title: Sensitive immunohistochemical detection of WT1 protein in tumors with anti-WT1 antibody against WT1 235 peptide.
abstract: The Wilms' tumor 1 (WT1) gene is overexpressed in leukemia and various types of solid tumor, such as lung and colorectal cancer, and plays an oncogenic role in their tumorigenesis. Recent studies have demonstrated the potential of WT1-targeting cancer immunotherapy in clinical settings. As expression of WT1 protein in tumor cells is a prerequisite for WT1-targeting immunotherapy, immunohistochemical methods to detect WT1 protein with high sensitivity and specificity are required. In the present study, we developed a rabbit polyclonal antibody (WT1-R) against the 9-mer WT1 235 peptide, which is used for vaccination. The specificity of WT1-R was confirmed by immunoprecipitation, western blotting analysis, and competitive enzyme-linked immunosorbent assay. Immunocytochemistry showed the same reactivity against five cell lines (K562, Daudi, HT-180, SW480, and PC-14), whereas levels of WT1 mRNA expression determined by real-time qPCR (RT-PCR) analysis were not equivalent. Next, we examined the reactivity of WT1-R in tissue samples compared with a previously developed anti-WT1 antibody, 6F-H2. WT1-R showed greater sensitivity for detecting WT1 protein expression in samples from four different breast cancer patients than 6F-H2 antibody. The discrepancy in WT1 expression between these methods suggested that immunohistochemical detection of WT1 peptide may be advantageous for predicting the efficacy of WT1 vaccine compared to RT-PCR, and the highly sensitive WT1 antibody, WT1-R, may be useful to detect WT1 protein in tumors.
authors.affiliation: Division of Hematopathology, Tohoku University Graduate School of Medicine, Sendai, Japan. ryo@mail.tains.tohoku.ac.jp
authors.forename: Ryo
authors.initials: R
authors.lastname: Ichinohasama
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Oji
authors.forename: Hisayuki
authors.initials: H
authors.lastname: Yokoyama
authors.forename: Kengo
authors.initials: K
authors.lastname: Takeuchi
authors.forename: Tohru
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ishizawa
authors.forename: Osamu
authors.initials: O
authors.lastname: Taniguchi
authors.forename: Akihiro
authors.initials: A
authors.lastname: Tsuboi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Oka
authors.forename: Haruo
authors.initials: H
authors.lastname: Sugiyama
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D009369:Neoplasms / Q000737:chemistry*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D025721:WT1 Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology
chemical_list: D025721:WT1 Proteins