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Journal of Histochemistry and Cytochemistry, Vol. 48, 755-760, June 2000, Copyright © 2000, The Histochemical Society, Inc.

ARTICLE

Thymidylate Synthase Expression in Patients with Colorectal Carcinoma Using a Polyclonal Thymidylate Synthase Antibody in Comparison to the TS 106 Monoclonal Antibody

Baukelien Van Triesta, Barbara M. Loftusc, Herbert M. Pinedoa, Harold H.J. Backusa, Pascale Schoenmakersa, Frank Tellemana, Thea Tademab, G. Wynne Ahernef, Cornelius J. Van Groeningena, Frans A.N. Zoetmulderd, Babs G. Taale, Patrick G. Johnstong, and Godefridus J. Petersa
a Departments of Medical Oncology, University Hospital VU, Amsterdam, The Netherlands
b Pathology, University Hospital VU, Amsterdam, The Netherlands
c Departments of Pathology, Netherlands Cancer Institute/Antoni Van Leeuwenhoekhuis, Amsterdam, The Netherlands
d Surgery, Netherlands Cancer Institute/Antoni Van Leeuwenhoekhuis, Amsterdam, The Netherlands
e Gastroenterology, Netherlands Cancer Institute/Antoni Van Leeuwenhoekhuis, Amsterdam, The Netherlands
f Department of Drug Development, Cancer Research Campaign, Institute for Cancer Research, Sutton, United Kingdom
g Department Oncology, Queen's University of Belfast, Belfast, United Kingdom

Correspondence to: Godefridus J. Peters, Dept. of Medical Oncology, University Hospital VU, PO 7057, 1007 MB Amsterdam, The Netherlands. E-mail: gj.peters@azvu.nl

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 avidin–biotin 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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