Wilms' Tumor 1 Overexpression Research Articles

Prognostic significance of Wilms' tumor gene 1 expression in children with B-cell precursor acute lymphoblastic leukemia.

The prognostic role of Wilms' tumor 1 (WT1) gene expression at diagnosis in children with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is still controversial. We detected the WT1 transcript levels of 533 de novo pediatric BCP-ALL patients using TaqMan-based real-time quantitative PCR and analyzed their clinical features. The WT1 transcript levels differed among the distinct molecularly defined groups, with the highest levels in the KMT2A rearrangements (KMT2A-r) group. According to the results of the X-tile software, all patients were divided into two groups: WT1/ABL ≥ 0.24% (group A) and <0.24% (group B). The proportions of patients whose age was ≥10 years old, with immunophenotype of Pro-B, belonging in high-risk group, or with minimal residual disease (MRD) ≥ 0.01% at week 12 were significantly higher in group A than in group B. In the B-other group, WT1 overexpression was an independent risk factor of overall survival (OS) rate (P = 0.042), and higher MRD ≥ 0.01% at week 12 was associated with lower OS rate (P<0.001) and event-free survival rate (P<0.001). Moreover, the subgroup analysis revealed that, in patients with initial WBC<50 × 109/L or MRD<0.1% at day 33 or MRD<0.01% at week 12 or in the standard-risk group, WT1 overexpression led to a poorer outcome in comparison with those with WT1 downexpression (P<0.05). Therefore, pediatric BCP-ALL with WT1 overexpression had unique clinico-pathological characteristics and poor treatment response. In B-other patients, WT1 overexpression at diagnosis predicted an inferior prognosis. The WT1 gene may serve as a biomarker for monitoring residual disease in the B-other population, especially in children in the standard-risk group.

WT1 Inhibits Human Renal Carcinoma Cell Proliferation and Induces G2/M Arrest by Upregulating IL-24 Expression.

The transcription factor Wilms' tumor 1 (WT1) is involved in development, tissue homeostasis, and disease. However, the exact roles and the mechanisms of WT1 in renal carcinoma are not well understood. Therefore, in this study, we evaluated the ability of WT1 to block proliferation in renal carcinoma cells in vitro. Experimental analysis showed that WT1 overexpression inhibited the proliferation of renal carcinoma A498 cells and promoted arrest at the G2/M checkpoint. RNA-Seq identified differentially expressed genes, including IL-24, related to both the cell proliferation and the cell cycle. WT1 overexpression upregulated IL-24 expression, and IL-24 overexpression induced G2/M arrest. ChIP-Seq identified JUN as a direct target of WT1 in A498 cells, in which positive regulation was shown by RT-qPCR. It has been shown that the transcription factor JUN can regulate IL-24 expression, and therefore, we hypothesize that WT1 might regulate the IL-24 through JUN. Furthermore, analysis based on TCGA datasets showed that the expression of WT1-regulated genes, including TXNIP and GADD45A, was significantly correlated with the stage and histological grade of tumors, with high levels linked to favorable prognoses. Our results demonstrated that the overexpression of WT1 upregulates IL-24, leading to G2/M checkpoint arrest to reduce proliferation. These results indicate that regulation of IL-24 by WT1 inhibits proliferation and may represent a potential target for treating renal carcinoma.

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

BackgroundVaccination (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 MethodsKey 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.ResultsA 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.ConclusionsThis 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. [Display omitted] DisclosuresDöhner: 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.

Overexpression of Wilms tumor 1 promotes IL-1β expression by upregulating histone acetylation in keratinocytes

Psoriasis is a common inflammatory skin disease. Infiltration of inflammatory cells and excessive proliferation of keratinocytes are the histopathological markers of psoriasis. The transcription factor Wilms Tumor 1 (WT1) is overexpressed in several tumor types, and plays an important part in the proliferation and apoptosis of cells. Studies have found that, compared with normal skin, WT1expression in the skin lesions of patients with psoriasis are increased significantly. Knockdown of WT1 inhibited the proliferation of a human epidermal keratinocyte cell line (HaCaT cells) and promoted their apoptosis, whereas WT1 overexpression exhibited the opposite effect. WT1 was overexpressed or inhibited in HaCaT cells by transfection with the WT1 plasmid or WT1 small interferring RNA (siRNA) using Lipofectamine 2000. Transcriptome sequencing and bioinformatics analysis revealed significant differences in IL-1β expression between the experimental group and control group. Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assays showed that expression of IL-1β and WT1 were consistent. Subsequently, IL-1β was demonstrated to be a target of WT1 by chromatin immunoprecipitation (ChIP)-sequencing and luciferase reporter assay. ChIP-qPCR showed that WT1 regulated IL-1β expression by altering acetylation. Expression of WT1 mRNA was positively correlated with expression of IL-1β mRNA in psoriatic skin lesions. Our study suggested that WT1 likely promotes psoriasis development by regulating its target gene IL-1β, which shows high expression in psoriatic lesions and is involved in psoriasis development. These findings provide a new target for psoriasis treatment.

The Prognostic Impact of WT1 Expression Levels, Mutations, and SNP rs16754 in AML Patients: A Retrospective Cohort Study

Background & Objective: The clinical outcomes and treatment options for acute myeloid leukemia (AML) patients are highly dependent upon molecular markers. In this study, Wilms tumor 1 (WT1) (exons 7 and 9) mutations, single-nucleotide polymorphism (SNP) rs16754, and WT1 expression levels in 130 random AML patients were screened; FMs-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD), nucleophosmin (NPM1), and CCAAT/enhancer-binding protein alpha (CEBPA) mutations were also evaluated. Materials & Methods: Overall, 130 AML patients were recruited for this study. WT1 mutations were determined by Sanger sequencing, and expression levels were determined by real-time polymerase chain reaction (PCR). The Kaplan-Meier method was used to calculate overall survival (OS) and disease-free survival (DFS). Results: The frequency of WT1 mutations in the study population was 5.4, and it did not affect OS (P=0.98), DFS (P=0.97), or complete remission (CR) rates in AML patients. The major allele of SNP rs16754 in the current study was A. No significant differences were found for OS (P=0.52), DFS (P=0.42), or CR rates among all SNP rs16754 genotypes. The overexpression of WT1 was observed in 83 of patients at diagnosis. No significant difference was found for OS (P=0.84), DFS (P=0.82), or CR rates between AML patients with high and low WT1 expression levels. Conclusion: The results of the current study do not support WT1 mutation, SNP rs16754, or WT1 overexpression at diagnosis, as they were found to be poor prognostic markers in AML patients. © 2021, Zanjan University of Medical Sciences and Health Services. All rights reserved.

Optimal time-points for detecting expression levels of BAALC, EVI1, and WT1 genes in patients with acute myeloid leukemia: a meta-analysis

ABSTRACT Objectives This meta-analysis examined the prognostic role of brain and acute leukemia, cytoplasmic (BAALC), Ecotropic virus integration site-1 (EVI1) and Wilms’ tumor 1 (WT1) genes at different time-points during conventional chemotherapy. Methods A systematic search of publications indexed in the electronic databases from January 1988 to October 2020 was performed. Over 7525 cases of AML from 25 studies were involved. Results At diagnosis, overexpression of either BAALC or EVI1 had a negative impact on complete remission achievement (Summary Odds ratios [SORs] for BAALC = 0.32; SORs for EVI1 = 0.49) and survival outcome. The summary hazard ratios of overall survival (OS) and disease-free survival (DFS) were 1.97 and 2.04 for BAALC and 1.33 and 1.86 for EVI1, respectively. The prognostic value of pretreatment WT1 levels was heterogeneous while subgroup analyses unveiled that overexpressed WT1 may correlate with a favorable outcome (summary hazard ratio [SHR] for OS = 0.42). Both WT1 and BAALC played a role in prognosis assessment at post-induction and the diagnostic performance of WT1 transcript reduction was superior to the absolute WT1 level. Post-consolidation WT1 overexpression consistently indicated an increased risk of relapse, while the combined HR for RFS was statistically insignificant (SHR = 4.22). Conclusion These findings confirm the application of BAALC and EVI1 at diagnosis, WT1 after induction chemotherapy in AML patients throughout conventional chemotherapy.

WT1 regulates cyclinA1 expression in K562cells.

The restricted expression of Wilms tumor1 (WT1) and cyclinA1 (CCNA1) in normal tissues, as opposed to their abnormal expression in leukemia demonstrates the applicability of WT1 and CCNA1 as cancer antigens for immunotherapy, and as markers for prognosis and relapse. In this study, the WT1 and CCNA1 mRNA levels were found to be elevated in bone marrow samples from pediatric acute promyelocytic leukemia (APL or AML‑M3) patients, and to be quite varied in pediatric acute lymphocytic leukemia (ALL) patients, compared to non‑leukemic bone marrow controls. Consistent with the observed upregulation of both WT1 and CCNA1 in APL, WT1 overexpression elevated the CCNA1 mRNA levels in K562 leukemia cells. Treatment with curcumin decreased the WT1 levels in K562cells, and also decreased CCNA1 protein expression. The examination of the CCNA1 promoter identified potential canonical WT1 binding sites within the 3‑kb region upstream of the transcription start site. Chromatin immunoprecipitation and luciferase reporter assays confirmed WT1 binding and the activation of the CCNA1 promoter. Furthermore, the GC‑rich core CCNA1 promoter region provided additional non‑canonical WT1 activation sites, as revealed by promoter assays. The importance of the GC‑rich core region of the CCNA1 promoter was confirmed by treating the K562cells with mithramycinA, which blocks the binding of zinc finger transcription factors to GC‑rich sequences. MithramycinA subsequently suppressed both CCNA1 promoter activity and protein expression in the K562cells. Taken together, the data from the WT1 overexpression, and curcumin and mithramycinA treatment experiments, as well as those from chromatin binding assays, along with inferences from patient RNA analyses, establish a plausible link between WT1 and CCNA1, and support the functional significance of an elevated WT1 expression in leukemia, which may also affect CCNA1 expression.

Study of Wilms’ tumor 1 gene expression in patients with acute myeloid leukemia

Background The Wilms’ tumor 1 (WT1) gene is overexpressed in patients with acute leukemias. Many studies have reported the importance of quantitative assessment of WT1 expression as a molecular marker of minimal residual disease monitoring. However, biological significance and the prognostic effect of WT1 overexpression in acute myeloid leukemia (AML) remain elusive. Subjects and methods A total of 42 patients with newly diagnosed AML were included in the study. Also, 42 individuals matched for age and sex were enrolled as controls. Immunophenotyping, cytogenetic analysis, and quantitative assessment of WT1 gene transcripts were performed using real-time PCR. Results WT1 overexpression was detected in 73.8% of our patient group. There was a statistically significant decrease in the probability of achieving complete remission with shorter overall survival and event-free survival in the WT1 overexpression group compared with the downexpression group (P=0.035, 0.045, 0.011, respectively). Application of multivariate analysis showed that WT1 overexpression by itself is an independent and negative indicator for predicting overall survival and disease-free survival of AML patients. Conclusion WT1 overexpression is an independent negative prognostic marker that could be used to evaluate response to induction chemotherapy and prognosis of AML patients.

The clinical significance of the alternative Wilms tumor gene overexpression-hypermethylation signature in acute myeloid leukemia.

Wilms tumor 1 (WT1) gene is overexpressed in numerous cancers, including acute myeloid leukemia (AML). The alternative WT1 gene (AWT1) is generated from alternative transcription start site in the WT1 first intron and encodes an N-terminal-truncated protein lacking the repressor domain. Although WT1 overexpression is a common feature in AML, the expression levels of the AWT1 and its underlying epigenetic alterations, as well as their clinical relevance in AML remain unknown. Quantitative assessment of AWT1 gene transcripts was performed using real-time polymerase chain reaction (PCR). Bisulfite PCR followed by pyrosequencing was done to determine the methylation status of the AWT1 promoter. The bone marrow samples were collected at diagnosis and after completion of induction chemotherapy from 80 newly diagnosed AML patients. Forty non-malignant BM samples were recruited as controls. The AWT1 was significantly overexpressed in AML patients. Robust hypermethylation of the AWT1 promoter was found to be a highly specific and sensitive marker for AML (p < 0.001). Significant positive correlations between the AWT1 expression and methylation levels with BM blast counts at both initial diagnosis and after induction therapy were observed (p < 0.001). AWT1 overexpression at the initial diagnosis of AML was found to be an independent negative factor for complete remission response after induction therapy (p = 0.014). The AWT1 gene overexpression-hypermethylation signature is a characteristic marker that positively correlates with the leukemic burden in AML. AWT1 overexpression at AML diagnosis is an independent negative predictor for CR after induction chemotherapy.

WT1 mRNA Expression in the Peripheral Blood of Patients with MDS Yields Additional Prognostic Information within the IPSS-R Risk Categories “Very Low”, “Low” and “Intermediate”

Introduction:The revised version of the International Prognostic Scoring System, (IPSS-R), is an accepted standard for assessing the prognosis of patients with MDS. Its usefulness may be further improved by integrating molecular findings. However, such efforts are impeded by limited access to molecular diagnostics, lack of standardized methodology, and a relatively low frequency of individual gene mutations in MDS. The Wilms' Tumor-1 (WT1) gene is overexpressed on mRNA level in the peripheral blood (PB) in about 50% of patients with MDS. The aim of this analysis was to determine whether PB WT1 expression status yields additional prognostic information.Methods:For this purpose, PB WT1 mRNA expression was measured in 91 newly diagnosed patients with MDS (WHO: MDS del5q, n=7; RARS, n=1; RCUD, n=4; RCMD, n=37; RAEB-I, n=16; RAEB-II, n=23; MDS/MPN unclassifiable, n=3 / IPSS-R: very low risk, n=3; low risk, n=28; intermediate risk, n=27; high risk, n=13; very high risk, n=20), using the Ipsogen® WT1 ProfileQuant® Kit. This standardized, commercially available assay uses a validated cut-off level of 50 WT1 copies/104ABL copies for discrimination between normal and overexpression of WT1 in PB. MDS patients in our study cohort were stratified accordingly (normal WT1 expression with <50 WT1 copies versus overexpression with >50 WT1 copies). WT1 expression status was correlated with clinical parameters and outcome.Results:Overall, 53 MDS patients (58%) showed WT1 overexpression, which correlated significantly with WHO 2008 disease category and IPSS-R risk groups, as indicated by both the absolute WT1 levels (correlation with WHO 2008 type, p=0.0028, and IPSS-R, p=0.0075) and the frequency of WT1-overexpressing patients within the respective MDS subgroup (correlation with WHO 2008 type, p=0.0029, and IPSS-R, p=0.0027). Regarding the entire cohort, patients with elevated WT1 expression had a significantly lower progression-free survival (PFS) and overall survival (OS) compared to those with normal WT1 expression (p<0.0001 and p=0.0306). Furthermore, within the IPSS-R risk groups ‘very low’, ‘low’ and ‘intermediate’, PFS differed significantly between patients showing normal vs. elevated WT1 expression status (IPSS-R very low/low: median PFS 30.1 months vs. not reached, for WT1 high vs normal, respectively, p=0.0127; IPSS-R intermediate: median PFS 14.4 months vs. 59.5 months, for WT1 high vs. normal, respectively p=0.0240). These differences in PFS retained their prognostic significance in multivariate analysis after adjusting for IPSS-R (HR 0.306; 95% CI 0,156-0,598, p=0.001). However, they did not translate into a difference in overall survival, which was probably due to a relevant number of patients proceeding to allogeneic stem cell transplantation. Given the large proportion of patients displaying WT1 overexpression in the IPSS-R high and very high risk groups, it was not surprising that no significant prognostic subdivision by WT1 expression level was seen in these risk categories.Conclusion:Our results show that PB WT1 expression offers additional prognostic information in patients belonging to the IPSS-R risk groups ‘very low’, ‘low’ and ‘intermediate’. Assessment of WT1 expression status at diagnosis is a relatively time and cost efficient method that can be performed without patient discomfort and may help to identify MDS IPSS-R low and intermediate patients at risk for early progression. DisclosuresRautenberg:Celgene: Honoraria. Germing:Janssen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kobbe:Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding. Schroeder:Celgene: Consultancy, Honoraria, Research Funding.

Overexpression of Wilms' tumor 1 in skin lesions of psoriasis is associated with abnormal proliferation and apoptosis of keratinocytes.

Psoriasis vulgaris (PV) is a chronic inflammatory skin disease, which is characterized by the abnormal proliferation and apoptosis of keratinocytes. Previous studies have demonstrated that transcription factor Wilms' tumor 1 (WT1) is involved in a number of pathophysiological processes, including organ development, tumorigenesis and cell proliferation. However, the role of WT1 in PV remains unclear. In the present study, WT1 expression was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blot analyses. WT1 was stably overexpressed or inhibited in HaCaT cells using Lipofectamine® 2000, and cell proliferation and apoptosis were determined using a Cell Counting Kit‑8 or Fluorescein Isothiocyanate Annexin V Apoptosis Detection kit II, respectively. We demonstrated that compared with normal controls, the mRNA and protein expression levels of WT1 were significantly increased in non‑lesional skins (human, P<0.0001 and P=0.0291, respectively; mouse, P=0.0020 and P=0.0150, respectively) and lesional skins (human, P<0.0001 and P=0.0060, respectively; mouse, P=0.0010 and P=0.0172, respectively) of patients with PV, in addition to the imiquimod (IMQ)‑induced psoriasis‑like mouse model. WT1 mRNA and protein expression levels in lesional skins were slightly increased compared with those in non‑lesional skins from patients with psoriasis (P=0.2510 and P=0.1690, respectively) and IMQ‑treated mice (P=0.9590 and P=0.2552, respectively), although there were no statistical differences. Knockdown of WT1 inhibited the proliferation of HaCaT cells [day (D)4, P=0.0454; D5, P=0.0021] and promoted their apoptosis (P=0.0007), while overexpressing WT1 exhibited the opposite effects (proliferation D3, P=0.0216; D4, P=0.0356; D5, P=0.0188; apoptosis, P=0.0003). Furthermore, it was identified that the inflammatory cytokines interleukin‑17A (IL‑17A), interferon‑γ and IL‑22 induced the overexpression of WT1 in HaCaT cells. The results of the present study suggested that inflammatory cytokine‑induced WT1 overexpression may promote the formation of psoriatic skin lesions via regulation of the proliferation and apoptosis of keratinocytes.

Wilms’ tumor 1 (WT1) as a prognosis factor in gynecological cancers

The oncogenic role of Wilms’ tumor 1 (WT1) which is regarded as a promising target antigen for cancer immunotherapy has been demonstrated in many types of cancer, but the relationship between expression of WT1 and the prognosis value in gynecological cancer reminds unclear.We performed a meta-analysis with thirteen published studies including 2205 patients searched from PubMed, EMBASE, Web of Science, and Google Scholar, whose results are expressed by overall survival (OS) or disease-specific survival (DSS) or disease-free survival or relapse/recurrence-free survival (RFS) or progression-free survival (PFS) in patients with gynecological cancer. The hazard ratio (HR) with its 95% confidence interval (CI) were calculated to investigate prognostic of WT1 expression in patients with gynecological cancer.Finally, the overexpression of WT1 was borderlinely associated with poor OS (metaHR = 1.51, 95% CI = 0.98–2.31) in univariate model. We found a significant association with poor DSS (metaHR = 1.61, 95% CI = 1.24–2.08) and DFS/RFS/PFS (metaHR = 2.06, 95% CI = 1.22–3.46). The subgroup analyses revealed that the expression of WT1 predicted the poor DSS (metaHR = 1.82, 95% CI = 1.42–2.73), and DFS/RFS/PFS (metaHR = 2.51, 95% CI = 1.81–3.48) in patients with ovarian cancer. In summary, WT1 overexpression indicates a poor prognosis in patients with some gynecological tumors, but more studies are needed to confirm these findings.

Cancer-related mRNA expression analysis using a novel flow cytometry-based assay.

Cancer-related gene expression data mostly originate from unfractionated bulk samples, leading to "expression averaging" of heterogeneous populations. Multicolor flow cytometry (FCM) may distinguish heterogeneous populations based on the phenotypic characterization of single-cells, but is not applicable for RNA targets. Here, we evaluated the PrimeFlow™ RNA assay, a novel FCM-based assay designed to measure gene expressions, in two cancer entities with high and low RNA target levels. Neuroblastoma (NB) cell lines were studied for MYCN gene expression by PrimeFlow™ and compared with the gold standard, RT-qPCR. Dilution series of NB cells (0.10-11%) were prepared to evaluate performance in small cell populations. Diagnostic material of de novo acute myeloid leukemia (AML) patients was used to measure Wilms' tumor 1 (WT1) expression in bulk leukemic cells and rare subsets, e.g. leukemic stem cells (LSCs). FCM analysis was performed on a FACSCanto II (BD Biosciences) using Infinicyt™ (Cytognos® ) for data analysis. mRNA expression was reported by normalized mean fluorescence intensity (MFI) values and staining indices. MYCN mRNA quantified by PrimeFlow™ significantly correlated with RT-qPCR and remained detectable in small (0.1%) populations. Using PrimeFlowTM , WT1 levels were shown to be significantly higher in AML patient samples with WT1 overexpression, previously defined by RT-qPCR. Moreover, WT1 overexpression was distinguishable between heterogeneous cell populations and remained measurable in rare LSCs. PrimeFlow™ is a sensitive technique to investigate mRNA expressions, with high concordance to RT-qPCR. High (MYCN) and subtle (WT1) overexpressed mRNA targets can be quantified in heterogeneous and rare subpopulations e.g. LSCs. © 2017 International Clinical Cytometry Society.

MicroRNA-193a inhibits breast cancer proliferation and metastasis by downregulating WT1.

In many cancers, microRNA-193a (miR-193a) is a suppressor miRNA, but its underlying anti-oncogenic activity in breast cancer is not known. In this study, we found decreased miR-193a (specifically, miR-193a-5p) expression not only in breast cancer cell lines but also in breast cancer tissues as compared with the adjacent non-tumor tissues. Ectopic miR-193a overexpression inhibited the proliferation, colony formation, migration, and invasion of MDA-MB-231 and BT549 cells. miR-193a reduced Wilms’ tumor 1 (WT1) expression and repressed luciferase reporter activity by binding WT1 coding region sequences; mutation of the predicted miR-193a binding site abolished this effect. miR-193a and WT1 expression were significantly inversely correlated in breast cancer tissues. Importantly, the anti-cancer activity induced by miR-193a was partially reversed by WT1 overexpression, indicating an important role for WT1 in such activity related to miR-193a. Our results reveal that miR-193a-WT1 interaction plays an important role in breast cancer metastasis, and suggest that restoring miR-193a expression is a therapeutic strategy in breast cancer.

Comparison Between Wilms' Tumor 1 (WT1) Expression Using a Standardized European Leukemia Net (ELN)-Certified Assay and Other Methods for Detection of Minimal Residual Disease (MRD) in MDS and AML Patients after Allogeneic Blood Stem Cell Transplantation

IntroductionAn "ideal" marker to monitor MRD after allo-SCT should be informative in the majority of pts and facilitate the use of a method with high sensitivity and specificity in a standardized manner. In addition, to allow repeated monitoring in timely tight intervals but also ensuring patients' comfort such marker should ideally be measurable in peripheral blood (PB).Despite recent identification of several molecular aberrations AML and MDS, many of these do not fulfil the above-mentioned requirements, as they are present only in small patient groups, their potential instability during disease course, the absence of standardized assays and the need for BM as optimal sample source.A molecular marker which might provide these properties is the WT1 gene, as it is overexpressed in the majority of AML pts and in about 50% MDS pts and is measurable in PB by a standardized assay. To estimate its value after allo-SCT we compared serial WT1 measurement with other methods used to monitor MRD in a real-life situation.Patients and MethodsFor this retrospective analysis all AML and MDS pts who underwent allo-SCT at our center between 2012 and 2016 were screened for PB WT1 mRNA overexpression using the ELN certified Ipsogen® WT1 ProfileQuant® Kit. Pts with WT1 overexpression, as defined by an validated cut-off level of 50 copies/104 ABL copies, were routinely monitored after transplant. In addition, in all pts STR-based chimerism analysis was performed. In pts with chromosomal aberrations existing prior allo-SCT metaphase and FISH analysis was performed, while XY FISH was additionally conducted in pts with sex-mismatched donor-recipient constellation. Furthermore, pts with molecular markers were regularly monitored by NGS or qPCR. Results of WT1 monitoring were correlated with clinical course and compared with results obtained from the other methods.ResultsOf 104 screened pts 59 (57%) showed an WT1 overexpression at diagnosis and underwent an allo-SCT. This included 40 AML pts (WT1 overexpressed in 66%) and 19 MDS pts (WT1 overexpressed in 44%).Chimerism analysis was accessible in all 59 pts (100%), while 20 pts (34%) could also be monitored by XY-FISH. Additionally, in 40 pts (68%) cytogenetics and FISH were applicable, while 22 pts (37%) could be investigated by NGS or qPCR. Overall, in 5 pts MRD could be monitored by WT1 and chimerism only, while in 29 pts MRD could be monitored by 1, in 22 pts by 2 and in 3 pts by 3 additional methods.With a median follow up of 13 months (2 - 51) we analyzed a total of 472 WT1 samples reflecting a median of 8 samples per patient (2 - 19). One month after allo-SCT 57 pts (97%) showed complete remission and a rapid decrease of WT1 expression below threshold. Only 2 pts had persistant hematological disease with sustained WT1 overexpression.Twenty-four pts (41%) experienced hematologic (62%) or molecular (38%) relapse at a median of 126 d (28 - 938 ) after allo-SCT. In 20 (83%) of these at least one WT1 value wasabove the cut-off before relapse. Median time from first elevated WT1 to relapse was 2 weeks (0 - 27). Only 4 pts (17%) with molecular relapse showed WT1 expression below cut-off.In contrast, known molecular aberrations were found again in 63% and loss of complete donor-chimerism or a positive signal in XY-FISH analyses were only seen in 46% and 57% before relapse. Furthermore, cytogenetics or FISH detected known or new aberrations in 39% before relapse.From 35 pts remaining in CR for a median of 13 months, only 1 (3%) had a transient increase of WT1 expression above the cut-off, whereas WT1 levels of the other 34 pts persisted below. Three pts (9%) with ongoing remission showed a transient decrease of donor-chimerism, whereas even 31% of pts accessible for XY-FISH showed temporary conspicuous results. Conventional cytogenetics and FISH in pts with CR showed transient abnormalities in 18%, whereas in 14% with molecular aberrations these were temporary detectable. ConclusionMeasurement of PB WT1 overexpression is an easy accessible method to monitor MRD after allo-SCT that can be employed by a standardized assay in the majority of AML and MDS pts independent from their individual leukemic genotype. Besides these advantages, the results from our real-life experience also suggest that WT1 overexpression allows sensitive detection of imminent relapse after allo-SCT. Taking into account the methodical restrictions of this retrospective analysis, our data requires confirmation in a prospective trial. DisclosuresGattermann:Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accomodation expenses, Research Funding. Kobbe:Jansen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support, Research Funding.

Expression of Wilms' tumor 1 (WT1) in ameloblastomas.

The Wilms' tumor 1 gene (WT1) was originally isolated and described as the gene responsible for Wilms' tumor. Although there is growing evidence linking the overexpression of WT1 to tumorigenesis, no reports on ameloblastoma are available at present. The aim of this study was to examine the expression of WT1 in various histological subtypes of ameloblastoma tissue specimens and in human ameloblastoma cell lines. Immunohistochemical analyses were performed on a total of 168 cases of ameloblastoma, one case of ameloblastic carcinoma, and five cases of tooth germs (control). Three immortalized human dental epithelial cell lines (HAM1, HAM2, and HAM3) derived from the same ameloblastoma patient were used for reverse transcription-polymerase chain reaction (RT-PCR) and western blot assays. The tooth germs did not express WT1 (0%), and more than half of the ameloblastoma cases showed WT1 overexpression (54.7%). Immunoreactivity of solid-type ameloblastoma (76.1%) was more evident than that of unicystic-type ameloblastoma (40.9%). The expression level of WT1 mRNA in HAM2 was higher than that in HAM1 (moderate) and HAM3 (weak), showing the heterogeneity of tumor cells. The WT1 protein was strongly detected in HAM2 and minimally detected in HAM1 and HAM3. Our results suggest that WT1 expression influences the pathogenesis of ameloblastoma by varying its expression level in different histological types. (J Oral Sci 58, 407-413, 2016).

Monitoring of Wilms’ Tumor 1 (WT1) Expression in Peripheral Blood (PB) of Patients with Myelodysplastic Syndromes (MDS) By a Standardized European Leukemia Net (ELN)-Certified Assay to Detect Minimal Residual Disease (MRD) and Guide Therapeutic Interventions Following Allogeneic Transplantation

Background:Since the success of therapeutic interventions to prevent or treat relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with MDS and secondary acute myeloid leukemia (sAML) especially depends on disease burden, early detection of MRD is essential. Despite the discovery of various mutations during the last years, no reliable MRD marker has been defined, which can be quantitatively measured by a standardized assay in a large proportion of MDS patients. Measurement of PB WT1 mRNA expression facilitates quantitative MRD monitoring in >90% of de novo AML patients in the pre- and posttransplant period. WT1 overexpression has also been reported in a proportion of MDS patients suggesting its potential use as MRD marker. Still, because of limited patients numbers, differing sample sources (PB or BM) and the use of in-house methods without reproducible cut-off levels, WT1 has been not established as MRD marker in MDS so far.In the current study we used a standardized ELN-certified assay (1) to measure WT1 expression in PB of a large group of all MDS subtypes enabling correlation with disease characteristics, (2) to determine its’ value as MRD marker in patients with MDS undergoing allo-HSCT, and (3) to monitor WT1 levels during salvage therapy.Patients and Methods:WT1 levels were measured in PB using the Ipsogen® WT1 ProfileQuant® Kit (Qiagen, Hilden, Germany) in accordance with the manifacturers’ instructions. This standardized assay has a validated PB cut-off level of 50 WT1 copies/104 ABL copies to distinguish between normal and overexpression of WT1.Our study included a total of 92 untreated MDS patients (7 RCUD, 30 RCMD, 7 MDS del5q, 7 CMML, 11 RAEB-1, 15 RAEB-2, 15 sAML). Samples of 12 healthy volunteers and 18 patients with non-MDS related cytopenias served as controls.In MDS patients, who had WT1 overexpression prior transplant, WT1 levels were serially monitored after allo-HSCT. Finally, in an independent group of patients with MDS or AML relapsing after allo-HSCT WT1 expression was measured during salvage therapy with Azacitidine (Aza) and donor lymphocyte infusions (DLI).Results:Overall, PB WT1 overexpression was found in 48 of 92 MDS patients (52%) and enabled not only to significantly discriminate between MDS and healthy controls (0/12 samples, 0%, p=0.0004), but also between MDS and cytopenias related to other causes (0/18 samples, 0%, p<0.0001)WT1 expression significantly correlated with disease category according to WHO classification as indicated by the frequency of WT1-overexpressing patients (RCUD 14%, MDS del5q 28%, RCMD 40%, CMML 57%, RAEB-1 55%, RAEB-2 87%, sAML 67%, p=0.0011) as well as by absolute WT1 levels of the respective subentities (p=0.0192). In addition, there was a clear association between WT1 expression and risk categories according to IPSS as well as IPSS-revised. While no differences were found with regard to WT1 expression between patients with a normal or abnormal karyotype, WT1 overexpression was significantly associated with increased BM blasts.In 16 patients with WT1 overexpression prior transplant, we monitored WT1 expression in the posttransplant period. All patients had a rapid decline to normal WT1 levels below the normal threshold after transplant. Nine patients exhibited low and stable WT1 levels and remained in remission throughout follow-up. In contrast, 7 patients relapsed after allo-HSCT and all of them had an increase of WT1 expression above the cut-off level at/or before relapse suggesting stable correlation between WT1 and disease burden after allo-HSCT.In line with this we found a 3,32-log-fold (range: 0,61 to 4,14-log) decrease of WT1 expression to normal levels in 8 patients achieving CR after salvage therapy with Aza and DLI. All 20 patients, who did not respond, remained above the normal WT1- threshold.Conclusion:Using a standardized assay with reproducible cut-off levels WT1 overexpression can be detected in PB of nearly half of MDS patients. WT1 overexpression was in particular found in those MDS patients with an increased BM blast count and expression levels correlated with disease burden in the posttransplant period. These findings suggest that WT1 expression might be used as MRD marker in a broader fraction of MDS patients for detection of relapse and for guidance of therapeutic interventions after allo-SCT. DisclosuresGattermann:Novartis: Honoraria, Research Funding. Germing:Novartis: Research Funding; Celgene: Honoraria, Research Funding; AMGEN: Research Funding; Janssen-Cilag: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other.

Wilms’ Tumor 1 Expression Levels in Bone Marrow after Induction and/or Consolidation Therapy Allow a Better Stratification of Patients and Improves Treatment in Adult AML

Introduction:The Wilms’ tumor 1 (WT1) gene, located on chromosome 11p13, encodes a transcription factor. WT1 is overexpressed in 90% of patients with acute myeloid leukemia (AML) and thus can be used for minimal residual disease (MRD) monitoring by quantitative RT-PCR. The aim of the present study was to analyze the usefulness of WT1 as a marker for MRD quantification in AML after chemotherapy.Patients and Methods: This retrospective and multicentric study included 148 patients with WT1-overexpressed AML. Quantitative assessment of WT1 transcript levels was performed by quantitative RT-PCR in 370 bone marrow (BM) samples at diagnosis (148), post-induction (125) and post-consolidation (97). WT1 gene expression was calculated by relative quantification using the normalized ratio of the target gene (WT1) related to a reference gene (GUS) and using cell line K562 as calibrator. Inter-laboratories methodological standardization was accomplished through a pilot study with 20 BM donor samples, 20 BM patient samples and commercial WT1 plasmids (ProfileQuant Kit, Ipsogen-Qiagen).Results: No significant differences in WT1 gene expression (cycle threshold, Ct) were observed between different laboratories in the pilot study (kappa coefficient >0.7). The cut-off value of WT1 over-expression in BM was established for each laboratory (median+2SD values from healthy donors, data not shown). Median WT1 expression in patient samples at diagnosis was 38% (range, 2-850). Differential expression at diagnosis did not correlate with age, gender, leukocytes, karyotype (risk). However a significantly higher expression in patients with AML-M1 and AML-M2 subtypes as well as patients with mutant NPM1 and/or ITD-FLT3 was observed (p<0.05). On the other hand, WT1 BM levels at diagnosis were not correlated with relapse free survival (RFS) and overall survival (OS). All patients received intensive chemotherapy as induction treatment. After induction, 84.5% (125/148) of patients had available WT1 data, of which 23.45% (45/125) showed overexpression. After consolidation, 65.5% (97/148) of patients had available WT1 data, of which 28.9% (28/97) showedoverexpression. WT1 post-induction and consolidation levels were correlated with RFS and OS (Figure 1). These differences were greater when patients who were treated withallogeneic stem cell transplantation (allo-SCT) were excluded from the analysis: lower RFS [post-induction (2 years 70% vs. 8% p<0.001) and post-consolidation (2 years 58% vs. 0% p<0.001)] and lower OS [post-induction (2 years 75% vs. 34% p=0.031) and post-consolidation (2 years 74% vs. 26% p=0.004)] were obtained. Univariate and multivariate analysis were performed; high risk cytogenetic (complex karyotype), not allo-SCT and post-induction WT1 levels remained as independent prognostic factors in terms RFS (Table 1).Conclusions: WT1 is a useful marker for MRD quantification in AML patients undergoing chemotherapy. Levels after induction and consolidation allow anticipation of relapse and survival. In the group of patients not treated with allo-SCT, results are especially relevant with greater statistically significant differences. Treatment intensification derived from positive post-chemotherapy WT1 levels with allo-SCT overcomes the poor prognosis. [Display omitted] Post-induction (A,C) and Post-consolidation (B,D). [Display omitted] Abbreviations: Neg. negative (normal levels o no overexpression; Pos: positive (overexpression). DisclosuresNo relevant conflicts of interest to declare.

A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS.

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

WT1 Overexpression Affecting Clinical Outcome in Non-Hodgkin Lymphomas and Adult Acute Lymphoblastic Leukemia

The Wilms tumor 1 (WT1) gene has a complex role as a transcriptional regulator, acting as tumor suppressor or oncogene in different malignancies. The prognostic role of its overexpression has been well-studied in leukemias, especially acute myeloid leukemia (AML), but not in lymphomas. For the first time to our knowledge, we present a study demonstrating the correlation of WT1 expression and survival in various non-Hodgkin lymphomas. We also studied the prognostic implications of WT1 overexpression in adult acute lymphoblastic leukemia (ALL). In our sample of 53 patients--25 with diffuse large B-cell lymphoma (DLBCL), 8 with mantle cell lymphoma (MCL), 9 with peripheral T-cell lymphoma (PTCL), 2 with Burkitt's lymphoma, 2 with mucosa-associated lymphoid tissue (MALT) lymphoma, and 7 with B-cell ALL--, we measured WT1 mRNA from blood samples by quantitative RT-PCR, and divided the patients into subgroups based on the level of expression. Kaplan-Meier survival curves were drawn and compared using the logrank test. In the sample of DLBCL patients, the difference in overall and disease-free survival between WT1-positive and negative subgroups was significant (p = 0.0475 and p = 0.0004, respectively), and in a few observed cases, a sudden increase in WT1 expression signified a relapse soon followed by death. Disease-free survival curves in MCL and ALL were similarly suggestive of a potential role played by WT1. In PTCL, though WT1-positivity was detected in 4 out of 9 cases, it did not seem to affect survival. The few cases of MALT and Burkitt's lymphoma all proved to be WT1-negative.