Japanese Journal of Clinical Oncology Advance Access originally published online on September 7, 2005
Japanese Journal of Clinical Oncology 2005 35(9):545-550; doi:10.1093/jjco/hyi146
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© 2005 Foundation for Promotion of Cancer Research
Expression of Double-stranded RNA-activated Protein kinase (PKR) and its Prognostic Significance in Lymph Node Negative Rectal Cancer
1 Department of Internal Medicine, 2 Department of Surgery, 3 Department of Radiation Oncology, 4 Department of Pathology and 5 Department of Pharmacology, Dong-A University, College of Medicine, Busan, Korea
For reprints and all correspondence: Hyo-Jin Kim, Department of Internal Medicine, Dong-A University College of Medicine, 3-1 Dongdaeshin-dong, Seo-gu, Busan, 602-715, Korea. E-mail: kimhj{at}dau.ac.kr
Received March 10, 2005; accepted July 20, 2005
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Abstract |
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Objective: The interferon-induced, double-stranded RNA-activated, protein kinase (PKR) is a key regulator of translational initiation, and plays an important role in the regulation of cell proliferation, apoptosis and transformation. The aim of this study was to evaluate the prognostic significance of PKR in lymph node negative rectal cancer.
Methods: Forty-three patients with stage II rectal carcinoma who underwent potentially curative resection followed by post-operative adjuvant chemoradiation and 5-fluorouracil-based chemotherapy were investigated immunohistochemically using the monoclonal antibody TJ4C4. Overall scores for PKR expression were calculated based on staining intensity and immunoreactive tumor cell fraction. Clinical information, including tumor grade, carcinoembryonic antigen (CEA), disease-free survival (DFS) and overall survival (OS) was evaluated and compared with the degree of PKR expression.
Results: The median follow-up duration was 53.2 months, and median patient age was 55 years (range 33–73). No relationships were found between PKR score and age, sex, tumor grade or CEA level; however, smaller tumors (
5 cm) were associated with high PKR score (P = 0.025). When patients were subdivided into two groups based on the PKR score, the relapse rate was lower for those with a high PKR score (7.4 versus 43.8%, P = 0.008), and a significant difference was found between these two groups in terms of 5 year DFS (92.6 versus 55.6%, P = 0.0072) and 5 year OS (92.6 versus 57.7%, P = 0.0459). Other clinicopathologic variables were not related to clinical outcome.
Conclusion: PKR expression levels were associated with disease recurrence, DFS and OS in lymph node negative rectal cancer patients.
Key Words: PKR • rectal cancer • immunohistochemistry • prognosis
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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The incidence of colorectal cancer is higher in the West than in Korea, where it is the fourth most common cancer in both sexes. However, its incidence in Koreans has shown a steep recent increase, from 6.6% among all malignancies in 1990 to 10.6% in 2001 in men, and from 7.2% in 1990 to 10.5% in 2001 in women (1). Thus, colorectal cancer has emerged as one of the greatest public health problems in Korea.
Approximately, one quarter of colorectal cancers are located in the rectum. Surgical resection remains the best curative treatment option and adjuvant therapy is of benefit after potentially curative resection in patients with stage II and III rectal cancer (2). Adjuvant radiotherapy alone decreases local recurrences without survival benefit (3). Only combined chemotherapy and radiotherapy has consistently demonstrated efficacy in terms of the incidence of pelvic recurrence, disease-free survival (DFS) and overall survival (OS) (4). It remains a priority to identify potential biomarkers that could predict recurrence, DFS and OS. Moreover, the majority of prognostic factors give no insight into the molecular events responsible for tumor invasion and/or metastatic behavior (5,6).
Double-stranded (ds) RNA-activated protein kinase, PKR, is an interferon-inducible enzyme of widespread occurrence in mammalian cells. Accumulating data suggests that PKR has additional substrates, and that this kinase may also regulate gene transcription and signal transduction pathways (7). It is interesting that PKR may be an important regulator of tumorigenesis and that increased levels of PKR in head and neck cancer and colon cancer are associated with improved survival (8,9).
The aim of this study was to determine the prognostic relevance of PKR expression in lymph node negative adenocarcinoma of the rectum.
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PATIENTS AND METHODS |
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ELIGIBILITY CRITERIA
All patients in this study had histologically confirmed lymph node negative (stage II) adenocarcinoma of the rectum. They had all undergone a potentially curative resection with neither gross nor microscopic evidence of residual disease. Patients had to have an adequate performance status, and normal hepatic, renal and bone marrow functions had to be documented before study entry. Patients who received earlier pelvic irradiation and/or chemotherapy, and those with a history of another malignant disease over the past 5 years, distant metastasis or severe coexistent disease were excluded. Written informed consent was obtained from each patient before study entry. The use of all patient material was approved by our institutional review board.
TREATMENT SCHEDULE
Post-operative chemoradiation followed by systemic therapy based on a 5-fluorouracil (5-FU) regimen was the adjuvant therapy protocol. Three weeks after surgery, patients received pelvic irradiation, which was delivered at 1.8 Gy per fraction for five times weekly. After 45 Gy had been administered to the entire pelvis, an additional boost field was applied to the primary tumor area, totaling 54 Gy. Concomitantly intravenous 5-FU (375 mg/m2/day) was administered for 3 days on the first and last weeks of the radiotherapy. After chemoradiation, patients received either the Mayo clinic regimen (5-FU 425 mg/m2/day and leucovorin 20 mg/m2/day intravenous bolus infusion for 5 days every 4 weeks) for six cycles or the oral doxifluridine (600 mg/m2/day) for 12 months.
PATIENT FOLLOW-UP
During adjuvant therapy, patients were monitored for signs of toxicity and appropriate adjustments were made to their chemotherapy and radiotherapies. Complete blood counts were determined weekly to detect myelosuppression. Patients were also evaluated by history taking, physical examination, complete blood counts and by blood chemistry studies before each chemotherapy cycle. After adjuvant treatment had been completed, we regularly evaluated patients for possible disease recurrence. History taking and physical examination, complete blood counts, blood chemistry studies, carcinoembryonic antigen (CEA) and chest radiography were repeated every 3 months for the first 24 months after surgery, and then every 6 months until 5 years after surgery. Computed tomography of the abdomen and pelvis were performed every 6 months for the first 2 years after surgery and then every 12 months. Colonofiberoscopy of the colon was performed every 12 months.
IMMUNOHISTOCHEMICAL ANALYSIS FOR PKR
This study was performed on formalin-fixed, paraffin-embedded, 4 µm thick tissue sections, using the avidin–biotin–peroxidase complex method. Mouse monoclonal antibody was used as the primary antibody directed against PKR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:50 dilution. Deparaffinization of all sections was performed through a series of xylene baths, with rehydration through graded alcohol solutions. To enhance immunoreactivity, microwave antigen retrieval was performed at 750 W for 30 min in citrate buffer (pH 6). After blocking endogenous peroxidase activity with 5% hydrogen peroxidase for 10 min, primary antibody incubation was performed for 30 min at room temperature. An EnvisionTMChemTM Detection Kit (Dako, Corporation, CA, USA) was used as the secondary antibody at room temperature for 30 min. After washing in Tris-buffered saline for 10 min, 3,3'-diaminobenzidine was applied as a chromogen, and this was followed by Mayer's hematoxylin counterstaining.
INTERPRETATION OF IMMUNOHISTOCHEMICAL STAINING
The evaluation of PKR expression was performed on a semiquantitative basis by assessing staining intensities and distributions. Staining intensity was scored as 3 (strong), 2 (moderate) or 1 (weak). Distribution was scored as 4 (diffuse, 
75%), 3 (regional, 50–74%), 2 (focal to regional, 25–49%) and 1 (focal, 1–24% tumor staining). For further analysis, we used the PKR score (defined as the product of the distribution and staining intensity scores). Tumors with PKR score 6 (i.e. strong staining with any distribution except focal, moderate staining with diffuse or regional distribution) were considered as having a high PKR score, whereas tumors with PKR score <6 (i.e. strong with focal, moderate with focal to regional or focal, weak staining with any distribution) were considered as low PKR score. The intensity of immunostaining was evaluated by a single pathologist (Mee Sook Roh).
STATISTICAL ANALYSIS
DFS was defined as the length of time from surgery to initial disease recurrence. OS was defined as the length of time from surgery to death. The Kaplan–Meier method was used to construct curves for DFS and OS. Data on patients who died without evidence of disease recurrence were censored at the time of death in DFS calculations. The log-rank test was used to compare distributions. To determine independent factors that were significantly related to the prognosis for patients with rectal cancer, multivariate analysis was performed using Cox's proportional hazards model with a stepwise procedure. To compare categorical variables, the Fisher's exact test was applied. All tests were two-sided and P < 0.05 was considered statistically significant. Analyses were done using SPSS version 10.0 (SPSS Inc., Chicago, IL, USA).
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RESULTS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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PATIENT CHARACTERISTICS
Forty-eight patients who registered between January 1995 and January 2001 were enrolled in this study. Five patients (10.4%) were ineligible for the following reasons; four patients refused their assigned treatment and one died due to a post-operative complication. Thus, the remaining 43 patients were retrospectively selected and were included in analyses. Patients' characteristics are presented in Table 1. Mean patient age was 55 ± 12.3 years (range 33–73).
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CORRELATION OF PKR SCORE AND CLINICOPATHOLOGIC PARAMETER
Immunohistochemical staining of PKR is shown in Fig. 1. PKR staining profiles of tissues were uneven, and showed strong and weak staining regions. Mean PKR score was 6.7 ± 3.5. A breakdown of clinical and pathologic parameters by PKR score is presented in Table 2. PKR score was neither related to age, sex, CEA level, histologic grade nor vascular invasion. However, smaller tumors (
5 cm) were associated with high PKR score (P = 0.025).
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DISEASE RECURRENCE AND SURVIVAL
The median follow-up duration was 53.2 months. Post-operative relapses occurred in nine patients (20.9%); five showed local recurrence and four distant metastases (three liver, one lung). The relapse rate was lower in the high PKR score group than in the low PKR score group (7.4 versus 43.8%, P = 0.008). After relapse, six patients were treated with a combination of oxaliplatin with 5-FU and leucovorin, and two patients were treated with capecitabline. Five year DFS and OS were 73.9 and 78.6%, respectively. One patient died due to advanced liver cirrhosis without relapse. Prognostic variables such as sex, age, preoperative CEA level, tumor size, histologic grade, number of lymph nodes, PKR score, type of surgical resection and chemotherapy were subjected to univariate analysis (Table 3). Five year DFS was significantly greater in the high PKR score group than in the low group (92.6 versus 55.6%, P = 0.0072) (Fig. 2), as was 5 year OS (92.6 versus 57.7%, P = 0.0459) (Fig. 3). Other variables were not related to clinical outcome. To verify the independent prognosis value of PKR expression, a multivariate Cox model was used to control for other prognosis factors. The relative risks for OS based on PKR expression was 1.42 (95% confidence interval = 1.19–1.91; P = 0.047).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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In the current pooled analysis, patients with T3N0 rectal cancer had a 5 year OS of 74–80% when treated with trimodal combinations of surgery and chemoradiation. However, 5 year DFS for T3N0 lesions was found to range from 63 to 75%, and local recurrence from 5 to 10% (10). The DFS and relapse data suggested that further improvements in outcome should be feasible, possibly by adding new agents or by evaluations of targeted therapy based on molecular markers.
The most important factor predicting outcome in rectal cancer is the pathologic evaluation, the results of a careful analysis of the circumferential margin can be used to predict patients at high risk of local failure (11). A thorough assessment of lymph nodes is also important. The Gastrointestinal Intergroup have previously reported on the importance of node number in specimens as a predictor of outcome. For patients classified as N0, outcome was far worse than for patients with <14 nodes in a specimen as determined by a pathologist (12). In the present study, the median number of lymph nodes examined was 13.0 ± 7.4. However, no association was found between the number of lymph nodes and the survival outcome, perhaps due to the relatively small number of patients.
Compared to colon cancer, molecular markers are rarely reported in rectal cancer. However, it was recently reported that the dynamic induction of p21WAF1/CIP1 is associated with lower proliferative activity, but with an ultimately worse treatment outcome following neoadjuvant radiochemotherapy and tumor resection (13).
The interferon-induced ds RNA-activated protein kinase, PKR, has been shown to play an important role in antiviral response and in the regulation of translation, transcription, cell growth and apoptosis (7). PKR inhibits translation initiation by phosphorylating the alpha subunit of the initiation factor eIF-2, eIF-2
, and also controls the activation of several transcription factors like NF-kappa B, p53 or STATs. Moreover, PKR is a target of caspase-3, -7 and -8, which cleave PKR and thus activate PKR without the need for earlier exposure to ds RNA (14).
PKR is also implicated in the regulation of cell proliferation in uninfected cells, and may also have a tumor suppressor function under normal physiologic conditions. Studies of human malignancies suggest that, in general, patients bearing tumors with a higher PKR content have a more favorable prognosis (8,9,15). High levels of PKR expression were found to be associated with a lower incidence of recurrent or residual disease and longer disease-free and total survival times in squamous cell carcinoma of the head and neck (8). One report showed a positive relationship between the PKR expression and the level of differentiation in colon cancer, and also found that patients with tumors expressing high levels of PKR had a 56% 5 year survival rate, compared to a 46% 5 year survival rate for patients with lower levels of PKR expression (9). These results concur with our data. Another important report demonstrated that PKR expression has prognostic significance during interferon therapy in patients with a carcinoid tumor, and showed that the PKR score was significantly elevated after treatment in patients with stable disease, but not in those with progressive disease. Moreover, a low PKR score during treatment was found as a predictor of a shorter response duration and poorer OS (15).
There was a report that was inconsistent with previous studies where PKR deficient or knock-out transgenic mice were found normal and showed no signs of neoplasia (16). The role of PKR in cell growth regulation is controversial, some studies support its tumor suppressor function while others suggest a growth-promoting role in breast cancer cell lines (17). Thus, it is possible that the functionality of PKR depends on cancer type.
The present study demonstrates a positive relationship between the expression of PKR and the survival in lymph node negative rectal cancer. A higher PKR score favored DFS and OS. Those patients with high PKR score expressing tumors had a 92.6% 5 year DFS and 92.6% 5 year OS, compared to a 55.6% DFS and 57.7% OS for those expressing low level (P = 0.0072, 0.0459, respectively). Moreover, these differences could not be explained by patient age, CEA level or treatment regimen, and could only be partly explained by tumor grade. We also evaluated PKR expression in stage III rectal cancer and found no significant correlation between the PKR score and the clinical outcome (data not shown). Moreover, PKR staining profiles in rectal cancer were heterogeneous; tissues showed strongly and weakly stained regions, but we are unable to propose a reasonable explanation for this patchy distribution. Therefore, in this study, PKR expression was scored on a subjective basis from 1 to 12, by combining staining intensity and the percentage of immunoreactive tumor cells present. It should be noted that this study does not include multivariate analysis results. Such data would be interesting in terms of intervariable interactions and interactions between the variables and the clinicopathological data. However, the number of patients enrolled in this study did not allow us to perform such an analysis, and thus these issues remain open for clarification by prospective study. In conclusion, we were able to find a correlation between the clinical outcome and the extent of PKR staining. By using a threshold PKR score level of 6 for immunostaining, we identified a relation between high PKR expression and DFS and OS. Therefore, we suggest that immunohistochemical methods of the type used in the present study may be applied to clinical specimens in a practical clinical manner to predict the therapeutic outcome in lymph node negative rectal cancer.
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Acknowledgments |
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This work was supported by the Dong-A University Research Fund in 2005.
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References |
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