Japanese Journal of Clinical Oncology 31:65-68 (2001)
© 2001 Foundation for Promotion of Cancer Research
Clinical Relevance of the Concentrations of Both Pyrimidine Nucleoside Phosphorylase (PyNPase) and Dihydropyrimidine Dehydrogenase (DPD) in Colorectal Cancer
First Department of Surgery, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
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ABSTRACT |
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Background: Pyrimidine nucleoside phosphorylase (PyNPase) converts 5'-deoxy-5-fluorouridine (5'-DFUR) to 5'-fluorouracil (5-FU), which exerts an anti-cancer effect before being catabolized by dihydropyrimidine dehydrogenase (DPD). We examined the possible correlation of the tissue concentrations of both PyNPase and DPD with the clinicopathological features of colorectal cancer.
Methods: In 36 cases of colorectal cancer, the concentrations of both PyNPase and DPD in fresh-frozen samples from either tumor or normal tissue were quantified using ELISA.
Results: The concentration of PyNPase was found to be significantly higher in the tumor than in the normal tissue (p = 0.001), whereas DPD showed no difference. The tumor/normal tissue ratio of PyNPase was higher in advanced stage cases, and also in the presence of liver metastasis, lymph node metastasis and vessel invasion (each p < 0.05). On the other hand, the tumor/normal tissue ratio of DPD was also higher in advanced stage cases and also in the presence of vessel invasion (each p < 0.05), thus indicating a poor response to 5-FU. The PyNPase/DPD ratio, which is known to be correlated with the tissue concentration of 5'-DFUR, was higher in the tumor than in the normal tissue (p = 0.001).
Conclusions: The tumor/normal tissue ratios of both PyNPase and DPD might be useful candidates for predicting the prognosis of colorectal cancer. The PyNPase/DPD ratio was higher in the tumor tissue than in the normal tissue; however, further investigations are needed to clarify the effectiveness of fluoropyrimidine therapy.
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INTRODUCTION |
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The effect of 5'-deoxy-5-fluorouridine (5'-DFUR, doxifluridine) is thought to be regulated by the balance of pyrimidine nucleoside phosphorylase (PyNPase), a converting enzyme of 5'-DFUR to 5'-fluorouracil (5-FU) (1–3) and dihydropyrimidine dehydrogenase (DPD), the major catabolic enzyme of 5-FU (4–6). In addition, a deviation in the 5-FU distribution in the cancer tissue is defined by the ratio of these enzyme activities between the cancerous tissue and normal tissue (7,8).
There is increasing evidence that PyNPase also participates in the growth and metastasis of cancer as an angiogenesis factor, platelet-derived endothelial cell growth factor (PD-ECGF) (9,10). In order potentially to predict the prognosis by 5'-DFUR sensitivity, we investigated the existence of a possible correlation between the tissue concentrations of both PyNPase and DPD, and the clinicopathological features in patients with colorectal cancer. The expression levels of both PyNPase and DPD were determined by ELISA, which has been reported to correlate well with the findings determined by a conventional enzyme activity assay (6,11).
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MATERIALS AND METHODS |
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Patients
Among 64 patients with colorectal cancer who underwent resections at our hospital during the 2-year period from February 1998 to January 2000, 36 patients were enrolled in this study. The 28 excluded cases were as follows. In 21 cases, the samples were not properly stored in liquid nitrogen within 30 min after the tumor was resected. In four cases, a sufficient volume of the specimen could not be obtained. In three cases, the clinicopathological features could not be evaluated because of a lack of detailed patient records. The clinicopathological features were evaluated according to the criteria of the Japanese Research Society for Cancer of the Colon and Rectum (12,13). The numbers of enrolled patients at each histological stage were as follows: stage 0, 2; stage I, 2; stage II, 11; stage IIIa, 4; stage IIIb, 5; and stage IV, 12.
Expression of PyNPase and DPD
After the colon had been resected, a specimen measuring >1 cm3 including both the mucosal and submucosal layers was obtained from either the tumor tissue or the normal tissue which was at least 10 cm away from the edge of the tumor, immediately frozen in liquid nitrogen and then stored at –80°C until analysis. The concentrations of both PyNPase and DPD in either tumor tissue or normal tissue were quantified with sandwich ELISA as described by Nishida et al. (11) and Mori et al. (6), in collaboration with the Nippon Roche Research Center (Kanagawa, Japan). In brief, the microplates were coated with 10 µg/ml of either anti-human dThdPase monoclonal antibody (MoAb) 104B or anti-DPD MoAb 4B9. Each sample was homogenized in a 10-fold volume of buffer, centrifuged at 10 000 g for 15 min and the supernatant was dispensed on to the plate and incubated at 37°C for 1.5 h. As the standard solution for dThdPase and DPD, serially diluted HCT116 tumor homogenate and HT-3 tumor homogenate were used, respectively. After washing, the plates were incubated at 37°C for 1 h with 1 µg/ml of second antibodies, either anti-dThdPase MoAb 232-2 or anti-DPD MoAb 3A5. The plates were further reacted with peroxidase-conjugated anti-mouse IgG antiserum and substrate solution. Each concentration obtained from ELISA was adjusted based on the whole protein concentration and was expressed as units/mg protein.
Statistics
The patients were divided into two groups based on the histological staging, between stage IIIa and stage IIIb, according to the presence of metastasis either in the lymph node further than N2, in the liver, in the peritoneum or in the other organs. The patients were also divided into two groups based on the presence of each histopathological feature, including liver metastasis, lymph node metastasis, vessel invasion and lymph vessel invasion. Each enzyme concentration between the tumor and normal tissue was compared using Wilcoxon’s rank test. The relevance between the enzyme concentration and each clinicopathological feature was analyzed by the Mann–Whitney rank test. A p value of <0.05 was considered to be significant.
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RESULTS |
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The profile of the PyNPase expressions in both the tumor tissue and the normal tissue is shown in Table 1, in relation to (a) the histological staging, (b) histology and (c) the macroscopic morphology. Statistical significance was not determined in this table because the number of subjects was insufficient to compare the multiple groups, even though both the poorly differentiated adenocarcinoma (Table 1b) and type 2 cancer (Table 1c) showed a relatively high PyNPase expression. A comparison of each enzyme concentration between the tumor and the normal tissue is shown in Table 2. The relevance between the enzyme concentration and each clinicopathological feature is shown in Table 3. The concentration of PyNPase was significantly higher in the tumor than in the normal tissue (p = 0.001) (Table 2). Although the concentration of PyNPase in either the tumor or normal tissue demonstrated no statistical correlations with the clinicopathological features, the ratio of tumor to normal tissue (T/N ratio) of the PyNPase concentration was higher in advanced histological stage cases (<stage IIIa, 2.3; >stage IIIb, 3.7; p = 0.01) and in each positive case of liver metastasis, lymph node metastasis and vessel invasion (liver metastasis, positive 3.4, negative 2.5, p = 0.04; lymph node metastasis, positive 3.5, negative 2.4, p = 0.02; vessel invasion, positive 3.6, negative 2.5, p = 0.03) (Table 3).
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On the other hand, no differences were observed regarding the DPD concentration between the tumor and normal tissue specimens (Table 2). The concentration of DPD also showed no correlation with the clinicopathological features; however, the T/N ratio of DPD was higher in cases of advanced histological stages (<stage IIIa, 1.0; >stage IIIb, 1.4; p < 0.05) and in positive cases for vessel invasion (positive 1.5, negative 0.9, p = 0.03) (Table 3). The ratio of PyNPase to DPD was statistically higher in the tumor specimens than in the normal tissue specimens (p = 0.001) (Table 2), whereas no statistical difference was observed between the PyNPase/DPD ratio in the tumor and the clinicopathological features (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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As reported previously (1–3,14), the PyNPase concentration was higher in the tumor specimens than in the normal tissue specimens. In addition, the T/N ratio of PyNPase increased in proportion to the progression of the histological staging and the presence of liver metastasis, lymph node metastasis and vessel invasion, although the PyNPase concentration either in the tumor or normal tissue showed no statistical correlation with the clinicopathological features. We calculated the T/N ratio, not only because it is considered to eliminate the differences of the basal levels between each individual, but also because it corrects errors due to the conditions of both sampling and preservation. Our findings thus suggested that T/N ratio of PyNPase is a potentially useful candidate for predicting prognosis.
In this study, we sampled ‘tumor tissue’ specimens from either the mucosal or submucosal layer, which might also include infiltrating cells around the cancer cells, thus resulting in a higher PyNPase concentration in the ‘tumor’, since these infiltrating cells are reported to be the main source of PyNPase (9). PyNPase has also been labeled PD-ECGF and has been shown to be involved in the angiogenesis of tumors (9,10) and is regulated by the inflammatory cytokines (15). These observations indicate that an upregulation of PyNPase may be related to any inflammation combined with the tumor progression and thus resulting in an increase in the T/N ratio of PyNPase. We could not clarify the direct relationship between the PyNPase expression and inflammation, although both poorly differentiated adenocarcinoma and type 2 cancer showed a relatively high PyNPase expression. It is therefore considered important to identify what type of cancer induces inflammatory changes in further investigations.
On the other hand, the effectiveness of 5'-DFUR is not defined only by PyNPase. It is also significantly affected by the DPD concentration in the tumor (16). To date, no relationship has yet been established between the DPD activity and the clinicopathological features (8). In this study, the DPD concentrations varied so extensively that no differences could be detected between the tumor and normal tissue specimens. However, the T/N ratio of DPD was higher in both the advanced stage cases and in the presence of vessel invasion. This may be related to the poor response to anticancer drugs (16), but further study is needed to prove this hypothesis conclusively.
The ratio of PyNPase to DPD, which may play a critical role in the concentration of fluorouracil after the administration of 5'-DFUR, was statistically higher in the tumor than in the normal tissue. We could not directly evaluate either the sensitivity of 5'-DFUR or the prognosis after 5'-DFUR administration in the present study. Nevertheless, a higher PyNPase/DPD ratio in the tumor would be an advantage to patients with colorectal cancer treated with 5'-DFUR. The PyNPase/DPD ratio was varied dramatically between patients and no correlation was observed with the clinicopathological features; however, further study is required to determine whether the PyNPase/DPD ratio is useful for selecting the most appropriate patients with colorectal cancer to undergo fluoropyrimidine therapy (6).
In summary, this study indicated that the tumor/normal tissue ratios of both PyNPase and DPD might be useful candidates for predicting the prognosis of colorectal cancer. In addition, the PyNPase/DPD ratio was observed to be higher in the tumor tissue than in the normal tissue.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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We thank Nippon Roche Research Center for assistance with ELISA analysis.
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FOOTNOTES |
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+ For reprints and all correspondence: Shungo Hiroyasu, First Department of Surgery, University of the Ryukyus, Uehara 207, Nishihara-cho, Okinawa 903-0125, Japan. E-mail: hiroyasu@med.u-ryukyu.ac.jp
Abbreviations: PyNPase, pyrimidine nucleoside phosphorylase; DPD, dihydropyrimidine dehydrogenase; 5'-DFUR, 5'-deoxy-5-fluorouridine/doxifluridine; 5-FU, 5'-fluorouracil; PD-ECGF, platelet-derived endothelial cell growth factor; MoAb, monoclonal antibody
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REFERENCES |
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1 Kono A, Hara Y, Sugata S, Karube Y, Matsushima Y, Ishitsuka H. Activation of 5'-deoxy-5-fluorouridine by thymidine phosphorylase in human tumors. Chem Pharm Bull 1983;31:175–8.
2 Ishitsuka H, Miwa M, Takemoto K, Fukuoka K, Itoga A, Maruyama HB. Role of uridine phosphorylase for antitumor activity of 5'-deoxy-5-fluorouridine. Gann 1980;71:112–23.[Web of Science][Medline]
3 Miwa M, Nishimura J, Kamiyama T, Ishitsuka H. Conversion of 5'-deoxy-5-fluorouridine to 5-FU by pyrimidine nucleoside phosphorylases in normal and tumor tissues from rodents bearing tumors and cancer patients. Gan To Kagaku Ryoho 1987;14:2924–9 (in Japanese).[Medline]
4 Fleming RA, Milano G, Thyss A, Etienne MC, Renee N, Schneider M, et al. Correlation between dihydropyrimidine dehydrogenase activity in peripheral mononuclear cells and systemic clearance of fluorouracil in cancer patients. Cancer Res 1992;52:2899–902.
5 Ishikawa T, Sekiguchi F, Fukase Y, Sawada N, Ishitsuka H. Positive correlation between the efficacy of capecitabine and doxifluridine and the ratio of thymidine phosphorylase to dihydropyrimidine dehydrogenase activities in tumors in human cancer xenografts. Cancer Res 1998;58:685–90.
6 Mori K, Hasegawa M, Nishida M, Toma H, Fukuda M, Kubota T, et al. Expression levels of thymidine phosphorylase and dihydropyrimidine dehydrogenase in various human tumor tissues. Int J Oncol 2000;17:33–8.[Web of Science][Medline]
7 Suzuki S, Hongu Y, Fukazawa H, Ichihara S, Shimizu H. Tissue distribution of 5'-deoxy-5-fluorouridine and derived 5-fluorouracil in tumor-bearing mice and rats. Gann 1980;71:238–45.[Web of Science][Medline]
8 McLeod HL, Sludden J, Murray GI, Keenan RA, Davidson AI, Park K, et al. Characterization of dihydropyrimidine dehydrogenase in human colorectal tumours. Br J Cancer 1998;77:461–5.[Web of Science][Medline]
9 Takahashi Y, Bucana CD, Liu W, Yoneda J, Kitadai Y, Cleary KR, et al. Platelet-derived endothelial cell growth factor in human colon cancer angiogenesis: role of infiltrating cells. J Natl Cancer Inst 1996;88:1146–51.
10 Furukawa T, Yoshimura A, Sumizawa T, Haraguchi M, Akiyama S, Fukui K, et al. Angiogenic factor. Nature 1992;356:668.[Medline]
11 Nishida M, Hino A, Mori K, Matsumoto T, Yoshikubo T, Ishitsuka H. Preparation of anti-human thymidine phosphorylase monoclonal antibodies useful for detecting the enzyme levels in tumor tissues. Biol Pharm Bull 1996;19:1407–11.[Web of Science][Medline]
12 Japanese Research Society for Cancer of the Colon and Rectum. General rules for clinical and pathological studies on cancer of the colon, rectum and anus. Part I. Clinical classification. Jpn J Surg 1983;13:557–73.[Medline]
13 Japanese Research Society for Cancer of the Colon and Rectum. General rules for clinical and pathological studies on cancer of the colon, rectum and anus. Part II. Histopathological classification. Jpn J Surg 1983;13:574–98.[Medline]
14 Watanabe K, Igarashi T, Suzuki M, Hayashi T, Yoshida K, Hanyu F. Study on the relationship between concentrations of 5-FU and PyNPase activity in tumor tissue during oral 5'-DFUR treatment in patients with advanced colorectal cancer. Gan To Kagaku Ryoho 1994;21:243–8 (in Japanese).
15 Eda H, Fujimoto K, Watanabe S, Ura M, Hino A, Tanaka Y, et al. Cytokines induce thymidine phosphorylase expression in tumor cells and make them more susceptible to 5'-deoxy-5-fluorouridine. Cancer Chemother Pharmacol 1993;32:333–8.[Web of Science][Medline]
16 Milano G, McLeod HL. Can dihydropyrimidine dehydrogenase impact 5-fluorouracil-based treatment? Eur J Cancer 2000;36:37–42.
Received August 10, 2000; accepted November 21, 2000.
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