Wilms Tumor 1 Expression Research Articles

Evidence that downregulation of Wilms' tumor 1 (WT1) is involved in cortical stromal cell differentiation into theca cells in adult bovine ovaries.

Bovine theca cells are thought to differentiate from cortical stromal cells, and ovary-derived Wilms' tumor 1+ (WT1+ ) cells are the primary source of mouse theca cells. However, it is not known whether the differentiation of cortical stromal cells is regulated by WT1. Here, we identified WT1 in the cortical stroma and theca layer of the bovine ovary and analyzed the theca cell functional markers in cortical stromal cells and theca cells; in addition, we determined the effects of this gene on the secretion of androstenedione and progesterone by cortical stromal cells and the responsiveness of cortical stromal cells to luteinizing hormone (LH) in vitro. We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR), western blot analysis,and immunohistochemistry to discover that the cortical stroma had higher WT1 expression than the theca layer. We used RT-qPCR and ELISA analyses to determine that the cortical stromal cells had lower levels of androstenedione and progesterone secretion and LHRmessenger RNA expression than the levels of the theca cells. In cultured bovine cortical stromal cells, we found that WT1 downregulation increased androstenedione and progesterone secretion but had no effect on the LH responsiveness. Notably, the increase in androstenedione and progesterone secretion was associated with an increase in 3-β-hydroxysteroid dehydrogenaseexpression. In conclusion, the results suggest that WT1 is involved in the differentiation of cortical stromal cells into cells with characteristics similar to theca cells of antral follicles in adult bovine ovaries.

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.

Effect of gestational exposure to perfluorononanoic acid on neonatal mice testes.

Perfluoroalkyl acids (PFAAs) are widely used in commercial products and are found in many goods of daily use. Perfluorononanoic acid (PFNA) is one of the PFAAs that possesses endocrine disrupting properties and we have recently shown that PFNA affects testicular functions in Parkes mice. Exposure to environmental endocrine disruptors during fetal life is believed to affect gonadal development and they might produce reproductive abnormalities in males. Therefore, the present study examined the effect of gestational exposure to PFNA on the testes of neonatal mice offspring. Pregnant Parkes mice were orally administered PFNA (2 and 5mg/kg body weight) or distilled water from gestational day 12 until parturition. Male pups were killed on postnatal day 3. PFNA treatment decreased testosterone biosynthesis by inhibiting expression of steroidogenic acute regulatory protein, cytochrome P450scc, and 3β- and 17β-hydroxysteroid dehydrogenase; proliferation of testicular cells was also affected in treated mice. Furthermore, a marked decrease in expression of Wilms tumor 1, steroidogenic factor 1 and insulin-like factor 3 was noted in neonatal mice testes, indicating that the PFNA treatment may affect the development of the testis. Moreover, observation of the dose-related expression of anti-müllerian hormone and c-Kit in neonatal mice testes is also suggestive of an interference with gonadal development by PFNA exposure. In conclusion, the results suggest that the gestational exposure to PFNA decreased testosterone biosynthesis and altered the expression of critical factors involved in the development of the testis, thereby advocating a potential risk of PFNA to male reproductive health.

Investigation of Insulin-Like Growth Factor-1 (IGF-1), P53, and Wilms’ Tumor 1 (WT1) Expression Levels in the Colon Polyp Subtypes in Colon Cancer

BackgroundThere is no study in the literature investigating the expression levels of WT1, p53, and IGF-1 in colon polyp subtypes. In this study, we aimed to investigate the expression levels of IGF-1, p53, and WT1 in colon polyp subtypes and to determine whether expression levels are correlated with each other.Material/MethodsTissue specimens were obtained from 105 patients (80 men, 25 women; age range, 30–91 years) who underwent surgical resection for colorectal cancer (CRC) at Ordu University School of Medicine, Department of Pathology between January 2015 and 2017. Parameters such as age, sex, region of origin, and pathological diagnosis type were determined. The preparations were immunohistochemically stained with corresponding markers.ResultsThe results of the study showed that there was a statistically significant relationship between WT1 expression (negative – positive) in polyps and the place where the sample was taken (P=0.011). There is a positive relationship between P53 staining score (0–3) and positive frequency of IGF-1 (60.9–85.7%). There was a statistically significant change in P53 scores and location (P=0.006, p=0.015, respectively). As the P53 score of the polyps increased (0 to 3), the rate of adenomatous (34.8–78.4%) increased, so a positive relationship was found. WT1 and IGF-1 gene expression was associated with tumor location, p53 staining score, and sex.ConclusionsWT1 and IGF-1 are appropriate markers for CRC, and WT1 expression in CRC primary tumors especially could be a novel independent marker for prognosis and tumor progression.

Corrigendum] Alterations in gene expression during sexual differentiation in androgen receptor knockout mice induced by environmental endocrine disruptors

Following the publication of the above article, an interested reader drew to our attention a number of issues and concerns related to the descriptions of the genes and mouse models in the above paper. After having consulted with the authors, they have acknowledged that their paper did indeed contain a number of errors, and these are listed below in this Corrigendum. First, the authors realized that an incorrect figure was included as Fig.1 in the paper. The correct (and entirely different) version of Fig.1, together with its figure legend, is shown opposite. The following textual errors in the paper should also be noted: i) In the Abstract, lines3‑4 in the left‑hand column (LHC): "androgen receptor (AR)‑/‑, AR+/‑ and AR+/+ male mice" should have been written as "androgen receptor (AR) conditional knockout mice"; line5 in the LHC: "flox‑AR" should have been written as "ARflox/+"; line6 in the LHC: "AR‑Cre" should have been written as "Amhr2‑Cre"; line22 in the LHC: "AR+/‑" should have been written as "ARflox/Y"; line1 in the right‑hand column (RHC): "heterozygous" should have been written as "ARflox/Y"; and line2 in the RHC, "heterozygous" should have been written as "ARflox/Y Cre+". ii) In the Introduction, paragraph 3, lines6‑7 in the LHC: "or female ARKO mice" should have been deleted; paragraph4, line5 in the LHC: "AR‑/‑", "AR+/‑" and "AR+/+" should have been deleted; paragraph4, line11 in the LHC: "AR‑/‑", and "AR+/‑" should have been written as "AR‑/Y", whereas "AR+/+" should have been written as "AR+/Y". iii) In the Materials and methods, paragraph3, the last sentence in the RHC {"The identification of the plasmid containing leydig cells expressing Cre recombinant enzyme and the specificity of the genetically modified (gm) sequence [Amhr2‑Cre (7.1)] are shown in Fig.1} should have been written as "The positive clones were identified by digestion and sequencing." iv) In the Results, the first subheading in the RHC ("...transgenic mouse model (Amhr2‑Cre)" should have been written as "...AR conditional knockout targeting vector"; paragraph1, lines1‑3 in the RHC: The sentence "The AR gene condition knockout plasmid was identified and confirmed by enzyme digestion with BamHI" should have been written as "The AR gene conditional knockout strategy is shown in Fig.1. The gene targeting vector was identified and confirmed by enzyme digestion with BamHI"; paragraph1, line4 in the RHC: "2.7kb" should have been written as "7.8 kb"; Table III, in the title and in the Table heading, "AR+/‑" should have been written as "AR+/Y", and "AR‑/‑" should have been written as "AR‑/Y"; in the subheading "Comparison between AR‑/‑ male mice and normal mice", "AR‑/‑" should have been written as "AR‑/Y", and "normal" should have been written as "AR+/Y"; and paragraph4, lines 3‑5 in the LHC: "AR+/‑" should have been written as "AR+/Y", and "AR‑/‑" should have been written as "AR‑/Y". All the authors thank the interested reader for drawing these matters to their attention, and agree to the contents of this Corrigendum. The authors also stress that these errors did not significantly influence either the results or the conclusions of the paper. Furthermore, the authors apologize to the Editor of International Journal of Molecular Medicine and to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 35: 399‑404, 2015; DOI: 10.3892/ijmm.2014.2015].

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages

Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6+ macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression oftwo rate-limiting enzymes in retinol metabolism. Depletion of Wt1+ stromal cellsreduced the frequency of GATA6+ macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1+mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages.

Predicted Markers of Overall Survival in Pancreatic Cancer Patients Receiving Dendritic Cell Vaccinations Targeting WT1

Background: We have developed a Wilms’ tumor 1 (WT1)-targeting dendritic cell (DC)-based cancer vaccine combined with standard chemotherapy for patients with advanced pancreatic ductal adenocarcinoma (PDA). Methods: We evaluated predictive markers of overall survival (OS) in PDA patients treated with multiple major histocompatibility complex class I/II-restricted, WT1 peptide-pulsed DC vaccinations (DC/WT1-I/II) in combination with chemotherapy. Throughout the entire period of immunochemotherapy, the plasma levels of soluble factors derived from granulocytes of 7 eligible PDA patients were examined. Moreover, systemic inflammatory response markers (neutrophil-to-lymphocyte ratio [NLR], monocyte-to-lymphocyte ratio [MLR], and granulocyte-to-lymphocyte ratio [GLR]) were assessed. In addition, cytoplasmic WT1 expression in PDA cells was examined. Results: Compared to the 4 non-super-responders (OS <1 year), the remaining 3 super-responders (OS ≥1 year) showed significantly decreased low plasma matrix metalloproteinase-9 levels throughout long-term therapy. The NLR, MLR, and GLR after 5 DC/WT1-I/II vaccinations and 3 cycles of gemcitabine were significantly lower in the super-responders than in the non-super-responders. Furthermore, the cytoplasmic WT1 expression in the PDA cells of super-responders was relatively weak compared to that in the PDA cells of non-super-responders. Conclusions: Prolonged low levels of a granulocyte-related systemic inflammatory response after the early period of therapy and low cytoplasmic WT1 expression in PDA cells may be markers predictive of OS in PDA patients receiving WT1-targeting immunochemotherapy.

WT1 Measurable Residual Disease Assay in Patients With Acute Myeloid Leukemia Who Underwent Allogeneic Hematopoietic Stem Cell Transplantation: Optimal Time Points, Thresholds, and Candidates.

The absence of relevant guidelines for Wilms tumor 1 (WT1) gene quantification as a measurable residual disease (MRD) assessment for patients with acute myeloid leukemia (AML) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) has limited the widespread use in practice. We investigated optimal time points, thresholds, and candidates for the bone marrow WT1 MRD assay in 425 consecutive patients with AML who underwent allo-HSCT. WT1 expression kinetics before allo-HSCT and at 1 or 3 months after allo-HSCT were determined by real-time PCR using the European LeukemiaNet (ELN) normalized method. Relapsed patients had significantly higher WT1 levels before allo-HSCT and at 3 months after allo-HSCT. The best time point for the WT1 MRD assay was before allo-HSCT by the receiver operating characteristic curve. Among various thresholds, 250 copies recommended from ELN researchers were mostly predictive of post-transplant relapse. In multivariate analysis, WT1 MRD positivity independently predicted relapse, resulting in inferior survival. In subgroup analyses, pretransplant WT1 MRD positivity was predictive of post-transplant relapse in the intermediate group, whereas WT1 MRD positivity occurred at 3 months after allo-HSCT in favorable and adverse risk groups. Among MRD-positive patients before allo-HSCT, all patients who were MRD positive at 3 months relapsed within 6 months. The WT1 MRD assay before allo-HSCT or 3 months after allo-HSCT is useful for predicting post-transplant relapse with a different significance in each risk group by time points, showing the benefit of multiple tests over time. Such monitoring is particularly available in patients with AML without specific molecular targets.

PF207 TARGETING WILMS TUMOR 1 WITH A T CELL BISPECIFIC ANTIBODY (WT1‐TCB): EX VIVO AND IN VIVO POTENCY BY BIVALENT RECOGNITION OF PEPTIDE‐MHC COMPLEXES FROM AN INTRACELLULAR TUMOR ANTIGEN

Background:Antibody‐based immunotherapy represents a promising strategy to target chemo‐resistant leukemic cells. However, classical antibody‐based approaches are restricted to cell surface antigens, even though targeting of intracellular antigens would allow further access to a large number of tumor‐specific target structures.Aims:In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody based on T cell receptor (TCR)‐like antibodies for immunotherapy of acute myeloid leukemia (AML). The TCB targets the intracellular tumor antigen Wilms Tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A∗02:01 (HLA‐A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR‐specificity.Methods:WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real‐time PCR, western blot and immunohistochemical staining. WT1‐TCB‐mediated cytotoxicity was analyzed by co‐cultivation of WT1‐expressing HLA‐A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCB‐mediated cytotoxicity against primary AML cells was evaluated in our pre‐established feeder layer‐based ex vivo long‐term culture system. For in vivo testing, NSG mice (NOD.Cg‐Prkdcscid‐Il2rgtm1Wjl/SzJ) were humanized with human HLA‐A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1‐expressing HLA‐A2+ SKM‐1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1‐TCB.Results:In accordance with previous reports, we observed WT1 expression in 79% (n = 38) of cancer cell lines and in 92% (n = 65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n = 26), whereas WT1 was expressed again at time of relapse (n = 21). WT1‐TCBs elicited antibody‐mediated T cell cytotoxicity against peptide‐pulsed T2 cells and AML cell lines in a WT1 and HLA‐restricted manner. Equally, TCR downstream signaling was observed in a WT1‐restrictive manner by co‐cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1‐TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67 ± 6% after 13–14 days; ± SEM; n = 18). Correspondingly, up‐regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3 ± 1.5, PD‐1: 5.1 ± 0.7, TIM‐3: 4.7 ± 0.6; ± SEM; n = 22). WT1‐TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38 ± 13% after 13–14 days; ± SEM; n = 5). In comparison with WT1RMF‐specific T cells, only bivalent binding by WT1‐TCB induced efficient lysis of primary AML cells. Furthermore, WT1‐TCB‐treated humanized mice bearing SKM‐1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control.Summary/Conclusion:TCR‐like TCBs targeting of intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide‐MHC complex on the cell surface validating WT1‐TCB as a promising therapeutic agent for the treatment of AML.

Expression of WT1 gene and its prognostic value in patients with acute myeloid leukemia

To investigate the expression of Wilms'tumor 1 (WT1) gene in patients with acute myeloid leukemia (AML), and to explore its application in predicting prognosis of AML in patients with wild or mutated nucleophosmin 1(NPM1) and Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD). One hundred and sixty-seven newly diagnosed AML patients(exclued M3 type) were enrolled in this study. The survival of patients were analyzed with Kaplan-Meier method. The clinical data, laboratory findings and the survival of patients were analyzed and compared between patients with high WT1 expression (high-WT1 group) and those with low WT1 expression (low-WT1 group), as well as among the patients with NPM1 or FLT3-ITD wild type and mutants. The overall response rates (ORR) in high-WT1 and low-WT1 groups were 65.9% (83/126) and 95.1% (39/41), respectively (P<0.01). Compared with the low-WT1 group, the high-WT1 group had lower 2-y overall survival (OS) rate (46.1% vs. 75.2%, P<0.05) and 2-y disease free survival (DFS) rate (43.5% vs. 68.5%, P<0.05). After induction chemotherapy, the patients with decreased WT1 gene expression ≥ 1log was associated with higher ORR and 2-y OS rate (all P<0.05), but the advantage of 2-y DFS rate was not shown (P>0.05). In patients with NPM1 wild type, the high-WT1 group had inferior ORR and 2-y OS rate (all P<0.05), while in the patients with FLT3-ITD wild type, the high-WT1 group had inferior ORR, 2-y OS rate and 2-y DFS rate (all P<0.05). In patients with NPM1 or FLT3 -ITD mutations, the WT1 expression had no significantly predicting values in treatment efficacy and survival (all P>0.05). WT1 gene overexpression indicated poor prognosis of AML patients; the patients with decreased WT1 gene expression ≥ 1 log after the first induction therapy show better prognosis than those with<1 log. The WT1 gene expression level at diagnosis can be used as an unfavorable prognostic factor for AML patients with NPM1 or FLT3-ITD wild types.

Renal-Protective Effects of the Peroxisome Proliferator-Activated Receptor-γ Agonist Pioglitazone in ob/ob Mice.

BackgroundThis study investigated the therapeutic effects of the peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone in ob/ob mice with obesity-related glomerulopathy (ORG).Material/MethodsA total of 24 mice were divided into 3 groups: wild-type C57BL/6 mice (n=8), ob/ob mice (n=8), and ob/ob mice receiving pioglitazone treatment (n=8). Body mass, blood glucose, serum adiponectin (ADP), and urine microalbumin (mALB) levels were determined. Renal histology was examined using light and electron microscopy. Wilms tumor 1 (WT1), Zonula occludens-1 (ZO-1), AMP activated protein kinase (AMPK), and NADPH oxidase-4 (NOX-4) expression were evaluated by immunohistochemistry and Western blot.ResultsSerum ADP did not alter between weeks 0 and 12 in the control group, while the ob/ob mice showed a time-dependent decrease that was prevented by pioglitazone. Urinary mALB did not alter between week 0 and 12 in the control group, but was higher in week 0 and week 12 in the ob/ob group. Pioglitazone prevented the rise in urinary mALB in week 12. Histology revealed glomerulomegaly, mesangial proliferation, focal segmental glomerulosclerosis, and foot processes fusion in the ob/ob group, which were ameliorated by pioglitazone treatment. Compared to the control group, ob/ob mice had a higher kidney index and glomerular diameter, which were reduced by pioglitazone treatment. Immunohistochemical and Western blot experiments revealed lower expression levels of WT1, ZO-1, and AMPK and higher NOX-4 expression level in the ob/ob group, which was prevented by pioglitazone treatment.ConclusionsPioglitazone, a PPARγ agonist, can prevent ORG, probably by reducing oxidative stress.

WT1 in astrocytomas: Comprehensive evaluation of immunohistochemical expression and its potential utility in different histological grades.

Wilms' tumor 1 (WT1) mutation has recently been detected in gliomas. Growing data indicate that WT1 mutation plays a causal role in gliomagenesis and is overexpressed in most glioblastomas. An emerging immunotherapy targeting WT1 has shown to be effective in resistant glioblastomas in clinical trials. WT1 expression and its potential utility in various grades of astrocytomas is still unclear and needs further elucidation. The evaluation of WT1 can be done by molecular or immunohistochemical methods. As immunohistochemistry is easier with wider routine use, immunoexpression of this biomarker was studied. The aim of this study was to characterize WT1 immunoexpression across different histological grades of astrocytomas to routinely aid in diagnosis and reproducibility and to assess the association between WT1 and immunomarker isocitrate dehydrogenase (IDH1). This was an observational prospective study on 79 cases of astrocytomas. Seventy-nine astrocytomas including 11 recurrent tumors were assessed for WT1 by immunohistochemistry. WT1 expression was detected in all astrocytomas (100%). The control group of reactive gliosis was negative. WT1 score correlated with histological tumor grades (P < 0.001) with higher score in higher grade. It was also observed that different tumor grades depicted two distinct expression patterns. WT1 score and pattern were valuable in differentiating high- and low-grade astrocytomas. This study supports the oncogenic role of WT1 in astrocytomas. WT1 was found to be valuable in distinguishing different grades of astrocytomas. WT1 can aid in differentiating neoplastic process from reactive gliosis, particularly in recurrent tumors. Higher expression in glioblastomas supports its immunotherapy potential.

Prognostic Impact of WT-1 and Survivin Gene Expression in Acute Myeloid Leukemia Patients.

Wilms Tumor 1 (WT1) and Survivin genes are important leukemia-associated antigens (LAAs) in AML with potential prognostic impact. We investigated WT1 and Survivin expression levels by RT-PCR in 61 AML patients in correlation with clinical characteristics and outcomes. WT1 was overexpressed in 45 patients (73.8%), associated with higher BM blasts (p = 0.017), lower incidence of favorable-prognosis cytogenetics (p = 0.035), and higher incidence of Flt3-ITD mutations (p = 0.026). Survivin was overexpressed in 17 patients (27.9%) with higher mean WBC count (p = 0.049). Patients with overexpression of either gene showed inferior complete remission (CR) rates and survival rates, patients with overexpression of both genes showed higher mean WBCs (p = 0.035) and higher BM blasts (p = 0.029) while the double negative group showed higher incidence of favorable cytogenetic events (p = 0.021), better CR rates and survival rates. Our findings support the introduced prognostic impact of WT1 and Survivin genes in AML patients and its potential use in MRD monitoring and immunotherapy.

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.

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 mRNA of HMGA2 and WT1 Genes in Whole Blood Samples As Potential Diagnostic Markers in MPN

BackgroundHigh-mobility group AT-hook protein 2 (HMGA2) gene codes for a non-histone protein which expressed during embryogenesis and early hematopoiesis but suppressed in differentiated cells. Overexpression of HMGA2 as well as transcription factor Wilms' tumor 1 (WT1) gene has been found in a variety of human cancers, but only a few reports has included myeloproliferative neoplasms (MPNs) (Guglielmelli P. et al., 2007; Chen C-C. et al., 2017; Tasdemir S. et al, 2015). Furthermore, there were no any data about co-expression of mRNA both genes in blood in patients with MPNs.Aims: evaluate of the diagnostic potential of the WT1 and HMGA2 mRNA level in MPN patients.MethodsIn our retrospective study was analysed 115 archived frozen peripheral blood samples with RNAse inhibitor from 62 MPN patients: 29 with polycythemia vera (PV), 16 with essential thrombocythemia (ET), and 17 with myelofibrosis (MF) were included. Samples of 28 healthy volunteers were tested as control group. Total RNA was isolated using “Ribo-zol-D” (Aplisens) and then were transcribed using “Reverta-L” (Aplisens). Synthesized cDNA was analysed by real-time PCR to detect of WT1 and HMGA2 expression levels on the CFX96 (Bio-Rad) housekeeping kits using. The results were calculated utilizing the delta Ct method in the software package of “R”. The threshold cycles (Ct) WT1 and HMGA2 genes and reference gene ABL determined using Cy0 method. Results are presented as median and range (25% and 75%). Comparisons between the patient and control group were made using the Mann-Whitney U test. Statistical analysis was performed using Statistica 12.0 software.ResultsThe level of HMGA2 mRNA in five samples of venous blood in PV patients and 12 samples in MF patients was higher than in healthy subjects (Figure 1). Expression of WT1 mRNA (≥ 0.01%) was observed only in samples among patients with MF (in 19 of 28). In 12 blood samples of patients with MF, transcripts of both genes were detected together (Table 1). The increased level of HMGA2 mRNA expression was associated with a higher level of erythrocytosis in patients with PV (p<0.05). The co-expression of HMGA2 mRNA and WT1 mRNA in patients with MF was associated with a low level of white and red blood cell (p<0.05). Interestingly that in MF patients the most pronounced thrombocytopenia was observed if only WT1 mRNA was detected. In all cases of repeated testing of patients, the growth of HMGA2 or WT1 mRNA level was accompanied by decline of the cellular blood composition. The level of HMGA2 mRNA and WT1 mRNA in our study did not depend on the driver mutations presence or their allele burden. It should be noted that 4 out of 12 patients with MF died within 5 months after the first detection of WT1 mRNA and one patient died more after 15 months. At that time only one MF patient without WT1 or HGMA2 transcripts died during of 20 months' observation.Summary and ConclusionsExpression of the WT1 and HMGA2 genes is involved in the pathogenesis and progression of the disease and can be used as diagnostic markers. The question of the independent prognostic significance of these transcripts in blood samples for various phenotypes of MPN is promising for further study. DisclosuresNo relevant conflicts of interest to declare.

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 Expression at Diagnosis Correlates With Genetic Abnormalities and Polymorphism But Is Not Independently Prognostic in Acute Myelogenous Leukemia: A Hokkaido Leukemia Net Study

BackgroundThe prognostic effect of Wilms tumor 1 (WT1) expression at the diagnosis of acute myelogenous leukemia (AML) has been controversial. The aim of the present study was to determine the correlations of WT1 expression at the diagnosis of AML with established prognostic alterations. Patients and MethodsWe analyzed diagnostic bone marrow samples from 252 patients. WT1 expression, single nucleotide polymorphism (SNP) in the WT1 gene (rs16754), and Fms-like tyrosine kinase receptor-3 internal tandem duplication (FLT3-ITD) mutation were analyzed for all patients. The nucleophosmin 1 (NPM1) mutation and CCAAT/enhancer-binding protein-α (CEBPA) double mutation were analyzed for cytogenetically normal (CN)-AML. The KIT mutation was analyzed for core-binding factor AML. ResultsWithin the cytogenetically favorable prognosis group, WT1 expression in AML with inv(16) or t(15;17) was significantly greater than that in AML with t(8;21). In cases with CN-AML, FLT3-ITD and NPM1 mutations both correlated with greater expression of WT1, and the CEBPA double mutation was related to lower WT1 expression. The existence of both FLT3-ITD and NPM1 mutations showed synergistically greater expression of WT1 in CN-AML. SNP in the WT1 gene (rs16754) was significantly associated with lower expression of WT1. The WT1 levels were not prognostic factors in the total cohort or any cytogenetic group or stratified by SNP status. ConclusionBecause WT1 expression has correlated with known prognostic factors, the prognostic effect of WT1 levels could be misunderstood depending on the distribution of the collaborative mutations in each cohort. We have concluded that the prognostic significance of WT1 at the diagnosis of AML is weak compared with the other established prognostic factors.

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.

Abstract 549: The CUG-translated WT1, not AUG-WT1, is an oncogene

Abstract The Wilms' tumor 1 (WT1) gene is believed to act as a canonical tumor suppressor. However, it has also been reported to function as an oncogene. Germline WT1 deletion is associated with Wilms' tumor, and exogenous WT1 cDNA introduction into cells induces the transcriptional suppression of its oncogenic target genes. In contrast, high WT1 expression is associated with poor prognosis in patients with various cancers. Why WT1 acts as a tumor suppressor under certain conditions but as an oncogene under other conditions is unknown. Here, we report that CUG initiation site for WT1 protein synthesis (CUG)-translated WT1 (cugWT1), an N-terminally extended form of canonical AUG initiation site for WT1 protein synthesis (AUG)-translated WT1 (augWT1), was overexpressed in most cancer cell lines and cancer tissues and functioned as an oncogene, whereas the classical augWT1 acted as a tumor suppressor as reported previously and inhibited the function of cugWT1. Translation of cugWT1 is initiated from a CUG codon upstream and in-frame with the coding region of augWT1. cugWT1 induced cell transformation and increased the gene expression of c-myc, bcl-2 and egfr, whereas overexpression of augWT1 repressed colony formation of cancer cells and inhibited the expression of the same target genes by recruiting histone deacetylase 1 (HDAC1). In addition, we found that protein kinase B (AKT)-phosphorylated cugWT1 on Ser62 and protected cugWT1 from proteasomal degradation induced by the F-box/WD repeat-containing protein 8 (FBXW8). These results provide an important breakthrough in the field of cancer biology and contribute significantly to the resolution of the chameleon function of WT1. Citation Format: Kun Yeong Lee, Hong-Gyum Kim, Joohyun Ryu, Do Young Lim, Hanyong Chen, Ann M. Bode, Zigang Dong. The CUG-translated WT1, not AUG-WT1, is an oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 549.