Tumor Gene WT1 Research Articles

WT1 epitope-specific IgG and IgM antibodies for immune-monitoring in patients with advanced sarcoma treated with a WT1 peptide cancer vaccine.

The Wilms' tumor gene WT1 is highly expressed in various malignancies and may be a common target antigen for cancer immunotherapy. In our group, peptide-based cancer vaccines targeting WT1 CTL epitopes were developed as an immunotherapy for these malignancies. In the present study, WT1 epitope-specific immune responses were analyzed in 31 patients with advanced sarcoma with human leukocyte antigen-A*24:02- and WT1-expressing tumors who received the WT1-235 peptide vaccine as monotherapy. The serum levels of IgG and IgM antibodies against the target epitope WT1-235 and the non-target epitopes WT1-332 and WT1-271 were measured using ELISA. IgM antibodies against WT1-235, WT1-332 and WT1-271 were detected in three (9.6%), four (12.9%) and 20 patients (64.5%), respectively, prior to vaccine administration, indicating immune recognition of the WT1 antigen prior to administering the vaccine. Of 15 patients who had completed the 3-month treatment protocol, WT1-235 IgG was positive in five (33.3%) patients. An enzyme-linked immunospot assay revealed that WT1-235 epitope-specific IL-10 production/secretion in peripheral blood mononuclear cells declined in the first month of vaccine administration in all three patients with positivity for WT1-235 IgM at the start of the vaccine. Furthermore, positivity for both WT1-235 and WT1-271 IgM antibodies at the start of treatment was associated with unfavorable tumor control at 3 months after vaccine administration. These results suggested that WT1 epitope-specific IgG and IgM antibodies may be utilized as immune-monitoring markers for WT1 peptide cancer vaccine immunotherapy. The trials were entered in the University hospital Medical Information Network (UMIN) Clinical Trials Registry (https://www.umin.ac.jp/ctr; no. UMIN000002001 on May 24, 2009 and no. UMIN000015997 on December 20, 2014).

Wilms' tumor (WT1) (±KTS) variants decreases the progesterone secretion of bovine ovarian theca cells

Wilms' tumor gene WT1 encodes a nuclear transcriptional factor, which has been shown to regulate granulosa cell steroidogenesis in bovine; however, it is not known whether the functions of theca cells are regulated by WT1. Here, we determined the effects of this gene on theca cell proliferation, apoptosis, and steroidogenesis in vitro. In cultured bovine theca cells, the downregulation of WT1 increased the secretion of progesterone but had no effect on proliferation and apoptosis. WT1 includes the variants WT1(+KTS) and WT1(−KTS), which differ by 3 amino acids KTS (lysine, threonine, and serine). WT1(±KTS) upregulation increased the messenger RNA (mRNA) expression of STAR and CYP17A1 and decreased the progesterone secretion and CYP11A1 mRNA expression. In contrast to WT1(+KTS), WT1(−KTS) upregulation also decreased the mRNA expression of 3β-HSD. In both variants, WT1(−KTS) has more obvious effects. In conclusion, WT1 can decrease progesterone secretion, likely due in part to the inhibition of CYP11A1 and 3β-HSD.

The Wilms tumor gene (WT1) (+/-KTS) isoforms regulate steroidogenesis by modulating the PI3K/AKT and ERK1/2 pathways in bovine granulosa cells†.

The Wilms tumor (WT) gene WT1 encodes the splicing variants WT1(+KTS) and WT1(-KTS). Recent data suggest that WT1 plays an important role in the development of mice follicles. However, the mechanism through which WT1 influences ovarian steroidogenesis remains unknown. This study identified WT1 and evaluated the impact of splicing variants WT1(+KTS) and WT1(-KTS) on steroidogenesis using adult bovine granulosa cells (GCs). Using RT-qPCR and western blotting, we found that the ratio between WT1(+KTS) and WT1(-KTS) was stabilized. WT1 expression, however, decreased gradually in bovine GCs in response to follicle enlargement or atresia. The downregulation of WT1 increased the secretion of basal and follicle-stimulating-hormone-induced progesterone (P4), but decreased the secretion of basal-induced estradiol (E2). This was associated with an increase in the expression of 3β-HSD, and a decrease in the expression of CYP19A1. In addition, WT1(-KTS) overexpression suppresses the secretion of E2 and P4 compared with WT1(+KTS) overexpression. This was associated with a decrease in the expression of CYP19A1, CYP11A1, and 3β-HSD in cultured bovine GCs. Of note, the downregulation of WT1 suppresses the phosphorylation levels of AKT and p-ERK1/2. However, WT1(-KTS) overexpression promotes the phosphorylation levels of AKT and suppresses p-ERK1/2 levels. LY294002 (AKT inhibitor) increases MKP3 mRNA expression levels but decreases the level of p-AKT and p-ERK1/2. Collectively, WT1 significantly suppresses the mRNA expression of CYP11A1 and 3β-HSD and the secretion of P4 in bovine GCs. Moreover, it regulates CYP19A1 mRNA expression and E2 secretion with complex networks, at least in part, by modulating AKT and ERK1/2 signaling. The effect of WT1(-KTS) was more pronounced than that exerted by WT1(+KTS).

Effect of antiepileptic drug (Topiramate) and cold pressed ginger oil on testicular genes expression, sexual hormones and histopathological alterations in mice

Sexual dysfunction in the epileptic patient is difficult to confirm whether it is ailment or therapy related. Antiepileptic drugs often use in reproductive age, through reproductive progress and maturation. On the other side, cold-pressed oils are rich in bioactive phytochemicals with health-promoting traits. The target of this work was to appraise the sexual dysfunction of antiepileptic Topiramate (TPM) and cold pressed ginger oil (CPGO) as antiepileptic alternative medicine in male mice. Fifty-four adult male albino mice were divided into nine groups (n = 6 mice). One group given saline and used as negative control; another one was given corn oil as vehicle. Six groups administered orally with TPM or CPGO at 100, 200 and 400 mg/kg. Moreover, group of animals co-administrated orally CPGO with TPM (400 mg/kg) to study their interaction. Fatty acid profile and tocols composition of CPGO were determined. in vitro assays were undertaken to evaluate radical scavenging traits of CPGO utilizing sable 1,1-diphenyl-2-picrylhydrazyl (DPPH·) and galvinoxyl radicals. The study investigated antioxidant and oxidative stress markers, sexual hormones levels, mRNA levels of vascular endothelial growth factor (Vegfa), synaptonemal complex protein (Sycp3), Wilms tumor gene (Wt1) as well as histopathological and immunohistochemical examination. Strong radical scavenging potential of CPGO against stable DPPH· and galvinoxyl radicals was recorded. The results revealed that TPM caused a dose-dependent reduction in the antioxidant activities and testosterone content, while, malonaldehyde (MDA) and nitric oxide (NO) as oxidative stress markers were elevated. Vegfa and Sycp3 mRNA expression down-regulated at all Topiramate tested doses, but Wt1 up-regulated at 400 mg/kg. TPM (400 mg/kg) revealed histological alterations associated with strong positive Bax immune reactive spermatogoneal and Leydig cells. Ginger oil elevated the CAT and SOD (antioxidant enzymes), serum testosterone and diminished the oxidative stress, up regulated the expression of Vegfa and Sycp3 and down-regulated the Wt1 expression. Meanwhile, CPGO revealed no histopathological alterations and no Bax immune-reactive cells. CPGO co-administration with TPM (400 mg/kg) attenuated the TPM toxicity. High doses of TPM may exhibit sexual dysfunction but CPGO is safe and has androgenic property. CPGO co-administration could protect the antiepileptic patient from the TPM sexual dysfunction.

A phase III, double-blind, randomized clinical trial comparing S-1 in combination with DC vaccine loaded with WT1 peptides (TLP0-001) or placebo for the patients with advanced pancreatic cancer refractory to standard chemotherapy.

TPS4153 Background: The development of new potent therapeutic is strongly called for treating pancreatic cancer. Dendritic cells (DCs) are a type of antigen-presenting cells that play an important role in adaptive immune system, and this property has prompted their recent application to therapeutic cancer vaccines. DCs loaded with tumor antigen ex vivo and administered as a cellular vaccine have been found to induce therapeutic anti-tumor immunity. Wilms' tumor gene WT1 is expressed in various kinds of cancers including pancreatic cancer. In pilot clinical trials of DC vaccination loaded with WT1 peptides for patients with advanced pancreatic cancer, induction of anti-tumor WT1 specific immune responses and tumor regressions have been observed (Kimura et al, Pancreas, 2012 / Kobayashi et al, Cancer Immunol Immunother, 2014 / Mayanagi et al Cancer Sci, 2015). TLP0-001 is activated Dendritic cells loaded with epitope peptides derived from WT-1. Methods: This is an investigator initiated, phaseIII, multicenter, double-blind, randomized trial of TLP0-001+S-1 versus placebo+S-1 for the patients with locally advanced or metastatic pancreatic cancer refractory to standard chemotherapy. Patients are allocated to either DC vaccine (TLP0-001) + S-1 group or placebo + S-1 in 1:1 ratio by dynamic allocation method. The primary endpoint is overall survival. Sample size is estimated presuming the effects will be observed from the time point of 50% cumulative survival rate. Assuming a type I error alpha (two-sided) level of 5% and a power of 80% or more for hazard ratio 0.644, sample size necessary is estimated as 174 patients. When the first 6 patients are administerd TLP0-001, the independent Data Monitoring Commitee checks safety data to assess continuation of the study. Enrollment begins in March 2017.

The Wilms' tumor (WT1) gene expression correlates with the International Prognostic Scoring System (IPSS) score in patients with myelofibrosis and it is a marker of response to therapy.

The Wilms tumor gene WT1 is a useful marker of clonal hematopoiesis and it has been shown to be a good marker of residual disease and it reflects the response to therapy. Although myelofibrosis is characterized by mutations of JAK2 and calreticulin (CALR), these mutations are not useful to monitor response to therapy. In this study we demonstrated that in patients affected by myelofibrosis WT1 correlates with the International Prognostic Scoring System (IPSS) score at diagnosis. Furthermore WT1 is a good marker of response to JAK2 inhibitors especially for patients without blasts and for patients who develop anemia or thrombocytopenia not for progression but as therapy related toxicity. Finally, WT1 transcript reduction can mirror a benefit of therapy on the disease burden. This study demonstrated that WT1 is a good marker for monitoring the response to therapy in patients affected by myelofibrosis.

Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms.

The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms.

Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

Wilms' tumor gene WT1 promotes homologous recombination-mediated DNA damage repair.

The Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(-)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(-)KTS(+)] isoform significantly inhibited apoptosis by DNA-damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)-mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(-)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(-)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(-)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(-)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(-)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non-small-cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(-)KTS(+)] isoform had a function to promote HR-mediated DNA repair.

Ubiquitin specific protease 18 (Usp18) is a WT1 transcriptional target

Wilms tumor gene WT1 encodes a zinc finger-containing transcription factor which is required for renal development. Mutations in WT1 are observed in 20% of Wilms tumors (a pediatric kidney cancer), but the in vivo WT1 targets and associated molecular pathways involved in the etiology of Wilms tumor are still elusive. To identify WT1 targets we performed genome-wide comprehensive expression profiling using Affymetrix Gene Chip Mouse Genome 430 2.0 Arrays, comparing E13.5 mouse kidneys in which Wt1 had been somatically ablated with littermate controls. We identified Usp18 as the most differentially expressed gene in mutant kidney. Using tetracycline inducible cells we demonstrated a repressive effect of WT1 on USP18 expression. Conversely, knockdown of WT1 led to the upregulation of Usp18. Furthermore, direct binding of WT1 to the Usp18 promoter was demonstrated by ChIP assay. Overexpression of USP18 in murine and human cell lines resulted in cell proliferation. Additionally, Usp18 upregulation was observed in a mouse model of Wilms tumor. Taken together our data demonstrate that Usp18 is a transcriptional target of WT1 and suggest that increased expression of USP18 following WT1 loss contributes to Wilms tumorigenesis.

T-cell Immune Abnormality and Novel Immunotherapeutic Approach by Blocking the B7-H1-PD-1 Pathway Combined with WT1 Tumor Vaccine in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are hematologic malignancies caused by the neoplastic transformation of hematopoietic stem or myeloid progenitor cells and characterized by cytopenias, excessive apoptosis of hematopoietic cells, and a high risk of progression to acute myeloid leukemia. During disease progression, clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Stem cell transplantation is the only curative treatment for MDS, but it is not suitable for elderly patients. B7-H1 (CD274) molecules, which are expressed on antigen-presenting cells and inhibit T-cell responses through programmed death-1 (PD-1) expressed on activated T cells, are expressed on various tumor cells, and B7-H1-expressing tumor cells evade attack by tumor-specific cytotoxic T lymphocytes (CTLs). We reported immune dysfunction associated with T cells in MDS, i.e., an increase in T-cell apoptosis, higher expression levels of PD-1 on circulating T cells, and higher levels of plasma-soluble interleukin-2 receptor. Furthermore, we demonstrated that MDS blasts overexpress Wilms tumor gene WT1 mRNA, suggesting that the anti-WT1 immune response elicited by WT1 peptide vaccine may induce tumor regression in some patients. In the current study, we investigated B7-H1 expression on MDS blasts and analyzed the characteristics and inhibitory effects on T-cell immune responses of B7-H1 blasts. Finally, to develop new strategies, we are now analyzing a novel immunotherapy involving blockade of the B7-H1-PD-1 pathway in combination with WT1 tumor vaccine and or inhibition of regulatory T cells. First, we analyzed B7-H1 expression in 3 MDS cell lines, i.e., F-36P, OIH-1, and SKM-1, and on MDS blasts from 29 MDS patients, 32 patients with acute myeloid leukemia transformed from MDS (AL-MDS), and 10 hematologically normal individuals. A high level of B7-H1 expression at the mRNA and protein levels was detected only in F-36P cells using reverse transcription-PCR and flow cytometry (FCM), respectively. B7-H1 protein was not detectable on OIH-1 and SKM-1. Blasts from patients with high-risk MDS and AL-MDS expressed B7-H1 molecules more often compared with those from low-risk MDS patients. B7-H1 molecules were expressed in fewer than 5% of blasts in normal individuals. Furthermore, we found that the cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which may be associated with MDS pathophysiology, induced B7-H1 expression on SKM-1 cells or blasts from MDS patients, and that B7-H1 induction by these cytokines was mediated by nuclear factor (NF)-κB activation, whose activation was observed in MDS patients, in particular in advanced disease. Second, to investigate the characteristics of B7-H1 blasts, we analyzed proliferative advantage, i.e., cell cycle and colony formation, in B7-H1 and B7-H1 cell fractions in MDS blasts using FCM and the methylcellulose assay, respectively. B7-H1 MDS blasts had greater intrinsic proliferative capacity than B7-H1 MDS blasts when examined in both assays. Third, to investigate the immunomodulatory effects of B7-H1 MDS blasts on T-cells, T-cell apoptosis and

Gemcitabine enhances Wilms' tumor gene WT1 expression and sensitizes human pancreatic cancer cells with WT1-specific T-cell-mediated antitumor immune response.

Wilms' tumor gene (WT1), which is expressed in human pancreatic cancer (PC), is a unique tumor antigen recognized by T-cell-mediated antitumor immune response. Gemcitabine (GEM), a standard therapeutic drug for PC, was examined for the regulation of WT1 expression and the sensitizing effect on PC cells with WT1-specific antitumor immune response. Expression of WT1 was examined by quantitative PCR, immunoblot analysis, and confocal microscopy. Antigenic peptide of WT1 presented on HLA class I molecules was detected by mass spectrometry. WT1-specific T-cell receptor gene-transduced human T cells were used as effecter T cells for the analysis of cytotoxic activity. GEM treatment of human MIAPaCa2 PC cells enhanced WT1 mRNA levels, and this increase is associated with nuclear factor kappa B activation. Tumor tissue from GEM-treated MIAPaCa2-bearing SCID mice also showed an increase in WT1 mRNA. Some human PC cell lines other than MIAPaCa2 showed up-regulation of WT1 mRNA levels following GEM treatment. GEM treatment shifted WT1 protein from the nucleus to the cytoplasm, which may promote proteasomal processing of WT1 protein and generation of antigenic peptide. In fact, presentation of HLA-A*2402-restricted antigenic peptide of WT1 (CMTWNQMNL) increased in GEM-treated MIAPaCa2 cells relative to untreated cells. WT1-specific cytotoxic T cells killed MIAPaCa2 cells treated with an optimal dose of GEM more efficiently than untreated MIAPaCa2 cells. GEM enhanced WT1 expression in human PC cells and sensitized PC cells with WT1-specific T-cell-mediated antitumor immune response.

Design and Construction of Multi-Epitope Fusion Gene Vaccine of Wilm Tumor Gene WT1 and Stimulating Epitope of Mycobacterium Tuberculosis Heat Shock Protein 70

AbstractAbstract 4713 ObjectiveDNA vaccination respresents a novel therapeutic strategy for cancer. Researchs have showed DNA vaccines encoding Wilms tumor gene WT1-Derived HLA *0201-restricted epitopes effectively induce epitope-specific human cytotoxic T-lymphocytes (CTLs) and kill human leukemia cells in vitro. Nevertheless, there is still a need to increase the potency of DNA vaccine and span the HLA diversity of a target population. In this study, we designed polytpoe WT1 DNA vaccine encoding multiple CTL and helper T-lymphocyte (HTL) epitopes and containing HSP70407-426 as immunoadjuvants for immunotherapy. MethodsOn the basis published data, To design a Multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted, one HLA *2402-restricted epitopes, two HTL epitopes and one universal Th Pan- DR epitope (PADRE) sequence. The construct also included IgG K chain leader sequence as signal peptide to facilitate transport into the endoplasmic reticulum and Kozak sequence at the N-terminus to support translation of the gene transcript. DNA-coding sequesces was used computer-based modeling to optimize proteasome mediated epitope processing through the introdcution of amino acid spacer sequences, then cloned into the eukaryotic vector to construct the plasmid PcDNA3.1(+). Mycobacterial HSP70 fragment including stimulatory domain P407-426 as immunotherapy was fused to the C-terminal of Multi-WT1 for enhancing CTL activity and then inserted to PcDNA3.1(+). Transfection HEK-293T cells with the Multi -WT1-HSP70 and identify the expressed product by Western bolt. ResultsMost of Multi -WT1 epitopes can be cleaved by software Net Chop 3.1 and PAPROC± analysis. Western blot showed correct expression of target gene in HEK293T cells. ConclusionMulti-WT1-mtHSP70 fusion gene eukaryotic expression vector was successfully designed, which prepared the materials for the research of DNA vaccine. Disclosures:No relevant conflicts of interest to declare.

Expression of the Wilm’s tumor gene WT1 during diaphragmatic development in the nitrofen model for congenital diaphragmatic hernia

The nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm's tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1(-/-) mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the nitrofen CDH model. Pregnant rats received vehicle or nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p<0.05. Diaphragmatic mRNA expression of WT1 was significantly decreased in the nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21. Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the nitrofen CDH model.

The Wilms' tumour gene WT1 is involved in bone metabolism

The Wilms' tumour gene WT1 is involved in bone metabolism

Clinical trials of WT1 peptide vaccine to gynecologic malignancies that are resistant to conventional therapies.

5081 Background: Wilms' tumor gene WT1, which has an oncogenic function, is expressed in various kinds of hematological malignancies and solid cancers. The WT1 protein is highly immunogenic and is considered to be a promising target of cancer treatment. On the clinical trials of WT1 peptide vaccine, the decrease of leukemic cells, shrinkage of solid tumor and long-term dormancy of malignancies are reported. We intend to report the clinical trials of WT1 peptide vaccine to gynecologic malignancies which are resistant to conventional therapies. Methods: Twenty one cases of gynecologic malignancy (12 cases of ovarian cancer, 4 of cervical cancer, 1 of endometrial cancer and 4 cases of other malignant tumor), which are resistant to first-/second-line therapy, were included to the trial. After obtainment of informed consent, modified WT1235 type peptide is injected intracutaneously to the HLA-A*2402- positive patients, whose tumor tissues expressed WT1 protein. The injection was repeated weekly to total of 12 times. Immune response to WT1 peptide was monitored with delayed type hypersensitivity (DTH). Side effect due tothe injection was also monitored. The antitumor effect was assessed based on Response Evaluation Criteria in Solid Tumors (RECIST). If any clinical effect is observed, the injection of the vaccine was continued biweekly. Results: In 15 (71%) of 21 patients, the 12 times of injection are completed. In remaining 6 cases, injection had been stopped due to worsening of performance status. Only 1 patient showed grade 3 vesicle at the injection site, however, no patient was cancelled the injection due to severe side effect. The SD status in RECIST criteria is observed in 5 (24%) patients and the injection is continued for these 5 patients, with the maximum of 29 times. Patients who is positive to DTH reaction during therapy, showed significantly longer overall survival than patients who are negative to DTH through the treatment. Conclusions: It was considered that the induction of WT1-specific immunological responses contributed to the occurrence of clinical responses. Some patients reached long-term dormancy of the tumor. We recognized the WT1 peptide vaccine is a new strategy for gynecologic malignancies. No significant financial relationships to disclose.

WT1 (Wilms' Tumor Gene 1): Biology and Cancer Immunotherapy

Wilms' tumor gene WT1 encodes a transcription factor and plays an important role in cell growth and differentiation. The WT1 gene is highly expressed in leukemia and various types of solid tumors, whereas WT1 is a tumor marker convenient for the detection of minimal residual disease of leukemia. The WT1 gene was originally defined as a tumor suppressor gene, but we proposed that it was, on the contrary, an oncogene. Furthermore, the WT1 protein has proven to be a promising tumor-associated antigen, in which many human leukocyte antigen class I- or II-restricted WT1 epitopes have been identified. Clinical trials of WT1-targeted immunotherapy have confirmed its safety and clinical efficacy. WT1-specific cytotoxic T lymphocytes and WT1 antibodies are spontaneously induced in tumor-bearing patients, probably because of high immunogenicity of the WT1 protein. WT1-specific cytotoxic T lymphocytes make a major contribution to the graft-versus-leukemia effect after allogenic stem cell transplantation. When 75 cancer antigens including WT1 were prioritized according to several criteria such as therapeutic function and immunogenicity, WT1 was ranked as the top antigen. These findings suggest that a new era of WT1 immunotherapy is imminent.

Real-Time Quantitative Polymerase Chain Reaction Detection of Minimal Residual Disease by Standardized WT1 Assay to Enhance Risk Stratification in Acute Myeloid Leukemia: A European LeukemiaNet Study

Risk stratification in acute myeloid leukemia (AML) is currently based on pretreatment characteristics. It remains to be established whether relapse risk can be better predicted through assessment of minimal residual disease (MRD). One proposed marker is the Wilms tumor gene WT1, which is overexpressed in most patients with AML, thus providing a putative target for immunotherapy, although in the absence of a standardized assay, its utility for MRD monitoring remains controversial. Nine published and in-house real-time quantitative polymerase chain reaction WT1 assays were systematically evaluated within the European LeukemiaNet; the best-performing assay was applied to diagnostic AML samples (n = 620), follow-up samples from 129 patients treated with intensive combination chemotherapy, and 204 normal peripheral blood (PB) and bone marrow (BM) controls. Considering relative levels of expression detected in normal PB and BM, WT1 was sufficiently overexpressed to discriminate > or = 2-log reduction in transcripts in 46% and 13% of AML patients, according to the respective follow-up sample source. In this informative group, greater WT1 transcript reduction after induction predicted reduced relapse risk (hazard ratio, 0.54 per log reduction; 95% CI, 0.36 to 0.83; P = .004) that remained significant when adjusted for age, WBC count, and cytogenetics. Failure to reduce WT1 transcripts below the threshold limits defined in normal controls by the end of consolidation also predicted increased relapse risk (P = .004). Application of a standardized WT1 assay provides independent prognostic information in AML, lending support to incorporation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification, and to identify patients who may benefit from allogeneic transplantation.

A WT1 protein‐derived, naturally processed 16‐mer peptide, WT1332, is a promiscuous helper peptide for induction of WT1‐specific Th1‐type CD4+ T cells

The Wilms' tumor gene WT1 is overexpressed in various tumors, and the WT1 protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. A WT1 protein-derived 16-mer peptide, WT1(332) (KRYFKLSHLQMHSRKH), which was naturally generated through processing in cells and could elicit Th1-type CD4(+) helper T cell responses with an HLA-DRB1*0405-restriction has previously been identified by us. In the present study, it has been demonstrated that WT1(332) can induce WT1(332)-specific CD4(+) T cell responses with the restriction of not only HLA-DRB1*0405 but also HLA-DRB1*1501, -DRB1*1502, or -DPB1*0901. These HLA class II-restricted WT1(332)-specific CD4(+) T cell lines produced IFN-gamma but neither IL-4 nor IL-10 with WT1(332) stimulation, thus showing a Th1-type cytokine profile. Furthermore, HLA-DRB1*1501 or -DRB1*1502-restricted WT1(332)-specific CD4(+) T cell lines responded to WT1-expressing transformed cells in an HLA-DRB1-restricted manner, which is consistent with our previous finding that WT1(332) is a naturally processed peptide. These results indicate that the natural peptide, WT1(332), is a promiscuous WT1-specific helper epitope. WT1(332) is expected to apply to cancer patients with various types of HLA class II as a WT1-specific helper peptide in combination with HLA class I-restricted WT1 peptides.

Expression of WT-1 mRNA in Peripheral Blood from Myelodysplastic Syndromes

(INTRODUCTION) The Wilms' tumor gene WT-1 is overexpressed in various types of solid tumor as well as in hematologic malignancies, i.e., acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myeloid leukemia, and myelodysplastic syndromes (MDS). It was reported that WT-1 overexpression in peripheral blood (PB) and bone marrow (BM) is useful for the monitoring of minimal residual disease and early detection of relapse in AML. MDS are clonal hematologic stem cell disorders characterized by cytopenias and a risk of progression to acute leukemia. It is important for decisions on treatment strategy to assess disease progression and predict the prognosis in MDS. The aim of this study was to investigate the clinical significance of WT-1 mRNA expression in PB obtained from patients with MDS.(METHODS AND RESULTS) PB was obtained from 92 patients with MDS or leukemia transformed from MDS (AL-MDS): 40 refractory anemia (RA), 5 RA with ringed sideroblasts (RARS), 27 RA with excess blasts (RAEB), 5 RAEB in transformation (RAEB-t), and 15 AL-MDS cases in the FAB classification (RA 27, RARS 5, refractory cytopenia with multilineage dysplasia 12, RAEB-1 13, RAEB-2 14, 5q- 1, and AL-MDS 20 cases in the WHO classification). RNA was isolated from PB mononuclear cells (PBMCs) and converted into cDNA. The levels of WT-1 mRNA expression were assessed using the real-time quantitative polymerase chain reaction. We analyzed whether the level of WT-1 mRNA expression in PBMCs was associated with MDS subtype in the FAB classification, clinical characteristics (hemoglobin value, white blood cell counts, neutrophil counts, lymphocyte counts, chromosomal abnormality, number of cytopenias, blast percentages in BM, lactate dehydrogenase values, C-reactive protein values), International Prognostic Scoring System (IPSS) score, survival, and time to leukemia transformation. High expression of WT-1 mRNA, which was defined as more than 50 copies/μg mRNA according to the results of normals, was observed in 42.5%, 40.0%, 85.2%, 80.0%, and 100% of RA, RARS, RAEB, RAEB-t, and AL-MDS patients, respectively. The WT-1 mRNA levels increased with the aggressiveness of disease subtype and IPSS. FAB subtypes included RARS (mean ± SD, 129 ± 111 copies/μg), RA (220 ± 134), RAEB (5554 ± 2593), RAEB-t (14284 ± 9056), and AL-MDS (51591 ± 30309). IPSS score were divided into low/intermediate-1 risk (316 ± 136) and intermediate-2/high risk (7901 ± 3035) (P < 0.05 for RA vs RAEB, RAEB-t or AL-MDS; RAEB vs AL-MDS; and low/intermediate-1 vs intermediate-2/high risk). Furthermore, the WT-1 mRNA level was inversely correlated with the neutrophil percentage in PB and positively correlated with the blast percentage in both PB and BM. Among RA patients, those with favorable cytogenetics had lower WT-1 mRNA values compared with other patients (P < 0.05). When patients were divided into three groups based on the log value of WT-1 mRNA (<2, 2–4, and >=4), the survival times of those groups were 73.1, 55.6, and 15.3 months, respectively (P < 0.005).(CONCLUSIONS) Higher quantitative expression of WT-1 mRNA in PBMCs is associated with aggressive disease behavior in MDS patients. This may justify anti-WT-1 immunotherapy under investigation for MDS treatment (PNAS 2004; 101:13885–13890).