Van Triest B, Loftus B M, Pinedo H M, Backus H H, Schoenmakers P, Telleman F, Tadema T, Aherne G W, Van Groeningen C J, Zoetmulder F A, Taal B G, Johnston P G, Peters G J
Departments of Medical Oncology, University Hospital VU, Amsterdam, The Netherlands.
J Histochem Cytochem. 2000 Jun;48(6):755-60. doi: 10.1177/002215540004800604.
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.
结直肠癌是人类最常见的癌症之一，5-氟尿嘧啶（5FU）通常是其治疗的一部分。胸苷酸合成酶（TS）是5FU的靶酶，可预测基于5FU治疗的结果。肿瘤样本中的TS水平可用放射化学酶法、逆转录聚合酶链反应（RT-PCR）和免疫组织化学染色来测定。我们使用抗生物素蛋白-生物素方法，用兔抗人TS多克隆抗体验证了TS免疫组织化学。该抗体可用于石蜡包埋、福尔马林固定的材料，采用柠檬酸盐缓冲液和微波处理的抗原修复方法。该抗体呈现颗粒状胞质染色模式。50例结直肠癌患者肿瘤横断面的重现性为90%，观察者间变异性可接受，kappa值为0.45。在蛋白质印迹法中，它能检测到36kD的纯化TS，而在5FU处理的细胞中，在38kD处可见FdUMP、TS和5,10-亚甲基四氢叶酸之间的三元复合物，无其他干扰条带。在另一组肿瘤中，将免疫染色与酶水平进行了比较；蛋白质印迹与酶水平相关。由于这种多克隆抗体和单克隆抗体TS-106都用于大规模研究，我们还确定了它们是否可以互换使用。未观察到差异。这种多克隆抗体具有特异性，结果可重现。一项更大规模的研究正在进行，以确定TS作为预测参数在接受术后辅助5FU/左旋咪唑治疗或仅接受手术治疗的结直肠癌患者中的作用。
