Bone Marrow WT1 Research Articles

Haploidentical versus Double-Cord Blood Stem Cells as a Second Transplantation for Relapsed Acute Myeloid Leukemia.

There are limited data on second stem cell transplantation (SCT2) outcomes with alternative donors for relapsed AML after the first stem cell transplantation (SCT1). We analyzed the outcomes of 52 adult AML patients who received SCT2 from haploidentical donors (HIT, N = 32) and double-cord blood (dCBT, N = 20) between 2008 and 2021. The HIT group received T-cell-replete peripheral blood stem cells after reduced-toxicity conditioning with anti-thymocyte globulin (ATG), while the dCBT group received myeloablative conditioning. For a median follow-up of 64.9 months, the HIT group, compared to the dCBT group, had earlier engraftment, superior 2-year overall survival (OS), disease-free survival (DFS), and non-relapse mortality (NRM) with similar relapse. Multivariate analysis demonstrated that HIT was significantly associated with better OS, DFS, and lower NRM than dCBT. Both longer remission duration after SCT1 and complete remission at SCT2 were significantly associated with a lower relapse rate. In addition, bone marrow WT1 measurable residual disease (MRD) positivity was significantly associated with inferior OS and higher relapse. This study suggests that T-cell-replete HIT with ATG-based GVHD prophylaxis may be preferred over dCBT as SCT2 for relapsed AML and that WT1-MRD negativity may be warranted for better SCT2 outcomes.

Bone Marrow WT1 mRNA Upregulation in Acute Myeloid Leukemia (AML) in Complete Molecular Remission (mCR): Understanding the Limitations and Advantages of WT1 As a Measurable Residual Disease (MRD) Target

Introduction WT1 mRNA levels assessed by q-PCR have been employed as a marker of remission in AML patients undergoing intensive chemotherapy (Nomdedeu J. et al. Leukemia 2013;27:2157) and hematopoietic cell transplantation (HCT) (Nomdedeu J et al. Biol Blood Marrow Transplant 2018;24:55). Patients with adjusted bone marrow WT1 mRNA levels greater than 100 copies showed a high probability of relapse. Despite the usefulness of WT1 quantification in this and other settings, this marker is scarcely employed as an MRD target because levels greater than >100 copies can also be observed in normal bone marrows. Furthermore, 10-20% of AML at diagnosis and most chronic myelomonocytic leukemia (CMML) cases lack WT1 upregulation. The high levels of bone marrow WT1 copy numbers observed in most AML, decline in responding patients, although the utility, if any, of WT1 levels in long-term survivors of AML is currently unknown. We investigated the frequency and biological findings of mCR AML samples showing WT1 upregulation(>100 copies) compared to that mCR AML with concordant low WT1 levels. Patients and methods The study included 717 bone marrow samples from 80 AML patients (23 patients with RUNX1-RUNX1T1, 18 pts with CBFb-MYH11, four pts with NPM1 mutations, and 35 pts with PML-RARa). Samples were obtained at diagnosis (58 samples) and the follow-up (659 samples, all in mCR and without relapses) from one month to 219 months after diagnosis. Chimeric transcripts and WT1 levels were investigated by standardized real-time methods (ELN). Hemoglobin and leukocyte counts were also recorded. Flow cytometry immunophenotyping was also performed using four-color flow cytometry. We analyzed the percentage of CD34+, CD117+, CD123+ cells, and the Myeloid/Lymphoid CD34 ratio. nCounter Stem cell panel (Nanostring) was used to investigate the differences between that AML in mCR, showing high (>100) and low (<100) WT1 levels. In an additional cohort (11 samples: 4 with WT1>100 and 7 with WT1 <100), we performed bulk RNAseq (Novogene) to investigate enriched genes in samples with high WT1 levels. Results Thirty-five patients had at least one sample with WT1 levels greater than 100 copies (44%) despite being in mCR. Twelve samples had WT1 levels greater than 300 copies. In one case, this level reached 770 copies. These WT1 "peak" samples were distributed as follows: 49/306 PML-RARa, 25/161 RUNX1-RUNX1T1, 10/174 CBFb-MYH11, and 1/18 NPM1. Patients showing WT1 peaks were older than those without elevations (47 years vs. 36 years, p:0.003) For PML-RARa samples, "peaking" was associated with low levels of hemoglobin (133 g/L for <100 copies vs. 125 g/L >100 copies, p:0.009). For RUNX1-RUNX1T1, "peaking" was linked to less platelets (132x109 vs. 197x109 p:0.05) , higher CD34 (1.5% vs. 1% p:0.05) and also higher CD117+ cells (1.6 vs.1%, p:0.01). Lastly, CBFb-MYH11+ samples with WT1 peaks had higher leukocyte counts that those showing low WT1 levels (4.5x109 vs. 2.9x109, p:0.006). Nanostring comparisons between mCR WT1 high vs. low samples showed upregulation of TOR1A, PIK3CB, PTPN6, and PGM1 with downregulation of HMGA1, HOXB2 and CDC6. RNA seq analysis detected an enrichment of SUZ12 targets in those samples with high WT1 levels. Conclusions Bone marrow mRNA WT1 levels commonly exceed the clinical relevant value of 100 copies in AML samples in mCR (13%). Peak samples were related to peripheral blood values and/or raised numbers of immature bone marrow cells (CD34, CD117). Bone marrow WT1 although unable to accurately predict relapses in long-term survivors of AML may be a non-invasive sensor of a yet undefined myeloid state.

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.

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

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

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.

Bone marrow WT1 levels in patients with myeloid neoplasms treated with 5-azacytidine: Identification of responding patients.

Increased levels of Wilms' tumor (WT1) mRNA have been used to establish risk categories in patients with acute myeloid leukemia (AML). Raised values of WT1 have been associated with progression in myelodysplastic syndrome (MDS). We retrospectively analyzed the available bone marrow (BM) samples from 115 patients with myeloid neoplasms obtained before and during treatment with 5-azacytidine. A threshold of 100 copies in BM was used to define risk groups: group 1: patients with WT1 levels always below<100 copies; group 2: cases with initial WT1 levels greater than 100 copies but with a conversion to sustained levels below 100; and group 3: cases with follow-up WT1 levels greater than 100. Twenty patients were included in group 1, 17 in group 2, and 78 in group 3. Survival analysis showed statistically significant differences in terms of OS between groups (p: 0.016). Patients in group 2 showed the best 5-year overall survival (OS). In multivariate analysis, only the cytogenetic risk category and receiving an allogeneic hematopoietic stem cell transplantation (HCT) independently predicted the survival. Further studies are needed to assess whether BM WT1 levels could be useful to predict the survival of patients with myeloid neoplasms treated with 5-azacytidine.

Significance of WT1 gene detection in the prognosis of acute myeloid leukemia patients with normal karyotype after hematopoietic stem cell transplantation

Objective To investigate the monitoring significance of WT1 gene level in the prognosis of acute myeloid leukemia (AML) patients with normal karyotype after hematopoietic stem cell transplantation (HSCT). Methods The clinical data of 115 AML patients with normal karyotype who were treated with HSCT from July 2009 to March 2017 in the First Affiliated Hospital of Soochow University were retrospectively analyzed. The dynamic detection of bone marrow WT1 gene was carried out by using reverse transcription- polymerase chain reaction (RT-PCR). According to the relative expression level median of WT1 gene before transplantation, the whole patients were divided into the two groups (< median group and ≥ median group) for survival analysis. Results There were 52 males and 63 females in 115 patients. The average age was (39±10) years old. The median white blood cell count at initial diagnosis was 20.45×109/L [(0.5-355.9)×109/L], the ratio of blast cells in the bone marrow was 0.60±0.28, and the relative expression level median of WT1 gene was 87×104, while the median time of the follow-up was 24 (3-79) months. Among 115 patients, 19 cases relapsed. Remission group (96 cases) and relapse group (19 cases) were followed up. The WT1 gene level was monitored by using bone marrow puncture in 1 month, 3 months, 6 months, 12 months after transplantation. It was found that the WT1 gene relative expression level of relapse group was higher than that of remission group, and the differences between the two groups at 6 month-point [remission group (187±50)×104, relapse group (871±211)×104, t= 2.519, P= 0.014] and 12 month-point [remission group (51±9)×104, relapse group (1 797±312)×104, t= 4.000, P < 0.05] were statistically different. The overall survival (OS) rate of 2-year, progression-free survival rate in WT1 gene relative expression level < 87×104 group were higher than those in WT1 gene relative expression level ≥ 87×104 group, the relapse rate in WT1 gene relative expression level < 87×104 group was lower than that in WT1 gene relative expression level ≥ 87×104 group, and the differences were statistically different (all P < 0.05). Multivariate analysis showed that the level of WT1 gene at 12 months after transplantation was an independent factor affecting OS (HR = 4.12, P = 0.046) and PFS (HR = 5.95, P = 0.001). There were 19 cases of recurrence (16.5%), with a median relapsed time of 11 (1-60) months. When WT1 gene relative expression level was significantly increased in 19 patients, firstly immunosuppressive agents were reduced, of which 6 patients were not influenced by this intervention; in other 13 cases, only 5 cases were influenced by intervention. Conclusions For CN-AML patients, the expression level of WT1 gene before transplantation has a negative correlation with the prognosis. The expression level of WT1 gene at 12 months after transplantation is an independent factor for affecting the survival. The relapsed patients have a higher WT1 expression level, and clinical interventions for relapsed patients have a favorable effect. Key words: Gene, WT1; Hematopoietic stem cell transplantation; Leukemia, myeloid, acute; Prognosis

Measurable Residual Disease Assay with WT1 Expression in Acute Myeloid Leukemia Who Underwent Allogeneic Hematopoietic Stem Cell Transplantation; Optimal Threshold, Time Points, and Candidates

BackgroundOverexpression of WT1 is a surrogate marker of abnormal myelopoiesis and has been evaluated as a potential tool to assess measurable residual disease (MRD) in myeloid malignancies. Given that lack of consensus on clinically relevant WT1 thresholds and time points in the allogeneic hematopoietic cells transplantation (allo-HSCT) setting, WT1 quantification has not yet gained widespread use despite several pieces of evidence demonstrating the possible role for MRD assessment with the limited numbers of patients. To investigate optimal threshold, time points, and candidates of WT1 quantification in AML, we retrospectively analyzed a large cohort of consecutive patients who underwent allo-HSCT at Catholic Hematology Hospital.Patients and methodsThis study included 425 consecutive patients with AML who underwent allo-HSCT at CR state from either a matched siblings (n=199), matched unrelated (n=117) or haploidentical family donors (n=109) from 2012 to 2016. Patients were in the first (n=400) or second (n=25) complete remission with a median age of 48 years (range, 18~70). Favorable, intermediate, poor risk groups by 2017 NCCN criteria were 28% (n=120), 49% (n=206), and 23% (n=99), respectively. Bone marrow WT1 levels before, and at 1 or 3 months after allo-HSCT were determined using real-time PCR using the ELN normalized method. We sought to clarify the prognostic relevance of the WT1 quantification regarding the cumulative incidence of relapse (CIR) and survival outcomes.ResultsWith a median follow-up of 39 months (range, two days to 73 months), the 4-year overall survival, disease-free survival, CIR and non-relapse mortality were 63.6%±2.6%, 61.5%±2.6%, 17.9%±2.1% and 24.7%±2.5%, respectively.Analysis of dynamic changes of WT1 levels demonstrated decreased levels at 1 (n=333, mean 86 copies, range 0~1800) and three months (n=346, mean 101 copies, range 0~1670) after allo-HSCT compared to before allo-HSCT (n=425, mean 219 copies, range 0~9630). Relapsed patients had significantly higher WT1 levels before (P=0.018) and at three months (P=0.041) after allo-HSCT, whereas no difference at one month after allo-HSCT (P=0.167). Even the ROC curve analysis revealed that WT1 levels before allo-HSCT were significantly available to predict CIR after allo-HSCT (P<0.001). Among various cut-off levels of WT1 expression (median, 25% from the top, and cut off by ELN), cutoff by ELN (250 copies) was most effective for predicting CIR. The CIR of MRD positive patients (³ 250 copies) before and at three months after allo-HSCT were 43% (vs. 14%, P<0.001) and 35% (vs. 11%, P<0.001), respectively. In multivariate analysis, the WT1-MRD positivity independently predicted the CIR (before, HR=3.5, P<0.001; at three months, HR=7.4, P<0.001), which translated into inferior disease-free survival (P<0.001) and overall survival (P<0.001). In a subgroup analysis with the WT1-MRD positive patients before allo-HSCT (n=44), the WT1-MRD positivity at three months was significantly effective to identify patients with a higher risk for relapse (100% vs. 26%, P<0.001).In subgroup analyses in each risk group by 2017 NCCN criteria, the WT1-MRD positivity before allo-HSCT was significantly effective to predict CIR in the intermediate risk group (57% vs. 12%, P<0.001), whereas no significance in both favorable and poor risk groups. On the other hand, the WT1-MRD positivity at three months after allo-HSCT was effective to predict CIR in the poor risk group (60% vs. 21%, P<0.001). In patients with normal karyotype without NPM1 mutations (n=117), the WT1-MRD positivity before allo-HSCT significantly predict CIR (32% vs. 8%, P=0.001), whereas no difference in patients with NPM1 mutations (n=66) or core-binding factor (CBF) AML (n=102).ConclusionsThese data suggest standardized bone marrow WT1 levels using the ELN threshold (250 copies) before and at three months after allo-HSCT provided relevant information to predict relapse in AML with intermediate and poor risk groups by 2017 NCCN criteria, respectively. The validated WT1 MRD assay by ELN was revealed to be particularly available in AML without specific MRD markers, such as NPM1 or CBF-AML, and different significance by times points should be considered for clinical applications to identify high-risk AML for relapse, potential candidates for various immunomodulatory approaches. DisclosuresKim:Pfizer: Research Funding; Ilyang: Research Funding; BMS: Research Funding; Novartis: Research Funding. Lee:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Long Non Coding RNA RP11-69I8.3 Expression in Acute Leukemia and Its Cinical Significance

To investigate the expression of long non coding RNA RP11-69I8.3 in acute leukemia and its clinical significance. lncRNA RP11-69I8.3 expression was detected by RT-PCR in bone marrow samples from 17 healthy controls, 32 newly diagnosed AML patients and 32 newly diagnosed ALL patients, and 25 ALL patients of complete remission after chemotherapy. Meanwhile, the clinical data were collected and the relation of lncRNA RP11-6918.3 expression with the clinical characteristics was analyzed. Compared with the control group, there was no significant difference in the expression of lncRNA RP11-69I8.3 in AML group(P>0.05). lncRNA RP11-69I8.3 lowly expressed in untreated ALL group(P=0.001). Compared with the de novo ALL group, lncRNA RP11-69I8.3 was highly expressed in complete remission ALL group (P<0.013). In 32 de novo ALL patients,the expression of lncRNA RP11-69I8.3 in children was significantly lower than that in adult(P=0.017). There was no correlation of the expression of lncRNA RP11-69I8.3 with the sex, WBC count, HB level, Plt count, LDH level, T or B type, ratio of bone marrow blast cell, BCR/ABL and WT1 fusion gene expression, chromosome karyotype, extramedullary infiltration, whether complete remission after one chemotherapy, whether relapse. In 26 B-ALL patients, there was no correlation between lncRNA RP11-69I8.3 and the immunophenotype. The expression of lncRNA RP11-69I8.3 in the untreated AML is not significantly different from the control group. lncRNA RP11-69I8.3 is low expressed in ALL group, highly expressed in ALL group with complete remission. In untreated ALL, the expression of lncRNA RP11-69I8.3 in children is significantly lower than that in adult. In B-ALL patients, the lncRNA RP11-69I8.3 is not relevant with the immunophenotype.

Bone Marrow WT1 Levels in Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myelogenous Leukemia and Myelodysplasia: Clinically Relevant Time Points and 100 Copies Threshold Value

The outcome of allogeneic hematopoietic stem cell transplantation (HCT) in patients with myeloid malignancies is better in those without minimal residual disease (MRD) than in those with MRD+, as assessed by multiparametric flow cytometry (MPFC). WT1 quantitation also has been used to assess the probability of relapse in acute myelogenous leukemia (AML) treated with chemotherapy. We analyzed the clinical value of normalized bone marrow WT1 levels as a measure of the expanded myeloid progenitor compartment in a consecutive series of 193 adult patients with myeloid malignancies who underwent HCT. Bone marrow WT1 levels before the HCT, at the first bone marrow aspirate after infusion, and in the follow-up samples after HCT were determined by means of real-time PCR using the European LeukemiaNet normalized method. We sought to clarify the prognostic relevance in terms of overall survival (OS), progression-free survival (PFS), and cumulative incidence of relapse (CIR). Based on earlier experience in AML, we selected a threshold of 100 copies, defining 2 groups: patients with <100 WT1 copies and those with ≥100 copies. Patients with <100 WT1 copies before HCT (median time, 36 days; range, 4 to 268 days) had a better OS, PFS, and CIR than those with ≥100 copies (40 ± 1 versus 29 ± 6 days, P = .004; 35 ± 9 versus 26 ± 6 days, P = .002; and 29 ± 7 versus 37 ± 6 days, P = .051). In the first bone marrow study after the HCT (median time, 42 days; range 14 to 157 days, respectively), patients with <100 WT1 copies also had better outcomes in terms of OS, PFS, and CIR (40 ± 7 versus 31 ± 9 days, P = .025; 36 ± 7 versus 30 ± 8 days, P = .004; and 29 ± 6 days versus 54 ± 9, P < .001, respectively). At this time point, bone marrrow samples with >100 copies also included patients who were negative for MRD as assessed by MPFC (19 of 32). During the HCT follow-up, patients with sustained WT1 levels <100 copies showed a marked benefit in terms of OS, PFS, and CIR even compared with those with only a single measurement >100 copies (mean, 68 ± 11 versus 26 ± 7 days, P < .001; 63 ± 11 versus 20 ± 8 days, P < .001; and 20 ± 8 vs. 71 ± 8 days, P < .001, respectively). Standardized bone marrow WT1 levels using a 100-copy threshold in samples obtained before HCT, at leukocyte recovery, and during follow-up provided relevant prognostic information in patients with myeloid malignacies submitted to HCT.

Abstract 3659: WT1 and PRAME mRNA transfected TLR 7/8-polarized fast DC vaccines in AML patients mount specific immune responses and impact progression free survival

Abstract Patients diagnosed with acute myeloid leukemia (AML) may not be eligible for curative intensive treatment due to co-morbidity factors or age. Here we report results of 5 AML patients in morphological remission after incomplete induction/consolidation chemotherapy treated with dendritic cells (DCs) targeting WT-1 and PRAME. DCs were produced as described previously (Subklewe et al 2014), using a maturation cocktail containing the TLR 7/8 ligand R848. These DCs show a polarized release of IL-12p70 combined with low IL-10 upon stimulation. 2.5 or 5E+6 DCs per antigen were injected intradermally once weekly for 4 weeks (wks), in wk 6 and thereafter at monthly intervals. Blood and bone marrow (BM) samples were collected at regular intervals. Minimal residual disease (MRD) was measured in BM by quantitative PCR of WT-1 and PRAME expression and by morphology. Immune responses were assessed by analysis of intracellular interferon gamma expression or proliferation following stimulation with peptides spanning WT-1, PRAME, hTERT and survivin or after stimulation with autologous WT-1 and PRAME DCs. A 57 year old woman with intermediate risk M4 AML was vaccinated over 22 months after chemotherapy. Five weeks after start of vaccination she mounted strong CD8 responses against PRAME combined with an increase in hTERT response, suggesting epitope spreading. WT-1 signals in BM showed low positive levels throughout vaccination, but she remains in morphological remission 33 months after end of chemotherapy (EoC). A 50 year old man with M2 intermediate risk AML, initially not eligible for BM transplantation, showed specific immune responses against WT-1 during 10 months of vaccination. Due to Bell’s Palsy he was treated with cortisone which immediately reduced the vaccine effect, accompanied by increase of blasts in the bone marrow. Following new induction therapy he received BM transplantation and is currently in CR. A 68 year old woman with M1 intermediate risk AML is under vaccination for 24 months. WT-1 signals in BM continue to be slightly elevated without any sign of morphological relapse. CD4 responses and low CD8 responses are detected against WT-1, PRAME and hTERT. A 73 year old woman with M1 good risk AML relapsed after 6 months of DC vaccination without mounting specific immune responses. DC treatment was combined with 5-Azacytidine without any immunological and clinical effects. A 59 year old woman with good risk AML has been vaccinated for 14 months and is in remission for 21 months since EoC. WT-1 in BM remains slightly elevated whilst the initially positive PRAME signal is negative. Our results show that in 4 out of 5 AML patients fast TLR-polarized DC vaccination induced or supported specific T cell responses. 3 patients continue to be in CR after 21, 25 and 33 months respectively, following suboptimal primary chemotherapy. Immune responses are under investigation and will be presented. Citation Format: Iris Bigalke, Guri Solum, Dag Josefsen, Yngvar Fløisand, Kirsti Hønnåshagen, Lisbeth Skoge, Signe Spetalen, Stein Sæbøe-Larssen, Dolores J. Schendel, Gunnar Kvalheim. WT1 and PRAME mRNA transfected TLR 7/8-polarized fast DC vaccines in AML patients mount specific immune responses and impact progression free survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2017-3659

Abstract B33: The zinc finger transcription factor, WT1, regulates growth control genes in leukemia cells.

Abstract The Wilms tumor gene, WT1, encodes multiple splice isoforms with varying functions including both transcriptional and post-transcriptional regulation mediated by DNA, RNA and protein binding domains. Involvement of WT1 in regulation of various growth control genes has been widely studied in many solid tumor types. However, evidence of its role in leukemia has been contradictory; although WT1 is both overexpressed and/or mutated in different subsets of leukemia patients, and these aberrations are linked to poor or intermediate prognosis. Our working hypothesis is that in leukemia WT1 regulates expression of genes that play mitogenic roles, such as Cyclin A1 (CCNA1) and Vascular Endothelial Growth Factor (VEGF). To demonstrate that WT1 regulates these genes we first identified potential WT1 binding sites in both CCNA1 and VEGF promoters, using the MatInspector Software. Three potential WT1 binding sites were located within both the CCNA1 and VEGF gene promoters and their functional significance was validated by chromatin immunoprecipitation (ChIP) assays. ChIP analysis of chromatin from K562 cells revealed WT1 binding at 2 of 3 sites within the CCNA1 promoter; and in chromatin of 293 Kidney cells and LNCaP prostate cancer (PC) cells, WT1 binding was observed in VEGF promoter. To demonstrate that these functional sites are involved in modulating transcription from these promoters, we performed luciferase assays using reporter constructs containing CCNA1 (-1180/+145) and VEGF (-411/+50) promoter regions. Co-transfection of WT1 with these reporter constructs demonstrated that both gene promoters were activated by WT1 over-expression in K562 cells. Transcriptional regulation of the endogenous genes was confirmed by Quantitative PCR in K562 cells transfected with WT1. Although CCNA1 mRNA levels increased as expected, no significant changes in the VEGF mRNA levels were observed. This absence of VEGF mRNA up-regulation in K562 cells differs from that observed in kidney and PC cells, and could be attributed to many factors, including a lack of necessary co-activators. To determine the biological relevance of WT1 mediated regulation, we measured the levels of WT1, CCNA1 and VEGF mRNA levels in pediatric leukemia bone marrow (BM) samples using Quantitative PCR. Overall, WT1 levels were higher in Acute Myelogeneous Leukemia- M3 than in Acute Lymphoblastic Leukemia BM samples and WT1 levels were low or undetected in non-neoplastic BM. The AML-M3s samples with high WT1 levels also had higher expression of CCNA1. Conversely, in ALL samples variation was seen in the expression of WT1 and CCNA1. VEGF transcript levels in leukemia BM were generally near or below those in normal BM. Taken together, these results suggest that WT1 transcriptionally up-regulates CCNA1, but the regulation of VEGF may be cell specific. Since CCNA1 is primarily expressed in leukemias and normal testes, this suggests that WT1 could be a significant factor controlling expression of this proliferative gene. Conversely,VEGF is expressed in many different tissues and under many different conditions, including hypoxia, suggesting that WT1 may be part of the many factors contributing to the complex orchestration of VEGF expression. Nonetheless when these proliferative factors are up-regulated in leukemia, WT1 may contribute to their altered expression levels, and therefore be a leukemogenic factor. Citation Format: Sony Pandey, Shorog Al omair, Mustafa Moazam, Kurtis Eisermann, Steven J. Kuerbitz, Gail C. Fraizer. The zinc finger transcription factor, WT1, regulates growth control genes in leukemia cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B33.

The level of bone marrow WT1 message is a useful marker to differentiate myelodysplastic syndromes with low blast percentage from cytopenia due to other reasons.

Myelodysplastic syndromes (MDS) are a group of hematological neoplasms associated with ineffective hematopoiesis and that transform to acute leukemia. Distinguishing MDS from other cytopenias is sometimes difficult even for trained hematologists. WT1, the gene mutated in Wilms' tumor, was found expressed in acute myeloid leukemia and MDS. The amount of WT1 in peripheral blood and bone marrow (BM) is low in low-risk MDS subtypes, and is high in high-risk MDS subtypes. However, the role of WT1 in the differential diagnosis between MDS and other diseases showing cytopenia has not been fully addressed. The present study evaluated whether WT1 expression level can assist in the differential diagnosis of MDS from other cytopenias. The amount of WT1 message was evaluated among 56 MDS patients and 47 patients with cytopenia for various other reasons (cytopenia VR) at the Nagasaki University Hospital. The level of WT1 was significantly related to the percentage of blasts in BM among MDS cases, and the type of French-American-British classification of MDS; refractory anemia (RA) cases showed significantly lower WT1 level than patients with RA with excess blasts. WT1 level was significantly related to the prognostic risk categories of MDS by the International Prognostic Scoring System (IPSS) and the revised IPSS. Although the blast percentage in the BM of RA and cytopenia VR were both less than 5%, there was a significant difference in the level of WT1 between MDS and cytopenia VR. WT1 might be a good marker to differentiate low blast percentage MDS and cytopenia VR.

Bone Marrow Wilms Tumor Gene 1 Expression before Allogeneic Hematopoietic Cell Transplantation Correlates with Relapse in Acute Myeloid Leukemia Patients

IntroductionEvaluation of WT1 expression is becoming an attractive marker in acute myeloid leukaemia (AML) for minimal residual disease (MRD) detection. Recent studies correlate therapy response with WT1 copy levels in the bone marrow (BM) offering an additional tool beside multiparameter flow-cytometry (MFC). No well-known data are available regarding its impact on the outcome after hematopoietic cell transplantation (HCT).Patients/MethodsOne hundred and two consecutive clinically and molecularly well characterized AML patients (pts) were transplanted in a single hematology center from 2004 to 2013. Indication for allogeneic HCT included high cytogenetic and molecular risk according to WHO criteria, high leukocytosis, extramedullary manifestations or secondary AML at diagnosis. Further, primary induction failure was considered as an indication for allogeneic HCT.WT1 expression was analyzed by real time polymerase chain reaction (RT-PCR), using the standardized European LeukemiaNet method on BM samples before HCT and during each follow up control. Only pts in first CR before HCT were included in the analysis.Cumulative incidence of relapse (CIR) was estimated considering death for other causes than relapse as a competing event. Univariate and multivariate Fine & Gray Regression models were used to test the association between CIR and pre-HCT BM WT1 levels. Linearity of the relationship between CIR and the WT1 level was investigated using a mathematical transformation (restricted cubic spline).Aim of this retrospective analysis was to investigate the impact of pre-HCT BM WT1 expression in first CR pts on predicting relapse after HCT.ResultsA BM WT1 evaluation pre-HCT was available in 89 out of 102 pts. Among them, 62 achieved a CR after induction treatment. Relapsed pts in CR after reinduction chemotherapy (n=16) or with disease persistence pre-HCT (n=2) and pts refractory to treatment (n=9) were excluded from analysis.The patient subgroup displaying a first CR before HCT had a median age at diagnosis of 49 years (range: 20-65). Patient/donor relationship involved 26 (42%) sibling, 32 (52%) matched unrelated and 4 (6%) haplo-identical donors. On the basis of standard cytogenetics, molecular biology and clinical criteria (global disease risk) pts were classified according to the following risk groups: 54 (87%) high, 5 (8%) intermediate and 3 (5%) low risk. Acute GVHD occurred in 24 (39%) whereas chronic GVHD was documented in 18 (28%) pts. Twenty-six (42%) deaths were documented after HCT, 21 (34%) due to relapse and 5 (8%) because of treatment related mortality (TRM).Pre-HCT BM WT1 expression was correlated with CIR. A cut point of 150 WT1 copies was applied according to the slope change of relapse hazard, and subsequently used for CIR analysis. Pts displaying a pre-HCT BM WT1 level > 150 copies (n=18) had a higher CIR (2-year CIR 52.5%, 95% CI: 27.1-77.8) compared to pts with a BM WT1 level ≤ 150 copies (n=44, 2-year CIR 27.7% (95% CI: 13.4-42.1). WT1 copy level > 150 showed a significantly higher risk of relapse in univariate analysis (HR 2.9, 95% CI 1.2-6.7, p=0.014). In multivariate analysis pre-HCT BM WT1 expression was confirmed as a significant independent risk factor for CIR when adjusted for patient/donor relationship, presence of GVHD, global disease risk and competitive risk of mortality due to TRM (HR 3.4, 95% CI 1.3-8.5, p=0,010), Figure.Conclusions In the present study, pre-HCT BM WT1 levels discriminated significantly for CIR in a cohort of AML in first CR. A cut off level of 150 BM WT1 copies pre-HCT had a powerful statistically significant discriminating value to predict the risk of relapse, independently from already established risk factors. The prognostic value of WT1 was confirmed also when TRM as competitive risk for mortality was added in the multivariable model. Based on these results, WT1 is a promising candidate as MRD tool. Further prospective studies are required to confirm these results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

WT1 Monitoring of Minimal Residual Disease (MRD) in Patients with Acute Myeloid Leukemia

Background: Although a complete remission (CR) can be achieved in 70-80% of newly diagnosed acute myeloid leukemia (AML) patients, relapses occur in up to the 50% of cases. Thus, minimal residual disease (MRD) monitoring is a major issue for early detection of patients at high-risk of treatment failure and relapse.Aim: to dynamically evaluate WT1 pan-leukemic molecular marker of MRD in patients with AML.Matherial and methods: 107 newly diagnosed AML patients consecutively treated between 2010 and 2013 were monitored with quantitative WT-1 from bone marrow (BM) and peripheral blood (PB) at baseline, after induction, after the first consolidation course, before allogeneic stem cell transplantation (allo-SCT), at the 3rd and the 6th month after transplantationResults: At diagnosis, 104/107 (97%) had increased PB and BM WT1 levels assessed according to the ELN assay. Eighty-eight out of 107 patients (82%) achieved a complete remission (CR) after induction, 30/88 (34%) relapsed during follow up and 24/107 (22%) were addressed to allogeneic stem cell transplantation (allo-SCT). By univariate analysis, PB-WT > 50x10^4/ABL and BM-WT1 > 250x10^4/ABL after induction (PB: p=0.02; BM: p=0.04), after consolidation (PB: p=0.003), at the end of treatment (PB and BM: p=0.001), at 3rd month of follow up (PB and BM: p=0.005) and at 6th month of follow up (PB: p=0.005) were associated with a reduced overall survival (OS). By multivariate analysis, a BM-WT1 > 250 x 10^4/ABL at the end of treatment was significantly associated with a reduced OS. In order to adapt the cut-off of WT1 in our series of patients, we considered WT1 levels as continuous variables and categorized them at approximately the 25th, 50th, and 75th percentile. A cut-off of PB-WT1 > 25x10^4/ABL and BM-WT1 > 125x10^4/ABL at the end of the treatment program was identified as correlated with reduced leukemia-free survival (LFS) and OS (p=0.001). Similarly, and restricting the analysis on the 24 patients allo-transplanted in CR, 8/11 (73%) with pre-transplant PB-WT1 ≥ 5 and 4/13 (31%) with PB-WT1 < 5 relapsed, respectively (p=0.04). The incidence of relapse was higher in AML patients with PB-WT1 ≥ 5 measured at 3rd (56% vs 38%; p=0.43) and 6th month (71% vs 20%; p=0.03) after allo-SCT. Interestingly, 5/5 (100%) patients with pre-transplant PB-WT1 ≥ 5 who never reduced this level at 3rd or 6th month after allo-SCT experienced a disease recurrence.Conclusions: our data, although retrospectively collected, show that WT1 monitoring may be useful to predict the relapse in AML patients.Acknowledgments: This work was supported in part by Banca di Credito Cooperativo di Pompiano e Franciacorta and Lions Club Bassa Bresciana Association. DisclosuresRusso:Celgene: Research Funding; Gilead: Research Funding; Novartis: Consultancy.

Expression of WT1 and PRAME gene in bone marrow and peripheral blood samples of patients with myelodysplastic syndrome

This study was aimed to explore the transcription level of WT1 and PRAME two genes in bone marrow and peripheral blood samples of patients with myelodysplastic syndrome(MDS) and their relationship with bone marrow dysplasia and karyotype. The quantitative expression of WT1 and PRAME transcripts detected by RQ-PCR in the bone marrow samples of 203 MDS patients and 19 aplastic anemia(AA), 6 other benign anemia(BA), 4 paroxysmal nocturnal hemoglobinuria(PNH) patients from July 2009 to June 2012 and 14 healthy donors, and in 92 peripheral blood samples. The results showed that WT1 and PRAME expression levels in both BM and PB samples of MDS group were higher than those in normal controls, AA, and BA patients (BM: WT1:P = 0.000, 0.000, 0.000, PRAME: P = 0.048, 0.000, 0.064; PB: WT1:P = 0.012, 0.000, 0.011, PRAME: P = 0.020, 0.004, 0.003). What is more, this expression in high risk MDS group (RAEB1, RAEB2, MDS-AML) were higher than those in low risk group (RCUD, RCMD, MDS-U) and AA and BA. The WT1 and PRAME mRNA expression levels in PB and BM were well correlated (WT1:r = 0.6028, P = 0.001; PRAME: r = 0.7628, P = 0.000), as well as the WT1 expression levels in BM samples with the Karyotype (P = 0.049). In addition, the same positive rate of WT1 or PRAME expression existed in BM and PB samples of MDS patients. It is concluded that the WT1 and PRAME gene expression levels in both BM and PB samples of MDS patients are higher than those in healthy controls, AA and other benign anemia patients, and increase with the progression of the disease. The WT1 and PRAME transcripts constitute good molecular markers for the clinical diagnosis and prognosis and monitoring minimal residual disease after treatment of MDS. What is more, when bone marrow is not so convenient to get, the transcript levels of PB samples can be detected.

Bone Marrow WT1 Levels In Long-Term Survivors Of Core-Binding Factor AML and Acute Promyelocytic Leukemia

BackgroundQuantitation of WT1 mRNA levels may be used in minimal residual disease studies in AML cases lacking a suitable molecular target. The normal thresholds of bone marrow WT1 levels at different time–points have not been clearly stated. Difficulties arise as a consequence of the relatively high WT1 expression in normal bone marrow and the unknown influence of chemotherapy. ObjectiveTo establish the reference range value of bone marrow WT1 levels in treated CBF-AML and APL in complete remission. Patients and MethodsPatients with bone marrow samples obtained from ≥2 years (2-11 y) after initial diagnosis of CBF-AML (AML1-ETO n:21 patients CBFb-MYH11 n:21 patients) or APL(n:22 patients) were included in the study (120 samples). Ages at diagnosis ranged from 1 to 76 years. Mean age was: AML1-ETO 36.8(2-76y),CBFb-MYH11 32.4(1-55y), PML-RARa 47.3(21-76y). Peripheral blood, reticulocyte count and a bone marrow aspirate were also available in 86 cases. WT1 was analyzed following the primers and conditions of the ELN group. Specific chimeric real time PCR was simultaneously performed in each sample in accordance with the BIOMED protocol. Each WT1 or chimeric PCR was analyzed in triplicate whereas the control gene (abl) was analyzed in duplicate. ResultsAll patients with an available BM study were in morphologic complete remission. Two samples corresponded to an anemic patient (2/86). Six cases had a reticulocyte count above 2%. Platelet counts were below 120x109/l in 3 cases (3/86). Leukocyte counts below 3.5x109/l were observed in 6 samples (6/86). The calculated mean WT1 copy number from the 120 bone marrow samples was 47.23 (AML1-ETO:61.33, CBFb-MYH11:43.51, PML-RARa:36.4 p:ns). The mean chimeric copy number was 0.75 (AML1-ETO:0.82, CBFb-MYH11:0.72, PML-RARa:0.69 p:ns). Only 5 samples had a specific copy number of chimeric transcripts above 5 (3 AML1-ETO,1 CBFb-MYH11 and one PMAL-RARa patient who had a molecular relapse one month later and was succesfully treated). In 6 samples, the specific chimeric copy number was between 1 and 5 copies ( 4 AML1-ETO and 2 CBFb-MYH11). In the remaining 109 BM samples, the specific copy number was <1. In 9 BM samples, the WT1 levels were in the range of 100-300 copies (7 AML1-ETO and 2 CBFb-MYH11). All these 9 samples had specific chimeric gene number below 1.5 copies. In three samples (1 AML1-ETO, 1 CBFb-MYH11 and 1 PML-RARa) peaks of WT1 levels of ≥300 copies were detected. In these three samples the specific chimeric gene was undetectable (<0.01). ConclusionsBone marrow WT1 copy number in treated CBF-AML and APL was 47.23. In ten percent of cases, the WT1 copy number was greater than 100 copies. Occasionally, WT1 peaks (≥300 ) were detected. The meaning of the differential bone marrow WT1 transcriptional activity in long-term survivors of AML remains to be investigated. Disclosures:No relevant conflicts of interest to declare.

Bone marrow WT1 levels at diagnosis, post-induction and post-intensification in adult de novo AML

We retrospectively assessed whether normalized bone marrow WT1 levels could be used for risk stratification in a consecutive series of 584 acute myeloid leukemia (AML) patients. A cutoff value of 5065 copies at diagnosis identified two prognostic groups (overall survival (OS): 44 ± 3 vs 36 ± 3%, P=0.023; leukemia-free survival (LFS): 47 ± 3 vs 36 ± 4%, P=0.038; and cumulative incidence of relapse (CIR): 37 ± 3 vs 47 ± 4%, P=:0.043). Three groups were identified on the basis of WT1 levels post-induction: Group 0 (WT1 between 0 and 17.5 copies, 134 patients, OS: 59 ± 4%, LFS:59 ± 4% and CIR: 26 ± 4%); Group 1 (WT1 between 17.6 and 170.5 copies, 160 patients, OS: 48 ± 5%, LFS:41 ± 4% and CIR: 45 ± 4%); and Group 2 (WT1 >170.5 copies, 71 patients, OS: 23 ± 6%, LFS: 19 ± 7% and CIR: 68 ± 8%) (P<0.001). Post-intensification samples distinguished three groups: patients with WT1 >100 copies (47 patients, 16%); an intermediate group of patients with WT1 between 10 and 100 copies (148 patients, 52%); and a third group with WT1 <10 copies (92 patients, 32%). Outcomes differed significantly in terms of OS (30 ± 7%, 59 ± 4%, 72 ± 5%), LFS (24 ± 7%, 46 ± 4%, 65 ± 5%) and relapse probability (CIR 72 ± 7%, 45 ± 4%, 25 ± 5%), all P<0.001. WT1 levels in bone marrow assayed using the standardized ELN method provide relevant prognostic information in de novo AML.

Normal Blood and Bone Marrow Wt1 Expression Level in Extramedullary Relapse in AML Patient

AbstractAbstract 4825Wilms' tumor gene 1 (WT1) that encodes a transcription factor has elevated expression in 90% of AML. High level of WT1 gene expression in bone marrow and peripheral blood of patients with AML indicates the presence of leukemia tumor burden. Thus, quantifying assessment of the level of WT1 could predict relapse. It is important to monitor a universal marker in the absence of target molecular genetic abnormalities in the disease onset. There are few data related to extramedullary relapses. Here we describe the patient with extramedullary relapse without concomitant elevation of WT1 expression.The patient is 22 year old male with the first early bone marrow and CNS relapse of AML. Cytogenetics- (45XY, t (3, 5) (q29; q15), −7, der (7), add (11) (p15)). Blast cells immunophenotype was CD45 + CD34 + CD38 + CD117 +. There was high blast count with high expression of WT1 level (Tabl 1). And there was no expression of other molecular genetic prognostic markers. Therapy with high dose of cytarabine with intrathecal administration of cytotoxic drugs was conducted. The complete hematological and cytogenetic remission was achieved after the first induction. Also there was no any blast cell in the CSF, WT1 expression decreased by 1,87 log (WT1 level became normal). Consolidation of remission with three similar courses with high doses of cytarabine was conducted. Extramedullary relapse during bone marrow and CNS remission with unilateral lesion of the lacrimal gland and the periorbital area( 2.8 × 3.4×4.6 cm )was diagnosed in 6 month. Relapse was confirmed by histological and immunochemical study of the left lacrimal gland biopsy, blasts phenotype was similar to the phenotype of bone marrow blasts at AML onset. WT1 level in bone marrow and peripheral blood remained within normal limits: 15.5 WT1/104 ABL copies and 13.4 WT1/104 ABL copies respectively. Therapy with high dose cytarabine and mitoxantrone was used as an induction. After this therapy a local remission was diagnosed according to MRI. Consolidation courses were followed by a course of radiotherapy to the area of the left orbit. Bone marrow remission and low-level expression of WT1 remained to be normal. Thereafter the patient has undergone unrelated allogeneic bone marrow transplantation. Conclusion.We have described isolated AML relapse without elevation of WT1. A possible reason for normal level of expression of WT1 in patients with extramedullary relapse may be a small amount of the extramedullary formation, its location and features of blood supply of the body area.Table 1Blast count in bone marrowWT1/104 ABL copies in bone marrowAt the diagnosis58%3560,6After the induction1%50,8After the first consolidation course1,4%26,7After the second consolidation course0,4%9,6After the third consolidation course0,4%3,04Follow-up2,4%14,3Follow-up1,4%17,7Extramedullary relapse1,4%15,5Day 14 of ≪HDAC≫3,4%29,4After ≪HiDAC≫6%49,2Follow-up before radiotherapy1,6%15Follow-up after radiotherapy32 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

WT1 Levels At Diagnosis and POST-Induction Provide Prognostic Information in Adult De Novo AML. Results From the Spanish Cetlam Group.

AbstractAbstract 2524▪▪This icon denotes a clinically relevant abstractWT1 monitoring is an almost universal target to follow de novo AML. Its exppression in myeloid malignancies is upregulated in parallel to the blast percentage. Recently, WT1 determination has been standardized as result of an European Leukemia Net initiative. Early reports have demonstrated that the best results are obtained when peripheral blood is used to establish clinical predictions.Pediatric studies in AML have shown that raised WT1 levels after induction associate with unfavourable outcome. Despite all the mentioned, WT1 quantitation has not yet gained widespread use, in part because some AML show normal WT1 levels at diagnosis.To investigate the prognostic impact of the normalized bone marrow WT1 levels at diagnosis and post-induction in a consecutive series of de novo AML patients enrolled in the CETLAM group trials.Available bone marrow samples at diagnosis (586 cases) and post induction (367 cases) were obtained in each participating center and sent to the CETLAM repository center at the Hospital de la Santa Creu i Sant Pau for complete immunophenotype and molecular analyses. One μg of RNA was reverse transcribed to cDNA in a total reaction volume of 20μl containing Cl2Mg 5mM, 10× Buffer, DTT 10mM, dNTP’s 10mM each, random hexamers 15μM, RNAsin 20 units (Promega) and 200 units of MMLV enzyme. WT1 expression levels were determined by real-time quantitative polymerase chain reaction (RQ-PCR) in an ABI PRISM 7700® Genetic Analyzer (Applied Biosystems, Foster City, CA) using the primers and conditions described by the ELN group (Cilloni et al J. Clin. Oncol 2009;27:5195-201). For WT1 copy number titration, the IPSOGEN® (Marseille, France) plasmid was employed. Results were expressed as copies and four normal bone marrow samples were used as test controls. Patients were treated between 2004 and 2011 according to the CETLAM03 protocol. Adults up to 70 years of age received induction chemotherapy with idarubicin, intermediate-dose cytarabine and etoposide, followed by consolidation with mitoxantrone and intermediate-dose ara-C. Subsequently, patients with favourable cytogenetics at diagnosis received one cycle of high-dose cytarabine.G-CSF priming during induction and consolidation was used. Patients with favorable cytogenetics and high leukocyte counts at diagnosis were treated with autologous transplantation instead of high-dose cytarabine. Furthermore, patients with a normal karyotype but an adverse molecular profile (FLT3 mutations or MLL rearrangements) were allocated to the treatment for unfavorable cases; this included allogeneic transplantation from an HLA-identical donor. Overall survival (OS) was measured from the date of enrolment until the date of death. Leukemia-free survival (LFS) for patients who achieved a CR was calculated from the date of CR to relapse or death. OS and LFS were plotted by the Kaplan-Meier method; differences between curves were analyzed by the log-rank test. The probability of relapse was calculated using cumulative incidence estimates and taking into account the competing risk of death in remission.A WT1 cut-off value of 5065.2 copies at diagnosis was obtained. Two hundred and four samples had WT1 levels greater than this value, whereas 382 samples showed levels below this cut-off. These groups had statistically different OS 55±3 vs 33±5 p<0.001, LFS 52±3 vs 30±6 p:0.004 and CIR 34±3 vs 56±6 p<0.001.As regards the post-induction results, four groups were established: Group 0 (135 patients) with WT1 levels between 0 and 17.5 copies, Group 1 (107 patients) with WT1 values ranging from 17.6 to 76 copies, Group 2 (54 patients) with WT1 between 76.1 and 170.5 copies and Group 3 (71 patients) with WT1 levels after induction greater than>170.6 copies. These groups showed statistically significant differences(p<0.001) in terms of OS: Group 0 59±4 months, Group 1 50±5 months, Group 2 45±7 months and Group 3 23±6 months. LFS was also statiscally different: Group 0: 58±4, Group 1: 46±5, Group 2: 39±8 and Group 3:19±8 (all p<0.001). Lastlly, CIR was markedly different between the four groups: Group 0:25±4, Group 1: 44±5, Group 2: 46±8 and Group 3: 68±8(p<0.001) .WT1 quantitation at diagnosis and post-induction provide a simple and well standardized measurement of the prognostic risk of adult AML patiens. Larger series need to be analyzed to ascertain whether this determination could be incorporated to initial AML risk stratification. Disclosures:No relevant conflicts of interest to declare.