Wilms Tumor 1 Levels Research Articles

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

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

Prediction of outcome in acute myeloid leukemia by measurement of WT1 expression as a basic marker of minimal residual disease

目的研究WT1基因作为微小残留病（MRD）监测指标在急性髓系白血病（AML）预后中的应用，并探索WT1 mRNA预测复发的阈值。方法回顾性分析121例诱导缓解并行巩固治疗的AML患者（非急性早幼粒细胞白血病）WT1 mRNA的动态表达水平。比较巩固治疗后不同转归组患者的WT1 mRNA表达水平，依据受试者工作特征（ROC）曲线确定可预测临床复发的WT1 mRNA阈值。WT1 mRNA水平采用实时定量聚合酶链反应（RQ-PCR）法检测。结果确立WT1 mRNA>2.98%提示高风险复发。为了临床应用方便，将提示复发的WT1 mRNA阈值设为3.00%。41例患者初诊时检测了WT1 mRNA水平，剔除3例初诊WT1 mRNA低于3.00%的患者，余下38例患者初诊WT1 mRNA中位值为44.09%（7.19%~188.06%）。缓解期351份标本WT1 mRNA检测值中位为0.48%（0~8.41%）。初诊WT1 mRNA水平高于缓解期水平。除外初诊WT1 mRNA水平低于3.00%的3例患者，巩固治疗开始后WT1阳性组（>3.00%）和阴性组（≤3.00%）复发率分别为70.0%（14/20）和12.2%（12/98）（P<0.001）。WT1 mRNA升高达阈值后到复发的中位时间为58 d。结论WT1基因可以作为AML患者巩固治疗开始后的MRD监测指标，WT1 mRNA>3.00%提示存在复发风险。

Clinical Utility of Wilms' Tumor 1 Monitoring in Patients with Myeloid Malignancy and Prior Allogeneic Hematopoietic Stem Cell Transplantation

Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is 1 of the standard treatments for myeloid malignancy, relapse remains a major obstacle to cure. Early detection of relapse by monitoring of minimal residual disease (MRD) may enable us to intervene pre-emptively and potentially prevent overt relapse. Wilms' tumor 1 (WT1) is well known as a pan-leukemic marker. We retrospectively examined serially monitored WT1 levels of peripheral blood in 98 patients (84 with acute myeloid leukemia and 14 with myelodysplastic syndrome). At the time of allo-HSCT, 49 patients (50%) were in complete remission. Patients were divided into 3 groups according to WT1 levels (<50 copies/µg RNA, 50 to 500 copies/µg RNA and >500 copies/µg RNA). The cumulative incidence of relapse (CIR) and overall survival (OS) differed statistically according to the WT1 levels before allo-HSCT and at days 30 and 60 after allo-HSCT. In multivariate analysis, WT1 >500 copies/µg RNA before and at day 60 after allo-HSCT and WT1 ≥50 copies/µg RNA at day 30 were correlated with CIR. Moreover, WT1 >500 copies/µg RNA at day 60 after allo-HSCT was only correlated with worse OS. Our data suggest that serial monitoring of WT1 levels in peripheral blood may be useful for MRD monitoring and as a predictor of hematological relapse in allo-HSCT.

Quantitative assessment of Wilms tumor 1 expression by real-time quantitative polymerase chain reaction in patients with acute myeloblastic leukemia.

Background:The Wilms tumor 1 (WT1) gene is originally defined as a tumor suppressor gene and a transcription factor that overexpressed in leukemic cells. It is highly expressed in more than 80% of acute myeloid leukemia (AML) patients, both in bone marrow (BM) and in peripheral blood (PB), and it is used as a powerful and independent marker of minimal residual disease (MRD); we have determined the expression levels of the WT1 by real-time quantitative polymerase chain reaction (RQ-PCR) in PB and BM in 126 newly diagnosed AML patients.Materials and Methods:This study was done in molecular pathology and cancer research center from April 2014 to June 2015, RQ-PCR method was used to determine the WT1 gene expression in BM and/or PB samples from 126 patients of AML, we cloned both WT1 and ABL genes for creating a standard curve, and we calculate copy number of WT1 genes in patients.Results:A total of 126 AML patients consist of 70 males (55.6%) and 56 females (44.4%), with a median age of 26 years; 104 (81%) patients out of 126 show overexpression of WT1 gene. We also concomitant monitoring of fusion transcripts (PML RARa, AML1-ETO, MLL-MLL, CBFb-MYH11, or DEK-CAN) in our patients, the AML1-ETO group showing remarkably low levels of WT1 compared with other fusion transcript and the CBFB-MYH11 showing high levels of WT1.Conclusion:We conclude that WT1 expression by RQ-PCR in AML patients may be employed as an independent tool to detect MRD in the majority of normal karyotype AML patients.

MiR-590 regulates WT1 during proliferation of G401 cells.

Nephroblastoma (Wilms' tumor) is frequently associated with mortality in children. MicroRNAs (miRNAs) are important for tumor development serving as oncogenes or tumor suppressors. In the present study, miRNA-590 (miR-590) was identified to be upregulated in Wilms' tumor tissues compared with the normal adjacent tissues. Additionally, the levels of miR-590 were consistent with their clinical stage. Wilms' tumor 1 (WT1) was considered to be a tumor suppressor in certain tumor types, and it has been detected at low expression levels in various types of cancer with high cell proliferation and aggressive behavior. The expression levels of miR-590 were quantified using reverse transcription-quantitative polymerase chain reaction. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine assays. The protein expression levels of WT1 were investigated by western blot analysis. To the best of our knowledge, the present study was the first to determine that WT1 was a target gene of miR-590 as miR-590 was able to negatively regulate WT1 expression level by binding to the specific target site within the 3′-untranslated region (3′-UTR) of WT1 in G401 cells. Additionally, overexpression of miR-590 promoted G401 cell proliferation which was consistent with the effect of small interfering RNA-WT1. Subsequently, the present study determined that the cell phenotype altered by miR-590 overexpression may be reversed by upregulation of WT1 in G401 cells. In conclusion, the observations indicated that miR-590 may function as an oncogene via targeting WT1 to induce G401 cell proliferation. These results may contribute to current understanding of the function of miR-590 in nephroblastoma.

Validation of a Molecular Risk Score for Prognosis of Patients with Acute Promyelocytic Leukemia

Introduction: Risk stratification in acute promyelocytic leukemia (APL) is based on clinical parameters, namely leukocyte and platelet counts at initial diagnosis as combined in the Sanz Score. However, during the last years the influence of additional molecular genetic markers on prognosis of APL patients has been postulated. In 2015 we published the results of a molecular risk score integrating expression data of the genes brain and acute leukemia, cytoplasmic (BAALC), ets' related gene (ERG) and Wilms' Tumor 1 (WT1) with strong independent influence on outcome and relapse risk of APL patients treated in the German AMLCG studies (Hecht et al., Leuk Res 2015). The aim of our study was to validate our data in an independent patient cohort.Methods: In cooperation with the German SAL (Study Alliance Leukemia) group we obtained a validation set of samples of mononuclear cells derived from the bone marrow of 76 patients with confirmed diagnosis of APL prior to therapy. The validation cohort consisted of 37 female and 39 male patients with a median age of 50 years (range 20-82 years; Table 1). Patients were diagnosed and treated between 2000 and 2014 mostly with an ATRA plus Idarubicin based induction therapy followed by Sanz score-dependent consolidation and maintenance chemotherapy. RNA was extracted using the AllPrep DNA/RNA Mini Kit and cDNA was synthesized using 500µg of RNA with a QuantiTect Reverse Transcription Kit (both Qiagen, Hilden, Germany). Expression levels of BAALC, ERG and WT1 were then analyzed using quantitative real-time RT-PCR with the same conditions and primers as used in the original test cohort and the same calibrator cDNA from cell lines was used for the analysis. The following gene expression levels were defined as negative risk factors in preceding studies: BAALC expression ≥25th percentile (BAALChigh), ERG expression ≥75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low or high). The integrative risk score was calculated as described before: For the presence of one of the mentioned risk factors, one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, ERGhigh and WT1low or high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors). Overall survival (OS), relapse free survival (RFS) and cumulative incidence of relapse (CIR) were calculated using the Kaplan-Meier method.Results: The expression of the three genes BAALC, ERG and WT1 compared to healthy controls were exactly the same as in the original cohort. Application of the molecular risk score on APL patients of the validation cohort clearly discriminated patients according to their risk comparable to the original APL cohort (Figure 1). Patients with 0 points had a very good prognosis with no deaths or relapses, whereas patients with 3 points (i.e. concurrent presence of all three risk factors) had a very poor outcome with an OS of 51%, a RFS of 50% and a CIR of 38%. The differences between patients with 1 and 2 points were less pronounced in the validation cohort. However, the statistical analyses in the validation cohort did not yield significance. As the main reason we assume that the validation cohort altogether showed a significantly improved OS and RFS compared to the original cohort which was treated a decade earlier, so differences between the subgroups could not be that pronounced. For both cohorts stratification by the Sanz Score did not show significant differences in outcome (analysis for RFS p=0.46 in original cohort and p=0.44 in validation cohort).Conclusion: A validated molecular-based integrative risk score was able to define subgroups of APL patients with different outcome independently of the Sanz score. The validation of the strong influence of the combination of molecular risk factors is particularly interesting as the new analyses of the three genes BAALC, ERG and WT1 separately did not confirm the strong results of the single analyses of each gene. The discrimination of patients according to the risk score is evident though. This argues for their integration in future studies. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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

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.

Wilms Tumor 1 Expression and Pre-emptive Immunotherapy in Patients with Acute Myeloid Leukemia Undergoing an Allogeneic Hemopoietic Stem Cell Transplantation

Minimal residual disease (MRD) was monitored by Wilms tumor 1 (WT1) expression in 207 patients with acute myeloid leukemia (AML) after an allogeneic hemopoietic stem cell transplantation (HSCT) as a trigger to initiate pre-emptive immunotherapy (IT) with cyclosporin discontinuation and/or donor lymphocyte infusion. The trigger for IT was WT1 ≥ 180 copies/104 Abelson cells in marrow cells in the first group of 122 patients (WT1-180) and ≥ 100 copies in a subsequent group of 85 patients (WT1-100). Forty patients received IT. The cumulative incidence (CI) of relapse was 76% in WT1-180 (n = 17) versus 29% in WT1-100 patients (n = 23) receiving IT (P = .006); the leukemia-free survival from MRD positivity was 23% versus 74%, respectively (P = .003). We then looked at the entire AML patient population (n = 207). WT1-180 and WT1-100 patients were comparable for disease phase and age. The overall 4-year CI of transplantation-related mortality was 13% in both groups; the CI of leukemia relapse was 38% in the WT1-180 and 28% in the WT1-100 patients (P = .05) and leukemia-free survival was 56% versus 48%, respectively (P = .07). In conclusion, we suggests that WT1-based pre-emptive immunotherapy is feasible in patients with undergoing an allogeneic HSCT. The protective effect on relapse is greater when IT is triggered at lower levels of WT1.

WT1 Expression in Circulating RNA as a Minimal Residual Disease Marker for AML Patients After Stem-Cell Transplantation.

Circulating RNA in plasma (CRNA) refers to soluble tumor-derived ribonucleic acids (RNA). The Wilms tumor 1 (WT1) gene appears to be a highly promising marker for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) patients after chemotherapy or hematopoietic stem-cell transplantation (HSCT). This study aimed to compare the relative expression level of the WT1 gene in CRNA, bone marrow (BM)- and peripheral blood (PB)-RNA for the monitoring of MRD in AML patients after HSCT. One hundred and eighteen AML patients were studied with WT1 expression assessed by quantitative polymerase chain reaction in plasma, BM- and PB-RNA. Correlation analysis was used to compare gene expression differences. The expression of the WT1 gene was successfully detected in 118 cases but was absent in controls (mean relative expression of WT1/ABL 8.77, range 0.5-56.0, P < 0.001) (WT1 in BM-RNA: mean relative expression 8.66, range 0.5-56.0, P < 0.001; WT1 in PB-RNA: mean relative expression 8.55, range 0.5-54.0, P < 0.001). WT1 expression in CRNA, BM-RNA and PB-RNA showed no difference at diagnosis (CRNA vs. BM-RNA, r = 0.999; CRNA vs. PB-RNA, r = 0.988). After HSCT, 62 patients achieved remission. The expression level of WT1 in CRNA and BM-RNA in nine patients who achieved permanent remission fluctuated within the normal range (WT1/ABL < 0.02). The other 53 patients who were predicted to relapse had elevated WT1 levels in CRNA and BM-RNA. Of these 53 patients, 48 had increased expression of the WT1 gene in both CRNA and BM-RNA at a median of 1 month prior to clinical relapse. In the other five patients (5/53) diagnosed with extramedullary relapse, the level of WT1 in CRNA was elevated prior to relapse. However, in these patients WT1 expression in both BM-RNA and PB-RNA was still negative (at a median of 1 month earlier than in BM-RNA). This study indicated that CRNA was no different from BM-RNA for determination of WT1 expression in AML patients (F = 0.260, P = 0.642). Analysis of WT1 expression in CRNA in AML patients could be a simple, convenient and noninvasive method to predict latent information about relapse.

Prognostic significance of diagnosed WT1 level in acute myeloid leukemia: a meta-analysis.

The Wilms' tumor 1 (WT1) expression has been recognized in a substantial number of acute myeloid leukemia (AML) patients. Some studies indicated the association of diagnosed WT1 higher expression (WT1(H)) and poor outcome in the AML patients, while other studies had different opinions. Therefore, we performed a meta-analysis to evaluate the controversial prognostic significance of diagnosed WT1(H) in AML. Eligible studies were identified from several databases including PubMed, Embase, Web of Science, and the Cochrane Library (up to September 2014). The primary end point was overall survival (OS) and disease-free survival (DFS) was chosen as secondary end point. If possible, we would pool estimate effects (hazard ratio [HR] with 95% confidence interval [CI]) of outcomes in bothfixed and random effects models. Eleven studies, covering 1497 AMLpatients, were included in this meta-analysis. Pooled HRs indicated that diagnosed WT1(H) had a poor impact on the survival of AML patients (HR for OS, 1.37; HR for DFS, 1.38). Furthermore, diagnosed WT1(H) appeared to be an adverse prognostic indicator in adult AML (HR for OS, 1.43; HR for DFS, 1.41) and non-promyelocytic AML (non-M3 AML) (HR for OS, 1.46; HR for DFS, 1.41). Diagnosed WT1(H) had slightly but significantly poor prognostic impact on OS and DFS of patients with AML in total population and some specific subgroups.

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

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

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

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

An Optimized Risk Stratification Model in Acute Promyelocytic Leukemia Identifies Patients with an Exceptionally Good Prognosis Regarding the Incidence of Relapse

Introduction: Risk stratification in acute promyelocytic leukemia (APL) is based on the easily accessible Sanz-Score, which combines leukocyte and platelet counts at initial diagnosis. This score showed significant differences in relapse-free survival (RFS) of APL patients in various studies and is currently used to determine whether a patient can be treated with ATRA and ATO alone or needs additional chemotherapy. However, to make therapeutic decisions based on a risk stratification system derived from the endpoint RFS bears the drawback that relapses are rare in APL and most events are deaths in complete remission (CR), which can be therapy related (e.g. toxicity). The cumulative incidence of relapse (CIR) therefore seems to be a better parameter for decision making with regard to therapy intensity. In this study, we optimized a risk score combining data on gene expression of BAALC (brain and acute leukemia, cytoplasmic), ERG (ets’ related gene) and WT1 (Wilms’ tumor 1) to retrospectively predict the CIR of APL patients.Methods: Data on BAALC, ERG and WT1 expression levels of 79 patients with newly diagnosed APL were obtained from bone marrow mononuclear cells using quantitative real-time RT-PCR in preceding studies. The following gene expression levels were identified as negative risk factors: BAALC expression ≥25th percentile (BAALChigh), ERG expression >75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low or high). As ERGhigh was the only independent predictor for relapse in multivariate analysis with a hazard ratio (HR) of 11.6, its predictive weight was regarded superior, respectively . Cut-off analyses were performed to determine the optimal ERG expression level cut-off for risk of relapse. Accordingly, the new cut-off for high ERG expression was set at ≥62nd percentile (optimized ERGhigh: optERGhigh; Sensitivity: 1.0, Specificity: 0.71). A combined risk score was developed as follows: For the presence of one of the mentioned risk factors, one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, optERGhigh and WT1low or high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors) could be allocated to one patient. Accordingly, patients were divided into two risk groups: 34 patients scored 0-1 points and 45 patients scored 2-3 points. CIR, overall survival (OS) and RFS were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the two risk groups (p<0.05).Results: Patients with 2-3 points had a CIR of 18% at 10 years of follow-up whereas none of the patients with 0-1 points suffered a relapse (CIR: 0%; p=0.02; Fig. 1). All relapses occurred between 8.4 months and 3.5 years after first CR. Moreover, OS and RFS also differed significantly between the two risk groups: OS was 53% for patients with 2-3 points vs. 85% for patients with 0-1 points (p=0.004); RFS was 49% vs. 93%, respectively (p<0.0001). In multivariate analysis the optimized combined risk score was the strongest independent risk factor for every endpoint.Conclusion: The combination of expression levels of BAALC, ERG and WT1 into a risk score identified a group of patients at high risk for relapse which could benefit from close monitoring resulting possibly in an early intervention when molecular relapse is detected. On the other hand, it identified a low risk group with very good outcome and no APL-related events after patients had achieved first CR. A molecular risk score focusing on relapse risk might be a promising approach to guide therapeutic decisions in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Wilms’ tumor 1 enhances Cisplatin-resistance of advanced NSCLC

Wilms’ tumor 1 (WT1) is an oncogene that has been correlated with tumor progression, bad prognosis and chemo-resistance in Non-Small-Cell lung cancer (NSCLC). Here, we found that WT1 expression is significantly higher in NSCLCs than in benign controls, and that Cisplatin-resistant patients display a notable increase in WT1 expression following relapse. In vitro, WT1 levels were associated with the IC50 of NSCLC cells and increased along with treatment time and dose of Cisplatin. Furthermore, WT1 enhanced Major Vault Protein (MVP) transcription via binding to its promoter. Therefore, WT1 may be a potential therapeutic target for solving resistance.

Role of the Wilms’ tumor 1 gene in the aberrant biological behavior of leukemic cells and the related mechanisms

The Wilms' tumor 1 (WT1) gene is one of the regulating factors in cell proliferation and development. It is a double-functional gene: an oncogene and a tumor suppressor. This gene was found to be highly expressed in many leukemic cell lines and in patients with acute myeloid leukemia. In the present study, we demonstrated that the WT1 gene was commonly expressed in leukemic cell lines apart from U937 cells. The K562 cell line which expresses WT1 at a high level (mRNA and protein) was used in the entire experiment. By MTT and colony formation assays, we found that curcumin, an inhibitor of the WT1 protein, inhibited cell proliferation and clonogenicity in a time- and dose-dependent manner. It also caused cell cycle arrest at the G2/M phase. We then designed specific short hairpin RNAs (shRNAs) which could downregulate WT1 by 70-80% at the mRNA and protein levels. Reduction in the WT1 levels attenuated the proliferative ability and clonogenicity. Cell cycle progression analysis indicated that the proportion of cells in the G0/G1 phase increased while the proportion in the S phase decreased distinctively. ChIP-DNA selection and ligation (DSL) experiment identified a cohort of genes whose promoters are targeted by WT1. These genes were classified into different cellular signaling pathways using MAS software and included the Wnt/β-catenin pathway, MAPK signaling pathway, apoptosis pathway, and the cell cycle. We focused on the Wnt/β-catenin signaling pathway, and compared expression of several genes in the K562 cells transfected with the control shRNA and WT1-specific shRNA. β-catenin, an important gene in the Wnt canonical pathway, was downregulated after WT1 RNAi. Target genes of β-catenin which participate in cell proliferation and cell cycle regulation, such as CCND1 and MYC, were also significantly downregulated. Collectively, these data suggest that WT1 functions as an oncogene in leukemia cells, and one important mechanism is regulation of the Wnt/β-catenin pathway.

A Risk Score Integrating BAALC, ERG and WT1 Expression Can Be Used To Improve Risk Stratification In Acute Promyelocytic Leukemia

IntroductionWith current therapy regimens over 75% of patients with de novo acute promyelocytic leukemia (APL) can be cured. Approaches to further improve patient outcome by stratifying patients at the time of initial diagnosis according to their individual risk and to adjust therapy accordingly have been based on clinical features only. Molecular markers have not been established for risk stratification as yet. Recently, we have shown that high expression levels of the genes brain and acute leukemia, cytoplasmic (BAALC) and ets related gene (ERG) are associated with inferior outcome in APL patients. In addition, data indicate that aberrant expression of the gene Wilms' tumor 1 (WT1) is a negative prognostic factor with regard to overall survival (OS) after complete remission (CR) and relapse free survival (RFS) in APL. In this study we evaluated the prognostic relevance of a combined score integrating the expression levels of the above mentioned genes to further improve risk stratification in APL patients. MethodsExpression levels of BAALC, ERG and WT1 of 62 patients with newly diagnosed APL were retrospectively analyzed in bone marrow mononuclear cells using multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR). Median age of patients was 47 years (range: 19 to 82y). All patients gave informed consent. Patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study with a treatment of simultaneous ATRA and double induction chemotherapy including high-dose ara-C, consolidation and maintenance chemotherapy. The following gene expression levels were identified as negative risk factors in preceding studies: BAALC expression ≥25th percentile (BAALChigh), ERG expression >75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low/high). A risk score was developed as follows: for the presence of one of the mentioned risk factors one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, ERGhigh and WT1low/high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors) could be allocated to one patient. Accordingly, patients were divided into four risk groups: 7 patients scored 0 points (= low risk), 27 patients scored 1 point (= intermediate 1 risk), 19 patients scored 2 points (= intermediate 2 risk) and 9 patients scored 3 points (= high risk). Subsequently, OS, RFS and relapse free interval (RFI) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the four risk groups (p<0.05). ResultsThe integrative risk score divided patients into four groups with significantly different outcome. The low risk group showed a RFS of 100% at 10 years of follow-up compared to the intermediate 1 risk group with 81%, the intermediate 2 risk group with 58% and the high risk group with a RFS of 42% only (median survival: 4.6y) (p=0.02). In accordance, the RFI differed significantly between the four groups: low risk 100%, intermediate 1 risk 100%, intermediate 2 risk 89% and high risk 71% (p=0.049). There was no statistically significant difference between the 4 groups with regard to OS in the entire patient cohort. However, there was a clear trend towards a difference in OS in patients who achieved a CR after induction therapy: low risk 100%, intermediate 1 risk 81%, intermediate 2 risk 68% and high risk 53% survival at 10 years of follow-up (p=0.09). ConclusionIntegration of expression levels of the genes BAALC, ERG and WT1 into a scoring system identifies 4 risk groups with significantly different outcome with regard to RFS and RFI. It might be a promising approach to guide therapeutic decisions in patients with APL. However, multivariate analyses and validation of these data in an independent patient cohort is warranted. Disclosures:No relevant conflicts of interest to declare.

Urinary exosomal Wilms' tumor-1 as a potential biomarker for podocyte injury

Renal Wilms' tumor-1 (WT-1) staining is used to detect podocyte loss in kidney biopsies. We aimed to determine if urinary exosomal WT-1 could serve as a noninvasive biomarker of podocyte injury. We examined WT-1 by Western blot in a human podocyte-like cell line, a mouse model of podocyte injury, and human subjects with podocyte disorders. WT-1 was detected in exosomal fraction of the conditioned media from podocytes and increased 48 h after hTGF-β1 stimulation. Cellular WT-1 decreased in podocytes following hTGF-β1 incubation. In mice with induced podocyte injury, urinary exosomal WT-1 was detected 1 wk earlier than albuminuria and also tracked the effects of angiotensin receptor blocker (ARB) treatment. In addition, urinary exosomal WT-1 levels at 1 wk post-injury correlated with the severity of glomerular injury at 3 wk later. In human subjects, urinary exosomal WT-1 was significantly increased in focal segmental glomerulosclerosis (FSGS) patients compared with healthy volunteers or steroid-sensitive nephrotic syndrome (SSNS) patients. Urinary exosomal WT-1 was also significantly decreased in patients in remission for either FSGS or SSNS or following steroid treatment in six SSNS subjects. We conclude that urinary exosomal WT-1 is a promising noninvasive biomarker with apparent podocyte specificity that can detect early progression and treatment-induced regression of podocyte injury in FSGS or SSNS. These results warrant longitudinal, prospective studies in a large cohort with a range of podocyte diseases.

Abstract 773: WT1 regulation of Cyclin A1 in leukemia.

Abstract We have observed high levels of Wilms Tumor 1 (WT1) expression in acute leukemias, suggesting that expression of this zinc finger transcription factor might contribute to leukemia development. WT1 has also been used as a marker for minimal residual disease, and patients who relapse often show high WT1 expression in peripheral blood lymphocytes. WT1 levels in the bone marrow of leukemia patients are found to be higher than in normal marrow or peripheral blood. Thus, we predict that WT1 plays a leukemogenic role by regulating expression of growth control genes. Cyclin A1 (CCNA1) is a cell cycle protein expressed in highly proliferative tissues such as the testis and bone marrow, where it is found in both hematopoietic stem cells and leukemia cells. We hypothesized that WT1 up-regulates the expression of CCNA1, potentially enhancing proliferation of leukemic blasts. To determine whether WT1 regulated CCNA1 expression we analyzed the CCNA1 promoter region using the MatInspector software and identified two possible WT1 binding sites. WT1 binding at the CCNA1 promoter in chromatin of K562 leukemic cells was demonstrated by Chromatin Immunoprecipitation (ChIP) analysis using both end point and quantitative real-time PCR (qRT-PCR). Transcriptional activation of the CCNA1 gene by WT1 was tested by transfection of K562 cells with the transcriptionally active WT1 isoform (-KTS) and analysis of RNA expression. Analysis of cDNA by qRT-PCR showed an upregulation of CCNA1 transcripts in WT1 transfected cells. However, upregulation was not observed following transfection with a mutant form of the WT1 that lacks the DNA binding domain (due to a frameshift mutation in exon 7). CCNA1 up-regulation may be relatively specific for leukemia cells, as prostate cancer cells transfected with WT1 showed no changes in CCNA1 levels. To address the clinical relevance of WT1 expression in pediatric acute leukemia, qRT-PCR was used to measure WT1 and CCNA1 transcript levels in leukemic bone marrow, relative to non-neoplastic bone marrow samples. Results showed higher levels of WT1 expression in Acute Myeloid Leukemia (AML) in comparison to Acute Lymphoid Leukemia (ALL). In particular, an examination of AML-M3 bone marrow samples showed high expression of both WT1 and CCNA1, consistent with WT1 upregulation of CCNA1. Additionally, in pediatric ALL samples with elevated CCNA1 levels, we also observed elevated WT1 levels. Overall these findings support the hypothesis that WT1 may up-regulate CCNA1 expression in bone marrow progenitors potentially promoting oncogenic transformation. WT1 may also contribute to leukemogenesis through transcriptional regulation of other growth control genes (such as VEGF, bcl2, and myc) shown to be regulated by WT1 in other tumor types. Ultimately this approach of identifying WT1 target genes up-regulated in leukemic blasts could reveal new biomarkers and identify biologically-defined therapeutic targets. Citation Format: Sony Pandey, Mustafa Moazam, Douglas Snyder, Steven J. Kuerbitz, Gail C. Fraizer. WT1 regulation of Cyclin A1 in leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 773. doi:10.1158/1538-7445.AM2013-773

Wilms’ tumor 1 gene modulates Fas-related death signals and anti-apoptotic functions in hepatocellular carcinoma

The Wilms' tumor 1 (WT1) gene is known to be overexpressed in hepatocellular carcinoma (HCC) and to upregulate tumor growth and oncogenic potential, although the detailed mechanisms remain to be elucidated. We identified host genes involved in WT1 gene modulation of human liver cancer cell lines in vitro, and further characterized genes related to apoptosis. Moreover, we evaluated the alteration of genes by WT1 in 40 HCC and 58 non-HCC human liver samples collected at resection. Analysis of the effect of small interfering RNAs-mediated knock-down of WT1 on apoptosis using an annexin V labeling assay, and on modulation of the activity of caspases-3, -8 and -9, indicated that WT1 has an anti-apoptotic role. We identified three apoptosis-related genes that were modulated by WT1; the cellular FLICE-inhibitory proteins (cFLIP) gene was upregulated, and Fas-associated death domain (FADD) and nuclear factor kappa B (NF-κB) were downregulated. Interestingly, knock-down of FADD or NF-κB resulted in the upregulation of WT1, and the expression of cFLIP changed in parallel with WT1 expression. We further evaluated WT1-mediated alteration of genes in HCC and non-HCC human liver samples. Both HCC and non-HCC tissues that expressed relatively high levels of WT1 showed cFLIP overexpression. WT1 modulates cFLIP, FADD and NF-κB, and has an anti-apoptotic role in HCC. This mechanism of action of WT1 could be related to the tumor growth and oncogenic potential of HCC.