High WT1 Expression Research Articles

The prognostic effect of high diagnostic WT1 gene expression in pediatric AML depends on WT1 SNP rs16754 status: Report from the Children's Oncology Group

WT1 is aberrantly over-expressed in most cases of AML. We recently demonstrated that WT1 SNP rs16754 correlates with favorable outcome and high diagnostic WT1 expression in childhood AML. We examined the clinical correlates of diagnostic WT1 expression within a contemporary COG trial and determined whether its prognostic impact differs between SNP+ and SNP- patients. WT1 mRNA expression was measured via qRT-PCR in diagnostic specimens obtained from 225 patients enrolled on COG-AAML03P1. Direct sequencing of WT1 exon 7 was performed to determine SNP rs16754 genotype. WT1 expression was correlated with disease characteristics, SNP status, and outcome. Patients were categorized into four groups (quartiles: Q1 through Q4) based on diagnostic WT1 expression for analysis. FLT3/ITD (P = 0.017) and WT1 mutations (P < 0.001) both occurred more frequently in patients with the highest WT1 expression. SNP rs16754 frequency did not vary significantly among the quartiles. When all patients were considered, survival outcomes were similar between quartiles. However, when only SNP- patients (n = 150) were analyzed, those with highest WT1 expression (Q4) had the poorest OS (51% vs. 72% for Q1-Q3, P = 0.006) and EFS (35% vs. 54% for Q1-Q3, P = 0.031). Among SNP+ patients (n = 75), survival did not vary significantly between WT1 expression quartiles. Although WT1 expression was not prognostic when all patients were considered together, stratifying patients by SNP rs16754 genotype revealed significant differences in outcome. In SNP- patients, high WT1 expression predicted decreased survival in univariate, but not multivariate, analysis, due to a preponderance of high-risk cyto/molecular abnormalities in the highest expression quartile.

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.

Prognostic Implications of WT1 and BAALC in Adult Patients with Core-Binding Factor Positive or Normal Karyotype AML: Consecutive Analyses From Diagnosis to Hematopoietic Stem Cell Transplantation

AbstractAbstract 1393 Background:Numerous molecular prognostic markers, in addition to specific chromosomal aberrations, have been identified to confer disease pathogenesis and survival outcomes in patients with adult acute myeloid leukemia (AML). Many efforts have been made to identify genetic mutations (e.g., c-kit, FLT3-ITD/TKD, NPM1, WT1, MLL-PTD) and modulated gene expression levels (e.g., BAALC and WT1) that can be used to predict outcomes in patients with cytogenetically normal AML (CN-AML). However, few data associated with core-binding factor AML (CBF-AML) and BAALC or WT1 expressions have been reported, and a consecutive analysis of BAALC and WT1 from diagnosis to hematopoietic stem cell transplantation (HSCT) has not yet been reported. We analyzed serial BAALC and WT1 data in patients with CN-AML and CBF-AML using the real-time quantitative polymerase chain reaction method. Methods:In this single center retrospective study, 1008 newly diagnosed patients with AML (age, 15–85 years; median 45.2 years) with variable karyotypes were initially enrolled from January 2005 to December 2010. Ultimately, we analyzed 335 patients with CN-AML and 156 with CBF-AML. Patients that underwent conservative treatment were excluded. Among 491 patients, we identified FLT3-ITD results in 316 patients (positive in 56 and negative in 260) and NPM1 results in 202 patients (positive in 50 for CN-AML only), and obtained BAALC and WT1 results at the initial diagnosis in 177 patients (126 in CN-AML and 51 in CBF-AML) since we started these laboratory examinations in 2008. In 126 of the patients with CN-AML, 68 underwent HSCT and 58 were treated with chemotherapy alone. In the CBF-AML group, 42 were treated with HSCT and 9 were treated with chemotherapy alone. We analyzed BAALC and WT1 serial data at the initial diagnosis, after the first cycle of induction chemotherapy, and before and after HSCT. An additional analysis included the decreased ratio in the logarithm between diagnosis, pre-HSCT, or post-HSCT. Overall survival (OS) and relapse free survival (RFS) were associated with BAALC and WT1 expression levels along with the treatment timeline, and concomitantly with FLT3, NPM1, and c-kit mutations. Survival and other clinical correlates and prognostic relevance of mutations in patients with CN-AML and CBF-AML were evaluated using the log-rank test, Cox proportional hazard model, and chi-square analyses. Results:In both the CN- and CBF-AML groups, BAALC and WT1 levels at the initial diagnosis had no definite influence on OS when patients were treated with HSCT. In patients with CN-AML, only the chemotherapy-treated group with higher BAALC expression at diagnosis showed poor remission status (p = 0.008) and inferior OS (p = 0.013). Higher BAALC expression at diagnosis resulted in inferior OS in patients with CN-AML and the NPM1 (n = 7 vs. 38, p = 0.002) and FLT3-ITD mutations (n = 14 vs. 19, p = 0.052) in each group, as well as those with both mutations (n = 4 vs. 15, p = 0.000). In contrast, higher WT1 expression at diagnosis resulted in inferior OS in patients with CN-AML without the NPM1 (n = 91, p = 0.022) and FLT3-ITD (n = 92, p = 0.012) mutations in each group, as well as without both mutations (n = 69, p = 0.092). Higher pre-HSCT WT1 expression also resulted in inferior OS (p = 0.002) and RFS (p = 0.015) after HSCT in patients with CN-AML. Pre-HSCT BAALC expression levels and the decrease in the BAALC ratio before HSCT was not significant, but the decrease in the WT1 ratio over 1 logarithm prior to HSCT resulted in superior OS (p = 0.001) and RFS (p = 0.002) in patients with CN-AML. In patients with CBF-AML, the c-kit mutation-positive group showed inferior OS (p = 0.044), and their mean BAALC expression level was exclusively higher than that of other karyotypes (73.4 vs. 19.8, p = 0.000). However, only higher BAALC expression levels at diagnosis were associated with an increased relapse rate and shortened RFS after HSCT (p = 0.023) in our cohort. Other BAALC and WT1 expression level parameters were not significant in patients with CBF-AML. Conclusion:The higher pre-HSCT WT1 expression and a decreased WT1 ratio before HSCT in patients with CN-AML, and higher BAALC expression at diagnosis in patients with CBF-AML resulted in significant changes in OS and RFS after HSCT. Therefore, the impact of combined molecular marker analysis on outcomes of allogeneic HSCT for AML should be considered intensively in order to produce a more defined HSCT plan. Disclosures:No relevant conflicts of interest to declare.

High WT1 Expression in 2nd CR of AML Is Predictive for Early Relapse (prospective study)

AbstractAbstract 4812 Backgound:The Wilms' tumor gene 1 (WT1), which is over-expressed in more than 90% of AML, is a useful marker for monitoring MRD. Quantification of WT1 transcript level in peripheral blood (PB) and bone marrow (BM) at recovery from chemotherapy reliably discriminates patients at different risks of relapse. It is known that WT1 expression may be a marker of future relapse in 1 CR AML patients, after allogenic BMT and WT1 reduction also is a prognostic during induction of the first episode of AML. But there no evidence of WT1 reduction value in AML relapse. Patients and methods:In our prospective study were 10 patients with the first AML relapse and 2nd bone marrow complete remission were studied. High level of WT1 at relapse and achieving 2nd remission were the main inclusion criteria. Median WT1 level at relapse was 4302,2 (range 540–13184) copies WT1/104 Abl. Monitoring of WT1 level were estimated in patients with median age 36,6 (range from 20 to 64). Median time from the first CR to the relapse was 16,47 months (range from 3 to 51 mo), so there were 6 patients with early relapse and 4 patients with late relapse respectively. Chemotherapy with high dose cytarabine (HiDAC, FLAG) used for 2nd CR induction. Results:Only 66,7% (6 patients) demonstrated WT1 normalization. All of patients (n=4) with high WT1 expression in bone marrow remission developed second relapse in a few months, median time to relapse was 4,45 months (range from 2,8 to 7,4 mo). While patients with normal WT1 level in 2nd remission had relapse free survival 83.3% at 20 months (p=0.048, Fisher exact test, two tailed). Only one of 6 patients developed an extramedullary relapse in 6 months. The groups of patients were comparable to cytogenetic and molecular risks. Conclusion:We have shown that high WT1 level in 2nd remission is predictive for early relapse. Thus, high WT1 level in 2nd remission is an indication for very early stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

High GATA2 expression is a poor prognostic marker in pediatric acute myeloid leukemia

AbstractIn acute myeloid leukemia (AML), aberrant expression and mutations of transcription factors have been correlated with disease outcome. In the present study, we performed expression and mutation screening of GATA2, which is an essential transcription factor for regulation of myeloid lineage determination, in de novo pediatric AML patients. GATA2 mutations were detected in 5 of 230 patients, representing a frequency of 2.2% overall and 9.8% in cytogenetically normal AML. GATA2 expression analysis demonstrated that in 155 of 237 diagnostic samples (65%), GATA2 expression was higher than in normal BM. In complete remission, normalization of GATA2 expression was observed, whereas GATA2 expression levels stayed high in patients with resistant disease. High GATA2 expression at diagnosis was an independent poor prognostic factor for overall survival (hazard ratio [HR] = 1.7, P = .045), event-free survival (HR = 2.1, P = .002), and disease-free survival (HR = 2.3, P = .004). The prognostic impact of GATA2 was particularly evident in specific AML subgroups. In patients with French-American-British M5 morphology, inv(16), or high WT1 expression, significant differences in survival were observed between patients with high versus normal GATA2 expression. We conclude that high GATA2 expression is a novel poor prognostic marker in pediatric AML, which may contribute to better risk-group stratification and risk-adapted therapy in the future.

High WT1 mRNA expression after induction chemotherapy and FLT3-ITD have prognostic impact in pediatric acute myeloid leukemia: a study of the Japanese Childhood AML Cooperative Study Group

The prognostic value of WT1 mRNA expression in pediatric acute myeloid leukemia (AML) remains controversial. A sample of newly diagnosed (n = 158) AML patients from the Japanese Childhood AML Cooperative Treatment Protocol, AML 99, were simultaneously analyzed for WT1 expression, cytogenetic abnormalities and gene alterations (FLT3, KIT, MLL, and RAS). WT1 expression (including more than 2,500 copies/μgRNA) was detected in 122 of the 158 (77.8 %) initial diagnostic AML bone marrow samples (median 45,500 copies/μgRNA). Higher WT1 expression was detected in French American British (FAB)-M0, M3, M7 and lower expression in M4 and M5. Higher WT1 expression was detected in AML with inv(16), t(15;17) and Down syndrome and lower in AML with 11q23 abnormalities. Multivariate analyses demonstrated that FLT3-internal tandem duplication (ITD), KIT mutation, MLL-partial tandem duplication were correlated with poor prognosis; however, higher WT1 expression was not. FLT3-ITD was correlated with WT1 expression and prognosis. Furthermore, 74 WT1 expression after induction chemotherapy was analyzed. Higher WT1 expression after induction chemotherapy was significantly correlated with M1 or M2/M3 marrow, FLT3-ITD and poor prognosis. Multivariate analyses in 74 AML patients revealed that FLT3-ITD, MLL-PTD, and KIT mutations were associated with poor prognosis; however, NRAS Mutation, KRAS mutation and high WT1 expression (>10,000 copies/μgRNA) did not show poor prognosis. Our findings suggest that higher WT1 expression at diagnosis does not correlate with poor prognosis, but that WT1 expression after induction chemotherapy is considered to be a useful predictor of clinical outcome in pediatric AML.

Abstract 2193: WT1 mediated regulation of growth control genes in leukemia

Abstract Prognosis and therapy of acute leukemia is influenced by leukemia-specific genetic alterations, thus highlighting the importance of identifying novel prognostic markers. One such potential marker, the Wilms’ tumor gene (WT1), a zinc finger transcription factor, is expressed in leukemic blasts and has been found to be mutated in approximately 10 percent of leukemia cases. Work in other tumor systems has shown that WT1 up-regulates expression of genes promoting cancer progression, including the angiogenic and mitogenic factor VEGF. Microarray studies have also correlated expression of JAG1 (Jagged 1) and CCNA1 (Cyclin A) with WT1 in Acute Myeloid Leukemia (AML) samples. We have identified potential WT1 binding sites within the promoters of both JAG1 and CCNA1 and have compared the expression of these genes to that of WT1, using real time quantitative PCR (QRTPCR), in pediatric leukemia and normal bone marrow. Compared to normal bone marrow, we observed lower than normal levels of WT1 in a majority of pediatric ALL samples, associated with lower than normal levels of VEGF, JAG-1 and CCNA-1. Conversely in pediatric AML(M3) we observed elevated levels of WT1 associated with elevated JAG-1 and CCNA-1. Mutations of the WT1 zinc finger (ZF) DNA binding domain have also been described in poor prognosis leukemias. To identify novel ZF mutations we sequenced WT1 in twelve pediatric acute leukemia samples. No ZF domain mutations were identified among these samples with high WT1 expression. However, a well-described SNP (rs 16754, also in exon 7), identified as a good prognostic marker in Cytogenetically Normal AML, was observed either as a homozygous or heterozygous variant of the WT1 gene. Because a majority of WT1 mutations identified in leukemias are frameshift mutations leading to a truncated protein lacking the ZF DNA binding domain, we created a truncation mutant of isoform A-WT1 lacking the ZF domain and assessed its function in K562 cells. Using QRTPCR we quantified the effect of over-expression of wild type and mutant WT1 on VEGF, JAG1 and CCNA1 expression. Compared to the vector control, wild type WT1 upregulated levels of JAG1 and CCNA1 but not VEGF mRNA. Mutant WT1 did not upregulate JAG1 or CCNA1. Our data and other studies have shown that JAG1 is elevated in AML patient samples, implicating the NOTCH 1 pathway that promotes cell proliferation and blocks differentiation. Thus, these results suggest that WT1 may act as an oncogene in K562 cells, in part by upregulating the growth promoting NOTCH-1 ligand, JAG1. Similarly, cell cycle regulation by WT1, demonstrated in other systems, may be mediated by transcriptional regulation of CCNA1. WT1 may contribute to leukemogenesis through transcriptional regulation of genes controlling cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2193. doi:1538-7445.AM2012-2193

Low Wilms’ Tumor Gene Expression in Tumor Tissues Predicts Poor Prognosis in Patients with Non-Small-Cell Lung Cancer

We elucidated the relationship between prognosis of non-small-cell lung cancer (NSCLC) and Wilms’ tumor gene (WT1) mRNA expression in tumor tissue. The WT1 mRNA expression levels of the fatal cases were lower as compared with those of the survival cases. Overall survival (OS) and disease-free survival (DFS) of the high WT1 expression group were longer than of the low expression group. As for squamous cell lung cancer (SQLC), low WT1 expression was significantly associated with lymph node metastasis. Cox analysis revealed that the gene level was a significant prognostic factor in OS and DFS. Low WT1 expression predicted poor prognosis in patients with NSCLC.

High Wilms’ tumour gene (WT1) expression and low mitotic count are independent predictors of survival in diffuse peritoneal mesothelioma

To evaluate the use of the Wilms' tumour gene (WT1) marker and histomorphological parameters as indicators of prognosis in malignant peritoneal mesothelioma (MPM). Histological samples of 31 MPM were stained immunohistochemically for the WT1 protein. The results were quantified by recording the number of stained nuclei, and then correlated with patient survival. Statistical correlation was evaluated for tumour histotype, mitotic count (MC), nuclear grade (NG), necrosis, lymphoid response (grade of inflammation) and desmoplasia with regard to survival. High-grade histology (solid epithelioid, pure sarcomatoid or biphasic tumours), high NG, MC more than five per 10 per high-power field (HPF), necrosis and desmoplasia were associated with a significantly worse prognosis. Patients with MPM with low WT1 expression (≤25% of positive cells) survived for a significantly shorter time compared to those with high WT1 expression (>25% of positive cells) (P = 0.0001). The 50% survival time of subjects with low WT1 expression was 2.9 months [95% confidence interval (CI): 2.05-3.71] versus 31.5 months (95% CI: 20.4-42.5) for those with high WT1 expression. On multivariate analysis, WT1 and MC were found to be associated independently with survival (P = 0.002; P = 0.005, respectively). Our study suggests that low WT1 expression and high MC may be indicative of an unfavourable prognosis in patients with advanced malignant peritoneal mesothelioma.

WT1 Protein Directly Regulates Expression of Vascular Endothelial Growth Factor and Is a Mediator of Tumor Response to Hypoxia

WT1 is a zinc finger transcription factor expressed at high levels in many types of solid tumors, and high WT1 expression is an adverse prognostic factor. How WT1 contributes to tumor growth and influences prognosis remains unclear. We investigated the hypothesis that WT1 up-regulates VEGF in solid tumors, augmenting the response to hypoxia. We found a correlation between levels of WT1 expression and VEGF expression in Ewing sarcoma cell lines. Transfecting WT1-null SK-ES-1 cells with WT1 up-regulated VEGF mRNA expression and resulted in increased angiogenic activity in vitro. Conversely, diminishing WT1 expression in WT1-positive cell lines using WT1-specific shRNA down-regulated VEGF mRNA expression and decreased angiogenic activity in vitro. Transient transfection assays demonstrated that WT1 can regulate the activity of the VEGF promoter, and chromatin immunoprecipitation assays showed that WT1 can bind directly to the VEGF promoter in intact cells. WT1 expression in Ewing sarcoma cells is up-regulated by hypoxia. Importantly, using shRNA to inhibit this up-regulation blunted the hypoxia-mediated increase in VEGF expression. Taken together, these data demonstrate that VEGF is a direct, bona fide WT1 target gene in sarcoma and that WT1 plays a key role in optimizing the response of tumor cells to hypoxia.

The Prognostic Effect of High WT1 Gene Expression in Pediatric AML Depends on WT1 SNP rs16754 Status: A Report From the Children's Oncology Group (COG)

Abstract 1444 Background:WT1 is aberrantly over-expressed in the blast cells of most AML patients. Several adult AML groups have reported poor outcome in association with high WT1 expression. In contrast, in previously the largest pediatric study, COG investigators found no association between diagnostic WT1 expression and survival in 155 patients treated on the legacy POG-9421 trial. We recently demonstrated that the WT1 synonymous SNP rs16754 correlates with favorable outcome in childhood AML; further, SNP+ patients had higher median WT1 expression. In the present study we examine the clinical correlates of diagnostic WT1 expression within a contemporary COG trial, accounting for SNP rs16754 genotypes. Methods:WT1 mRNA expression was measured via qRT-PCR in diagnostic specimens obtained from 225 patients enrolled on the pediatric trial COG-AAML03P1. Expression levels were normalized against pooled normal bone marrow (NBM) controls. Direct sequencing of WT1 exon 7 was performed to determine SNP rs16754 genotype. WT1 expression was correlated with disease characteristics, SNP status, and clinical outcome. Results:WT1 expression varied over 4 log folds across the study cohort (range: 0.00 to 3148.27 fold NBM, median: 75.91). In 29 (13%) patients, WT1 expression was undetectable. These patients had distinct biologic characteristics compared to the remainder of the cohort, as they were predominantly FAB class M5 (56%, p<0.001) and the majority (52%, p<0.001) had 11q23 rearrangements. This group was also younger (median age 5.1 years), and rarely harbored favorable cytogenetics (Inv(16): 1 patient; t(8;21): 1 patient) or molecular abnormalities (FLT3/ ITD: 1 patient; NPM1- mutated: 1 patient; no patients with mutated CEBPA or WT1). Survival outcomes for this group did not differ significantly from the study cohort at large. WT1 expression higher than NBM was detected in 183/196 of the remaining patients. We grouped patients into WT1 expression quartiles (Q1-Q4) for comparison of clinical characteristics and outcome. FLT3/ ITD (23% vs. 9%, p=0.017) and WT1 mutations (22% vs. 4%, p<0.001) occurred more frequently in those within the highest quartile (Q4; WT1 expression >302.97 fold NBM) compared to the lower 3 quartiles. Infant patients were also less common in Q4 (p=0.010). WT1 SNP rs16754 was equally distributed among quartile groups. Survival outcomes did not differ significantly between the quartiles, confirming that diagnostic WT1 expression has no prognostic impact in contemporarily-treated pediatric AML patients, when all patients were considered. However, given our recent findings regarding the clinical relevance of the WT1 SNP, we examined the prognostic impact of WT1 expression in SNP+ and SNP- subgroups. Median WT1 expression for SNP+ and SNP- patients was 149.76 (range: 0 – 2104.44) vs. 74.40 (range: 0 – 3148.27) respectively (p=0.299). When restricting analysis to SNP rs16754 negative patients (n=150), those in the highest quartile had significantly worse 3-year overall survival (OS) from study entry (51% vs. 72%, p=0.034) and event-free survival (EFS, 35% vs. 54%, p=0.033) than those in lower quartiles. When comparing SNP- patients with higher vs. lower than median WT1 expression (Q3 + Q4 vs. Q1 + Q2), patients with higher expression again had inferior EFS (39% vs 59%, p=0.008) with a corresponding relapse rate from CR of 45% vs. 25% (p=0.011). Notably, similar comparisons in WT1 SNP+ patients with high vs. low WT1 expression did not demonstrate significant outcome differences. Surprisingly, SNP+ patients within the lowest expression quartile had the poorest outcome, while those in the highest quartile had no relapses from CR. Conclusion:WT1 expression levels vary across cyto-molecular subgroups of AML. WT1 expression was not prognostic when all AML patients were considered; however, stratifying patients by SNP rs16754 genotype revealed that high WT1 expression was significantly associated with poor clinical outcome in SNP- patients. Conversely, although the small sample size of SNP+ patients precluded significant comparisons, increasing WT1 expression trended towards improved outcome in the SNP+ group. The clinical and biologic significance of WT1 over-expression appears to differ depending on the presence or absence of SNP rs16754. WT1 expression must be considered in the context of SNP status, to determine which patients would most benefit from novel WT1 -targeted therapies. Disclosures:No relevant conflicts of interest to declare.

WT1 overexpression at diagnosis may predict favorable outcome in patients with de novo non-M3 acute myeloid leukemia

We reviewed the frequency and prognostic significance of FLT3 (fms-like tyrosine kinase receptor-3) and NPM (nucleophosmin) gene mutations and WT1 (Wilms’ tumor) and BAALC (brain and acute leukemia, cytoplasmic) gene expression in 100 consecutive patients with intermediate and poor cytogenetic risk de novo acute myeloid leukemia (AML) receiving conventional anthracycline–AraC based therapy. We observed a strict relationship between unfavorable karyotype and BAALC >1000 (p = 0.0001). Multivariate analysis of 81 patients with intermediate karyotype revealed that younger age (p = 0.00009), NPM gene mutation (p = 0.002), and WT1 >75th percentile (>2365) (p = 0.003) were independent, positive factors for complete remission (CR). WT1 expression above 2365 was correlated also to longer event-free survival (EFS) and overall survival (OS) in the same subset of patients (p = 0.003 and p = 0.02, respectively); the same finding occurred in younger patients with AML with intermediate karyotype (p = 0.008 and p = 0.01, respectively). In patients with intermediate karyotype, FLT3 internal tandem duplication (ITD) negatively affected EFS (EFS at 30 months: 30% vs. 6% in FLT3-ITD negative and FLT3 positive patients, respectively; p = 0.01) and OS (OS at 30 months: 38% vs. 20%, p = 0.03). The positive prognostic value of high WT1 expression does not have a clear explanation; it may be implicated either with WT1 anti-oncogenic function, or with the stimulating effect of WT1 oncogene on the leukemic cellular cycle, possibly associated with an enhanced response to chemotherapy.

Abstract 5007: WT1 promotes tumor growth and vascularization by regulating matrix metalloproteinase 9 in Ewing's sarcoma xenografts

Abstract WT1, a zinc-finger transcription factor, is expressed in tumors of varied origins including sarcomas. High level WT1 expression often correlates with poor patient survival. The role of WT1 in sarcoma biology and the mechanism by which high WT1 expression confers a poor prognosis are both unclear. The aim of this study is to investigate the effect of WT1 expression on sarcoma growth and angiogenesis. We transfected the WT1-null Ewing's sarcoma cell line SK-ES-1 with one of two major WT1 isoforms, and used shRNA to silence WT1 expression in the WT1-positive Ewing's sarcoma cell line MHH-ES. Cells were injected into the flanks of immune deficient mice and tumor growth was monitored. Tumors arising fromWT1-expressing SK-ES-1 cells grew faster than control, while tumors arising from WT1-silenced MHH-ES cells grew more slowly than control. Because our laboratory and others have shown that WT1 regulates VEGF expression, we evaluated tumor vascularity using the endothelial cell marker CD31 and the pericyte marker αSMA. In xenografted SK-ES-1 cells, quantification of CD31 positive area in WT1 expressing tumors showed 60 -70 % more compared to the controls. In contrast, CD31 expression is abolished in tumors arising from WT1-silenced MHH-ES cells. To investigate the mechanism by which WT1 affects tumor growth and vascularity, we performed a gene expression profiling in SK-ES-1 cells expressing different isoforms of WT1. We identified a number of genes up- or downregulated by WT1, including matrix metalloproteinase-9 (MMP9). Luciferase reporter assays showed that WT1 can upregulate MMP9 promoter activity in transiently transfected NIH3T3 cells. Also, immunohistochemical analysis showed increased MMP9 expression in WT1-expressing tumors. Finally, imunohistochemical analysis of primary Ewing's sarcoma samples showed correlated expression of WT1, VEGF, and MMP9. Taken together, our data suggest that WT1 promotes tumor growth and angiogenesis, perhaps via upregulation of MMP9. Increased tumor angiogenesis may lead to more aggressive tumors and a worse outcome for patients, explaining the effect of WT1 on prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2011-5007

Wilms’ tumor protein and FLT3‐internal tandem duplication expression in patients with de novo acute myeloid leukemia

Bone marrow samples of 30 patients with de novo adult acute myeloid leukemia (AML) were analyzed for Wt1 and FLT3‐internal tandem duplication (FLT3‐ITD) expression measured by western blot and reverse transcription‐polymerase chain reaction analysis, respectively. Wt1 was detected in 53·3% of AML patients (16/30), while FLT3‐ITD in 23·3% (7/30). The high Wt1 expression correlated with the presence of FLT3‐ITD (P = 0·014) and lower rate of complete remission (P = 0·023). The cumulative survival in AML patients was affected significantly by the presence of FLT3‐ITD, being lower in the FLT3‐ITD (+) group (6·0±2·4 months) compared to the FLT3‐ITD (−) patients (17·9±3·3; P = 0·04). The expression of FLT3‐ITD could probably activate Wt1 expression in AML blast cells and thus might contribute to its oncogenic function to provide cells with survival advantages in vivo. The detection of both molecular markers (Wt1 and/or FLT3‐ITD) may be helpful in defining high risk AML patients that need special therapeutic strategies.

WT1 Expression for Monitoring Minimal Residual Disease In Acute Lymphoblastic Leukemia Patients

AbstractAbstract 3234 Background.Wilms' tumor 1 (WT1) gene encodes a transcriptional factor involved in normal cellular development. It is over-expressed in most cases of acute myeloid leukemia (AML). Aim.To estimate the usefulness of WT1 expression quantitative assessment for monitoring MRD and for relapse prediction in acute lymphoblastic leukemia (ALL) patients. Methods.Real time quantitative RT-PCR was made according to the Europe Against Cancer Protocol. WT1 transcript level was determined in bone marrow (BM) samples of adult patients with AML (except acute promyelocytic leukemia) and ALL. We examined the samples of 77 patients with AML and 34 patients with ALL at diagnosis. We also analyzed 190 specimens of AML patients and 150 specimens of ALL patients during follow-up. The obtained WT1 copy numbers were normalized with respect to the β-GUS transcripts. Results.The median value of WT1 level at diagnosis was 581 (range 1,0-8462) in AML patients and 1161 (range 1,5-14879) in ALL patients (no statistical difference in Student's t-test). The median value of WT1 level of patients in complete remission was 4,3 (range 1,1-9,7) for AML and 5,2 (range 1,0-16,8) for ALL. At diagnosis 84% of AML patients and 61% of ALL patients showed the expression of WT1 one log higher than the median values in complete remission. In 7 assessed T-ALL patients the over-expression of WT1 at diagnosis was observed (the median value 1188, range 206–6972). The B-ALL patients with high WT1 expression at diagnosis (range 441–14879) and the B-ALL patients with low WT1 expression at diagnosis (range 1,5-63) differ in fusion genes. There were 2 patients with BCR-ABL (one p190 (7%) and one p210) within the B-ALL subgroup with high WT1 expression at diagnosis whereas within the B-ALL patients with low WT1 expression at diagnosis were 7 ones (6 patients with p190 (46%) and 1 with p210) (7% versus 46%, p=0,033 by Fisher Exact test). In the patients bearing a fusion gene transcript we performed a simultaneous analysis of WT1 and fusion gene transcript expression: in 19 patients with AML (8 AML1-ETO, 2 CBFβ-MYH11, 2 BCR-ABL, 1 DEK-CAN, 1 MLL-AF6, 1 MLL-AF9, 4 MLL duplications) and in 15 patients with ALL (9 BCR-ABL, 3 MLL-AF4, 1 MLL-ENL, 2 E2A-PBX1). It was found the parallelism between the dynamics of WT1 level and fusion-gene transcript level during follow-up. The haematological relapse was correlated with the increase of WT1 expression: 8 AML patients had a median of 1545 (range 140–2578) and for 6 ALL patients a median value was 1050 (range 31–2665) in relapse (no statistical difference in Mann-Whitney U test). We observed the increase of WT1 level was occurred earlier than the relapse was stated for AML patients and for ALL patients with high WT1 expression at diagnosis. Conclusion.The level of WT1 expression may be used as additional nonspecific marker for MRD detection and for imminent relapse prediction during follow-up in patients without additional molecular markers in ALL with high WT1 expression at diagnosis. Disclosures:No relevant conflicts of interest to declare.

High WT1 Expression Is Associated with a Shorter Time to Complete Remission After ATRA Based Induction Therapy In Acute Promyelocytic Leukemia

AbstractAbstract 1694 Introduction:In acute promyelocytic leukemia (APL) the t(15;17) is detectable in the vast majority of cases and leads to the fusion transcript PML-RARα. The introduction of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) into therapeutic management of APL resulted in long term remissions in about 80% of patients. However, relapse occurs in about 10–15% of cases and remains a major cause of treatment failure. The Wilms Tumor 1 gene (WT1) encodes for a transcription factor that is involved in normal and malignant hematopoiesis. Aberrant expression of WT1 has been shown to be associated with an adverse outcome in different types of hematological malignancies such as acute myeloid leukemia, acute lymphoblastic leukemia and myelodysplastic syndromes. To date only small series exist on aberrant WT1 expression in APL. Therefore, the aim of this study was to further elucidate the incidence of aberrant WT1 expression in APL and its prognostic significance for clinical endpoints. Methods:Bone marrow (BM) mononuclear cells were retrospectively evaluated in 89 APL patients (53 female, 36 male) and 21 healthy controls (15 female, 6 male) after informed consent. The median age of APL patients and healthy controls was 46 (range: 19–82 years) and 60 years (range: 21–84 years), respectively. All patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study. The treatment consisted of simultaneous ATRA and double induction (TAD/HAM) chemotherapy including high-dose ara-C, one cycle of consolidation chemotherapy (TAD) and 3 years monthly maintenance chemotherapy. BM cells were analyzed by multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR) in triplicates on a LightCycler 480 (Roche, Mannheim, Germany). Beta–glucuronidase (GUS) served as a housekeeping gene for normalization. For quantification of relative expression values a modified delta-delta CT calculation model according to Pfaffl was used (Pfaffl MW, Nucleic Acids Res, 2001) after determination of PCR efficiencies. WT1 expression groups were defined as follows: WT1 expression higher than the 75th percentile (WT1high) and WT1 expression lower than the 25th percentile (WT1low). Time to complete remission, disease free survival (DFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the groups (p < 0.05). Results:Normalized WT1 expression was significantly higher in APL samples (median: 0.1129, range: 0.00001–1.414) as compared to normal controls (median: 0.00135, range: 0.00001–0.01105) (p=0.0011). WT1high was not significantly associated with high white blood cell counts (WBC). In univariate analysis time to complete remission was significantly shorter in patients showing high WT1 expression as compared to patients with low WT1 expression (47 days vs. 58 days, p=0.0051). However, this did not translate into a longer DFS or OS. In contrast, there was a trend toward an impaired DFS and OS in the WT1high group. Interestingly, analyzing deaths prior to complete remission WT1high was significantly associated with early death as compared to the WT1low group (median 6 days vs. 17 days, p=0.0197). Conclusion:WT1 is highly expressed in APL as compared to healthy controls but is not associated with increased WBC. WT1high was significantly associated with a shorter time to CR. Since WT1 plays a critical role in differentiation and survival in myeolopoiesis this finding might be due to a higher succeptibility of WT1high APL patients to ATRA induced differentiation as compared to the WT1low group. Moreover, in vitro studies have shown that in NB4 cells WT1 mRNA levels decrease upon treatment with ATRA, this provides further evidence that WT1 might be an additional target for ATRA in APL (Gu W, Haematologica, 2005). Interestingly, WT1high is significantly correlated with early death prior to CR achievement. We hypothezise that this might also be an effect of an increased toxicity of the ATRA based induction therapy in patients having high WT1 expression. Disclosures:Lengfelder:Cephalon: Research Funding.

High WT1 Expression Has An Independent Positive Influence On CR Rate and EFS in Non M3 AML Patients Treated with Chemotherapy.

Abstract 4675The prognostic value of WT1 expression at diagnosis is still controversial. It has been retrospectively evaluated in 99 consecutive non pretreated non M3 AML patients who had undergone a complete prognostic work up at diagnosis and had received intensive chemotherapy.Biological markers were evaluated on fresh marrow samples collected at diagnosis. WT1 expression was evaluated using TaqMan Gene Expression Assays as described.All patients received induction therapy with combination of fludarabine, Ara-C and anthracycline ± low dose gemtuzumab ozogamicin (n. 59) or with a conventional combination of Ara-C and anthracycline (n. 40) A conventional post-induction chemotherapy including intermediate dosage Ara-C was administered to all responding patients.Univariate comparisons between patients in CR vs non CR were performed using chi-square analysis or Fisher's exact test for categorical variables and t-test for continuous variables. P values < 0.05 were considered statistically significant. Analyses were performed using SPSS. The prognostic impact of WT1 expression was evaluated using quartiles as cut off point and selecting the one with the lowest p value. The event free survival and OS were calculated using the Kaplan Meier method. Non CR after the first induction course, relapse and death due to any cause were considered events. OS and EFS duration were calculated from start of treatment. The impact of multiple predictor variables was assessed by multivariate analyses according to the Cox regression model for OS and EFS while for the evaluation of RC was used the Logistic regression model.Median age of patients was 59 years (range 17-81). Cytogenetic alterations were prognostically favorable in 3 patients and belonged to the intermediate prognostic group in 77 patients (normal karyotype in 75 patients and +8 in two). Nineteen patients had a poor prognosis cytogenetics. For statistical analyses we considered two karyotipic groups: unfavorable (19 patients) and not unfavorable (80 patients).CRs were 60/99 (60%), of which 40 in 51 patients aged 60 or less (78%) and 20 in 48 older than 60 years (41%). Twenty-six patients relapsed, 54 are alive, 45 have died, with a median follow up of 360 days (range 20-2300).In Table 1 are reported clinical indicators of outcome being patients grouped according to the percentile of WT1 expression with the lowest p value (75th). Statystical analysis showed that all WT1 quartiles were balanced for other prognostic factors, such as cytogenetics, BAALC expression, FLT3 and NPMA and B mutations, age, blast count and therapy.The lack of consense on the role of WT1 level at diagnosis in the prognostic stratification indicate that further clinical studies are required. The clear correlation between the level of WT1 transcript and the tumor burden explains why WT1 is used in the follow up of leukemic patients as universal marker of residual disease, also in patients with specific chimeric products. On the contrary, the biological explanation of the prognostic impact of WT1 transcript level at diagnosis remains uncertain. Over the years WT1 gene has been considered as an oncogene or a tumor suppressor gene. In our experience the protective influence of high WT1 expression cannot be explained with an association with good prognosis biological features (such as mut NPM and / or low BAALC). The positive prognostic value of high WT1 expression might be implicated either with WT1 antioncogenic function, or with the stimulating effect of WT1 oncogene on leukemic cellular cycle, possibly associated with an enhanced response to chemotherapy.Table 1WT1 <= 2400 N./N.pts (%)WT1 > 2400 N./N.pts (%)p univ,p multiv.*RR (95% CI)CR (all karyotypes)41/ 75 (54)19/24 (82)0,0260.063.364 (0.927-12.202)CR (int/good karyot.)36/59 (61)19/210.010,0276.649 (1.240-35.645)CR (denovo AML int kar)31/45 (69)14/15 (98)0.020,03412.557 (1.218-129.446)CR (denovo, N.K.)26/40 (65)15/16 (94)0.0250.0413.430 (1.111-162.318)EFS at 24 months (all karyotypes)8%6%0.0020.050.486 (0.235-1.007)EFS at 24 months (int / good karyot.)9%64%0.0010.0230.360 (0.150-0.866)EFS at 24 months (de novo, N.K.)5%70%0.0010.0070.227 (0.077-0.671)OS (all karyot)15%55%0,110,660.837 (0.371-1.890)OS (int/good kar.)18%63%0,050,180.507 (0.186-1.381)Table 1 legend: * for multivariate analysis age, karyotype, FLT3, NPM mutation, BAALC expression, denovo/secondary disease were considered. Disclosures:No relevant conflicts of interest to declare.

High WT1 gene expression before haematopoietic stem cell transplant in children with acute myeloid leukaemia predicts poor event‐free survival

WT1 gene expression has been proposed as a useful marker of minimal residual disease in leukaemia. Its utility in paediatric haematopoietic stem cell transplantation (HSCT) has not been studied. We studied the prognostic value of WT1 expression in peripheral blood prior to HSCT in 36 children with acute myeloid leukaemia (AML). Samples were obtained 2 weeks pre-transplant to determine the level of WT1 expression. WT1 expression was normalized using K562 cells as a control and a relative value of 0.5 was chosen as the cut-off point between high and low WT1 expression. The median level of pre-transplant WT1 expression in the 36 patients was 0.09 (range 0.0001-11.0), with 11 patients having WT1 >or= 0.5 and 25, WT1 < 0.5. After HSCT, 76% of patients with high pre-transplant WT1 expression relapsed, in contrast to 0% of the patients with low WT1 expression. Those with high WT1 expression had significantly lower 5-year event-free survival (EFS) (18%, 95% CI 0-40%) as compared to those with low WT1 expression (68%, 95% CI 50-86%, P = 0.007). Multivariate analysis showed that pre-transplant WT1 level is the only significant prognostic factor for the difference in EFS. Our finding suggests that elevated WT1 gene expression before HSCT in paediatric AML predicts relapse and poor long-term EFS. A larger prospective study is warranted to compare the value of high WT1 expression and other markers of minimal residue disease in predicting clinical outcomes after HSCT.

RQ‐PCR based WT1 expression in comparison to BCR‐ABL quantification can predict Philadelphia negative clonal evolution in patients with imatinib‐treated chronic myeloid leukaemia

A retrospective comparison of WT1 and BCR-ABL1 expression was performed in 40 imatinib-treated chronic myeloid leukaemia patients. The overall correlation of WT1 and BCR-ABL1 was low. In two patients WT1 expression was increasing despite very low BCR-ABL1 levels. As both revealed Ph-negative aberrant clones, a second independent cohort of 20 cases, all with Ph-negative clonal evolution, was analysed. High WT1 expression (5.0-177.0%) was detected in a case with +11 and in four of eight cases with +8, but not in cases with del(20q) or -Y. Thus, increasing WT1 levels in molecular responders may indicate Ph-negative clonal cytogenetic evolution during imatinib treatment.

Increased p16 Expression in High-grade Serous and Undifferentiated Carcinoma Compared With Other Morphologic Types of Ovarian Carcinoma

There are several morphologic types of ovarian carcinoma. It has been shown that p16 is overexpressed in high-grade serous carcinoma but there has been little detailed comparison of p16 expression in the common types of ovarian carcinoma. The aim of this study was to compare p16 expression in ovarian carcinomas of serous, endometrioid, clear cell, and mucinous type with a view to ascertaining whether high expression in a primary ovarian carcinoma is specific for a serous neoplasm. We included problematic cases, which are difficult to type, such as poorly differentiated and undifferentiated carcinomas and serous carcinomas with clear cells. In these problematic groups, we compared p16 expression with that of WT1, which is known to be relatively specific for a serous phenotype. Cases of ovarian high-grade serous carcinoma (n=38), endometrioid carcinoma (n=15), clear cell carcinoma (n=12), and mucinous carcinoma (n=10) were stained with p16. Cases were scored both with respect to distribution of immunoreactivity (0-5) and intensity (0-3). An immunohistochemical composite score was also calculated (0-15) by multiplying the distribution and intensity scores. Serous carcinomas typically exhibited high p16 expression; there was statistically significant higher p16 expression in serous carcinomas compared with the other morphologic types. There was high p16 and WT1 expression in most undifferentiated carcinomas and in serous carcinomas with clear cells, suggesting that these represent variants of serous carcinoma. We have demonstrated that p16 is highly expressed in high-grade serous and undifferentiated carcinomas compared with other morphologic types of ovarian carcinoma. This may be useful, in conjunction with WT1, in the classification of problematic neoplasms. p16 may be involved in the pathogenesis of high-grade ovarian serous carcinomas, possibly through inactivation of retinoblastoma protein.