Thymidylate synthase expression in patients with colorectal carcinoma using a polyclonal thymidylate synthase antibody in comparison to the TS 106 monoclonal antibody

Colorectal cancer is one of the most common human cancers, for which 5-fluo rouracil (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 en zyme assays, RT-PCR, and immunohistochemical staining. We validated TS immu nohistochemistry with a polyclonal rabbit antihuman TS antibody using the a vidin-biotin method. This antibody can be used on paraffin-embedded, formal in-fixed material using an antigen retrieval method with citrate buffer and microwave treatment. The antibody shows a granular cytosolic staining patt ern. The reproducibility in cross-sections from colorectal tumors from 50 p atients was 90% and the interobserver variability was acceptable with a kap pa of 0.45. On Western blotting it detects purified TS at 36 kD, while in 5 FU-treated cells the ternary complex between FdUMP, TS, and 5,10-methylene- tetrahydrofolate is clearly visible at 38 kD, with no other interfering ban ds. In a separate set of tumors, immunostaining was compared with enzyme le vels; Western blots correlated with enzyme levels. Because both this polycl onal antibody and the monoclonal antibody TS-106 are being used for large-s cale studies, we also determined whether they could be used interchangeably . No differences were observed. This polyclonal antibody is specific and gi ves reproducible results. A study on a larger scale is ongoing to determine the role of TS as a predictive parameter in patients with colorectal cance r treated either with postoperative adjuvant 5FU/levamisole or with surgery only.