Wilms Tumor 1 mRNA Expression Research Articles

Downregulation of the Mutated WT1 Alleles in Malignant Hematopoietic Cells Via Nonsense-Mediated mRNA Decay Contradicts Their Prognostic Impact

Background The Wilms Tumor 1 (WT1) gene is highly expressed in almost all types of malignancies including acute myeloid leukemia (AML). It is also mutated in up to 20% of AML blasts in pediatric and adult patients. Both, the increased expression and also the presence of mutations are correlated negatively with chance of survival in different AML patient cohorts. We and others have previously showed devastating survival outcomes in children with AML blasts harboring WT1 mutations in combination FLT3-ITD and NUP98-NSD1 (Niktoreh et al., 2019). As WT1 can act as both, oncogene and tumor suppressor gene, this study was designed to analyze the WT1 genomic locus mutations in acute leukemia blasts. Methods and Results Screening for WT1 sequence alterations. Using the TruSight Myeloid Sequencing PanelTM (Illumina), we initially screened 35 leukemia and lymphoma cell lines for genomic alterations in 54 genes. This panel allowed us to detect even low variant-allele-frequency (VAF) alterations in the mutational hotspots of WT1, exons 7 and 9. In addition to a known WT1 mutation in CTS cells (AML M1), we discovered novel alterations in three other malignant cell lines. KASUMI-6 cells (AML M2) harbored the identical mutation as CTS cells, a 2-bp deletion in WT1 exon 7 leading to frameshift with a premature termination codon (c.1202_1203delGA, p.Arg401IlefsTer3). The U937 histiocytic lymphoma cells carried the known TP53 mutation and also an unknown heterozygous nonsense mutation in WT1 exon 7, changing the arginine 369 into a premature stop codon (c.1105C>T, p.Arg369Ter). Interestingly, MOLM-1 cells (CML in blast crisis) harbored a p.Arg462Gln alteration. Next, we performed Sanger sequencing for the complete coding region of WT1, including the upstream promoter as well as the 5'UTR and 3'UTR regions, all 10 exons and the adjacent 150 bp intronic sequences at the exon-intron boundaries. Sanger sequencing confirmed the mutations discovered via NGS and revealed single bp alterations in the WT1 promoter region of CTS cells and in the 5'UTR of K-562 cells. We also detected a recurring variation in intron 3 of WT1 (c.872+16G>A) in the cell lines K-562, NB-4, M07e and UOC-M1; the functional implications of the alterations are yet to be determined. Functional impact of WT1 mutations. To understand the potential impact of the WT1 sequence alterations, we studied the WT1 mRNA expression in these cells in the presence of cycloheximide (CHX), an inhibitor of the nonsense-mediated mRNA decay (NMD) machinery. As shown in figure 1A, U937 cells were used as negative control cells, as they are known to have no expression of the WT1 mRNA. Despite the presence of mutations in CTS, KASUMI-6 and MOLM-1 cells, all three cell lines expressed elevated levels of WT1, well within the range of 30 other malignant cell lines without any alterations/mutations in the WT1 locus (data not shown). To differentiate the WT1 expression between the mutated and the wildtype allele, we performed a fragment length analysis on a capillary electrophoresis system using the complimentary DNA of CHX-treated cell lines. For these analyses, we employed a multiplex PCR strategy using ABL1 as housekeeping gene and a primer pair binding in exons 4 and 10 of WT1. As delineated in figure 1B, the mutated alleles in CTS and KASUMI-6 cells, which are 2-bps shorter, could be easily separated from the wildtypes (data for KASUMI-6 not shown). Additionally, since our primer pair also amplified the alternatively spliced exon 5 and the alternative splice donor at the end of exon 9, leading to inclusion or exclusion of three amino acids (KTS), we were able to separately analyze the four major WT1 isoforms from both alleles. As shown in figure 1B, the expression levels of the WT1 mutated alleles of CHX-treated cells increased 2- to 3-fold compared to DMSO-treated cells in CTS and 3- to 4-fold in KASUMI-6 (data not shown). Conclusion Careful analyses of the WT1 genomic locus in 35 malignant hematological cell lines revealed previously unknown heterozygous loss-of-function WT1 mutations in KASUMI-6, MOLM-1 and U-937 cells. mRNA expression analysis demonstrated that these mutations are strongly regulated by NMD machinery, leading to (almost) complete downregulation of the mutated WT1 allele. These results strongly question any direct effect of WT1 mutations in the leukemic blasts and therefore cannot explain the role of mutations in WT1 as a prognostic factor in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Possible prognostic impact of WT1 mRNA expression at day + 30 after haploidentical peripheral blood stem cell transplantation with posttransplant cyclophosphamide for patients with myeloid neoplasm: a multicenter study from the Okayama Hematological Study Group.

Previous studies have revealed that relapse of myeloid neoplasms after allogeneic hematopoietic stem cell transplantation (allo-HSCT) could be predicted by monitoring Wilms' tumor 1 (WT1) mRNA expression. However, only a few studies have investigated patients who received human leukocyte antigen-haploidentical stem cell transplantation with posttransplant cyclophosphamide (PTCY-haplo). In this study, we investigated the relationship between WT1 mRNA levels and clinical outcomes in the PTCY-haplo group, and compared them with those in the conventional graft-versus-host disease prophylaxis group (conventional group). We retrospectively analyzed 130 patients who received their first allo-HSCT between April 2017 and December 2020, including 26 who received PTCY-haplo. The WT1 mRNA expression level at day + 30 after allo-HSCT associated with increased risk of 1-year cumulative incidence of relapse (CIR) was ≥ 78 copies/μg RNA in the conventional group (p < 0.01) and ≥ 50 copies/μg RNA in the PTCY-haplo group (p = 0.03). The appropriate cutoff level of WT1 mRNA at day + 30 after allo-HSCT for predicting prognosis in patients treated with PTCY-haplo may be < 50 copies/μg RNA.

Expression of the Tumor Suppressor WT1 Is Induced By PML-Rara in Acute Promyelocytic Leukemia. Â

Inactivating mutations in Wilms Tumor 1 (WT1) are found in 5-10% of cases of acute myeloid leukemia (AML). Paradoxically, wild type WT1 is expressed highly in most cases of AML compared to normal hematopoietic progenitors, and high levels of WT1 have been associated with poor outcomes. Therefore, it is not yet clear whether loss-of-function WT1 mutations-or paradoxically, wild type WT1 overexpression-both promote disease progression in AML. We performed a genome-wide screen for mutations in acute promyelocytic leukemia (APL), a morphologic subtype of AML characterized by the PML-RARA fusion. We identified WT1 mutations in 11/42 patients, suggesting that WT1 mutations may cooperate specifically with PML-RARA in APL. Further, expression analysis demonstrated a high level of wild type WT1 expression in non-mutated cases. To test the effect of Wt1 inactivation in a mouse model of APL, we bred mice carrying a floxed Wt1 allele (give Baylor reference) to transgenic mice expressing tamoxifen-inducible Cre recombinase (ERT2-Cre). Tamoxifen treatment leads to deletion of the C-terminal zinc finger domains of Wt1, mimicking some of the mutations that are commonly found in AML/APL cases. These mice were crossed with mice expressing the PML-RARA fusion cDNA in early myeloid progenitors with Cathepsin G (Ctsg) regulatory sequences (Ctsg-PML-RARA). Bone marrow cells from Ctsg-PML-RARA x Wt1fl/fl x ERT2-Cre mice were transplanted into irradiated recipients. Tamoxifen-treated mice (i.e. Wt1 deficient) developed APL with a significantly shortened latency compared to untreated controls (198 days vs. endpoint not reached, N=8-10 each group, p=.0001), suggesting that Wt1 loss-of-function mutations can cooperate with PML-RARA to drive APL. Next, to test the effect of wild type Wt1 overexpression on hematopoietic cells, bone marrow cells from wild type or Ctsg-PML-RARA mice was transduced with retroviruses expressing full length Wt1 cDNA or an empty vector control, and subjected to serial replating. As previously reported, untransduced bone marrow cells from Ctsg-PML-RARA mice were able to form colonies in methylcellulose week after week, while cells overexpressing wild type Wt1 were selected against during serial replating, as demonstrated by a gradual reduction in GFP percentage and Wt1 expression, as measured by western blotting. Interestingly, in contrast to human AML/APL, Wt1 expression could not be detected in a panel of murine tumors with initiating mutations in DNMT3A, PML-RARA, or MLL-AF9 . Taken together, these findings suggest that Wt1 expression suppresses APL development in mice.To reconcile these findings with the high level of WT1 expression in human AML/APL, we speculated that in human progenitor cells, WT1 expression may be induced by leukemia-associated oncogenes (like PML-RARA) as a physiologic, inhibitory response to oncogenic stress. In this case, subsequent inactivating mutations in WT1 would provide an additional growth advantage. Given the observed interspecies difference between WT1/Wt1 expression levels in mouse and human leukemia cells, we used human umbilical cord blood-derived CD34+ cells transduced with retroviruses expressing PML-RARA to model early events in human APL development. CD34+ cells expressing PML-RARA share some characteristics of fully transformed APL cells, including enhanced proliferation in liquid culture, and reduced expression of the myeloid maturation markers CD11b and CD14. WT1 mRNA expression is low in freshly isolated CD34+ cells, but rapidly increases in cells expressing PML-RARA over two weeks in culture (10.5-fold increase compared to empty-vector transduced controls, N=3 separate experiments, p<0.05). Western blotting confirmed robust induction of WT1 protein in transformed cells compared to empty-vector transduced controls, suggesting that WT1 expression is a normal consequence of PML-RARA activation in human hematopoietic progenitor cells. In summary, these findings support a cooperating role for WT1 mutations in APL development, and a potential mechanism to explain the high level of wild type WT1 expression observed in APL cells. Further experiments will be required to determine the downstream mechanisms by which WT1 antagonizes APL development, and whether these observations are generalizable to other AML subtypes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BASP1 Suppresses Cell Growth and Metastasis through Inhibiting Wnt/β-Catenin Pathway in Gastric Cancer.

Objective Our research is designed to explore the function of brain acid soluble protein 1 (BASP1) in the progression of gastric cancer (GC) and its underlying molecular mechanisms. Methods In this study, the expression of BASP1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) in both GC tissue and GC cells. The cell cloning, proliferation, apoptosis, migration, and invasion potential of AGS and HGC-27 cells were, respectively, determined using colony formation assay, 5-ethynyl-20-deoxyuridine (EDU) assay, flow cytometry, and Transwell assay. The protein expressions of Bax, caspase-3, Bcl-2, matrix metalloproteinases 2 (MMP-2), MMP-9, Wilms tumor 1 (WT1), Wnt, and β-catenin in AGS and HGC-27 cells were measured by western blot. In addition, the mRNA expressions of WT1, Wnt, and β-catenin in AGS and HGC-27 cells were detected by qRT-PCR. Results BASP1 expression was significantly downregulated in both GC tissue and GC cells. BASP1 overexpression markedly repressed proliferation, migration, and invasion and facilitated apoptosis in AGS and HGC-27 cells. In addition, BASP1 overexpression notably promoted the protein expression of Bax and caspase-3 in AGS and HGC-27 cells and inhibited the expression of Bcl-2, MMP-2, and MMP-9. Moreover, BASP1 overexpression significantly inhibited the mRNA and protein expression of WT1, Wnt, and β-catenin in AGS and HGC-27 cells. Conclusion BASP1 could significantly suppress cell proliferation, migration, and invasion and promote apoptosis through inhibiting the activation of the Wnt/β-catenin pathway in GC.

Changes of WIF-1 and WT-1 genes expression following the anti-cancer effects of omega-3 and omega-6 on gastric cancer cells

Gastric cancer is one of the most common cancers. Environmental factors, especially diet, play an important role in causing gastric cancer. The omega-3 (ω−3) and omega-6 (ω−6) from the polyunsaturated fatty acids (PUFAs) group as the natural compounds indicated significant effects on inhibiting various types of tumors. In this study, we investigated the anti-cancer properties of ω−3 and ω−6 on gastric cancer cells with focusing on WNT signaling pathway via WNT inhibitory Factor-1 (WIF-1) and The Wilms Tumor-1 (WT-1) genes.AGS cells were used to investigate the effects of ω−3 and ω−6 on cell viability, apoptosis and the expression of WIF-1 and WT-1 genes in gastric cancer. AGS cells were treated with different concentrations of ω−3 and ω−6 for 24, 48, and 72 h, and then cell viability was assessed by MTT assay. The apoptosis rate was assessed using the flow cytometery test in every three times. The mRNA expression of WIF-1 and WT-1 were examined by quantitative real-time PCR (qRT-PCR).Our study revealed that ω−3 and ω−6 provide various antitumor activities such as inhibition of cell viability in a dose and time-dependent manner. The results of flow cytometery showed that cytotoxicity effects are due to the ability of ω−3 and ω−6 to induce apoptosis. In addition, ω−3 and ω−6 significantly increased the expression of WIF-1, whereas WT-1 level decreased in AGS cells compared to the control group.These results suggested that ω−3 and ω−6 can serve as therapeutics in against gastric cancer by targeting of the WNT signaling pathway.

Influence of single nucleotide polymorphism of Wilms tumor 1 gene on the efficacy and clinical prognosis of elderly patients with acute myeloid leukemia

Objective To evaluate the influence of single nucleotide polymorphism of Wilms tumor 1(WT1)gene rs16754 on the chemosensitivity and clinical outcomes of elderly patients with acute myeloid leukemia(AML). Methods A total of 178 AML patients aged 60 years and over who received cytarabine-based chemotherapy were enrolled in this retrospective study.The peripheral blood was extracted from 178 AML patients receiving chemotherapy for DNA preparation and study.And bone marrow specimens were collected in 65 AML patients before chemotherapy.The Wilms' Tumor-1(WT1)rs16754 polymorphism was detected by PCR-RFLP method.The association between genotypes and other variables were analyzed by using Logistic regression model.Variables were adjusted by Cox regression analysis. Results The locus of WT1 gene rs16754 is located in coding region of WT1 gene.The genotype frequency and distribution of the studied population were 55.62%(99/178)in GG, 37.64%(67/178)in GA, and 6.74%(12/178)in AA, with minimum allele frequencies of 0.26.The distributions of the three genotypes were in accordance with Hardy-Weinberg Equilibrium(P=0.884). There was no statistical difference in the data distribution of the genotypes on clinical indexes at baseline.Overall survival time(OS)was longer in patients with allele A and genotype GA plus AA[2.73 years(95%CI: 1.03-5.11 years)]than in patients with GG genotype[1.64 years(95%CI: 0.71-4.34), (P=0.003)]. The replase free survival(RFS)was longer in patients with allele A and genotype GA plus AA[2.06 years(95%CI: 0.95-4.87)]than in patients with GG genotype[1.12 years(95%CI: 0.56-4.11), P=0.032)]. Adjusted by using multivariate Cox regression analysis, GA plus AA genotypes still showed a better effect on OS(HR=0.51, P=0.013)than did GG genotypes.In the 65 pretreatment bone marrow specimens, the expression level of WT1 mRNA in bone marrow cells was higher in patients with GG genotype than in patients with GA plus AA genotype(P<0.001). Conclusions Among elderly AML patients treated by cytarabine-based chemotherapy, the WT1 rs16754 may impact the clinical prognosis of AML patients by influencing the mRNA expression of WT1. Key words: Leukemia, myeloid; Polymorphism, single nucleotide; Treatment outcome

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) mRNA Expression Level at Diagnosis Is a Significant Prognostic Marker in Elderly Patients with Myelodysplastic Syndrome

Background/Aims: A high expression of Wilms tumor 1 (WT1) mRNA occurs in most cases of acute leukemia and myelodysplastic syndrome (MDS). Although there are many reports suggesting that acute myeloid leukemia patients with high expression levels of WT1 mRNA have a relatively poor long-term survival, there are few reports addressing the relationship between WT1 levels and prognosis in MDS. Methods: We retrospectively analyzed 42 elderly patients with MDS whose WT1 levels at diagnosis were available, and we assessed the relationships between WT1 levels in peripheral blood and preexisting prognostic factors such as World Health Organization prognostic scores and Revised International Prognostic Scoring System risk categories, bone marrow blast percentages, and chromosomal abnormalities linked to a poor prognosis. We also evaluated the relationship between WT1 levels and prognosis. Results:WT1 levels were significantly different between high- and low-risk MDS patients (p < 0.05). There was a trend towards a significant difference between those with and those without poor prognostic chromosomal rearrangements (p = 0.051). Moreover, the overall survival and progression-free survival were significantly worse in elderly patients with higher levels of WT1 (p = 0.00039 and p = 0.00077, respectively). Conclusions: The WT1 mRNA expression level at diagnosis may be a significant independent prognostic marker for elderly patients with MDS.

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.

Frequent hypermethylation of a CTCF binding site influences Wilms tumor 1 expression in Wilms tumors

The Wilms tumor 1 (WT1) gene plays an essential role in early development and differentiation of the urinary tract, particularly the kidneys. Aberrant transcriptional activity of WT1 is a key finding in the genesis of Wilms tumors (WTs). However, the mechanisms responsible for this alteration remain poorly understood. In the present study, we examined the methylation pattern of a putative CCCTC-binding factor (CTCF) binding site downstream of the WT1 gene as a potential cause of WT1 misregulation in 44 native WT specimens. We found that 16 WT cases exhibited a much higher WT1 expression compared to normal kidney tissue, and that the high mRNA expression of WT1 is strongly correlated with a high degree of DNA methylation of the CTCF binding site near the WT1 promoter. However, there was no correlation between the KTS+/KTS- splicing variants of WT1 and the methylation status of the CpGs of the CTCF binding site. Our results demonstrated an aberrant methylation pattern at a CTCF binding site downstream the WT1 gene, which is associated with an elevated WT1 transcriptional activity. Thus, methylation of the CTCF binding site may be partially responsible for the transcriptional activation of the WT1 locus and hypermethylation of this site may be an important oncogenic mechanism in the genesis of WT.

Analysis of mutational status, SNP rs16754, and expression levels of Wilms tumor 1 (WT1) gene in acute promyelocytic leukemia

Overexpression, polymorphisms, and mutations of the WT1 gene have been reported in several human tumors including acute myeloid leukemia (AML) and variably correlated with prognosis. Acute promyelocytic leukemia (APL) represents the AML subset disclosing higher WT1 expression levels; however, no WT1 studies specifically focused on APL have been conducted. We screened for the presence of mutations, SNP rs16754, and expression levels of WT1 gene in 103 adult patients with newly diagnosed APL. Fms-like tyrosine kinase (FLT3) mutations were analyzed as well. WT1 mutations were identified in four (4 %) patients. At least one copy of the minor SNP rs16754 allele (WT1(AG) or WT1(GG)) was detected in 30 (29 %) patients. Six patients (6 %) were homozygous for the minor allele (WT1(GG)) and this genotype was associated with higher WT1 mRNA copies (p = 0.018). FLT3 mutations were found in 37 % of patients and correlated with high WT1 mRNA expression (p = 0.004). Patients heterozygous or homozygous for the minor allele and patients homozygous for major (WT1(AA)) allele did not differ in terms of presenting features. In adult APL, WT1 gene mutational and polymorphic profile shows similarities with pediatric AML rather than with adult AML.

High Wilms’ tumor 1 mRNA expression correlates with basal-like and ERBB2 molecular subtypes and poor prognosis of breast cancer

The role of Wilms' tumor 1 (WT1) in breast cancer and the relationship between WT1 expression and clinicopathological factors, molecular subtypes and prognosis of breast cancer patients have not been clarified to date. We used publicly available microarray datasets of 266 early breast cancer patients to perform bioinformatics analysis on the relationship between WT1 mRNA expression and breast cancer. Results showed that WT1 mRNA expression was correlated with higher histological grades, ER-negative and basal-like and ERBB2 molecular subtypes in breast cancer. With regard to disease-free survival analysis, the WT1 high expression group showed worse prognosis than the low expression group in univariate analysis, and WT1 was demonstrated to be an independent prognostic indicator in multivariate analysis. This study confirms an oncogenic role of WT1 and demonstrates a possible relation between WT1 and progression of breast cancer.

Expression of Wilms tumor 1 gene in a variety of pediatric tumors

Background/Purpose Wilms tumor 1 ( WT1) gene is overexpressed in many types of neoplasms, thus suggesting that WT1 has oncogenic properties. Therefore, WT1 is a molecular target for cancer therapy. The objectives of this study were to evaluate the WT1 gene expression in various pediatric tumors and to elucidate that WT1 can be a target of cancer therapy in pediatric malignancies. Patients and Methods The expression of WT1 protein was examined in 60 cases of primary pediatric tumors. The levels of WT1 messenger RNA (mRNA) expression were examined by a quantitative real-time reverse transcriptase polymerase chain reaction analysis in frozen tissue samples from 56 cases with pediatric tumors. Results Immunohistochemical staining revealed that WT1 protein was widely detected in pediatric malignancies. The alveolar subtype of rhabdomyosarcoma showed more intensive staining than the embryonal subtype. The positive rate of the alveolar type was significantly higher than that of the embryonal type. The expression of WT1 mRNA in the tumor samples varied widely. However, no significant correlation was observed between WT1 mRNA expression and clinical factors. Conclusion The WT1 expression was broadly detected in various pediatric neoplasms. These results indicate that WT1 may therefore be a potentially useful therapeutic target in most of pediatric malignancies.

T-cell immune responses to Wilms tumor 1 protein in myelodysplasia responsive to immunosuppressive therapy

AbstractClinical observations and laboratory evidence link bone marrow failure in myelodysplastic syndrome (MDS) to a T cell–mediated immune process that is responsive to immunosuppressive treatment (IST) in some patients. Previously, we showed that trisomy 8 MDS patients had clonally expanded CD8+ T-cell populations that recognized aneuploid hematopoietic progenitor cells (HPC). Furthermore, microarray analyses showed that Wilms tumor 1 (WT1) gene was overexpressed by trisomy 8 hematopoietic progenitor (CD34+) cells compared with CD34+ cells from healthy donors. Here, we show that WT1 mRNA expression is up-regulated in the bone marrow mononuclear cells of MDS patients with trisomy 8 relative to healthy controls and non–trisomy 8 MDS; WT1 protein levels were also significantly elevated. In addition, using a combination of physical and functional assays to detect the presence and reactivity of specific T cells, respectively, we demonstrate that IST-responsive MDS patients exhibit significant CD4+ and CD8+ T-cell responses directed against WT1. Finally, WT1-specific CD8+ T cells were present within expanded T-cell receptor Vβ subfamilies and inhibited hematopoiesis when added to autologous patient bone marrow cells in culture. Thus, our results suggest that WT1 is one of the antigens that triggers T cell–mediated myelosuppression in MDS.

Sensitive immunohistochemical detection of WT1 protein in tumors with anti‐WT1 antibody against WT1 235 peptide

The Wilms' tumor 1 (WT1) gene is overexpressed in leukemia and various types of solid tumor, such as lung and colorectal cancer, and plays an oncogenic role in their tumorigenesis. Recent studies have demonstrated the potential of WT1-targeting cancer immunotherapy in clinical settings. As expression of WT1 protein in tumor cells is a prerequisite for WT1-targeting immunotherapy, immunohistochemical methods to detect WT1 protein with high sensitivity and specificity are required. In the present study, we developed a rabbit polyclonal antibody (WT1-R) against the 9-mer WT1 235 peptide, which is used for vaccination. The specificity of WT1-R was confirmed by immunoprecipitation, western blotting analysis, and competitive enzyme-linked immunosorbent assay. Immunocytochemistry showed the same reactivity against five cell lines (K562, Daudi, HT-180, SW480, and PC-14), whereas levels of WT1 mRNA expression determined by real-time qPCR (RT-PCR) analysis were not equivalent. Next, we examined the reactivity of WT1-R in tissue samples compared with a previously developed anti-WT1 antibody, 6F-H2. WT1-R showed greater sensitivity for detecting WT1 protein expression in samples from four different breast cancer patients than 6F-H2 antibody. The discrepancy in WT1 expression between these methods suggested that immunohistochemical detection of WT1 peptide may be advantageous for predicting the efficacy of WT1 vaccine compared to RT-PCR, and the highly sensitive WT1 antibody, WT1-R, may be useful to detect WT1 protein in tumors.

Wilms' Tumor 1 (WT1) Gene Mutations in Pediatric T-Acute Lymphoblastic Leukemia.

Abstract 3075Poster Board III-12The Wilms' tumor 1 (WT1) gene, located at chromosome band 11p13, encodes a transcriptional regulator involved in normal hematopoietic development. WT1 mutations have been identified in approximately 10 % of acute myeloid leukemia (AML), where it has recently been found to predict poor outcome, but also in T-cell acute lymphoblastic leukemias (T-ALL). Our aim was to evaluate the frequency, the main associated features and the prognostic significance of WT1 mutations in a cohort of pediatric patients with T-ALL treated according to EORTC-CLG trials. A total of 146 children, aged 7 months to 17 years, with newly diagnosed T-ALL were included in this study. Patients were treated according to 2 consecutive EORTC trials: 58 881 and 58 951. Immunophenotypic subtypes were classified according to the EGIL. Standard karyotype as well as molecular screening of HOX11/TLX1, HOX11L2/TLX3 and HOXA10 overexpression, SIL-TAL, NUP214-ABL, CALM-AF10 fusions were performed at diagnosis. WT1 transcript level was quantified by real-time PCR (RQ-PCR). Mutations of NOTCH1 exons 26, 27, 34, FBXW7 exons 9, 10, and WT1 exon 7, 9 were screened by direct sequencing. At least one WT1 mutation was found at diagnosis in 15 out of 146 (10%) T-ALL. WT1 mutations were predominantly exon 7 frameshift mutations (14/15 cases), consisting of small duplications, deletions or combined insertions/deletions, and were predicted to result in the production of a truncated protein missing the normal zinc finger domain. The remaining mutated patient harbored a somatically acquired missense mutation in exon 9 (C388Y), previously described in Denys-Drash syndrome. Only 4 out of 15 (27%) patients had 2 WT1 mutations and all WT1 mutations identified showed retention of the wild-type allele. Clonal evolution was investigated by analysis of 12 diagnostic-relapse pairs. Identical WT1 mutation was found at relapse in 3/4 mutated patients whereas 1/4 patients acquired an additional WT1 exon 7 mutation at relapse. One of the 8 patients with WT1 wild-type T-ALL at diagnosis acquired a WT1 exon 7 mutation at relapse. WT1 mutated and wild-type patients did not significantly differ in terms of age, gender, white blood cell count, or mediastinal involvement. Interestingly, WT1 mutated patients had significantly higher WT1 mRNA expression levels (median: 84% [25-837] for WT1 mutants vs 17% [0.007-657] for WT1 wild-type cases, p=0.005). This is in line with the trend for earlier developmental stage arrest observed in our WT1 mutated T-ALL as compared with WT1 wild-type T-ALL. Indeed, WT1 is preferentially expressed in immature hematopoietic progenitors and down-regulated in more differentiated cells. No association was found between the presence of WT1 mutations and NOTCH1 activating lesions. WT1 mutation was associated with HOX genes deregulation. HOX11 or HOX11L2 were overexpressed in 10/15 (67%) WT1 mutated ALL versus 29/123 (24%) WT1 wild-type ALL (p=0.001). In addition, HOXA overexpression and MLL-AF6 were found in one WT1 mutated T-ALL each. Overall, HOX deregulation was demonstrated in 12/15 (80%) WT1 mutated ALL at diagnosis and was also found in the T-ALL that acquired WT1 mutation at relapse. Despite being subclonal lesions strongly associated with HOX11 and HOX11L2 overexpression in T-ALL, WT1 mutations and NUP214-ABL fusion were found independent. A possible impact of WT1 mutation on outcome was investigated. The incidence of very high risk features was similar for patients with WT1 mutated and wild-type T-ALL. No significant differences were found between the WT1 mutated and wild-type group regarding 5-year event free survival (71.6% vs 74.1%; Wald test stratified for protocol: p=0.8) and overall survival (81.8% vs 81.3%; p=0.9). Notably, HOX112 overexpression, which is found in half of WT1 mutated T-ALL, has no pejorative impact either on outcome in EORTC trials. In conclusion, our study confirms that the type and incidence of WT1 mutations are very similar in pediatric T-ALL and AML, although the frequency of bi-allelic alterations may be lower in T-ALL. However, in contrast with AML, no pejorative outcome was associated with WT1 mutation. Moreover, we found that WT1 mutations are highly associated with direct or indirect aberrant HOX genes expression. DisclosuresNo relevant conflicts of interest to declare.

GATA-1 and GATA-2 binding to 3′ enhancer of WT1 gene is essential for its transcription in acute leukemia and solid tumor cell lines

Although oncogenic functions and the clinical significance of Wilms tumor 1 (WT1) have been extensively studied in acute leukemia, the regulatory mechanism of its transcription still remains to be determined. We found a significant correlation among the amounts of WT1, GATA-1 and GATA-2 mRNAs from leukemia and solid tumor cell lines. Overexpression and small interfering RNA (siRNA) transfection experiments of GATA-1 and GATA-2 showed that these GATA transcription factors could induce WT1 expression. Promoter analysis showed that the 5' promoter did not explain the different WT1 mRNA levels between cell lines. The 3' enhancer, especially the distal sites out of six putative GATA binding sites located within the region, but not the intron 3 enhancer, were essential for the WT1 mRNA level. Electrophoretic mobility shift assay (EMSA) showed both GATA-1 and GATA-2 bound to these GATA sites. Besides acute leukemia cell lines, solid tumor cell lines including, TYK-nu-cPr also showed a high level of WT1 mRNA. We showed that GATA-2 expression is a determinant of WT1 mRNA expression in both TYK-nu-cPr cells and HL60 cells without GATA-1 expression. Taken together, these results suggest that GATA-1 and/or GATA-2 binding to a GATA site of the 3' enhancer of WT1 played an important role in WT1 gene expression.

Effect of pure curcumin, demethoxycurcumin, and bisdemethoxycurcumin on WT1 gene expression in leukemic cell lines

Leukemias are groups of hematological malignancies with high incidence and mortality rates in patients worldwide. There have been shown in many studies that Wilms' tumor1 (WT1) gene were highly expressed in leukemic blast cells. Curcuminoids, major active components of the spice turmeric, are well known for its anticancer. Curcuminoids consist of pure curcumin, demethoxycurcumin, and bisdemethoxycurcumin. In this study, the effect of each curcuminoids'components on WT1 gene expression in leukemic cell lines (K562, HL60, U937, and Molt4) was investigated. The levels of WT1 mRNA and WT1 protein in leukemic cell lines were assessed by RT-PCR and Western blot analysis, respectively. It was found that the WT1 mRNAs were detected in all 4 types of leukemic cell lines. However, the WT1 protein levels were found only in the cell lines K562 and Molt4. Pure curcumin exhibited a strong inhibitory effect on WT1 mRNA and WT1 protein expression. The treatment of leukemic cell lines with non-cytotoxic doses (5, 10, and 15 microM) of pure curcumin for 2 days reduced the level of WT1 mRNA expression and WT1 protein in a dose-dependent manner. In addition, pure curcumin at 10 microM significantly decreased the level of WT1 mRNA and protein in a time-dependent manner. Pure curcumin, an excellent curcuminoid derivative, decreased WT1 gene expression in both transcriptional and translational levels. Thus, pure curcumin is one of a potential chemotherapeutic agent used for treatment of human leukemia. However, its chemotherapeutic property will need to be studied more in future.

Lack of clinical utility of minimal residual disease detection in allogeneic stem cell recipients with childhood acute lymphoblastic leukemia: multi-institutional collaborative study in Japan.

The clinical utility of minimal residual disease (MRD) measurements following allogeneic stem cell transplantation (SCT) in childhood ALL is controversial. We therefore performed a multi-institutional study of MRD in bone marrow samples taken before SCT and at 1, 3, 6 and 12 months after SCT. Case-specific clonal rearrangements of IgH and TCR genes and expression levels of Wilms' tumor 1 (WT1) mRNA were determined by PCR or RT-PCR methods. In total, 95 cases met all criteria for analysis of informative IgH/TCR markers and quantitative WT1 mRNA expression levels. During the 2-year (median 414 days) study period, 20 patients relapsed. Although the proportion of patients with a positive IgH/TCR result before SCT was significantly reduced at 1 month after treatment (P<0.001), attesting the efficacy of SCT, serial measurements of IgH/TCR rearrangements did not correlate with leukemic relapse. Clonal switch was demonstrated in 11 of the 14 patients with bone marrow relapse, indicating that the poor predictive power of the MRD assay most likely reflected the loss of PCR targets. WT1 expression was not related to either MRD detection by IgH/TCR assays or to clinical leukemic relapse. The clinical value of serial MRD monitoring would be limited in ALL patients undergoing SCT.