Ki67 Labelling Index predicts clinical outcome and survival in oral squamous cell carcinoma

Abstract Objective To investigate the Ki 67 expression and its correlation with clinicopathological features and 3 years as well as 5 years survival rate in oral squamous cell carcinoma (OSCC). Methodology Total 217cases of OSCC primarily treated with surgery with or without radiation were included. All patients were followed up for 3 years and 150 were followed up of 5 years for disease free survival. The immunohistochemistry was carried out on neutral buffered formalin fixed paraffin embedded tissue to evaluate the expression of Ki67. Results The Ki67 labeling index (LI) was significantly higher with respect to adverse clinicopathological parameters such as histopathological grading (p<0.001), clinical TNM staging (p<0.001) and nodal metastasis (p<0.001). The OSCC patients survived for less than 3 and 5 years were showed significantly higher Ki67 LI as compared to diseases free survived more than 3 and 5 years(p<0.001). The three years survival rate of OSCC patient significantly higher with low Ki67 LI (≤45) 96.2%, followed by moderate Ki67 LI (46 to 60) 60.7% and high Ki67 LI (≥61) 37.7% (p<0.001). The five years survival rate of OSCC patient statistically significantly higher with low Ki67 LI (≤45)93.3%, followed by moderate Ki67 LI (46 to 60) 46.8% and Ki67 LI (≥61) 23.3% (p<0.001). Conclusion The measurement of cell proliferative activity by using Ki67 antigen expression in individual OSCC might provide unique, predictive information on clinical outcome, prognosis and deciding treatment modalities in OSCC.


Introduction
Globally, oral squamous cell carcinoma (OSCC) incidence is 2.7 in 100000 populations. In southcentral Asia, the incidence of OSCC cases was highest that is40.9% of all incident cases of OSCC and often associated poor prognosis due to high morbidity and mortality rate. 1

Immunohistochemistry
The standard procedure of immunohistochemistry (IHC) was done for Ki67, by utilizing appropriate controls on neutral buffered formalin fixed paraffin embedded tissue. The tissue sections were treated with 01 mol/L sodium citrate buffer (pH 6.0) in microwave oven for 10 minute followed by bench-cooled for 20 min, and again the same cycle was repeated for the antigen retrieval. Endogenous peroxidase activities of the tissue were blocked by incubating the tissue sections with 3% H2O2 in methanol for 30 minutes. Thus, based on the KI67 LI, the Ki67 expression were categorized in to low: Ki67 LI≤45, moderate: Ki67 LI 46 to 60 and high: Ki67 LI ≥61.

Statistical analysis
The data were statistically analyzed using SPSS, version 17.0 for Windows. One-way ANOVA and Tukey's HSD test were utilized to find out the differences of The relationship between Ki67 LI with 3 and 5 years disease free survival of oral squamous cell carcinoma patients  However, use of cost effective, easy and widely use In this study, we assess the cut of range value for Ki67 LI as low, moderate and high with respect to three  and five years disease free survival of OSCC patients.
The three year disease free survival rate of OSCC patient significantly higher with low Ki67 LI (96.2%), followed by moderate KI67 LI (60.7%) and high Ki67 LI (37.7%). The five year disease free survival rate of OSCC patient significantly higher with low Ki67 LI (93.3%), followed by moderate Ki67 LI (46.8%) and high Ki67 LI (23.3%). In this study for the first time we introduce the range value for ki67 LI in OSCC that could be use as prognostic indicator for survival and to decide the treatment regime.
The mean of Ki-67 LI, was significantly higher in OSCC patients survived for less than 3 years as compared to diseases free survived OSCC patients more than 3 years. Similarly, the mean of Ki-67 LI, was significantly higher in OSCC patients survived for less than 5 years as compared to diseases free survived OSCC patients more than 5 years. It has been noticed in previous studies 9,24-26 that K67 expression in OSCC was identified as prognostic factor for survival rate.
However, several other studies 27-30 have described no association between higher cell proliferation and survival. Recently, Jing,et al. 9 (2019) studied Ki67 expression in large cohort of 298 OSCC and found that Ki67 expression was independent prognostic marker in OSCC patients. Ki67 expression was also found to be prognostic marker in oropharyngeal and OSCC with a lager cohort of 239 cases. 26 Therefore, it is summarize that the increase in Ki67 expression associated with a poor prognosis of OSCC. It was also suggested that Ki67 is an indicator for treatment failure in OSCC. 9,26 Thus, it was further strengthen that the high cell proliferation identified by Ki67 antigen expression is a reliable prognostic marker in oral cancer.
In this study, a significant increase in Ki-67 LI was observed from WDSCC to MDSCC to PDSCC. This results was in agreement with previous studies 9,[31][32][33][34][35] and revealed that as the Ki67 LI increases with poorer the degree of tumor cell differentiation. In contrast, few studies 29,36 failed to show correlation of Ki67 LI and degree of differentiation. Disparity in the methodology, assessment of Ki67 immunoreactivity, and sample size may result in these conflicting results.
Higher expression of Ki67 protein was discovered in undifferentiated/poorly differentiated squamous cells carcinoma. Therefore, results of the present study further strengthen the fact that, the Ki67 LI could be considered as useful potential biomarker in grading the OSCC.
The mean of Ki-67 LI was significantly higher in metastatic as compared to non-metastatic OSCC.
Other studies 3,25,32,36 found insignificant correlation with cell proliferation and cervical lymph node metastasis, which are not in accordance with this study results. In this study large cohort of OSCC was used and results are in accordance with previous studies. 9,28,33,37,38 This finding may implicate that tumors with high cell proliferation index may point out towards aggressive behavior of a tumour and a potential for metastasis.
In the present study, Ki67 LI was significantly higher in TNM stage IV as compared to stage III.
However, non-significant difference of ki67 LI was found amongst stage I, stage II and stage III. The previous studies 3, 30,32,34,36 showed no significant association between Ki67LI and clinical TNM staging. Interestingly, on broadly categories TNM staging, the mean Ki67 LI was significantly higher in advanced stage OSCC as compared to early stage OSCC. These findings are in agreement with previous studies 28,33 suggesting that the increased Ki67 LI indicates the higher intrinsic growth potential of the tumor resulting in advance TNM staging.
In accordance with previous studies 37,38 the result of this study have also showed non-significant difference of Ki67 LI amongst site of OSCC.
In this study the Ki67 LI was significantly higher with respect to adverse clinicopathological parameters such as histopathological grading, clinical TNM staging, cervical lymph node metastasis and 3 as well as 5 years disease free survival of OSCC.
Thus, it can be suggested that assessment of cell

Conflict of interest
All the authors associated with present manuscript declared no potential conflicts of interest.

Funding source
The author received no specific funding for this work.

Acknowledgement
We would like to thank authorities of SharadPawar Dental College and Hospital, Sawangi (Meghe), Wardha for providing the infrastructure and related resources that immensely helped in execution of the present study.We also acknowledge that the samples and marker used in the present paper were taken from our already published work (cited in the paper). However, the objectives of the present study are totally different and are not related to our previously published work.