WT1 Levels Research Articles

Effect of the microtransplantation of allogeneic hematopoietic stem cells as maintenance therapy for elderly patients with acute leukemia.

The incidence of acute myeloid leukemia (AML) increases with age. Elderly patients with AML are less tolerant to high-intensity consolidation therapy than younger patients, with significantly worse prognoses. Induction and consolidation therapy combined with allogeneic hematopoietic stem cell microtransplantation may improve the prognosis of elderly patients with AML. The present study reports the effect of maintenance therapy with low-dose chemotherapy treatment combined with microtransplantation in elderly patients with AML following consolidation. Between January 2011 and April 2014, three elderly patients (>55 years old), including one 58-year-old patient with acute mixed lineage leukemia (AMLL) and two patients with AML aged 59 years and 62 years, underwent microtransplantation maintenance therapy. Following a complete response to induction chemotherapy and consolidation chemotherapy with two to four cycles of medium dose Ara-c (auto transplantation was performed in the patient with AMLL), maintenance therapy was performed using low-dose Ara-c combined with human leukocyte antigen haploidentical allogeneic hematopoietic stem cell microtransplantation, which was repeated every 3 to 6 months. The patients were followed up for 27, 20 and 16 months, respectively, and all three patients achieved disease-free survival. The bone marrow Wilms' tumor suppression gene (WT1) level of the case with AMLL was dynamically monitored. The results showed that the WT1 level was abnormally high prior to microtransplantation and gradually declined to normal levels subsequent to the process. None of the patients suffered from graft versus host disease during the microtransplantation process. In conclusion, microtransplantation maintenance therapy following consolidation therapy is feasible in elderly patients with AML, and is expected to be able to further remove residual lesions and improve treatment efficacy. A large-scale clinical trial is required to confirm the effect of maintenance therapy in elderly patients with AML.

Combined Assessment of WT1 and BAALC Expression Levels Improves Risk Stratification in Myelodysplastic Syndromes

BACKGROUND AND AIMSIn patients with myelodysplastic syndromes (MDS) several validated prognostic scores, such as IPSS and R-IPSS, are available to assess the risk of AML progression and predict overall survival (OS) as well as leukemia-free survival (LFS).A number of molecular aberrations can be identified in MDS. However, differently from AML, none of the current prognostic indexes takes into account molecular profile at diagnosis.WT1 expression has often been evaluated in acute leukemias and MDS. High WT1 expression levels on bone marrow at diagnosis have been reported to identify MDS patients who are at high risk of progression to AML. BAALC (Brain And Acute Leukemia Cytoplasmic) hyper-expression has been associated with a poor prognosis in AML patients, whereas its prognostic value in MDS is not yet clearly defined. The aim of our study was to determine if combined assessment of WT1 and BAALC expression levels at diagnosis could be predictive of leukemic evolution.MATERIALS AND METHODSWe selected 86 patients with available WT1 and BAALC expression levels on BM samples at diagnosis.According to IPSS score, 22 patient were considered low-risk, 27 intermediate-1 and 28 intermediate-2 or high risk. Patients underwent different treatment schedules including supportive care, erythropoietin, hypomethylating and immunomodulating agents, according to their risk group. Median follow-up was 36 months (range 4 -121 months).Leukemia-free survival (LFS) was calculated from the diagnosis until last follow-up or documented leukemic progression as defined in literature. LFS was estimated using the Kaplan–Meier method.All Real-Time PCR were performed on DNA Engine 2 (Opticon®, MJ Research®). WT1 copy number/Abl copy number 1000x104 was used as cut-off value for high WT1 expression, a level of 1000x104 BAALC copy number/Abl copy number was set as cut-off for BAALC hyper-expression.RESULTSAfter a median time of 32 months, 43 patients died. The main cause of death was leukemic evolution (accounting for 31/43 deaths, 72%), other causes were cardiovascular events and infections (data not shown).The risk of death by any cause was significantly affected by leukemic evolution, diagnosis according to WHO classification and molecular expression profile at diagnosis. Multivariate analysis showed that leukemic evolution was an independent predictor of death (p <0.001).Twenty-nine leukemic evolutions were observed. Median LFS was 34 months. The probability of leukemic evolution was significantly affected by karyotype, IPSS and R-IPSS scores, diagnosis according to WHO classification, and molecular profile at diagnosis.According to our data WT1 and BAALC combined expression levels further enhanced prognostic stratification. In IPSS Int-1, Int-2/high and in R-IPSS high risk groups, low levels of expression resulted in significantly lower probability of leukemic progression, whereas high levels predicted poor outcome. Furthermore, in patients assigned to IPSS unfavorable prognostic groups, low levels of WT1 and BAALC seemed to predict a significantly longer LFS.In the univariate analysis LFS duration was significantly affected by WT1 and BAALC expression levels (fig. 1), IPSS and R-IPSS scores, karyotype and WHO classification at diagnosis. A multivariate Cox Regression model showed that LFS duration was significantly influenced only by molecular profile at diagnosis and R-IPSS risk group (p <0.001 and p <0.01, respectively).Median OS was 32 months. In univariate analysis OS was significantly influenced by diagnosis according to WHO classification, karyotype, R-IPSS score, leukemic evolution and molecular profile expression at diagnosis. The multivariate model disclosed molecular expression profile, R-IPSS score and leukemic evolution as independent predictor of OS (p <0.02, <0.03 and <0.01, respectively).CONCLUSIONSIn MDS patients combined WT1 and BAALC expression levels on bone marrow samples at diagnosis is a reliable predictor of risk of AML progression, LFS and OS. This can improve risk stratification especially in intermediate and high risk groups and may lead to a risk tailored therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Wilms Tumor Gene 1 Expression before Allogeneic Hematopoietic Cell Transplantation Correlates with Relapse in Acute Myeloid Leukemia Patients

IntroductionEvaluation of WT1 expression is becoming an attractive marker in acute myeloid leukaemia (AML) for minimal residual disease (MRD) detection. Recent studies correlate therapy response with WT1 copy levels in the bone marrow (BM) offering an additional tool beside multiparameter flow-cytometry (MFC). No well-known data are available regarding its impact on the outcome after hematopoietic cell transplantation (HCT).Patients/MethodsOne hundred and two consecutive clinically and molecularly well characterized AML patients (pts) were transplanted in a single hematology center from 2004 to 2013. Indication for allogeneic HCT included high cytogenetic and molecular risk according to WHO criteria, high leukocytosis, extramedullary manifestations or secondary AML at diagnosis. Further, primary induction failure was considered as an indication for allogeneic HCT.WT1 expression was analyzed by real time polymerase chain reaction (RT-PCR), using the standardized European LeukemiaNet method on BM samples before HCT and during each follow up control. Only pts in first CR before HCT were included in the analysis.Cumulative incidence of relapse (CIR) was estimated considering death for other causes than relapse as a competing event. Univariate and multivariate Fine & Gray Regression models were used to test the association between CIR and pre-HCT BM WT1 levels. Linearity of the relationship between CIR and the WT1 level was investigated using a mathematical transformation (restricted cubic spline).Aim of this retrospective analysis was to investigate the impact of pre-HCT BM WT1 expression in first CR pts on predicting relapse after HCT.ResultsA BM WT1 evaluation pre-HCT was available in 89 out of 102 pts. Among them, 62 achieved a CR after induction treatment. Relapsed pts in CR after reinduction chemotherapy (n=16) or with disease persistence pre-HCT (n=2) and pts refractory to treatment (n=9) were excluded from analysis.The patient subgroup displaying a first CR before HCT had a median age at diagnosis of 49 years (range: 20-65). Patient/donor relationship involved 26 (42%) sibling, 32 (52%) matched unrelated and 4 (6%) haplo-identical donors. On the basis of standard cytogenetics, molecular biology and clinical criteria (global disease risk) pts were classified according to the following risk groups: 54 (87%) high, 5 (8%) intermediate and 3 (5%) low risk. Acute GVHD occurred in 24 (39%) whereas chronic GVHD was documented in 18 (28%) pts. Twenty-six (42%) deaths were documented after HCT, 21 (34%) due to relapse and 5 (8%) because of treatment related mortality (TRM).Pre-HCT BM WT1 expression was correlated with CIR. A cut point of 150 WT1 copies was applied according to the slope change of relapse hazard, and subsequently used for CIR analysis. Pts displaying a pre-HCT BM WT1 level > 150 copies (n=18) had a higher CIR (2-year CIR 52.5%, 95% CI: 27.1-77.8) compared to pts with a BM WT1 level ≤ 150 copies (n=44, 2-year CIR 27.7% (95% CI: 13.4-42.1). WT1 copy level > 150 showed a significantly higher risk of relapse in univariate analysis (HR 2.9, 95% CI 1.2-6.7, p=0.014). In multivariate analysis pre-HCT BM WT1 expression was confirmed as a significant independent risk factor for CIR when adjusted for patient/donor relationship, presence of GVHD, global disease risk and competitive risk of mortality due to TRM (HR 3.4, 95% CI 1.3-8.5, p=0,010), Figure.Conclusions In the present study, pre-HCT BM WT1 levels discriminated significantly for CIR in a cohort of AML in first CR. A cut off level of 150 BM WT1 copies pre-HCT had a powerful statistically significant discriminating value to predict the risk of relapse, independently from already established risk factors. The prognostic value of WT1 was confirmed also when TRM as competitive risk for mortality was added in the multivariable model. Based on these results, WT1 is a promising candidate as MRD tool. Further prospective studies are required to confirm these results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

WT1 Monitoring of Minimal Residual Disease (MRD) in Patients with Acute Myeloid Leukemia

Background: Although a complete remission (CR) can be achieved in 70-80% of newly diagnosed acute myeloid leukemia (AML) patients, relapses occur in up to the 50% of cases. Thus, minimal residual disease (MRD) monitoring is a major issue for early detection of patients at high-risk of treatment failure and relapse.Aim: to dynamically evaluate WT1 pan-leukemic molecular marker of MRD in patients with AML.Matherial and methods: 107 newly diagnosed AML patients consecutively treated between 2010 and 2013 were monitored with quantitative WT-1 from bone marrow (BM) and peripheral blood (PB) at baseline, after induction, after the first consolidation course, before allogeneic stem cell transplantation (allo-SCT), at the 3rd and the 6th month after transplantationResults: At diagnosis, 104/107 (97%) had increased PB and BM WT1 levels assessed according to the ELN assay. Eighty-eight out of 107 patients (82%) achieved a complete remission (CR) after induction, 30/88 (34%) relapsed during follow up and 24/107 (22%) were addressed to allogeneic stem cell transplantation (allo-SCT). By univariate analysis, PB-WT > 50x10^4/ABL and BM-WT1 > 250x10^4/ABL after induction (PB: p=0.02; BM: p=0.04), after consolidation (PB: p=0.003), at the end of treatment (PB and BM: p=0.001), at 3rd month of follow up (PB and BM: p=0.005) and at 6th month of follow up (PB: p=0.005) were associated with a reduced overall survival (OS). By multivariate analysis, a BM-WT1 > 250 x 10^4/ABL at the end of treatment was significantly associated with a reduced OS. In order to adapt the cut-off of WT1 in our series of patients, we considered WT1 levels as continuous variables and categorized them at approximately the 25th, 50th, and 75th percentile. A cut-off of PB-WT1 > 25x10^4/ABL and BM-WT1 > 125x10^4/ABL at the end of the treatment program was identified as correlated with reduced leukemia-free survival (LFS) and OS (p=0.001). Similarly, and restricting the analysis on the 24 patients allo-transplanted in CR, 8/11 (73%) with pre-transplant PB-WT1 ≥ 5 and 4/13 (31%) with PB-WT1 < 5 relapsed, respectively (p=0.04). The incidence of relapse was higher in AML patients with PB-WT1 ≥ 5 measured at 3rd (56% vs 38%; p=0.43) and 6th month (71% vs 20%; p=0.03) after allo-SCT. Interestingly, 5/5 (100%) patients with pre-transplant PB-WT1 ≥ 5 who never reduced this level at 3rd or 6th month after allo-SCT experienced a disease recurrence.Conclusions: our data, although retrospectively collected, show that WT1 monitoring may be useful to predict the relapse in AML patients.Acknowledgments: This work was supported in part by Banca di Credito Cooperativo di Pompiano e Franciacorta and Lions Club Bassa Bresciana Association. DisclosuresRusso:Celgene: Research Funding; Gilead: Research Funding; Novartis: Consultancy.

Prognostic Significance of WT1 and PRAME Gene Expression in Bone Marrow Samples of MDS and AML Patients Treated with Azacytidine

Introduction: The Wilms’ tumor gene (WT1) and preferentially expressed antigen of melanoma (PRAME) gene are frequently overexpressed in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) Combined quantitation of PRAME and WT1 transcript levels could provide a powerful molecular marker in newly diagnosed MDS patients, enabling the early identification of relapse through minimal residual disease monitoring and also to enable alternate treatment strategies (Qin et al, 2012).In this study, we investigated the prognostic impact of WT1 and PRAME gene expression on MDS and AML patients treated with azacytidine.Methods:In this ethically approved, retrospective study, we studied WT1 and PRAME gene expression in the bone marrow of 20 patients with MDS and AML (unsuitable for more intensive chemotherapy) prior to starting epigenetic therapy with azacytidine (100mg/M2 sc for 5 days every 28 days). 20 patients with lymphoma not involving the bone marrow were used as a control group. Quantitative assessment of WT1 and PRAME transcript levels was performed on formalin fixed paraffin-embedded trephine biopsies, using the WT1 Ipsogen ProfileQuant and a ‘homebrew’ kit respectively. A control GADPH was used. Overall survival was extimated using the Kaplan-Meier method and compared using the log-rank test. The logistic regression model was used to correlate IPSS-R with WT-1 and PRAME gene expression levels.Results: The median age of the patient group was 71. 16 patients had MDS (RCMD: 3; RARS: 2; RAEB1: 2; RAEB2: 5; CMML: 4) and 4 had AML. Median age of the control group was 61. Median number of cycles of azacytidine was 4 (range 1-21). In 11 of the patient group and in all of the controls, neither PRAME nor WT1 gene expression was detected.. PRAME gene expression without WT1 gene expression was detected in 5 patients, whilst, in the remaining 4 patients, both PRAME and WT1 gene expression was detected. There was no significant difference in mean percentage of bone marrow blasts between patients with detectable PRAME +/- WT1 gene expression (16.67%) and those without (12.64%).Median gene expression levels of PRAME +/- WT1 significantly correlated with IPSS-R (p=0.003).Overall survival (OS) was significantly inferior in patients who were positive for PRAME +/- WT1 (p=0.0103). Median OS for patients positive for both PRAME and WT1 was 270 days and for patients who were PRAME positive and WT1 negative was 300 days, whilst patients negative for both had a median OS of 630 days (figure 1).Conclusions: Our study results show that patients who have detectable WT1 and PRAME expression in their bone marrow prior to azacytidine therapy have more advanced disease in terms of IPSS-R score and have a much inferior OS compared to patients without detectable WT1 and PRAME. Such patients should be considered for either additional or alternate therapy to single agent azacytidine. DisclosuresNo relevant conflicts of interest to declare.

DNA Hydroxymethylation Profiling Reveals That WT1 Mutations Result in Loss of TET2 Function in Acute Myeloid Leukemia

Mutations in TET2 and IDH1/2, which result in reduced conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), are observed in a significant proportion of patients with acute myeloid leukemia (AML). Hence these mutations define a novel class of AMLs with alterations in DNA methylation. However, it has been shown that not all AML cases with low levels of 5-hmC harbor somatic mutations in TET2 and IDH1/2. We hypothesized there are additional somatic mutations that can lead to alterations in TET enzyme function and resultant changes in the epigenetic state of hematopoietic cells.Mutational studies of the ECOG 1900 trial allowed us to discover that somatic mutations in the WT1 gene were mutually exclusive of IDH1/2 mutations and negatively correlated with TET2 mutations (Figure 1A). 28/313 (9%) of TET2/IDH-wild-type patients had somatic WT1 mutations, whereas 2/85 (2%) TET2/IDH1/2-mutant patients had co-occurring WT1 mutations (p=0.026, Fisher’s Exact test). Analysis of combined data from the ECOG 1900 study and the AML TCGA dataset confirmed a significant anti-correlation between WT1 mutations and TET2/IDH1/2 mutations (p=0.016). We next compared the DNA methylation profiles of WT1, TET2, and IDH1/2 mutant AML patients to AML patients wild-type for these disease alleles. Comparative analysis of the three hypermethylation profiles revealed a near-complete overlap of TET2 and WT1 hypermethylated loci within the IDH1/2 hypermethylation signature, and a highly significant overlap between the TET2 and WT1 mutant signatures (p-value< 0.001 for all comparisons), consistent with convergent, site-specific effects on DNA methylation.These genetic observations suggested a shared functional role for WT1, TET2, and IDH1/2 mutations in AML in regulating 5-hmC. WT1-mutant AML patients had significantly reduced 5-hmC (Liquid chromatography-electron spray ionization-tandem mass spectrometry) compared to AML patients wild-type for WT1, TET2 or IDH1/2 (p=0.016 T-test Figure 1B), similar to the reduction in 5-hmC observed in IDH1/2-mutant and TET2-mutant AML patient samples. In order to determine the impact of IDH1/2, WT1 and TET2 mutations on the distribution of epigenetic marks throughout the genome more precisely, we examined 5-hmC localization in primary AML specimens with next-generation sequencing. The average number of 5-hmC peaks present in samples from AML patients with TET2, WT1, IDH1 or IDH2 mutations was significantly reduced compared to AML patients wild-type for these disease alleles (t-test p-values between 0.0005 and 0.003). We observed highly significant overlap of differential 5-hmC peaks lost in WT1-mutant AML and TET2-mutant AML (Hypergeometric test p-value < 10-133). In a manner analogous to the findings for DNA methylation, the regions with reduced 5-hmC in WT1/TET2 mutant AML were a subset of those found in IDH1/2 mutant AML patients.We next investigated whether perturbations in WT1 expression can directly alter 5-hmC levels. Knockdown of Wt1 significantly decreased 5-hmC levels in M15 cells (p<0.01) (Figure 1C). Conversely, overexpression of WT1 in 32D cells significantly increased 5-hmC levels compared to cells expressing a control vector or WT1-mutant (p<0.05) (Figure 1D). Thus, WT1 can directly regulate 5-hmC levels. Given the effects of WT1 on 5-hmC levels and the inverse correlation between WT1 and TET2 mutations in AML, we hypothesized that WT1 might modulate TET2 function through direct interaction.Co-immunoprecipitation experiments demonstrated WT1 interacts with TET2 and with TET3. Consistent with these effects, WT1 expression inhibited colony forming ability of TET2-deficient cells, but could not attenuate the colony growth of TET2/TET3-deficient cells in vitro.Taken together, our genetic and functional studies demonstrate an inverse correlation between WT1 mutations and TET2/IDH1/2 mutations in AML and a functional role for WT1 in regulating DNA methylation. WT1 mutant AML is characterized by similar global and site-specific alterations in 5-hmC/5-mC as observed in TET2/IDH1/2 mutant AML. Furthermore, we demonstrate that alterations in WT1 levels directly regulate 5-hmC levels, which is due to an interaction between TET2/TET3 and WT1. These data provide a novel role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/2, and WT1 mutations define a novel AML subtype. [Display omitted] DisclosuresLevine:Novartis: Consultancy, Grant support Other.

Optimal time-points for minimal residual disease monitoring change on the basis of the method used in patients with acute myeloid leukemia who underwent allogeneic stem cell transplantation: A comparison between multiparameter flow cytometry and Wilms’ tumor 1 expression

Minimal residual disease (MRD) of 30 adult AML patients was monitored by multiparameter flow cytometry (MFC) and WT1 expression before and after allogeneic stem cell transplantation (allo-SCT). Diagnostic performance of pre-transplant MRD measured by MFC was higher than that obtained by WT1 expression. Comparable results were displayed at day +30 post-transplant, while better values by WT1 compared to MFC were found at day +90. Positive MRD by MFC predicted a shorter disease free survival (DFS) before and 1 month after transplant (p=0.006 and p=0.005), while only high WT1 levels at 1 month from the transplant significantly impacted on DFS (p=0.010). Our results support the idea that MRD monitoring by MFC should be suggested before and 30 days after the transplant, while WT1 expression should be preferred after this procedure. The assessment of MRD at day +30 from allo-SCT is recommended as post transplant check-point for the predictive role displayed, independently of the method used.

Influence of Bushenhuoxue on podocytes of focal segmental glomerulosclerosis mice

ObjectiveTo observe the effects and mechanisms of Bushenhuoxue on desmin and nephrin expression in mice podocytes, and to investigate its effects on wt1 expression in Wilms' tumor. MethodsAdriamycin (ADR) was used to induce focal segmental glomerulous sclerosis (FSGS) in mice. Bushenhuoxue was used to treat FSGS for 6 weeks. We measured body mass and right renal mass, and determined serum albumin (ALB) levels, protein content in urine, and urinary protein and albumin creatinine ratio (UACR). Changes in renal tissue morphology were evaluated by microscopy. wt1 and nephrin expression in podocytes were detected using immunofluorescence. Expression levels of desmin, wt1 and nephrin mRNAs in renal tissue were determined using reverse transcription polymerase chain reaction assays. ResultsProtein levels in urine and UACR were significantly increased in FSGS model mice compared with Bushenhuoxue-treated and control mice. Body mass and ALB levels were decreased in FSGS mice compared with control and Bushenhuoxue-treated mice. Expression of the wt1 protein was observed in control mice. Compared with controls, wt1 expression levels were reduced in Bushenhuoxue-treated mice, and to a greater extent in FSGS mice. Nephrin protein expression was widespread in FSGS mice, and significantly reduced in control and Bushenhuoxue mice. Expression levels of wt1 and nephrin mRNAs in FSGS mice were lower compared with those in control and Bushenhuoxue-treated mice. Desmin mRNA levels in FSGS mice were reduced compared with those in control and Bushenhuoxue-treated mice. ConclusionBushenhuoxue ameliorated albuminuria in FSGS mice; this was possibly related to the up-regulation of wt1 and nephrin, and down-regulation of desmin.

MicroRNA-191, by promoting the EMT and increasing CSC-like properties, is involved in neoplastic and metastatic properties of transformed human bronchial epithelial cells.

Lung cancer is the leading cause of cancer mortality worldwide. A common interest in lung cancer research is the identification of biomarkers for early diagnosis and accurate prognosis. There is increasing evidence that microRNAs (miRNAs) are involved in lung cancer. To explore new biomarkers of chemical exposure in risk assessment of chemical carcinogenesis and lung cancer, we analyzed miRNA expression profiles of human bronchial epithelial (HBE) cells malignantly transformed by arsenite. High-throughput microarray analysis showed that 51 miRNAs were differentially expressed in transformed HBE cells relative to normal HBE cells. In particular, miR-191 was up-regulated in transformed cells. In HBE cells, arsenite induced increases of miR-191 and WT1 levels, decreased BASP1 expression, and activated the Wnt/β-catenin pathway, effects that were blocked by miR-191 knockdown. In addition, a luciferase reporter assay indicated that BASP1 is a direct target of miR-191. By inhibiting the expression of BASP1, miR-191 increased the expression of WT1 to promote activation of Wnt/β-catenin pathway. In transformed cells, inhibition of miR-191 expression blocked the epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-like properties of cells and decreased their migratory capacity and neoplastic properties. Thus, these results demonstrate that miR-191 modulates the EMT and the CSC-like properties of transformed cells and indicate that it is an onco-miR involved in the neoplastic and metastatic properties of transformed cells.

Real-time assessment of relapse risk based on the WT1 marker in acute leukemia and myelodysplastic syndrome patients after hematopoietic cell transplantation

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (alloHCT) for acute leukemia and myelodysplastic syndrome (MDS). Wilms' tumor antigen (WT1) is overexpressed in the majority of acute leukemia and MDS patients and has been proposed as a universal diagnostic marker for detection of impending relapse. Comprehensive studies have shown that WT1 transcript levels have predictive value in acute leukemia patients in complete remission after chemotherapy. However, the focus of this study is the period after alloHCT for predicting relapse onset. We analyzed the accumulation of WT1 mRNA transcripts in peripheral blood of 82 leukemia and MDS patients and defined specific molecular ratios for relapse prediction. The extensively validated WT1/c-ABL ratio was used to normalize increases in WT1 transcript levels. The observed lead time of crossing or exceeding set WT1 levels is presented along with linear interpolation to estimate the calculated day the WT1 thresholds were crossed. The WT1/c-ABL transcript ratio of 50 or above yielded 100% specificity and 75% sensitivity reliably predicting future relapse with an observed average of 29.4 days (SD=19.8) and a calculated average of 63 days (SD=29.3) lead time before morphologic confirmation. A lower ratio of 20 or above gave lower specificity but higher sensitivity (84.8 and 87.5%, respectively) identified more patients that relapsed, at earlier times, providing an earlier warning with actual average lead time of 49.1 days (SD=30.8) and calculated average of 78 days (SD=28.8). WT1 transcript levels serve as a diagnostic relapse test with greater sensitivity than the morphologic approach used in the clinic as a readout.

MRD assessed by WT1 and NPM1 transcript levels identifies distinct outcomes in AML patients and is influenced by gemtuzumab ozogamicin.

We analysed the prognostic significance of minimal residual disease (MRD) level in adult patients with acute myeloid leukemia (AML) treated in the randomized gemtuzumab ozogamicin (GO) ALFA-0701 trial. Levels of WT1 and NPM1 gene transcripts were assessed using cDNA-based real-time quantitative PCR in 183 patients with WT1 overexpression and in 77 patients with NMP1 mutation (NPM1mut) at diagnosis. Positive WT1 MRD (defined as > 0.5% in the peripheral blood) after induction and at the end of treatment were both significantly associated with a higher risk of relapse and a shorter overall survival (OS). Positive NPM1mut MRD (defined as > 0.1% in the bone marrow) after induction and at the end of treatment also predicted a higher risk of relapse, but did not influence OS. Interestingly, the achievement of a negative NPM1mut MRD was significantly more frequent in patients treated in the GO arm compared to those treated in control arm (39 % versus 7% (p=0.006) after induction and 91% versus 61% (p=0.028) at the end of treatment). However, GO did not influence WT1 MRD levels. Our study supports the prognostic significance of MRD assessed by WT1 and NPM1mut transcript levels and show that NPM1 MRD is decreased by GO treatment.

Early B-cell factor 1 is an essential transcription factor for postnatal glomerular maturation

The coordination of multiple cytokines and transcription factors with their downstream signaling pathways have been shown to be integral to nephron maturation. Here we present a completely novel role for the helix-loop-helix transcription factor Early B cell Factor 1 (Ebf1), originally identified for B cell maturation, for the proper maturation of glomerular cells from mesenchymal progenitors. The expression of Ebf1 was both spatially and temporally regulated within the developing cortex and glomeruli. Using Ebf1-null mice we then identified biochemical, metabolic, and histological abnormalities in renal development that arose in the absence of this transcription factor. In the Ebf1 knockout mice the developed kidneys show thinned cortices and reduced glomerular maturation. The glomeruli showed abnormal vascularization and severely effaced podocytes. The mice exhibited early albuminuria and elevated blood urea nitrogen levels. Moreover, the GFR was reduced over 66 percent and the expression of podocyte-derived VEGF-A was decreased compared to wild type control mice. Thus, Ebf1 has a significant and novel role in glomerular development, podocyte maturation, and the maintenance of kidney integrity and function.

Expression of WT1 and PRAME gene in bone marrow and peripheral blood samples of patients with myelodysplastic syndrome

This study was aimed to explore the transcription level of WT1 and PRAME two genes in bone marrow and peripheral blood samples of patients with myelodysplastic syndrome(MDS) and their relationship with bone marrow dysplasia and karyotype. The quantitative expression of WT1 and PRAME transcripts detected by RQ-PCR in the bone marrow samples of 203 MDS patients and 19 aplastic anemia(AA), 6 other benign anemia(BA), 4 paroxysmal nocturnal hemoglobinuria(PNH) patients from July 2009 to June 2012 and 14 healthy donors, and in 92 peripheral blood samples. The results showed that WT1 and PRAME expression levels in both BM and PB samples of MDS group were higher than those in normal controls, AA, and BA patients (BM: WT1:P = 0.000, 0.000, 0.000, PRAME: P = 0.048, 0.000, 0.064; PB: WT1:P = 0.012, 0.000, 0.011, PRAME: P = 0.020, 0.004, 0.003). What is more, this expression in high risk MDS group (RAEB1, RAEB2, MDS-AML) were higher than those in low risk group (RCUD, RCMD, MDS-U) and AA and BA. The WT1 and PRAME mRNA expression levels in PB and BM were well correlated (WT1:r = 0.6028, P = 0.001; PRAME: r = 0.7628, P = 0.000), as well as the WT1 expression levels in BM samples with the Karyotype (P = 0.049). In addition, the same positive rate of WT1 or PRAME expression existed in BM and PB samples of MDS patients. It is concluded that the WT1 and PRAME gene expression levels in both BM and PB samples of MDS patients are higher than those in healthy controls, AA and other benign anemia patients, and increase with the progression of the disease. The WT1 and PRAME transcripts constitute good molecular markers for the clinical diagnosis and prognosis and monitoring minimal residual disease after treatment of MDS. What is more, when bone marrow is not so convenient to get, the transcript levels of PB samples can be detected.

Transcriptional Repression of Tumor Suppressor CDC73, Encoding an RNA Polymerase II Interactor, by Wilms Tumor 1 Protein (WT1) Promotes Cell Proliferation: IMPLICATION FOR CANCER THERAPEUTICS

The Wilms tumor 1 gene (WT1) can either repress or induce the expression of genes. Inconsistent with its tumor suppressor role, elevated WT1 levels have been observed in leukemia and solid tumors. WT1 has also been suggested to act as an oncogene by inducing the expression of MYC and BCL-2. However, these are only the correlational studies, and no functional study has been performed to date. Consistent with its tumor suppressor role, CDC73 binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex and causes transcriptional repression of oncogenes MYC and CCND1. It also represses β-catenin-mediated transcription. Based on the reduced level of CDC73 in oral squamous cell carcinoma (OSCC) samples in the absence of loss-of-heterozygosity, promoter methylation, and mutations, we speculated that an inhibitory transcription factor is regulating its expression. The bioinformatics analysis predicted WT1 as an inhibitory transcription factor to regulate the CDC73 level. Our results showed that overexpression of WT1 decreased CDC73 levels and promoted proliferation of OSCC cells. ChIP and EMSA results demonstrated binding of WT1 to the CDC73 promoter. The 5-azacytidine treatment of OSCC cells led to an up-regulation of WT1 with a concomitant down-regulation of CDC73, further suggesting regulation of CDC73 by WT1. Exogenous CDC73 attenuated the protumorigenic activity of WT1 by apoptosis induction. An inverse correlation between expression levels of CDC73 and WT1 was observed in OSCC samples. These observations indicated that WT1 functions as an oncogene by repressing the expression of CDC73 in OSCC. We suggest that targeting WT1 could be a therapeutic strategy for cancer, including OSCC.

Peripheral blood WT1 expression predicts relapse in AML patients undergoing allogeneic stem cell transplantation.

To evaluate if WT1 expression may predict relapse after allo-SCT, we analyzed WT1 levels on peripheral blood (PB) and bone marrow (BM) before and after allo-SCT in 24 AML patients with WT1 overexpression at diagnosis. Five copies of WT1/ABL × 104 from PB were identified as the threshold value that correlated with relapse after allo-SCT. The same correlation was not identified when WT1 expression was assessed from bone marrow (BM). Eight out of 11 (73%) patients with a pre-allo-SCT PB-WT1 ≥ 5 and 4/13 (31%) patients with a pre-allo-SCT PB-WT1 < 5 relapsed, respectively (P = 0.04). The incidence of relapse was higher in patients with PB-WT1 ≥ 5 measured after allo-SCT, at the 3rd (56% versus 38%; P = 0.43) and at the 6th month (71% versus 20%; P = 0.03). Patients with pretransplant PB-WT1 < 5 had significantly better 2-year OS and LFS than patients with a PB-WT1 ≥ 5 (81% versus 0% and 63% versus 20%) (P = 0.02). Our data suggest the usefulness of WT1 monitoring from PB to predict the relapse in allotransplanted AML patients and to modulate the intensity of conditioning and/or the posttransplant immunosuppression in an attempt to reduce the posttransplant relapse risk.

Soluble fas and the -670 polymorphism of fas in lupus nephritis.

This study was performed to clarify the role of soluble Fas (sFas) in lupus nephritis (LN) and establish a potential relationship between LN and the −670 polymorphism of Fas in 67 patients with systemic lupus erythematosus (SLE), including a subset of 24 LN patients with proteinuria. Additionally, a group of 54 healthy subjects (HS) was included. The allelic frequency of the −670 polymorphism of Fas was determined using PCR-RFLP analysis, and sFas levels were assessed by ELISA. Additionally, the WT-1 protein level in urine was measured. The Fas receptor was determined in biopsies by immunohistochemistry (IHC) and in situ hybridization (FISH) and apoptotic features by TUNEL. Results. The −670 Fas polymorphism showed that the G allele was associated with increased SLE susceptibility, with an odds ratio (OR) of 1.86. The sFas was significantly higher in LN patients with the G/G genotype, and this subgroup exhibited correlations between the sFas level and proteinuria and increased urinary WT-1 levels. LN group shows increased expression of Fas and apoptotic features. In conclusion, our results indicate that the G allele of the −670 polymorphism of Fas is associated with genetic susceptibility in SLE patients with elevated levels of sFas in LN with proteinuria.

Effect of NSAIDs and Diuretics on Nephrogenesis in an Ex Vivo Embryogenic Kidney Model

The kidney is one of the key organs in clearing foreign compounds. The effects of drugs on the developing kidney are relatively unknown. We studied the direct effect of furosemide, hydrochlorothiazide, ibuprofen, and indomethacin on kidney development in an ex vivo embryonic kidney model. At embryonic day 13, metanephroi were dissected from mice and cultured in control media or media supplemented with various clinically relevant concentrations of drugs. The ureteric tree was visualized by whole-mount staining and branching was evaluated by counting. Additionally, gene expression levels of Wt1, Sox9, Bmp7, Fgf8, and Gdnf were investigated. No distinct differences were noted on either ureteric tip development or gene expression analysis for each drug after 24 hr of exposure. Even though short-term exposure to clinically relevant concentrations seems not to disturb renal development, future research is needed to study prolonged or repeated exposures.

Bone Marrow WT1 Levels In Long-Term Survivors Of Core-Binding Factor AML and Acute Promyelocytic Leukemia

BackgroundQuantitation of WT1 mRNA levels may be used in minimal residual disease studies in AML cases lacking a suitable molecular target. The normal thresholds of bone marrow WT1 levels at different time–points have not been clearly stated. Difficulties arise as a consequence of the relatively high WT1 expression in normal bone marrow and the unknown influence of chemotherapy. ObjectiveTo establish the reference range value of bone marrow WT1 levels in treated CBF-AML and APL in complete remission. Patients and MethodsPatients with bone marrow samples obtained from ≥2 years (2-11 y) after initial diagnosis of CBF-AML (AML1-ETO n:21 patients CBFb-MYH11 n:21 patients) or APL(n:22 patients) were included in the study (120 samples). Ages at diagnosis ranged from 1 to 76 years. Mean age was: AML1-ETO 36.8(2-76y),CBFb-MYH11 32.4(1-55y), PML-RARa 47.3(21-76y). Peripheral blood, reticulocyte count and a bone marrow aspirate were also available in 86 cases. WT1 was analyzed following the primers and conditions of the ELN group. Specific chimeric real time PCR was simultaneously performed in each sample in accordance with the BIOMED protocol. Each WT1 or chimeric PCR was analyzed in triplicate whereas the control gene (abl) was analyzed in duplicate. ResultsAll patients with an available BM study were in morphologic complete remission. Two samples corresponded to an anemic patient (2/86). Six cases had a reticulocyte count above 2%. Platelet counts were below 120x109/l in 3 cases (3/86). Leukocyte counts below 3.5x109/l were observed in 6 samples (6/86). The calculated mean WT1 copy number from the 120 bone marrow samples was 47.23 (AML1-ETO:61.33, CBFb-MYH11:43.51, PML-RARa:36.4 p:ns). The mean chimeric copy number was 0.75 (AML1-ETO:0.82, CBFb-MYH11:0.72, PML-RARa:0.69 p:ns). Only 5 samples had a specific copy number of chimeric transcripts above 5 (3 AML1-ETO,1 CBFb-MYH11 and one PMAL-RARa patient who had a molecular relapse one month later and was succesfully treated). In 6 samples, the specific chimeric copy number was between 1 and 5 copies ( 4 AML1-ETO and 2 CBFb-MYH11). In the remaining 109 BM samples, the specific copy number was <1. In 9 BM samples, the WT1 levels were in the range of 100-300 copies (7 AML1-ETO and 2 CBFb-MYH11). All these 9 samples had specific chimeric gene number below 1.5 copies. In three samples (1 AML1-ETO, 1 CBFb-MYH11 and 1 PML-RARa) peaks of WT1 levels of ≥300 copies were detected. In these three samples the specific chimeric gene was undetectable (<0.01). ConclusionsBone marrow WT1 copy number in treated CBF-AML and APL was 47.23. In ten percent of cases, the WT1 copy number was greater than 100 copies. Occasionally, WT1 peaks (≥300 ) were detected. The meaning of the differential bone marrow WT1 transcriptional activity in long-term survivors of AML remains to be investigated. Disclosures:No relevant conflicts of interest to declare.

Combined overexpression of WT1 and BAALC genes May Predict AML evolution in MDS Patients

Background and aimsMyelodisplastic syndromes (MDS) are a group of hemopoietic disorder characterized by an impaired blood cell production, morphologic dysplasia and peripheral cytopenias; they are the most common hematologic neoplastic disorder and its diagnosis relays on morphologic evaluation, associated to a karyotypic assay.In order to predict the outcome of patients affected from these disorders, knowing that the order of survival can be extremely variable, several prognostic index were developed such as International Prognostic Score Sistem (IPSS) or the most usefull WHO-Prognostic Score Sistem (WPSS).On the contrary of acute leukemia, these disorders have not a biomolecular profile evalutation of intrinsic markers able to stratify patients in different prognostic risk groups.The aim of our study is to assess the risk of leukemic evolution, in MDS patients, on the basis of the levels expression of WT1 and BAALC at disease diagnosis, and to evaluate the leukemia free survival (LFS) at 6-12-24-36 months of follow up, among the different risk category according to IPSS and irrespectively of treatment. Materials and methodIn five years we analized 102 patients with a diagnosis of MDS divided according to the WHO classification such as follows: 38 AR, 1 AR with del(q5), 21 aREB-1, 23 AREB-2, 3 chronic myelomonocitic leukemia, 1 RARS, 1 MDS, 11 RCMD, 1 5q- syndrome, 2 suspected MDS.According to IPSS 58 belonged to the low risk category, 21 to the intermediate-1, 23 to the intermediate-2/high risk.Cytogenetic assay showed 20 people with an abnormal karyotype, 8 of them fallen into the high risk class and 12 into the intemediate risk.Low risk and intermediate-1 patients were treated only with supportive care; high risk patients were treated with hypomethylating agents. Iron chelation were used when necessary. Lenalidomide was used in the only case of 5 q- syndromeSamples of bone marrow were analized with Real-Time quantitave PCR and levels of WT1 and BAALC expression were determinated.Molecular datas were analized with X-square Test and a significant association was recorded between overexpression of the genes evaluated (WT1 higher than 100 copy numbers and BAALC higher than 1000 copy numbers) and the probability of develop acute myeloid leukemia ( AML ). ResultsNine out of 102 patients showed an isolated WT1 hyperexpression (3 of them developed an AML ), in 15 cases we reported an isolated BAALC overexpression (3 of them developed an AML ), while 13 out 18 patients ( 72% ) with combined WT1 and BAALC overexpression developed AML within an average time of 6 months; instead only 5% of patients, which expressed low levels of WT1 and BAALC, developed AML within the interval of observation.In particulary a combined high expression of WT1 and BAALC were strongly associated with an high risk to develop leukemia and a short LFS, especially in INT-1 subset.After that we calculated the LFS, divided for the risk category at 6-12-24-36 months of follow up.Patients with combined overexpression of WT1 and BAALC showed a LFS of 40% at 6 months of follow up and 0% at 24 months. ConclusionMDS have a great variable survival, and the current approach to these diseases relays on morphological evaluation, karyotypic assay and need of transfusional support; gene expression could be a promising system to predict the prognosis in these patients.Analysis of gene expression, which belong to AML evaluation, allows to divide patients in several risk groups; furthermore is not the single gene evaluation that is more predictable but a combined assay.With this method, which seems to be more realiable than IPSS, we could find that a great percentage of patients with levels of WT1>100 and BAALC >1000, indipendently from karyotypic status and treatment, developed AML and have a shorter LFS than the population with WT1 <100 and BAALC < 1000. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Clinical and Prognostic Influence Of Mutations In The Cohesin Complex In Acute Myeloid Leukemia

BackgroundThe cohesin complex presents a ring structure that regulates chromosome segregation during meiosis and mitosis and is thus essential for cell division. Genes that belong to the cohesin complex in somatic vertebrate cells are SMC1A, SMC3, RAD21 (SCC1), STAG2 (SA-2) and STAG1 (SA-1). Recently, mutations in the cohesin complex have been reported to occur in AML. The aim of this study was to evaluate the clinical and prognostic implications of cohesin mutations in 389 uniformly treated younger AML patients. Patients and MethodsDiagnostic bone marrow or peripheral blood samples were analyzed from 389 younger adult patients with de novo (n=348) or secondary AML (n=41) with French-American-British (FAB) classification M0-M2 or M4-M7, who were entered into the multicenter treatment trials AML SHG 0199 (n=276) or AML SHG 0295 (n=113). Patient samples were assessed for frequently occurring mutations including FLT3-ITD, NPM1, DNMT3A, IDH1, IDH2. In the subgroup of cytogenetically normal AML (CN-AML), additional mutation analyses were performed for CEBPA, MLL-PTD, WT1 and WT1 SNP rs16754, NRAS, and expression levels of BAALC, ERG, EVI1, MN1, MLL and WT1. Sequencing of all coding exons of the cohesin genes was performed with the SOLiDTM system (Life Technologie, Darmstadt, Germany). Sequences were analyzed twice separately using the DNAnexus software and a separate pipeline of bioinformatics software. Identified mutations were validated by Sanger sequencing. Results27 patients (7%) were identified to carry a mutation in one of the cohesin genes. 10 patients harboured a mutation in STAG1. The second most frequently mutated gene in the cohesin complex was SMC3 with 7 patients showing this mutation. 5 patients harboured a mutation in RAD21, 3 patients in STAG2 and 2 patients in SMC1A. All mutations were mutually exclusive. The majority of mutations were missense mutations, apart from 2 frameshift mutations and 3 nonsense mutations. All evaluable patients lost their cohesin mutation during remission and with the exception of one patient regained their mutation during relapse.Patients with mutations in the cohesin genes were older (47 vs 44 years, P=.04), had a higher white blood cell count (WBC) (P=.046) and a higher peripheral blast count (P=.048). We observed a strong correlation between cohesin gene and NPM1 mutations (P=.007). BAALC expression was lower in patients with a mutation compared to patients without a mutation in the cohesin genes (P=.026).In order to get a better understanding of when mutations in the cohesin complex occur during clonal evolution we evaluated the allelic burden of mutations in the cohesin complex. Because of the strong association between NPM1 and cohesin mutations, the allelic ratio of mutated and wildtype NPM1 was compared to the allelic ratio of mutated and wildtype cohesin genes. Interestingly, we found a similar mutation burden between NPM1 and genes of the cohesin complex (Pearson correlation coefficient R=0.67), suggesting that cohesin gene mutations occurred in the same clone as NPM1 mutations.When considering all mutations in the cohesin complex as one group, overall survival (OS), relapse free survival (RFS) and complete remission rate (CR) were not influenced by the presence of cohesin mutations (OS: HR 1.23; 95%CI 0.77-1.99; P=.4; RFS: HR 0.92; 95%CI 0.51-1.65; P=.77; CR: mutated 77% vs wildtype 76%, P=.83). We next evaluated the prognostic influence of each gene in the cohesin complex separately in all patients. STAG1, SMC3 and RAD21 mutations had no influence on OS and RFS. A trend for a reduced OS was observed for STAG2 mutated patients (OS: HR 3.02; 95%CI 0.97-9.46; P=.057), although the analysis is limited by the small number of mutated patients. In the subgroup of patients with cytogenetically normal AML we did not identify a difference in OS, RFS or CR rates for patients with (n=18) or without mutations (283) in the cohesin complex (OS: HR 1.00; 95%CI 0.52-1.92; P=.99; RFS: HR 0.64; 95%CI 0.30-1.39; P=.26; CR rates: mutated 80% vs wildtype 73%, P=.64). ConclusionMutations in the cohesin genes represent novel, recurrent mutations in AML. We found a strong association between these mutations and mutations in NPM1. Cohesin complex mutations as a group had no prognostic impact in all and in cytogenetically normal AML patients. Disclosures:Schlenk:Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Chugai: Research Funding; Ambit: Honoraria.