed 8 well-differentiated and 2 moderately differentiated specimens. The degree of differentiation was significantly lower in MOSCC than in POSCC.
2Immunohistochemical staining
1)Expressions of Ki-67 in POSCC and MOSCC
The mean expression values of Ki-67 in POSCC and MOSCC were 35.1% and 29.1%, respectively. The moderate expressions of Ki-67 were observed in 8 out of 10 cases in POSCC and 7 out of 10 cases in MOSCC. There was no significant correlation between Ki-67 expression in POSCC and MOSCC. (Table 2)
2)The expression of P53 in POSCC and MOSCC
The mean expression values of p53 in POSCC and MOSCC were 25.6% and 23.6%, respectively. The moderate expressions of p53 were found in 5 out of 10 cases in POSCC and 5 out of 10 cases in MOSCC. The result showed that there was no statistically significant correlation between p53 expression in POSCC and MOSCC. (Table 3)
3)The expressions of Ki-67 and p53 in POSCC
The mean expression values of Ki-67 and p53 in POSCC were 35.1% and 25.6%, respectively. The immunohistochemical staining of 8 out of 10 cases in POSCC showed an increased pattern of expression of p53 in areas with proliferative cell activity. Despite the strong tendency, there was no statistically significant correlation between expressions of Ki-67 and p53 in POSCC.
4)The expressions of Ki-67 and p53 in MOSCC
The mean expression values of Ki-67 and p53 in MOSCC were 29.1% and 23.6%, respectively. The immunohistochemical staining showed similar patterns in 8 of 10 cases in MOSCC. There was no statistical significance between expressions of Ki-67 and p53 in MOSCC.
5)The relationship between expression of Ki-67 and p53 and the differentiation of tumor cells
The degree of differentiation of POSCC differed from that of MOSCC. Higher expression of Ki-67 was significantly correlated with poorer degree of differentiation. There was a statistically significant correlation between the expression and differentiation of Ki-67 in both POSCC (p<0.01) and MOSCC(p<0.05). (Table 4) However, there was no statistical significance between the expression and differentiation of p53 in both POSCC and MOSCC. (p>0.05) Fig.1, Fig.2
ⅣDISCUSSION
Ki-67, first introduced in 1984, has become a relatively popular immunohistochemical tool to estimate the tumor growth rate. The proliferation activity is reflected by the Ki-67 antibodies by recognizing a cell nucleus antigen that is expressed maximally in the G2 and M phases and absent in the non-proliferating cells (phase G0).12) The immunoreactivity of Ki-67 of the patients with a neck metastasis was higher than those of the patients without a metastasis. Thus, Ki-67 provides useful information for assessing the prognosis of OSCC patients.13) Taken together, these findings indicated that the expression of Ki-67 with regard to proliferative activity of tumor cells is one of the indicators for the potential of tumor invasion and invasive activity of cancers related to degree of malignant neoplastic cells.14) Coutinho-Camillo et al. demonstrated the association between the enhanced expression of Ki-67 with the presence of lymph node metastasis, advanced stages of disease (III/IV), tumors occurring in the floor of mouth, and moderately/well-differentiated tumors in OSCC.15) Pich et al. suggested that a high cell proliferation is a reliable prognostic factor in head and neck tumors and is a significant indicator determining the possible failure of treating either recurrent or non-recurrent oral and oropharyngeal carcinomas.7) Most authors have established significant correlations between lower histologic differentiation and poorer prognosis. But, the clinical application of Ki-67 still remains controversial. Several reports have shown that Ki-67 does not have any prognostic value in terms of predicting outcome in OSCC. Gonzalez-Moles MA et al. reported that Ki-67 expression was significantly higher in well-differentiated carcinomas compared to that in poorly-differentiated carcinomas. However, the result may be explained by the fact that Ki-67 expression indicates the total fraction of proliferative cells in a tumor appears to account for its lack of association with tumor prognosis.11) In this study, the expression of Ki-67 in OSCC was significantly correlated with degree of differentiation. The elevated expression of Ki-67 was related to poorer degree of differentiation. Further study would be needed to understand the relationship between Ki-67 expression and the prognosis by analyzing additional clinical information.
p53 is a nuclear phosphoprotein inhibiting cell cycle progression and inducing apoptosis, both regulated by several target genes. It ultimately functions as a tumor suppressor gene, inducing an arrest of the G1-S transition in response to mitogenic stimuli. p53 mutations are extremely frequent in human cancers.
Studies suggest that the overexpression of p53 is highly correlated with the metastasis to lymph nodes in oral cavity carcinomas as well as with the neoplasias of other primary sites, such as the esophagus and the brain.16) Oliveira et al. reported that the presence of p53 is correlated with metastasis to lymph nodes and worse prognosis.17) Nonetheless, in this study, the expression of p53 in OSCC was not significantly associated with any of the clinicopathologic parameters analyzed.
Numerous studies on the relationship between p53 expression and cell proliferation in SCC with immunohistochemical markers such as Ki-67 and PCNA have been conducted. Most authors found p53 to show a similar immunolocalisation as for PCNA or Ki-67, indicating that p53 levels increased in areas with proliferative activity.18,19) Patel et al. reported p53 and Ki-67 were co-expressed in 94.87% of their cases and Raju et al. found it to be 80% in their study.20, 21) Consistent with these findings, our studies showed 80% of similar immunohistochemically staining pattern between the expression of p53 and Ki-67. However, despite the strong tendency, there was no statistically significant between expressions Ki-67 and p53 in POSCC or in MOSCC.
In the present study, we have successfully investigated the expression of Ki-67 and p53 in POSCC and MOSCC despite the limited sample size. We, however, found no statistically significance between Ki-67 or p53 expression in POSCC or MOSCC. Our results showed a correlation between the degree of differentiation of the tumor and the level of expression of Ki-67 in OSCC. Higher expression of Ki-67 was significantly correlated with poorer degree of differentiation. Overall, our results suggest that the expression of Ki-67 may be a predictable factor for the degree of differentiation of the tumor, indicating its role as an important prognosis marker in POSCC and MOSCC.
