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Introduction It was proposed that peripheral blood (PB) monocyte subset analysis evaluated by flow cytometry, hereafter referred to as "monocyte assay", could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay required a large multicenter validation. Methods PB and/or bone marrow (BM) samples from adult patients displaying monocytosis were assessed with the "monocyte assay" by ten ELN iMDS Flow working group centers (6 equipped with BD FACSCanto™ II (BD Biosciences), 3 with Navios™ (Beckman Coulter) and one with BD™ LSRII (BD Biosciences)) with harmonized protocols. The corresponding files were reanalyzed in a blind fashion by a skilled operator and the cMo (CD14 ++CD16 -) percentages obtained by both analyses were compared. Information regarding age, gender, complete blood count, marrow cytomorphology, cytogenetics and molecular analysis was collected. Confirmed diagnoses were collected when available as well as follow-up for CMML patients. Results The comparison between cMo percentages from 267 PB files provided by the 10 centers and the centralized cMo percentages showed a good global significant correlation (r=0.88; p The second aim of this multicenter study was to assess the feasibility of the monocyte assay on 117 BM samples provided by 7 out of the 10 ELN centers, 43 of which being paired to PB samples. The comparison between cMo percentages provided by the 7 centers and the centralized cMo percentages showed a lower global significant correlation compared to PB samples (r=0.74; p Conclusions This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership.

Multidimensional flow cytometry (MFC) is routinely used for the diagnosis and follow-up of hematolymphoid neoplasms but its contribution to the identification of non-hematolymphoid malignant tumors is limited.The presence of non-hematolymphoid cells in clinical samples obtained via minimally invasive methods was ascertained by using a panel of monoclonal antibodies previously developed in our laboratory comprising a mixture of antibodies: CD9-PacB/CD45-OC515/CD57-FITC/CD56-PE/CD3-PerCP-Cy5.5/CD117-PE-Cy7/CD326-APC/CD81-APC-C750. Histopathological studies were performed using standard techniques.164 specimens of different origins were included. Malignancy was finally confirmed in 142 (86.5%), while 22 non neoplastic samples were identified. The most frequent diagnosis was small cell lung carcinoma (SCLC) (50%). High sensitivity (S = 98.6%) was reached combining MFC and conventional pathology. Individual markers differed according to the cellular origin of the neoplasm, with neuroendocrine tumors showing a unique immunophenotypic profile (CD56+ CD326+ CD117-/+ and variable tetraspanins expression). Principal component analysis efficiently distinguished SCLC from other tumor samples. In immune cell populations, differences between reactive and malignant biopsies were found in different cell compartments, especially in B cells and Plasma cells. Differences also emerged in the percentage of CD4+ CD8- T cells, CD4-CD8+ T cells and NK cells and these were dependent on the origin of the tumor cells.These results support the use of MFC as a rapid and valuable technique to detect non-hematolymphoid tumoral cells in clinical specimens, providing an initial orientation to complement hystopathological studies and allow a more precise diagnosis, especially in neuroendocrine neoplasms. The impact of different immune cell patterns warrants further research.

[Background]: Several risk stratification models have been proposed in recent years for systemic mastocytosis but have not been directly compared. Here we designed and validated a risk stratification model for progression-free survival (PFS) and overall survival (OS) in systemic mastocytosis on the basis of all currently available prognostic factors, and compared its predictive capacity for patient outcome with that of other risk scores., [Methods]: We did a retrospective prognostic modelling study based on patients diagnosed with systemic mastocytosis between March 1, 1983, and Oct 11, 2019. In a discovery cohort of 422 patients from centres of the Spanish Network on Mastocytosis (REMA), we evaluated previously identified, independent prognostic features for prognostic effect on PFS and OS by multivariable analysis, and designed a global prognostic score for mastocytosis (GPSM) aimed at predicting PFS (GPSM-PFS) and OS (GPSM-OS) by including only those variables that showed independent prognostic value (p, [Findings]: Our GPSM-PFS and GPSM-OS models were based on unique combinations of independent prognostic factors for PFS (platelet count ≤100 × 109 cells per L, serum β2-microglobulin ≥2·5 μg/mL, and serum baseline tryptase ≥125 μg/L) and OS (haemoglobin ≤110 g/L, serum alkaline phosphatase ≥140 IU/L, and at least one mutation in SRSF2, ASXL1, RUNX1, or DNMT3A). The models showed clear discrimination between low-risk and high-risk patients in terms of worse PFS and OS prognoses in the discovery and validation cohorts, and further discrimination of intermediate-risk patients. The GPSM-PFS score was an accurate predictor of PFS in systemic mastocytosis (C-index 0·90 [95% CI 0·87–0·93], vs values ranging from 0·85 to 0·88 for pre-existing models), particularly in non-advanced systemic mastocytosis (C-index 0·85 [0·76–0·92], within the range for pre-existing models of 0·80 to 0·93). Additionally, the GPSM-OS score was able to accurately predict OS in the entire cohort (C-index 0·92 [0·89–0·94], vs 0·67 to 0·90 for pre-existing models), and showed some capacity to predict OS in advanced systemic mastocytosis (C-index 0·72 [0·66–0·78], vs 0·64 to 0·73 for pre-existing models)., [Interpretation]: All evaluated risk classifications predicted survival outcomes in systemic mastocytosis. The REMA-PFS and GPSM-PFS models for PFS, and the International Prognostic Scoring System for advanced systemic mastocytosis and GPSM-OS model for OS emerged as the most accurate models, indicating that robust prognostication might be prospectively achieved on the basis of biomarkers that are accessible in diagnostic laboratories worldwide., Carlos III Health Institute, European Regional Development Fund, Spanish Association of Mastocytosis and Related Diseases, Rare Diseases Strategy of the Spanish National Health System, Junta of Castile and León, Charles and Ann Johnson Foundation, Stanford Cancer Institute Innovation Fund, Austrian Science Fund.

Background Diagnosis of hematolymphoid neoplasm (HLN) requires different technologies which are performed on a patient basis instead of per protocol. We hypothesize that integration of hematimetric and cytological analysis along with multiparametric flow cytometry (MFC) provides a framework to evaluate peripheral blood (PB) samples from Primary Care. Methods Samples from patients with persistent (>3 months) lymphocytosis (>5 × 109/L) and/or monocytosis (>109/L) or the presence of atypical and/or blast cells upon the smear review were analyzed by MFC concurrent to cytological analysis. MFC studies were carried out following standardized procedures. Results In a 3-year period, smear review and MFC were performed simultaneously in 350 samples, demonstrating HLN in 194 cases (55.4%). In 156 cases, reactive cell populations were found. The combination of age, absolute lymphocyte count (ALC), hemoglobin and platelets provided the best correlation with MFC for the presence of a chronic lymphoproliferative disorder (CLPD) in lymphocytosis [area under the curve (AUC) 0.891, p Conclusions A strategy to perform MFC studies following standardized procedures has proven to be useful to evaluate samples from patients in Primary Care centers for HLN diagnosis or reactive conditions, providing a sensitive and rapid clinical orientation and avoiding unnecessary consultations in routine clinical practice. The probability for the presence of CLPD in PB can be calculated and help guide decision-making regarding further testing.

Background: Evaluation of MRD is standard in patients with AML. However, the role of decentralized MRD assessment for risk stratification in AML remains largely unknown, and so it does which methodological aspects are critical to empower the evaluation of MRD with prognostic significance, particularly if using MFC. Aim: To evaluate the role of decentralized MRD assessment using MFC for risk stratification and putative treatment individualization of patients with AML. Methods: This study was performed on 1,076 AML patients in complete remission (CR) after 7+3 induction chemotherapy, in whom MRD was evaluated by MFC in local laboratories over a period of 20 years in the PETHEMA group. We conducted a survey of technical aspects of MFC based MRD testing in the laboratories of the 60 participating Hospitals, to determine the impact of methodological heterogeneity in the prognostic value of MFC. Results: We first investigated the most effective MRD cutoff to stratify patients' risk at first remission. Patients were segmented into progressively higher cutoffs, starting at 0.01% followed by 0.05%, 0.1%, 0.5% and 1%. Our results showed that 0.1% reached higher statistical significance to discriminate patients with different relapse-free survival (RFS, HR: 0.77; P = .001) and overall survival (OS, HR: 0.73; P = .001). In multivariate analyses together with patients' age, WBC, genetic risk and post-consolidation therapy, MRD status was selected as an independent prognostic factor for OS. To further define the utility of "real-world" MRD assessment using MFC in risk stratification of AML, recursive partitioning was performed using the prognostic and treatment related factors selected in the multivariate Cox model for OS. Of the four variables evaluated, hematopoietic stem cell transplantation (HSCT, regardless of autologous or allogeneic source) vs no transplant emerged as the best single discriminator for OS, followed by genetic risk, age and MRD status. There were two branching points defined by MRD status; the first in patients ≤60 years with intermediate genetic risk who were not transplanted and the second in patients with adverse genetics who were not transplanted, in whom Forty-nine of the 60 hospitals (82%) responded to the survey on questions regarding the measurement of MRD using MFC in the PETHEMA LMA 1999, 2007 and 2010 protocols, providing information corresponding to 966 of the 1,076 (90%) patients regarding the number of markers, preparation of samples, instruments, approach (ie, LAIP, DfN or LAIP+DfN), number of cells to define a cluster, etc. The survey revealed significant heterogeneity intra- and inter-protocols that reflected improvement in MFC assessment of MRD over time, in the absence of harmonization nor standardization at the national level. Accordingly, we investigated if the heterogeneity in methodological, interpretation and reporting aspects of MFC based MRD testing were hampering its ability to predict outcome independently of other patient and treatment related factors. Strikingly, our results showed that except for the denominator used to calculate MRD burden (ie, total nucleated cells vs leukocytes), lack of standardization in all other parameters had an impact on the ability of MFC to predict outcomes in AML (Figure). Namely, panels with ≤4 markers or ≤2 combinations failed to identify patients with significantly different RFS according to MRD status, and MFC-based MRD monitoring was prognostic only when >500,000 cells were measured. Only MRD assessment using patient-specific panels was predictive of outcome. Conclusions: We report here one of the largest studies investigating the role of MRD monitoring using MFC. Our results confirmed that detection of MRD identifies patients in CR/CRi with inferior survival, but uncovered that decentralized MRD testing lacks significance when compared to other baseline risk factors and in the context of risk-adapted post-consolidation strategies. Thus, while this study demonstrated that "real-world" decentralized assessment of MRD using MFC does provide prognostic information in AML patients at first remission, our results question its readiness for risk stratification towards clinical decisions outside trials, at least until adequate standardization of this technique is achieved. Figure Disclosures Paiva: SkylineDx: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Roche: Research Funding; Adaptive: Honoraria; Amgen: Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Kite: Consultancy; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding. Alonso Dominguez:Celgene: Research Funding; Incyte: Research Funding; Pfizer: Research Funding. Martinez-Lopez:Janssen: Speakers Bureau; Altum: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Hosea: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Roche: Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Vivia Biotech: Honoraria; Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau. Sossa:Astellas: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novo: Honoraria. San-Miguel:Roche, AbbVie, GlaxoSmithKline, and Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb, Celgene, Novartis, Takeda, Amgen, MSD, Janssen, and Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees.

CD47 over expression has been reported in several tumor subtypes. CD47 interacts with SIRPalpha on macrophages inhibiting phagocytic signal, providing a survival advantage to tumor. CD47, therefore, represents a valuable target for immunotherapy and is currently under clinical investigation. We aimed to study CD47 expression in Hodgkin Reed Sternberg cells (HRS). We tested a polyclonal CD47 antibody (LifeSpan Biosciences, Seattle, WA) expression along with classical HRS cell markers on a tissue array of 16 classical Hodgkin Lymphoma (CHL) tumor biopsies obtained from newly diagnosed, non-selected patients (8 Female, 8 Male patients) in our institution from October 2016 to January 2018. Histologic subtypes were nodular sclerosis in 11 cases, mixed Cellularity in 3 cases and lymphocyte rich in 2 additional cases. Median age was 53 years (Range: 8, 74). Early stage disease was found in three patients without unfavorable prognostic factors according to EORTC and GHSG criteria, one patient with unfavorable prognostic factors and nine patients had advanced disease. Bulk disease was present in one patient. Normal lymphoid tissue and normal prostate epithelium were used as normal controls as recommended by manufacturer. Approval from the Local Ethical committee was obtained before any analysis. CD47 was overexpressed on all HRS cells with a characteristic dot-like pattern in 13/13 cases of CHL. HRS clearly expressed CD47 more intensely than infiltrating T and stromal cells. We propose that HRS cells, by up-regulating CD47, might avoid innate immunity check on tumor growth, which could be circumvented using blocking monoclonal antibodies.

Background: Flow cytometry (FC) is a valuable tool for the diagnosis of myelodysplastic syndromes (MDS). We present results of a survey carried out to evaluate FC current practice for MDS diagnosis in Latin America (LA), focusing on markers used and characteristics of the clinical diagnostic report. Compliance to IMDSflow recommendations was also evaluated. These practices were then compared with those used in other countries. Methods: An online survey was sent through the Grupo Latino-Americano de Mielodisplasia to LA cytometrists and other international scientific societies. Results: 91 responses from 15 LA countries were received. The median of the number of markers used was 20 +/- 4.5, but only 8.1% of participants adopted the complete panel proposed by the International/European LeukemiaNet Working Group (IMDSflow). We received 140 eligible answers from regions other than LA (66 Europe, 59 USA-Canada, 8 Oceania, 6 Asia and 1 Africa). LA utilized more markers for MDS diagnosis than USA/Canada (p = 0.006), but similar to Europe. The use of MDS scoring systems differed among regions: 10.3% in LA, 0% USA/Canada and 25.7% Europe reported the "Ogata score". Finally, 52.0% of all participants included a general interpretation statement in the final report about the consistency of the FC results with MDS diagnosis, with no statistical differences between regions. Conclusions: This survey shows a low compliance with the IMDSflow recommendations and a scarce use of the scoring systems proposed in the literature. However, the number of surface markers used is high. We will work to develop a FC consensus for MDS diagnosis adapted to the clinical practice requirements in LA.

The role of decentralized assessment of measurable residual disease (MRD) for risk stratification in acute myeloid leukemia (AML) remains largely unknown, and so it does which methodological aspects are critical to empower the evaluation of MRD with prognostic significance, particularly if using multiparameter flow cytometry (MFC). We analyzed 1076 AML patients in first remission after induction chemotherapy, in whom MRD was evaluated by MFC in local laboratories of 60 Hospitals participating in the PETHEMA registry. We also conducted a survey on technical aspects of MRD testing to determine the impact of methodological heterogeneity in the prognostic value of MFC. Our results confirmed the recommended cutoff of 0.1% to discriminate patients with significantly different cumulative-incidence of relapse (-CIR- HR:0.71, P, This study was supported by the Centro de Investigación Biomédica en Red – Área de Oncología - del Instituto de Salud Carlos III (CIBERONC; CB16/12/00369, CB16/12/00233, CB16/12/00284 and CB16/12/00400), Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS No. PI16/01661, PI16/00517 and PI18/01946), Gerencia Regional de Salud de CyL (GRS 1346/A/16) and the Plan de Investigación de la Universidad de Navarra (PIUNA 2014-18). This study was supported internationally by the Cancer Research UK, FCAECC and AIRC under the Accelerator Award Program EDITOR.

B7-H6, a ligand for the NK activating receptor NKp30, has been identified as a biomarker of poor prognosis in several solid cancers. However, little is known about the role of B7-H6 and the mechanisms that control its expression in acute myeloid leukemia (AML). Epigenome modulation, including epigenomic reader dysregulation, is one of the hallmarks of AML. Bromodomain-containing protein 4 (BRD4), the best-known member of the BET family of epigenetic readers, is overexpressed in AML cells and regulates the transcription of genes involved in the pathogenesis of AML, as MYC oncogene. Here, we analyze the role of BRD4 in regulating B7-H6 in AML cells. Results demonstrated that the specific inhibition of BRD4 drastically reduces the expression of B7-H6 in AML cells. Histone acetylation mediated by CBP30/P300 facilitates the binding of BRD4 to the B7-H6 promoter, which recruits the P-TEFb elongation factor that phosphorylates RNA polymerase II, thereby activating B7-H6 transcription. BRD4 also co-bounded with JMJD6 at the distal enhancer of the B7-H6 gene. Metabolic modulation with metformin modifies the acetylation pattern in the B7-H6 promoter, impairing BRD4 binding, thereby inhibiting B7-H6 expression. B7-H6 knockdown induces the apoptosis in HEL-R cell line. Moreover, a high level of B7-H6 expression in AML patients is related to increased BRD4 levels, myelodysplastic-derived AML, and del5q, the two latter being associated with poor prognosis. Our data show that BRD4 is a positive regulator of the pro-tumorigenic molecule B7-H6 and that the blockage of the B7-H6 is a potential therapeutic target for the treatment of AML.

Not available.

This study was supported by the Ministerio de Economía y Competitividad, Grant n. SAF2015-67633-R ,and PI16/00420 which are part of Plan Nacional de I+D+I and are co-financed by the European Regional Development Fund (FEDER-“Una manera de hacer Europa”) and the CERCA program from Generalitat Catalunya. European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n. 306240; Generalitat de Catalunya Suport Grups de Recerca AGAUR 2017-SGR1009, and Departament de Salut (SLT002-16-00350), Instituto de Salud Carlos III (ISCIII) International Cancer Genome Consortium for Chronic Lymphocytic Leukemia (ICGC-CLL Genome Project), and project PM15/00007, which is part of Plan Nacional de I+D+I and are co-financed by FEDER. NG is a recipient of a predoctoral fellowship from Agaur and EC is an Academia Researcher of the "Institució Catalana de Recerca i Estudis Avançats" (ICREA) of the Generalitat de Catalunya., Giménez, N., Martínez-Trillos, A., Montraveta, A., Lopez-Guerra, M., Rosich, L., Nadeu, F., Valero, J.G., Aymerich, M., Magnano, L., Rozman, M., Matutes, E., Delgado, J., Baumann, T., Gine, E., González, M., Alcoceba, M., Terol, M.J., Navarro, B., Colado, E., Payer, A.R., Puente, X.S., López-Otín, C., Lopez-Guillermo, A., Campo, E., Colomer, D., Villamor, N.

Introduction: Acute myeloblastic leukaemia (AML) is an heterogeneous disease with different molecular and prognostic characteristics. According to the comorbidities and the revised 2017 European Leukaemia genetic risk stratification (ELN17), allogeneic hematopoietic cell transplantation (HCT) is the best therapeutic option for many patients with AML (Grimm, Blood Adv 2020). However, relapse remains the main cause of mortality after transplantation. Impact of MRD on the outcome of patients is well recognized and ELN2017 introduced the new response category complete remission (CR) without MRD (Döhner H, Blood 2017). Detection of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) in AML before allogeneic HCT could be a powerful predictor of outcome and decisive when establishing strategies that modify the prognosis of these patients. Methods: Retrospective multicentre analysis of MRD by MFC of patients undergoing transplantation allogeneic in 4 centres during the period from 2012 to 2020. Both Leukaemia Associated Aberrant Immunophenotype (LAIP) and different from normal (DFN) approach were used to analyse the MRD. The MRD was carried out with 8-color panels based on Euroflow protocols. The samples were acquired in 8-color digital cytometers (FACSCanto II) calibrated and compensated according to Euroflow protocols. Results: 295 of 318 patients were evaluated. Table 1 shows the characteristics of the patients. 285 (96.7%) were in complete remission (CR), 207 had negative MRD, in 21 MRD was less than 0.1% (MRD-low) and in 57 greater than or equal to 0.1% (MRD-high). At 2 years, the overall survival (OS) and leukaemia-free survival (LFS) in the whole group were 69% (95% CI 63.18-74.18) and 58.4% (95% CI 52.4-63.9) respectively. In CR patients, MRD levels significantly influenced on outcomes, with OS and LFS of 76.7% and 67.6% for negative MRD, 68.5% and 49.7% MRD-low and 50 % and 36.6% in MRD-high, p Figure 1 Figure 1. Disclosures Caballero: Celgene: Consultancy. Belén Vidriales: Roche: Consultancy; Novartis: Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Astellas: Consultancy, Speakers Bureau. Montesinos: Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline/Menarini: Consultancy; Tolero Pharmaceutical: Consultancy; Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Forma Therapeutics: Consultancy; Glycomimetics: Consultancy; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau. Perez-Simon: JANSSEN, TAKEDA, PFIZER, JAZZ, BMS, AMGEN, GILEAD: Other: honorarium or budget for research projects and/or participation in advisory boards and / or learning activities and / or conferences.

The clinical consequences of the infectious events in patients receiving azacitidine are poorly documented. Likewise, the role of primary antimicrobial prophylaxis is unknown. In this retrospective, single-center study, we compare the impact of prophylaxis on the incidence of infection and morbidity in all consecutive higher-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients, during the first 4 azacitidine cycles. Seventy-six patients, corresponding to 283 azacitidine cycles, were studied. There were infectious events in 43% of the patients. Development of infections led to more hospital admissions, increased red blood cells and platelet requirements, and a delay in subsequent cycles. Median overall survival was comparable between patients with or without infections. In the multivariate analysis, a neutrophil count below 0.5 × 109/L (OR 12.5 [2.6–50]) and antimicrobial prophylaxis (OR 0.1 [0.02–04]) were independent factors for the development of infection. We conclude that infectious events have a significant impact in the early clinical course of azacitidine-treated patients by increasing hospital admissions and transfusion requirements. Antimicrobial prophylaxis may prevent infections, leading to a decreased need for supportive care in these patients with poor outcome.

// Aroa Baragano Raneros 1 , Alfredo Minguela Puras 2 , Ramon M. Rodriguez 1 , Enrique Colado 3 , Teresa Bernal 3 , Eduardo Anguita 4 , Adela Vasco Mogorron 2 , Alberto Chaparro Gil 4 , Jose Ramon Vidal-Castineira 1 , Leonardo Marquez-Kisinousky 1 , Paula Diaz Bulnes 1 , Amelia Martinez Marin 5 , Maria Carmen Garcia Garay 6 , Beatriz Suarez-Alvarez 1, * , Carlos Lopez-Larrea 1, * 1 Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, Spain 2 Immunology Service, Instituto Murciano de Investigacion Biosanitaria (IMIB), Hospital Clinico Universitario Virgen de la Arrixaca, Murcia, Spain 3 Department of Hematology, Hospital Universitario Central de Asturias, Oviedo, Spain 4 Hematology Department, Hospital Clinico San Carlos, Instituto de Investigacion Sanitaria San Carlos (IdISSC), Department of Medicine, Universidad Complutense de Madrid (UCM), Madrid, Spain 5 Hematology Service, Hospital Clinico Universitario Virgen de la Arrixaca, Murcia, Spain 6 Hematology Service, Hospital General Universitario Santa Lucia, Cartagena, Murcia, Spain * These authors have contributed equally to this work Correspondence to: Carlos Lopez-Larrea, email: inmuno@hca.es Beatriz Suarez-Alvarez, email: bsuarez@hca.es Keywords: acute myeloid leukemia (AML), DNA methylation, NKG2DL, NKG2D, TIMP3 Received: September 16, 2016 Accepted: March 16, 2017 Published: March 29, 2017 ABSTRACT Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza ® , AZA) and decitabine (Dacogen ® , DAC) have been approved for the treatment of AML patients, but their mechanisms of action are poorly understood. Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.

Background Although consensus guidelines have been proposed in 2010 for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometry (FCM), so far no study has investigated the efficiency of such medical indications in multicentric vs. reference laboratory settings. Methods Here we evaluate the efficiency of consensus medical indications for PNH testing in 3,938 peripheral blood samples submitted to FCM testing in 24 laboratories in Spain and one reference center in Brazil. Results Overall, diagnostic screening based on consensus medical indications was highly efficient (14% of PNH+ samples) both in the multicenter setting in Spain (10%) and the reference laboratory in Brazil (16%). The highest frequency of PNH+ cases was observed among patients screened because of bone marrow (BM) failure syndrome (33%), particularly among those with aplastic anemia (AA; 45%) and to a less extent also a myelodysplastic syndrome (MDS; 10%). Among the other individuals studied, the most efficient medical indications for PNH screening included: hemolytic anemia (19%), hemoglobinuria (48%) and unexplained cytopenias (9%). In contrast, only a minor fraction of the patients who had been submitted for PNH testing because of unexplained thrombosis in the absence of cytopenia, were positive (0.4%). Conclusions In summary, our results demonstrate that the current medical indications for PNH screening by FCM are highly efficient, although improved screening algorithms are needed for patients presenting with thrombosis and normal blood cell counts. © 2016 International Clinical Cytometry Society

[ES] A pesar de los avances en el conocimiento de la patogenia de las leucemias agudas mieloblásticas, su tratamiento especifico no ha sido modificado substancialmente en las últimas décadas y una parte importante de los enfermos presentan resistencia terapéutica primario o falleciendo de su enfermedad. Basándose en esta hipótesis, esta tesis establece la eficacia antileucémica de fármacos clásicos como Arac- C, Fludarabina y Doxorubicina, con el fin de disponer de un patrón de referencia para la ulterior comparación con nuevos fármacos en fase preclínica/experimental. A continuación investiga la eficacia y mecanismo de acción de tres fármacos en fase preclínica: el inhibidor del proteasoma Bortezomib (PS-341), el inhibidor Pan-acetilasa: Panobinostat (LBH589) y la Zalypsis, un nuevo fármaco de origen marino. Y de cada fármaco se estudiará: el efecto antitumoral y el perfil de toxicidad, el posible sinergismo de las combinaciones de estos agentes con gentes clásicos, el efecto sobre el ciclo celular y apoptosis y los cambios que inducen en el perfil de expresión génica., [EN] Despite advances in understanding the pathogenesis of acute myeloblastic leukemia, no specific treatment has been modified substantially in recent decades and an important part of primary therapy patients show resistance or died of their disease. Based on this hypothesis, this thesis establishes the antileukemic efficacy of drugs such classics as Arac-C, fludarabine and doxorubicin, in order to provide a baseline for later comparison with new drugs in preclinical / experimental. It then investigates the efficacy and mechanism of action of three drugs in preclinical phase: the proteasome inhibitor Bortezomib (PS-341), the Pan-acetylase inhibitor: Panobinostat (LBH589) and Zalypsis, a new drug of marine origin. And for each drug will be studied: the antitumor activity and toxicity profile, the possible synergism of combinations of these agents with traditional people, the effect on the cell cycle and apoptosis and inducing changes in gene expression profiling.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death., Plan Nacional de I+D+I co-funding FEDER [PI15/00892 and PI18/01527 to M.F.F. and A.F.F.; PI16/01318 and PI14/01244 to C.L.]; ISCIII-Subdireccion General de Evaluacion y Fomento de la Investigacion, and Plan Nacional de I+D+I 2008–2011/FEDER [CP11/00131 to A.F.F.]; IUOPA (to G.F.B. and M.I.S.); Fundacion Cientifica de la AECC (to R.G.U.); Ministry of Economy and Competitiveness Juan de la Cierva postdoctoral fellowships [FJCI-2015-26965 to J.R.T., IJCI-2015- 23316 to V.L.]; Fundacion Ramon Areces (to M.F.F); FICYT (to E.G.T., M.G.G., A.C.); Asturias Regional Government [GRUPIN14-052 to M.F.F.]; Gobierno del Principado de Asturias, PCTI-Plan de Ciencia, Tecnologia e Innovacion co-funding Fondos FEDER (grant number IDI/2018/146 to M.F.F. and IDI/2018/144 to C.L.); Deutsche Forschungsgemeinschaft (DFG) [SFB960 to A.V.G., R.D.]; European Research Council [CoG-2014-646903]; Spanish Ministry of Economy and Competitiveness [SAF-SAF2013-43065 to P.M.]; Asociacion Española Contra el Cancer [AECC-CI-2015]; FERO Foundation, and the ISCIII [PI14-01191 to C.B.]; P.M. acknowledges financial support from The Obra Social La Caixa Fundacio Josep Carreras and The Generalitat de Catalunya (SGR330). P.M. an investigator from the Spanish Cell Therapy cooperative network (TERCEL). The IUOPA is supported by the Obra Social Liberbank-Cajastur, Spain. Funding for open access charge: Plan Nacional de I+D+I co-funding FEDER [PI18/01527].

The authors uncover a role for the proteostasis modulator AIRAPL as a tumor suppressor in myeloproliferative malignancies, through its regulation of IGFR stability. The results ascribe a biological function to AIRAPL, and they implicate prosteostatic deregulation as an oncogenic mechanism in myeloid transformation, thus suggesting potential novel therapeutic strategies. AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown1,2,3. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2V617F mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms4,5,6. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.

Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza®, AZA) and decitabine (Dacogen®, DAC) have been approved for the treatment of AML patients, but their mechanisms of action are poorly understood. Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.

Introduction: Genomic studies of chronic lymphocytic leukemia (CLL) have uncovered >80 potential driver mutations. The vast majority of these mutations affect coding regions, and just two potential drivers have been identified in non-coding elements. Aim: To describe the biological and clinical impact of a recurrent A>C mutation at the third base of the small nuclear RNA U1, the non-coding component of the spliceosome involved in the recognition of the 5' splice site (5'SS). Methods: Whole-genome sequencing (WGS) and RNA-seq from 318 CLL patients were used to identify and characterize a highly recurrent A>C point mutation occurring at position 3 of the U1 snRNA gene (g.3A>C mutation). The U1 wild-type and mutant forms were introduced into three CLL cell lines (JVM3, HG3, MEC1) to validate in vitro the predicted effect of this alteration. We screened two independent cohorts including a total of 1,314 CLL patients for the presence of the mutation using the rhAmp SNP genotyping assay, and integrated the U1 mutational status with well-known driver alterations, IGHV and epigenetic subgroups, and clinical parameters. Results: The U1 mutation was found in 8/78 (10.3%) CLL cases analyzed by WGS. Given its role in 5'SS recognition by base-pairing, we reasoned that this mutation was likely to alter the splicing and expression patterns of CLL. We were able to confirm widespread specific alterations in the transcriptome by comparing RNA-seq data between wild-type and g.3A>C mutated samples. Applying this knowledge to an algorithm aimed to infer the U1 mutational status from expression data, we were able to identify 4 mutated cases among 240 additional cases that had RNA-seq but no WGS. In total, 12/318 (3.8%) CLL patients analyzed by WGS and/or RNA-seq harbored this mutation. This g.3A>C U1 mutation changes the preferential A-U base-pairing between U1 and 5'SS to C-G base-pairing, creating novel splice junctions and altering the splicing pattern of 3,193 introns in 1,519 genes. In addition to altered splicing, 869 genes were differentially expressed between mutated and wild-type cases. We identified specific cancer genes (e.g. MSI2, POLD1, or CD44) and pathways (B-cell receptor signaling, promotion of apoptosis, telomere maintenance, among others) altered by the U1 mutation. To confirm a causal link between this mutation and splicing changes, we introduced exogenous U1 genes with or without the mutation into three cell lines. Subsequent RNA-seq of these cell lines recapitulated the altered splicing and expression patterns observed in CLL patients. We next screened for the presence of the U1 mutation 1,057 patients (cohort 1) using the rhAmp assay and it was found in 30 (2.8%) cases. The distribution of the mutation was similar in Binet stages and CLL vs monoclonal B-cell lymphocytosis. However, the U1 mutation was almost always found in IGHV unmutated CLL (29/30, p=9.0e-11) and within the naïve-like CLL epigenetic subgroup (p=3.7e-7). None of the U1 mutated cases had mutations in the SF3B1 splicing factor. Considering only pre-treatment CLL samples, U1 mutation was associated with a shorter time to first treatment independently of the Binet stage, IGHV mutational status, epigenetic subgroups, and mutations in the well-known CLL drivers SF3B1, NOTCH1, ATMor TP53. In cohort 2 (n=257), this mutation was found in 13 (5.1%) patients, confirming its enrichment in IGHV unmutated cases, naïve-like epigenetic subgroup, and splicing modulation. Despite the relatively small number of pre-treatment samples carrying the U1 mutation (7/178) and short follow-up of the patients (median 2.6 years), the effect of this mutation on time to first treatment in cohort 2 was compatible with the one observed in cohort 1. Finally, we screened for the U1 mutation a cohort of diffuse large B-cell lymphoma (n=108), mantle cell lymphoma (n=101), follicular lymphoma (n=87), splenic marginal zone lymphoma (n=12), acute myeloid leukemia (n=52), and myelodysplastic syndrome (n=67). The mutation was not present in any of the samples analyzed. Conclusions: Here we have reported that the third base of the small nuclear RNA U1 is recurrently mutated in CLL, proved its effect in splicing and gene expression, and shown that this mutation is independently associated with faster disease progression. The g.3A>C U1 mutation represents a novel non-coding driver alteration in CLL with potential clinical and therapeutic implications. Disclosures Ramirez Payer: GILEAD SCIENCES: Research Funding. Terol:Astra Zeneca: Consultancy; Gilead: Research Funding; Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Roche: Consultancy. Lopez-Guillermo:Celgene: Consultancy, Research Funding; Janssen: Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Research Funding.

Introduction. Nucleophosmin gene mutation (NPM1mut) occurs in around 30% of acute myeloid leukemia (AML) patients, frequently linked with favourable prognosis in the absence of FLT3-ITDmut, which occurs in around 40% of NPM1mutAML. Therefore, more expeditious diagnostic approaches may contribute to early diagnosis and prognostic stratification of these patients. Herein, we investigated the association of immunophenotypic features of leukemic and monocytic cells with the presence of NPM1mut in AML. Methods. A total of 404 bone marrow (BM) samples from newly-diagnosed AML patients according to WHO 2017 classification were retrospectively studied by 8-color flow cytometry, including 225 AML with NPM1mut and 179 cases wild type gene (NPM1wt). Information on FLT3-ITD could be obtained from 397/404 cases. Thus, FLT3-ITDmut was present in 85/397 (21%), being concomitant with NPM1mutin 62/85 AML cases (73%). Logistic regression analysis was used to identify predictive phenotypes for the presence of NPM1mut. Results. Overall, blast cell with immunophenotypic features of monocytic differentiation (corresponding to FAB M4 and M5 AML subtypes) were observed among 135/225 (60%) and 69/179 (38.5%) AML patients with NPM1mutand NPM1wt, respectively (p Among AML with monocytic blast cell differentiation, altered monocytic phenotypes were more frequent among NPM1mut vs. NPM1wtcases (98% vs. 36%) (p Noteworthy, in AML cases without blast cell monocytic differentiation, remaining monocytic cells showed similar asynchronous phenotypic patterns, which were also more frequent among NPM1mut cases (78% vs. 23% of NPM1wtcases, respectively; p In addition, aberrant CD9 blast cell expression was found in a significant proportion of all AML cases studied (124/222, 56%). However, altered CD9 was more frequent on (either monocytic or immature/myeloid) blast cells from AML cases with NPM1mut (76% vs. 46% NPM1wtcases; p0.05). In turn, aberrant CD25 expression on blast cells was otherwise linked to FLT3-ITDmut (61% vs. 20% of FLT3-ITDwtcases; p In multivariate analysis, baseline detection of monocytic-lineage blast cells with asynchronous expression of CD300 prior CD14 -C-index= 0.954, odds ratio (OR), 78.8; 95% confidence interval (CI), 13.1-471; p Conclusions. Detection of specific aberrant immunophenotypic patterns among blast cells and/or remaining monocytic cells from AML patients is highly predictive for NPM1mut, which may contribute to early diagnosis and follow-up of these patients. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Severe hemorrhagic events occur in a significant fraction of acute promyelocytic leukemia patients, either at presentation and/or early after starting therapy, leading to treatment failure and early deaths. However, identification of independent predictors for high-risk of severe bleeding at diagnosis, remains a challenge. Here, we investigated the immunophenotype of bone marrow leukemic cells from 109 newly diagnosed acute promyelocytic leukemia patients, particularly focusing on the identification of basophil-related features, and their potential association with severe bleeding episodes and patient overall survival. From all phenotypes investigated on leukemic cells, expression of the CD203c and/or CD22 basophil-associated markers showed the strongest association with the occurrence and severity of bleeding (p ≤ 0.007); moreover, aberrant expression of CD7, coexpression of CD34+/CD7+ and lack of CD71 was also more frequently found among patients with (mild and severe) bleeding at baseline and/or after starting treatment (p ≤ 0.009). Multivariate analysis showed that CD203c expression (hazard ratio: 26.4; p = 0.003) and older age (hazard ratio: 5.4; p = 0.03) were the best independent predictors for cumulative incidence of severe bleeding after starting therapy. In addition, CD203c expression on leukemic cells (hazard ratio: 4.4; p = 0.01), low fibrinogen levels (hazard ratio: 8.8; p = 0.001), older age (hazard ratio: 9.0; p = 0.002), and high leukocyte count (hazard ratio: 5.6; p = 0.02) were the most informative independent predictors for overall survival. In summary, our results show that the presence of basophil-associated phenotypic characteristics on leukemic cells from acute promyelocytic leukemia patients at diagnosis is a powerful independent predictor for severe bleeding and overall survival, which might contribute in the future to (early) risk-adapted therapy decisions., This work was supported by the Fundación Científica de la Asociación Española Contra el Cáncer (AECC, Madrid, Spain) and the Fundación Rafael del Pino (Madrid, Spain) and both CIBERONC (CB16/12/00400, CB16/12/00233, CB16/12/00480) and grant PI16/00787 from Instituto de Salud Carlos III (Ministerio de Economía y Competitividad, Madrid, Spain).

This study was supported by the Instituto de Salud Carlos III (ISCIII) PMP15/00007; Ministerio de Economía y Competitividad (MINECO) SAF2015-64885-R; Generalitat de Catalunya AGAUR 2014-SGR-795; and Gilead Spain (GLD15/00288). ECa is an Academia Researcher of the 'Institució Catalana de Recerca i Estudis Avançats' (ICREA) of the Generalitat de Catalunya. FN is supported by a pre-doctoral fellowship of the MINECO (BES-2016-076372). We are indebted to the Genomics core facility of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). We are grateful to N Villahoz and MC Muro for their excellent work in the coordination of the CLL Spanish Consortium, and also thank C Capdevila, S Guijarro, L Pla and M Sánchez for their excellent technical assistance. We are also very grateful to all patients with CLL who have participated in this study., Nadeu, F., Clot, G., Delgado, J., Martín-García, D., Baumann, T., Salaverria, I., Beà, S., Pinyol, M., Jares, P., Navarro, A., Suárez-Cisneros, H., Aymerich, M., Rozman, M., Villamor, N., Colomer, D., González, M., Alcoceba, M., Terol, M.J., Navarro, B., Colado, E., Payer, Á., Puente, X.S., López-Otín, C., López-Guillermo, A., Enjuanes, A., Campo, E.

Solitary plasmacytoma represents a heterogeneous group of patients; approximately half develop multiple myeloma (MM) in 2 or 3 years,whereas others remain disease-free at 10 years. By definition, these patients do not have morphologic bone marrow (BM) plasma cell (PC) infiltration. Here, we investigated whether sensitive BM evaluation of patients withsolitaryboneplasmacytoma(SBP; n = 35) andextramedullary plasmacytoma (EMP; n = 29) through multiparameter flow cytometry (MFC) would unravel the presence of clonal PCs in otherwise disease-free BM, and whether BMclonality predicted higher risk of progression. BMclonal PCs were detected in 17 of 35 SBP (49%) and 11 of 29 EMP (38%) patients. Seventy-one percent of flow-positive vs only 8% of flow-negative SBP patients evolved to MM (median time to progression of 26 months vs not reached; hazard ratio, 17.4; P < .001). No significant differences were observed among EMP cases. Our results highlight the importance of MFC for sensitive BM evaluation of SBP patients, to predict risk of developing treatment-requiring MM and to plan disease monitoring., This work was supported by the Cooperative Research Thematic Network grants RD12/0036/0058, RD12/0036/0069 and RD12/0036/0052 of the Red de Cancer (Cancer Network of Excellence), Instituto de Salud Carlos III, Spain, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS: PI060339, 06/1354, 02/0905, 01/0089/01-02, PS09/01897/01370, G03/136, Sara Borrell: CD13/00340), Consejería de Educacion, Junta de Castilla y León Valladolid, Spain (HUS396A12-1), and Asociación Española Contra el Cáncer (AECC, GCB120981SAN), Spain.

Mutations in Toll-like receptor (TLR) and myeloid differentiation primary response 88 (MYD88) genes have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics, and outcome of patients with TLR/MYD88 mutations in 587 CLL patients. Twenty-three patients (3.9%) had mutations, 19 in MYD88 (one with concurrent IRAK1 mutation), 2 TLR2 (one with concomitant TLR6 mutation), 1 IRAK1, and 1 TLR5. No mutations were found in IRAK2 and IRAK4. TLR/MYD88-mutated CLL overexpressed genes of the nuclear factor κB pathway. Patients with TLR/MYD88 mutations were significantly younger (83% age ≤50 years) than those with no mutations. TLR/MYD88 mutations were the most frequent in young patients. Patients with mutated TLR/MYD88 CLL had a higher frequency of mutated IGHV and low expression of CD38 and ZAP-70. Overall survival (OS) was better in TLR/MYD88-mutated than unmutated patients in the whole series (10-year OS, 100% vs 62%; P = .002), and in the subset of patients age ≤50 years (100% vs 70%; P = .02). In addition, relative OS of TLR/MYD88-mutated patients was similar to that in the age- and gender-matched population. In summary, TLR/MYD88 mutations identify a population of young CLL patients with favorable outcome.

We evaluated the impact of detection of minimal residual disease by flow cytometry (FCMRD) and CD3 chimerism in relapse in a cohort of 87 patients with acute myeloid leukemia or myelodysplastic syndrome undergoing stem cell transplantation. Patients with a positive FCMRD at day +100 after transplantation showed higher relapse rates and worse overall survival. In multivariate analysis, a positive FCMRD after transplantation was a significant predictor of relapse. Mixed chimerism showed a trend to statistical signification. We conclude that FCMRD at day 100 after SCT is the best predictor of relapse after SCT in patients with aggressive myeloid malignancies.

Introduction Several studies have shown that the minimal residual disease (MRD) in acute myeloid leukemia (AML) patients has a prognostic value after induction and consolidation therapy. Nevertheless the relapse is the most important cause of treatment failure in these patients, although they achieved a negative MRD, and even after an allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nowadays, the value of the MRD before allogeneic BMT is still controversial. Method Multicentric study where we have studied correlative AML patients who went under an allo-HSCT in a situation of complete response, between 2012 and April'18. The MRD was analyzed by 8-coloured multiparametric flow cytometry, at least with 2 tubes per patient and 1,000,000 events per tube. We evaluated the prognostic value of the MRD before allo-HSCT. Results Between January'12 and April'18 we have gathered 90 allogeneic BMT in AML patients who were in CR, with a median age of 45 years old (17 - 66). The pre-HSCT situation was 1st complete remission (CR) in 75 patients and 2nd CR in 15. In 45 patients the conditioning regimen was myeoablative. In the group of patients (67) where we could know the risk group at diagnosis, the distribution was: low risk 18%, intermediate risk 59.7% and high risk 22.4%. The 46.7% of the donors were not related. In the last follow-up after allo-HSCT 24 patients have suffered a relapse (26.7%) and 41 (45.5%) have died (17 cases of mortality related to the transplant and 24 not related). In the global analysis the median follow-up of the overall survival (OS) was 37.5 months. Among the 90 patients, MRD was valuable in 86. Ten of 59 patients (16.9%) with negative MRD relapsed vs 12/27 (44.4%) with positive MRD, p= 0.016. If we consider only patients in 1st CR, 9/50 (18%) patients with negative MRD relapsed vs 10/22 (45.5%) with positive MRD, p= 0.02. This statistically significant difference does not exist if we consider only patients in 2nd CR. The median follow-up of OS and event free survival (EFS) was not reached in the negative MRD group and 571 days and 299 days in the positive MRD group. OS and EFS at 2 years after transplantation were 65% and 64% in the negative MRD group and 42% and 37% in the positive MRD group, p= 0.03 and p= 0.008 respectively (figure 1). Conclusions The detected MRD by 8-colour multiparametric flow cytometry previous an allo-HSCT in patients with AML in 1st CR is a prognostic factor in terms of relapse. Patients with a positive MRD before the allo-HSCT have a poorer OS and EFS than the patients with a negative MRD. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

This study was supported by research funding from (Spanish Ministry of Science and Innovation MICINN) through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTICC) (RD06-0020-0039 to E Campo and RD06-0020-0051 to AL-G), Fondo de investigaciones sanitarias (PI07/0301, MA) and Botín Foundation (CL-O)., Villamor, N., Conde, L., Martínez-Trillos, A., Cazorla, M., Navarro, A., Beà, S., López, C., Colomer, D., Pinyol, M., Aymerich, M., Rozman, M., Abrisqueta, P., Baumann, T., Delgado, J., Giné, E., González-Díaz, M., Hernández, J.M., Colado, E., Payer, A.R., Rayon, C., Navarro, B., JoséTerol, M., Bosch, F., Quesada, V., Puente, X.S., López-Otín, C., Jares, P., Pereira, A., Campo, E., López-Guillermo, A.

Matrix metalloproteases (MMPs) regulate innate immunity acting over proinflammatory cytokines, chemokines, and other immune-related proteins. MMP-25 (membrane-type 6-MMP) is a membrane-bound enzyme predominantly expressed in leukocytes whose biological function has remained largely unknown. We have generated Mmp25-deficient mice to elucidate the in vivo function of this protease. These mutant mice are viable and fertile and do not show any spontaneous phenotype. However, Mmp25-null mice exhibit a defective innate immune response characterized by low sensitivity to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules. Moreover, these immune defects can be tracked to a defective NF-κB activation observed in Mmp25-deficient leukocytes. Globally, our findings provide new mechanistic insights into innate immunity through the activity of MMP-25, suggesting that this proteinase could be a potential therapeutic target for immune-related diseases.

An increase of stroke incidence occurs in women with the decline of estrogen levels following menopause. This ischemic damage may recur, especially soon after the first insult has occurred. We evaluated the effects of estrogen and phytoestrogen treatment on an in vitro recurrent stroke model using the HT22 neuronal cell line. HT22 cells were treated with 17β-estradiol or genistein 1 h after the beginning of the first of two oxygen and glucose deprivation/reoxygenation (OGD/R) cycles. During the second OGD, there was a deterioration of some components of the electron transport chain, such as cytochrome c oxidase subunit 1 with a subsequent increase of reactive oxygen species (ROS) production. Accordingly, there was also an increase of apoptotic phenomena demonstrated by poly(ADP-ribose) polymerase 1 cleavage, Caspase-3 activity, and Annexin V levels. The recurrent ischemic injury also raised the hypoxia-inducible factor 1α and glucose transporter 1 levels, as well as the ratio between the lipidated and cytosolic forms of microtubule-associated protein 1A/1B-light chain 3 (LC3-II/LC3-I). We found a positive effect of estradiol and genistein treatment by partially preserving the impaired cell viability after the recurrent ischemic injury; however, this positive effect does not seem to be mediated neither by blocking apoptosis processes nor by decreasing ROS production. This work contribute to the better understanding of the molecular mechanisms triggered by recurrent ischemic damage in neuronal cells and, therefore, could help with the development of an effective treatment to minimize the consequences of this pathology.

Despite the advantage observed with novel drugs such as bortezomib, thalidomide, or lenalidomide, multiple myeloma (MM) remains incurable and there is a clear need for new drugs or combinations based on the pathogenetic mechanism of MM. One of the proposed mechanisms in MM pathogenesis is the involvement of kinase molecules in the growth and survival of myelomatous cells. In this study, we have explored the optimal combination for dasatinib, a tyrosine kinase inhibitor, in MM cells. A clear synergistic effect was observed with the triple combination of dasatinib with bortezomib and dexamethasone which was evident even in the presence of bone marrow microenvironment. Experiments performed on freshly isolated patients' cells also demonstrated potentiation of response in the triple as compared with the agents alone or in double combinations. Gene expression profiling experiments provided some clues on the transcriptional rationale underlying this potentiation, as the triple combination led to significant deregulation of genes involved in cell death, cell growth, proliferation, DNA replication, repair and recombination, and cell-cell signaling. Some of these results were further confirmed by apoptosis and cell cycle experiments and also by Western blot and PCR. These data provide the rationale for the use of this novel combination in MM patients. © 2011 Springer-Verlag., This work was supported by grants from the Ministerio de Ciencia e Innovación - ISCIII (PI081825); Fundación de Investigación Médica Mutua Madrileña (AP27262008); Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León; the Spanish Myeloma Network Program (RD06/0020/0006), and Grupos de Excelencia de Castilla y León (Ref. GR33). The CIC receives support from the European Community through the regional development funding program (FEDER). AG-G was supported by the “Proyecto Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León (Consejería de Sanidad JCyL–ISCIII)”.

La hemoglobinuria paroxistica nocturna (HPN), tambien conocida como sindrome de Marchiafava-Michelli, es una hemopatia adquirida poco frecuente de la que aun quedan aspectos por conocer. Se presenta principalmente en el adulto joven, aunque existen casos que aparecen en la infancia o en la senectud. La HPN es consecuencia de la expansion clonal no maligna de celulas progenitoras hematopoyeticas que han adquirido una mutacion somatica en el gen PIG-A (fosfatidil inositol glucano A), situado en el brazo corto del cromosoma X. Como consecuencia de la misma, las celulas afectas son deficientes en una serie de proteinas que se anclan a la membrana a traves del glucosil fosfatidil-inositol (GPIAP). Entre ellas, se encuentran MIRL (inhibidor de la lisis reactiva de la membrana; CD59) y DAF (factor acelerador de la degradacion del complemento; CD55). Estas glucoproteinas son reguladoras fisiologicas de la actividad litica del complemento y su deficit provoca la existencia de hemolisis intravascular cronica, caracteristica de la HPN. Mas compleja es la explicacion fisiopatologica de la hipercoagulabilidad, la insuficiencia medular y la distonia de la musculatura lisa que presentan estos pacientes. La expresividad clinica de la HPN es muy variable, desde casos con escasa sintomatologia hasta casos muy graves e incapacitantes. La supervivencia media de los pacientes con la HPN se situa en torno a los 10-15 anos tras el diagnostico. Los fenomenos tromboembolicos, caracteristicamente recurrentes y de localizacion atipica (abdominal, visceral, cerebral, cutanea), ocurren en

[Background]: Proteasome inhibition represents a promising novel anticancer therapy, and bortezomib is a highly selective reversible inhibitor of the proteasome complex. Acute myeloid leukemia (AML) is an immnunophenotypically heterogeneous group of diseases, with CD34+ cases being associated with drug resistance and poor outcome. We investigated the effects of bortezomib on the growth and survival of AML cells. [Design and Methods]: We studied the in vitro activity and mechanism of action of bortezomib on both cell lines and fresh cells from 28 AML patients including CD34+ and CD34- cases. [Results]: Bortezomib showed potent anti-AML activity (IC50 < 50 nM), which was greater than that of conventional agents (doxorubicin, cytarabine and fludarabine). Moreover, synergistic effects were observed when bortezomib was adminstered in combination with doxorubicin and cytarabine. Mechanistically, bortezomib induced accumulation of cells in the G2/M phase, with up-regulation of p27, together with cell death through an increase in the mitochondrial outer membrane permeability involving caspase-dependent and -independent pathways. The apoptotic activity of bortezomib on fresh CD34 + blast cells from patients was similar to that observed on CD34 - blast cells. Importantly, bortezomib was significantly more active than doxorubicin in the immature CD34+ cells, while there were no differences in its action on CD34- cells. [Conclusions]: Bortezomib induces apoptosis in acute myeloid leukemia cells in vitro. Whether this drug might be useful in the treatment of patients with acute myeloid leukemia can be established only in ad hoc clinical trials. ©2008 Ferrata Storti Foundation., We thank Johnson and Johnson Pharmaceutical Research and Development (JJPRD).

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of the haematopoietic progenitor cells due to a somatic mutation in theX-linked phosphatidylinositol glycan class A gene. The disease is characterized by intravascular haemolytic anaemia, propensity to thromboembolic events and bone marrow failure. Other direct complications of haemolysis include dysphagia, erectile dysfunction, abdominal pain, asthenia and chronic renal failure (65% of patients). The disease appears more often in the third decade of life and there is no sex or age preference. Detection of markers associated with glucosyl phosphatidyl inositol deficit by flow cytometry is currently used in the diagnosis of PNH. For years, transfusions have been the mainstay of therapy for PNH. A breakthrough in treatment has been the approval of the humanized monoclonal antibody eculizumab, which works by blocking the C5 complement protein, preventing its activation and therefore haemolysis. Several studies have confirmed that treatment with eculizumab avoids or decreases the need for transfusions, decreases the probability of thrombosis, improves the associated symptomatology and the quality of life in patients with PNH, showing an increase in survival. Because of rapid advances in the knowledge of the disease and its treatment, it may become necessary to adapt and standardize clinical guidelines for the management of patients with PNH.

Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients.

Hypomethylating agents are able to prolong the overall survival of some patients diagnosed with acute myeloid leukemia. The aim of this study was to evaluate the clinical use of azacitidine as front-line therapy in unfit acute myeloid leukemia (AML) patients and to develop a clinical prediction model to identify which patients may benefit more from the drug. One hundred and ten untreated unfit AML patients received front-line azacitidine therapy in Spain, and response and survival were evaluated in them following European LeukemiaNet (ELN) guidelines. A clinical prediction rule was obtained from this population that was validated and refined in 261 patients treated in France, Austria and Italy. ELN response was achieved in 21.0% of the 371 patients (CI95% 17.0-25.5) and did not depend on bone marrow blast cell percentage. Median overall survival was 9.6 months (CI95% 8.5-10.8) and 40.6% of the patients were alive at 1 year (CI95% 35.5-45.7). European ALMA score (E-ALMA), based on performance status, white blood cell counts at azacitidine onset and cytogenetics, discriminated three risk groups with different survival and response rates. Azacitidine seems a reasonable therapeutic option for most unfit AML patients, i.e. those displaying a favorable or intermediate E-ALMA score. (C) 2014 Elsevier Ltd. All rights reserved.

Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3′ region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥ 4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia., This work was funded by Spanish Ministry of Economy and Competitiveness through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTICC). C.L.-O. is an Investigator of the Botin Foundation supported by Banco Santander through its Santander Universities Global Division, and E.Ca. and D.T. are Institució Catalana de Recerca i Estudis Avançats-Academia investigators. We acknowledge Partnership for Advanced Computing in Europe (PRACE) for awarding us access to resource Marenostrum based in Spain at the BSC, the Pershing Square Sohn Cancer Research Alliance and European Union’s FP7 through the Blueprint Consortium.

The study was partially funded by the “Accion Transversal del Cancer”, approved on the Spanish Ministry Council on the 11th October 2007, by the Instituto de Salud Carlos III-FEDER (PI08/1770, PI08/0533, PI08/1359, PS09/00773, PS09/01286,PS09/01903, PS09/02078, PS09/01662, PI11/01403, PI11/01889,PI11/00226, PI11/01810, PI11/02213, PI12/00488, PI12/00265,PI12/01270, PI12/00715, PI12/00150), Castaño-Vinyals, G., Aragonés, N., Pérez-Gómez, B., Martín, V., Llorca, J., Moreno, V., Altzibar, J.M., Ardanaz, E., de Sanjosé, S., Jiménez-Moleón, J.J., Tardón, A., Alguacil, J., Peiró, R., Marcos-Gragera, R., Navarro, C., Pollán, M., Kogevinas, M., Alonso, M.T., Amiano, P., Arias, C., Azpiri, M., Benavente, Y., Boldo, E., Bueno, A., Bustamante, M., Caballero, F.J., Campo, E., Cantón, R., Capelo, R., Carmona, C., Casabonne, D., Chirlaque, M.D., Cirac, J., Clofent, J., Colado, E., Costas, L., Crous, M., Del Campo, R., Santos, M.D., Dierssen-Sotos, T., Ederra, M., Espinosa, A., Cabrera, M.F., Somoano, A.F., Villa, T.F., García-Esquinas, E.G., Martín, P.G., Gómez-Acebo, I., Puga, C.G., Gràcia, E., Eslava, M.G., Guinó, E., Huerta, J.M., Lope, V., López-Abente, G., Lopez-Otín, C., Argüelles, B.M., Salas, S.M., Pozo, B.M., de la Torre, A.J., Moreno, E., Iribas, C.M., Olea, N., Gelis, G.O., Paré, L., Porta, M., Puig, M., Del Fresno, M.R., Robles, C., Suarez, M.M., Romero, B., Castillo, A.I., Serra, M.S., Trejo, D.S., Santaballa, A., Santibáñez, M., Sierra, Á., Souto, A., Villanueva, C.M., Carrasco, E., Sabaté, Y., Persavento, C., García, M., Carrasco, G., Expósito, A., Andreu, M., Bessa, X., Piracés, M., Lorente, J.A., Tusquets, I., Collet, I., Bory, F., Pera, M., Abella, E., Garcia, F., Salar, A., Piñol, M., Fernandez-Llamazares, J., Martín, M.V., Garsot, E., Servio, L.I., Arzoz-Fabregas, M., Suarez, L., Ruiz, J.M., Castells, A., Serradesanferm, A., Bosch, A., Muñoz, M., Fontanillas, M., Alcaraz, A., Mengual, L., Duran, E., Izard, C., López, C., Benítez, J.M., Massanes, A.B., Ferrer, O.G., Almeda, J., Sarret, S., Rodriguez, M.A., Valmaseda, A.B., Liceran, M., Petitbó, D., Nolla, J.I., Pérez, S., Martínez, S., Vilaseca, J.M., Sebastián, L., Quesada, P., Sequera, G., Barca, E.G., Domingo-Domenech, E., Oliveira, A., Alonso, E., de la Banda, E., Sabater, Y., Vergara, M., Exposito, A., Alonso, T., Padrol, I., Klaustermeier, J., Florencia, Y., Camon, V., Esteban, A., Aymerich, M., Lopez-Otin, C., Muñoz, A., Torralba, Y., Dot, D., Mercadal, S., Sarra, J., Medina, P., Atienza, C., Biondo, S., de Oca, J., Ferran, L., Rodriguez-Moranta, F., Soriano, A., Guardiola, J., Urruticoechea, A., Galan, M., Banda, J.M., Zumel, Á., Saez, A., Garrido, J.A., Lacasaña, M., Ariza, J.L., García, T., García, M.Á., Hidalgo, M.Á., Asuero, M., Bayo, J., Vázquez, V.C., Franco, F., Gurucelain, J.L., Frías, M.J., Van de Haar, R., Viñas, J., Jiménez, H., Aguayo, M., Pereira, A., Gutiérrez, S.P., Rada, R., Candón, J., Domínguez, J., Ramos, M., Pedraza, G., Braulio, J., Salas, J., Labrero, D., Muñoz, D., Barrero, F., Delgado, S., Galisteo, L., Camacho, A., Elena, H., Caballero, J., Muñoz, M.J., Arredondo, F., Linares, R., Tejada, A., Tinto, H., Alés, J.D., Coronado, M.V., Bernal, M.L., Ortiz, J.L., Ocaña, M.N., Cavanillas, A.B., Miguel, Mejías, E.J., Abril, O.M., Requena, R.O., Morata, J.L., Echezarreta, E.G., López, L.M., Martínez, M.M., Sedano, P.P., de la Pedraja Pavón, A., López-Rojo, C., Mendiola, J., Pellicer, E., Caparrós, J.M., Oltra, E.G., Martínez, J.M., Molina, A., Villanueva, V., Miranda, M.J., Abril, C., Martinez, J., Salas, D., Ruiz, J.L., Ponce, M., Noos, P., Cervera, J., Del Val, A., Segura, A., Jiménez, N., Bellmunt, E., Aznar, I., Ramos, D., Montón, T., Solera, M.C., Mora, E.M., Estañ, N., Camarasa, N., Solanas, J.V., Ripoll, J., Cantero, J., Díaz, C.A., García, A.S., Avellón, S.M., Neumann, M., González, M.J., Suárez, M.M., Fernández, G., Fresno, M.R., Gómez, A., Garín, U., de Gros, A., Tamayo, E., Rua, M., Lasarte, C.S., Jauregi, M., Recio, J., Fernández, M., Múgica, M., Ciria, J.P., Guimón, E., Adúriz, C., Lacasta, A., Francisco, J.S., Alvarez, I., Enriquez-Navascués, J.M., Ruiz, I., Michelena, M., Alberro, J.A., Almansa, A.M., Goñi, L.M., Elizagaray, M.I., Técnico, M.O., Pérez, R.B., De Miguel Medina, M.C., Repáraz Romero, M., de Azúa Ciria, A.Y., Díaz, M.M., Lizárraga, M.S., Jiménez, Y.L., López, C.E., Legaz, S.O., Lezaun, R.A., Hurtado, H.O., De Miguel Velasco, M., Rubio, P.A., Cunchillos, F.D., de Liaño Arguelles, Á.D., Peñuela, J.M., Dorronsoro, M.L., Celaya, F.B., García, D.R., de la Higuera Carnicer, B.G., Tricas, J.M., Rivera, E.M., García, F.V., Noain, J.J., Erdozain, E.A., Ulibarrena, C.I., Lopeandía, J.G., Caden, M.A., Moraza, F.A., Domench, J.J., Arteaga, A.A., Navarro, K.A., Del Burgo Tajadura, M.A., Irastorza, F.C., Urrisarri, M.J., Zubicoa, B.F., Lorente, P.G., Sevilla, P.H., Echamendi, M.L., Díaz, Á.M., Salaverri, J.M., Gorricho, F.J., Del Valle, M.P., de Prado Marcilla, J., Garcésducar, M., Herce, P.A., Barasoain, J.E., Campos, I.A., Vergara, E.A., Beraza, B.C., Blasco, L.F., Díaz, L.G., Larrañeta, J.G., Ruiz, N.G., García, J.G., Orduna, M.S., Larena, D.I., Fernández, M.P., de Ciriza Pejenaute, F.J., Escuin, R., Puertas, I.R., Samper, I.A., Sesma, M.E., López, M.J., Apestegui, C.Z., Linares, C., Cervantes, M., Ferreras, E., García-Pérez, J., Fernández-Navarro, P., Pastor, R., Ramis, R., González, Á., Ruiz, T., Muñoz, V., Delgado, R., Lanza, M., Marín, M., Posada, M., Cosmen, J., Villanueva, A., Castaño, A., Jiménez, J.A., Rin, A., Díaz, G., Herreros, M., Pedraza, V., López, P., de la Fuente, M., de Castro, M.F., Sobrino, J., Calvo, R., Carrascal, I., Bernal, R., Mateo, A., García-Olmo, D., Zarazaga, A., Gombau, M.Á., Díaz, J., Gómez, T., Sánchez, T., de la Quintana, P., Ordás, J., Cuevas, P., Sánchez-Pastor, M., de la Peña, J.J., Tabernero, Á., Cámara, N., Hardisson, D., Suárez, A., Burgos, E., Alves, J., Miguel, M., Feliú, J., Belda, C., Zamora, P., Sánchez, A., Hernández, A., Beato, T., Arroba, A., Ballesteros, A., Canora, S., García, C., Romero, M., José, T.S., Espinel, M., Mañas, A., Sánchez, M., Ramón, H., Morillo, J.L., Cuchí, M., García, M.J., Ramos, J.M., Rojo, R., Sanjuanbenito, A., Villanueva, A.G., Gras, M., Cabañas, J.L., Collado, V., Arano, I., Capela, I., Mojarrieta, C., Peñas, B., Rodríguez, M., Ramos, A., Sancho, S., Hervás, A., Moreno, P., Sánchez, C., DoloresRubio, Montoya, L., Sancho, P., Rodríguez, L., Durán, E., Morel, S., Burgos, J., García, R., Del Cañizo, C.G., Díaz, A., González, F., Cristóbal, E., Barahona, C., Vázquez, S., Esteban, V., Pedrera, M., Martínez, J., Carrato, A., Ponce, C.G., Santos-Olmo, A., Barrientos, R.R., López, G., Fernández, A., García, A.B., Noriega, A., Campo, M., Varona, E., López, F., Montero, M., Gómez, M.T., Bartibas, P., Sanz, R., Montes, T., Salinero, M.Á., Herrero, M., Igea, M., Calvo, C., Pérez-Pellón, C., Maestre, M., Agora, M.A., Martín, M.G., Velázquez, D., Fernández, B., Castro, M., Ayuso, E., Masa, R., Antelo, C., García, S., Herrero, S., Delgado, A., Vilardell, L., Puig-Vives, M., Osca-Gelis, G., Buxo, M., Izquierdo, A., Carmona-Garcia, M.C., Romanos, R.R., Galindo, C.T., Bargalló, P.M., Sanchez, E.R., Saez, M., Roncero, J.M., Gallardo, D., Coll, R., Blanco, I., Ruano, L.M., Castaño, E.A., Rusiñol, J.M., Perez, R.J., Pujol, I.M., Tuero, G.C., de Barrio Lera, J.P., Carral, J.M., Pérez, C.A., Puente, M.E., Balbuena, S.P., Presa, J.M., Ramírez, J.A., Martínez, A.Á., de Francisco, T.G., Elosua, T.G., Teso, E.P., Fueyo, J.F., Guadarrama, O.A., Turienzo Frade Mdel, A., De la Hoz Riesco, M., Fernández, J.J., Fernández, V.S., Fernández, R.C., Santamaría, M.V., Simón, J.A., Ganso, A.M., Pacho, A.V., Alegre, S.V., Plaza, F.J., Cuenllas, B.Á., Franco, E.H., Martín, M.H., Ariño, M.T., Tascón, C.D., Palomo, J.A., Castañón López Mdel, C., Lorenzo, M.P., González, I.A., Rivero, F.G., Román, C.H., Fernández, E.Á., Bayón, T.R., Fernández, A.G., Municio, F., de Celis, M.G., Ingelmo, L.Á., Herrero, J.G., Carbajo, M.J., Esteras, T.R., Baz, B.V., Baticón, C.H., Baza, E.Á., Urdiales, A.U., Gil, J.I., López, M.A., Ule, E.C., Diez, C.B., Marín, A.I., Bernabeu, M.Á., de Abajo Olea, S., Rojo, C.V., Fernández, M.Á., González, F.G., Martínez, L.G., Del Huertotrancón Moratiel, M., Ramos, N.C., Fidalgo, S.P., Coque, P.R., González, Á.G., Díez, A.B.