WT1 Expression Levels Research Articles

290 WT1 expression level in peripheral blood: an early marker of myelodysplastic syndromes evolution?

290 WT1 expression level in peripheral blood: an early marker of myelodysplastic syndromes evolution?

Integrative prognostic risk score in acute myeloid leukemia with normal karyotype

AbstractTo integrate available clinical and molecular information for cytogenetically normal acute myeloid leukemia (CN-AML) patients into one risk score, 275 CN-AML patients from multicenter treatment trials AML SHG Hannover 0199 and 0295 and 131 patients from HOVON/SAKK protocols as external controls were evaluated for mutations/polymorphisms in NPM1, FLT3, CEBPA, MLL, NRAS, IDH1/2, and WT1. Transcript levels were quantified for BAALC, ERG, EVI1, ID1, MN1, PRAME, and WT1. Integrative prognostic risk score (IPRS) was modeled in 181 patients based on age, white blood cell count, mutation status of NPM1, FLT3-ITD, CEBPA, single nucleotide polymorphism rs16754, and expression levels of BAALC, ERG, MN1, and WT1 to represent low, intermediate, and high risk of death. Complete remission (P = .005), relapse-free survival (RFS, P < .001), and overall survival (OS, P < .001) were significantly different for the 3 risk groups. In 2 independent validation cohorts of 94 and 131 patients, the IPRS predicted different OS (P < .001) and RFS (P < .001). High-risk patients with related donors had longer OS (P = .016) and RFS (P = .026) compared with patients without related donors. In contrast, intermediate-risk group patients with related donors had shorter OS (P = .003) and RFS (P = .05). Donor availability had no impact on outcome of patients in the low-risk group. Thus, the IPRS may improve consolidation treatment stratification in CN-AML patients. Study registered at www.clinicaltrials.gov as #NCT00209833.

Monitoring of minimal residual disease by quantitative WT1 gene expression following reduced intensity conditioning allogeneic stem cell transplantation in acute myeloid leukemia

WT1 is well-known to be a panleukemic marker that is expressed in 90% of acute myeloid leukemias (AML). Quantification of WT gene expression in bone marrow (BM) samples may be useful as a marker of minimal residual disease (MRD) during and after treatment for early prediction of relapse. We evaluated the validity of this AML-MRD marker after reduced intensity conditioning (RIC) allogeneic stem cell transplantation (SCT). The quantitative assessment of WT1 expression by real-time quantitative PCR (RQ-PCR) was measured in 25 patients (pts) with AML at diagnosis, at the time of RIC-SCT and after transplant at precise time points. All pts showed high WT1 levels at diagnosis with a mean of 4895 (SD 4462) and a median of 3679 (range 454-16,853) copies WT1/10(4) ABL. At transplant, 18/25 pts (72%) were in complete cytologic remission (CcR) and 7/25 (28%) had refractory AML. At the pre-SCT evaluation, BM samples from pts transplanted in CcR showed significantly lower WT1 expression levels compared to the samples from pts with refractory AML (p = 0.002). Median follow-up after RIC-SCT was 18 months (range 2-54). On 18 pts transplanted in CcR, those (17/18) who maintained CcR after RIC-SCT displayed a WT1 copy number persistently low during all the follow-up period. In patients who received RIC-SCT with active disease obtaining a sustained CcR after transplant (3/25), WT1 levels decreased to normal range in the first two months after RIC-SCT and remained low through the entire study period. All pts who relapsed after RIC-SCT (4/25) had a high WT1 copy number before the cytologic relapse. In 50% of these cases, an increase in WT1 expression was documented before molecular chimerism decreasing. With this experience, taking into account the limited number of pts, we confirmed a concordance between WT1 expression levels (measured by RQ-PCR at precise and sequential time points) and status of AML before and after RIC-SCT and we found a concordance between WT1 expression levels and hematopoietic chimerism status. Our data suggest that, in the RIC-SCT setting, the sequential and quantitative analysis of WT1 may be useful as a leukemia marker for monitoring MRD and as a predictor of overt AML cytologic relapse.

Role of hTERT and WT1 gene expression in disease progression and imatinib responsiveness of patients with BCR–ABL positive chronic myeloid leukemia

Most cases of leukemia show hTERT and WT1 gene overexpression. However, the role of these genes in the progression and monitoring of chronic myeloid leukemia (CML) has not been very well explored. Two hundred and eight patients diagnosed as having CML in chronic phase (CP), accelerated phase (AP), and blast crisis (BC) were studied. Real-time polymerase chain reaction (PCR) was performed for comparative quantification of BCR–ABL, hTERT, and WT1 gene expression. The expression levels of the three genes were compared among the three phases and seven imatinib-naive and -treated subgroups. Among the three major groups, the gene expression levels of hTERT (p < 0.01) and WT1 (p < 0.01) were significantly different. Freshly diagnosed, imatinib-responsive, and -unresponsive patients among these revealed interesting changes in hTERT and WT1 gene expression profiles, especially in 25 patients with CML in whom sequential samples were analyzed. Our results showed that hTERT and WT1 gene expression analyses provided relevant information for the understanding of disease progression and indicate their possible usefulness as surrogate markers for treatment monitoring.

Opposite regulation of estrogen receptor-α and its variant ER-α36 by the Wilms’ tumor suppressor WT1

The genomic and non-genomic signaling pathways are well-known estrogen signaling pathways. The 66-kDa estrogen receptor-α (ER-α66) is a typical ligand-inducible transcription factor that mainly mediates genomic estrogen signaling. Recently, we identified and cloned a 36-kDa variant of ER-α66, known as ER-α36. This variant lacks intrinsic transcription activity and predominantly mediates non-genomic estrogen signaling. Thus, the expression of ER-α66 and ER-α36 should be firmly regulated and carefully correlated to maintain a balance between genomic and non-genomic estrogen signaling. However, the molecular mechanisms underlying this correlation remain poorly understood. The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger protein WT1 that functions as a dual transcription regulator to activate or suppress gene transcription. High levels of WT1 expression are associated with breast cancer malignancy. In the present study, high-passage ER-positive breast cancer MCF7 cells were found to express ER-α66 and WT1 at higher levels and ER-α36 at a very low level. Using the small hairpin RNA method, stable MCF7 cells were established that expressed knocked-down levels of WT1. The cells expressed a reduced level of ER-α66 but an increased level of ER-α36, suggesting that WT1 regulates the expression of ER-α66 and ER-α36 oppositely. Further co-transfection assays showed that all isoforms of WT1 directly activated the promoter activity of the ER-α66 gene while suppressing ER-α36 promoter activity. Our results therefore indicate that WT1 acts as a dual transcription factor that regulates the promoter activity of ER-α66 and ER-α36 oppositely, implicating WT1 as one of the coordinators that orchestrate genomic and non-genomic estrogen signaling.

Increased excretion of urinary podocytes in lupus nephritis

Podocytes are highly specialized epithelial cells that form part of the filtration barrier in the kidney, and their loss reflects a malfunction in glomerular filtration, which is usually associated with the progression of the disease. Glomerulonephritis is a serious complication that develops in about 50% of the lupus patients and is characterized by proteinuria arising from direct or indirect podocyte injury. To assess the possible role of podocytes in the pathogenesis of lupus nephritis (LN). Urinary and glomerular podocytes were detected in the kidney biopsies of patients (n = 17) with lupus nephritis, and from control biopsies obtained during autopsies. The WT-1 protein was used as a podocyte marker. The cumulative excretion of urinary podocytes was detected in the urinary sediments of LN patients and normal healthy controls, and the specimens were analyzed by immunohistochemistry, immunofluorescence, and enzyme-linked immunosorbent assay. The apoptotic index was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Gross proteinuria in lupus patients was determined via 24-hour urine samples, and the results were analyzed by Student t test. Biopsy specimens from 17 patients with class-III or IV LN had lower levels of glomerular WT-1 expression than the levels found in normal kidneys (P < 0.0001). The reduction of glomerular podocytes in patients with lupus nephritis correlated with the cumulative excretion of urinary podocytes (P < 0.0001) and proteinuria. There was no correlation between the urinary podocytes and the apoptotic index in the LN urinary sediments. A decrease in glomerular podocytes is associated with their cumulative excretion in urinary sediments; therefore, such findings correlate with proteinuria in lupus nephritis patients.

The WT1 synonymous SNP rs16754 Is Associated with Higher mRNA Expression and Predicts Significantly Improved Outcome In Favorable-Risk Pediatric AML: a Report From the Children's Oncology Group

AbstractAbstract 950SNP rs16754, a synonymous single nucleotide polymorphism of the WT1 gene, has recently been reported to have prognostic implications in adult normal-karyotype acute myeloid leukemia (AML). We sought to determine the prevalence and clinical implications of WT1 SNP rs16754 in a large cohort of unselected pediatric AML patients. Available diagnostic bone marrow specimens (N=790) from patients treated on 3 consecutive CCG / COG trials (CCG-2941, CCG-2961, and COG AAML03P1) were analyzed for the presence of SNP rs16754 via direct sequencing of exon 7 of the WT1 gene. SNP status was correlated with disease characteristics, WT1 quantitative mRNA expression level, and clinical outcome. At least 1 copy of the minor SNP allele was detected in 229 (29.0%) patients, 38 (16.6%) of whom were homozygous for the SNP. SNP rs16754 was significantly more common in Asian and Hispanic patients than in Caucasian patients (P<0.001). The SNP was also less common in patients with inv(16) (P=0.043) and more common in patients with -5/del(5q) (P=0.047). Although FLT3/ITD, NPMc, and CEBPA mutations occurred at comparable frequencies in patients with or without the WT1 SNP, WT1 mutations were significantly less common in SNP-positive patients (3.2% vs. 10.4%, P=0.002). WT1 expression levels were evaluated by quantitative RT-PCR in 114 unselected patient samples with known WT1 SNP and mutation status (SNP-positive, n=31; WT1-mutation positive, n=13; one patient harbored both the SNP and a WT1 mutation). Median WT1 expression in WT1 wild-type patients (without SNP rs16754 and negative for a WT1 mutation, n=71) was 40.61 times that of normal marrow controls (range 0.00–2949.42). Median WT1 expression levels were significantly higher in both patients with SNP rs16754 (215.76 fold normal marrow, range 0.00–1399.23, P=0.0214) and in patients with a WT1 mutation (327.14 fold normal marrow, range 54.17–902.3, P=0.0005). Five-year overall survival (OS) for patients with or without the SNP was 60% ± 7% and 50% ± 5%, respectively (P=0.031). In multivariate analysis, the presence of the SNP was an independent predictor of improved OS (HR 0.64, 95% CI 0.47–0.86, P=0.004). Relapse rates were comparable between the 2 groups, but OS after relapse was significantly higher in SNP-positive patients (31% ± 11% vs. 24% ± 6% at 5 years, P=0.034) suggesting a higher rate of salvage success. The prognostic impact was most pronounced in patients with low-risk disease (those with CBF-AML, CEBPA mutation, or NPMc mutation), where five-year OS for those with or without the SNP was 90% ± 8% vs. 64% ± 9% (P=0.001) with a corresponding disease-free survival of 72% ± 13% vs. 53% ± 10% (P=0.041). The high prevalence of WT1 SNP rs16754, and its association with improved outcome, identifies WT1 SNP rs16754 as a potentially important molecular marker of prognosis in pediatric AML. WT1 SNP status, WT1 mutation status, and WT1 expression levels should be evaluated together prospectively in future pediatric AML clinical trials. Disclosures:No relevant conflicts of interest to declare.

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.

Apoptosis of Leukemic Cells by Using tRNase ZL Under the Direction of Oncogene-Targeting Small Guide RNA.

AbstractAbstract 3763 Introduction:tRNase ZL is one of the enzymes for tRNA maturation and removes a 3′ trailer of pre-tRNA by recognizing four leaves-clover-like structure of pre-tRNA. In addition, we found that tRNase ZL can recognize 12-bases hairpin structures and cleaves 3′ portion of them. Furthermore, we have demonstrated that tRNase ZL could cleave 3′ portion of target mRNA by recognizing a complex consisting of both the target mRNA and artificially designed small guide RNA (sgRNA) which possesses the complementary sequences with target RNA and form a four leaves-clover-like structure or 12-bases hairpin structures with target mRNA. Currently, sgRNA is divided into five categories; 3′-truncated tRNA (form a four leaves-clover-like structure with target mRNA), 5′-half tRNA (form a half of four leaves-clover-like structure with target mRNA), 14-nucleotides linear type sgRNA (form a 14-bases stem with target mRNA), hook type sgRNA (containing a single loop structure and form 12-bases hairpin structure with target mRNA) and heptamer sgRNA (7-bases RNA structure complementary to target mRNA and form a 12-bases hairpin structure with target mRNA). The use of sgRNA, especially heptamer sgRNA, to knockdown specific mRNAs has several advantages over long form RNAs (antisense, shRNA or siRNA); 1) heptamer sgRNA can be introduced into cells without any transfection operation, 2) less off-target effect, 3 less interferon responses, 4) the synthesis of heptamer sgRNA is inexpensive than that of long form RNAs. Thus, gene silencing methods using tRNase ZL and sgRNA are presumed to induce effective and specific target RNA inhibition.In the present study, in order to explore the application of sgRNA (especially heptamer sgRNA) as novel nucleic acid medicine, we investigated the effects of heptamer sgRNA targeting WT1 gene (WT1sgRNA) and PRMAE gene (PRAMEsgRNA) on apoptosis, proliferation and mRNA suppression in leukemic cell lines. Materials and Methods:The 5′-phosphorylated 2′-O-methyl heptamer sgRNAs, which were designed to form 12-bases hairpin structure with target mRNA of leukemia associated antigens (LAA) were synthesized and purified by high-performance liquid chromatography. The leukemic cell lines (HL60, C2F8 and U937) were cultured with heptamer sgRNA at the concentration of 1 mM. Introduction of heptamer sgRNA into cells was investigated by using FITC-conjugated heptamer sgRNA and evaluated by flow cytometry and fluorescence microscopy. Quantification of target mRNA was determined by real-time quantitative PCR. Effects of heptamer sgRNA on apoptosis of leukemic cells were evaluated by flow cytometry. Inhibition of cell proliferation was evaluated by TetraColor ONE reagent. Results:Heptamer sgRNA could be introduced into cells by just adding them to culture medium by both flow cytometry and fluorescent microscopy. Expression level of WT1 and PRAME mRNA in HL60 cells was reduced by culturing HL60 cells with WT1sgRNA and PRAMEsgRNA (up to 75% and 30% reduction, respectively) compared to control EGFPsgRNA (estimated as 0% reduction). Likewise, WT1sgRNA suppressed WT1 mRNA expression in C2F8 cells by just adding WT1sgRNA to culture medium (30% reduction). In addition, WT1sgRNA and PRAMEsgRNA could induce apoptosis in HL60 cells but not in WT1(–)PRAME(–) U937 cells. Control EGFPsgRNA did not induce apoptosis. WT1sgRNA and PRAMEsgRNA could inhibit cell proliferation in HL60 cells (70% and 40% inhibition) respectively, compared to control EGFPsgRNA (estimated as 0% inhibition). WT1sgRNA could also inhibit cell proliferation in C2F8 cells (70% inhibition). Conclusion:These data indicate that WT1sgRNA and PRAMEsgRNA specifically bind to target mRNA and forms 12-bases hairpin structure with target mRNA, which complex is recognized by tRNase ZL. Then 3′ portion of target mRNA is cleaved by tRNase ZL by target-specific manner. Taken together, these findings suggested a possibility that oncogene-targeting sgRNA (especially heptamer sgRNA) could be applied as a novel nucleic acid medicine against leukemia and other tumors. Disclosures:No relevant conflicts of interest to declare.

Molecular Characterization of Acute Undifferentiated and Biphenotypic Leukemia

AbstractAbstract 1692 Background:Acute leukemias of ambiguous lineage account for only 3–5% of all cases of acute leukemia and represent a heterogeneous group of rare, poorly characterized leukemia with adverse outcome. The aim of this study was to further molecularly characterize this rare subtype to gain insights into the pathogenesis of acute undifferentiated leukemia (AUL) and biphenotypic acute leukemia (BAL). These molecular insights may then lead to targeted treatment strategies for specific molecular subgroups. Design and Methods:The European Group for Immunologic Classification of Leukemia (EGIL) defines BAL as a single blast population with co-expression of myeloid- and lymphoid-associated antigens according to a scoring system. Cases without expression of lineage-specific antigens are designated as AUL. Based on the EGIL scoring system, we identified 11 and 20 adults with newly diagnosed AUL and BAL, respectively. In all samples, flow cytometry was performed using a panel of monoclonal antibodies for precursor cells (CD34, TdT), lymphoid-associated antigens, including surface and cytoplasmic antigens (CD79a, CD22, IgM, CD19, CD20, CD10, CD24, CD3, TCR, CD2, CD5, CD8, CD7, CD1a), and myeloid-associated antigens including myeloperoxidase, CD13, CD33, CD65s, CD117, CD14, CD15, CD64. In order to further molecularly characterize AUL and BAL, samples were studied for expression and genotype alterations of candidate genes with prognostic impact or with relevance in the pathogenesis of acute leukemias. The mRNA expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by quantitative real-time PCR. In addition, samples were studied for WT1 mutations in exons 7 and 9, for FLT3 mutations [internal tandem duplications (ITD) and mutations in the tyrosine kinase domain (TKD835)], as well as for BCR-ABL (breakpoints: e1/a2; b3/a2; b2/a2) and MLL-AF4 rearrangements. Results were than compared to data generated from acute myeloid leukemia (AML; n = 75), acute T-lymphoblastic leukemia (T-ALL; n = 242) and B-precursor ALL (n = 368) patients. Results:AUL patients were characterized by a specific gene expression pattern. In particular, in AUL over-expression was shown for IGFBP7 (P = 0.01) and MN1 (P = 0.04) compared to BAL; for BAALC (P = 0.001), IGFBP7 (P = 0.0001) and MN1 (P = 0.02) compared to T-ALL; for BAALC (P = 0.02) and IGFBP7 (P = 0.001) compared to AML; as well as for IGFBP7 (P = 0.0001) compared to B-precursor ALL. Genotype alterations (WT1, FLT3, BCR-ABL, MLL-AF4) were not detectable in AUL samples.The majority of BAL cases expressed either T-lineage plus myeloid antigens (n = 9; 45%) or B-lineage plus myeloid markers (n = 9; 45%), while co-expression of T/B lymphoid antigens was only seen in 2 samples of BAL. Cell surface expression of CD34 (≥ 20%) was most frequently found in BAL (17 of 20 cases; 85%). Gene expression studies revealed over-expression of BAALC and IGFBP7 in BAL compared to T-ALL, AML and B-precursor ALL, but less pronounced as shown for AUL. Moreover, BAL samples were characterized by frequent WT1 mutations in exon 7 (sufficient DNA was available in 14 samples of which 4 had WT1 mutations; 29%). In contrast, WT1 mutations were only found in 8% of T-ALL and have previously been reported in about 10% of AML patients with normal cytogenetics (Paschka et al, 2008). In concordance with published data, the BCR-ABL fusion gene was detected in 32% of BAL samples (19 samples studied of which 9 were positive for BCR-ABL). MLL-AF4 translocations and FLT3 mutations were not detectable in this BAL cohort. Conclusion:Here we present a comprehensive molecular study investigating mRNA expression and genotype alterations in the poorly characterized subgroup of acute leukemias with ambiguous or lacking lineage commitment. We show that AUL and BAL patients share important prognostic features of both myeloid and lymphoid leukemias. Over-expression of stem cell associated genes (BAALC and IGFBP7) and genes associated with an adverse prognosis (BAALC and MN1) was most prominent in AUL. As lineage-directed therapies are likely ineffective for these high-risk patients and the fact that AUL lack known specific molecular targets, further studies are warranted to unravel molecular aberrations, which might allow the development of more specific therapies. BAL patients however, were characterized by genetic aberrations including BCR-ABL rearrangements and WT1 mutations, which may imply targeted therapy options. Disclosures:No relevant conflicts of interest to declare.

WT1 Expression at the Diagnosis of Childhood AML Has No Prognostic Value but Corresponds with the Biological Characteristics of Leukemic Cells - Results From European Multicenter Study.

AbstractAbstract 1684▪ Introduction:The Wilms' tumor gene 1 (WT1) is highly expressed in a large proportion of human acute leukemias and other hematological malignancies. Therefore, a lot of studies have been performed to determine the role of WT1 in hematopoiesis and leukemogenesis. Despite the large number of studies, the results still remain controversial. WT1 expression was repeatedly proved to be a strong prognostic factor as well as a marker for minimal residual disease (MRD). However, other studies did not confirm these results. A lack of standardized and universal method for the detection of WT1, together with different treatment regimens, has been proposed to explain these discrepancies. Nevertheless, the role of WT1 gene in normal and malignant hematopoiesis still remains to be elucidated. Materials and methods:To investigate the prognostic significance of WT1 expression at diagnosis of childhood acute myeloid leukemia (AML), we analyzed bone marrow samples (BM) from 211 patients with childhood AML enrolled in AML-BFM 04 treatment protocol. The analysis of WT1 expression level was performed in 3 participating centers (Frankfurt, Jena – Germany and Prague – Czech Republic) using the standardized and quality-controlled qPCR method published in 2009 (Willasch et al. 2009). Results:With respect to a different treatment strategy for M3 patients, we performed the analysis of entire cohort (excluding 2 patients with Down syndrome from the survival analysis) as well as non-M3 patients separately (n=195). The median of WT1 level at diagnosis was 2014 WT1/ABLx104 NCN and 1779 WT1/ABLx104 NCN for all patients and non-M3 patients, respectively. We found significantly lower WT1 level in high risk group (HR) compared to standard risk group (SR) (p<0.0001 and p=0.0014 for all pts and non-M3 pts resp.). The highest WT1 expression was detected in patients with M3 AML (n=16; 7959 WT1/ABLx104 NCN) and the lowest WT1 in M5 AML (n=44; 10 WT1/ABLx104 NCN). The presence of t(15;17), t(8;21) or inv16 was strongly associated with higher WT1, whereas patients with MLL rearrangements had lower WT1 expression (p<0.0001). FLT3/ITD positive patients (n=23) expressed significantly higher WT1 levels in comparison with FLT3/ITD negative pts (p=0.0001 for all and non-M3 pts). We proved a relation of WT1 expression with gender and WBC at diagnosis neither in the group of all patients nor in non-M3 pts. There was a non-significant trend (p>0.05) in the HR group for pts with > 5% of blasts at d15 to a higher WT1 expression. The survival analysis showed correlation of WT1 expression level and Event Free survival (EFS) or Cumulative Incidence of Relapse (CIR) neither in the entire cohort (3-year pEFS for patients with </>=140 WT1/ABLx104 NCN: 61% SE 4% vs. 52% SE 7%, p=0.12; 3-year CIR 39% SE 7% vs 35% SE 4%, p=0.45; n=59 vs 150) nor in the cohort of non-M3 patients or in patients with normal cytogenetics (n=43, data not shown). Conclusion:We report the first results of an international multicenter study on WT1 expression in childhood AML, based on the standardized, universal and quality-controlled method for quantitative assessment of WT1. Our patients were treated according to a concordant treatment protocol, thus representing a well-characterized group of AML patients. Based on our results we can conclude that the initial WT1 expression in patients with childhood AML is not an independent prognostic factor for the outcome. Conversely, WT1 expression pattern in morphologically resp. cytogenetically defined subgroups of AML indicates, that WT1 level is strongly related to the biological characteristics of malignant cells. These findings should be taken into consideration when proposing WT1 as a prognostic marker in childhood AML. Supported by grant MSM0021620813, GAUK 81709 and IGA NS10488-3/2009 Disclosures:No relevant conflicts of interest to declare.

THE SIGNIFICANCE OF DETECTING WT1 EXPRESSION IN CHILDHOOD ACUTE LEUKEMIAS

WT1 (Wilms' tumor gene 1) overexpression is implicated in the prognosis of acute leukemia. The purpose of this study was to investigate WT1 expression and its clinical implication in childhood acute leukemia (AL) in Chinese population. Bone marrow specimen from 200 children at different stages of acute leukemia and from 21 children without leukemia were studied. The WT1 expression at diagnostic marrow specimen in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) was higher than control group, whereas WT1 expression in AML was higher than in ALL, and WT1 expression level in relapse in ALL increased more significantly than in AML. The WT1 expression level showed positive correlation with the hypodiploidy and BCR-ABL fusion gene in acute leukemia. A rapidly decrease of WT1 expression level predicted a good response to the induction therapy and low expression of WT1 correlates with remission status. This study suggested that WT1 expression levels in acute leukemia can potentially be a marker for evaluating therapeutic efficacy, correlating with monitoring minimal residue disease, and predicting hematological relapse in children acute leukemia.

Wilms’ tumor 1 gene (WT1) is overexpressed and provides an oncogenic function in pediatric nephroblastomas harboring the wild-type WT1

Wilms' tumor 1 gene (WT1) is known to be a tumor suppressor gene in the subset of nephroblastomas that harbors WT1 mutations. However, its role in nephroblastomas without mutations remains unclear. This study aimed to evaluate the expression of WT1 and its potential oncogenic role in pediatric nephroblastoma with wild-type WT1. A total of 24 nephroblastomas were studied for WT1 mRNA expression by quantitative reverse-transcription polymerase chain reaction. The expression levels were compared between nephro-blastomas with and without WT1 mutations, as well as to normal kidney tissue, other pediatric renal tumors and neuroblastomas. Immunohistochemistry was used to evaluate expression patterns at the tissue level. Post-transcriptional inhibition of WT1 was performed in primary cultures of wild-type nephroblastoma using WT1 siRNA. The average WT1 expression level in nephroblastoma tissue was significantly higher than that in normal kidney tissue and neuroblastomas. Expression at the mRNA level was not different between nephroblastomas with WT1 mutations (4 cases) and those with wild-type WT1 (20 cases). However, while WT1 immunoreactivity was positive in all of the nephroblastoma components in the tumors with wild-type WT1, the protein expression was weaker and limited to stromal components in the tumors with mutated WT1, where it co-localized with β-catenin nuclear accumulation. The post-transcriptional inhibition of WT1 resulted in growth retardation and a significantly increased apoptotic fraction. Our study found overexpression of the WT1 gene in pediatric nephroblastomas with wild-type WT1. Moreover, the study suggests an oncogenic role of WT1 in this tumor subset.

Expression of WT1 gene in multiple myeloma patients at diagnosis: is WT1 gene expression a useful marker in multiple myeloma?

Monitoring patients with multiple myeloma during and after treatment for the presence of residual myeloma cells (minimal residual disease – MRD) has been shown to give a major insight into the effectiveness of treatment. It has been reported that Wilms' tumor gene (WT1) expression levels measured by real-time quantitative polymerase chain reaction was useful as an indicator of minimal residual disease in leukemia and myelodysplastic syndrome. The aim of this study was to measure levels of WT1 expression, in order to find a possible association between the expression of this gene and multiple myeloma at diagnosis. If an association was found, the WT1 gene could be evaluated as an MRD marker by comparison with other prognostic factors. We investigated peripheral blood WT1 expression level measured by real-time light cycler quantitative polymerase chain reaction in 50 newly diagnosed multiple myeloma patients. The normal WT1 gene copy number was found to be <23/μl cDNA and all patients with myeloma were found to have normal WT1-mRNA levels. On this basis WT1 expression analyses is unlikely to be a useful genetic marker for routine clinical use in multiple myeloma patients at diagnosis.

Mutation or loss of Wilms' tumor gene 1 (WT1) are not major reasons for immune escape in patients with AML receiving WT1 peptide vaccination

BackgroundEfficacy of cancer vaccines may be limited due to immune escape mechanisms like loss or mutation of target antigens. Here, we analyzed 10 HLA-A2 positive patients with acute myeloid leukemia (AML) for loss or mutations of the WT1 epitope or epitope flanking sequences that may abolish proper T cell recognition or epitope presentation.MethodsAll patients had been enrolled in a WT1 peptide phase II vaccination trial (NCT00153582) and ultimately progressed despite induction of a WT1 specific T cell response. Blood and bone marrow samples prior to vaccination and during progression were analyzed for mRNA expression level of WT1. Base exchanges within the epitope sequence or flanking regions (10 amino acids N- and C-terminal of the epitope) were assessed with melting point analysis and sequencing. HLA class I expression and WT1 protein expression was analyzed by flow cytometry.ResultsOnly in one patient, downregulation of WT1 mRNA by 1 log and loss of WT1 detection on protein level at time of disease progression was observed. No mutation leading to a base exchange within the epitope sequence or epitope flanking sequences could be detected in any patient. Further, no loss of HLA class I expression on leukemic blasts was observed.ConclusionDefects in antigen presentation caused by loss or mutation of WT1 or downregulation of HLA molecules are not the major basis for escape from the immune response induced by WT1 peptide vaccination.

WT1 expression as a marker of minimal residual disease, predicts outcome in acute myeloid leukaemia when measured at post-consolidation

Aims (1) Validate and establish a WT1 expression assay from peripheral blood and bone marrow. (2) Investigate prognostic relevance of WT1 expression levels at: diagnosis, post-induction and post-consolidation. Methods The study is prospective and longitudinal, in which WT1 expression from 107 patients with de novo acute myeloid leukaemia (AML) are measured from bone marrow ( WT1 _bm) and peripheral blood ( WT1 _pb) at various clinical time points. Results The cohort had a median age 56 years, 56% male, and 96 patients were treated with curative intent, achieving complete response (CR) in 79%, refractory disease in 7% and 14% induction death. At median follow-up time of 36 months, the median leukaemia free survival (LFS) was 12 months and median overall survival (OS) was 24 months. Median WT1 _pb level at diagnosis was 4558 and varied according to marrow blast percentage ( p = 0.022). Multi-variate analysis showed increased WT1 _pb at diagnosis is predictive of decreased LFS ( p = 0.033). Lower post-consolidation WT1 _pb significantly correlated with better LFS when measured as a continuous variable ( p = 0.006) or undetected versus detected (p = 0.02) and continued to be significant in multivariate analysis. Discussion A correlation between WT1 expression levels and known risk factors for early relapse is demonstrated. In the minimal residual disease setting, detectable WT1 levels in peripheral blood post-induction and post-consolidation predicted very poor leukaemia free survival.

Single Nucleotide Polymorphism in the Mutational Hotspot of WT1 Predicts a Favorable Outcome in Patients With Cytogenetically Normal Acute Myeloid Leukemia

We assessed the prognostic impact of a known single nucleotide polymorphism (SNP) located in the mutational hotspot of WT1 in patients with cytogenetically normal acute myeloid leukemia (CN-AML) in the context of other prognostic markers. WT1 exons 7 and 9 from 249 CN-AML patients from multicenter treatment trials AML-SHG Hannover 0199 (Clinical Trials Identifier NCT00209833) and 0295, and 50 healthy volunteers were analyzed by direct sequencing. NPM1, FLT3, CEBPA, and MLL were assessed for mutations and WT1 expression was quantified. The minor allele of SNP rs16754 (WT1(AG/GG)) was found in 25.7% of CN-AML patients' blasts and germline DNA and in 36% of healthy volunteers. Patient characteristics, frequencies of mutations, or WT1 expression levels were similarly distributed between patients homozygous for the major allele compared with patients heterozygous or homozygous for the minor allele. SNP rs16754 status was an independent predictor of relapse-free survival (RFS; hazard ratio [HR], 0.49; 95% CI, 0.3 to 0.81; P = .005) and overall survival (OS; HR, 0.44; 95% CI, 0.27 to 0.74; P = .002) in multivariate analysis. The favorable effect of SNP rs16754 was stronger in NPM1/FLT3-ITD (internal tandem duplication of the FLT3 gene) high-risk patients compared with NPM1/FLT3-ITD low-risk patients. Favorable prognosis could not be identified by any other known low-risk marker in half the patients with at least one minor allele (13% of all patients). No difference for complete remission rate, RFS, or OS was found between patients with or without acquired WT1 mutations. WT1 SNP rs16754 may be a novel independent favorable-risk marker in CN-AML patients that might improve risk and treatment stratification.

Clinical significance of Wilm tumor gene(WT1) expression in breast cancer

Objective To investigate the clinical significance of Wilm tumor gene (WT1) expression in breast cancer. Methods Real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and GAPDH mRNA expression levels in 110 cases of various breast tumor and the corresponding adjacent normal breast tissue. Normalized WT1 expression level (WT1_N)was determined as a ratio between WT1 and GAPDH for each case. The tumor tissue WT1_N over the normaltissue WT1_N of the same case was calculated as T/N_(WT1) ratio, and T/N_(WT1) value was analyzed with the clinicalpathological parameters. Results The WT1_N expression levels of the 102 breast cancer tissues were significantly higher than those of the adjacent normal breast tissues, with the median WT1_N of 2.38 (ranged from 0.12 to 112.3) and 0.81 (ranged from 0.03 to 11.65) for each (P <0.01), but there were no statistical differences between the WT1_N of 8 benign breast tumors and the nearing normal tissues, with the median WT1_N of 0.46 (ranged from 0.16 to 5.04) and 0.53 (ranged from 0.14 to 4.94) for each. Furthermore the WT1_Nas well as the T/N_(WT1) ratio of the malignant breast cancer tissues were significantly higher than those of the benign tumor tissues, with the median T/N_(WT1) value of 2.54 (ranged from 0.28 to 172.88) and 1.17 (ranged from 0.09 to 2.63) for each. Non-parameter correlation analysis showed that the T/N_(WT1) in breast cancers were of no relevance to lymph node metastasis, clinical-pathological types, estrogen receptor and progestone receptor status, but positively correlated with the expression level of IL-8 gene which calculated with T/N IL-8(r =0.723, P <0.01). Conclusion The WT1 gene is highly expressed in breast cancer which suggests that WT1 level assessed by RQ-RT-PCR could be a novel marker of disease progression and poor prognosis. Key words: Breast neoplasms; Gene; wilms tumor; RT-PCR; quantitative; real-time

WT1 Transcripts Is a Powerful Leukemia Marker to Predict Early Relapse After Allogeneic Haematopoietic Stem Cell Transplantation.

Abstract 4488▪ IntroductionDefinition of leukemia remission requires the lack of disease markers at sub-microscopic level. This is highly important after a potentially curative approach as allogeneic hematopoietic stem cell transplant (HSCT), where early detection of relapse at molecular level may lead to modulation of immunosuppressive therapy or donor lymphocyte infusion (DLI). In AML various approaches has been used to define MRD, but still the majority of AML cases do not have a useful and sensitive MRD marker. Over-expression of Wilms' tumor gene 1 (WT1) in leukemic blasts has been reported in >80% of AML and >40% of MDS. Physiologic hematopoietic stem cell compartments also express WT1, however, a ‘malignant' WT1 expression can be clearly distinguished based on quantitative detection methods such as semiquantitative RT-PCR. Quantification of WT1 expression levels can detect frequencies of leukemic cells in bone marrow (BM) and peripheral blood (PB) as low as 10-3 and 10-5, respectively. Therefore WT1 expression levels provide a new marker for leukemic blast cells regardless of the type of leukemia especially in the relevant percentage of patients that lacks a specific molecular marker of their disease and thus may be a useful marker MRD and may predict the relapse after allogeneic HSCT. Materials and methodsWe measured quantitative expression of WT1 at diagnosis, before and after allogeneic transplant monthly for the first six months and then every three months. The quantitative assessment of the WT1 transcript amount was performed by real-time quantitative polymerase chain reaction (RQ-PCR). ResultsOur study included 19 AML and 6 MDS pts who underwent allogeneic HSCT in our Institute between 12/2007 and 6/2009. Median age at diagnosis was 49 years (range 22-68). Bone marrow samples at diagnosis showed a WT1 median expression level of 5851.66 copies (range 77.98-31203.57). Fifteen pts (60%) had a specific cytogenetic marker that could allow MRD monitoring. At HSCT 17 pts (68%) were in CR, 5 (20%) had a refractory or relapsed disease, while 3 (12%) were transplanted upfront. Six pts (24%) received grafts from a matched sibling donors, 6 (24%) were transplanted from a matched unrelated donor (MUD), 10 (40%) from a familiar haploidentical donor and 3 (12%) received a cord blood unit. Myeloablative conditioning regimen consisted of Treosulfan 42 g/sqm, Fludarabine 150 mg/sqm, ALG 30 mg/kg and Rituximab 500 mg (last two drugs only for alternative donors). After HSCT a rapid decline of WT1 expression levels was observed in all pts that obtained or maintained a condition of CR. Two pts (8%) relapsed and both had an increase in WT1 expression before relapse. In the first relapsed pt, analysis of WT1 showed a dramatically increase between pre transplant level and day +28, while STR showed 100% donor chimerism. Relapse occurred on day +43, still on IST, with 69% of blast at AM evaluation and 40% donor chimerism. This pt was successfully reinduced with chemotherapy followed by allogeneic PBSC infusion without GvHD profilaxys obtaining a rapid reduction of WT1 (43.66). The pt developed GvHD that required a new IST, confirming a strong immune-surveillance of HSCT. The second pt was still on immunosuppressive treatment (IST) at the time of relapse (+136). WT1 in BM on day +30 was 30 cp and then increased gradually to 167 and 190 cp on day +67 and +102, respectively. Cytogenetic analysis and STR chimerism still showed a cCR with 100% donor chimerism. At relapse AM showed 10% blasts, WT1 expression level was 7447 cp with >95% donor chimerism. IST was discontinued and one month later WT1 expression level decreased to 1640 with >95% donor chimerism. In this situation we reasoned that DLI was the best available treatment. The total dose of donor T cell infused was 5 ×105 CD3/Kg. This procedure allowed an immune-mediated leukemia control with reduction of WT1 (1.58), cCR and 100% donor chimerism. ConclusionsThese data show that WT1 may be useful as a non-specific leukemia marker for monitoring MRD in AML and MDS after allogeneic HSCT and should enable the detection of early relapse allowing intervention at a more favourable stage than at overt relapse. We observed a complete concordance between WT1 expression levels and status of AML before and after allogeneic HSCT. Based on these results cases with increase of WT1 levels after HSCT and without GVHD may be candidate to immune intervention such as discontinuation of immunosuppression and/or DLI. Disclosures:Bonini:MolMed S.p.A.: Consultancy.

WT1 Expression Levels at Diagnosis and as a Marker of Minimal Residual Disease (MRD) in Patients with Acute Myeloid Leukaemia (AML).

Abstract 2639▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-615 Background.-As wt1 expression is elevated in the majority (80%) of acute myeloid leukemias at diagnosis and relapse, a validated wt1 assay would be a powerful adjunctive tool for evaluation and monitoring of patients during their treatment. Monitoring of disease status with wt1 is applicable to more AML patients in a single assay than any other leukemic marker and includes patients that are cytogenetically normal and may not otherwise be amenable to molecular MRD evaluation. We aimed to analyse wt1 expression in the peripheral blood and bone marrow of patients with de novo AML, when measured at diagnosis, post-induction and post-consolidation; and to correlate these wt1 expression levels with patient characteristics and outcome. Methods-This is a prospective, longitudinal study in which the levels of wt1 expression from 99 patients with de novo AML are measured from bone marrow (59 patients) and peripheral blood (94 patients) specimens at the time of diagnosis and at various time-points along their treatment and clinical courses. The specimens were prospectively stored in the PwC Australasian Leukaemia and Lymphoma Group (ALLG) Tissue Bank. Samples analysed included: 59 bone marrow (bm-wt1) and 94 peripheral blood (pb-wt1) at diagnosis; 54 bm-wt1 and 57 pb-wt1 post-induction; and 46 bm-wt1 and 41 pb-wt1 post-consolidation. 90 of the 99 patients were treated with standard induction chemotherapy on a variety of protocols. RNA was extracted from 107 leukocytes purified from blood and bone marrow using TRIzol® (Invitrogen) extraction. Reverse transcription was performed using Superscript ® (Invitrogen) and RQ-PCR was performed using multiplexed 5' nuclease assay with Black Hole Quencher labelled probes according to published methods. Copy number of wt1 was normalised to 104 copies of ABL. Baseline patient characteristics were correlated with baseline wt1 expression levels and the impact of baseline and post-treatment wt1 expression on leukemia-free survival (LFS) was assessed. Results-99 AML patients were studied: median age 55years, 53% male. Cytogenetic prognostic groups (SWOG) were favourable – 12%, intermediate – 59% and poor – 29%. 90 patients were treated with curative intent and 83% achieved CR, 6% had refractory disease and 11% died during induction. With a median follow-up of 33 months, the median LFS was 12 months and median OS was 24 months. Median pb-wt1 levels at diagnosis were 5122 (range, 1 to 36125) and varied significantly according to: cytogenetic risk group (good risk 12745, intermediate risk 2632, poor risk 4554 (P = 0.009)) and marrow blast percentage (P = 0.02). Diagnostic bm-wt1 showed similar correlations with baseline parameters, but did not reach statistical significance. Median pb-wt1 at relapse was 3781. One of 21 relapsed patients with elevated pb-wt1 expression at diagnosis did not express wt1 at relapse. In a multivariate Cox regression analysis model including age and cytogenetic risk-group, increased expression of wt1 from peripheral blood at diagnosis is predictive of decreased LFS (P=0.04). This correlation was not seen with wt1 measured from bone marrow aspirates. The wt1 expression levels were reduced following induction chemotherapy with a median 2.6 log reduction (range, 0 to 4.2) seen in marrow aspirate and 3.2 log reduction (range, 0 to 4.3) seen in peripheral blood. There was a trend for lower post-induction peripheral blood wt1 to correlate with better LFS when measured as a continuous variable (P=0.067) or undetected vs detected (P=0.051). Lower post-consolidation pb-wt1 significantly correlated with better LFS when measured as a continuous variable (P=0.002) or undetected vs detected (P=0.02) and continued to be significant in multivariate analysis. Bone marrow aspirate wt1 levels post-induction or post-consolidation did not correlate with LFS. Conclusion-This study demonstrates a correlation between wt1 expression levels and known risk factors for early relapse. In the MRD setting, detectable wt1 levels in peripheral blood post-induction and post-consolidation predicted very poor leukaemia free survival. Analysis is ongoing to validate these findings in a uniformly treated cohort of patients enrolled on the current ALLG clinical trial. [Display omitted] Disclosures:No relevant conflicts of interest to declare.