WT1 mRNA Expression Research Articles

Wilms’ tumour gene 1 (WT1) enhances non-small cell lung cancer malignancy and is inhibited by microRNA-498-5p

BackgroundWilms’ tumour gene 1 (WT1) is clearly recognized as a tumour promoter in diversiform of human malignancies. Nevertheless, knowledge of its expression, functions and potential molecular mechanisms in non-small cell lung cancer (NSCLC) remains elusive.MethodsDifferential expression of WT1 mRNA and protein between NSCLC and normal tissues were assessed by analyzing RNA-seq data from Oncomine and protein data from Human Protein Atlas, respectively. Subsequently, prognosis significance and immune cell infiltration were analyzed by Kaplan-Meier plotter and CIBERSORT. 60 pairs of local NSCLC tissues were involved to validate WT1 expression by quantitative PCR (qPCR) and Western blot. Moreover, Cell Counting Kit-8 (CCK-8), colony formation, transwell, dual luciferase reporter assays and in vivo xenograft tumour growth experiments were conducted to explore the function and mechanism of WT1 in NSCLC.ResultsOur solid data indicated that WT1 was increased in NSCLC tissues and cell lines in comparison with their matched controls. In particular, its upregulation correlated with worse prognosis and immune infiltration of the patients. Functional assays demonstrated that knockdown of WT1 inhibited NSCLC malignancy, including inhibiting cell proliferation, survival and invasion. Further exploration discovered that microRNA-498-5p (miR-498-5p) was the upstream suppressor of WT1 by directly targeting the 3’ untranslated region (UTR) of WT1 mRNA. Moreover, expression of miR-498-5p was notably decreased and inversely correlated with WT1 in NSCLC tissues. Finally, we proved that miR-498-5p was a potent tumour suppressor in NSCLC by suppressing cell proliferation, survival and invasion, while WT1 restoration could in turn disrupt this suppression both in vitro and in vivo.ConclusionThe abnormal increase in WT1 contributes to the malignant properties of NSCLC cells, and miR-498-5p is a natural inhibitor of WT1. Our findings might facilitate the development of novel therapeutic strategies against NSCLC in the future.

Study of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples.

Simultaneous quantitative measurement of mRNA of the WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples reflects the specific pathological proliferative activity in acute leukemia and their ratio is promising as a diagnostic marker. The transcriptome profile of acute leukemia cells is usually assessed using NGS or microarray techniques after a preliminary procedure for isolation of mononuclear cells. However, the results of using the multiplex PCR reaction for the simultaneous determination of all above mRNAs in whole blood samples have not been published so far. Determination of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in venous blood level samples by multiplex RT-PCR. The study included 127 blood samples from patients who diagnosis of acute leukemia was subsequently confirmed. In the comparison group, 87 samples of patients without oncohematological diagnosis were selected, including 31 samples (K1) with a normal blood formula and 56 samples (K2) with a violation of the cellular composition - anemia, leukocytosis and thrombocytopenia. RNA isolation and reverse transcription were performed using the Ribozol-D and Reverta-L kits (TsNIIE, Russia). Determination of the mRNA expression level of the WT1, BAALC, EVI1, PRAME and HMGA2 genes by multiplex real-time PCR using a homemade multiplex PCR kit. The mRNA level was characterized by high interindividual variation and did not correlate with the rate of circulating leukocytes or blood blasts. Expression of WT1 mRNA was observed in whole blood only in one patient from the control group and in 112 (88%) patients with leukemia and was combined with a decrease in the level of HMGA2 mRNA expression and BAALC mRNA values. In contrast to the control groups, patients with leukemia had higher levels of BAALC mRNA in AML and ALL, increased PRAME mRNA in AML and APL, but lower levels of HMGA2 in APL.

The nephroprotective action of Passiflora edulis in streptozotocin-induced diabetes

In the present study, we aimed to evaluate the therapeutic effect of Passiflora edulis fruit peel aqueous (AFA) extract as an adjuvant to insulin to confer nephroprotection against streptozotocin-induced diabetes. Male Wistar rats were divided into four groups based on treatment received for 60 days: diabetic (DB), control (CTL), insulin (INS), and insulin + AFA extract (INS + AFA). mRNA and protein expression levels of podocyte (nephrin, podocin, and WT1) and tubular (megalin) proteins were measured in kidney tissue specimens and urine. Biochemical parameters and kidney histopathology were also examined. Herein, the INS + AFA group showed superior glycemic control, which resulted in the reduction of urinary albumin/creatinine ratio, maintenance of baseline levels of Nphs1, Nphs2, Wt1, and Lrp2 mRNA expression, prevention of protein loss from the kidney tissue into the urinary space, along with the maintenance of glomerular basement membrane thickness, hyalinization, glomerular and tubulointerstitial fibrosis at values approximating those of the CTL group and significantly lower than those in the DB group. Therefore, these results suggest that, as an anti-diabetic agent, the AFA extract adjuvant to insulin could reduce and potentially prevent diabetic kidney disease.

Overexpression of Prohibitin 2 Protein is Associated with Adverse Prognosis in Cytogenetically Normal Acute Myeloid Leukemia.

Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for 40%–50% of all AML cases. Despite advances in understanding the molecular pathophysiology of CN-AML, its clinical outcome remains unsatisfactory and unpredictable. To investigate its clinical implication in CN-AML, we measured the expression of prohibitin 2 (PHB2) using immunohistochemical staining (IHCS) of paraffin-embedded bone marrow sections from 134 CN-AML patients. IHCS results were semi-quantitatively scored. Clinical outcome was analyzed in comparison with other prognostic markers, including NPM1 polymorphism and FLT3 internal tandem duplication, and WT1 and BAALC mRNA expression. Except for BAALC mRNA expression, the known molecular markers showed no prognostic effect in the CN-AML patients. PHB2 protein overexpression was significantly associated with adverse prognosis in CN-AML patients. The PHB2 protein expression status may serve as an independent prognostic indicator in CN-AML.

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

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

Stratification of Intermediate-Risk Acute Myeloid Leukemia According to the Expression Level of WT1 mRNA at Diagnosis

IntroductionDespite advances in the genetic analysis of acute myeloid leukemia (AML), Wilms' tumor oncogene 1 (WT1) mRNA remains an important pan-marker of AML. A log reduction in WT1 mRNA expression after chemotherapy is a predictor of prognosis, and WT1 mRNA can be used as a marker of minimal residual disease or relapse. In the 1990s, a high expression level of WT1 mRNA at diagnosis was considered a poor prognostic factor. However, recent analyses have found that WT1 mRNA expression varies with AML type. Since the prognosis is affected by cytogenetic abnormalities and therapeutic intensity, we re-evaluated the clinical significance of WT1 mRNA expression at diagnosis in the context of the European Leukemia Net (ELN) risk classification and treatment.MethodWe retrospectively analyzed 216 patients at five institutions between 2011 and 2020. The expression level of WT1 mRNA was measured for all patients at diagnosis using bone marrow (BM) samples from 191 patients and peripheral blood (PB) samples from 25. WT-1 mRNA expression was measured using real-time quantitative polymerase chain reaction and the measured values were converted in normal logarithm. The median age at diagnosis was 62 (range: 23-93) years. The cytogenetic risk of ELN was classified as favorable (n = 41), intermediate (n = 123), and adverse risk (n = 41). Selected therapeutics were standard chemotherapy (n = 182, 84.3%, including CAG regimen; cytarabine, aclarubicin and G-CSF), hypomethylating agents or low-dose cytarabine (n = 19, 8.8%), and best supportive care (n = 15, 6.9%). Also, 143 patients (66.2%) received one or more courses of standard consolidation chemotherapy and 61 (28.3%) underwent allogeneic hematopoietic stem cell transplantation (allo-HCT). The median observation period was 518 [1-3418] days (Table 1). The overall survival (OS) and event-free survival (EFS) rates were assessed. Relapse or death was defined as an event, and OS was evaluated on the date of death.ResultThe median expression level of WT1 mRNA was 4.68 (range: 1.0-5.72) [log copies/µg RNA, units are omitted thereafter] in BM and 3.66 (range: 1.34-5.20) in PB (Table 1). Favorable-risk AML had the highest WT1 mRNA expression level in BM (4.95, p = 0.0054), whereas there was no difference between the expression levels in intermediate- and adverse-risk AML in BM (4.63 and 4.47, p = 0.711, Fig. 1A). WT1 mRNA expression in PB were 4.04, 3.84, and 3.05 for favorable-, intermediate-, and adverse-risk AML, respectively, and were higher for those with a favorable-risk AML (vs. adverse risk, p = 0.048, Fig. 1B). When WT1 mRNA expression level in BM was compared between the two groups, i.e., <4.0 (BM-WT1 low) vs. ≥4.0 (BM-WT1 high), 2-year EFS rates were 26.8% and 49.7% (p = 0.0035) and 2-year OS rates were 44.9% and 61.5% (p = 0.00053), respectively. When WT1 mRNA expression level in PB was compared between the two groups, i.e., <3.0 (PB-WT1 low) vs. ≥3.0 (PB-WT1 high), 2-year EFS rates were 0% and 51.5% (p = 0.023) and 2-year OS rates were 0% and 73.1% (p = 0.01), respectively. PB- or BM-WT1 low showed a worse prognosis at diagnosis.Since AML prognosis is affected by ELN risk and selected therapeutics, we evaluated 109 intermediate-risk patients who had received at least one course of standard chemotherapy. Fifty of them (45.9%) underwent subsequent allo-HCT. The 2-year EFS rates of PB or BM-WT1 low (n = 25, PB = 2 and BM = 23) vs. PB or BM-WT1 high (n = 84, PB = 23 and BM = 61) were 23.9% and 50.4%, respectively (p = 0.023, Fig. 2A) and the 2-year OS rates were 51.2% and 64.6%, respectively (p = 0.03, Fig. 2B). PB or BM-WT1 low of intermediate-risk AML had a poor prognosis even with standard chemotherapy. Multivariate analysis adjusted for ages, the reduction rate of WT1 mRNA after induction chemotherapy, duration of receiving consolidation chemotherapy, allo-HCT, and PB or BM-WT1 low at diagnosis were independent poor prognosis factors for EFS in intermediate-risk AML (HR: 3.32, 95%CI: 1.29-8.53, p = 0.013). The OS rate of PB or BM-WT1 low also worsened (HR: 2.33, 95%CI: 0.88-6.18, p = 0.089) (Table 2). Whereas, the outcome of adverse-risk AML was not affected by PB or BM-WT1 low/high.ConclusionThe expression level of WT1 mRNA at diagnosis was negatively correlated with prognosis. Favorable-risk AML had higher expressions of WT1 mRNA. PB or BM-WT1 low in intermediate-risk AML was associated with a poor prognosis. In standard-risk AML, WT1 mRNA may be a useful pan-marker to predict prognosis at diagnosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

WT1 Expression Levels Combined with Flow Cytometry Blast Counts for Risk Stratification of Acute Myeloid Leukemia and Myelodysplastic Syndromes.

Wilm’s tumor 1 (WT1), a zinc-finger transcription factor and an epigenetic modifier, is frequently overexpressed in several hematologic disorders and solid tumors, and it has been proposed as diagnostic and prognostic marker of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, the exact role of WT1 in leukemogenesis and disease progression remains unclear. In this real-world evidence retrospective study, we investigated prognostic role of WT1-mRNA expression levels in AML and MDS patients and correlations with complete blood counts, flow cytometry counts, and molecular features. A total of 71 patients (AML, n = 46; and MDS, n = 25) were included in this study, and WT1 levels were assessed at diagnosis, during treatment and follow-up. We showed that WT1 expression levels were inversely correlated with normal hemopoiesis in both AML and MDS, and positively associated with blast counts. Flow cytometry was more sensitive and specific in distinguishing normal myeloid cells from neoplastic counterpart even just using linear parameters and CD45 expression. Moreover, we showed that a simple integrated approach combining blast counts by flow cytometry, FLT3 mutational status, and WT1 expression levels might be a useful tool for a better prognostic definition in both AML and MDS patients.

WT-1, BAALC, and ERG Expressions in Iranian Patients with Acute Myeloid Leukemia Pre- and Post-chemotherapy.

Purpose: Acute myeloid leukemia (AML) is the most prevalent acute leukemia in adults. It possesses different cytogenetic and molecular features. The expression of Wilms tumor-1 (WT1), brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) might be considered as prognostic factors in AML patients. The aim of this study was to determine the mRNA expressions of WT-1, BAALC and ERG genes in bone marrow of mononuclear cells and their effects on complete remission in the Iranian AML patients, pre- and post- chemotherapy. Methods: Forty AML patients with normal karyotype were evaluated. The mRNA gene expressions were measured with quantitative real-time PCR in bone marrow of mononuclear cells of AML patients at the baseline and after chemotherapy. The subtypes of AML and flow cytometry panel were also assessed. Complete remission (CR) after the treatment was addressed for all patients. Results: The mRNA expressions of WT-1, BAALC and ERG were significantly decreased after the treatment (p = 0.001, 0.017, 0.036). WT-1 mRNA expression was inversely correlated with CR after chemotherapy (P =0.024). There was also significant correlation between baseline expression of BAALC and CR (P =0.046). No significant correlation was observed between ERG and CR pre- and post- chemotherapy (P =0.464 and 0.781). There was also significant correlation between BAALC mRNA expression and CD34+ (P <0.001). Conclusion: The present study showed that WT-1 decreased significantly after standard chemotherapy which could have favorable effects on CR. Also, the high expression of BAALC could have a poor prognostic role in AML patients. The identification of these gene expressions can be an efficient approach in targeted therapy among AML patients.

Dynamic Change of Peripheral Blood WT1-mRNA within Three Cycles of Azacitidine Therapy Predicts Treatment Response in High-Risk Myelodysplastic Syndrome

Dynamic Change of Peripheral Blood WT1-mRNA within Three Cycles of Azacitidine Therapy Predicts Treatment Response in High-Risk Myelodysplastic Syndrome

WT1, PRAME, and PR3 mRNA Expression in Acute Myeloid Leukemia (AML).

Several tumor-associated antigens (TAAs) were recently identified, that could qualify as targets for immunotherapy, they could qualify (on RNA-level) for monitoring of tumor load. Here, we studied the expression levels of the immunogenic antigens PRAME (preferentially expressed antigen of melanoma), WT1 (Wilms' tumor gene), and PR3 (proteinase 3) on myeloid blasts by real-time quantitative polymerase chain reaction and correlated these data to the state and course of disease and to the defined subgroups of acute myeloid leukemia (AML). At first diagnoses, 41 of 47 patients tested showed overexpression of PRAME (87%), 38 of WT1 (81%), and 26 of PR3 (55%), with the highest expression levels for PRAME (2048-fold), followed by WT1 (486-fold) and PR3 (196-fold). Thereby, with 70%, the most frequent combination at first diagnoses was detected to be PRAME and WT1 (33/47 patients). Overall, 21 patients (45%) revealed overexpression for all 3 TAAs. Moreover, the highest expression levels of PRAME were found to be correlated with the FAB subtype M5, cytogenetic unfavorable risk groups, and AMLs arising from myelodysplasia (secondary AML; P=0.02). To compare TAA expression levels in the course of disease, expression data were calculatory adjusted to 100% blasts, revealing a relative increase in the PRAME expression levels during the course of persistent disease (3/4 cases). Independent of stage of disease, by trend, higher TAA expression levels were found on blasts derived from peripheral blood than those derived from the bone marrow. In conclusion, it is suggested that vaccine strategies for cancer immunotherapy should comprise different TAA peptides anticipating the diverse TAA expression levels on blasts evolving during the course of disease or treatment.

DC Vaccination Induces Antigen Specific Immune Responses in AML Patients: A 1-Year Interim Assessment

Acute myeloid leukemia (AML) is a lethal hematological malignancy characterized by high rates of relapse (50%). Studies of immune responses in AML patients led to identification of two leukemia-associated antigens (Wilms' tumor protein 1 (WT1) and preferentially expressed antigen of melanoma (PRAME) for which cellular and humoral immune responses were detected. Based on expression profiles in AML and normal tissues, WT1 and PRAME were selected as target antigens for a DC vaccine, aiming to elicit adaptive and innate immune responses in AML patients. The primary objective of the trial (EudraCT No.: 2014-003520-44) is to evaluate safety and feasibility of active immunotherapy with antigen-loaded autologous DCs. The secondary objectives are to assess DC vaccination-induced T cell responses against WT1 and PRAME and to evaluate the clinical outcome over a period of 2 years or until disease progression/relapse. WT1-positive AML patients, with or without PRAME positivity were included, if they had a morphological remission with or without hematological recovery (CRi) after induction chemotherapy and were not eligible for allogeneic hematopoietic stem cell transplantation. DCs were administered intradermally (2.5x106 WT1 plus 2.5x106 PRAME DCs) on a weekly basis for 4 consecutive weeks, followed by administration on week 6 and then every 4 weeks until 2 years, tumor progression or patient withdrawal. This report focusses on the immune-monitoring data collected during the first year of treatment. Multiple parameters were evaluated, including WT1- and PRAME specific T cell responses in peripheral blood (IFN-g ELISPOT), the recurrence of WT1- and/or PRAME-positive AML blasts in bone marrow (qRT-PCR) and peripheral blood (flow cytometry), and occurrence of AML-specific mutations in bone marrow (NGS). DCs were assessed for expression of WT1 and PRAME and costimulatory molecules by flow cytometry, while secretion of IL10 and IL12 was evaluated by double-color ELISPOT. Twenty CR/CRi AML patients were vaccinated, and vaccination was well tolerated, and no signs of autoimmunity were observed. 12/20 patients remained stable throughout the first year of treatment, while 8/20 relapsed. 2/8 relapsed patients died due to progression of disease during the first year. In patients in stable remission, 7/12 patients had no detectable IFN-γ ELISPOT response in peripheral blood, 3/12 mounted a response and 2/12 were not evaluable due to high background production of IFN-γ. Positive IFN-γ ELISPOT responses were observed within 1-30 weeks of treatment and associated with a decrease over time in levels of WT1 (3/3) and/or PRAME mRNA (1/3) expression. Except for 2 patients who remained MRD-negative throughout the first year of treatment, ELISPOT-negative patients in remission (5/7) exhibited low, relatively stable levels of target antigens. Stable remissions were associated with one (3/12) or no (8/12) AML-related mutations while one patient was not evaluable. AML relapses were observed within 50 days in 4 patients, within 51-100 days in 1 patient, and after more than 150 days in 3 patients. Overall, 6/8 relapsing patients exhibited positive IFN-γ ELISPOT responses, while 2 patients relapsing in &lt;50 days failed to respond and one patient relapsing on day 77 exhibited an IFN-γ response at baseline. In all other relapsing patients, IFN-γ ELISPOT responses were detected within 6-30 weeks after vaccination, in association with rapid and progressive increases in levels of WT1 and/or PRAME. AML-related gene mutations were detected in 6/8 relapses, while one patient was not evaluable, and one had no mutation; In 4 of the relapsing patients, 3 to 6 mutations were detected, while in 2/8 a single mutation was observed. In conclusion, IFN-γ responses to WT1 and/or PRAME were detected in peripheral blood of 75% (6/8) of relapsing patients, but only 25% (3/12) of the patients in remission. It is tempting to speculate that detection of IFN-γ response in peripheral blood is linked to the concomitant presence of AML blasts in the periphery of relapsing patients. Stable or decreasing levels of WT1 and/or PRAME mRNA in bone marrow of most patients in remission is compatible with the hypothesis that a local response against AML antigens may be ongoing, despite a negative IFN-γ response in peripheral blood. Monitoring of the patients currently in remission may shed some light on the role of DC vaccination on the prevention of AML recurrence. Disclosures Eckl: Medigene Immunotherapies GmbH: Employment. Raffegerst:Medigene Immunotherapies GmbH: Employment. Schnorfeil:Medigene Immunotherapies GmbH: Employment. Prinz-Schulz:Medigene Immunotherapies GmbH: Employment. Fingerhut:Medigene Immunotherapies GmbH: Employment. Bigalke:BioNTech Innovative Manufacturing Services GmbH: Employment. Pinkernell:Medigene AG: Employment. Schendel:Medigene AG: Employment, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Tafuri:Medigene Immunotherapies GmbH: Employment.

Post-Induction Minimal Residual Disease Measured By Flow Cytometry and Deep Sequencing of Mutant GATA1 Are Both Significant Prognostic Factors for Children with Myeloid Leukemia and Down Syndrome: A Nationwide Prospective Study of the Japanese Pediatric Leukemia/Lymphoma Study Group

Background: Myeloid leukemia in Down syndrome (ML-DS) is associated with good response to chemotherapy thus results in a favorable outcome. However, relapsed and refractory cases are rarely salvageable, regardless of receiving hematopoietic stem cell transplantation. Several factors such as certain chromosomal abnormalities and age at diagnosis are somewhat prognostic, but no universal prognostic factor has been found to date. In order to identify a subgroup with high risk of treatment failure, the role of minimal residual disease (MRD) with three methods were explored in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) trial AML-D11. Procedure: AML-D11 is a nationwide single-arm clinical trial for children (4 months to 17 years old) with ML-DS. All patients received an identical chemotherapy to the previous AML-D05 study (Taga T. Pediatr Blood Cancer 2016). MRD was evaluated at two time points, one after the induction therapy and another at the end of whole chemotherapy, using 3 different methods; flow cytometric MRD (FCM-MRD), deep sequencing MRD of mutant GATA1 (GATA1-MRD) and PCR MRD of WT1 mRNA expression (WT1-MRD). WT1-MRD was measured in both bone marrow (BM) and peripheral blood (PB) samples, while FCM- and GATA1-MRD were measured only in BM samples. Results: A total of 78 patients were eligible and followed-up with a median of 47.6 months (range, 8 to 68.8 months). Seventy-six patients were stratified to the standard risk (SR) and one patient to the high risk (HR) group by morphological response. One patient died of sepsis during initial induction therapy. Three-year event-free survival (EFS) and overall survival (OS) rates were 87.2% (95%CI, 77.5 to 92.9%) and 89.7% (95%CI, 80.5 to 94.7%), respectively. FCM-MRD and GATA1-MRD after initial induction therapy were positive in 5/65 and 7/59 patients, respectively, which were both significantly prognostic (Fig.1). Prognostic significance of WT1-MRD could not be evaluated due to a limited number of collected samples. Conclusions: MRD detections by FCM and targeted deep sequencing of GATA1 after initial induction therapy are both significant prognostic factors for predicting relapse. Risk stratification using FCM-MRD is currently incorporated in the on-going Japan Children's Cancer Group ML-DS trial (AML-D16; jrct.niph.go.jp, jRCTs041190047). Figure 1 Disclosures No relevant conflicts of interest to declare.

Prognostic impact of peripheral blood WT1-mRNA expression in patients with MDS

Few reports suggested a prognostic impact of Wilms‘Tumor-1 (WT1)-mRNA overexpression in MDS, but translation into clinical routine was hampered by limited patients numbers, differing sample sources, non-standardized methods/cut-offs. To evaluate whether WT1-mRNA expression yields additional prognostic information, we measured peripheral blood (PB) WT1-mRNA expression in 94 MDS using a standardized assay offering a validated cut-off to discriminate between normal and WT1-mRNA overexpression. Overall, 54 patients (57%) showed WT1-mRNA overexpression, while 40 patients (43%) had normal WT1-mRNA expression. This enabled discrimination between MDS and both healthy controls and non-MDS cytopenias. Furthermore, WT1-mRNA expression correlated with WHO 2016 subcategories and IPSS-R as indicated by mean WT1-mRNA expression and frequency of WT1-mRNA overexpressing patients within respective subgroups. Regarding the entire group, PB WT1-mRNA expression was associated with prognosis, as those patients showing WT1-mRNA overexpression had higher risk for disease progression and AML transformation and accordingly shorter progression-free, leukemia-free and overall survival in univariate analysis. In multivariate analysis, prognostic impact of PB WT1-mRNA expression status was independent of IPSS-R and enabled more precise prediction of PFS, but not OS, within IPSS-R very low/low and intermediate risk groups. Overall, measuring PB WT1-mRNA appears valuable to support diagnostics and refine prognostication provided by the IPSS-R.

Changes of WT1 mRNA expression level in patients with myelodysplastic syndromes after hypomethylating agents and its prognostic significance

目的监测骨髓增生异常综合征（MDS）患者去甲基化治疗后WT1 mRNA水平的变化，评估WT1 mRNA水平变化在判断患者疗效、区分不同疾病稳定（stable disease, SD）状态中的意义。方法以2009年11月至2018年3月56例在广东省人民医院接受去甲基化治疗（≥4个疗程）的MDS患者为研究对象，实时定量PCR检测其骨髓或外周血WT1 mRNA的表达，观察WT1 mRNA表达水平的动态变化与患者临床疗效和预后的相关性。结果去甲基化治疗3个疗程后，MDS患者WT1 mRNA水平较治疗前显著降低。20例疾病进展患者病情进展或转为急性髓系白血病（AML）后WT1 mRNA表达水平较治疗前明显升高。45例SD的MDS患者，根据SD期间WT1 mRNA表达水平变化分为WT1升高与未升高两组，SD期间WT1 mRNA表达水平升高的患者中病情进展或转AML的比例为95.65%（22/23），而SD期间WT1 mRNA表达水平未升高的患者中病情进展或转为AML的比例为9.09%（2/22）（χ2=33.852，P<0.001）。与SD期间WT1 mRNA表达水平未升高的患者相比，SD期间WT1 mRNA表达水平升高的患者的总生存期[17（95%CI 11~23）个月对未达到，P<0.001]和无进展生存期[13（95%CI 8~18）个月对未达到，P<0.001]明显缩短。结论WT1 mRNA表达水平是评估MDS患者去甲基化治疗后疗效变化有效的实验室指标，尤其对于SD的患者，动态检测WT1 mRNA表达水平，可预测病情演变，从而指导临床治疗。

The Proportional Association between WT1 mRNA Level in Peripheral Blood before Allogeneic Hematopoietic Cell Transplantation and Risk of Mortality in Acute Myeloid Leukemia Not in Remission

Abstract INTRODUCTION The prognosis for allogeneic hematopoietic cell transplantation (allo-HCT) is relatively poor in patients with acute myeloid leukemia (AML) who are not in remission (non-CR). Wilms' tumor-1 gene messenger ribonucleic acid (WT1 mRNA), which is overexpressed in almost all AML, has been used as an indicator of monitoring minimal residual disease (MRD) and has been regarded as one of the prognostic factors in AML patients with complete remission (CR). However, the association between the expression levels of WT1 mRNA in peripheral blood (WT1) before allo-HCT and risk of mortality in AML patients in non-CR remains quite elusive. We therefore assessed the effect of the pre-transplant WT1 level on survival after allo-HCT in AML patients in non-CR. METHODS We retrospectively analyzed data from consecutive patients undergoing allo-HCT at our institution between November 2007 and January 2017. We excluded patients who did not have an available WT1 value within 28 days before the start of conditioning. RESULTS A total of 125 AML patients, including 46 non-CR patients (36.8 %), were eligible for this study. The median age was 48 years (range: 17-68 years) and the number of males was 69 (55.2%). Forty-two patients (33.6%) had AML with myelodysplasia-related changes. This study included 34 patients (27.2%) who had undergone multiple transplantation. Myeloablative and reduced-intensity conditioning were used in 73 (58.4%) and 52 (41.6%) patients, respectively. In 63 patients who received transplants from either a related peripheral blood stem cell or bone marrow (BM) donor, four pairs (6.3%) were serologically mismatched for one antigen of HLA and 45 pairs (71.4%) received transplants from HLA haploidentical donors. In 32 patients who received transplants from an unrelated BM donor, seven pairs (21.9%) were serologically mismatched for one HLA antigen. Twenty-nine patients (23.2%) received unrelated cord blood transplantation. Among 46 non-CR patients, the median follow-up period among survivors was 1,092 days (range: 688-2,607 days) following allo-HCT. Receiver operating characteristic analysis for prediction of composite events including any cause of death or subsequent transplantation indicated that the best cutoff value for WT1 was 5,000 copies/ μg RNA in non-CR AML patients. Estimated two-year, event-free survival of non-CR patients with WT1 levels of less than 5,000 copies/ μg RNA was significantly higher than that of those with WT1 levels of 5,000 copies/ μg RNA or more (41.2% [95% confidence interval (CI), 18.6-62.6] vs 10.3% [95% CI, 2.6-24.3]; P=0.006). On multivariate analysis of the non-CR patients, WT1 5,000 copies/ μg RNA or more was significantly associated with increased risk of mortality (hazard ratio (HR), 2.7; 95% CI, 1.3-5.5; P=0.008). Log10-transformed WT1 (continuous scale) was also significantly related to increased risk of mortality (HR, 1.5; 95% CI, 1.0-2.1; P=0.037). In the entire cohort, log10-transformed WT1 (divided into four categories) before allo-HCT was found to be the most powerful prognostic factor of survival among remission status, cytogenetic risk group, and revised disease risk index, applying Akaike's information criterion. Moreover, under the assumption of a non-linear model, as the log10-transformed WT1 level rose, the HR of mortality increased monotonically; a non-linear fit was obtained using a restricted cubic spline function (three knots; Figure 1). CONCLUSION Our data showed that WT1 levels obtained before allo-HCT monotonically increased HR of mortality in a non-linear manner regardless of CR status. Of importance is the finding that WT1 levels before allo-HCT were proportionally correlated with probability of survival after allo-HCT in non-CR AML patients. This suggests that the level of expression of WT1 mRNA in peripheral blood might reflect tumor burden and that there may be a WT1 threshold in non-CR patients for benefiting from allo-HCT. Further prospective studies of WT1 are necessary to determine acceptable or feasible levels of disease activity for receiving allo-HCT in relapsed or refractory AML patients. Disclosures Nakamae: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding. Hino:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Novartis: Research Funding. Nakamae:Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding.

Differential expression of Wilms' tumour 1 gene in porcine urogenital organs during development.

Wilms' tumour 1 gene (WT1) is essential for the development of mammalian urogenital system. However, the expression pattern of WT1 in the development of porcine urogenital organs is still unclear. Here, we examined the expression of WT1 mRNA and protein in porcine kidneys, ovaries and testes from embryonic days 35 and 60 (E35d, E60d, n=3) to the newborn (0d, n=4) and adult (210d, n=3) stages, using real-time PCR and immunofluorescent staining. Real-time PCR analysis showed that porcine kidneys, ovaries and testes all expressed high level of WT1 mRNAs, especially in adult testes (p<0.05 or 0.01 vs. kidney and ovary, respectively). Morphologically, characteristic microstructures of the kidneys, ovaries and testes were observed and discerned at all four stages. Immunofluorescently, WT1 expression was detected in a dynamic and context-specific pattern during the development of these organs. Taken together, porcine urogenital organs express relatively high levels of WT1 mRNA. Dynamical and context-specific expression profile of WT1 in these organs occurs during their development, implying its close association with the development and function of porcine kidney, ovary and testis.

A study of the WT1 and HMGA2 mRNA expression in myeloproliferative neoplasms

A study of the WT1 and HMGA2 mRNA expression in myeloproliferative neoplasms

229 - WT1 mRNA Expression is a Good Laboratory Indicator for Diagnosis of Disease Progression in MDS Patients with Stable Disease

Malignant pleural mesothelioma (MPM) is a locally aggressive neoplasm, principally linked to asbestos fibres exposure. Strong evidences associate this pollutant with induction of DNA breaks, aberrant chromosomes segregation and important chromosomal rearrangements, considered crucial events in malignant transformation. A considerable contribution to cellular transformation in MPM is also given by the presence of high genomic instability, as well as by the increased DNA methylation, and consequent decreased expression, of tumor-suppressor genes. In this study we first demonstrated that MPM cells are characterized by a decreased methylation level of pericentromeric DNA sequences which can justify, at least in part, the genomic instability observed in this neoplasia. Concomitantly, we found a paradoxical increased expression of DNMT1, the most expressed DNA methyltransferases in MPM cells, DNMT3a and all five isoforms of DNMT3b. Thus, we compared two experimental strategies, DNMT1 silencing and usage of a demethylating agent (5-aza-2′-deoxycytidine or Decitabine), both theoretically able to revert the locally hypermethylated phenotype and considered potential future therapeutic approaches for MPM. Interestingly, both strategies substantially decrease cell survival of MPM cells but the antitumor activity of Decitabine, differently from DNMT1 silencing, is mediated, at least in part, by a p53-independent p21 upregulation, and is characterized by the arrest of MPM cells at the G2/M phase of the cell cycle. These results indicate that the two approaches act probably through different mechanisms and, thus, that DNMT1 silencing can be considered an effective alternative to Decitabine for cancer treatment.

The effects of dual PPARα/γ agonism compared with ACE inhibition in the BTBRob/ob mouse model of diabetes and diabetic nephropathy.

The leptin‐deficient BTBRob/ob mouse develops progressive albuminuria and morphological lesions similar to human diabetic nephropathy (DN), although whether glomerular hyperfiltration, a recognized feature of early DN that may contribute to renal injury, also occurs in this model is not known. Leptin replacement has been shown to reverse the signs of renal injury in this model, but in contrast, the expected renoprotection by angiotensin‐converting enzyme (ACE) inhibition in BTBRob/ob mice seems to be limited. Therefore, to investigate the potential renal benefits of improved metabolic control in this model, we studied the effect of treatment with the dual peroxisome proliferator‐activated receptor (PPAR) α/γ agonist AZD6610 and compared it with the ACE inhibitor enalapril. AZD6610 lowered plasma glucose and triglyceride concentrations and increased liver size, but had no significant effect in reducing albuminuria, whereas enalapril did have an effect. Nephrin and WT1 mRNA expression decreased in the kidneys of BTBRob/ob mice, consistent with podocyte injury and loss, but was unaffected by either drug treatment: at the protein level, both nephrin and WT1‐positive cells per glomerulus were decreased. Mesangial matrix expansion was reduced in AZD6610‐treated mice. GFR, measured by creatinine clearance, was increased in BTBRob/ob mice, but unaffected by either treatment. Unexpectedly, enalapril‐treated mice showed intrarenal arteriolar vascular remodeling with concentric thickening of vessel walls. In summary, we found that the BTBRob/ob mouse model shows some similarities to the early changes seen in human DN, but that ACE inhibition or PPAR α/γ agonism afforded limited or no kidney protection.

Oxidative Stress Levels Are Correlated with Disease Progression and Iron Overload in MDS Patients with Excess Blasts

Introduction: Oxidative stress is closely related to iron overload in myelodysplastic syndrome (MDS) and induces DNA damage. A recent study has suggested that oxidative stress leads to increased mutation frequency in a murine model of MDS, and that additional mutations lead to the progression of MDS. We have recently reported that oxidative stress marker reflects not only iron overload but also disease progression in a MDS patient (2016, Shimizu et al). So there is a possibility that the increase in oxidative stress markers may have not only caused the iron overload but also resulted in the disease progression of MDS. To clarify this point, we evaluated the association among oxidative stress marker and disease progression in eight patients with MDS. In this study, we first examined the oxidative stress marker, derivatives of reactive oxygen metabolite (dROM), ferritin in serum and Wilms Tumor 1 gene (WT1) in the peripheral blood throughout the course of treatment. Then, we analyzed the correlations of the levels of dROM, ferritin and WT1. Next, we investigated ROS expression in each fraction of WBC by flow cytometry in a healthy donor and some MDS patients.Materials and methods: Levels of serum dROM were measured by colorimetry, using samples of MDS (RA n=3, RAEB-1 n=4, RAEB-2 n=1). Ferritin in serum and WT1 in peripheral blood were also measured. Statistical analyses were performed by Pearsonfs test. The ROS expression was evaluated during the clinical course by flow cytometry.Results: Table 1 shows the relationship between dROM, ferritin and WT1 in MDS patients throughout the treatment. Case 1 to 3 patients showed disease progression with excess blasts in spite of azacitidine treatment. These patients showed a correlation tendency between dROM and ferritin, dROM and WT1. Case 1 showed close relationship between ferritin and WT1. Case 3 shows slight relation between ferritin and WT1. Case 4 to 8 patients showed no disease progression. They were treated with supportive therapy, including blood transfusion or cytokine therapy. These patients showed no relation between dROM and the elevation of ferritin and WT1. In these patients, there was no relation between ferritin and WT1 except Case 6. We also evaluated the score of WPSS, IPSS and dROM at diagnosis. dROM did not correlate with WPSS and IPSS. Figure 1 shows increased ROS expression during disease progression in a MDS patient compared with healthy control. Considering that other MDS patients with excess blasts showed the similar pattern, the present result suggested that the oxidative stress markers were produced by tumor-derived cells.Conclusions: dROM levels were slightly related with the ferritin and WT1 mRNA expression levels in MDS patients with excess blasts. We speculate that the increase in the oxidative stress markers is a cause of not only iron overload but also the disease progression of MDS. Oxidative stress markers may contribute to evaluate the progression of treatment efficacy for MDS patients. [Display omitted] [Display omitted] DisclosuresNakaseko:BMS: Honoraria, Research Funding; PFIZER: Honoraria, Research Funding; NOVARTIS: Honoraria.