Japanese Journal of Clinical Oncology 31:500-505 (2001)
© 2001 Foundation for Promotion of Cancer Research
Expression of Pyrimidine Nucleoside Phosphorylase (PyNPase) in Colorectal Cancer
Digestive Surgery, Tokyo Medical and Dental University, Graduate School, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: We investigated the expression of PyNPase both in cancer cells and in stroma cells to clarify the correlation between PyNPase expression and the prognosis of patients with colorectal cancer.
Methods: Using immunohistochemical staining with an anti-PyNPase antibody, the PyNPase expression in tissues from 114 patients with stage II or III colorectal cancer was examined. From the correlation between PyNPase expression and the clinicopathological findings, the prognosis for survival was analyzed.
Results: The expression of PyNPase was classified as negative or positive on the basis of staining. No relationship between PyNPase expression and any of the clinicopathological findings was identified. However, a relationship was observed regarding positive staining between cancer cells and stroma cells. The prognosis of patients with positive staining in both cancer cells and stroma was worse than that of other patients. In multivariate analyses, expression in cancer cells was the strongest predictor of prognosis.
Conclusions: PyNPase expression appears to be a relevant factor for predicting the prognosis of colorectal cancer.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The prognosis of patients with colorectal cancer is predicted by its TNM (tumor, nodes and metastasis) stage (1). It is generally accepted that the prognosis of patients with stage I colorectal cancer is better than the prognosis of patients with stage II or III cancer after surgery. The National Institutes of Health consensus statement recommends no adjuvant therapy for stage I colorectal cancer (2). In the management of node-positive (stage III) colon cancer, several adjuvant treatments have been reported as effective, with a reduction of 30% in the risk of recurrence and a decrease of 5–12% in the mortality rate (3–9). The assertion that all patients with stage II or III colorectal cancers would live longer if they received systemic therapy after surgery is challenged because the treatment for node-negative (stage II) cancer remains controversial (9,10).
To improve the outcome for patients with colorectal cancers, the tentative identification of high-risk patients is important. Recent advances in tumor biology have uncovered many biological factors related to prognosis prediction (11–14). Angiogenesis is one of the biological factors for predicting high-risk tumors because carcinomas need neovascularization for growth and metastasis (15,16).
One indicator of angiogenesis is the enzyme pyrimidine nucleoside phosphorylase (PyNPase). Involved in pyrimidine metabolism, PyNPase catalyzes the reversible phosphorylation of thymidine to deoxyribose-1-phosphate and thymine (17–19). Reportedly, PyNPase is identical with platelet-derived endothelial cell growth factor (PD-ECGF) (19–21), which stimulates chemotaxis and thymidine incorporation by endothelial cells in vitro and has angiogenic activity in vivo (22,23). Increased PyNPase expression in human breast cancer cells was found to correlate with an increased number of microvessels in breast cancer tumors (24). Increased PyNPase activity was seen in tissues of various kinds of tumors compared with that in normal tissue (24–33). In colorectal cancers, stromal cells showed greater PD-ECGF staining intensity than cancer cells (34). Takebayashi et al. reported that PyNPase expression, assessed by immunohistochemistry, is associated with angiogenesis and the clinical outcome in cases of colorectal cancer (35).
In addition to its role in angiogenesis, PyNPase also has a role in nucleic acid metabolism. Although PyNPase is probably involved in the nucleic acid metabolism of cancer cells, no studies have analyzed PyNPase expression in cancer cells and stromal cells separately to determine whether the PyNPase expression in cancer cells has any correlation with that in stroma cells.
In this study, we analyzed PyNPase expression in colorectal cancer cells and in stroma cells separately and investigated the influence of PyNPase on the clinicopathological findings and prognosis of stage II and III colorectal cancers.
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MATERIALS AND METHODS |
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Patients and Tumors
Consecutively enrolled in this study were 70 men and 44 women with colorectal cancer who underwent surgical treatment between January 1986 and July 1996. Of these 114 patients, 53 were in stage II and 61 in stage III cancer, according to the UICC TNM classification (1). The patients’ ages ranged from 25 to 75 years, with an average of 59.8 years. No patient had received chemotherapy or radiation therapy before surgery. Following surgery, the patients all received adjuvant therapy with 5-FU 200 mg/day p.o. for 2 years.
The patients were followed from surgery until death; the median follow-up period was 6.8 years with a range from 3.1 to 13.2 years (Table 1). Letters were sent to surgeons and local tumor registers to obtain up-to-date information on patients’ survival or causes of death.
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Pathological Review
Histological sections from the 114 surgical specimens were reviewed without information on clinical outcome. The sections were routinely prepared in paraffin, then stained with hematoxylin and eosin. To compare the significance of PyNPase expression on prognosis with other known predictive parameters, the following morphological details were recorded: grade of tumor differentiation (grading), depth of invasion, lymph node involvement and venous and lymphatic infiltration.
The M category (the absence or presence of distant metastasis) was determined by clinical data including intraoperative findings, chest radiography, ultrasonography and computed tomography.
The histopathological staging of tumors was determined according to the TNM classifications (1).
Immunohistochemical Staining
From each tumor, the area of deepest invasion was selected for PyNPase immunohistochemical staining.
The paraffin sections (4 µm thick) were dewaxed in three changes of xylene, rinsed in graded ethanol and finally rehydrated in two changes of phosphate-buffered saline (PBS). Two microwave treatments, each for 5 min at 500 W in citrate buffer, pH 6.0, were performed to unmask the antigen and yield a more reproducible reaction. Endogenous peroxidase was blocked by incubating the sections with 0.3% hydrogen peroxide in absolute methanol for 20 min at room temperature (RT). After the sections had been washed three times with PBS for 5 min, the non-specific reaction was blocked by incubating the sections in a solution containing 1% normal mouse serum for 20 min at RT. After the sections had been rinsed three times with PBS, they were incubated for 90 min at RT with appropriate dilutions of the primary antibody, anti-human PyNPase mouse monoclonal antibody clone 654-1, established by Nishida et al. (36) and provided by Nippon Roche Research Cancer, Kamakura, Kanagawa, Japan. Small intestine mucosa specimens known to express PyNPase were used as positive controls and non-specific mouse IgG as primary antibody, at the same IgG concentration as the test antibody, was used as the negative control. The sections were incubated with the secondary antibody, anti-mouse IgG, for 45 min at RT. The sections were washed three times with PBS, then incubated with avidin–biotin peroxidase complex diluted with PBS for 30 min. After the sections had been washed three times with PBS for 15 min, the sections were incubated with 0.5 mg/ml diaminobenzine and 0.03% (v/v) H2O2 in PBS for 5 min and finally counterstained with hematoxylin prior to mounting.
Evaluation of Immunostaining
For microscopic analysis of sections stained with PyNPase antibody, we examined 200 cells to determine positivity for PyNPase. In the normal colonic mucosa, staining for PyNPase was found in the cytoplasm of the part of intestinal crypt cells (crypt of Lieberkürn) and weakly positive in the cytoplasm of the stromal cells. The average expression in the normal stromal cells was <5%. The cancer cells were considered to be PyNPase positive when >5% of them contained stain either in the cytoplasm or in the nucleus (Fig. 1). Similarly, the stromal cells were considered to be PyNPase positive when >5% of them contained stain either in the cytoplasm or in the nucleus (Fig. 2). The staining intensity was not evaluated.
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Expression of PyNPase was scored as positive or negative for all cell samples. The samples were then classified into four groups as follows: both cancer cells and stroma cells were positive (group I); cancer cells were positive and stroma cells were negative (group II); cancer cells were negative and stroma cells were positive (group III); and both cancer cells and stroma cells were negative (group IV).
These evaluations were done by three researchers/authors without any histopathological information. In cases in which the results were not unanimous, the majority opinion was taken.
Statistical Analyses
Student’s t-test was used to assess means and the chi-squared test to assess distributions. Survival curves were calculated using the Kaplan–Meier method and evaluated by the log-rank test. P values <0.05 were considered to be significant. The independent value of the variables in predicting survival was evaluated using the Cox proportional hazard regression model.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the normal colonic mucosa, staining for PyNPase was found in the cytoplasm of the crypt cells and weakly positive in the cytoplasm of the stromal cells. The average expression in the normal stroma cells was <5%.
In colorectal cancer tumors, staining for PyNPase was widely distributed in the cytoplasm and rarely seen in the nuclear compartments. The stroma cells in the cancer tissues showed various staining patterns and PyNPase was stained in the cytoplasm but rarely seen in the nuclear compartments.
Of the 114 tumor samples, 36 (31.6%) were PyNPase positive in the cancer cells: 16 (30.2%) were in stage II and 20 (32.8%) were in stage III. A positive relationship was observed between the PyNPase expression in the cancer cell and that in the stromal cell (P=0.0011) (Table 2). This relationship was stronger in stage III (P=0.0048) than in stage II (P=0.067).
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There was no significant difference between the PyNPase expression in the stromal cells and the following clinicopathological findings: patient age, tumor location, tumor size, histological grade, depth of invasion, cancer stage, lymph node metastasis, lymphatic invasion and venous invasion.
The overall survival curves of the 114 patients in this study are shown in Fig. 3. The 3-year over-all survival rate was 89.7% and the 5-year over-all survival rate was 79.6%.
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The 3-year survival rate was 78.6% for patients whose cancer cells were PyNPase positive and 94.6% for patients whose cancer cells were PyNPase negative. The 5-year survival rate dropped to 56.6% for patients whose cancer cells were PyNPase positive and 90.2% for patients whose cancer cells were PyNPase negative. The prognosis of patients whose cancer cells were PyNPase positive was significantly worse than that of patients whose cancer cells were PyNPase negative (log rank P = 0.0009) (Fig. 4).
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The 3-year survival rate was 83.9% for patients whose stroma cells were PyNPase positive and 93.6% for patients whose stroma cells were PyNPase negative. The 5-year survival rate dropped to 65.9% for patients whose stroma cells were PyNPase positive and 88.5% for patients whose stroma cells were PyNPase negative. The prognosis of patients whose stroma cells were PyNPase positive was significantly worse than that of patients whose stroma cells were PyNPase negative (log rank P = 0.0128) (Fig. 5).
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There was no significant difference between each of the four groups classified by staining patterns and the following clinicopathological findings: patient age, tumor location, tumor size, histological grade, depth of invasion, cancer stage, lymph node metastasis, lymphatic invasion and venous invasion.
Of the 114 tumors, 24 were classified in group I, 12 in group II, 26 in group III and 52 in group IV. The 3-year survival rates was 70.9% for patients with tumors in group I, 91.7% for patients with tumors in group II, 95.7% in group III and 94.1% in group IV. The 5-year survival rates was 44.4% for patients with tumors in group I, 78.6% for patients with tumors in group II, 87.7% in group III and 90.8% in group IV. There was no significant difference between group I and group II (P = 0.0617). However, there was a significant difference between group I and group III (P = 0.0018) (Fig. 6). The 5-year survival rate dropped to 46.7% for patients with tumors in group I and 87.6% for patients with tumors in other groups. The prognosis of patients with tumors in group I was significantly worse than that of patients with tumors in other groups (log rank P = 0.0003) (Fig. 7).
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The multivariate model is shown in Tables 3 and 4. Table 3 shows that the expression of PyNPase in cancer cell is confirmed as the factor with the strongest independent effect on survival. Table 4 shows that the expression of PyNPase in both cancer cells and stromal cells is confirmed as the factor with the strongest independent effect on survival.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, the prognosis of patients with stage II or III colorectal cancer with group I tumors, in which both the cancer cells and the stroma cells were positive for PyNPase expression, was significantly worse than the prognoses of the other patients.
The enzymatic activity of PyNPase is reported to be indispensable for angiogenic activity in carcinomas (37). It is well known that the growth and metastasis of malignant tumors depend on neovascularization (38,39). New vessels facilitate entry of tumor cells into the bloodstream and subsequent metastasis (40,41). Reportedly, PyNPase is identical with PD-ECGF (19–21), which has angiogenic activity in vivo (22,23). However, the exact role of PyNPase in the proliferation of cancer cells has not been entirely defined. The enzyme may have a role in nucleic acid metabolism in the cancer cells. Monden et al. (42) reported the expression of PyNPase in macrophages of the stroma. We found that the portion of colonic crypt in normal mucosa was positively stained for PyNPase. The crypt has the function of proliferation of colonic epithelium and it shows the most active cytokinesis in normal colorectal mucosa. It is conceivable that PyNPase is expressed in normal colorectal crypt as part of nucleic acid metabolism.
The correlation between PyNPase activities in angiogenesis and nucleic acid metabolism is unclear. However, in the present study, a positive relationship was observed between PyNPase expression in cancer cells and that in stroma cells.
To improve the outcome for patients with colorectal cancers, the tentative identification of high-risk patients is important. This study suggests that positive staining of PyNPase in both cancer cells and stroma cells may be an important predictor of high-risk patients. From a clinical perspective, high-risk patients identified by positive staining of PyNPase in both cancer cells and stroma cells may improve their prognosis with postoperative systemic chemotherapy.
More studies are needed to assess which patients are high-risk and should receive postoperative chemotherapy. The present study suggests that PyNPase expression is an important consideration for determining high-risk patients.
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FOOTNOTES |
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+ For reprints and all correspondence: Takayuki Osanai, Digestive Surgery, Tokyo Medical and Dental University, Graduate School, 1–5–45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: t-osanai.srg2@tmd.ac.jp
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Received March 21, 2001; accepted July 17, 2001.
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