TECHNICAL NOTE |
Correspondence to: Toru Kono, Second Dept. of Surgery, Asahikawa Medical College, 2-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510, Japan. E-mail: kono@asahikawa-med.ac.jp
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Summary |
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Thymidine phosphorylase (dThdPase) is an essential enzyme for activation of the oral cytostatic drug capecitabine and its intermediate metabolite, doxifluridine, to 5-fluorouracil in tumors. Methods to estimate dThdPase expression in tumor tissue might be useful to predict the efficacy of capecitabine and doxifluridine in cancer patients. We established a new monoclonal antibody (MAb), 1C6-203, applicable for dThdPase immunohistochemistry and compared its staining characteristics with those of a previously established MAb, 654-1. In 4% paraformaldehyde-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 19/30 and 9/30 patients, respectively. In 10% formalin-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 18/30 and 6/30 patients, respectively. In negative 10% formalin-fixed tissues, microwave treatment improved the positivity of 654-1-stained cancer cells. These results suggest that an epitope recognized by 1C6-203 is resistant to epitope masking by formaldehyde fixation, whereas that for MAb 654-1 is sensitive. Therefore, MAb 1C6-203 might be more suitable than MAb 654-1 for evaluating dThdPase expression in colorectal carcinoma. (J Histochem Cytochem 49:131137, 2001)
Key Words: thymidine phosphorylase, monoclonal antibody, Western blotting, immunohistochemistry, human, colorectal cancer, cancer cell, stromal cell
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Introduction |
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THYMIDINE PHOSPHORYLASE (dThdPase) is an essential enzyme for activating the oral cytostatic drug capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda) and its intermediate metabolite doxifluridine [5'-deoxy-5-fluorouridine (5'-dFUrd, Furtulon)] to 5-fluorouracil (5-FU) in tumors (
On the other hand, dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and is reported to promote angiogenesis (
However, in colorectal carcinoma tissues, the frequency of positive dThdPase expression in tumor cells and tumor stroma remains to be investigated. A monoclonal antibody (MAb) against dThdPase, 654-1 (
Recently, we developed a new anti-human dThdPase MAb by using a recombinant dThdPase as an antigen for staining formalin-fixed sections. In this study, we compared our new antibody with those currently in use in clinical research.
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Materials and Methods |
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Immunization and Hybridoma Preparation
BALB/c mice were immunized with 50 µg of recombinant dThdPase, prepared as described elsewhere (
Antibody Capture Assay
A 96-well microtiter plate (Nunc-immunoplate Maxisorp; Nunc, Roskilde, Denmark) was incubated with 0.25 µg/ml of the recombinant dThdPase in 10 mM PBS (pH 7.6) overnight at 4C. The coated plate was then incubated with 3% (w/v) skim milk in PBS (blocking buffer) for 1 hr at room temperature (RT). The plate was washed with PBS containing 0.05% Tween-20 and 0.05% sodium azide and kept at 4C until used. Hybridoma culture supernatants were dispensed onto a plate coated with the recombinant dThdPase. The plate was (a) incubated at 37C for 1 hr and washed with PBS containing 0.05% Tween-20 (washing buffer), (b) incubated with 1000-fold diluted anti-mouse IgG conjugated with horseradish peroxidase (KPL; Gaithersburg, MD) for 1 hr at 37C and washed, and (c) incubated with a substrate solution containing 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 (TMB microwell peroxidase substrate system; KPL) for 1020 min at RT. The peroxidase reaction was stopped by addition of 1 M phosphate solution, and absorbance was read at 450 nm with a plate reader (BIO-RAD Model 3550; Bio-Rad Laboratories, Hercules, CA).
Western Blotting Analysis
Fifty µg of human tumor homogenates was electrophoresed on 520% SDS-polyacrylamide gradient gels (PAGEL; ATTO, Tokyo, Japan) according to the method of
Patients and Methods
All tissues used in this study were obtained from surgically resected specimens. Carcinomas were analyzed from colorectal adenocarcinoma (n = 30), gastric adenocarcinoma (n = 5), and breast cancer (n = 5). The histopathological diagnosis was confirmed in all patients by the Department of Surgical Pathology (Asahikawa Medical College Hospital).
Specimens were washed in ice-cold 0.1 M PBS (pH 7.4) and immersed in either 4% paraformaldehyde or 10% formaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). After incubation for 12 hr at 4C, the tissues were kept at 4C in 25% sucrose in PB (pH 7.4) until they sank. Then tissue specimens were embedded in paraffin and consecutively cut into 46-µm-thick sections from each tumor block, and mounted on gelatin-coated glass slides. The sections were deparaffinized with xylene and rehydrated with 98% ethanol. In dThdPase-negative cases, consecutive deparaffinized sections were placed in 0.1 M citrate buffer (pH 6.0) and antigen retrieval was performed by heating in a microwave oven for 5 min twice (500 W). After soaking in 0.1 M PB containing 3% blocking serum and 0.3% Triton X-100, the slides were incubated with mouse MAb 654-1 against human dThdPase (10 µg/ml) or MAb 1C6-203 (10 µg/ml), overnight at 4C. The slides were washed with PBS, incubated with secondary antibodies, stained with the ABC staining technique (Vector Laboratories), and developed with diaminobenzidine tetrahydrochloride in PBS containing 0.03% H2O2. The sections were counterstained with hematoxylin and viewed and photographed using a Nikon microscope. As a control for nonspecific staining, mouse IgG1 (ICN Pharmaceuticals; Aurora, OH) was used as primary antibody.
We examined 200 cells, including cancer cells or various stromal cells in tumor tissue, and normal mucosal cells or various stromal cells in normal tissue, to determine whether the cells were dThdPase-positive. Because more than 90% of normal colorectal tissues showed less than 5% epithelial cell staining for dThdPase, specimens were regarded as dThdPase-negative when less than 5% of 200 cells were stained and as positive when more than 5% of them were stained. Specimens were evaluated independently by two experienced pathologists, who were blinded to the dThdPase antibody type and the fixation.
Statistics
The two-sided Fisher exact test was used to assess the difference in the frequency of dThdPase immunostaining among the various groups shown in Table 1. p<0.05 was regarded as statistically significant.
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Results |
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Specificity of the Anti-dThdPase MAb
Anti-dThdPase MAb 1C6-203 is a mouse IgG1. Fig 1 shows Western blotting analysis of various human cancer tissue homogenates with the MAb 1C6-203. 1C6-203 reacted only with a protein band having a molecular mass of 55 kD, which corresponds to dThdPase protein in breast, gastric, and colorectal cancer tissue homogenates. The specificity of 1C6-203 was consistent with that of the previously reported anti-dThdPase antibody 654-1 (
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dThdPase Expression in Colorectal, Breast, and Gastric Carcinoma Tissues
In 10% formalin-fixed colorectal tumor tissues, 1C6-203 stained both tumor cells and stromal cells, whereas 654-1 stained mainly stromal cells and not tumor cells in many cases (Fig 2). However, in breast and gastric tumor tissues, both 1C6-203 and 654-1 stained tumor cells and stromal cells (Fig 3).
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Most histologically normal colorectal mucosal cells were not stained with MAb 1C6-203, similar to the results with MAb 654-1, in both 4% paraformaldehyde-fixed and 10% formalin-fixed tissues. The percentage of cells expressing dThdPase was less than 5% of 200 cells in 28 (93.3%) of 30 4% paraformaldehyde-fixed normal tissues, and in 29 (96.7%) of 30 10% formalin-fixed tissues when stained with MAb 1C6-203. Various stromal dThdPase-expressing cells were detected in normal interstitum. Of 30 normal tissues, 14 (47%) and 12 (40%) showed more than 5% of examined stromal immune cells in 4% paraformaldehyde-fixed and 10% formalin-fixed tissues, respectively.
dThdPase-expressing cancer cells in colorectal carcinoma were detected by MAb 1C6-203 in 19 (63.3%) of 30 paraformaldehyde-fixed tissues and 18 (60%) of 30 formalin-fixed tissues, respectively (Table 1). In dThdPase-expressing cancer cells, dThdPase staining was observed in nucleus and/or cytoplasm (Fig 2A). Although various degrees of stromal dThdPase expression were detected in most colorectal cancer specimens, including immune cells (macrophages and lymphocytes), fibroblasts, and endothelial cells, they occurred predominantly in immune cells (macrophages and lymphocytes) in the interstitum adjacent to cancer cells (Fig 2B). Of 30 colorectal tumors, 21 (70%) and 19 (63%) showed more than 5% of positive stromal immune cells in 4% paraformaldehyde-fixed and 10% formalin-fixed tissues, respectively (Table 1).
We compared frequencies of dThdPase staining in colorectal carcinoma using different fixations and different antibodies (Table 1). In 10% formalin-fixed colorectal cancer tissue, 18 (60%) of 30 cases displayed positive dThdPase immunoreactivity in carcinoma cells with MAb 1C6-203, in contrast to six (20%) of 30 with MAb 654-1. Twelve of the 24 negative specimens stained with MAb 654-1 were positive with MAb 1C6-203 (Fig 2). Therefore, the frequency of dThdPase expression was significantly reduced when MAb 654-1 was used in formalin-fixed tissues (p<0.05). Despite this, there was no significant difference between 4% paraformaldehyde and 10% formalin with MAb 1C6-203.
In dThdPase-negative 10% formalin-fixed and 4% paraformaldehyde-fixed tissues, antigen retrieval was performed by microwave heating and slides were reexamined. MAb 654-1, but not MAb 1C6-203, gave a higher frequency of dThdPase expression in dThdPase-negative cases (Table 1).
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Discussion |
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Thymidine phosphorylase is an essential enzyme for activating the oral cytostatic drug capecitabine and its intermediate metabolite, doxifluridine, in tumors (
There are indications that dThdPase immunoexpression in colorectal tumor cells of formalin-fixed tissue is weak when MAb 654-1 (
On the other hand, in contrast to colorectal cancers, dThdPase positive frequency and staining intensity of tumor cells in breast and gastric cancers estimated by two MAbs, 1C6-203 and 654-1, were similarly high in a preliminary analysis. In colorectal cancer tissue, dThdPase was expressed not only in tumor cells but also in tumor stromal cells in the present study. These findings are consistent with those previously reported (
The above observations indicate that MAb 1C6-203 may be better than MAb 654-1 when formalin-fixed colorectal cancer tissue is used for dThdPase immunohistochemical analysis. However, further studies are needed to clarify whether MAb 1C6-203 can be used for clinical histological studies in a long-term formalin-fixed samples. At this time, we do not have enough information to correlate the site of dThdPase expression, such as tumor cell or stromal cell cytoplasmic staining or nuclear staining, with the antitumor activity of capecitabine/doxifluridine. This question requires further studies to investigate the correlation of expression levels and localization of dThdPase with the antitumor activity of these drugs.
In conclusion, MAb 1C6-203 for human dThdPase was newly developed and revealed a high frequency of dThdPase expression in formalin-fixed colorectal cancer tissues. Therefore, 1C6-203 should be useful for future clinical studies.
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Acknowledgments |
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We thank Mr Yoshiyasu Satake and Ms Yoko Okada for expert technical assistance.
Received for publication March 10, 2000; accepted August 8, 2000.
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