ARTICLE |
Correspondence to:
Godefridus J. Peters, Dept. of Medical Oncology, University Hospital VU, PO 7057, 1007 MB Amsterdam, The Netherlands. E-mail:
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Summary |
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Colorectal cancer is one of the most common human cancers, for which 5-fluorouracil (5FU) is usually part of the treatment. Thymidylate synthase (TS), the target enzyme for 5FU, can be predictive for the outcome of 5FU-based therapy. TS levels in tumor samples can be determined with radiochemical enzyme assays, RT-PCR, and immunohistochemical staining. We validated TS immunohistochemistry with a polyclonal rabbit anti-human TS antibody using the avidinbiotin method. This antibody can be used on paraffin-embedded, formalin-fixed material using an antigen retrieval method with citrate buffer and microwave treatment. The antibody shows a granular cytosolic staining pattern. The reproducibility in cross-sections from colorectal tumors from 50 patients was 90% and the interobserver variability was acceptable with a kappa of 0.45. On Western blotting it detects purified TS at 36 kD, while in 5FU-treated cells the ternary complex between FdUMP, TS, and 5,10-methylene-tetrahydrofolate is clearly visible at 38 kD, with no other interfering bands. In a separate set of tumors, immunostaining was compared with enzyme levels; Western blots correlated with enzyme levels. Because both this polyclonal antibody and the monoclonal antibody TS-106 are being used for large-scale studies, we also determined whether they could be used interchangeably. No differences were observed. This polyclonal antibody is specific and gives reproducible results. A study on a larger scale is ongoing to determine the role of TS as a predictive parameter in patients with colorectal cancer treated either with postoperative adjuvant 5FU/levamisole or with surgery only.
Key Words: thymidylate synthase, immunohistochemistry, polyclonal antibody, colorectal cancer
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Introduction |
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Thymidylate synthase (TS; EC 2.1.1.45) is the limiting irreversible step in de novo DNA synthesis because it catalyzes the methylation of dUMP to dTMP. It is the target for the widely used anticancer agents 5-fluorouracil (5FU) and fluorodeoxyuridine (
Clinical drug resistance at the TS level is of relevance for response to 5FU; increased TS mRNA, insufficient TS inhibition by 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP), increase of TS levels after treatment are all mechanisms of 5FU resistance related to TS (reviewed by
In a previous study we measured TS levels enzymatically in patients with advanced colorectal cancer and found a significant relationship between the response to 5FU therapy and the TS levels (
TS levels in patient samples can be measured by various methods. With enzymatic assays (
In this article we describe the methodology and validation of the use of a polyclonal TS antibody for quantitation of TS levels in paraffin-embedded material from 50 representative patients with colorectal cancer. A secondary aim of the study was a comparison of the polyclonal antibody with the monoclonal TS-106 to establish their interchangeability. We also tested the intensity of the (immuno)staining compared with TS enzyme levels. Samples were obtained from patients who were entered in the Netherlands Adjuvant Colorectal Cancer Project (NACCP) trial in which postoperative 5FU/levamisole is compared to surgical treatment only in patients with colorectal cancer.
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Materials and Methods |
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Tissues
Formalin-fixed, paraffin-embedded material from the primary tumors from 50 randomly selected patients with colorectal cancer were used. These patients were entered in the NACCP trial. In this trial, patients with colon and rectal cancer were randomized between surgical treatment only or surgery followed by 5FU combined with levamisole (
Immunohistochemical Staining
Tissue sections were cut at 4 µm, and mounted on poly-L-lysine-coated slides, and dried. The sections were deparaffinized and rehydrated by incubation twice for 3 min in 100% xylene, followed by incubation in decreasing ethanol concentrations. Antigen retrieval was necessary (
To compare the polyclonal TS antibody with the TS monoclonal antibody TS 106 (kindly provided by Dr. P.G. Johnston; then at the National Cancer Institute, Bethesda, MD) which has already been extensively characterized, we used the same protocol as above. The monoclonal antibody was 1:50 diluted in 1% BSA/PBS and incubated for 1 hr followed by incubation with the second goat anti-mouse biotin-labeled antibody (1:50). As a negative control, a mouse IgG1 antibody was used.
Western Immunoblotting and Enzyme Assays
For further characterization of the antibody with Western immunoblotting, monolayer SW1573 cancer cells in exponential growth phase were treated for 24 hr at 50% growth-inhibitory concentrations of either 5FU or the TS inhibitors AG377 and ZD1694. Treated and control cells were harvested, washed, and centrifuged, and the pellets were immediately frozen in liquid nitrogen and processed essentially as described previously (
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Results |
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Immunohistochemistry
Antigen retrieval with citrate buffer, pH 6.0, was essential for positive staining of the formalin-fixed, paraffin-embedded tumor samples. Titration of the primary TS antibody concentration between 1:50 and 1:500 resulted in optimal staining when a concentration of 1:100 was used. Antigen retrieval was necessary; omission of this step resulted in nonevaluable samples. After the retrieval step, all samples could be evaluated. An example of a colon tumor stained with the TS polyclonal antibody is shown in Fig 1. Positive staining with the polyclonal TS antibody gave a granular cytosolic staining pattern in the tumor cells. In most tumor samples the staining was diffuse, although heterogeneity was also observed. The intensity of the staining was scored as low, intermediate, or high (1+ to 3+) and in Fig 1 an example is shown. Fig 1A is high intensity, Fig 1B intermediate, and Fig 1C low intensity. Normal colon epithelial cells showed weak positive staining, but this was much weaker than in tumor cells (Fig 1D). There was more intense staining at the proliferative zone. A colon tumor sample of a patient with high TS expression was always used as a positive control and gave a similar staining pattern in each separate experiment. The results of the immunostaining were as follows: 17 patients had 1+, 24 had 2+, and nine patients had a high TS level of 3+. Therefore, the majoritiy of the patients had an intermediate TS intensity. The reproducibility of this staining was tested by replicate staining of 50 samples and was 90%. The interobserver variability was tested between two observers (BvT and BML) and was acceptable, with a kappa of 0.45. On 36 of the 50 samples the two observers agreed. When the observers disagreed on the results of the TS staining, the debate was on differences between 1+ or 2+ or differences between 2+ and 3+. The slides were then re-examined by both observers and agreement was obtained. This antibody was also suitable for immunostaining on cytospins of tumor cell lines and on frozen tissue (not shown). The polyclonal and monoclonal TS antibodies showed comparable results. TS staining was identical concerning the intensity of the tumor and normal tissue in 12 different samples from paraffin-embedded tumor sections. No differences in background staining could be observed between the polyclonal and the monoclonal antibody.
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Western Immunoblotting and TS Activity Level
To evaluate the specificity of the antibody, TS Western blotting was performed in the human non-small-cell lung cancer cell line SW1573 (Fig 2) and in a panel of 13 human colon cancer cell lines (
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Discussion |
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In this article we describe the results of the validation of a polyclonal antibody for TS staining in paraffin-embedded colon or rectal tumor material. This polyclonal antibody was highly specific and could detect the TS protein using Western immunoblotting and immunohistochemistry. A discrimination between high and low TS levels could easily be measured in 50 samples of patients with colon tumors. In the tumor material, the method for the TS immunostaining was reproducible and showed acceptable intraobserver variability.
For immunostaining with the polyclonal TS antibody, various methods of detection have been used. For detection of TS in tumor cell lines we initially used a direct peroxidase-labeled secondary antibody (
A comparison with the TS monoclonal antibody developed by Johnston et al. was undertaken to determine whether these antibodies could be used interchangeably. Both antibodies showed similar results for the intensity of staining of tumor and normal tissue, the background and on TS Western immunoblotting (
Received for publication December 29, 1999; accepted January 12, 2000.
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Literature Cited |
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Aherne GW, Hardcastle A, Newton R (1992) Measurement of human thymidylate synthase (hTS) in cell lines using ELISA. Ann Oncol 3(suppl 5):77[Medline]
Brown RW, Chirala R (1995) Utility of microwave citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol 8:15-20
Drake J, Allegra CJ, Johnston PG (1993) Immunological quantitation of thymidylate synthase-FdUMP-5,10-methylenetetrahydrofolate ternary complex with the monoclonal antibody TS-106. Anti-cancer Drugs 4:431-435[Medline]
Edler D, Blomgren H, Allegra CJ, Johnston PG, Lagerstedt U, Magnusson I, Ragnhammar P (1997) Immunohistochemical determination of thymidylate synthase in colorectal cancermethodological studies. Eur J Cancer 33:2278-2281[Medline]
Findlay MPN, Cunningham D, Morgan G, Clinton S, Hardcastle A, Aherne GW (1997) Lack of correlation between thymidylate synthase levels in primary colorectal tumors and subsequent response to chemotherapy. Br J Cancer 75:903-909[Medline]
Freemantle SJ, Aherne GW, Hardcastle A, Lunec J, Calvert AH (1991) Increases in thymidylate synthase protein levels measured using newly developed antibodies. Proc Am Assoc Cancer Res 32:360
Holgate CS, Jackson P, Cowen PN, Bird CC (1983) Immunogold-silver staining: new method of immunostaining with enhanced sensitivity. J Histochem Cytochem 31:938-944[Abstract]
Jackman AL, Calvert AH (1995) Folate-based thymidylate synthase inhibitors as anticancer drugs. Ann Oncol 6:871-881[Abstract]
Johnston PG, Drake JC, Trepel J, Allegra CJ (1992) Immunological quantitation of thymidylate synthase using the monclonal antibody TS-106 in 5-fluorouracil-sensitive and -resistant human cancer cell lines. Cancer Res 52:4306-4312[Abstract]
Johnston PG, Fisher ER, Rockette HE, Fisher B, Wolmark N, Drake JC, Chabner BA, Allegra CJ (1994) The role of thymidylate synthase expression in prognosis and outcome of adjuvant chemotherapy in patients with rectal cancer. J Clin Oncol 12:2640-2647[Abstract]
Johnston PG, Liang C-M, Henry S, Chabner BA, Allegra CJ (1991) Production and characterization of monoclonal antibodies that localize human thymidylate synthase in the cytoplasm of human cells and tissue. Cancer Res 51:6668-6676[Abstract]
Johnston PG, Mick R, Recant W, Behan KA, Dolan ME, Ratain MJ, Beckmann E, Weichselbaum RR, Allegra CJ, Vokes EE (1997) Thymidylate synthase expression and response to neoadjuvant chemotherapy in patients with advanced head and neck cancer. J Natl Cancer Inst 89:308-313
Leichman L, Lenz H-J, Lechman CG, Groshen S, Danenberg K, Baranda J, Spears CP, Boswell W, Silberman H, Ortega A, Stain S, Beart R, Danenberg P (1995) Quantitation of intratumoral thymidylate synthase expression predicts for resistance to protracted infusion of 5-fluorouracil and weekly leucovorin in disseminated colorectal cancers: preliminary report from an ongoing trial. Eur J Cancer 31:1306-1310
Lenz H-J, Hill C, Danenberg KD, Leichmann LL, Priest DG, Danenberg PV (1995) Rapid quantitative PCR for determination of relative gene expressions in tissue specimens. PCR Methods Applications 4:305-308[Medline]
Lenz H-J, Leichman CG, Danenberg KD, Danenberg PV, Groshen S, Cohen H, Crookes P, Silberman H, Baranda J, Garcia Y, Li J, Leichman L (1996) Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival. J Clin Oncol 14:176-182[Abstract]
Merritt AJ, Potten CS, Watson AJM, Loh DY, Nakayama K-I, Nakayama K, Hickman JA (1995) Differential expression of bcl-2 in intestinal epithelia; correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia. J Cell Sci 106:2261-2271
Peters GJ, Ackland SP (1996) New antimetabolites in preclinical and clinical development. Exp Opin Invest Drugs 5:637-679
Peters GJ, Jansen G (1996) Resistance to antimetabolites. In Schilsky RL, Milano GA, Ratain MJ, eds. Principles of Antineoplastic Drug Development and Pharmacology. New York, Marcel Dekker, 543-585
Peters GJ, Laurensse E, Leyva A, Pinedo HM (1987) Purine nucleosides as cell-specific modulators of 5-fluorouracil metabolism and cytotoxicity. Eur J Cancer Clin Oncol 23:1869-1881[Medline]
Peters GJ, Van der Wilt CL, Van Groeningen CJ, Smid K, Meijer S, Pinedo HM (1994) Thymidylate synthase inhibition after administration of fluorouracil with or without leucovorin in colon cancer patients: implications for treatment with fluorouracil. J Clin Oncol 12:2035-2042[Abstract]
Peters GJ, Van Groeningen CJ, Laurensse E, Pinedo HM (1991) Thymidylate synthase from untreated human colorectal cancer and colonic mucosa: enzyme activity and inhibition by 5-fluoro-2'-deoxyuridine-5'-monophosphate. Eur J Cancer 27:263-267[Medline]
Pinedo HM, Peters GJ (1988) Fluorouracil: biochemistry and pharmacology. J Clin Oncol 6:1653-1664[Abstract]
Reynolds T (1995) Dutch trial casts doubt on colorectal adjuvant therapy. J Natl Cancer Inst 87:476-479[Medline]
Rustum YM, Harstrick A, Cao S, Vanhoefer U, Yin M-B, Wilke H, Seeber S (1997) Thymidylate synthase inhibitors in cancer therapy: direct and indirect inhibitors. J Clin Oncol 15:389-400[Abstract]
Schneider HJ, Sampson SA, Cunningham D, Norman AR, Andreyev HJN, Tilsed JVT, Clarke PA (1997) Bcl-2 expression and response to chemotherapy in colorectal adenocarcinomas. Br J Cancer 75:427-431[Medline]
Taylor CR, Shi S-R, Chaiwun B, Young L, Imam SA, Cote RJ (1994) Strategies for improving immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections. Hum Pathol 25:263-270[Medline]
Van der Wilt CL, Smid K, Aherne GW, Pinedo HM, Peters GJ (1993) Evaluation of immunohistochemical staining and activity of thymidylate synthase in cell lines. Adv Exp Med Biol 338:605-608[Medline]
Van der Wilt CL, Smid K, Noordhuis P, Aherne GW, Peters GJ (1997) Biochemical mechanisms of interferon modulation of 5-fluorouracil activity in colon cancer cells. Eur J Cancer 33:471-478[Medline]
Van Triest B, Pinedo HM, Telleman F, Van der Wilt CL, Jansen G, Peters GJ (1997) Cross-resistance to antifolates in multidrug resistant cell lines with P-glycoprotein or multidrug resistance protein expression. Biochem Pharmacol 53:1855-1866[Medline]
Van Triest B, Pinedo HM, Van Hensbergen Y, Smid K, Telleman F, Schoenmakers PS, Van der Wilt CL, Van Laar JAM, Noordhuis P, Jansen G, Peters GJ (1999) Thymidylate synthase level as the main predictive parameter for sensitivity to 5-fluorouracil, but for folate-based thymidylate synthase inhibitors in 13 non-selected colon cancer cell ines. Clin Cancer Res 5:643-654
Van Triest B, Pinedo HM, Blauwgeers JLG, Van Diest PJ, Schoenmakers PS, Voorn DA, Smid K, Hoekman K, Hoitsma HFW, Peters GJ (in press) Prognostic role of thymidylate synthase, thymidine phosphorylase/platelet derived endothelial cell growth factor, and proliferation markers in colorectal cancer. Clin Cancer Res