Abstract
Aim: To evaluate the usefulness of Ki-67 index and survivin as predictive prognostic factors for rectal cancer treated with preoperative chemoradiotherapy. Patients and Methods: The Ki-67 index and survivin expression were examined in patients with stage II/III rectal cancer (n=46) by immunohistochemistry. Patients were divided into a high-group and a low-group for the Ki-67 index, and positive and negative groups for survivin expression. Overall and disease-free survival were compared between the groups, and the correlation between Ki-67 index and survivin expression was assessed. Results: The 5-year disease-free survival rate of the group with high Ki-67 index was significantly lower than that of the group with low Ki-67 index (53% and 88%, p=0.03), as was the 5-year overall survival rate (68% and 100%, p=0.03). Findings for survivin were not significant. Conclusion: Ki-67 index and survivin may be useful biomarkers for rectal cancer with preoperative CRT.
Chemoradiotherapy (CRT) is being increasingly used in neoadjuvant therapy for rectal cancer. Preoperative CRT can preserve the anal sphincter, reduce the risk of local recurrence, and circumvent the need for invasive treatments, such as lateral lymph node dissection. Our group has used a novel protocol for neoadjuvant CRT combining S-1, which is a novel oral fluoropyrimidine, tegafur, 5-chloro-2,4-dihydroxypyridine (gimeracil; CDHP), and potassium oxonate (oteracil potassium). Tegafur is a prodrug for 5-FU (fluorouracil) that acts as an effector. CDHP reversibly inhibits the degradation of 5-FU by dihydropyrimidine dehydrogenase, resulting in prolonged high concentrations of 5-FU in the blood. S-1 has radiosensitizing effects and enhances the effects of radiation. However, recent data have shown that preoperative CRT may result in a broad variety of tumor responses in rectal cancer. The success of radiation therapy is dependent on its ability to control tumor proliferation and induce cell death by causing irreparable cellular damage that ultimately triggers apoptosis. For patients with rectal cancer treated by neoadjuvant CRT, some reports have shown that a high number of apoptotic tumor cells in pretreatment biopsy specimens is a significant predictor for tumor response and long-term local control. The mechanisms behind the responses to CRT are not yet fully understood, although the ability of CRT to eradicate tumor cells appears to depend upon cell-cycle arrest, apoptosis, and DNA repair (1).
There are various markers that can be used to show the proliferative activity of cells. One of these markers is Ki-67, and the Ki-67 index is the most commonly used indicator for measuring proliferation. Ki-67 is a nuclear non-histone protein that has been used to identify high-grade tumors in several cancer types. It is stably expressed in the G1, S, G2, and M phases, yet is absent in quiescent and resting cells (G0). As such, Ki-67 expression has been reported to correlate with tumor cell proliferation rate (2). To date, many studies that have investigated the role of Ki-67 as a prognostic marker in various cancer types have shown that Ki-67 positivity is an indicator of a higher risk of recurrence and a worse survival (1-4). To the best of our knowledge, there have been few reports of a relationship between Ki-67 and the prognosis of rectal cancer treated with CRT.
Survivin is a structurally unique member of the inhibitor of apoptosis protein family that regulates both cell-cycle progression and cell survival. It acts as a suppressor of apoptosis by inhibiting the activation of caspase-3/7, and it regulates the cell cycle at the G2/M phase. It is expressed in various human cancer types, such as lung, breast, prostate, ventricle, pancreas, and colon cancer, but it is not expressed in most normal adult human tissues; as such, it has attracted a great deal of interest as a potential drug target (5).
Although there are many markers that can be used to predict the response to preoperative CRT, few have been reported to be useful as predictive markers of prognosis. The aim of this study was to investigate the usefulness of the Ki-67 index and the expression of survivin as predictive prognostic factors for rectal cancer treated with preoperative CRT.
Patients and Methods
Patients. CRT is routinely offered to patients with locally advanced (T3 or node-positive) cancer at the time of diagnosis or to those who have very distal T2N0 cancer close to or involving the sphincter. All patients received CRT with a total dose of 4,000 cGy of pelvic irradiation, which was administered five times weekly, with a daily fraction of 200 cGy utilizing a four-field technique. The top of the radiation field consisted of the bifurcation point of the aorta, and the bottom of the field was marked at least 4 cm below the tumor. The radiation field included the lateral nodes. Radiation was delivered concomitantly with S-1 (80-120 mg/day) on the same days. Surgical treatment was performed 6 to 8 weeks after the completion of preoperative CRT. Repeat examination was performed for post-neoadjuvant CRT evaluation. All patients completed the neoadjuvant treatment and underwent curative radical resection no earlier than 6 weeks after the completion of CRT.
Patients with stage II/III disease (n=46) were examined in this study. All of the patients underwent surgical treatment at Tokushima University Hospital between 2003 and 2012, and all of them provided informed consent for participation in this study. The staging was principally based on the Japanese Classification of Colorectal Cancer.
Immunohistochemical staining for Ki-67. The resected tissue specimens were fixed in 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections that were deparaffinized with xylene, dehydrated with ethanol, and then treated with 0.03% hydrogen peroxide in methanol for 10 min. For antigen retrieval, the sections were heated in 10 mM citrate buffer (pH 6) in a microwave. After cooling to room temperature, the sections were washed three times for 5 min each in phosphate-buffered saline, and then they were incubated with 1% goat serum to block nonspecific reactions. Next, the sections were incubated with primary antibodies against Ki-67 [anti-human Ki-67 antigen clone MIB-1 (M7240) diluted 1:100; Dako, Tokyo, Japan] for 60 min at room temperature. After washing three times for 5 min each in phosphate-buffered saline, the sections were subjected to a Dako REAL EnVision/HRP detection system (Dako) for 60 min at room temperature. The sections were then washed three times for 5 min each in phosphate-buffered saline, the peroxidase reaction was developed with 3,3’-diaminobenzidine (Sigma-Aldrich, St Louis, MO, USA), and the sections were counterstained with 10% Mayer's hematoxylin. The sections were then dehydrated, treated with xylene, and coverslipped. A cut-off value of 30% was used.
Immunohistochemical staining for survivin. Slides were prepared as described above with the addition of 0.01% saponin in the permeabilization step. A 1:50 dilution of the primary antibody [polyclonal rabbit anti-survivin antibody (ab469); Abcam Cambridge, UK] was applied overnight in phosphate-buffered saline containing 5% bovine serum albumin. After two washes with a buffer, a 1:200 dilution of the secondary antibody (AlexaFluor 488 anti-rabbit antibody; Invitrogen, Karlsruhe, Germany) was added in a buffer with 5% albumin and the nuclei were stained with DAPI (0.2 mg/ml). Slides were covered with 25% glycerol in the buffer. The mean percentage of positively stained tumor cells was determined using an Image System (Nikon Digital Camera, DXM1200F; Nikon Tokyo, Japan) and assigned to one of the following categories: 0 (≤5%), 1 (5% to ≤25%), 2 (25% to ≤50%), 3 (50% to ≤75%), or 4 (>75%). The intensity of survivin immunostaining was scored as: 1 (weak), 2 (moderate), or 3 (intense). The score for the percentage of positively stained tumor cells and the score for the staining intensity for cytoplasm and nucleus were then added to obtain the total score for each case, ranging from 0 to 10. The immunostaining results were evaluated by two independent pathologists in a blinded fashion without any knowledge of the clinicopathological or biological information of the cases. Because of the limited number of patients in this study and to facilitate further statistical analyses, the weighted survivin scores were arbitrarily dichotomized: negative-survivin expression was classified as a survivin score of 4 or below, and positive-survivin expression was classified as a score of 5 or above.
Statistical analyses. Fisher's exact test and the Wilcoxon test were performed as appropriate. Continuous variables are expressed as the means±standard deviation (SD). JMP 8 software (SAS Institute, Cary, NC, USA) was used for all statistical analyses. Survival endpoints measured from the operation for cancer. Continuous variables were compared by Mann–Whitney U-tests, and categorical data were compared by chi-square tests. Survival curves were calculated by the Kaplan–Meier method and compared by log-rank tests. The prognostic potentials of the parameters were analyzed by univariate analysis. Relative risk and 95% confidence intervals were estimated with the Cox proportional hazards model with stepwise forward selection. Statistical significance was defined as p<0.05.
Results
Immunohistochemistry. Examples of the immuno histochemical analysis of Ki-67 and survivin expression are shown in Figure 1.
Immunohistochemical analysis of Ki-67 expression. There were 17 patients in the group with low Ki-67 index and 29 patients in the group with high Ki-67 index. There was no significant difference between the two groups in clinicopathological factors (Table I).
Survival curves according to Ki-67 expression. Only high Ki-67 expression was found to be a prognostic factor for overall survival. The group with high Ki-67 index had a significantly poorer prognosis than the group with low Ki-67 index; the 5-year survival rate of the group with low Ki-67 index was 100%, while that of the group with high Ki-67 index was 68% (p=0.03) (Figure 2A; Table II). For disease-free survival, univariate analysis revealed that the tumor size and Ki-67 expression were prognostic factors, while multivariate analysis revealed that Ki-67 expression was an independent prognostic factor (hazard ratio=2.46, 95% confidence interval=1.08-5.87, p=0.03). The group with high Ki-67 index had a significantly poorer prognosis than that with low Ki-67 index; the 5-year disease-free survival rate of the group with low Ki-67 index was 88%, while that of the group with high Ki-67 index was 53% (p=0.03) (Figure 2B; Table III).
Examples of samples with positive immunohistochemistry results. A: Ki-67. B: Survivin (score 7).
Correlation between Ki-67 and survivin. There were 23 patients with survivin-positive disease and 23 with survivin-negative. There was no significant difference in disease-free or overall survival between the two groups. A significant correlation was found between high Ki-67 expression and survivin: 78% of the patients with survivin-positive disease had high Ki-67 expression, while only 47% of those with survivin-negative disease had high Ki-67 expression (p=0.03) (Figure 3).
Discussion
The results showed that a high Ki-67 index may be used to predict a poor prognosis for patients with rectal cancer undergoing CRT therapy, and that the expression of Ki-67 is correlated with survivin.
Preoperative CRT was introduced to improve the prognosis of surgery. The use of preoperative CRT for locally advanced rectal cancer has been strongly supported by the results of the German Rectal Cancer Trial, which demonstrated that preoperative CRT reduced the occurrence of local recurrence, lowered the rates of short-term and chronic toxicities, and improved the rate of sphincter preservation (3). Additionally, preoperative CRT for advanced low rectal cancer has been shown to increase the probability of tumor resectability, reduce the risk of local recurrence, and improve both disease-free and overall survival. Ki-67 antigen is expressed during all of the active phases of the cell cycle (G1, S, G2, and mitosis) and has become a widely used marker for determining the proliferative potential of tumors. Rapidly proliferating cells typically respond more dramatically to radiation-induced cell damage (4). Ki-67 has been used to assess cell proliferation and activity, and a correlation between Ki-67 and chemosensitivity has been reported (5-8). However, to the best of our knowledge, this is the first report on the expression levels of Ki-67 in rectal cancer treated with CRT; we found that a high expression of Ki-67 is correlated with a poorer prognosis in patients with rectal cancer treated with CRT.
Clinicopathological factors of patients included in this study by Ki-67 expression.
In addition, we examined the expression of survivin as it regulates both cell-cycle progression and cell survival. We found a positive correlation between Ki-67 and survivin. This significant direct correlation between Ki-67 and survivin suggests that the latter may have a pro-mitotic function. As a member of the inhibitor of apoptosis protein family, survivin may inhibit apoptosis in two ways: direct inhibition of caspase-3/7 activity, and negative regulation of cell apoptosis induced by various stimulation processes. Survivin with cyclin-dependent kinase CDK4/2 can block the apoptosis signaling transduction pathway. It can act directly on caspases by inhibiting caspase-3/7 activity, and also indirectly by inhibiting caspases through P21 interactions (9-11). Studies have shown that survivin along with cell-cycle regulatory factors can induce CDK/cyclin-E activation and ribosomal phosphorylation through CDK4 interactions. Ribosomal phosphorylation rapidly induces cells to enter the cell cycle and accelerates the progression of the G1/S phase, causing P21 release from the surviving–CDK4 complex combined with mitochondrial pro-caspase-3 to inhibit caspase-3 activity and prevent mitochondrial release of cytochrome c, thereby inhibiting apoptosis (12). Studies have also shown that survivin can inhibit the BCL2-associated X, apoptosis regulator (BAX)- and FAS-mediated apoptosis pathways. In addition, Ki-67 and survivin form interaction networks, and survivin and p53 are linked through cell-cycle regulatory signaling pathways (12, 13).
Survival curves. A: Overall survival (OS). B: Disease-free survival (DFS).
Overall survival (OS) of patients included in this study.
Disease-free survival (DFS) of patients included in this study.
There are currently many markers that predict the response to preoperative CRT, e.g., p21, survivin, Ki-67, BAX, and thymidylate synthase (13). However, there has been a few reports of predictive marker of prognosis. In this study, although survivin was not found to be a predictive factor of prognosis, Ki-67 was; this represents a novel finding. Predictive factors that can be assessed in surgically resected specimens are expected to be very useful in identifying individuals with a poor prognosis. Although a clear cut-off value for Ki-67 in rectal cancer treated with CRT has not yet been established, this study successfully distinguished the patients with a better prognosis from those with a worse prognosis after CRT using a cut-off value of 30%. A large prospective study is required to re-evaluate the validity of Ki-67 as a prognostic factor and its cut-off value.
Correlation between Ki-67 and survivin.
Limitations of this study include its retrospective design and the fact that the specimens were obtained after CRT. CRT may influence the cell cycle of rectal cancer cells, so a pre-CRT specimen should also be examined, but the amount of the tissue was limited in this study because the biopsy material obtained was small.
Conclusion
A high Ki-67 index and the expression of survivin may be useful prognostic biomarkers for rectal cancer treated with preoperative CRT.
Footnotes
Conflicts of Interest
Drs. Kozo Yoshikawa, Mitsuo Shimada, Jun Higashijima, Toshihiro Nakao, Masaaki Nishi, Chie Takasu, Syohei Eto, Hideya Kashihara have no conflicts of interest or financial ties to disclose.
- Received December 12, 2017.
- Revision received January 19, 2018.
- Accepted January 24, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
