WT1 Transcript Levels Research Articles

High WT1 expression predicted induction chemotherapy failure in acute myeloid leukemia patients with non-favorable cytogenetic risk.

The prognostic significance of WT1 expression at diagnosis in acute myeloid leukemia (AML) remains obscure, and subgroup analysis is the way for clarification. We previously reported the results in t(8;21) AML. In this study, 437 consecutive adult AML patients with non-favorable cytogenetic risk were enrolled. All patients were tested WT1 transcript levels using real-time quantitative PCR at diagnosis; AML-related common fusion genes, KMT2A-PTD, FLT3-ITD, NPM1, CEBPA and TP53 mutations were simultaneously tested. 92.4% of patients overexpressed WT1 compared to normal bone marrow. The existence of FLT3-ITD, NPM1 mutation and the absence of CEBPA biallelic mutation were significantly related to higher WT1 expression. The cutoff value for WT1 was determined by performing receiver operating characteristic curve analysis in regard to complete remission (CR) achievement and was used to categorize patients into low-expression (WT1-L) and high-expression (WT1-H) groups. In the entire cohort, WT1-H was significantly associated with a lower 1-course and 2-course CR rate (P < 0.0010 and P = 0.0060) but was not related to relapse-free survival (RFS). Multivariate analysis showed that WT1-H was an independent adverse prognostic factor for both 1-course and 2-course CR achievement. Subgroup analysis was further performed. WT1-H had a significant adverse impact on CR achievement within intermediate-cytogenetic risk, high-cytogenetic risk, ELN-defined-intermediate-risk, normal karyotype, KMT2A rearrangement, FAB-M2, FAB-M5 and NPM1 mutation (+) subgroups, whereas it had no impact within ELN-defined-low-risk, ELN-defined-high-risk, FAB-M4, FLT3-ITD mutation (+) and CEBPA biallelic mutation (+) subgroups. Moreover, WT1-H patients had a significantly lower RFS rate than WT1-L patients within both FAB-M5 and KMT2A rearrangement subgroups (P = 0.010 and 0.028), whereas WT1 had no impact on RFS within other subgroups mentioned above (all P > 0.05). Therefore, high WT1 expression at diagnosis independently predicted induction chemotherapy failure in AML patients with non-favorable cytogenetic risk, and it was related to relapse just within FAB-M5 and KMT2A rearrangement subgroups.

P790: THE EXPRESSION LEVEL OF WT1 MRNA IN PATIENTS WITH IDIOPATHIC CYTOPENIA OF UNDETERMINED SIGNIFICANCE (ICUS) PROGRESSING TO MYELODYSPLASTIC SYNDROME (MDS)

Background: Idiopathic cytopenia of undetermined significance (ICUS) is defined as patients with one or more unexplained cytopenia who do not meet diagnostic criteria for myelodysplastic syndrome (MDS) or another hematologic disorder. In the clinical course of ICUS, some patients eventually have typical MDS changes. Until now, there is no effective method to predict the development of the disease. Recent studies have shown that the expression level of WT1 mRNA in peripheral blood has important clinical significance in the diagnosis, occurrence, development and prognosis of MDS. At the same time, some studies have proved that there is a significant difference in the expression level of WT1 mRNA between MDS and hemocytopenia caused by other reasons. Aims: In order to clarify the clinical significance of WT1 mRNA expression in the progression of ICUS to MDS. Methods: Between April 2017 and January 2020, untreated adult patients with ICUS or lower-risk MDS were enrolled in the study. All patients detected the WT1 mRNA expression of bone marrow and peripheral blood at diagnosis. Then patients with ICUS were followed up. Every three months, the patients with ICUS detected the WT1 mRNA expression of peripheral blood, until the patient progressed to MDS. The results of WT1 transcript levels are depicted as WT1 (copies/mL)/c-ABL (copies/mL) as a percentage. Results: Total of 80 patients with ICUS and 40 patients with lower-risk MDS were enrolled. At diagnosis, the median expression level of WT1 mRNA in MDS group was 5.99 (0.08-79.27), and that in ICUS group was 0.50 (0.16-22.48). There was significant difference between them (P = 0.000). Then 80 patients with ICUS were followed up to January 2022. The median follow-up time was 30 months (11-54 months). 8 patients developed MDS, the progression rate was 10%, and the median progression time was 6.5 months (2-19 months). The median expression level of WT1 mRNA at the initial diagnosis in ICUS group without progressing to MDS was 0.44 (0.16-7.58), and that in progressing to MDS group was 9.44 (0.26-22.48). There was significant difference between the two groups (P = 0.031). The median expression level of WT1 mRNA was 19.185 (0.73-42.30) when MDS was diagnosed. The median maximum expression level of WT1 mRNA in peripheral blood of ICUS group without MDS was 0.98 (0.22-7.58). There was significant difference between the two groups (P = 0.007). Summary/Conclusion: The study clarified the WT1 mRNA expression status of ICUS patients. There were significant differences in WT1 mRNA expression level between patients with ICUS and lower-risk MDS, and the WT1 mRNA expression level of patients with lower-risk MDS was relatively high. The study confirmed that patients with high WT1 mRNA expression level in the patients with ICUS (WT1 mRNA expression level > 9.44) are very likely to progressing to MDS; Dynamic monitoring of WT1 mRNA expression in peripheral blood is an effective method for following-up after ICUS diagnosis. The progressive increase of WT1 mRNA expression is one of the indicators for researchers to judge the progress of ICUS to MDS.

WT1 overexpression predicted good outcomes in adult B-cell acute lymphoblastic leukemia patients receiving chemotherapy

ABSTRACT Objectives: The prognostic role of WT1 in acute lymphoblastic leukemia (ALL) is still controversial. No study has focused on the prognostic role of WT1 expression in adult B-ALL patients receiving chemotherapy only. Methods: Using TaqMan-based real time quantitative PCR (RQ-PCR), we detected the WT1 transcript levels of 162 de-novo adult B-ALL patients at the time of diagnosis and analysed their clinical features. Results: WT1 overexpression was defined as a transcript level higher than 0.50%, which is the upper limit in normal bone marrow. WT1 overexpression was identified in 66.0% of the patients and was an independent positive prognostic factor for CIR, RFS and OS in patients who received chemotherapy only (CIR: HR = 0.236 [95% confidence interval 0.094–0.592]; P = 0.002; RFS: HR = 0.223 [0.092–0.543]; P = 0.001; OS: HR = 0.409 [0.214–0.783]; P = 0.007) and in patients who did not have BCR-ABL fusion or KMT2A rearrangements (CIR: HR = 0.431 [0.201–0.921]; P = 0.030; RFS: HR = 0.449 [0.224–0.899]; P = 0.024; OS: HR = 0.521 [0.278–0.977]; P = 0.042). However, WT1 overexpression had no prognostic value in patients who received allogenic hematopoietic stem cell transplantation (allo-HSCT). Furthermore, allo-HSCT could improve the prognosis of patients with low WT1 expression. Conclusion: Therefore, testing for WT1 expression at the time of diagnosis may predict outcomes in adult B-ALL patients who receive only chemotherapy and who do not have the BCR-ABL fusion gene or KMT2A rearrangements. Allo-HSCT may improve the prognosis of patients with low WT1 transcript levels.

Subgroup Analysis Can Optimize the Relapse-Prediction Cutoff Value for WT1 Expression After Allogeneic Hematologic Stem Cell Transplantation in Acute Myeloid Leukemia

High WT1 expression after allogeneic hematologic stem cell transplantation (allo-HSCT) can strongly predict relapse in acute myeloid leukemia (AML). However, the cutoff values obtained have been inconsistent. Precise cutoff values may be optimized through subtype analysis; the RUNX1-RUNX1T1 fusion transcript provides an ideal reference. RUNX1-RUNX1T1 and WT1 transcript levels were simultaneously measured in 1299 bone marrow samples serially collected from 176 t(8;21) AML patients after allo-HSCT. The upper limit of the normal bone marrow WT1 level was 0.6%, which we previously reported to be the cutoff value for significant relapse prediction in AML as a whole. WT1 cutoff values of 0.6%, 1.2%, and 1.8% significantly differentiated patients in relapse after allo-HSCT. Nonetheless, patients with WT1 levels of 0.6% to 1.2% and those with levels of >1.2% and 1.8% after HSCT had rates of cumulative incidence of relapse similar to those with a continuous WT1 level of ≤0.6%, and both were significantly lower than that in patients with a WT1 level of >1.8%. WT1 expression was significantly related to RUNX1-RUNX1T1 transcript levels at WT1 levels of >1.8% but not at levels of 0.6% to 1.2% or >1.2% to 1.8%. Therefore, subgroup analysis can optimize the relapse-prediction cutoff value of WT1 expression. A cutoff level of 1.8% more accurately differentiates t(8;21) AML patients in relapse after allo-HSCT than does a cutoff level of 0.6%.

PF185 WT1 OVEREXPRESSION PREDICTED GOOD OUTCOMES IN ADULT B‐CELL ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS RECEIVING CHEMOTHERAPY

Background:B‐cell acute lymphoblastic leukemia (B‐ALL) is a clinically and biologically heterogeneous disease that is characterized by the clonal expansion of developmentally arrested B‐cell precursors. Despite significant improvements in the management of this disease in children, the prognosis of adult patients remains poor. Identifying new prognostic markers is necessary to help select the best therapeutic schedules. Wilms’ tumor gene 1 (WT1) is a transcription factor that is overexpressed in diverse neoplasms, including acute leukemia. The prognostic role of WT1 in ALL is still controversial. No study has focused on the prognostic role of WT1 expression in adult B‐ALL patients receiving chemotherapy only.Aims:To investigate the prognostic value of WT1 expression in adult patients with B‐ALL.Methods:The WT1 transcript levels of 162 de‐novo adult B‐ALL patients were determined by TaqMan‐based real time quantitative PCR (RQ‐PCR) at the time of diagnosis. Eighty‐eight patients received allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Survival functions were estimated by the Kaplan‐Meier method and compared by the log‐rank test. Cumulative incidences were estimated for relapse to accommodate competing risks. A Cox proportional hazard regression model was used to determine the associations between WT1 transcript levels and CIR, RFS and OS.Results:WT1 overexpression was defined as a transcript level higher than 0.50%, which is the upper limit in normal bone marrow. WT1 overexpression was identified in 66.0% of the patients. WT1 overexpression was an independent positive prognostic factor for the cumulative incidence of relapse (CIR), relapse‐free survival (RFS) and overall survival (OS) in patients who received chemotherapy only (CIR: HR = 0.236 [95% confidence interval 0.094–0.592]; P = 0.002; RFS: HR = 0.223 [0.092–0.543]; P = 0.001; OS: HR = 0.409 [0.214–0.783]; P = 0.007) and in patients who did not have BCR‐ABL fusion or KMT2A rearrangements (CIR: HR = 0.431 [0.201–0.921]; P = 0.030; RFS: HR = 0.449 [0.224–0.899]; P = 0.024; OS: HR = 0.521 [0.278–0.977]; P = 0.042). However, WT1 overexpression had no prognostic value in patients who received allogenic hematopoietic stem cell transplantation (allo‐HSCT). Furthermore, allo‐HSCT could improve the prognosis of patients with low WT1 expression.Summary/Conclusion:Therefore, testing for WT1 expression at the time of diagnosis may predict outcomes in adult B‐ALL patients who receive only chemotherapy and who do not have the BCR‐ABL fusion gene or KMT2A rearrangements. Allo‐HSCT may improve the prognosis of patients with low WT1 transcript levels.

WT1 gene is overexpressed in myeloproliferative neoplasms, especially in myelofibrosis

Classical Philadelphia-negative myeloproliferative neoplasms include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). They are characterized by the presence of driver mutations of JAK2, CALR or MPL genes. Overexpression of WT1 is used as a marker of minimal residual disease in acute myeloid leukemia, especially after allogeneic stem cell transplantation (SCT). We investigated WT1 expression at diagnosis in 152 MPN patients and showed that the WT1 transcript was overexpressed in PMFs and PVs compared to controls. In particular, WT1 transcript levels were higher in PMF than in ET and PV. WT1 transcript levels were significantly increased during myelofibrotic transformation of ET or PV. Using multivariate linear regression, high WT1 transcript levels in PMF were associated with age over 65, splenomegaly and thrombocytopenia. The ROC curve analysis showed that a level of WT1 transcript >10 WT1 copies/104ABL1 enabled the diagnosis of PMF with a specificity of 95.8% (PMF vs ET; ROC AUC = 0.91). In myelofibrosis, studying follow-ups of WT1 transcript showed that this marker is of interest after allogeneic SCT. These results demonstrate that WT1 overexpression is a simple marker of myelofibrosis in MPN and could be used during patient follow-up.

Early Reduction of WT1 Transcript Level During Induction Chemotherapy Predicts for Longer Relapse-Free and Overall Survival in De Novo Intermediate Risk Acute Myeloid Leukemia

Early Reduction of WT1 Transcript Level During Induction Chemotherapy Predicts for Longer Relapse-Free and Overall Survival in De Novo Intermediate Risk Acute Myeloid Leukemia

High EVI1 Expression Predicts Poor Outcomes in Adult Acute Myeloid Leukemia Patients with Intermediate Cytogenetic Risk Receiving Chemotherapy.

BackgroundAcute myeloid leukemia with intermediate cytogenetic risk (ICR-AML) needs to be stratified. The abnormal gene expression might be prognostic, and its cutoff value for patient grouping is pivotal.Material/MethodsEcotropic viral integration site 1 (EVI1) transcripts were assessed in 191 adult ICR-AML patients at diagnosis who received chemotherapy only. MLL-PTD, WT1 transcript levels, FLT3-ITD, and NPM1 mutations were simultaneously evaluated, and 27 normal bone marrow samples were tested to define normal threshold.ResultsThe normal upper limit of EVI1 transcript levels was 8.0%. Receiver operating characteristic curve analysis showed that 1.0% (a 0.9-log reduction from the normal limit) was the EVI1 optimal cutoff value for significantly differentiating relapse (P=0.049). A total of 23 patients (12%) had EVI1 levels ≥1.0%. EVI1 ≥1.0% had no effect on CR achievement, whereas it was significantly associated with lower 2-year relapse-free survival (RFS), disease-free survival (DFS), and overall survival (OS) rates in the entire cohort (P=0.0003, 0.0017, and 0.0009, respectively), patients with normal karyotypes (P=0.0032, 0.0047, and 0.0007, respectively), and FLT3-ITD (−) patients (all P<0.0001). Multivariate analysis showed that EVI1 ≥1.0% was an independent adverse prognostic factor for RFS, DFS, and OS in the entire cohort. In addition, patients with EVI1 transcript levels between 1.0% and 8.0% had 2-year RFS rates similar to those with EVI1 ≥8.0%, and they both had significantly lower RFS rates than those with EVI1 <1.0% (P=0.0005 and 0.027).ConclusionsHigh EVI1 expression predicts poor outcome in ICR-AML patients receiving chemotherapy. The optimal cutoff value for patient stratification is different from the normal limit.

Wilms’ tumor gene 1 expression can predict sudden disease progression to blast crisis in patients with chronic myeloid leukemia receiving imatinib therapy

Background The Philadelphia chromosome is the hallmark of chronic myeloid leukemia (CML). As the disease progresses to accelerated phase and blast crisis (BC), clonal evolution may occur with the emergence of additional chromosomal abnormalities. Wilms’ tumor 1 gene (WT1) plays an important role in leukemogenesis, and its expression could represent a useful molecular marker of hematological malignancies. Aim The aim of this study was to evaluate WT1 expression and compare its kinetics with that of BCR-ABL1 expression in peripheral blood of patients with CML, to explore the utility of WT1 as an alternative marker for prediction of early disease progression and sudden transformation to BC. Patients and Methods A total of 49 newly diagnosed patients with CML, and 20 normal individuals as controls were enrolled in this study. WT1 and BCR-ABL1 expression was evaluated by quantitative real-time PCR. Results There was significant correlation between the expression of WT1 and BCR-ABL1 only at diagnosis and after 3 and 6 months of therapy. At 12 months of follow-up, an increase in %WT1 associated with low BCR-ABL1 was noticed in two of cases. One case suddenly progressed to BC at 18 months, and the other case showed emergence of clonal chromosome abnormality (+8) and progressed to accelerated phase. Conclusion Our results suggested that serial assessment of WT1 transcript level in patients with CML may be a useful marker for predicting early and sudden disease progression into advanced stages.

WT1 expression is increased in primary fibroblasts derived from Dupuytren's disease tissues.

Dupuytren's disease (DD) is a fibroproliferative and contractile fibrosis of the palmar fascia that, like all other heritable fibroses, is currently incurable. While DD is invariably benign, it exhibits some molecular similarities to malignant tumours, including increased levels of ß-catenin, onco-fetal fibronectin, periostin and insulin-like growth factor (IGF)-II. To gain additional insights into the pathogenesis of DD, we have assessed the expression of WT1, encoding Wilm's tumour 1, an established tumour biomarker that is syntenic with IGF2, the gene encoding IGF-II in humans. We found that WT1 expression is robustly and consistently up regulated in primary fibroblasts derived from the fibrotic palmar fascia of patients with DD (DD cells), whereas syngeneic fibroblasts derived from the macroscopically unaffected palmar fascia in these patients and allogeneic fibroblasts derived from normal palmar fascia exhibited very low or undetectable WT1 transcript levels. WT1 immunoreactivity was evident in a subset of cells in the fibrotic palmar fascia of patients with DD, but not in macroscopically unaffected palmar fascia. These findings identify WT1 expression as a novel biomarker of fibrotic palmar fascia and are consistent with the hypothesis that the pathogeneses of DD and malignant tumours have molecular similarities.

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.

Vaccination with WT1 mRNA-Electroporated Dendritic Cells: Report of Clinical Outcome in 66 Cancer Patients

Tumor recurrence and lack of tumor control are major problems in cancer treatment. In order to control malignant disease, we performed phase I/II dendritic cell (DC) vaccination studies in an adjuvant setting in 30 patients with acute myeloid leukemia (AML) and in 36 patients with solid tumors. Following chemotherapy, the patients underwent leukapheresis; CD14+ monocytes were isolated, cultured into clinical-grade mature DC, electroporated with mRNA encoding the Wilms’ tumor protein WT1 and injected intradermally (Van Tendeloo et al. PNAS 2010;107:13824-9). No major DC-related systemic toxicity was observed.DC vaccination as a post-remission treatment was evaluated in 30 AML patients following chemotherapy; 27 patients were in complete remission (CR) but at very high risk of relapse and 3 in partial remission (PR). WT1 mRNA levels in blood and marrow were followed as a measure of residual disease. Clinical and molecular response, as determined by normalization of WT1 transcript levels in blood and/or marrow, occurred in 8/23 patients who had increased levels of that marker at the start of DC vaccination. Of these 8 responding patients, 5 are still in complete and molecular remission, all of them now more than 5 years after diagnosis and most probably cured; 1 of those 5 patients was in PR following chemotherapy and was brought into complete and molecular remission by the DC vaccination only. There was a possible effect of DC vaccination in 6 additional patients: 3 with stable disease, some of it late; and 3 at high risk of relapse but without increased WT1 mRNA levels before DC vaccination: 1 patient with erythroleukemia and 2 patients with initial leucocytosis >20,000/µL have remained in CR, now at respectively 57, 52 and 45 months post-diagnosis. Overall 8/30 patients have not relapsed yet, with a median follow-up from diagnosis and start of DC vaccination of respectively 70 months (range 45 - 92 months) and 63 months (range 39-90 months). Delayed type hypersensitivity (DTH) testing showed immunoreactivity to the DC vaccine in all patients tested. WT1 epitope tetramer+ CD8+ T-cells were evaluated in 13 HLA-A2+ patients: an increase following DC vaccination in tetramer+ T-cells for at least 2/4 epitopes tested was only observed in patients with long-standing CR.Ten patients with unresectable, epithelial-type malignant pleural mesothelioma and non-progressive disease after platinum/pemetrexed-based chemotherapy received DC vaccination. Evaluation of response according to RECIST criteria showed 7 patients with stable disease and 3 with progressive disease. DTH testing showed vaccine-elicited immunity in 9/10 patients. Median overall survival (OS) from start of chemotherapy was 32 months; this compares with an OS of 22 months reported in the literature for a similar subgroup of patients treated with chemotherapy only (Hillerdal et al. J Thorac Oncol 2008).Twenty-six patients with other advanced and pre-treated cancers also underwent DC vaccination (13 with breast cancer (12 with metastatic disease), 5 with glioblastoma multiforme (GBM), 1 with brain stem astrocytoma and 1 each with metastatic melanoma, Ewing sarcoma, esophageal, colon, pancreatic, renal cell and ovarian cancer). Significant DTH responses were recorded in all patients. At a median follow-up from start of DC vaccination of 23.3 months, 8/26 patients (31%) are still alive and median OS was 23.5 months. Evaluation of response showed 3 patients with PR (1 brain stem astrocytoma, 1 GBM and 1 breast cancer) and 1 patient with CR (1 GBM). In the breast cancer patient subgroup, 5/13 patients are still alive (38%) and median OS was 33.5 months after start of DC vaccination; this compares with OS data from the literature of 21.7 months after diagnosis (Kiely et al. J Clin Oncol 2011;29:456-63). In the GBM (n=5) patient subgroup, 1/5 patients is alive in CR 26 months and median OS was 14.7 months after start of DC vaccination; this compares with OS data from the literature of 14.6 months after diagnosis (Stupp et al. N Engl J Med 2005;352:987-96).In conclusion, WT1-targeted DC vaccination is feasible, safe and immunogenic in cancer patients. In AML, metastatic breast cancer and malignant glioma, there is evidence of objective response. In addition, OS data in solid tumors compare favorably with the best data reported so far for similar cohorts of patients, suggesting that adjuvant WT1/DC-based immunotherapy provides a clinical benefit to these patients. DisclosuresOff Label Use: Dendritic cells as immunotherapy.

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.

Wilms' Tumor Gene 1 Transcript Levels in Leukapheresis of Peripheral Blood Hematopoietic Cells Predict Relapse Risk in Patients Autografted for Acute Myeloid Leukemia

Autologous hematopoietic stem cell transplantation (ASCT) is a curative option alternative to allogeneic transplantation for patients with acute myeloid leukemia (AML). Relapse after ASCT can be due to contamination with leukemic blasts of autologous peripheral blood stem cells (PBSCs) collected by leukapheresis (LK). Identification and quantification of a minimal residual disease (MRD) marker in PBSCs could be relevant in determining the relapse risk after ASCT. High levels of the WT1 gene transcript in bone marrow of AML patients after treatment completion predict disease relapse. We evaluated WT1 transcript levels in autologous PBSC from LK used for ASCT in 30 consecutive AML patients in complete remission (CR) and established a correlation with clinical outcome. At diagnosis, all patients had WT1 overexpression. All patients were in morphological and genetic CR at the time of PBSC collection and before ASCT. Real-time quantitative PCR of WT1 was performed in samples of each LK, using TaqMan technology on RNA from mononucleated cells. The median WT1 transcript level in the PBSC graft (WT1-LK) of patients who relapsed was significantly higher than of those who did not relapse after transplantation (P <.0001). We defined a cut-off level of 80 WT1-LK copies/ABL 10e4 copies to discriminate between positive and negative PBSC grafts. The cut-off level was strongly associated with disease recurrence, DFS and OS. Our study represents the largest series of patients evaluating WT1 as a marker of MRD in PBSC LK products using a completely standardized real-time WT1-reverse transcriptase-PCR based assay. These data, if confirmed by prospective study, will help to determine an individual patient's adapted postremission allocation strategy.

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.

Low WT1 Expression At Diagnosis Is a Strong Predictor On Poor Outcome In Patients With t(8;21) Acute Myeloid Leukemia

IntroductionAcute myeloid leukemia (AML) with t(8;21) is a heterogeneous disease. The dynamic patterns of AML1-ETO transcript levels during treatment and clinical outcome of patients vary greatly. Our AML05 trial revealed that minimal residual disease (MRD)status and treatment strategy were independent risk factors for outcome and that risk stratification treatment directed by MRD may improve the outcome of t(8;21) AML in CR1 (Blood 2013; 121:4056). As for pretreatment parameters, c-KIT mutation is a well-established adverse predictor on survival. However, a subset of t (8;21) AML patients without c-KIT mutation still showed poor clinical outcome. The prognostic value of WT1 transcript levels at diagnosis in AML has been investigated and the results were controversial. We wondered if WT1 expression associated with outcome in t (8;21) AML patients. MethodsA total of 101 patients were included. They all were eligible cases who enrolled into AML05 trial from June 2005 to December 2012, and had available bone marrow samples at diagnosis. After 1 or 2 induction therapy and 2 cycles of intermediate-dose cytarabine-based consolidation therapy, fifty-seven patients continued cytarabine-based consolidation chemotherapy or received autologous-hematopoietic stem-cell transplantation (auto-SCT) and were defined as CT group, the remaining 44 patients received allogeneic SCT (allo-SCT) and defined as SCT group.WT1 and ABL transcript were tested by real time quantitative PCR, and WT1 transcript levels were calculated as WT1copies/ABL copies in percentage. The upper limit of normal bone marrows (NBMs) was 0.5%. c-KIT mutations in exon 8 and 17 were screened by direct sequencing. ResultsThe median follow-up time was 25 (6-93) months for 73 alive patients. The cumulative incidence of relapse (CIR) at 3 years was 35.3%. The 3-year disease free survival (DFS) and overall survival (OS) rates were 57.2% and 62.8%, respectively. The median WT1 transcript levelsssof all patients were 9.1% (0.02%-99.3%). c-KIT mutation was detected in 31 patients. Receiver operating characteristics (ROC) curves revealed that WT1 transcript levels of 5.0% (1-log increase compared to the upper limit of NBM) were the best cutoff values to discriminate patients with different outcome. WT1 transcript levels of ≤5.0% were significantly associated with c-KIT mutation (23/42 vs 8/59, P<0.001), but didn't related to other pretreatment parameters (all P>0.05). In CT group, patients with WT1≤5% (n=19) had significantly higher CIR rate at 3-year, lower 3-year DFS and OS rate than those with WT1>5% (n=38), respectively (89.5% vs 27.9%, P<0.0001; 10.5% vs 66.1%, P<0.0001; 28.5% vs 66.5%, P=0.0062). Furthermore, patients with WT1≤5% and c-KIT mutation (-) had similar CIR, DFS and OS rate to both patients with WT1≤5% and c-KIT mutation (+) and patients with WT1>5% and c-KIT mutation (+) (all P >0.05, Figure 1). Therefore, they were merged into one group (n=24). Thus, patients with WT1≤5% and/or KIT mutation (+) had significantly higher CIR rate at 3-year, lower 3-year DFS and OS rate than those with WT1>5% and c-KIT mutation (-) (n=33) in CT group, respectively (89.4% vs 27.4%,P<0.0001; 10.6% vs 72.6%, P<0.0001; 26.1% vs 72.2%, P=0.0013). For patients ith WT1≤5%, allo-SCT significantly lowered CIR rate at 3-year, improved 3-year DFS rate and tended to improve OS rate compared to chemotherapy/auto-SCT by landmark analysis, respectively (11.5% vs 88.2%, P<0.0001; 65.4% vs 11.8%, P=0.0001; 60.6% vs 32.6%, P=0.10. Figure 2). Multivariate analysis revealed that WT1 transcript levels (≤5% vs >5%) and treatment (chemotherapy/auto SCT vs allo-SCT) instead of other pretreatment parameters were independent prognostic factors for relapse (hazard ratio (HR) 0.20, 95% CI 0.093¨C0.44; 0.096, 95% CI 0.033¨C0.28. all P<0.0001) , DFS (HR 0.22, 95% CI 0.11¨C0.44; 0.23, 95% CI 0.11¨C0.49. all P<0.0001) and OS (HR 0.30, 95% CI 0.14¨C0.66, P=0.003; 0.37, 95% CI0.16¨C0.82, P=0.014).▪▪ ConclusionLess than 1-log increase of WT1 transcript levels at diagnosis is a strong predictor on poor outcome in patients with t (8;21) AML, and allo-SCT could significantly improve outcome of such patients. Grant SupportBejing Municipal Science & Technology Commission(Z111107067311070) and Nature Science Foundation of China (81170483). Disclosures:No relevant conflicts of interest to declare.

Quantitative Monitoring Of Wilms' Tumor 1 Expression In Predicting Relapse After Allogeneic Hematopoietic Stem Cell Transplantation In Patients With Acute Leukemia and Myelodysplastic Syndrome

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (HCT) for leukemia and myelodysplastic syndrome (MDS). In order to improve the outcome of leukemia patients after allogeneic HCT, it is imperative to identify reliable markers to predict impending relapse. Wilms' tumor antigen (WT1) is overexpressed in the majority of leukemia and MDS patients and is being considered as a possible universal diagnostic marker for minimal residual disease (MRD), especially since no chromosomal translocation has nearly the frequency of association as WT1 does with leukemia.In this study we prospectively evaluated the prognostic value of MRD monitoring by qRT-PCR for WT1 transcripts. WT1 transcript levels in peripheral blood mononuclear cells (PBMC) were measured utilizing SYBR-Green qRT-PCR on the ABI7300 instrument (Applied Biosystems, Carlsbad, CA) and results were expressed as a ratio of WT1/c-ABL transcript copies normalized by 104 (WT1 ratio: WT1/c-ABLx104). PBMC samples were obtained monthly for 6 months post-HCT, then on alternating monthly schedule until 3 years post-HCT. Patients >18y.o. with confirmed diagnosis of MDS with <20% blasts, AML/ALL in 1st or 2ndCR, and CML in chronic phase undergoing HCT were eligible for the study.A total of 83 patients (median age: 54, range: 19-74) with AML (n=39), ALL (n=24), MDS (n=17), or CML (n=3) received allogeneic HCT after fully ablative (n=39) or reduced-intensity (n=44) conditioning. Donor sources were matched related (n=33), unrelated (n=50), or umbilical cord blood (n=2). Fifty-one patients were considered low-risk (AML/ALL in CR1, CML in 1stchronic phase, or MDS-RA/RARS subtypes) while the remaining 32 patients were considered high risk.Sixteen of 83 patients relapsed with a median time of 238 days post-HCT (range: 76-747). The minimum WT1 ratio that gave specificity of 100% in predicting relapse was 50 (95% binomial exact CI: 92.5-100%), as none of the non-relapsed patients crossed this level. Of 16 patients who relapsed, 12 crossed the WT1 ratio of 50, providing a sensitivity of 75% (95% binomial exact CI: 48- 93%). The positive predictive value (PPV) and the negative predictive value (NPV) performance parameters for the WT1 ratio of 50 were 100% and 94.4%, respectively (Table 1). There was an average number of 63 days (SD=29.3) from crossing the WT1 ratio of 50 to hematologic relapse for the 12 relapsed patients. Since PBMC samples from healthy donors consistently demonstrated a WT1 ratio <10, we also examined different WT1 cutoff ratios (10 to 50) for their performance characteristics (Table 1). Compared with the WT1 ratio of 50, a cutoff ratio at 20 resulted in an increased sensitivity (87.5%) for relapse prediction and days to relapse (78 days), while the specificity and PPV decreased to 85% and 58.3%, respectively. The performance of the cutoff ratio of 20 was improved on PPV (69%) and days to relapse (85 days) in a subgroup of patients with high-risk disease while maintaining good sensitivity and specificity above 80%. Univariate analysis showed WT1 ratios (as a continuous variable), crossing the WT1 ratio of 20 (as a time-dependent variable), high risk disease, and donor age were significantly associated with relapse. In multivariate analysis, crossing the WT1 ratio of 20 remained the only significant factor predicting relapse (HR 56.9 [18-189], p<0.0001).In summary, our data demonstrate that the quantitative measurement of WT1 transcripts is a reliable marker to assess MRD post-HCT for leukemia patients, and its real-time prospective monitoring provides a 2-3 month window of opportunity to introduce medical/immunologic interventions prior to overt hematologic relapse.Table 2WT1 ratio Specificity Sensitivity Positive Predictive Value Negative Predictive Value Days to Relapse (SD)50100%75%100%94.4%63 days (29)4095.5%75%80%94.1%66.9 days (29)3094%75%75%94%71.5 days (30)2085%87.5%58.3%96.6%78 days (28)1056.7%93.8%34.1%97.4%107.5 days (58) Disclosures:No relevant conflicts of interest to declare.

Prevention Of Relapse In Acute Myeloid Leukemia By Dendritic Cell Vaccination: Report on a Phase II Study With 29 Patients

Relapse remains a major problem in acute myeloid leukemia (AML) and has an important impact on survival, especially in the majority of older patients who cannot undergo allogeneic hematopoietic stem cell transplantation. In a phase II study, we investigated the impact on relapse of dendritic cell (DC) vaccination as a post-remission treatment in 29 AML patients; 26 patients were in complete remission and 3 in partial remission following chemotherapy. DC were derived from blood monocytes, electroporated with mRNA encoding the Wilms' tumor 1 protein (WT1) and administered via the intradermal route. Three different WT1 constructs were used to generate mRNA by in vitro transcription: 'construct 1' encoding full-length WT1, 'construct 2' with a Sig-DC-LAMP major histocompatibility complex (MHC) class II-skewing signal and a deletion of the WT1 nuclear localization signal and 'construct 3', a codon-optimized version of construct 2. WT1 mRNA levels in blood and marrow were followed as a measure of minimal residual disease.DC electroporated with mRNA derived from constructs 1, 2 and 3 were used to vaccinate respectively 13, 6 and 10 AML patients at very high risk of relapse. In those 3 groups, clinical and molecular response, as determined by normalization of WT1 transcript levels in blood and/or marrow, occurred in respectively 7/13, 1/4 and 0/6 patients who had increased levels of that marker at the start of DC vaccination. Of these 8 responding patients, 3 relapsed and died; the other 5 are still in complete remission, 4 of them now more than 5 years after diagnosis and most probably cured; 1 of those 4 patients was in partial remission following chemotherapy and was brought into complete and molecular remission by the DC vaccination only. All 15 patients who did not normalize WT1 mRNA levels following DC vaccination relapsed and/or died. There was a possible effect of DC vaccination in 6 additional patients: 3 with stable disease, some of it late; and 3 at high risk of relapse but without increased WT1 mRNA levels before DC vaccination: 1 patient with erythroleukemia and 2 patients with initial leucocytosis > 20,000/µL have remained in complete remission, now at respectively 46, 42 and 35 months post-diagnosis. Overall 8/29 patients have not relapsed yet, with a median follow-up from diagnosis and start of DC vaccination of respectively 60 months (range 35 - 84 months) and 53 months (range 27-78 months).Delayed type hypersensitivity (DTH) testing showed immunoreactivity to the DC vaccine components in all patients tested. There was also evidence of natural killer (NK) cell activation following DC vaccination. WT1 epitope tetramer+ CD8+ T-cells were evaluated in 13 HLA-A2+ patients: an increase following DC vaccination in tetramer+ T-cells for at least 2/4 epitopes tested was only observed in patients with long-standing complete remission.In conclusion, DC vaccination had a demonstrable antileukemic effect in 8/29 AML patients and a possible effect in another 6. Contrary to expectations, the WT1 constructs with MHC class II-skewing signal did not seem to have superior activity over the full-length WT1 construct without that signal. Vaccination with WT1 mRNA-transfected DC is emerging as a non-toxic strategy to prevent or delay relapse in AML. Disclosures:Off Label Use: Dendritic cell vaccination.

Prognostic significance of WT1 expression at diagnosis and end of induction in Egyptian adult acute myeloid leukemia patients

BackgroundWilms' tumor (WT1) gene overexpression has been reported in the majority of acute myeloid leukemia (AML) patients at diagnosis and has been evaluated as prognostic and minimal residual disease (MRD) marker.Patients and methodsWT1 overexpression was evaluated in 68 adult AML patients at diagnosis and at the end of induction using quantitative real-time polymerase chain reaction (PCR).ResultsNo significant associations were encountered between WT1 overexpression at diagnosis and other prognostic factors. Complete remission (CR) was achieved in 74% of the patients with WT1 overexpresssion compared to 80% of patients with normal levels (P = 0.5). No significant associations were encountered between WT1 overexpression at diagnosis and disease-free survival (DFS) or overall survival (OS) (P = 0.6 and 0.3, respectively). At the end of induction, the median duration of DFS in patients achieving ≥2 log reduction was not reached compared to only 5 months (range: 2.1–7.9 months) in those attaining <2 log reduction (P = 0.2). The median duration of OS in patients achieving ≥2 log reduction was 13 months (range: 0–33.3 months) compared to 7.5 months (5.4–9.6 months) in those attaining <2 log reduction (P = 0.2). The survival at 1 year in patients achieving ≥2 log was double the group with <2 log reduction (67% compared to 33%).ConclusionOur results, although not reaching the level of significance, probably due to the small sample size, still suggest that the early assessment of WT1 transcript level at the end of induction in patients in CR may have a prognostic significance on clinical outcome and may thus be a useful marker for risk stratification especially in patients lacking disease-specific marker.