Low WT1 Expression Research Articles

Wilms’ tumor 1 expression combined with genetic mutations for prognostic assessment in MDS

Overexpression of Wilms’ tumor (WT1) is frequently observed in myelodysplastic syndrome (MDS), which has been proposed as a prognostic marker. However, the prognostic role of WT1 expression in different contexts remains to be fully elucidated. We retrospectively assessed the relationships between WT1 levels and preexisting prognostic factors to further investigate its prognostic role under different contexts. In our study, WT1 expression was positively correlated with WHO 2016 classification and IPSS-R stratification. Lower WT1 expression was found in relation to TET2, TP53, CD101, or SRSF2 mutations, while mutant NPM1 patients possessed higher level. Notably, WT1 overexpression maintained its inferior prognostic effect on overall survival (OS) in TP53-wild patients but not in TP53-mutated group. In multivariate analysis, higher WT1 expression was a risk factors for OS in EB patients without TP53 mutations. Overall, WT1 expression was useful to predict prognosis for MDS and its prognostic role was impacted by some gene mutations.

Reduced Toxicity FluBu3 Regimen Showed a Comparable Post-Transplant Outcomes to a Myeloablative BuCy Conditioing in Acute Myeloid Leukemia Patients Receiving First Allogeneic Hematopoietic Stem Cell Transplantation in MRD Negative CR1

Introduction: In a previous report, we found that post-transplant clinical outcomes were not significantly different between MAC (BuCy, CyTBI) and RIC (FluBu2TBI400) groups in AML CR1 with pre-transplant low WT1 expression. Based on this result, we expanded the use of conditioning regimen with reduced dose intensity for patients achieving deep response by minimal residual disease (MRD) assessment with available assays. FluBu3 regimen, consisting of fludarabine for 5 days (30mg/m2/day, D-6 to D-2) and busulfan for 3 days (3.2mg/kg/day, D-5 to D-3), was chosen and applied as reduced toxicity conditioning (RTC) in our institution since Jan 2017. Method: In this analysis, we compared MAC and RTC regimens; BuCy (busulfan 3.2 mg/kg/day for 4 days from D-7 to D-4 and cyclophosphamide 60 mg/kg/day for 2 days from D-3 to D-2) and FluBu3 protocols were used as MAC and RTC regimen, respectively. The inclusion criteria were set as follows: (1) adult (age ≥ 18 years) AML patients who were registered in a prospective cohort study; (2) receiving first allogeneic hematopoietic stem cell transplantation (HSCT) in CR1 between Aug 2017 and Nov 2021; (3) MRD negative at pre-transplant; (4) transplantation from either HLA-matched (8/8) sibling donor (MSD), matched unrelated donor (MUD) or 1-locus mismatched (7/8) unrelated donor (MMUD); (5) the use of either BuCy or FluBu3 conditioning. MRD negativity was defined by the absence of abnormalities in the following order; (i) <3 log reduction in RUNX1-RUNX1T1 and CBFB-MYH11, (ii) <4 log reduction of NPM1 level from diagnosis, and (iii) pre-transplant WT1 levels ≥ 250 copies for RUNX1-RUNX1T1, CBFB-MYH11, or NPM1 mutation negative AML. Peripheral blood was used as the source of stem cells in all patients, and the data was analyzed based on information available as of Jun 2022. Results: A total of 124 patients met the inclusion criteria, and 78 (62.9%) and 46 (37.1%) patients received BuCy and FluBu3 conditioning, respectively. The median age was 45 years and the patients with intermediate cytogenetics comprised the majority (71.8%). By the donor type, 64 (51.6%), 48 (38.7%) and 12 (9.7%) patients underwent MSD, MUD and MMUD transplantation. Between the two conditioning groups, patients receiving FluBu3 were older (median 39.5 vs 53 years, p<0.001) and significantly more patients in the FluBu3 group had received ATG (37.9% vs 93.5%, p<0.001). In each of the BuCy and FluBu3 groups, after median follow up of 39.5 and 53 months (p<0.001), the 2-year overall survival (OS) of 78.3% and 84.5% (p=0.358), relapse free survival (RFS) of 78.0% and 76.3% (p=0.806), cumulative incidence of relapse (CIR) of 7.7% and 21.5% (p=0.074) and non-relapse mortality (NRM) of 14.3% and 2.2% (p=0.0324) were observed. In addition, the probabilities of grade II-IV acute graft versus host disease (aGVHD) and cGVHD of moderated to severe at 2-year for BuCy and FluBu3 were 46.2% and 39.2% (p=0.306), and 59.5% and 36.0% (p=0.008), respectively. At the time of data cut-off, relapse was the main cause of HSCT failure in FluBu3 group (n=8) while NRM contributes most to HSCT failure in BuCy group (n=11). Six of 8 relapsed patients receiving FluBu3, compared to 1 out of 6 in BuCy group, had high-risk features of either poor cytogenetics or FLT3-ITD mutation. The majority of NRM in the BuCy group, 8 out of 11 cases, was due to GVHD and 4 of them received transplantation from MMUD. Conclusion: Among the AML patients in MRD negative CR1, the FluBu3 RTC regimen showed a comparable post-HSCT outcomes to a BuCy conditioing in terms of similar OS and RFS, while associated with significant reduced NRM that was offset by trend towards higher risk of relapse.

PB1895: ANALYSIS REVEALS CANCER-ASSOCIATED GENE MUTATIONS IN CHRONIC MYELOID LEUKEMIA PATIENTS ：A CHINESE MONOCENTRIC RETROSPECTIVE STUDY

Background: Recent genetic studies have shown that tyrosine kinase inhibitors (TKI) resistant or intolerant Chronic myeloid leukemia (CML) patients often carry one or more genetic changes related to myeloid malignancies, suggesting that additional mutations other than ABL1 mutation may lead to TKI treatment failure or disease progression. Aims: We performed sequencing of 22 CML patients using next-generation sequencing (NGS). 23 CML patients tested WT1 expression detection. Methods: The second cohort (No. 1) tested by second-generation sequencing included 22 CML patients. The second cohort (No. 2) tested by WT1 expression detection included 23 CML patients. The study has been approved by the institutional ethics committee and patients provided written informed consent in accordance with the Fourth Affiliated Hospital of Zhejiang University School of Medicine.Definitions Response was defined as BCR-ABL1 transcript levels according to the International Scale (IS) ≤10%, ≤1% and ≤0.1% at 3, 6 and 12 months after initiating TKI-therapy. Molecular response with 4.0 log reduction (MR4.0) was defined as 0.01%. Molecular response with 4.5 log reduction (MR4.5) was defined as 0.0032% or less BCR-ABLtranscript level. The comparison between groups of measurement data showed that the data with normal distribution were analyzed by one-way ANOVA or t-test. Results: Overall, a total of 51 mutations involving 25 genes were detected most frequently in the ASXL1 (40.9%), KMT2C (27.3%), DIS3 (18.2%), ATM (13.6%), DNMT3A (13.6%) and NOTCH3 (13.6%) genes. NOTCH3 and RELN mutations were only 3/20 (10%) in those who did not reach 12M-MMR group. Although the difference was not statistically significant (P = 0.214, P = 0.476). The number of median mutant genes in reaching MR4.5 group was 1 (0-3). The number of median mutant genes in not reaching MR4.5 group was 4 (0-6), and the difference was statistically significant (P = 0.033). ASXL1 mutation in reaching MR4.0 group and non reaching MR4.0 group was statistically significant (P = 0.023). With KMT2C mutation group had lower PFS (P = 0.079). There was a potential statistical difference between low WT1 expression group and high WT1 expression group in early EMR after a 3-month treatment (P = 0.089).The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011; the 2019–2024 Academician Workstation of the Fourth Affiliated Hospi-tal of the Zhejiang University School of Medicine; the 2019–2022 Key Medical Dis-cipline (Hematology) Fund of Jinhua, China. *Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China.E-mail: [email protected] Image:Summary/Conclusion: Genomic risk assessment can improve the risk stratification of clinical diagnosis, and can more accurately identify patients with poor effect of TKI treatment in the era of TKI treatment. The treatment effect of CML patients with NOTCH3, RELN and ASXL1 mutations and more than two gene mutations is poor. CML patients with high expression of WT1 at diagnosis have poor therapeutic effect in the early stage.

Absence of BCL-2 Expression Identifies a Subgroup of AML with Distinct Phenotypic, Molecular, and Clinical Characteristics.

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the rapid and uncontrolled clonal growth of myeloid lineage cells in the bone marrow. The advent of oral, selective inhibitors of the B-cell leukemia/lymphoma-2 (BCL-2) apoptosis pathway, such as venetoclax, will likely induce a paradigm shift in the treatment of AML. However, the high cost of this treatment and the risk of additive toxicity when used in combination with standard chemotherapy represent limitations to its use and underscore the need to identify which patients are most—and least—likely to benefit from incorporation of venetoclax into the treatment regimen. Bone marrow specimens from 93 newly diagnosed AML patients were collected in this study and evaluated for BCL-2 protein expression by immunohistochemistry. Using this low-cost, easily, and readily applicable analysis method, we found that 1 in 5 AML patients can be considered as BCL-2−. In addition to a lower bone marrow blast percentage, this group exhibited a favorable molecular profile characterized by lower WT1 expression and underrepresentation of FLT3 mutations. As compared to their BCL-2+ counterparts, the absence of BCL-2 expression was associated with a favorable response to standard chemotherapy and overall survival, thus potentially precluding the necessity for venetoclax add-on.

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

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

The assessment of minimal residual disease versus that of somatic mutations for predicting the outcome of acute myeloid leukemia patients

BackgroundIn addition to morphological and cytogenetic features, acute myeloid leukemias are characterized by mutations that can be used for target-therapy; also the minimal/measurable residual disease (MRD) could be an important prognostic factor. The purpose of this retrospective study was to investigate if somatic mutations could represent an additional prognostic value in respect of MRD alone.MethodAt baseline, 98 patients were tested for NPM1, FLT3, and for WT1 expression; 31 for ASXL1, TET2, IDH1, IDH2, N-RAS, WT1, c-KIT, RUNX1, and DNMT3A. The same genes have been also tested after induction and consolidation.ResultsOverall, 60.2% of our patients resulted mutated: 24.5% carried mutations of FLT3-ITD, 38.7% of NPM1, 48.4% of c-KIT, 25.8% of N-RAS and 19.3% of IDH2. The probability of achieving a complete response (CR) was higher for younger patients, with low ELN risk score, NPM1-mutated, with low WT1 levels, and without FLT3. The presence of additional mutations represented a poor predictive factor: only 19% of these cases achieved CR in comparison to 43% of subjects without any of it. Concerning survival, it was conditioned by a lower ELN risk score, younger age, reduction > 1 log of the NPM1 mutational burden, disappearance of FLT3 mutations and lower WT1 expression. Regarding the role of the additional mutations, they impaired the outcome of 20% of the already MRD-negative patients. Concerning the possibility of predicting relapse, we observed an increase of the NPM1 mutational burden at the time-point immediately preceding the relapse (about 2 months earlier) in 50% of subjects. Similarly concerning WT1, an increase of its expression anticipated disease recurrence in 64% of cases.ConclusionsWe demonstrated that additional somatic mutations are able to impair outcome of the already MRD-negative subjects. About MRD, we suggest a prognostic role also for the WT1 expression. Finally, we considered as relevant the assessment of NPM1 quantity clearance instead of the presence/absence of mutations alone. Still remains in doubt the utility in terms of long-term prognosis of a baseline more complex mutational screening; we could hypothesize that it would be useful for those patients where other markers are not available or who reached the MRD negativity.

Assessment of WT1 Expression as a Marker of Treatment Outcome in Karyotype Normal Acute Myeloid Leukemia Patients in Pakistan.

Currently, there is an effort to predict relapse by follow-up monitoring of MRD and subsequently to begin the treatment of the patients during their clinical and hematological remission prior to overt hematological relapse. Expression of WT1 in AML is known to be independently associated with significant inferior response to therapy and short survival outcome. Follow-up monitoring of WT1 gene expression during or after therapy would be a valuable predictive marker for early recurrence or relapse of AML disease. This pilot study evaluated newly diagnosed and post-induction or consolidation chemotherapy of AML patients who were registered with the Oncology Clinics of the Aga Khan University Hospital, Karachi. High WT1 burden (> 5000 copies/ml) in 2 patients was indicative of early recurrence of the disease along with shorter disease-free and overall survival. Low WT1 expression (< 200 copies/ml) in 2 patients after induction and consolidation therapy, respectively, was suggestive of better prognosis.

Expression of WT1 gene in acute leukemia and its significance

Objective To explore the relationship between WT1 and prognosis of patients with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), and to evaluate the possibility of WT1 as a potential marker for monitoring the minimal residual disease (MRD). Methods Bone marrow mononuclear cells from 58 patients with primary AML, 32 patients with primary ALL, 40 patients with AML-complete remission (CR), 28 patients with ALL-CR and 31 patients with trilineage hyperplasia (control group) were collected. Real-time fluorescent quantitative PCR method was used to detect the expression of WT1 in all patients. The expression threshold of WT1 in each group was established. WT1 copy number/ABL copy number ratio×100% denotes the relative expression level of WT1 gene. Results Median relative expression level of WT1 in the control patients was much lower than that in primary AML patients [0.026% (0-0.240%) vs. 20.880% (3.550%-48.500%), Z=-7.74, P 20.880%) was 60.7% (17/28), while the CR rate was 76.7% (23/30) in those with lower WT1 expression. WT1 expression was increased dramatically in recurrent AML patients. Relative expression level of WT1 was significantly higher in primary ALL patients [0.350% (0.021%-10.780%)] compared with that in the control group Z=-2.58, P<0.05. There was no significant difference in relative expression level of WT1 between ALL and ALL-CR patients [0.038% (0-2.800%), P=0.065]. Conclusion WT1 expression level in AML patients is relatively high, which could be used as an effective index of prognosis evaluation and MRD monitoring for AML patients, but not for ALL patients. Key words: Leukemia, acute; Gene, WT1; Polymerase chain reaction

Does the level of WT1 expression predict the outcome in Philadelphia-negative myeloproliferative neoplasms?

Despite the clinical importance of the leukemic transformation of chronic myeloproliferative neoplasms (MPNs), very little is known about markers that predict leukemic transformation. We studied WT1 expression in 37 MPN patients diagnosed as bcr-abl negative and JAK2 (V617F) positive with a molecular genetic test, and 23 healthy controls. WT1 expression is higher in MPN patients compared with normal controls (p=0.002). According to the WT1 expression levels, patients were divided into two groups: high (≥0.205) and low (0-0.205) WT1 expression. Two out of six patients with a high WT1 expression level transformed to myelodysplastic syndrome at a 42- and 46-month follow-up, respectively. Our results suggest that the overexpression of WT1 may play an important role in the leukemic transformation of MPNs.

Prognostic importance of expression of the Wilms’ tumor 1 gene in newly diagnosed acute promyelocytic leukemia

Wilms’ tumor 1 gene (WT1) is known to be highly expressed in acute promyelocytic leukemia (APL) but information on its impact on prognosis is lacking. WT1 expression was analyzed in bone marrow samples of 79 patients with APL at initial diagnosis. Patients had a differing outcome according to their level of WT1 expression. In patients who achieved a complete remission (CR), low or high WT1 expression was significantly associated with inferior overall survival (OS) compared to intermediate WT1 expression (49% for WT1high vs. 63% for WT1low vs. 93% for WT1int; p = 0.008). Moreover, there were significant differences in relapse-free survival (RFS) between the three expression groups (42% for WT1high vs. 63% for WT1low vs. 83% for WT1int; p = 0.047). In multivariable analysis WT1 expression showed an independent prognostic impact on OS of responders to induction therapy. In conclusion, the level of WT1 expression can add prognostic information in APL risk stratification.

WT1 Expression In Acute Myeloid Leukaemia: A Useful Marker For Improving Therapy Response Evaluation

IntroductionWilms Tumor gene 1 (WT1) is overexpressed in the vast majority of patients (pts) in Acute Myeloid Leukemia.(1) Recently the decrease of WT1 expression was suggested to reflect cytoreduction after chemotherapy in AML.(2) However, despite its potential role in minimal residual disease (MRD) assessment, at the moment there is no consensus on its clinical application. Patients/Methods233 clinically and molecularly well characterized consecutive AML pts were treated with intensive induction chemotherapy according to age, performance status and co-morbidities in a single hematology center. WT1 expression was analyzed by Real Time Polymerase Chain Reaction (RT-PCR), using the standardized European LeukemiaNet (ELN) method, at baseline and after induction chemotherapy. Further, WT1 expression was compared with several demographic (sex, age), clinical (organomegaly and leucocytosis), biological (genetic and genomic aberrations, immunophenotype) and therapy related variables.Aim of this retrospective analysis was to investigate the role of WT1 expression in risk stratification of de novo AML, its potentially added value to already established risk factors and its possible role for response evaluation. ResultsWT1 expression at diagnosis was determined in the bone marrow of all pts with a mean value of 9379 copies (range 1-83200 copies). There was no statistical significance in the difference of WT1 expression when pts were divided on the basis of demographic or clinical variables. Interestingly with increasing age of pts at diagnosis WT1 expression decreased significantly, for instance the patient group 19-30 years (yrs) showed higher copies than the 41-50 yrs- or the 51-60 yrs group, p=0.019 and p=0.004, respectively. Organomegaly or amount of blasts in peripheral blood didn't show any correlation, whereas a high number of blasts in the bone marrow expressing AML specific antigens displayed significant correlation with high WT1 expression (p=0.024).There was no significant correlation between WT1 expression and cytogenetic abnormalities, whereas there was a significant correlation between WT1 expression and the presence or absence of FLT3. Patients with FLT3 mutations (n=38) had significantly higher WT1 expression levels than pts with FLT3 wild type (wt) (n=170) at baseline, 58709,4 vs 8710,3 copies (p=0.0008). Significant correlation was also obtained when subdividing the pts on the basis of NPM1 mutations. Patients presenting a NPM1 mutation showed higher WT1 expression than NPM1 wt pts (p=0.018). Even the combination of FLT3 and NPM1 yielded statistically significant correlation with WT1 expression. Patients with FLT3 and NPM1 mutations had higher WT1 copies than FLT3 and NPM1 wt pts (p=0.028).Bone marrow analysis of WT1 expression was repeated after induction chemotherapy in 133 pts (57%) with a mean value of 891 copies (range 0-23261 copies).The achievement of complete remission (CR) according to ELN criteria (3) in 110 pts was related to significant decrease of WT1 expression (p<0,0001), whereas 23 non-responders didn't show significant diminution in WT1 expression (p=0,1) after induction chemotherapy.Moreover, 17 CR pts with high WT1 expression (>350 copies) after first induction chemotherapy showed reduced disease-free survival (DFS) compared to 89 pts in CR with low WT1 expression (<350 copies, median 16 months vs 40 months, p=0.01).In conclusion, in the present cohort high WT1 expression correlates significantly with high disease burden as expressed by a high number of AML blasts in the bone marrow at diagnosis as well as acknowledged bad prognosis markers such as FLT3 mutation at diagnosis. Additionally, significant reduction of the WT1 expression level is detected in the patient group that shows complete response after intensive induction chemotherapy. The subgroup of pts with CR after induction chemotherapy who failed to reduce significantly WT1 expression had reduced DFS, probably because of higher burden of residual disease.These results may support the value of the quantitative assessment of WT1 expression in the bone marrow as an additional potential marker for therapy response evaluation in AML and warrant a prospective study in order understand better its value.(1) Cilloni D, Saglio G. Acta Haematol 2004;112:79-84(2) Grimwalde H, Vyas P, Freeman S. Curr Opin Oncol 2010;22:656-663(3) Dohner H, Estey EH, Amadori S et al. Blood 2010;115:453-474 Disclosures:No relevant conflicts of interest to declare.

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.

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.

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 Mutation and 11P15 Loss of Heterozygosity Predict Relapse in Very Low-Risk Wilms Tumors Treated With Surgery Alone: A Children's Oncology Group Study

Children's Oncology Group defines very low-risk Wilms tumors (VLRWT) as stage I favorable histology Wilms tumors weighing less than 550 g in children younger than 24 months of age. VLRWTs may be treated with nephrectomy alone. However, 10% to 15% of VLRWTs relapse without chemotherapy. Previous studies suggest that VLRWTs with low WT1 expression and/or 11p15 loss of heterozygosity (LOH) may have increased risk of relapse. The current study validates these findings within prospectively identified children with VLRWT who did not receive adjuvant chemotherapy. Fifty-six VLRWTs (10 relapses) were analyzed for mutation of WT1, CTNNB1, and WTX; for 11p15 LOH using microsatellite analysis; and for H19DMR and KvDMR1 methylation. 11p15 LOH was identified in 19 (41%) of 46 evaluable VLRWTs and was significantly associated with relapse (P < .001); 16 of 19 were isodisomic for 11p15. WT1 mutation was identified in nine (20%) of 45 evaluable VLRWTs and was significantly associated with relapse (P = .004); all nine cases also had 11p15 LOH. All evaluable tumors showing LOH by microsatellite analysis also showed LOH by methylation analysis. Retention of the normal imprinting pattern was identified in 24 of 42 evaluable tumors, and none relapsed. Loss of imprinting at 11p15 was identified in one of 42 tumors. WT1 mutation and 11p15 LOH are associated with relapse in patients with VLRWTs who do not receive chemotherapy. These may provide meaningful biomarkers to stratify patients for reduced chemotherapy in the future. VLRWTs show a different incidence of WT1 mutation and 11p15 imprinting patterns than has been reported in Wilms tumors of all ages.

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.

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.

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.