A Novel WT1 Mutation Identified in a 46,XX Testicular/Ovotesticular DSD Patient Results in the Retention of Intron 9

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for −KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no −KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/−KTS ratio.

Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion

Background. The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results. Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen ( p ≤ 0.05 ). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions. Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

First-in-Human Study of WT1 Recombinant Protein Vaccination in Elderly Patients with AML in Remission: A Single-Center Experience

Background Vaccination (vac) strategies to maintain remissions in AML have been pursued for decades. The usage of recombinant proteins instead of peptides allows a potential immune response to multiple epitopes, hence could be offered to all patients (pts) independent of HLA expression. Wilms' tumor 1 (WT1) protein is highly immunogenic and frequently overexpressed in AML, thus ranked as a very promising target for novel immunotherapies. Here we report a single-center experience of a phase I/II clinical trial (NCT01051063) of a first-in-human vac strategy based on WT1 recombinant protein (WT1-A10) together with vaccine adjuvant AS01 B in 5 elderly AML pts. Patients and Methods Key eligibility criteria: overexpression of WT1 transcripts in AML blasts at diagnosis (qRT-PCR); 1 or 2 induction chemotherapies, with partial remission (PR) or morphologic complete remission with incomplete blood count recovery (CRi). The vaccine consisted of WT1-A10, a truncated WT-1 protein retaining the N-terminus (amino acids 2-281) of full length WT1 protein (429 aa) linked to the first 11 amino acids of trimethylamine N-oxide reductase signal peptide via one histidine residue combined with the liquid AS01 B adjuvant. AS01 B is an Adjuvant System containing MPL (3-O-desacyl-4´-monophosphoryl lipid A), QS-21 (Quillaja saponaria Molina, fraction 21) and liposome (50µg MPL and 50µg QS-21). One human dose of WT1-A10 + AS01 B contained 200 μg of WT1-A10 antigen. Pts received the vaccine by i.m. injection. To assess cellular response, antigen-specific stimulation of cultured PBMCs was performed with a pool of 123 15mer peptides covering the entire WT1 (1μg/ml/peptide), together with irrelevant re-stimulation plus negative control peptide. CD4 + and CD8 + T cells were serially assessed by intracellular flow cytometry for their ability to produce both IFN-γ and TNF upon antigen stimulation. Results A total of 5 pts (median age 69, range 63-75) were enrolled on the WT1 protein-based vac study at our institution (Table 1), receiving a total of 62 vac after a median of 3 courses (1-5) of standard chemotherapy. The repeated vac had an acceptable safety profile and were thus well tolerated. 2/5 pts experienced therapy-related toxicity, injection site pain (CTCAE v.3, grade 2) and injection site inflammation (CTCAE v.3, grade 1). Symptoms were of mild / moderate severity and resolved completely. No hematologic toxicity was noted. With a median progression-free survival of 28.8 mths (range 1-59) and median overall survival (OS) of 35.4 mths (range 3-75) from the 1st vac, this older patient cohort showed above-average clinical outcome (Table 1), pointing to a potential clinical efficacy of WT1-based vac therapy. All vaccinated pts showed highly elevated WT1 ratios before WT1-based vac therapy and normal levels after vac (Fig. 1A). Two pts demonstrated early relapse after 3 WT1 protein-based vac, and clinical benefit was observed in 3 pts: one achieved complete and sustained measurable residual disease clearance (NPM1 ratios) during WT1 vac, resulting in molecular CR at the 18th vac. The pt died from unrelated reasons 5.5 years after initial diagnosis of AML, 3.5 years after the last WT1 vac, with continued molecular CR. One pt maintained long-term hematological and molecular remission over 59 mths, until molecular relapse occurred 11 mths after the final, 21 st vac. Interestingly, in one case, a complete clonal switch occurred at hematologic relapse following 18 vac, with loss of WT1 overexpression: while the clone at initial diagnosis harbored FLT3, NPM1 and SRSF2 mutations, BRAF, KRAS and STAG2 mutations were detected at relapse (Fig. 1B), pointing to an ongoing suppression of the WT1 expressing AML clone. Flow cytometry studies were conducted in one pt to elucidate specific cellular immune responses. We noted CD4 + T cell immune responses by strong IFN-γ and TNF expression (Fig. 1C), suggestive of efficient immune stimulation post-vac, while CD8 + T cells failed to upregulate these key cytokines. Conclusions This vac strategy showed good feasibility, with a very acceptable safety profile, and appeared to extend remissions beyond the expected duration, together with MRD clearance. Thus, our data provide evidence of potential clinical efficacy of WT1 protein-based vac therapy in AML pts, making this maintenance approach an attractive alternative to more complex strategies, particularly in elderly pts with comorbidities. Figure 1 Figure 1. Disclosures Döhner: Abbvie: Consultancy, Honoraria; Jazz Roche: Consultancy, Honoraria; Daiichi Sankyo: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Celgene/BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Astellas: Research Funding; Agios and Astex: Research Funding. Schmitt: MSD: Membership on an entity's Board of Directors or advisory committees; TolerogenixX: Current holder of individual stocks in a privately-held company; Bluebird Bio: Other: Travel grants; Hexal: Other: Travel grants, Research Funding; Novartis: Other: Travel grants, Research Funding; Kite Gilead: Other: Travel grants; Apogenix: Research Funding. Lübbert: Teva: Research Funding; Janssen: Research Funding; Cheplapharm: Research Funding; Aristopharm: Research Funding; Syros: Honoraria; Pfizer: Honoraria; Janssen: Honoraria, Research Funding; Imago BioSciences: Honoraria; Hexal: Honoraria; Astex: Honoraria; Abbvie: Honoraria.

Expression of the Wilms’ tumour gene and its association with PPARβ/δ in healthy skin and melanoma of horses

The Wilms’ tumour gene (WT1) has previously been described as an oncogene in several neoplasms of humans, including melanoma, and its expression increases cancer cell proliferation. Recent reports associate the expression of the PPARβ/δ gene (peroxisome proliferator-activated receptor beta/delta) with the downregulation of WT1 in human melanoma and murine melanoma cell lines. The aim of this work was to analyse the expression of WT1 and its association with PPARβ/δ in samples of healthy and melanoma-affected skin of horses by immunohistochemistry. WT1 protein expression was detected in healthy skin, mainly in the epidermis, hair follicle, sebaceous gland and sweat gland, while no expression was observed in equine melanoma tissues. Moreover, it was observed that PPARβ/δ has a basal expression in healthy skin and that it is overexpressed in melanoma. These results were confirmed by a densitometric analysis, where a significant increase of the WT1-positive area was observed in healthy skin (128.66 ± 19.84 pixels 106) compared with that observed in melanoma (1.94 ± 0.04 pixels 106). On the other hand, a positive area with an expression of PPARβ/δ in healthy skin (214.94 ± 11.85 pixels 106) was significantly decreased compared to melanoma (624.86 ± 181.93 pixels 106). These data suggest that there could be a regulation between WT1 and PPARβ/δ in this disease in horses.

Cytotoxic and Antiproliferative Effects of Diarylheptanoids Isolated from Curcuma comosa Rhizomes on Leukaemic Cells

Curcuma comosa belongs to the Zingiberaceae family. In this study, two natural compounds were isolated from C. comosa, and their structures were determined using nuclear magnetic resonance. The isolated compounds were identified as 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (1) and trans-1,7-diphenyl-5-hydroxy-1-heptene (2). Compound 1 showed the strongest cytotoxicity effect against HL-60 cells, while its antioxidant and anti-inflammatory properties were stronger than those of compound 2. Compound 1 proved to be a potent antioxidant, compared to ascorbic acid. Neither compounds had any effect on red blood cell haemolysis. Furthermore, compound 1 significantly decreased Wilms’ tumour 1 protein expression and cell proliferation in KG-1a cells. Compound 1 decreased the WT1 protein levels in a time- and dose- dependent manner. Compound 1 suppressed cell cycle at the S phase. In conclusion, compound 1 has a promising chemotherapeutic potential against leukaemia.

Wilms' Tumor 1 (WT1): The Vaccine for Cancer

ABSTRACT Vaccines have been used to fight and protect against infectious diseases for centuries. With the emergence of immunotherapy in cancer treatment, researchers began investigating vaccines that could be used against cancer, especially against tumors that are resistant to conservative chemotherapy, surgery, and radiotherapy. The Wilms' tumor 1 (WT1) protein is immunogenic, has been detected in almost all types of malignancies, and has played a significant role in prognosis and disease monitoring. In this article, we review recent developments in the treatment of various types of cancers with the WT1 cancer vaccine; we also discuss theoretic considerations of various therapeutic approaches, which were based on preclinical and clinical data.

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms’ tumor 1 (WT1) gene

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining geneSRYis present in many cases, the etiology is unknown in mostSRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms’ tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P= 4.4 × 10−6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P< 1.8 × 10−4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts andWt1Arg495Gly/Arg495GlyXX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor β-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Antileukemic Cell Proliferation of Active Compounds from Kaffir Lime (Citrus hystrix) Leaves

Kaffir lime (Citrus hystrix) is a plant member of family Rutaceae, and its leaves are commonly used in folk medicine. The present study explores antileukemic effects of the extracts and purified active compounds from the leaves. The antileukemic activity was investigated via inhibition of Wilms’ tumor 1 (WT1), which is a protein that involves in leukemic cell proliferation. In addition, the compounds were investigated for their effects on WT1 gene expression using real time RT-PCR and Western blotting. Cell cycle arrest and total cell number were investigated using flow cytometry and trypan blue exclusion method, respectively. The results demonstrated that the hexane fractionated extract had the greatest inhibitory effect on WT1 gene expression of many leukemic cell lines and significantly decreased WT1 protein levels of K562 cells (representative of the leukemic cells), in a dose- and time-dependent manner. Subfraction No. 9 (F9) after partial purification of hexane fractioned extract showed the highest suppression on WT1 protein and suppressed cell cycle at G2/M. The organic compounds were isolated from F9 and identified as phytol and lupeol. The bioassays confirmed antiproliferative activities of natural products phytol and lupeol. The results demonstrated anticancer activity of the isolated phytol and lupeol to decrease leukemic cell proliferation.

Immunohistochemical Expression of Wilms’ Tumor 1 Protein in Human Tissues: From Ontogenesis to Neoplastic Tissues

The human Wilms’ tumor gene (WT1) was originally isolated in a Wilms’ tumor of the kidney as a tumor suppressor gene. Numerous isoforms of WT1, by combination of alternative translational start sites, alternative RNA splicing and RNA editing, have been well documented. During human ontogenesis, according to the antibodies used, anti-C or N-terminus WT1 protein, nuclear expression can be frequently obtained in numerous tissues, including metanephric and mesonephric glomeruli, and mesothelial and sub-mesothelial cells, while cytoplasmic staining is usually found in developing smooth and skeletal cells, myocardium, glial cells, neuroblasts, adrenal cortical cells and the endothelial cells of blood vessels. WT1 has been originally described as a tumor suppressor gene in renal Wilms’ tumor, but more recent studies emphasized its potential oncogenic role in several neoplasia with a variable immunostaining pattern that can be exclusively nuclear, cytoplasmic or both, according to the antibodies used (anti-C or N-terminus WT1 protein). With the present review we focus on the immunohistochemical expression of WT1 in some tumors, emphasizing its potential diagnostic role and usefulness in differential diagnosis. In addition, we analyze the WT1 protein expression profile in human embryonal/fetal tissues in order to suggest a possible role in the development of organs and tissues and to establish whether expression in some tumors replicates that observed during the development of tissues from which these tumors arise.