Abstract
The prognostic relevance of Ki-67 expression in oral squamous cell carcinoma (OSCC) is still controversial. As proliferating cells are more susceptible to ionizing radiation, the authors investigated if a high proliferation rate reflected by Ki-67 expression, predicts radiosensitivity in OSCC patients. In 52 patients with OSCC who received primary surgery followed by radiation therapy, the proliferation rate was assessed by Ki-67 immunhistochemistry and correlated to recurrent free survival and overall survival. Low proliferative carcinomas showed a significantly shorter mean time to recurrence of 27.5 months compared to 49.5 months of high proliferative tumours ( p = 0.048). The 5-year survival rate of low proliferative tumours was 49% compared to 80% for high proliferative tumours ( p = 0.042). This study indicates that tumours with high proliferative activity are more susceptible to radiation therapy. Ki-67 might be used as a marker to predict the response to radiation therapy in patients with OSCC.
Despite advances in the multimodal treatment of oral squamous cell carcinoma (OSCC), little improvement in the 5-year survival rate has been achieved over the past 20 years. This is partially due to the fact that the prediction of the individual clinical course, including response to radiotherapy (RT), of a patient with OSCC remains difficult considering classical histopathological parameters only. Although several markers have been linked to radioresistance, the exact mechanisms responsible for radioresistance are poorly understood and the lack of an accurate system to predict the response to RT for individual patients with OSCC remains a major concern. As actively proliferating cells are more susceptible to radiation than quiescent tissues, there might be a correlation between the proliferation rate of a tumour and the response to RT. There are several approaches for measuring the proliferation rate of tumour cells, including determination of the mitotic index, the AgNOR-method or analysing the cell fraction in the S-phase by flowcytometry, and certain cell cycle proteins including the Ki-67 antigen are suitable for determining proliferative activity. As the Ki-67 antigen is present in every phase of the cell cycle except the G 0 and the early G 1 phase, measuring Ki-67 expression is suitable for determining the proliferative cell population of a tumour. The aim of this study was to investigate if patients with OSCC showing a high proliferative activity, reflected by high Ki-67 expression, might be more susceptible to RT and show a better clinical outcome after RT, compared to patients with low proliferative tumours.
Materials and methods
For immunohistochemical evaluation, the routinely processed paraffin blocks of formalin fixed specimens from 56 patients were retrospectively retrieved from the pathology archives. The criteria for inclusion in this study were: a histopathological diagnosis of OSCC; no preoperative therapy; potentially curative radical resection; postoperative radiation therapy (52–63 Gy in 25–30 fractions, 5 times per week); clinical follow-up data available; and satisfactory tissue preservation. All specimens were obtained from patients who gave written informed consent in studies reviewed and approved by the Institutional Review Board. The patients’ clinico-pathological parameters are summarized in Table 1 .
| Characteristics | n = 52 |
|---|---|
| Age at diagnosis | |
| Median (years) | 57.1 |
| Range (years) | 35.5–84.8 |
| Gender | |
| Male | 42 (80.8%) |
| Female | 10 (19.2%) |
| UICC-stage | |
| III | 21 (40.4%) |
| IV | 31 (59.6%) |
| Histological grade | |
| Well differentiated | 04 (7.7%) |
| Moderately differentiated | 43 (82.7%) |
| Poorly differentiated | 05 (9.6%) |
| 5-Year survival | |
| Alive | 34 (65.4%) |
| Dead | 18 (34.6%) |
| Recurrence | |
| Locoregional | 13 (25.0%) |
| No recurrence | 39 (75.0%) |
| Ki-67 LI | |
| Median | 20.53% |
| Range | 0.53–59.62% |
Slides of paraffin and formaldehyde fixed tissue of the resection specimens were cut into 4 μm thick sections and captured on poly- l -lysine-coated slides (DAKO Diagnostics, Hamburg, Germany). For immunohistochemical staining, sections were deparaffinized and rehydrated via serial passage through xylene and a graded series of ethanol. Microwave oven antigen retrieval was used to unmask the Ki-67 epitope. Slices were treated with 0.3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, followed by incubation with 10% normal goat serum, to block nonspecific binding. Sections were incubated with the primary mouse monoclonal MIB-1 antibody (Dianova, Hamburg, Germany) at a final dilution of 1:500 at 4 °C overnight in a humidified chamber. After several washing steps, sections were incubated with a biotinylated rabbit anti-mouse secondary antibody (DAKO Diagnostics, Hamburg, Germany) for 25 min at room temperature, followed by a avidin–biotin complex (DAKO Diagnostics, Hamburg, Germany) for 60 min at room temperature. Diaminobenzidine chromogen (DAKO Diagnostics, Hamburg, Germany) was used for visualization. Nuclei were counterstained with haematoxylin. Omission of the primary antibody served as a negative control.
Cells were counted in 5 fields of view for each slide at a magnification of x40 and the Ki-67 labelling index (LI) was determined as the percentage of Ki-67 positive cells among the total number of cells counted in each OSCC specimen.
Ki-67 expression comparisons were made by dividing the Ki-67 LI into two categorical groups, with the median being the cutoff point. A tumour was defined as high proliferative if Ki-67 LI was above the median and low proliferative if Ki-67 LI was below the median. Survival analyses for both groups were performed using the Kaplan–Meier method and statistical significance was calculated using the Breslow–Gehan–Wilcoxcon test.
Results
Positive staining for Ki-67 was easily and clearly identified by the brown nuclear staining in the tumour cells and could be detected in all 52 tumour samples. For the whole study population the Ki-67 LI were calculated, with a median of 20.53% (range 0.53–59.62%) and tumours were divided into two groups of high and low Ki-67 expression. Figure 1 A illustrates a low proliferative tumour (Ki-67 LI < 20.53%) and Fig. 1 B demonstrates a high-proliferative tumour (Ki-67 LI ≥ 20.53%). The relation between Ki-67 LI and clinicopathological parameters is summarized in Table 2 . The rate of high-proliferative tumours (Ki-67 LI ≥ 20.53%) in patients with tumours ≤4 cm in diameter (stages T1 and T2) was not statistically different from tumours >4 cm in diameter (stages T3 and T4) ( p = 0.406). High-proliferative tumours seemed to increase in cases without lymph node metastasis (70.1%), but there was no significant difference compared to those with lymph node metastasis (40.0%) ( p = 0.075).
| Variable | Ki-67 labelling index (LI) | p -Value a | |
|---|---|---|---|
| Ki-67 LI < 20.53% | Ki-67 LI ≥ 20.53% | ||
| T 1,2 ( n = 26) | 15 (57.7%) | 11 (42.3%) | 0.406 |
| T 3,4 ( n = 26) | 11 (42.3%) | 15 (57.7%) | |
| N 0 ( n = 17) | 5 (29.4%) | 12 (70.1%) | 0.075 |
| N + ( n = 35) | 21 (60.0%) | 14 (40.0%) | |
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