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-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.
Van Triest
B
B
Departments of Medical Oncology, University Hospital VU, Amsterdam, The Netherlands.
Loftus
B M
BM
Pinedo
H M
HM
Backus
H H
HH
Schoenmakers
P
P
Telleman
F
F
Tadema
T
T
Aherne
G W
GW
Van Groeningen
C J
CJ
Zoetmulder
F A
FA
Taal
B G
BG
Johnston
P G
PG
Peters
G J
GJ
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western / Q000379:methods; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000473:pathology; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008875:Middle Aged; D011817:Rabbits; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D013940:Thymidylate Synthase