ANTICANCER RESEARCH 36: 3715-3724 (2016) Immunohistochemical Analysis of WT1 Antigen Expression in Various Solid Cancer Cells KEIKO NAITOH, TAKASHI KAMIGAKI, ERIKO MATSUDA, HIROSHI IBE, SACHIKO OKADA, ERI OGUMA, YOSHIHIRO KINOSHITA, RISHU TAKIMOTO, KAORI MAKITA, SHUN OGASAWARA and SHIGENORI GOTO Seta Clinic, Tokyo, Japan Abstract. For a peptide-pulsed dendritic cell (DC) vaccine cancer vaccines with various cancer antigens in treatment of to work effectively in cancer treatment, it is significant that solid tumors (3-6). For DC-based cancer vaccines, some the target protein is expressed in cancer cells. Wilms’ tumor reports have described insufficient clinical responses despite 1 (WT1) has been identified as a molecular target for the good immunoresponses indicating delayed-type immune cell therapy of cancer. We evaluated the protein hypersensitivity (DTH) (7). Immune check-point inhibitors, expression levels of WT1 in various solid tumors, as well as such as antibodies against programmed cell death protein 1 mucin 1 (MUC1) or major histocompatibility complex (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (MHC) class l molecules. Seven hundred and thirty-eight (CTLA4), are clinically used for patients with advanced or patients whose tissue samples were examined by recurrent melanoma and non-small cell lung cancer to immunohistochemical analysis agreed to undergo DC reverse immune suppression and activate effector T cells (8, vaccine therapy. The positive staining of WT1 in tumor cells 9). Furthermore, the efficacy of immune check-point was observed in 25.3% of patients, with only 8.5% of them inhibitors was reported to correlate with disorders related to showing moderate to strong expression; moreover, WT1 TSAs, oncogenic viral proteins or DNA repair pathway tended to localize in the nucleus and cytoplasm. A positive mutations (10). staining of tumor cells by an anti-MHC class l monoclonal Tumor antigens can be categorized as TSAs, cancer/testis antibody was observed in 98.6% and by an anti-MUC1 (CT) antigens or tumor-associated antigens (TAAs) (11, 12). monoclonal antibody in 76.8% of the patients. In relation to TSAs are abnormal proteins that arise from non-synonymous the application of cancer-specific immunotherapy, these somatic mutations in tumor cells; however, such antigens are findings provide useful information for determining the not expressed in normal cells. CT antigens can be potential efficacy of MUC1- and WT1-targeted therapy. targets as cancer vaccines because their expression is normally restricted to the germ cells of the testis or ovary or Dendritic cell (DC)-based vaccines pulsed with various certain tumor cells (11, 13). TAAs are overexpressed normal tumor-specific antigens (TSAs) have been developed for proteins, such as Wilms’ tumor 1 (WT1) (14, 15) or mucin 1 cancer immunotherapy (1). Our previous data suggest that (MUC1) that regulate growth-promoting functions (16). The immunotherapy with tumor antigen-pulsed immature DCs antigenicity of TAAs was reported to depend on the levels with zoledronate leads to activation of Vγ9γδ T cells and of abnormal expressions (17) because of the lower affinity induction of CD40L on Vγ9γδT cells (2). The activated of the T cell receptor (TCR) against TAAs than of TCR Vγ9γδT cells secret T helper (Th)1-cytokines, such as against TSAs (18). interferon (IFN)-γ, and enhance the expansion of tumor For DC vaccines loaded with various TAAs peptides, their antigen-specific CD8+ cells by tumor antigen-pulsed phase I/IIa clinical trial for immunotherapy was carried out immature DCs. We utilized zoledronate-pulsed DCs as in elderly patients with acute myeloid leukemia (AML), using pulsed DCs with a modified WT1 peptide and zoledronate (19). In that trial, three human leukocyte antigen (HLA)- A2402-positive elderly patients with AML were enrolled. The Correspondence to: Keiko Naitoh, Senior Chief Researcher of induction of immunoresponses to the WT1 peptide detected Clinical Research Center, Seta Clinic, 2-1-45 Kanda-surugadai, as DTH was indicated in two of the three patients, with a Chiyoda, Tokyo 101-0062, Japan. Tel: +81 352445751, Fax: +81 transient decrease in the number of leukemic cells being 332190750, e-mail: [email protected] observed in these two patients. Unfortunately, a rapid Key Words: Immune cell therapy, immunohistochemistry, WT1, expansion of leukemic cells was observed in the patient dendritic cell vaccine therapy. showing no immunoresponses to the WT1-specific peptide 0250-7005/2016 $2.00+.40 3715 ANTICANCER RESEARCH 36: 3715-3724 (2016) after the third vaccination. Recently, we studied a DC vaccine Evaluation method. The relative ratio (proportion) and positive pulsed with zoledronate and an overlapping pool of peptides reaction strength (i.e., staining intensity) were determined to analyze derived from the full length of WT1 for patients with WT1- antigen expression. The level and distribution of expression were subjectively estimated and positive reaction strength was described expressing solid tumors as both CD8+ cytotoxic T as -, +, ++ and +++ (Table ll). lymphocytes (CTLs) and CD4+ T-helper cells against WT1 can be potentially induced without HLA restriction. However, in order to be recognized by CTLs with low-affinity TCRs, it Results is essential that a sufficient amount of TAAs, such as WT1 or MUC1, should be presented by major histocompatibility Immunohistochemical findings of WT1, MUC1 and MHC complex (MHC) class I on tumor cells (20). class I protein expressions in tumor cells. We studied WT1 In the present study, we examined the protein expression protein expression in various solid tumors by levels of WT1 in various solid tumors, as well as MUC1 or immunohistochemical staining using monoclonal antibodies MHC class I molecules by immunohistochemical analysis. against WT1, MUC1 and MHC class I molecules. The We also analyzed the organ and histopathological profiles of results of the immunohistochemical staining of WT1 WT1 protein expression in various malignancies classified expressed in various solid tumors are shown in Figure 1. For on the basis of ICD10 and ICD-O-3. malignant pleural mesothelioma, a strong expression of the WT1 protein was observed in the nucleus of tumor cells Materials and Methods (Figure 1A). A weak expression of WT1 protein was observed only in the cytoplasm of cancer cells and in both Patients’ background. Between October 2007 and September 2014, the nucleus and cytoplasm of breast cancer and malignant tumor samples were obtained intraoperatively or by biopsy from pleural mesothelioma cells, respectively (Figure 1B and C). 738 patients who provided their informed consent to four facilities of the Seta Clinic Group. The patients agreed to undergo DC It is shown in Figure 1D that no WT1 protein expression was vaccine therapy and their tissue samples were examined by found in one patient with adenocarcinoma of the pancreas. immunohistochemical analysis for the expression levels of WT1, Immunohistochemical staining showed MUC1 expression at MUC1 and MHC class I molecules. The mean age of the 738 the luminal and/or apical site of cancer cells (Figure 2A and patients was 58.6 years (range=6-87) and the male-to-female ratio B). MHC class I molecules were expressed in most of the was 1:1.10 (352:386). In this study, 55 cancer types (ICD-10) and solid tumors; however, loss or down-regulation of the 24 histological types (ICD-O-3M) were included (Table I). expression of MHC class I molecules was observed in few Specimens. A total of 738 specimens were examined, comprising of tumors (Figure 2C and D). 72 stomach cancers, 68 colon cancers, 47 rectum cancers, 63 pancreas cancers, 120 lung cancers, 48 breast cancers and 63 Analysis of WT1, MUC1 and MHC class I protein expression ovarian cancers. The remaining tumor types are summarized in in various tumors classified on the basis of ICD10. We Table I. The prepared paraffin-embedded or formalin-fixed tissues categorized the WT1 expression patterns in tumors classified were examined at Tokyo Central Pathology Laboratory Company. on the basis of ICD10 and ICD-O-3. Additionally, we also The histological diagnosis of each tumor was confirmed on the basis studied the expression patterns of MUC1, as one of the of hematoxylin and eosin staining results and the pathological reports provided by each medical facility. TAAs, and MHC class I molecules, as antigen presentation- associated molecules in various tumors. The expression Immunohistochemistry. A mouse monoclonal anti-human WT1 levels of WT1, MUC1 and MHC class I in various tumors protein antibody (immunogen:human WT1 protein consisting of N- classified on the basis of ICD10 are shown in Table III. The terminal amino acids 1-181, clone 6F-H2; Dako, Glostrup, expression of WT1 substantially differed depending on the Denmark), an anti-MUC-1 glycoprotein mouse monoclonal antibody tumor site, classified on the basis of ICD10. On the other (Novacastra Laboratories Ltd., Newcastle, UK) and an anti-HLA hand, MUC1 expression was observed in most solid tumors. class I-A, B, C mouse monoclonal antibody (Hokudo Co., Ltd., Hokkaido, Japan) were used for the detection of WT1, MUC1 and For malignant mesothelioma, WT1 expression was found in MHC class l antigens, respectively. all tumors. Additionally, a high frequency of WT1 expression was shown (39.3%; 46/117) for the malignancies of female WT1 immunohistochemical staining method. The mouse monoclonal genital organs (ICD10; C52-C59), including cancers of the anti-WT1 antibody (clone 6F-H2; Dako), an enzyme antibody ovary (52.4%; 33/63). The frequency of WT1 expression was LSAB method (labeled streptavidin-biotin) (21), and a Ventana also relatively high in cancers of the bile duct (C23-24, Benchmark XT (Ventana Medical Systems, Inc., Arizona, USA) 41.2%; 7/17), lung (C34, 35.0%; 42/120), breast (C50, device were used for the immunohistochemical staining. Tissue sections were prepared as follows: enzyme and heat treatment for 8 25.0%; 12/48) and prostate (C61, 28.6%; 4/14).