IHC, WT1 Wilm's Tumor Protein Test

The WT1 gene was identified as a tumor suppressor gene located at 11p13 and is involved in the development of Wilm’s tumor. Ever since WT1 has been identified as a molecular target for cancer immunotherapy, immunohistochemical detection of WT1 protein in tumor cells has become an essential part of routine practice. WT1-positive tumors includes tumors of the stomach, prostate, biliary and urinary systems and malignant melanomas. However, in recent studies it was found out that WT1 protein plays an oncogenic role rather than tumor-suppressive role in human cancers.

Immunohistochemistry (IHC) is the method which combines anatomical, immunological and biochemical techniques to image discrete components in tissues by using appropriately-labeled antibodies to bind specifically to their target antigens. Immunohistochemical staining is widely used in the diagnosis of cancer cells. It is crucial in IHC tests that the cell morphology, tissue architecture and the antigenicity of target epitopes of the samples are maintained. The tissue collected for testing should be rapidly preserved to prevent the breakdown of cellular protein and degradation of the normal tissue architecture.

Immunohistochemical study showed the cytoplasmic expression of WT1 in a large proportion of various kinds of human cancers. In the present study, the expression of WT1 was examined in 494 cases of human cancers, including tumors of the gastrointestinal and pancreatobiliary system, urinary tract, male and female genital organs, breast, lung, brain, skin, soft tissues and bone done by immunohistochemistry. It is also to be noted that WT1 vaccination induced a reduction in tumor size or decrease in tumor marker levels in breast cancer, lung cancer and leukaemia. The results of the present study provided a rationale for immunotherapy targeting Wt1 as a new treatment strategy for the various kinds of tumors resistant to conventional surgery or chemo radiotherapy.

Immunohistochemistry is done by using polylconal (C-190) and monoclonal (6F-H2) antibodies against the WT1 protein. After dewaxing and rehydration, 3-μm-thick sections were subjected to heat-induced epitope retrieval by microwaving them for 15 min in 1 mM citrate buffer (pH 6.0), followed by incubation with anti-WT1 antibody diluted 1:100 at 4°C overnight. For 6F-H2, a positive signal is detected using the ENVISION+ kit (Dako cytomation). For C-19, after incubation with biotinylated anti-rabbit or anti-mouse secondary antibody, sections were treated with a 3% H₂O₂ solution to reduce endogenous peroxidise activity. Visualization was performed by a standard avidin-biotin complex method using a Vectastain ABC elite kit.