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Background: Pyruvate kinase deficiency (PKD) is a rare inherited hemolytic anemia caused by mutations in the PKLR gene resulting in decreased red cell pyruvate kinase activity and impaired erythrocyte metabolism. Manifestations include anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current treatments are palliative and limited to blood transfusions, chelation therapy, and splenectomy which are associated with significant side effects. Preclinical studies in a clinically relevant PKD murine model have demonstrated that infusion of gene-modified Lin− bone marrow (BM) cells may ameliorate PKD phenotype. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adult and pediatric subjects with severe PKD. Methods: Six adult and pediatric patients with severe PKD (defined as severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled. Peripheral blood (PB) hematopoietic stem cells (HSCs) are collected on 2 consecutive days via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. HSCs are enriched, transduced with PGK-coRPK-WPRE lentiviral vector (LV), and cryopreserved. Following release testing of the investigational product (IP), RP-L301, myeloablative therapeutic drug monitoring (TDM) busulfan is administered over 4 days. RP-L301 is then thawed and infused. Patients are followed for safety assessments, including replication competent lentivirus (RCL) and insertion site analysis (ISA), and for efficacy parameters including PB and BM genetic correction, decrease in transfusion requirements, clinically significant improvement in anemia, and reduction of hemolysis for 2 years post-infusion. Results: As of May 2021, 2 adult patients with severe anemia have received RP-L301. Patient 1 (age 31 years) received 3.9x106 CD34+ cells/kg with mean vector copy number (VCN) of 2.73. Patient 2 (age 47 years) received 2.4x106 CD34+ cells/kg with mean VCN of 2.08. Despite baseline hemoglobin (Hb) levels in the 7.0-7.5 g/dL range, both patients displayed normal-range hemoglobin (Hb), improved hemolysis markers, and have required no red blood cell transfusions post-engraftment at 9- and 6- months follow-up. Both report improved quality of life. PB mononuclear cell VCNs for both patients were >2.0 at last evaluated timepoint (6- and 3-months post-treatment, respectively). No serious adverse events have been attributed to RP-L301. Updated safety and efficacy data will be presented. Conclusions: Hematopoietic stem cell mobilization using G-CSF and plerixafor is feasible and effective in adult PKD patients. RP-L301 was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications. Efficacy was demonstrated by normalized Hb associated with engraftment confirmed by PB and BM VCN. Disclosures Shah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: Dr. Navarro has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla: Miltenyi: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Sevilla is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, and may be entitled to receive financial benefits from the licensing of such patents.; SOBI: Consultancy. Glader: Agios: Consultancy. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company. Beard: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Rao: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Bueren is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, may be entitled to receive financial benefits from the licensing of such patents and receives funding for research., Patents & Royalties, Research Funding. Schwartz: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding.

INTRODUCTION: The use of Peripherally Inserted Central Catheters (PICCs) has increased significantly in the last years due to their advantages compared to the other types of central catheters: easier and protocolized insertion by specialized nurse-led teams; cost-effectiveness; ease of management, ... However, an increase in the incidence of Catheter Related Thrombosis (CRT) has been observed with this type of device, especially in cancer patients and in the critical care setting. The main objective of this study is to determine the incidence of PICC related deep venous thrombosis in oncologic and onco-hematologic patients at the Donostia University Hospital, in Spain. The secondary outcome is the identification of possible risk factors associated with this event. METHODS: Using the database created and handled by the nurse-led Intravenous Therapy Team (ITT) in our center, in which all inserted PICCs are prospectively and consecutively included since 2011, a retrospective analysis was conducted on oncology and hemato-oncology-derived adult patients (over 18 years old) with a PICC inserted between May 15th 2018 and December 15th 2019. In the total population, several characteristics of the patient, of the PICC and of the thrombotic event were descriptively analyzed, and a bivariant analysis of four potential risk factors was carried out using Pearson's Chi-squared test. Patient and CRT treatment-associated risk factors were more exhaustively analyzed in the subgroup of patients with CRT. The missing data were obtained from the electronic clinical history records. RESULTS: The final study sample consisted of 1024 PICCs (n=1024), 19,10% (n=313) derived from the Hematology department and 43,62% (n=715) from the Oncology department (tables 1 and 2). The global incidence of CRT was 4,9% (n=50): 5.8% in hematologic patients and 4.5% in patients derived from Oncology. In the bivariant analysis no significant association was found between the selected potential risk factors (department of origin, PICC lumen number, PICC material and the catheter-to-vessel ratio) and CRT (table 3). In terms of the treatment administered to patients presenting CRT, in 80% of the cases (n=40) a Low Molecular Weight Heparin (LMWH) at therapeutic dose was initiated; in 10% (n=5) LMWH at a lower dose, and in 2 patients treatment could not be initiated because of thrombopenia. Finally, the PICC was withdrawn in only 8 patients after the diagnosis of the thrombotic event. CONCLUSIONS: The majority of the studies on PICC associated venous thrombosis in cancer patients are small, observational, retrospective, and without comparison groups. Here we present a work with an important sample size, a homogeneous population and with a prospective data collection. The CRT incidence has been similar to that described in the literature and significant association has not been found between the included potential risk factors and CRT. In conclusion, this study reflects the need of more trials on this subject, in particular to identify CRT risk factors in order to design effective prevention strategies. Disclosures No relevant conflicts of interest to declare.

Introduction: Pyruvate Kinase Deficiency (PKD) is a rare inherited hemolytic anemia that is caused by mutations in the PKLR gene leading to decreased red cell pyruvate kinase (RPK) activity and impaired erythrocyte metabolism. The disorder is characterized by anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current therapies are palliative and limited to chronic blood transfusions, iron chelation therapy, and splenectomy. The side effects of these supportive treatments include iron overload, end-organ damage and increased infection risks. AG-348, an allosteric activator of RPK, is under evaluation in clinical trials, predominantly in less severely-afflicted transfusion-independent patients. Allogeneic hematopoietic stem cell transplantation (HSCT) has been performed in selected cases and resulted in transfusion independence, suggesting that the disorder may be reversed when an adequate level of hematopoietic stem and progenitor cells (HSPCs) harboring a corrected PKLR gene engraft in the bone marrow (BM). The therapeutic efficacy of allogeneic transplant is limited by the availability of a suitable donor and transplant-associated toxicities. Preclinical studies conducted in a clinically relevant PKD murine model have demonstrated the safety and efficacy of Lin- BM cells transduced with the therapeutic lentiviral vector, PGK-coRPK-WPRE, in ameliorating the PKD phenotype. More specifically, transplantation of transduced cells resulted in increased erythrocyte survival, decreased reticulocytosis, and improvement in the secondary manifestations of hemolytic anemia, including splenomegaly and hepatic iron overload. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (clinicaltrials.gov#NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adults and pediatric subjects with severe PKD. Methods: 6 subjects with severe PKD (defined as having a history of severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled in the Phase 1 study; the first 2 subjects will be adults (age ≥18- Results: An adult female PKD subject (age 31 years) with significant anemia and transfusion requirement has received treatment as of July 2020. Mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target area under the curve (AUC). IP consisted of 3.9×106 CD34+ cells/kg body weight, with a mean vector copy number (VCN) of 2.73. Safety and preliminary efficacy results will be available at the time of presentation. Conclusions: Efficacy in pre-clinical models indicates promising potential for clinical gene therapy in severe PKDHematopoietic stem cell mobilization using G-CSF and plerixafor appears feasible and effective in adult PKD patientsIP was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications Disclosures Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Glader:Agios Pharmaceuticals, Inc.: Consultancy. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Acute myeloid leukemia (AML) is a malignant disease characterized by uncontrolled proliferation, differentiation arrest and accumulation of immature myeloid progenitors. Despite recent developments and the approval of new therapeutic agents in the last few years, long term survival of AML, particularly in elderly patients remains an unmet medical need.The use of all-trans retinoic acid (ATRA) in Acute Promyelocytic Leukemia has proven that differentiation therapy may significantly change the survival of AML patients, however the success in APL has not been translated to other groups of AML. Therefore, the identification of new therapeutic agents that may induce the differentiation of AML blasts represents an attractive new target. Furthermore, it is well known that epigenetic alterations have an important role in the development and maintenance of cancer and AML in particular. Thus, our aim was to develop new small molecules targeting epigenetic modifying enzymes like DNA methyltransferases (DNMT), histone methyltransferases or histone deacetylase (HDAC) with the aim of inducing differentiation in AML. We performed a screening of over 50 small molecules synthesized by our group. The design was performed in-house using a knowledge and structure based strategy and the read out of the screening was based on changes in expression of CD11b (a well described marker of myeloid differentiation) after in vitro treatment of AML cells lines. Interestingly, we found several compounds with high capacity to promote the differentiation of leukemic cells in AML cells lines at low non-cytotoxic doses, selecting CM-444 and CM-1758 as our lead compounds (Figure 1a).A complete biochemical characterization showed that both compounds are specific pan-HDACs inhibitors (HDACi). CM-444 and CM-1758 induced in vitro cell differentiation in all subtypes of AML, independently of the AML genetic subgroups or the presence of mutations, which was significantly more pronounced that differentiation induced by reference compounds such as Panobinostat, Vorinostat, Entinostat, Tubastatin or Quisinostat, previously described HDACi. CM-444 and CM-1758 also induced in vivo differentiation in xenogeneic models of AML. AML differentiation was associated with induction of CD11b, downregulation of c-MYC, overexpression of transcription factors that govern the myeloid differentiation and morphologic changes. In addition, these compounds promoted in vitro differentiation of patient-derived AML blasts. The complete transcriptome analysis by RNA-Seq carried out in AML cell lines after CM-444, CM-1758, Panobinostat or Vorinostat treatment showed changes in genes implicated in differentiation, but without explaining the differences among the different HDACi. Analysis of the complete acetylome and proteome before and after treatment with CM-444 and CM-1758 in comparison with other HDACi showed differential acetylation of non-histone proteins included in the GO categories of Zn binding proteins and nucleic acid binding proteins (Figure 1b). Most of these proteins are epigenetic enzymes and have been related to AML and myeloid differentiation, such as MLL2, EP300 or BRD4. In summary, we have developed and characterized novel epigenetic small molecules with a high in vitro and in vivo capacity of differentiating AML cells. These compounds might be an effective differentiation-based therapy to be tested in AML. Besides, the mechanism of differentiation of these compounds is due, at least in part, to the acetylation of non-histone epigenetic proteins, which are key in the myeloid differentiation. Disclosures Paiva: Celgene, Janssen, Sanofi and Takeda: Consultancy; Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche and Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria.

Background Cytogenetics at diagnosis is the most important prognostic factor in acute myeloid leukemia (AML). Of note, intermediate cytogenetic risk group (IR-AML) is a very heterogeneous subset including normal karyotypes and all the cytogenetic abnormalities not included in the favorable or the adverse groups. Molecular alterations affecting NPM1, FLT3 and CEBPA show a prognostic impact in IR-AML. MLL partial tandem duplications (MLL-PTD) have also been described in this group of AML, but their prognostic impact have not been well established. Aim To analyze the prognostic relevance of MLL-PTD in the subset of patients diagnosed with IR-AML since 2003, and included in the CETLAM protocols LMA-2003 and LMA-2012. Methods Between 2003 and 2004 MLL-PTD were analyzed by Southern Blot (enzymes employed BglII, EcoRI, BamHI). Since 2004, a long PCR strategy was used to identify this abnormality. Results NPM1 mutations (NPM1mut), FLT3 internal tandem duplications (FLT3-ITD) and MLL-PTD were available for 893 patients. No MLL-PTD was found among 111 and 161 patients of the good and poor cytogenetic risk groups, respectively. The IR-AML group included 621 patients, and 37 carried a MLL-PTD (6%), thus only this cytogenetic group of patients was analyzed. NPM1mut were found in a 41% of patients and none of them had a concomitant MLL-PTD (p Conclusions MLL-PTD is a genetic alteration found in a 6% of IR-AML. Patients with this abnormality have a worse LFS and OS than the rest of patients of the IR-AML group. Based on these results, patients with MLL-PTD should be considered as patients with poor cytogenetic risk AML for treatment allocation. Disclosures No relevant conflicts of interest to declare.

BACKGROUND Cure rates of pediatric acute lymphoblastic leukemia (ALL) have reached 90% in the developed countries. However, toxicity due to chemotherapeutic regimens occurs frequently but with great heterogeneity. This suggests that genetic variation is involved. In order to identify determinants of adverse effects, recent studies have investigated pharmacogenetic features in relation to toxicity. Most of these studies examined coding regions of the genome. Recently, it has been described that epi genetic regulators, such as micro-RNA's (miRNA), might also have an important regulatory function in genes involved in drug related toxicity. In a recent study 25 miRNA SNPs were found to be related to toxicity in pediatric ALL treatment (Lopez-Lopez, PLoS ONE, 2014). In pediatric ALL mucositis is one of the most frequent side effects during high dose methotrexate (MTX) treatment. AIM The aim of this study was to detect novel, epigenetic biomarkers that predict MTX related oral mucositis in pediatric acute lymphoblastic leukemia (B- and T cell) by studying single nucleotide polymorphisms (SNP) involved in miRNA levels and function. METHODS DNA was isolated from whole blood of 118 pediatric ALL patients that were treated with high dose MTX (5 gr/m2) according to the Dutch Childhood Oncology Group ALL-10 protocol. The recently published 25 SNPs, involved in miRNA function and located in the DROSHA, CNOT1, CNOT4, EIF2C1, GEMIN3, GEMIN4, MIR604, MIR453, MIR2110, MIR2053, MIR1294, MIR1206, DICER, XPO5 and TNRC6B genes, were selected for genotyping. Toxicity data during the consolidation phase were prospectively collected and documented according to the National Cancer Institute (NCI) v.3.0 score system. Mucositis NCI grade ≥ 3 (grade 3: confluent ulcerations, bleeding with minor trauma), was considered as clinical significant toxicity and was used as endpoint. RESULTS Mucositis was the only recurring toxicity in this prospectively well-documented cohort and therefore used as endpoint of this study. A selection of 20 of the previously identified 25 candidate SNPs was studied based on technical feasibility. In addition, 1 SNP in the XPO 5 gene was not considered for analysis because it was not in Hardy Weinberg equilibrium. Mucositis occurred in 19% of the patients in at least one of the MTX courses. Only the TT genotype of rs11866002 in the CNOT1 (CCR4-NOT complex, subunit 1) gene was associated with a higher risk of developing mucositis (NCI ≥ 3) compared to carries of CC/CT. The other 18 candidate SNPs analyzed did not show statistically significant associations. CONCLUSION The inter-patient variability of mucosal toxicity was not associated with most of our investigated SNPs which are involved in miRNA transcription and function. CNOT1 rs11866002 C>T was the only single nucleotide polymorphism associated with the occurrence of oral mucositis during pediatric acute lymphoblastic leukemia treatment. We acknowledge the Foundation Children Cancerfree (KiKa), Amstelveen, The Netherlands, for funding this research. Disclosures No relevant conflicts of interest to declare.

Introduction: Methotrexate (MTX) is an important and effective chemotherapeutic drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). However MTX can induce toxicity, which can lead to amendments of treatment and subsequent impaired survival. The aim of this study was to identify metabolic and genetic determinants of MTX toxicity. Patients and Methods: In this prospective study, 134 Dutch pediatric ALL patients were treated with four high dosages MTX (HD-MTX: 5 g/m2) every other week according to the DCOG-ALL10 protocol. Toxicity was prospectively scored and a National Cancer Institute (NCI) grade ≥3 was considered clinically relevant toxicity. Plasma MTX levels were measured at 24 and 48 hours after each HD-MTX infusion. Erythrocyte folate, plasma folate and plasma homocysteine levels were determined at the start of protocol M. Seventeen single nucleotide polymorphisms (SNPs) in 7 candidate genes in the MTX pathway were analyzed. Results: Grade ≥3 mucositis occurred in 20% of the patients, skin toxicity in 7%, diarrhea in 3%, and neurotoxicity in 3%. Mucositis occurred especially after the first course compared to the other courses (p=0.006). Mucositis was not associated with plasma MTX, plasma folate or plasma homocysteine levels. Patients with mucositis had higher baseline levels of erythrocyte folate (median 1.2 μmol/L vs. 1.4 μmol/L, p Conclusion: Mucositis is the most frequent occurring toxicity during the HD-MTX phase in pediatric ALL treatment, and occurs especially after the first MTX course. Only a higher baseline erythrocyte folate, which reflects the accumulation of MTX polyglutamates in mucosal cells, and the wild-type variant of rs7317112 SNP in ABCC4 were determinants of mucositis in pediatric ALL during MTX-HD treatment. Disclosures No relevant conflicts of interest to declare.

Background Quantitation 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. Objective To establish the reference range value of bone marrow WT1 levels in treated CBF-AML and APL in complete remission. Patients and Methods Patients 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. Results All 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 Conclusions Bone 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.

Abstract 2524 WT1 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 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 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.

Abstract 4813 Accurate cytogenetic and molecular diagnosis of acute myeloid leukemia help to establish the prognosis and to select the best postconsolidation treatments. This genetic characterization is currently being performed by means cytogenetic techniques and molecular methods mainly based in PCR, capillary electrophoresis and Sanger sequencing. Recently, large and homogeneous series of AML patients have been analyzed by means expression arrays. These studies have helped to standardize the method and they are becoming an alternative to traditional methods in terms of simplicity and time. Objectives To assess the usefulness of the AMLprofiler™ in genetic stratification of de novo AML enrolled in a cooperative Group. To investigate the applicability of the array diagnostic platform in multicenter trials. To compare the AMLprofiler™ with the standard approach. Patients and methods Twenty one consecutive de novo AML cases enrolled in the Spanish CETLAM protocol were included in the study. Patients were recruited during 2012. Cytogenetic analysis was performed in each participating center and the results were sent to the HSCSP. Complete immunophenotype and molecular studies were performed centrally at the HSCSP. Purified DNA was employed to test FLT3-ITD and TKD mutations, NPM1, CEBPa and MLL. For each FLT3-ITD positive case, the allelic ratio was calculated. Given that FLT3 mutations are not detected by the AML-profiler™, this determination was common to both approaches. Purified RNA was used to assess the presence of the AML1-ETO and CBFb-MYH11 rearrangements and to test RNA integrity. We used the AMLprofiler™ assay which detects chromosomal aberrations (t(8;21), t(15;17), inv(16), mutations (CEBPA dm, NPM1 A/B/D) and genetic expresion of BAALC and EVI1. Only those cases fulfilling the quality required by the manufacturer were hybridized the AMLprofiler™array. Based on this requirement one case was ruled out. Results Twenty cases were analyzed with the AMLprofiler™ and with the conventional methods (21 intended/20 hybridized). There were 4 cases with FLT3-ITD. One case had a t(8;21) and it has been succesfully detected by both methods. There were 4 inv(16), in one case the RNA quality was not satisfactory and was not hybridized, in the remaining 3 cases the AMLprofiler detected the genetic lesion including one case with a cryptical translocation and in one case with a typical inv(16) but with a complex CBFb-MYH11 transcript. In 8 cases a NPM1 mutation was detected by molecular methods, in all NPM1 A, B or D, the AMLprofiler™ gave concordant results, one case with a non-ABD mutation was not detected. When considering technician times, AMLprofiler™ compared favorably with the conventional molecular biology techniques (mean times: 2958 min vs 3422 min). This figure does not take into consideration the cytogenetic workload performed in each participating center. Conclusions The AMLprofiler™ could be easily used to stratify de novo AML patients enrolled in multicenter trials. It provides accurate results and requiring less time than the currently employed methods in the CETLAM group. The AMLprofiler™ seems to be specially useful in the core binding-factor leukemias. The most important limitations are related to the need of an excellent RNA sample and the presence of rare NPM1 mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract 4886 The Wilms tumor 1 (WT1) gene encodes for a zinc-finger protein that may act both as a tumor suppressor and as an oncogene. It is widely expressed in normal tissues, especially in the urogenital system during fetal development. WT1 is a transcriptional regulator of genes involved in the cell growth and metabolism, such as c-Myc, bcl-2 and epidermal growth factor receptor (EGFR). Its abnormal mRNA overexpression in myeloid malignancies has been exploited as minimal residual disease marker. WT1 mutations have also been described in a percentage of acute myeloid leukemia (AML) with a normal karyotype. It has been reported that these patients had a bad outcome. Few data are available on WT1 protein expression in myeloid neoplasms. WT1 expression was investigated in bone marrow samples obtained from 61 patients (54 AML, 1 MPN, 5 MDS and one case with myeloid sarcoma) and 5 controls. Blast cell immunophenotype and cytogenetics were available for each case. WT1 mutations were investigated in 14 AML cases. Immunohistochemistry procedures were performed on formalin-fixed paraffin embedded whole tissue sections with primary monoclonal antibody against WT1 (WT1 MxH 6FH2 clone, DAKO, Glostrup, DK). The staining intensity was graded on a 0 to 3 scale (0, negative; 1, weak; 2, moderate; and 3, intense staining) and the percentage of stained cells was scored. To obtain the final score, both values (staining intensity and percentage of positive cells) were multiplied. Cases with WT1 score under 10 were considered as negative. We also analyzed WT1 mRNA gene expression on the same cases by real-time PCR as previously described (Cilloni D et al, J Clin Oncol 2009).Relationship between quantitative variables was evaluated by non-parametric correlation analysis (Spearman correlation test). Two-sided p value under 0.05 was considered significative. In normal bone marrow, WT1 immunohistochemical expression was restricted to the cytoplasm of occasional megakaryocytes and endothelial cells, whereas no nuclear staining was observed neither in hematopoietic cells nor in endothelial and circulating lymphoid cells. Immunohistochemical WT1 expression was found in 43 out of 61 (70.5%) cases. The mean value of mRNA WT1 expression was 3812.3 copies (standard deviation (SD):7163.9). We observed a positive correlation between WT1 score and mRNA WT1 expression (p In myeloid malignancies, WT1 protein expression showed some degree of correlation with the WT1 mRNA levels. It remains to be investigated the mechanisms which can explain the discordances and if these findings could provide prognostically relevant information. Disclosures: No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by an abnormal proliferation of the myeloid precursors and a maturation block. A large proportion of AML cases have either a normal karyotype or non-recurrent chromosomal alterations. Underlying genetic lesions of some of these cases have been characterized with the discovery of MLL-internal tandem duplications, activating FLT3 mutations and NPM mutations. Loss of heterozygosity (LOH) derives from the loss of one of the two alleles at a given locus and can be a sign of inactivation of tumor-suppressor genes. We performed a high-resolution genotype analysis on DNA obtained from 19 AML patients with a normal karyotype, both at diagnosis and in samples obtained in complete remission(assessed by multiparametric flow cytometry) using the 10K SNP Array (Affymetrix). Both LOH and copy number analysis, as well as visualization of these analysis were performed by means of the dChip software (M. Lin et al., Bioinformatics (2004), 20:1233–40). A mean call rate of 96.8%. SNP array-based LOH analysis revealed that 4 patients presented large regions of homozygosity at diagnosis which were absent from samples in complete remission. In all four patients copy number analysis indicated no gross chromosomal losses or gains, as was confirmed by conventional cytogenetic analysis. Therefore, it can concluded that the LOH observed in these four patients was due to the presence of uniparental disomy. Simultaneous analysis of FLT-3 internal tandem duplications (FLT-3/ITD), FLT3- D835 mutations, NPM mutations and MLL rearrangements was performed using conventional molecular methods. Two of these patients (UPN2 and UPN12) had FLT-3/ITD in association with NPM mutations. UPN4 had a mutated form of NPM whereas in patient UPN16 FLT-3 and NPM genes were in the germ line configuration. All four cases were negative for MLL rearrangements and FLT-3-D835 mutations. These results suggest that NPM and FLT3 mutations may be associated with acquired somatic recombinations. It remains to be investigated whether there are loci preferentially involved by these events. | Patient | LOH | FLT3 | NPM | D835 | MLL | |:------- | ------------------------ | --------- | --------- | --------- | --------- | | UPN2 | 13q | Mutated | Mutated | Germ line | Germ line | | UPN4 | 6pter-p12.212q13.12-qter | Germ line | Mutated | Germ line | Germ line | | UPN12 | 2p | Mutated | Mutated | Germ line | Germ line | | UPN16 | complex | Germ line | Germ line | Germ line | Germ line | Uniparental disomy and genetic lesions in normal karyotype AML