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Severe ADAMTS13 deficiency defines thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is responsible for VWF cleavage. In the absence of this enzyme, widespread thrombi formation occurs, causing microangiopathic anemia and thrombocytopenia, and leading to ischemic organ injury. Understanding ADAMTS13 function is crucial to diagnose and manage TTP, both in the immune and the hereditary form.ADAMTS13 role in coagulation homeostasis and the consequences of its deficiency are detailed. Other factors that modulate the consequences of ADAMTS13 deficiency are explained, such as complement system activation, genetic predisposition or the presence of an inflammatory status. Clinical suspicion of TTP is crucial to start prompt treatment and avoid mortality and sequelae. Available techniques to diagnose this deficiency and detect autoantibodies or gene mutations are presented, as they have become faster and more available in the last years.A better knowledge of TTP pathophysiology is leading to an improvement in diagnosis and follow-up, as well as a customized treatment in patients with TTP. This scenario is necessary to define the role of new targeted therapies already available or coming soon and the need to better diagnose and monitor at the molecular level the evolution of the disease.

Introduction: Transfusion plays a main role in supportive treatment for patients who receive an allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we compare the transfusion requirements of patients undergoing different modalities of HSCT according to different time periods. The objective is to assess the evolution of HSCT transfusion requirements over time, from a single institution. Methods: The clinical charts and transfusion records of patients who underwent HSCT of different modalities at La Fe University Hospital during a twelve-year period were reviewed (2009–2020). For analysis, we divided the overall time into three periods: 1 from 2009 to 2012, 2 from 2013 to 2016 and 3 from 2017 to 2020. The study included 855 consecutive adult HSCT: 358 HLA-matched related donors (MRD), 134 HLA-matched unrelated donors (MUD), 223 umbilical cord blood transplantation (UCBT) and 140 haploidentical transplants (Haplo-HSCT). Results: There were no significant differences in RBC and PLT requirements or transfusion independence among the three time periods for MUD and Haplo-HSCT. However, the transfusion burden increased significantly for MRD HSCT during the 2017–2020 period. Conclusion: despite HSCT modalities having evolved and changed over time, overall transfusion requirements have not significantly decreased and continue to be a cornerstone of transplantation-supportive care.

Acquired thrombocytopenic thrombotic purpura (aTTP) is an autoantibody-mediated disease against the enzyme A Disintegrin and Metalloprotease domain with ThromboSpondin-1 type motif 13, which until now has been treated with plasma exchange (PEX) and corticosteroids. A 29-year-old female patient, who presented with aTTP in the context of pregnancy, has developed multiple relapses after treatment with PEX, corticosteroids, and rituximab. Recently, caplacizumab, a nanobody against von Willebrand factor, has been approved for the treatment of aTTP. In our patient, caplacizumab achieved better disease control, with a lower platelet count restoration time, days of PEX and hospitalization duration, as compared to standard therapy, reproducing the results of clinical trials. Caplacizumab represents a significant advance in the treatment of aTTP, especially in cases of recurrent relapses.

Introduction Thrombotic thrombocytopenic purpura (TTP) is a rare life-threatening thrombotic microangiopathy (TMA) characterized by the severe deficiency of ADAMTS13 activity ( Methods A comparison method was performed to compare HemosIL Acustar® with an in-house FRETS-VWF73 assay and two commercial ELISA assays: the TECHNOZYM® ADAMTS13 Activity (Technoclone GmbH, Vienna, Austria) and the DG-EIA ADAMTS-13 Activity (Diagnostic Grifols, SA, Barcelona, Spain). A set of 241 frozen plasma samples with known ADAMST13 levels was used. Agreement between methods was assessed with focus on two cut-off ADAMTS13 activity values: Results HemosIL AcuStar® showed high agreement with the other methods in correctly classify patients with ADAMTS13 values below 10% (Kappa = 0.89) and even below 5% (Kappa = 0.94) with no false negatives and few false positives (5.40%; 95% CI: 2.20 to 8.60%). However, it also tended to underestimate ADAMTS13 levels, especially for the high assay range values (>40%) (absolute mean bias of 8.40% (95% CI: 6.53 to 10.42%)) when compared to other assays. Conclusions HemosIL AcuStar® is highly sensitive to detect ADAMTS13 values below 10% and 5%. A large prospective validation study is needed to corroborate its utility in clinical practice.

Introduction: Acquired thrombotic thrombocytopenic purpura (aTTP) is a thrombotic microangiopathy caused by inhibitory autoantibodies against ADAMTS13 protein. Until recently, the combination of plasma exchange (PEX) and immunosuppression has been the standard front-line treatment in this disorder. However, aTTP-related mortality, refractoriness, and relapse are still a matter of concern. Areas covered: The better understanding of the pathophysiological mechanisms of aTTP has allowed substantial improvements in the diagnosis and treatment of this disease. Recently, the novel anti-VWF nanobody caplacizumab has been approved for acute episodes of aTTP. Caplacizumab is capable to block the adhesion of platelets to VWF, therefore inhibiting microthrombi formation in the ADAMTS13-deficient circulation. In this review, the characteristics of caplacizumab together with the available data of its efficacy and safety in the clinical setting will be analyzed. Besides, the current scenario of aTTP treatment will be provided, including the role of other innovative drugs. Expert opinion: With no doubt, caplacizumab is going to change the way we treat aTTP. In combination with standard treatment, caplacizumab can help to significantly reduce aTTP-related mortality and morbidity and could spare potential long-term consequences by minimizing the risk of exacerbation.

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we perform a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identify a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10−8; KMT5B). We also identify a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N = 1287) at 6p21.32 (rs3916765; P = 1.51 × 10−10; HLA). Our results inform on AML etiology and identify putative functional genes operating in histone methylation (KMT5B) and immune function (HLA)., Genome wide association studies in cancer are used to understand the heritable genetic contribution to disease risk. Here, the authors perform a genome wide association study in European patients with acute myeloid leukemia and identify loci associated with risk of developing the disease.

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we performed a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identified a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10-8; KMT5B). We also identified a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N=1287) at 6p21.32 (rs3916765; P = 1.51 × 10-10; HLA). Our results inform on AML etiology by identifying putative functional genes operating in histone methylation (KMT5B) and immune function (HLA).

Introduction Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy characterized by microangiopathic hemolytic anemia, thrombocytopenia, and organ injury. TTP pathophysiology is based on a severe ADAMTS13 deficiency, and is a medical emergency with fatal outcome if appropriate treatment is not initiated promptly. Areas covered Authors will review the best options currently available to minimize mortality, prevent relapses, and obtain the best clinical response in patients with immune TTP (iTTP). Available bibliography about iTTP treatment has been searched in Library's MEDLINE/PubMed database from January 1990 until April 2021. Expert opinion The generalized use of plasma exchange marked a paradigm in the management of iTTP. In recent years, strenuous efforts have been done for a better understanding of the pathophysiology of this disease, improve diagnosis, optimize treatment, reduce mortality, and prevent recurrences. The administration of front-line rituximab and, more recently, the availability of caplacizumab, the first targeted therapy for iTTP, have been steps toward a further reduction in early mortality and for the prevention of relapses.

Minimal residual disease (MRD) assessment is an essential tool in contemporary acute lymphoblastic leukemia (ALL) protocols, being used for therapeutic decisions such as hematopoietic stem cell transplantation in high‐risk patients. However, a significant proportion of adult ALL patients with negative MRD still relapse suggesting that other factors (ie, molecular alterations) must be considered in order to identify those patients with high risk of disease progression. We have identified partial IKZF1 gene deletions and CDKN2A/B deletions as markers of disease recurrence and poor survival in a series of uniformly treated adolescent and adult Philadelphia chromosome‐negative B‐cell progenitor ALL patients treated according to the Programa Español de Tratamientos en Hematología protocols. Importantly, CDKN2A/B deletions showed independent significance of MRD at the end of induction, which points out the need for treatment intensification in these patients despite being MRD‐negative after induction therapy., Fundació Internacional Josep Carreras; Generalitat de Catalunya, Grant/Award Number: 2017 SGR 288 GRC; Instituto de Salud Carlos III; Ministerio de Salud Carlos III RTICC‐FEDER, Grant/Award Numbers: RD12/0036/0029, RD/0036/044; Obra Social “La Caixa”; Sociedad Española de Hematología y Hemoterapia; Fondo de Investigaciones Sanitarias, Grant/Award Numbers: PI14/01971, PI10/01417

Therapeutic plasma exchange (TPE) is the extracorporeal technique performed in an apheresis device were patient's plasma is separated from whole blood and removed, while the cellular blood components are returned to the patient together with a replacement fluid. By the extracorporeal removal of pathological substances and the replacement of deficient plasma components, it constitutes an important tool for the management of several disorders and it is a well-known and established treatment for numerous diseases. Additionally, overall available data confirm the safety and efficacy of TPE. Nevertheless, the quality of the evidence supporting the utility and efficacy of the procedure is diverse. This review attempts to compile the current indications of TPE in different disorders according to an extensive and updated literature review, with special focus on its present role and its validity in the twenty-first century medicine.

Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections is on focus of research. We evaluate herein the feasibility of expanding virus‐specific T cells (VST) against SARS‐CoV‐2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS‐CoV‐2 asymptomatic infection/negative serology, (b) SARS‐CoV‐2 symptomatic infection/positive serology, and (c) no history of SARS‐CoV‐2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 62.7%, respectively) with CD4 + dominance (60% in both donors). Higher numbers of VST were obtained from the donor 2 as compared to donor 1. T‐cell clonality test showed oligoclonal reproducible peaks on a polyclonal background for both donors. In contrast, VST could be neither expanded nor primed in a donor without evidence of prior infection. This proof‐of‐concept study supports the feasibility of expanding ex vivo SARS‐CoV‐2‐specific VST from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS‐CoV‐2‐specificity for future adoptive transfer to immunosuppressed patients., The neutralization antibody assay was supported by Valencian government grant Covid_19-SCI as well as the Spanish National Research Council grants CSIC-COV19-082 and CSIC-COV-19-104 to RG.

Wra is the most common LIA in white population. The incidence of Wra antigen in Spanish population has been estimated to be 1 in 785 in blood donors, while anti-Wra was found in 2.7 % and 3.6 % of healthy donors and transfused patients respectively. Severe, even fatal hemolytic transfusion reactions and hemolytic disease of the newborn caused by anti-Wra have been reported. Since the reagent red blood cells used for antibody screening usually lack Wra antigen, the anti-Wra is not detected and hemolytic reaction could occur if transfusion is performed by type and screen approach. We report an acute hemolytic reaction due to anti-Wra in a patient with negative antibody screening. We have also reviewed the records of the hospital hemovigilance database in order to collect the previous hemolytic cases due to anti-Wra. During a 21-year period 461,539 red blood cell units have been transfused to 81,614 patients in our hospital. Alloimmnunization was detected in 3840 patients (0.83 %) and anti-Wra was detected in 22 patients (1/3709), 10 of whom had other alloantibodies, and only in 1 occasion (this case) has been implicated in mild hemolytic acute transfusion reaction. In our experience, the risk of fatal hemolytic reaction due to LIA in hospitals with blood services using the type and screen policy is extremely low.

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA) characterized by the development of microangiopathic haemolytic anaemia, thrombocytopenia, and ischaemic organ dysfunction associated with ADAMTS13 levels lower than 10% in most cases. Recently there have been numerous advances in the field of PTT, new, rapid and accessible techniques capable of quantifying ADAMTS13 activity and inhibitors. The massive sequencing systems facilitate the identification of polymorphisms in the ADAMTS13 gene. In addition, new drugs such as caplacizumab have appeared and relapse prevention strategies are being proposed with the use of rituximab. The existence of TTP patient registries allow a deeper understanding of this disease but the great variability in the diagnosis and treatment makes it necessary to elaborate guidelines that homogenize terminology and clinical practice. The recommendations set out in this document were prepared following the AGREE methodology. The research questions were formulated according to the PICO format. A search of the literature published during the last 10 years was carried out. The recommendations were established by consensus among the entire group, specifying the existing strengths and limitations according to the level of evidence obtained. In conclusion, this document contains recommendations on the management, diagnosis, and treatment of TTP with the ultimate objective of developing guidelines based on the evidence published to date that allow healthcare professionals to optimize TTP treatment.

Thrombotic thrombocytopenic purpura (TTP) is a rare life-threatening thrombotic microangiopathy (TMA) characterized by the severe deficiency of ADAMTS13 activity (10%). Rapid ADAMTS13 testing is crucial for early diagnosis and optimal management of TTP patients and other TMAs. The objective of this study was to retrospectively evaluate the performance of the recently commercialized HemosIL AcustarA comparison method was performed to compare HemosIL AcustarHemosIL AcuStarHemosIL AcuStar

Pure red cell aplasia (PRCA) is a complication related to major or bidirectional ABO mismatched hematopoietic stem cell transplantation. This disorder is characterized by anemia, reticulocytopenia, and the absence or virtual absence of erythroid progenitors, other causes such as infections, hemolysis, disease relapse, or drug toxicity having been excluded. Patients with PRCA may become RBC transfusion dependent for long periods, suffering an important long-term iron overload, alloimmunization, and transfusion reactions. The persistence of recipient isoagglutinins against donor ABO antigens produced by host residual plasmatic cells has been considered as the immunological cause of the prolonged erythroid aplasia. PRCA behaves in many cases as a self-limited condition and resolution may occur spontaneously within weeks, months, and even years. Many different therapeutic approaches have been reported for posttransplant PRCA as plasmapheresis, high doses of erythropoietin, donor lymphocyte infusions, anti-thymocyte globulin, Rituximab and steroids, among others. However, to date there is no standard of care and the question if patients with PRCA should be treated and at which point remains. The objective of this article is to review the natural evolution of PRCA, and the treatments that have been used over time focusing on their suitability and efficacy.

Este artículo se encuentra disponible en la siguiente URL: https://www.nature.com/articles/s41598-020-61589-9.pdf En este artículo también participan: David Hervás-Marín, Eva Barragán, Rosa Ayala, Marta LLop, María López-Pavía, Inmaculada Rapado, Alex Neef, Alejandra Sanjuan-Pla, Claudia Sargas, Elisa Gonzalez-Romero, Mireia Boluda-Navarro, Rafa Andreu, Leonor Senent, Pau Montesinos, Joaquín Martínez-López, Miguel Angel Sanz, Guillermo Sanz y José Cervera. Nearly 50% of patients with de novo acute myeloid leukemia (AML) harbor an apparently normal karyotype (NK) by conventional cytogenetic techniques showing a very heterogeneous prognosis. This could be related to the presence of cryptic cytogenetic abnormalities (CC A) not detectable by conventional methods. The study of copy number alterations (CNA) and loss of heterozygozity (LOH) in hematological malignancies is possible using a high resolution SNP-array. Recently, in clinical practice the karyotype study has been complemented with the identification of point mutations in an increasing number of genes. We analyzed 252 de novo NK-AML patients from Hospital La Fe (n = 44) and from previously reported cohorts (n = 208) to identify CCA by SNP-array, and to integrate the analysis of CCA with molecular alterations detected by Next-Generation-sequencing. CCA were detected in 58% of patients. In addition, 49% of them harbored CNA or LOH and point mutations, simultaneously. Patients were grouped in 3 sets by their abnormalities: patients carrying several CCA simultaneously, patients with mutations in FLT3, NPM1 and/or DNMT3A and patients with an amalgam of mutations. We found a negative correlation between the number of CCA and the outcome of the patients. This study outlines that CCA are present in up to 50% of NK-AML patients and have a negative impact on the outcome. CCA may contribute to the heterogeneous prognosis.

Introduction: Autoimmune thrombotic thrombocytopenic purpura (aTTP) is a severe disease caused by the production of autoantibodies against von Willebrand factor (vWF)-cleaving ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin-1 motifs; 13th member of the family). Without functional ADAMTS13, endothelial cells-derived unusually large vWF (ULVWF) multimers are present and are responsible for platelet clumping in the microvasculature. Plasma exchange (PE) and immunosuppressive therapy with steroids and rituximab are the milestones of the treatment of this disease. Moreover, in 2018, nanobody caplacizumab was approved for the treatment of adult patients with an acute episode of aTTP. Our objective was to retrospectively review the efficacy and safety of the current use of caplacizumab in Spain. Methods: We collected demographic, clinical, and laboratory data of patients with aTTP who were reported in the TTP Spanish Registry from 15 centres between July 2018 and July 2020. All patients were diagnosed with aTTP because of the presence of microangiopathic hemolytic anemia (MAHA), thrombocytopenia, organ dysfunction, ADAMTS13 activity less than 5% and the presence of ADAMTS13 autoantibodies. Qualitative data are presented as number (frequencies). Quantitative data are presented as mean ± standard deviation (SD) or median (interquartile range -IQR-) when appropriate. A correlation test was performed to determine the linear relationship between number of days since diagnosis to caplacizumab start and number of days since diagnosis to the first day with >150x109/L platelets. Results: Our series comprises 30 patients: 22 (73%) females and 8 (27%) males with a median age of 45 (IQR: 31-55). At presentation, neurologic abnormalities were seen in 18 (60%) patients, symptoms and signs of anemia were present in 15 (50%) patients, and bleeding was present in 10 (33%) patients. Fever was present in 1 (3%) patient. Laboratory tests showed hemoglobin 92±28 g/L, platelet count 19±16 x109/L, unconjugated bilirubin 2.33±1.76 mg/dL, LDH 1,467±1,428 IU/L, and creatinine 1.0±0.45 mg/dL. Median ADAMTS13 activity was 0 (IQR: 0-0.5) and ADAMTS13 autoantibodies were positive in all cases. After diagnosis, treatment was started with PE and steroids in all patients after a median of 0 days (IQR: 0-0) and patients received a median of 10 PE procedures (IQR: 7-13). Caplacizumab was administered to all patients with a median of 3 (IQR: 1-9) days after diagnosis and it was administered during 36 days (IQR: 31-39). No severe adverse events were reported with the use of caplacizumab. Rituximab was added in 19 (63%) patients after a median of 4 days (IQR: 14-21). Time to platelet normalization (>150x109/L) was 5.5 days (IQR: 4-17) after diagnosis. The longer the delay in administering caplacizumab after diagnosis, the longer the delay in platelet normalization (Pearson's correlation coefficient, r=0.94 (95% CI: 0.86 to 0.98; R2=0.89; p Conclusion: Caplacizumab was an efficacious and safe drug to treat adult patients with acute episodes of aTTP. A statistically significant strong positive linear correlation was observed between number of days from diagnosis to caplacizumab start and number of days from diagnosis to the first day with >150x109/L platelets. Disclosures No relevant conflicts of interest to declare.

The incidence of SF3B1 mutations in patients with RARS is high. Recently, it has been shown that SF3B1 and DNMT3A mutations overlap more often than expected, although it is not clear how this could affect the disease. We studied SF3B1 and DNMT3A in 123 RARS patients: 101 out of 123 samples (82%) had somatic mutations in SF3B1, and 13 of them (13%) showed a co-mutation (SF3B1mutDNMT3Amut). All co-mutated patients had a normal karyotype, and 12 of them (92%) were lower-risk patients (IPSS and IPSS-R). Despite their favorable profile, SF3B1mutDNMT3Amut patients showed a higher RBC transfusion dependency (92% versus 48%, p = .007), a shorter overall survival (OS) (median, 30 versus 97 months, p = .034), and a higher risk of progression to acute myeloid leukemia (AML) at 5 years (25% versus 2%, p = .023) than SF3B1mutDNMT3Awt patients. In conclusion, DNMT3A mutations are present in a significant proportion of SF3B1mut patients with a negative clinical impact.

Context IKZF1 (Ikaros) deletion has been proposed as a poor prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP ALL) in children and adults. Objective To analyze the frequency and prognostic impact of IKZF1 deletions in adult BCP ALL patients. To identify the IKZF1 gene expression signature to find patients with different deletion isoforms and therapeutic opportunities. Patients and methods MLPA or SNP array samples of 151 (109 Ph-negative and 42 Ph+) adult BCP ALL patients treated with MRD-oriented protocols from the PETHEMA Group. RNAseq was performed in 48 of them (27 Ph-negative and 21 Ph+). Results Median age was 40 [15–72] years. Ph+ patients showed older age (52 [20;72] vs. 36 [15;68] years, p 1.5 in RNAseq data analysis, we identified a robust IKZF1 deletion gene expression profile. This resulted in 119 significantly upregulated genes after multi-comparison adjustment (i.e. CCND1, LAMA3, SLC2A9, SNAI1, LDHC, CD34, ID3, CDH2, MAF) and 39 downregulated genes (i.e. ROBO1, HES6, KREMEN1, DHCR24, ABHD15). Downregulated genes were involved in Slit/Robo/EMT, Notch, Wnt/beta-catenin, and glucose and fatty acid metabolism pathways, while upregulated genes were involved in focal adhesion, ROS homeostasis, histone modification, anaerobic metabolism, stem cell quiescence, and IL-6/STAT pathways. A significant number of dysregulated gene targets of chemotherapeutic agents (retinoic acid, doxorubicin, cisplatin, gemcitabine) and targeted therapies, such as FAKi, ERKi, BCL2i, mTORi, JAKi, BRKi, EGFRi and CDKi, were identified. Conclusions Adult BCP ALL patients with IKZF1 partial gene deletions showed poor prognosis. Gene expression analysis enables the identification of potentially targetable lesions. Funding Supported in part by a grant from the Instituto de Salud Carlos III, Ministerio de Economia y Competividad, Spain (PI14/01971); 2017 SGR288 (GRC) Generalitat de Catalunya; and support from CERCA Programme/Generalitat de Catalunya, Fundacio Internacional Josep Carreras. The research leading to this invention has received funding from “la Caixa” Foundation.

Objective: To assess if infusion of human bone marrow-derived stem cells (BMDSCs) could promote follicle development in patients with impaired ovarian functions. Design: Experimental design. Setting: University research laboratories. Animal(s): Immunodeficient NOD/SCID female mice. Intervention(s): Human BMDSCs were injected into mice with chemotherapy-induced ovarian damage and into immunodeficient mice xenografted with human cortex from poor-responder patients (PRs). Main Outcome Measure(s): Follicle development, ovulation, and offspring. Apoptosis, proliferation, and vascularization were evaluated in mouse and human ovarian stroma. Result(s): Fertility rescue and spontaneous pregnancies were achieved in mice ovaries mimicking PRs and ovarian insufficiency, induced by chemotherapy, after BMDSC infusion. Furthermore, BMDSC treatment resulted in production of higher numbers of preovulatory follicles, metaphase II oocytes, 2-cell embryos, and healthy pups. Stem cells promoted ovarian vascularization and cell proliferation, along with reduced apoptosis. In xenografted human ovarian tissues from PRs, infusion of BMDSCs and their CD133+ fraction led to their engraftment close to follicles, resulting in promotion of follicular growth, increases in E-2 secretion, and enhanced local vascularization. Conclusion(s): Our results raised the possibility that promoting ovarian angiogenesis by BMDSC infusion could be an alternative approach to improve follicular development in women with impaired ovarian function. (C) 2018 by American Society for Reproductive Medicine.

BACKGROUND Some copy number alterations (CNAs) have independent prognostic significance for adults with acute lymphoblastic leukemia (ALL). METHODS This study analyzed via multiplex ligation–dependent probe amplification the frequency and prognostic impact of CNAs of 12 genetic regions in 142 adolescents and adults with de novo precursor B-cell ALL. RESULTS The cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion (59 of 142 or 42%) was the most frequent CNA, and it was followed by Ikaros family zinc finger 1 (IKZF1) losses (49 of 142 or 35%). IKZF1 deletions were more prevalent in Philadelphia chromosome (Ph)–positive ALL and were associated with advanced age and high white blood cell (WBC) counts. The multivariate analysis showed that advanced age and early B-cell factor 1 (EBF1) deletions were associated with chemotherapy resistance in both the whole series (hazard ratios, 0.949 and 0.135, respectively) and the Ph-negative subgroup (hazard ratios, 0.946 and 0.118, respectively). High WBC counts and focal IKZF1 deletions correlated with disease recurrence (hazard ratios, 1.005 and 1.869, respectively), whereas advanced age and CDKN2A/B losses influenced overall survival in both the whole series (hazard ratios, 1.038 and 2.545, respectively) and the Ph-negative subgroup (hazard ratios, 1.044 and 2.105, respectively). CONCLUSIONS Deletions of EBF1, IKZF1, and CDKN2A/B have an independent adverse prognosis for adolescents and adults with B-precursor ALL, and this suggests that these CNAs should be included in the initial risk assessment of ALL. Cancer 2015;121:3809–3817. © 2015 American Cancer Society.

Objective: To evaluate effects of autologous stem cell ovarian transplant (ASCOT) on ovarian reserve and IVF outcomes of women who arc poor responders with very poor prognosis. Design: Prospective observational pilot study. Setting: University hospital. Patient(s): Seventeen women who are poor responders. Intervention(s): Ovarian infusion of bone marrow-derived stem cells. Main Outcome Measure(s): Serum antimullerian hormone levels and antral follicular count (AFC), punctured follicles, and oocytes retrieved after stimulation (controlled ovarian stimulation) were measred. Apheresis was analyzed for growth factor concentrations. Result(s): The ASCOT resulted in a significant improvement in AFC 2 weeks after treatment. With an increase in AFC of three or more follicles and/or two consecutive increases in antimullerian hormone levels as success criteria, ovarian function improved in 81.3% of women. These positive effects were associated with the presence of fibroblast growth factor-2 and thrombospondin. During controlled ovarian stimulation, ASCOT increased the number of stimulable antral follicles and oocytes, but the embryo euploidy rate was low (16.1%). Five pregnancies were achieved: two after ET, three by natural conception. Conclusion(s): Our results suggest that ASCOT optimized the mobilization and growth of existing follicles, possibly related to fibroblast growth factor-2 and thrombospondin-1 within apheresis. The ASCOT improved follicle and oocyte quantity enabling pregnancy in women who are poor responders previously limited to oocyte donation. (C) 2018 by American Society for Reproductive Medicine.

Introduction: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare disease characterized by a severe deficiency of the enzymatic activity of ADAMTS13 caused by autoantibodies, with an incidence of 3-4 x106inhabitants per year according to the few published data available. Accurate estimates of the incidence of aTTP are important to assess the resources required for current treatments and to anticipate the need to develop new treatments. The aim of this study was to determine the actualincidence of aTTP in Spain, as well as its diagnosis, management, and associated complications. Material and methods:A cross-sectional surveywascarried out among hematologists working in Spanish hospitals by means of an email that was sent to all members of the three main hematological scientificsocieties of Spain. All participants were asked to report the number of patients over the age of 16 years with a de novodiagnosis and relapses examined between Jan 2015 and Dec 2017. They were also asked about the number of patients that were known and alive in each hospital without having experienced any episode during such period. The population area of each participating hospital was consideredto calculate the incidence and prevalence of the disease. We also estimated the hospitalization service, mean hospital stay, percentage of ADAMTS13 activity at diagnosis and during follow-up, initial management, refractory cases and exacerbations (as defined by Scully et al.), treatment-related complications, and sequelae of aTTP. The median, interquartile ranges, and percentages were used for the descriptive analysis. Given that no personal data were treated, this study did not require the approval of a Research Ethics Committee. Results:A response was received from 42 centers (Figure 1). All hospitals except a private one belonged to the Spanish public health system, which provides health coverage to the entire Spanish population.A total of 203 episodes were reported (138 new episodes). The calculated population of the participating centers was nearly 21 x 106inhabitants. The incidence was 2.25 x106inhabitants per year, and the prevalence 19 x106inhabitants. Six patients died before they could start treatment (all but one in first episodes) and five were sent to other hospitals; thus, a total of 192 episodes were eventually treated. Table 1 and 2 show the data of the enzymatic activity of ADAMTS13 and the ADAMTS13 inhibitor at diagnosis, as well asthe complications. Plasma exchange (PEX) was performed by the Hematology and Nephrology Departments of 29 (70.7%) and 12 (29.3%) hospitals, respectively. The median hospital stay was 14 days (IQR: 10-20). Seventy-five episodes (39.1%) required admission to the intensive care unit with a median stay of 4 days (IQR: 3-7). During first-time episodes, a median of 12 PEX procedures (IQR: 8-19) were performed per patient, whereas in the case of relapses, a median of 9 (7-10) PEX procedures were carried out per patient. One plasma volume (PV) was used in the PEX procedures performed in 34% of the episodes, while 1.5 PVs were used in 56% of the episodes, and other PVs were used in the remaining 9.8%. The median duration of the PEX procedures was 121 minutes (IQR: 118-180). PEX and corticosteroids were the initial treatments administered in 98.4% of the episodes. Rituximab was used as a first-line treatment for new episodes in 18 of the 127 patients (14.1%), as a second-line treatment in 34 patients (26.6%), and as a prophylactic treatment (followingremission) in 4 patients (3.5%). In addition to the 6 early deaths, 9 patients died despite receiving the treatment (15 of 203 episodes, accounting for a mortality rate of 7.3%). Refractoriness to the PEX + corticoids was observed in 31 episodes of the 192 ones treated (16.1%), and at least one exacerbation (26.5%) took place in 51 episodes. Conclusion.Wecalculated the incidence ofclinically diagnosed aTTP associated with a severe ADAMTS13 deficiency inalmost half of the Spanish population which provided a high accuracy to our findings. These data are concordant with those published previously in other countries. Despite the currently available therapies, considerable rates of refractoriness and mortality still persist.Our data will be very useful for estimating the budget invested in this pathology and proposing standards for the diagnosis and treatment of this disease in our region. Disclosures Pascual Izquierdo: Novartis: Consultancy; Sanofi: Consultancy. De La Rubia:AMGEN: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; AbbVie: Consultancy; Janssen: Consultancy. Mingot-Castellano:Novartis: Consultancy; Novonordisk: Consultancy; Roche: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Amgen: Consultancy; CSL Behring: Consultancy; Sobi: Consultancy.

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.

Here we report whole-exome sequencing of individuals with various myeloid malignancies and identify recurrent somatic mutations in SETBP1, consistent with a recent report on atypical chronic myeloid leukemia (aCML). Closely positioned somatic SETBP1 mutations encoding changes in Asp868, Ser869, Gly870, Ile871 and Asp880, which match germline mutations in Schinzel-Giedion syndrome (SGS), were detected in 17% of secondary acute myeloid leukemias (sAML) and 15% of chronic myelomonocytic leukemia (CMML) cases. These results from deep sequencing demonstrate a higher mutational detection rate than reported with conventional sequencing methodology. Mutant cases were associated with advanced age and monosomy 7/deletion 7q (-7/del(7q)) constituting poor prognostic factors. Analysis of serially collected samples indicated that SETBP1 mutations were acquired during leukemic evolution. Transduction with mutant Setbp1 led to the immortalization of mouse myeloid progenitors that showed enhanced proliferative capacity compared to cells transduced with wild-type Setbp1. Somatic mutations of SETBP1 seem to cause gain of function, are associated with myeloid leukemic transformation and convey poor prognosis in myelodysplastic syndromes (MDS) and CMML.

To elucidate differential roles of mutations in myelodysplastic syndromes (MDS), we investigated clonal dynamics using whole-exome and/or targeted sequencing of 699 patients, of whom 122 were analyzed longitudinally. Including the results from previous reports, we assessed a total of 2,250 patients for mutational enrichment patterns. During progression, the number of mutations, their diversity and clone sizes increased, with alterations frequently present in dominant clones with or without their sweeping previous clones. Enriched in secondary acute myeloid leukemia (sAML; in comparison to high-risk MDS), FLT3, PTPN11, WT1, IDH1, NPM1, IDH2 and NRAS mutations (type 1) tended to be newly acquired, and were associated with faster sAML progression and a shorter overall survival time. Significantly enriched in high-risk MDS (in comparison to low-risk MDS), TP53, GATA2, KRAS, RUNX1, STAG2, ASXL1, ZRSR2 and TET2 mutations (type 2) had a weaker impact on sAML progression and overall survival than type-1 mutations. The distinct roles of type-1 and type-2 mutations suggest their potential utility in disease monitoring.

Preliminary Acute Promyelocytic Leukemia (APL) whole exome sequencing (WES) studies have identified a huge number of somatic mutations affecting more than a hundred different genes mainly in a non-recurrent manner, suggesting that APL is a heterogeneous disease with secondary relevant changes not yet defined. To extend our knowledge of subtle genetic alterations involved in APL that might cooperate with PML/RARA in the leukemogenic process, we performed a comprehensive analysis of somatic mutations in APL combining WES with sequencing of a custom panel of targeted genes by next-generation sequencing. To select a reduced subset of high confidence candidate driver genes, further in silico analysis were carried out. After prioritization and network analysis we found recurrent deleterious mutations in 8 individual genes (STAG2, U2AF1, SMC1A, USP9X, IKZF1, LYN, MYCBP2 and PTPN11) with a strong potential of being involved in APL pathogenesis. Our network analysis of multiple mutations provides a reliable approach to prioritize genes for additional analysis, improving our knowledge of the leukemogenesis interactome. Additionally, we have defined a functional module in the interactome of APL. The hypothesis is that the number, or the specific combinations, of mutations harbored in each patient might not be as important as the disturbance caused in biological key functions, triggered by several not necessarily recurrent mutations.

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs) and T-cell large granular lymphocytic leukemias (T-LGLs) are clonal lymphoproliferations arising from either natural killer cells or cytotoxic T lymphocytes (CTLs). We have investigated for distribution and functional significance of mutations in 50 CLPD-NKs and 120 T-LGL patients by direct sequencing, allele-specific PCR, and microarray analysis. STAT3 gene mutations are present in both T and NK diseases: approximately one-third of patients with each type of disorder convey these mutations. Mutations were found in exons 21 and 20, encoding the Src homology 2 domain. Patients with mutations are characterized by symptomatic disease (75%), history of multiple treatments, and a specific pattern of STAT3 activation and gene deregulation, including increased expression of genes activated by STAT3. Many of these features are also found in patients with wild-type STAT3, indicating that other mechanisms of STAT3 activation can be operative in these chronic lymphoproliferative disorders. Treatment with STAT3 inhibitors, both in wild-type and mutant cases, resulted in accelerated apoptosis. STAT3 mutations are frequent in large granular lymphocytes suggesting a similar molecular dysregulation in malignant chronic expansions of NK and CTL origin. STAT3 mutations may distinguish truly malignant lymphoproliferations involving T and NK cells from reactive expansions.

The prognostic value of cytogenetics in adult acute lymphoblastic leukemia (ALL) is not as established as in childhood ALL. We have analyzed the outcome and prognostic value of karyotype in 84 adults diagnosed with Philadelphia-negative ALL from a single institution that received induction chemotherapy and had successful karyotype performed. The most frequent finding was normal karyotype in 35 (42%) cases, followed by aneuploidies in 20 cases (24%) and t(4;11)(q21;q23)/MLL/AF4 in 5 (6%), and the remaining 24(27%) cases carried miscellaneous clonal abnormalities. The group of patients with t(4;11)(q21;q23)/MLL/AF4, hypodiploidy and low hyperdiploidy (less than 50 chromosomes) showed a worse outcome than those with normal karyotype and miscellaneous abnormalities in terms of overall survival (OS) (3 years OS; 47% vs. 13%, p = 0.014) and relapse-free survival (RFS) (3 years RFS; 44% vs. 27%, p = 0.005). Other cytogenetic prognostic classifications reported to date were tested in our series, but any was fully reproducible. In conclusion, karyotype is a useful tool for risk assessment in adult ALL. We have confirmed the bad prognosis of t(4;11)(q21;q23)/MLL/AF4 and hypodiploidy. Besides, low hyperdiploidy could also define a high-risk group of patients who might be candidates for more intensive treatment.

Resumen Antecedentes y objetivo El termino microangiopatia trombotica (MAT) incluye un grupo heterogeneo de enfermedades potencialmente mortales o invalidantes, rapidamente evolutivas, caracterizadas por anemia hemolitica microangiopatica y trombocitopenia. La actuacion en las primeras horas es crucial para mejorar el pronostico de los pacientes. El objetivo de esta revision es proporcionar recomendaciones orientadas a optimizar el tratamiento inicial de la MAT y agilizar el diagnostico etiologico. Pacientes y metodos Se disena una guia practica en la cual se diferencian cuatro apartados en el abordaje inicial de las MAT: sospecha diagnostica, confirmacion sindromica, tratamiento urgente y estudios complementarios. Resultados La deteccion de anemia hemolitica microangiopatica (caracterizada por aumento de reticulocitos, LDH y bilirrubina indirecta, Coombs directo negativo y esquistocitos en el frotis de sangre periferica) y trombocitopenia no justificable por otras causas secundarias confirma el diagnostico sindromico de anemia hemolitica microangiopatica y trombocitopenia (AHMAT). Estos pacientes requieren ingreso en la unidad de cuidados intensivos para iniciar lo antes posible el recambio plasmatico, preferiblemente en las primeras 4-8 h. Antes de realizar el recambio plasmatico deben extraerse las muestras para el estudio de ADAMTS13 y de complemento. Finalmente, es importante solicitar las pruebas complementarias necesarias para obtener un correcto diagnostico etiologico. Conclusiones La puesta en practica de las recomendaciones consensuadas en esta guia permitira mejorar los resultados terapeuticos al facilitar la cooperacion de los distintos especialistas implicados en la atencion de las MAT.

Background and aim The term thrombotic microangiopathy (TMA) involves a heterogeneous group of diseases that can be overwhelming or invalidating, with an acute development, characterized by microangiopathic haemolytic anaemia and thrombocytopaenia. Its management during its initial hours is essential to improving the prognostic of these patients. The aim of this review is to give recommendations about the optimization of TMA initial treatment and to accelerate the aetiological diagnosis. Patients and methods We provide a practice guideline based on four steps for the initial management of TMA: diagnosis of suspicion, syndromic confirmation, emergent treatment and complementary tests. Results The detection of microangiopathic haemolytic anaemia (characterized by elevated reticulocytes, LDH and indirect bilirubin, negative direct Coombs test and schistocytes in peripheral blood), and thrombocytopaenia not explained by other secondary aetiologies confirm the syndromic diagnosis of microangiopathic haemolytic anaemia and thrombocytopaenia (MAHAT). These patients require admission to an Intensive Care Unit to initiate plasma exchange therapy as soon as possible, ideally within the first 4–8 h. Prior to this, samples for ADAMTS13 and complement study should be obtained. Finally, it is important to request the complementary tests necessary to have a correct aetiological diagnosis. Conclusions Adherence to the agreed recommendations in this guideline will improve therapeutic results by facilitating cooperation between different specialists involved in TMA management.

Some copy number alterations (CNAs) have independent prognostic significance for adults with acute lymphoblastic leukemia (ALL).This study analyzed via multiplex ligation-dependent probe amplification the frequency and prognostic impact of CNAs of 12 genetic regions in 142 adolescents and adults with de novo precursor B-cell ALL.The cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion (59 of 142 or 42%) was the most frequent CNA, and it was followed by Ikaros family zinc finger 1 (IKZF1) losses (49 of 142 or 35%). IKZF1 deletions were more prevalent in Philadelphia chromosome (Ph)-positive ALL and were associated with advanced age and high white blood cell (WBC) counts. The multivariate analysis showed that advanced age and early B-cell factor 1 (EBF1) deletions were associated with chemotherapy resistance in both the whole series (hazard ratios, 0.949 and 0.135, respectively) and the Ph-negative subgroup (hazard ratios, 0.946 and 0.118, respectively). High WBC counts and focal IKZF1 deletions correlated with disease recurrence (hazard ratios, 1.005 and 1.869, respectively), whereas advanced age and CDKN2A/B losses influenced overall survival in both the whole series (hazard ratios, 1.038 and 2.545, respectively) and the Ph-negative subgroup (hazard ratios, 1.044 and 2.105, respectively).Deletions of EBF1, IKZF1, and CDKN2A/B have an independent adverse prognosis for adolescents and adults with B-precursor ALL, and this suggests that these CNAs should be included in the initial risk assessment of ALL.

Introduction In the last years genome wide profilings have identified recurrent Copy Number Alterations (CNA) in genes potentially involved in the pathogenesis of Acute Lymphoblastic Leukemia (ALL). These studies have identified deletions in B-cell development genes (IKZF1, EBF1, PAX5, TCF3, etc.), cell cycle regulation genes (CDKN2A/B, RB1, TP53, etc.), glucocorticoid resistance genes (BTG1, CREBBP) and growth factor receptors genes (CRLF2, CSF2RA, IL3RA) among others. Some of these CNA (i.e. IKZF1, CDKN2A, CRLF2) have been reported to have prognostic significance in several pediatric series but there are very few data regarding their impact in B-lineage adult ALL. Our aim was to analyze the frequency and prognostic significance of CNA in a series of 125 B-lineage adult ALL patients treated according to risk-adapted protocols from the Spanish PETHEMA Group. Methods Bone marrow or peripheral blood (with significant blast burden) samples from 125 B-lineage adult ALL patients enrolled in risk-adapted protocols from the PETHEMA Group were analyzed at diagnosis. MLPA assays (MRC-Holland) were performed for the following genes: IKZF1, IKZF2, IKZF3, EBF1, CDKN2A/B, PAX5, ETV6, BTG1, RB1, hsa-miR-31, X/Y PAR1 region genes (CRLF2, CSF2RA, IL3RA) and 14q32.33 region genes (IGH D, MTA1, KIAA0284). Fragment analysis was made by Genescan in an ABI-3130 sequencer (Applied Biosystems). Data normalization provided a value indicative of the presence or absence of CNA: 0-0.20 homozygous deletion, 0.21-0.70 heterozygous deletion, 0.71-1.30 normal, 1.31-1.70 heterozygous duplication and 1.71-2.20 homozygous duplication. Results The median age [range] was 40 [15-74] years, 71 (57%) males, median WBC count 12.11 x109/L [0.4-388]. Immunophenotype: pro-B 14 (11%), common 71 (58%), pre-B 26 (21%), mature-B 10 (8%), unavailable 2 (2%). Cytogenetics: normal 16 (13%), hyperdiploid 6 (5%), hypodiploid 2 (2%), t(9:22) 20 (16%), t(1;19) 8 (6%), 11q23/MLL 11 (9%), 8q24/C-MYC 7 (5%), complex 1 (1%), iAMP21 2 (2%), other translocations or deletions 31 (25%), no growth 20 (16%). CNA frequencies of the 125 patients are shown in the table. IKZF1 deletions were significantly associated with EBF1 deletions, high WBC count and Philadelphia (Ph) chromosome. In the IKZF1 deleted cohort whole gene deletions were as frequent as Ik6 isoforms (28% each). A high codeletion rate was detected in genes located in 9p (CDKN2A/B with PAX5, CDKN2A/B with hsa-miR-31 and PAX5 with hsa-miR-31). CDKN2A/B also showed concomitant deletions with ETV6 while PAX5 showed codeletions with BTG1. CDKN2A/B and PAX5 deleted patients had higher WBC counts than non-deleted individuals. Clinical follow-up data was available for 123 patients of the whole series and for the 105 patients of the Ph-negative cohort. Multivariate analysis showed that advanced age, BTG1 deletions and EBF1 deletions were negative prognostic factors for achieving Complete Remission (CR) and WBC count and IKZF1 deletions significantly reduced CR duration in both cohorts. Interestingly, there were significant differences in relapse rates between whole and partial gene IKZF1 deletions. IKZF1 haploinsufficient patients had a probability of CR duration at 3 years of 83% ± 30% vs. 6% ± 12% of partial gene deletion carriers. Advanced age and IKZF1 deletions were predictors for overall survival in the Ph-negative cohort and age>30 years, IKZF1 deletions and hsa-miR-31 deletions were associated with poor prognosis in the whole series. Conclusions In B-lineage adult ALL, deletions of IKZF1, EBF1, BTG1 or hsa-miR-31 are markers with prognostic significance in addition to age and WBC count. Patients with partial IKZF1 gene deletions have a significantly higher probability of relapse than those with whole gene loss. These genetic abnormalities could help to better define prognostic subgroups in adult patients with B-lineage ALL. Supported by the grants PI10/01417 and RD12-0036-0029 from Instituto Carlos III and a grant from the Spanish Society of Hematology and Hemotherapy (2012). Disclosures: No relevant conflicts of interest to declare.

WT1 plays a dual role in leukemia development, probably due to an imbalance in the expression of the 4 main WT1 isoforms. We quantify their expression and evaluate them in a series of AML patients. Our data showed a predominant expression of isoform D in AML, although in a lower quantity than in normal CD34+ cells. We found a positive correlation between the total WT1 expression and A, B and C isoforms. The overexpression of WT1 in AML might be due to a relative increase in A, B and C isoforms, together with a relative decrease in isoform D expression.

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic myeloid neoplasms, in which disease progression is quite common, eventually terminating in secondary acute myeloid leukemia (sAML). To elucidate differential roles of mutations in MDS progression and sAML evolution, we investigated clonal dynamics of somatic mutations using targeted sequencing of 699 MDS patients, of which 122 were analyzed for longitudinally collected samples. Combining publicly available data, mutational data in a total of 2,250 MDS cases were assessed for their enrichment in specific disease subtypes. All samples were obtained after informed consent. Genotyping data from samples with low- (n=1,207) and high-risk (n=683) MDS as well as sAML (n=360) were available for most prevalently mutated 25 driver genes. In univariate comparison between low- and high-risk MDS, the majority of differentially mutated genes were enriched in high-risk MDS, except for SF3B1, which was more frequently mutated in low-risk MDS. Multivariate analysis was performed using a least absolute shrinkage and selection operator model. As a result, mutations in 7 genes (FLT3, PTPN11, WT1, IDH1, NPM1, IDH2,and NRAS) designated as 'Type-1' mutations, were significantly enriched in sAML compared to high-risk MDS. When comparison was made between high- and low-risk MDS, mutations in 10 genes, including GATA2, NRAS, KRAS, IDH2, TP53, RUNX1, STAG2, ASXL1, ZRSR2, and TET2, were enriched in high-risk MDS. The latter mutations are designated as 'Type-2' mutations, excluding NRAS and IDH2 mutations, which were already assigned to the Type-1 category. To characterize the chronological behavior of Type-1 and Type-2 mutations, we performed longitudinal analyses of 122 cases, of which 90 progressed to sAML. Overall, driver mutations tended to increase their clone sizes between two time points. In accordance with their significant enrichment in sAML, Type-1 mutations were more likely to be newly acquired at the second time points, compared to Type-2 and other mutations (P=0.0001). By contrast, in patients with high-risk MDS at the second time point, Type-2 mutations were more dominant than Type-1 mutations, and most of the Type-2 mutations (88%) increased their clone sizes at the second sampling. Similarly, Type-2 mutations found in high-risk MDS or sAML evolving from low-risk MDS increased their clone sizes more frequently (30 out of 38 mutations (79%)) than Type-2 mutations in stable low-risk MDS without disease progression over time (4 out of 11 (36%)) (P=0.02). These findings suggest that Type-1 and Type-2 mutations might be associated with progression from high-risk MDS to sAML and low- to high-risk MDS, respectively. To further clarify the effects of the different classes of mutations on progression to sAML, 429 patients with MDS were analyzed for progression free survival (or PFS). Patients with Type-1 mutations (Group-I) had a significantly shorter PFS, compared to those who had Type-2 mutations but lacked Type-1 mutations (Group-II) (HR=1.82, 95% CI:1.08−3.05; P=0.025). Nevertheless, PFS in Group-II cases was still significantly shorter than that in other cases (HR=2.46, 95% CI:1.43−4.23; P=0.001). Of note, some Group-II cases subsequently acquired Type-I mutations during progression to sAML. By contrast, SF3B1-mutated patients tended to show slower progression to sAML, unless they carried either of Type-1 or 2 mutations (Group-III). Finally, the effects of these mutations on overall survival (OS) were assessed in a larger cohort of patients with MDS (n=1,347). Group-I cases were shown to have a significantly shorter OS than Group-II cases (HR=1.50, 95% CI:1.20−1.86; P In conclusion, our study has elucidated clonal dynamics associated with MDS progression and sAML evolution. Close monitoring of these sets of distinct mutations in the prospective fashion may help in the prediction of the clinical outcome in MDS. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Introduction Intensive consolidation chemotherapy in acute myeloid leukemia (AML) patients induces important hematologic toxicity with potential life-threatening infections that can lead to delays in further treatment or death in complete remission (CR). Recent single and multi-center studies (Guo et al. Blood 2011, JCO 2012, ASH 2015) have shown that the infusion of HLA-mismatched peripheral stem cell without immunosuppressive prophylaxis can accelerate hematologic recovery after chemotherapy, without developing graft versus host disease (GVHD). Objectives The primary objective of this phase I-II trial is to confirm the safety (absence of GVHD) and efficacy (reduction of neutropenia duration) of HLA-mismatched stem cells infusion after consolidation chemotherapy with idarubicine and cytarabine (3+7) in 50 younger patients with intermediate/high-risk AML. Herein we present the preliminary results of the phase I trial with 9 adult patients (safety cohort). Methods Patients younger than 65 years old with AML in CR after induction therapy that were assigned to receive consolidation course with idarubicin (12 mg/m2/day IV 1-3) and cytarabine (200 mg/m2/day IV 1-7) according to PETHEMA protocol were enrolled in this study in a single Spanish institution. To determine safety of HLA-mismatched stem cells infusion a standard 3+3 design was used in this preliminary study: cohorts of 3 patients were established with decreasing immunosuppressive prophylaxis, 3 additional patients would be enrolled with the same immunosuppression if limiting toxicity (LT) was observed in 1 out of 3 patients. LT was defined as global GVHD>grade 2 or grade 4 infusion related reactions. The first cohort of 3 patients was assigned to receive immunosuppression with cyclosporine (1-1.5 mg/kg/bid) and prednisone (0.5 mg/kg/qid), the second cohort to receive only prednisone (0.5 mg/kg/qid), and the third would not receive immunosuppressive treatment. The immunosuppression resulting of this phase would be used in an expansion cohort. Stem cells were obtained after mobilization with G-CSF and apheresis from HLA-mismatched family-related donors and were infused the day 9 of the consolidation course. The donor and recipient HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 alleles were genotyped using a PCR-SSP method. Hematologic recovery was defined as days from start of chemotherapy to neutrophil count >0.5x109/L and to platelet count >50x109/L. G-CSF was administered only in case of severe infection. This study was approved by the Ethical Committee, and inform consent was obtained from all patients and donors. Results From March 2015 to June 2016, 9 patients were enrolled in this study. Median age was 46 (28-64) and 6 were male. All were in CR after induction therapy, 6 had intermediate risk cytogenetic, and 3 high risk cytogenetic/molecular AML. All the donors were family-related and HLA compatibility was 3/6 for 8 patients and 5/6 in one patient. HLA-mismatched stem cells infusion characteristics were: median number of mononuclear cells, CD34+ and CD3+ T cells infused per course was 4.5 (2.1-6.6)x108/kg, 3.3 (0.7-4.9)x106/kg and 1.7 (0.8-2.3)x108/kg, respectively. LT was not reached and no diagnosis of GVHD was made. Two patients developed cytarabine related rash and other 2 patients infectious diarrhea. No patient needed further immunosuppressive treatment. Median duration of neutropenia and thrombocytopenia was 30 (27-50) and 44 (22-51) days, respectively. 3 patients received G-CSF and 2 developed severe infections. Median blood cell unit and platelet units transfused was 4 (2-20) and 8 (2-32). These results are similar to those observed in a historical cohort (non-matched) of 59 patients with AML who received consolidation with 3+7 at the same institution between January 2010 to January 2015 (median duration of neutropenia and thrombocytopenia was 29 (17-57) and 36 (18-206) days, respectively). Conclusion The infusion of HLA-mismatched stem cell is safe after consolidation with idarubicin and cytarabine in younger patients. The methodology and in consequence the results of our safety cohort (with immunosuppressive prophylaxis) are not comparable to the previous experience reported by other groups. The reduction of hematologic recovery remains to be confirmed with this schedule in a larger cohort without immunosuppressive prophylaxis. Research granted by IIS La Fe (2014/0368) Disclosures Boluda: Instituto de Investigación Sanitaria La Fe: Employment.

BRAF V600E is a point mutation located in exon 15 of the serine/threonine protein kinase B-Raf (BRAF) gene, and was originally discovered to recurrently occur in melanoma [1]. Because of this acqui...