Wilms Tumor 1 Gene Expression Research Articles

Prognostic Impact of WT1 Mutation on AML of Different Risk Groups Based on 2022 European Leukemianet (ELN) Risk Classification

Background The Wilms tumor 1 (WT1) gene, located on chromosome 11p13 exists in multiple isoforms. It has been implicated in the regulation of cell survival, proliferation and differentiation, and may function both as a tumor suppressor and an oncogene. Approximately 15% of acute myeloid leukemia (AML) patients carry WT1 mutations, and WT1 mutation have been shown to be independent predictor of worse clinical outcome in some but not all adult AML studies. Some studies suggested that NPM1-AML & AML with CEBPA double-mutation accompanied with WT1 mutation can alter their original favorable prognosis. Recently, a revised European LeukemiaNet (ELN) genetic risk classification has been published and the prognostic significance of WT1 mutation in three risk groups according to this 2022 ELIN risk classification is still unclear. Objective To investigate the prognostic significance of WT1 gene mutations in acute myeloid leukemia (AML) patients with different risk of the 2022-ELN risk classification. Methods 502 patients de novo AML diagnosed between March 2015 and July 2021 who underwent next-generation sequencing (NGS) tests and had MICM precise typing at the Peking University Institute of Hematology were enrolled in this study. The patients received chemotherapy ± hematopoietic stem cell transplantation (HSCT), and they were stratified into three groups according to the 2022 version of ELN-AML risk stratification. Results Among 502 patients, a total of 71 (14.1%) patients carried WT1 mutations, of which 57 patients had mutations at a single locus, 8 patients had mutations at double sites, 5 patients had co-mutations at 3 sites in the WT1 gene, and 1 patient had mutations at 4 sites. Comparing the incidence of AML-related mutations between the WT1 mutation group and the wild-type group, the results showed that the concomitant rate of NPM1 mutations was lower in the WT1 mutation group than in the WT1 wild-type group (14.1% vs 27.1%, p=0.028), and the concomitant rate of CEBPA-bZIP mutations was higher in the WT1 mutation group than in the WT1 wild-type group (29.6% vs 10.9%, p<0.001). The concomitant rate of CEBPA double mutation was also higher in the WT1 mutation group than in the WT1 wild-type group (29.6% vs 10.4%, p<0.001), and the concomitant rate of GATA2 mutation was higher in the WT1 mutation group than in the WT1 wild-type group (14.1% vs. 4.4%, p=0.004). In addition, the WT1 gene expression level (WT1/ABL1) was higher in the WT1 mutation group than in the WT1 wild group at diagnosis (22.0% vs 13.5%, p=0.004). All patients were further divided into 6 groups based on 2022 ELIN risk classification and whether they received HSCT. There were 158 (31.5%) patients in the low-risk chemotherapy group, 73 (14.5%) in the low-risk transplantation group, 72 (14.3%) in the intermediate-risk chemotherapy group, 58 (11.6%) in the intermediate-risk transplantation group, 78 (15.5%) in the high-risk chemotherapy group, and 63 (12.5%) in the high-risk transplantation group. Univariate and multivariate COX regression analyses were performed including WT1 mutation, FAB classification, age, sex, and karyotype stratification indicators. In 58 patients who were intermediate-risk and received HSCT, patients with WT1 mutation resulted in higher cumulative incidence of relapse(CIR) in compared with those without WT1 mutation (52.3% vs 33.5%, HR=9.28, p<0.001). In the remaining 5 groups of patients, WT1 mutation seemed have no significant effect on overall survival(OS), event-free survival (EFS), or CIR in patients (p-values were all less than 0.05). Conclusion Among intermediate-risk AML patients in the 2022 version of ELN stratification who received transplantation, the CIR after transplantation was higher in AML patients with WT1 mutation. It seemed that WT1 mutation was an independent risk factor for post-transplantation recurrence, and allogeneic HSCT cannot efficiently overcome the poor prognosis of WT1 mutation in the intermediate-risk group. Intermediate-risk AML with WT1 mutations still requires more active strategies to prevent relapse. Of course, the impact of WT1 mutation on prognosis needs to be further confirmed by expanding the sample size.

Clinical significance of the increased expression of the WT1 gene in peripheral blood of patients with acquired aplastic anemia.

To determine the significance of increased Wilms tumor 1 (WT1) gene expression in the peripheral blood of patients with acquired aplastic anemia (AA), we analyzed serial changes in WT1 mRNA copy number (WT1cn) in 63 patients with AA as well as in five patients with myelodysplastic syndromes (MDS) and seven patients with paroxysmal nocturnal hemoglobinuria (PNH). WT1cn was higher than the cut-off (≥50 copies/μg RNA) at the time of the first measurement in 41% of untreated (60-190 copies/μg RNA [median 130]) and 59% of treated (59-520 copies/μg RNA [median 150]) AA patients. Although WT1cns gradually increased in most AA patients during the 2-105 months follow-up period, they did not lead to clonal evolution except in three patients in whom the maximum change ratio of WT1cn (WT1cn-change max), defined as the ratio of WT1cn at the first examination to that of the maximum value, exceeded 20.0 and who developed MDS at 2, 46, and 105 months. Increased WT1 gene expression was enriched in granulocytes rather than in mononuclear cells in most WT1-positive AA patients and did not correlate with mutations of genes associated with myeloid malignancy. WT1cns were high at 690-5700 (median 2000) in MDS patients and remained high thereafter, while WT1cns in PNH patients (77-200; median 96) were similar to those in AA. Thus, moderate increases in WT1cns up to 600 are common in AA patients in stable remission. An increase in the WT1cn-change max over 20.0 may portend transformation from AA to MDS.

Significance of WT1 and multiparameter flow cytometry assessment in patients with chronic myelomonocytic leukemia receiving allogeneic hematopoietic stem cell transplantation.

The objective of this study was to investigate the clinical significance of minimal residual disease (MRD) monitoring through Wilms tumor 1 (WT1) gene expression and multicolor flow cytometry (FCM) in patients with chronic myelomonocytic leukemia (CMML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). For this purpose, WT1 gene expression and the CMML-related abnormal immunophenotype were examined using real-time quantitative polymerase chain reaction and FCM, respectively. In total, 59 patients with CMML who underwent allo-HSCT were enrolled in this study. Thirteen cases (22.0%) developed hematological relapse, and 15 patients (25.4%) expired during the follow-up period. Thirty-four patients (37.6%) were positive for WT1 (WT1+), and 44 patients (74.6%) were positive for FCM prior to allo-HSCT. After allo-HSCT, there were 21 WT1+ patients (35.6%) and 10 patients (16.9%) who were positive in FCM (FCM+). Post-transplant WT1+ (post-WT1 0.6+; 50.7% vs. 7.6%, p<.001) and post-transplant FCM+ (post-FCM+; 90.0% vs. 8.8%, p<.001) indicated a higher 3-year cumulative incidence of relapse (CIR) compared with the WT1- or FCM-patients. Multivariate analysis of event-free survival (EFS), overall survival (OS), and CIR showed that the FCM status after transplantation was an independent prognostic factor for relapse (p<.05). Both FCM and WT1 after HSCT were identified as important predictors of recurrence of CMML following transplantation and may be useful in guiding interventions against disease relapse.

AML-237: Wilms Tumor 1 (WT-1) Gene Methylation as an Epigenetic Biomarker in Acute Myeloid Leukemia

Context: Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy characterized by different genetic and epigenetic aberrations. Several epigenetics modifications were observed in the promoter regions of the cellular epigenome, including hypomethylation or hypermethylation of CpG islands during the onset and progression of AML. DNA methylation is an epigenetic modification that regulates gene expression and plays a pivotal role in tumorigenesis, including AML. Wilms tumor 1 (WT-1) is a transcription factor involved in the differentiation and proliferation of AML. The clinical relevance of WT-1 gene expression and its underlying epigenetic alterations has not been fully addressed in AML. Objective: This work aimed to investigate promoter methylation and mRNA expression of WT-1 gene in AML. Patients: Bone marrow samples were collected at the time of diagnosis and after completion of induction chemotherapy from 10 newly diagnosed AML patients. Five non-malignant BM samples were recruited as controls. Methods: Quantitative assessment of WT-1 gene transcripts was performed using real-time PCR. In addition, methylation-specific PCR was done to evaluate the promoter methylation status of the WT-1 gene in AML. Results: Robust promoter hypermethylation and higher expression of the WT-1 gene were observed in AML patients at time of diagnosis. In contrast, hypomethylation and lower WT-1 gene expression were observed after completion of induction therapy (p<0.001). Conclusions: This is the first report, to our knowledge, regarding WT-1 gene overexpression/hypermethylation signature as a characteristic marker that correlates with leukemic burden in AML among the Indian population. Increased expression and methylation signature of the WT-1 gene can be used as a promising molecular marker for diagnosis, clinical progression of the diseases, or monitoring response to treatment, as well as a target for the development of novel therapeutic approaches. Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy characterized by different genetic and epigenetic aberrations. Several epigenetics modifications were observed in the promoter regions of the cellular epigenome, including hypomethylation or hypermethylation of CpG islands during the onset and progression of AML. DNA methylation is an epigenetic modification that regulates gene expression and plays a pivotal role in tumorigenesis, including AML. Wilms tumor 1 (WT-1) is a transcription factor involved in the differentiation and proliferation of AML. The clinical relevance of WT-1 gene expression and its underlying epigenetic alterations has not been fully addressed in AML. This work aimed to investigate promoter methylation and mRNA expression of WT-1 gene in AML. Bone marrow samples were collected at the time of diagnosis and after completion of induction chemotherapy from 10 newly diagnosed AML patients. Five non-malignant BM samples were recruited as controls. Quantitative assessment of WT-1 gene transcripts was performed using real-time PCR. In addition, methylation-specific PCR was done to evaluate the promoter methylation status of the WT-1 gene in AML. Robust promoter hypermethylation and higher expression of the WT-1 gene were observed in AML patients at time of diagnosis. In contrast, hypomethylation and lower WT-1 gene expression were observed after completion of induction therapy (p<0.001). This is the first report, to our knowledge, regarding WT-1 gene overexpression/hypermethylation signature as a characteristic marker that correlates with leukemic burden in AML among the Indian population. Increased expression and methylation signature of the WT-1 gene can be used as a promising molecular marker for diagnosis, clinical progression of the diseases, or monitoring response to treatment, as well as a target for the development of novel therapeutic approaches.

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

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

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].

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.

Desmoplastic small round cell tumor of the parotid gland-report of a rare case and a review of the literature

BackgroundDesmoplastic small round cell tumor (DSRCT) is a rare soft tissue tumor that generally involves the retroperitoneum, pelvis, omentum and mesentery in younger patients. However, extra-abdominal DSRCT is very rare.Case presentationA 49-year-old Japanese man noticed a mass in the right parotid gland. Ultrasound examination revealed a solid tumor about 2 cm in diameter. Computed tomography (CT) of the whole body revealed no other tumors or lymph node swelling. Superficial parotidectomy was performed. Histologically, the tumor was composed of various-sized tumor cell nests in an abundant fibromyxoid and collagenous background. The tumor cells were small to medium-sized. Immunohistochemistry showed that the tumor cells were immunoreactive for epithelial markers and desmin. They also showed strong nuclear staining with a Wilms tumor 1 (WT1) antibody detecting the C-terminal region (C-WT1), but not the N-terminal region (N-WT1). We also performed 3′/5′ expression imbalance assay based on reverse transcription polymerase chain reaction (RT-PCR) to determine whether aberrant WT1 gene expression was present. This tumor was found to lack 5′-regional expression of the WT1 gene, as well as immunoreactivity with the N-WT1 antibody. Finally, fluorescence in situ hybridization (FISH) and RT-PCR analyses revealed the presence of a gene showing fusion between exon 7 of EWSR1 and exon 8 of WT1. The tumor was diagnosed as a DSRCT of the right parotid gland. The patient has been followed for 3 years without recurrence or metastasis.ConclusionsAlthough DSRCT in the salivary gland is extremely rare, it should be included in the differential diagnosis of poorly differentiated salivary gland neoplasms, especially with a fibromyxoid background. Pathologists should bear in mind that DSRCT may occur in major salivary glands and should perform immunohistochemistry with appropriate antibodies, not only those against keratin and desmin, but also one detecting the C-terminal region of WT-1. Furthermore, molecular detection of EWSR1-WT1 fusion gene conclusively confirmed the diagnosis of DSRCT in this uncommon location.

Outcomes of Measurable Residual Disease in Pediatric Acute Myeloid Leukemia before and after Hematopoietic Stem Cell Transplant: Validation of Difference from Normal Flow Cytometry with Chimerism Studies and Wilms Tumor 1 Gene Expression

We enrolled 150 patients in a prospective multicenter study of children with acute myeloid leukemia undergoing hematopoietic stem cell transplantation (HSCT) to compare the detection of measurable residual disease (MRD) by a “difference from normal” flow cytometry (ΔN) approach with assessment of Wilms tumor 1 (WT1) gene expression without access to the diagnostic specimen. Prospective analysis of the specimens using this approach showed that 23% of patients screened for HSCT had detectable residual disease by ΔN (.04% to 53%). Of those patients who proceeded to transplant as being in morphologic remission, 10 had detectable disease (.04% to 14%) by ΔN. The disease-free survival of this group was 10% (0 to 35%) compared with 55% (46% to 64%, P < .001) for those without disease. The ΔN assay was validated using the post-HSCT specimen by sorting abnormal or suspicious cells to confirm recipient or donor origin by chimerism studies. All 15 patients who had confirmation of tumor detection relapsed, whereas the 2 patients with suspicious phenotype cells lacking this confirmation did not. The phenotype of the relapse specimen was then used retrospectively to assess the pre-HSCT specimen, allowing identification of additional samples with low levels of MRD involvement that were previously undetected. Quantitative assessment of WT1 gene expression was not predictive of relapse or other outcomes in either pre- or post-transplant specimens. MRD detected by ΔN was highly specific, but did not identify most relapsing patients. The application of the assay was limited by poor quality among one-third of the specimens and lack of a diagnostic phenotype for comparison.

Regulation of HtrA2 on WT1 gene expression under imatinib stimulation and its effects on the cell biology of K562 cells.

The aim of the present study was to investigate the regulation of Wilms Tumor 1 (WT1) by serine protease high-temperature requirement protein A2 (HtrA2), a member of the Htr family, in K562 cells. In addition, the study aimed to observe the effect of this regulation on cell biological functions and its associated mechanisms. Expression of WT1 and HtrA2 mRNA, and proteins following imatinib and the HtrA2 inhibitor 5-[5-(2-nitrophenyl) furfuryl iodine]-1, 3-diphenyl-2-thiobarbituric acid (UCF-101) treatment was detected with reverse transcription-quantitative polymerase chain reaction and western blot analysis. Subsequent to treatment with drugs and UCF-101, the proliferative function of K562 cells was detected using MTT assays, and the rate of apoptosis was detected using Annexin V with propidium iodide flow cytometry in K562 cells. The protein levels in the signaling pathway were analyzed using western blotting following treatment with imatinib and UCF-101. In K562 cells, imatinib treatment activated HtrA2 gene at a transcription level, while the WT1 gene was simultaneously downregulated. Following HtrA2 inhibitor (UCF-101) treatment, the downregulation of WT1 increased gradually. At the protein level, imatinib induced the increase in HtrA2 protein level and concomitantly downregulated WT1 protein level. Subsequent to HtrA2 inhibition by UCF-101, the WT1 protein level decreased temporarily, but eventually increased. Imatinib induced apoptosis in K562 cells, but this effect was attenuated by the HtrA2 inhibitor UCF-101, resulting in the upregulation of the WT1 protein level. However; UCF-101 did not markedly change the proliferation inhibition caused by imatinib. Imatinib activated the p38 mitogen activated protein kinase (p38 MAPK) signaling pathway in K562 cells, and UCF-101 affected the activation of imatinib in the p38 MAPK signaling pathway. Imatinib inhibited the extracellular signal-related kinase (ERK1/2) pathway markedly and persistently, but UCF-101 exhibited no notable effect on the inhibition of the ERK1/2 pathway. HtrA2 and its regulatory effect on WT1 may affect the sensitivity of BCR/ABL(+) cell lines to target therapy drugs through different mechanisms. Regulation of WT1 by HtrA2 occurs in K562 cells, and the regulation may affect the apoptosis of K562 cells under the stress caused by chemotherapeutic treatment. The p38 MAPK signaling pathway, which serves an important role in cell apoptosis, is a downstream pathway of this regulation.

Use of Wilms Tumor 1 Gene Expression as a Reliable Marker for Prognosis and Minimal Residual Disease Monitoring in Acute Myeloid Leukemia With Normal Karyotype Patients.

Acute myeloid leukemia with normal karyotype (AML-NK) represents the largest group of AML patients classified with an intermediate prognosis. A constant need exists to introduce new molecular markers for more precise risk stratification and for minimal residual disease (MRD) monitoring. Quantitative assessment of Wilms tumor 1 (WT1) gene transcripts was performed using real-time polymerase chain reaction. The bone marrow samples were collected at the diagnosis from 104 AML-NK patients and from 34 of these patients during follow-up or disease relapse. We found that overexpression of the WT1 gene (WT1high status), present in 25.5% of patients, was an independent unfavorable factor for achieving complete remission. WT1high status was also associated with resistance to therapy and shorter disease-free survival and overall survival. Assessment of the log reduction value of WT1 expression, measured in paired diagnosis/complete remission samples, revealed that patients with a log reduction of< 2 had a tendency toward shorter disease-free survival and overall survival and a greater incidence of disease relapse. Combining WT1 gene expression status with NPM1 and FLT3-ITD mutational status, we found that the tumor behavior of intermediate patients (FLT3-ITD-/NPM1- double negative) with WT1high status is almost the same as the tumor behavior of the adverse risk group. WT1 expression status represents a good molecular marker of prognosis, response to treatment, and MRD monitoring. Above all, the usage of the WT1 expression level as an additional marker for more precise risk stratification of AML-NK patients could lead to more adapted, personalized treatment protocols.

Down-regulatory mechanism of mammea E/BB from Mammea siamensis seed extract on Wilms' Tumor 1 expression in K562 cells.

BackgroundWilms’ tumor 1 (WT1) is a biological marker for predicting leukemia progression. In this study, mammea E/BB, an active compound from Saraphi (Mammea siamensis) seed extract was examined for its effect on down-regulatory mechanism of WT1 gene expression, WT1 protein and mRNA stability, and cell proliferation in K562 cell line.MethodsM. siamensis seeds were obtained from the region of Chiang Mai (North of Thailand). Mammea E/BB was extracted from seeds of M. siamensis. WT1 protein expression and stability were evaluated by Western blot analysis. WT1 mRNA stability was assessed by qRT-PCR. WT1-DNA binding and WT1 promoter activity were assayed by ChIP assay and luciferase-reporter assay, respectively. Cell cycle arrest was studied by flow cytometry.ResultsTreatment with mammea E/BB led to down-regulation of WT1 expression. The suppression of WT1 expression did not involve protein and mRNA degradation. Rather, WT1 protein was down-regulated through disruption of transcriptional auto-regulation of the WT1 gene. Mammea E/BB inhibited WT1-DNA binding at the WT1 promoter and decreased luciferase activity. It also disrupted c-Fos/AP-1 binding to the WT1 promoter via ERK1/2 signaling pathway and induced S phase cell cycle arrest in K562 cells.ConclusionMammea E/BB had pleotropic effects on kinase signaling pathways, resulting in inhibition of leukemia cell proliferation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1107-z) contains supplementary material, which is available to authorized users.

The relationship between WT1 expression level and prognosis in patients of acute T lymphoblastic leukemia following allogeneic hematopoietic stem cell transplantation

目的探讨监测WT1基因在急性T淋巴细胞白血病(T-ALL)患者异基因造血干细胞移植(allo-HSCT)后预后的意义。方法回顾性分析2009年1月至2012年3月在我单位行allo-HSCT，且移植后连续监测WT1基因表达水平的68例T-ALL患者。留取初治，移植前，+30、+60、+90、+180、+270、+360 d骨髓标本，用实时定量聚合酶链反应(RQ-PCR)方法监测WT1基因表达水平，同时经流式细胞术(FCM)监测微小残留病(MRD)。结果①移植后WT1基因低水平表达与较低复发风险相关；②+60、+90 d WT1基因升高与较高累计复发率相关(P<0.001、P=0.003)，与较低的无病生存率(P=0.004、P=0.006)和总生存率相关(P=0.004、P=0.007)；③移植后微小残留病(MRD)阳性是T-ALL移植后复发的独立危险因素；④联合WT1基因和FCM可用于移植后复发的监测。结论+60、+90 d WT1基因升高与T-ALL预后显著相关，应尽早予以干预，减少复发甚至死亡风险。T-ALL移植后WT1基因表达低于0.6%，复发风险较低；WT1基因表达超过0.6％需密切随访，并结合FCM监测，达到MRD阳性标准需临床干预，降低复发率。

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.

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

In the present study, we aimed to explore the effect of environmental endocrine disruptors (EEDs) on sexual differentiation in androgen receptor (AR)-/-, AR+/- and AR+/+ male mice. By using a Cre-loxP conditional knockout strategy, we generated AR knockout mice. By mating flox-AR female mice with AR-Cre male mice, the offspring male mice which were produced were examined. Mice not subjected to any type of intervention were used as the controls. Furthermore, male mice of different genotypes were selected and further divided into subgroups as follows: the control group, bisphenol A (BPA) group and the dibutyl phthalate [corrected] (DBP) group. The expression of the Wilms tumor 1 (WT1), lutropin/choriogonadotropin receptor (LHR), 17-β-hydroxysteroid dehydrogenase type 3 (17βHSD3) and steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2) genes was determined by RT-qPCR and western blot analysis. There was no statistically significant difference in the weight of the mice between the control group and the knockout group (P>0.05). The results revealed that, compared with the control group, in the knockout group, anogenital distance was shortened, and testicular weight and testosterone levels were decreased; estradiol levels were elevated; the differences were statistically significant (P<0.05). In the group of AR+/- male mice exposed to 100 mg/l EEDs, hypospadias was successfully induced, suggesting that EEDs are involved in the embryonic stage of sexual development in male mice. The quantitative detection of WT1, LHR, 17βHSD3 and SRD5A2 gene expression by RT-qPCR and western blot analysis indicated that these genes were significantly downregulated in the mice in the BPA group. In conclusion, exposure to EEDs induces hypospadias in heterozygous and wild-type male mice offspring during sexual differentiation, but has no effect on homozygous offspring. Therefore, EEDs play an important role during the third stage of sexual differentiation.

WT1 gene expression difference in leukemia and non-leukemia and its clinical significance

This study was aimed to investigate the expression level of Wilms' tumor 1( WT1) gene in hematologic neoplasm (leukemia, multiple myeloma and lymphoma) patients and its clinical significance. Real-time quantitative polymerase chain reaction (RQ-PCR) was used to detect the copy number of WT1 gene and reference gene (ALB) in bone marrow cells of 228 patients with hematologic neoplasm in our hospital. The gene expression level was determined by using the ratio of the copy number of WT1 gene and reference gene. The results showed that the WT1 expression level between male and female patients was not statistically significantly different (P > 0.05). All the patients were divided into 3 groups: the group aged under 19, the group aged between 19-50, and the group aged over 50; the WT1 expression level among the three groups were not statistically significantly different (P > 0.05) . The above-mentioned patients were redivided into the groups aged under 45 and over 45, the difference between them was not statistically significant (P > 0.05). The difference of WT1 expression level between newly diagnosed patients and treated patients with hematologic neoplasm was statistically significant (P < 0.01), but no statistically significant difference of WT1 expression was found (P > 0.05) at each stage within 3 years after treatment, however, among them the difference between newly diagnosed leukemia patients and treated leukemia patients was very statistically significant (P < 0.01), while the difference between newly diagnosed and treated non-leukemia patients was not statistically significant (P > 0.05). The expression difference of WT1 between leukemia and non-leukemia patients was very statistically significant (P < 0.01), the difference between the newly diagnosed leukemia and non-leukemia patients also was very statistically significant (P < 0.01). The difference of WT1 expression between treated leukemia and non-leukemia patients was not statistically significant (P > 0.05). It is concluded that the WT1 expression level in leukemia patients can be a reliable marker to evaluate the prognosis of newly diagnosed leukemia and the curative effect for minimal residual disease. No WT1 expression difference has been found before and after treatment among the patients with non-leukemia, such as multiple myeloma and lymphoma, therefore, which should be furtherly explored.

Exogenous expression of WT1 gene influences U937 cell biological behaviors and activates MAPK and JAK-STAT signaling pathways

Wilms’ tumor 1 (WT1) gene plays important roles in leukemogenesis. To further explore its underlying mechanisms, we transfected two WT1 isoforms, WT1(+17AA/−KTS) and WT1(+17AA/+KTS) into U937, a WT1-null monoblastic cell line, studied their effects on migration, colony formation, apoptosis, gene expression and pertinent signaling pathways of U937 cells. The results showed that WT1(+17AA/−KTS), but not WT1(+17AA/+KTS), enhanced migration and colony forming abilities of U937 cells, and suppressed etoposide-induced U937 cell apoptosis. Transfection of WT1 isoforms activated gene expressions of chemokine, and induced up-regulation of signaling molecules involved in JAK-STAT and MAPK signaling pathways. This study showed that exogenous expression of WT1 gene remarkably affected biological behaviors of U937 cells, and these effects are possibly mediated by up-regulation of genes related to chemokine, JAK-STAT and MAPK signaling pathways.

Activated Omentum Slows Progression of CKD

Stem cells show promise in the treatment of AKI but do not survive long term after injection. However, organ repair has been achieved by extending and attaching the omentum, a fatty tissue lying above the stomach containing stem cells, to various organs. To examine whether fusing the omentum to a subtotally nephrectomized kidney could slow the progression of CKD, we used two groups of rats: an experimental group undergoing 5/6 nephrectomy only and a control group undergoing 5/6 nephrectomy and complete omentectomy. Polydextran gel particles were administered intraperitoneally before suture only in the experimental group to facilitate the fusion of the omentum to the injured kidney. After 12 weeks, experimental rats exhibited omentum fused to the remnant kidney and had lower plasma creatinine and urea nitrogen levels; less glomerulosclerosis, tubulointerstitial injury, and extracellular matrix; and reduced thickening of basement membranes compared with controls. A fusion zone formed between the injured kidney and the omentum contained abundant stem cells expressing stem cell antigen-1, Wilms' tumor 1 (WT-1), and CD34, suggesting active, healing tissue. Furthermore, kidney extracts from experimental rats showed increases in expression levels of growth factors involved in renal repair, the number of proliferating cells, especially at the injured edge, the number of WT-1-positive cells in the glomeruli, and WT-1 gene expression. These results suggest that contact between the omentum and injured kidney slows the progression of CKD in the remnant organ, and this effect appears to be mediated by the presence of omental stem cells and their secretory products.

The Presence Of Residual Disease After Induction and/Or Consolidation Therapy Based On Wilms' Tumor 1 (WT1) Expression Is a Strong Prognostic Factor For Relapse and Survival In AML

IntroductionThe Wilms' tumor 1 (WT1) gene, located on chromosome 11p13, encodes a transcription factor with both oncogene and tumor suppressor functions. WT1 is reportedly 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 in AML after chemotherapy and as a predictor of relapse and survival. Patients and MethodsThis retrospective and multicentric study included 114 patients with WT1-overexpressed AML (Table 1). Quantitative assessment of WT1 transcript levels was performed by quantitative RT-PCR in 283 bone marrow (BM) samples at diagnosis, post-induction and post-consolidation. 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 10 BM donor samples, 20 BM patient samples and commercial WT1 plasmids (ProfileQuant Kit, Ipsogen-Qiagen). ResultsNo significant differences in WT1 gene expression (cycle threshold, Ct) were observed between different laboratories in the pilot study. The cut-off value of WT1 over-expression in BM was established in 0.55% (median+2SD values from healthy donors). Median WT1 expression in patient samples at diagnosis was 29.5% (range, 2-1220). Differential expression at diagnosis was not correlated with age, sex, leukocytes, karyotype (risk), however a higher expression in patients with AML-M1 and AML-M2 subtypes as well as patients with mutant NPM1 and/or ITD-FLT3 was observed. Most patients (88.6%; 101/114) received intensive chemotherapy as induction treatment. After induction, 80.2% (81/101) of patients had available WT1 data, of which 23.45% (19/81) were positive. In addition, 79.8% (91/114) received intensive treatment during consolidation. WT1 results were available for 75.5% (66/91), of which 22.7% (15/66) were positive. Post-induction WT1 positivity was correlated with a higher cumulative incidence of relapse (CIR; 2 years 76% vs. 28.2% p=0.002) and a lower overall survival (OS; 2 years, 44.9% vs. 78.2% p=0.022; Figure 1a,b). Similar results were obtained when patients intensified with allogeneic stem cell transplantation (allo-SCT) were excluded from the analysis: higher CIR (2 years 88.9% vs. 32.5%; p=0.005) and lower OS (40% vs. 76.2% p=0.17). Post-consolidation WT1 positivity was correlated with a trend to a higher CIR (2 years 60.3% vs. 41.4% p=0.21) and a lower OS (2 years 44.9% vs. 66% p=0.09; Figure 1c,d). Statistically significant results were obtained after consolidation when patients treated with allo-SCT were excluded from the analysis: higher CIR (2 years 100% vs. 40.1% p=0.005) and lower OS (2 years 20% vs. 66% p=0.003). ConclusionsWT1 is a useful marker for MRD in AML patients undergoing chemotherapy (induction and consolidation) which allows anticipation of relapse and survival. Post-induction results were a strong risk factor of relapse and survival in all patients, including those intensified with allo-SCT. By contrast post-consolidation results are especially relevant in the group of patients not treated with allo-SCT. Intensification with allo-SCT overcomes the poor prognosis derived from positive post-consolidation WT1 results.Paper presented on behalf of the Hematological Molecular Biology Group (GBMH) of the Spanish Society of Hematology (SEHH).▪ Disclosures:No relevant conflicts of interest to declare.