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Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to secondary acute myeloid leukemia (sAML). However, the mutational dynamics and clonal evolution underlying disease progression are poorly understood at present. To elucidate the mutational dynamics of pathways and genes occurring during the evolution to sAML, next generation sequencing was performed on 84 serially paired samples of MDS patients who developed sAML (discovery cohort) and 14 paired samples from MDS patients who did not progress to sAML during follow-up (control cohort). Results were validated in an independent series of 388 MDS patients (validation cohort). We used an integrative analysis to identify how mutations, alone or in combination, contribute to leukemic transformation. The study showed that MDS progression to sAML is characterized by greater genomic instability and the presence of several types of mutational dynamics, highlighting increasing (STAG2) and newly-acquired (NRAS and FLT3) mutations. Moreover, we observed cooperation between genes involved in the cohesin and Ras pathways in 15-20% of MDS patients who evolved to sAML, as well as a high proportion of newly acquired or increasing mutations in the chromatin-modifier genes in MDS patients receiving a disease-modifying therapy before their progression to sAML., This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS PI18/01500, PI17/01741, Instituto de Salud Carlos III (ISCIII), Fondo de Investigación Sanitaria (Instituto de Salud Carlos III – Contratos Río Hortega (CM17/0017), European Regional Development Fund (ERDF), Una manera de hacer Europa, European Union Seventh Framework Programme [FP7/2007-2013] under Grant Agreement nº306242-NGS-PTL, SYNtherapy: Synthetic Lethality for Personalized Therapy-based Stratification in Acute Leukemia (ERAPERMED2018-275); ISCIII (AC18/00093), Proyectos de Investigación del SACYL, Gerencia Regional de Salud de Castilla y León: GRS1850/A18, GRS1653/A17, and Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233). MMI is supported by a predoctoral grant from the Junta de Castilla y León, and by the Fondo Social Europeo (JCYL-EDU/556/2019 PhD scholarship) and JMHS is supported by a research grant from Fundación Española de Hematología y Hemoterapia.

[Background]: Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored. [Methods]: Amplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19− early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities. [Results]: NGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19− early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation. [Conclusions]: Our study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients., This work was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI12/00281, PI15/01471, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) “Una manera de hacer Europa,” Consejería de Educación, Junta de Castilla y León (SA085U16), Proyectos de Investigación del SACYL, Spain: GRS 1172/A/15, BIO/SA10/14, GRS 1343/A/16, and Fundación Española de Hematología y Hemoterapia (FEHH) and by a grant (RD12/0036/0069) from the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) “Una manera de hacer Europa” (Innocampus; CEI-2010-1-0010), Fundación “Memoria Don Samuel Solórzano Barruso” 2016, and the European Union Seventh Framework Programme [FP7/2007–2013] under Grant Agreement no. 306242-NGS-PTL. MHS is fully supported by an “Ayuda predoctoral de la Junta de Castilla y León” by the Fondo Social Europeo (JCYL-EDU/346/2013 PhD scholarship).

[Background]: The presence of genetic changes is a hallmark of chronic lymphocytic leukemia (CLL). The most common cytogenetic abnormalities with independent prognostic significance in CLL are 13q14, ATM and TP53 deletions and trisomy 12. However, CLL displays a great genetic and biological heterogeneity. The aim of this study was to analyze the genomic imbalances in CLL cytogenetic subsets from both genomic and gene expression perspectives to identify new recurrent alterations. [Patients and methods]: The genomic imbalances and expression levels of 67 patients were analyzed. The novel recurrent abnormalities detected with bacterial artificial chromosome array were confirmed by FISH and oligonucleotide microarrays. In all cases, gene expression profiling was assessed. [Results]: Copy number alterations were identified in 75% of cases. Overall, the results confirmed FISH studies for the regions frequently involved in CLL and also defined a new recurrent gain on chromosome 20q13.12, in 19% (13/67) of the CLL patients. Oligonucleotide expression correlated with the regions of loss or gain of genomic material, suggesting that the changes in gene expression are related to alterations in copy number. [Conclusion]: Our study demonstrates the presence of a recurrent gain in 20q13.12 associated with overexpression of the genes located in this region, in CLL cytogenetic subgroups., This work was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias (02/1041 and FIS 09/01543); Fondo Social Caja de Burgos de Investigación Clínica, Proyectos de investigación del SACYL (106/A/06) and by the ‘Acción Transversal del Cáncer’ project, through an agreement between the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation and the Cancer Research Foundation of Salamanca University and the Redes de Investigación RTIIC (FIS). AER is fully supported by an ‘Ayuda predoctoral FIS de formación en investigación’ by the Spanish Fondo de Investigaciones Sanitarias.

Idiopathic hypereosinophilic syndrome (HES) is a rare disorder characterized by unexplained, persistent hypereosinophilia associated with multiple organ dysfunctions. The cause of HES is unknown and shows clinical heterogeneity. FIP1L1-PDGFRA fusion is a clonal marker for the diagnosis and treatment of HES. We prospectively studied 78 patients with chronic eosinophilia. In all cases, the most salient clinical and biological characteristics as well as the response to the therapy were analyzed. In addition, we performed conventional cytogenetics and fluorescent in situ hybridization (FISH) with three BACs covering the FIP1-like-1 (FIP1L1)/platelet-derived growth factor receptor-α gene (PDGFRA) fusion. Nineteen of 78 patients (24 %) presented criteria of HES. The majority of patients were male (18) with median age of 49 years (range 19-84 years). FIP1L1-PDGFRA fusion was found in eight patients. Patients with FIP1L1-PDGFRA fusion presented with more bone marrow eosinophils and peripheral blood eosinophilia as well as anemia, leukocytosis and thrombocytopenia. Using of low-dose imatinib mesylate (100 mg/day) a hematological and molecular remission in all patients displaying the FIP1L1-PDGFRA fusion gene was observed. Therefore, imatinib may be effective for use in the treatment of chronic eosinophilic leukemia, and patients should be treated before tissue damage.

Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are hematological disorders at high risk of progression to acute myeloid leukemia (sAML). Previous high-throughput sequencing studies have provided insight into the mutational dynamics and clonal evolution underlying disease progression. However, large serial sequencing studies are still required to define which type of mutations alone or in combination contribute to leukemic transformation. To assess the mutational profiles and mutational dynamics underlying progression from MDS to sAML, a targeted-deep sequencing (TDS) of 117 MDS/AML related-genes was performed in 110 bone marrow serial samples from 50 MDS/CMML patients who evolved to sAML and 5 patients who did not evolved (controls), at two different time-points: at the time of diagnosis and at sAML progression or after a median of 3 year follow-up, respectively. A total of 269 mutations in 57 different genes were identified at second sampling. At diagnosis, all patients, progressing and not progressing (controls), presented similar number of mutations (p=0.15). Moreover, patients evolving to sAML were then divided by FAB/WHO subtypes at diagnosis (CMML, low-risk and high-risk MDS subgroups) and no differences were observed in the number of mutations (p=0.71) and variant allele frequency (VAF) between each group (p=0.63). It should be noted that mutations in the splicing pathway were significantly more frequent in low-risk MDS patients (89% low-risk MDS vs. 56% high risk MDS, p=0.038). However, after progression, those patients who evolved to sAML displayed a statistically significant increase of mutations (p=0.001) at the leukemic phase, while controls did not at the follow-up sample (p=0.88). This higher number of mutations at second sampling in patients who evolved to sAML, independently of their diagnostic subtype, may be indicative of a higher genomic instability during disease evolution. To study the mutational dynamics and what mutations could be important during disease evolution, the VAFs of mutations detected at both time-points in each patient of transformation cohort were compared. We observed that some mutations identified at the sAML stage (163 mutations) were already present at the MDS stage, at clonal or subclonal levels, and were retained during evolution, for example in genes such as SRSF2 and DNMT3A. However, 106 mutations increased in clonal size or were newly acquired. Interestingly, most of mutations in Ras signaling pathway showed a same pattern: they were not present at time of diagnosis and appeared at sAML. In fact, mutations in this pathway were detected in 25 of 50 patients (50%) included in this cohort and in 22 of them (88%) mutations displayed this dynamic. Therefore, in this study, Ras signaling was the most common pathway involved in the progression from MDS to sAML. Of note, 9 of these patients (18% of the whole cohort) presented, independently of diagnosis, a co-occurring cohesin mutation, that was already present at diagnosis and, in most cases, markedly increased in clonal size at sAML. Thus, the combination of mutations in these two pathways could play an important role during disease evolution. In addition, 22 of 50 patients were treated with a disease-modifying agent (18 azacytidine and 4 lenalidomide) before they progressed to sAML, while the remaining 28 patients received no treatment or supportive care and were considered as non-treated. Thus, we studied the effect of disease-modifying therapy on mutational dynamics in this cohort of patients progressing to sAML. In the treated patients, a higher proportion of newly acquired or increasing mutations at sAML in chromatin modifiers was observed, while in non-treated patients most mutations remained stable (61% vs. 28.6%, p=0.013). By contrast, regarding treatment, no differences were detected in the mutational dynamics of cohesin (p=0.56) or Ras pathway (p=1.00). MDS progression to sAML was characterized by a higher genomic instability, independently of MDS subtypes of patients at diagnosis. Ras signaling was the most frequent affected pathway during disease evolution in this cohort and, interestingly, the co-occurrence of Ras signaling and cohesin mutations could play an important role in the progression. Moreover, mutations in chromatin modifiers genes could be related to the evolution of patients who received disease-modifying treatment before progression to sAML. Disclosures Olivier: Celgene: Honoraria; Jassen: Honoraria. Díez-Campelo:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background Among patients with B-cell chronic lymphoid leukemia, those with 13q14 deletion have a favorable outcome. However, whether the percentage of cells with 13q- influences the prognosis or the biological characteristics of this disease is unknown. We analyzed the clinico-biological characteristics and outcome of patients with B-cell chronic lymphoid leukemia with loss of 13q as the sole cytogenetic aberration. Design and Methods Three hundred and fifty patients with B-cell chronic lymphoid leukemia were studied. Clinical data were collected and fluorescence in situ hybridization and molecular studies were carried out. In addition, a gene expression profile was obtained by microarray-based analysis. Results In 109 out of the 350 cases (31.1%) loss of 13q was the sole cytogenetic aberration at diagnosis. In the subgroup of patients with 80% or more of cells with loss of 13q (18 cases), the overall survival was 56 months compared with not reached in the 91 cases in whom less than 80% of cells had loss of 13q ( p < 0.0001). The variables included in the multivariate analysis for overall survival were the percentage of losses of 13q14 ( p =0.001) and B symptoms ( p =0.007). The time to first therapy in the group with 80% or more vs. less than 80% of losses was 38 months vs. 87 months, respectively ( p =0.05). In the multivariate analysis the variables selected were unmutated status of IgVH ( p =0.001) and a high level of β2microglobulin ( p =0.003). Interestingly, these differences regarding overall survival and time to first therapy were also present when other cut-offs were considered. The gene expression profile of patients with a high number of losses in 13q14 showed a high proliferation rate, downregulation of apoptosis-related genes, and dysregulation of genes related to mitochondrial functions. Conclusions Patients with B-cell chronic lymphoid leukemia with a high number of losses in 13q14 as the sole cytogenetic aberration at diagnosis display different clinical and biological features: short overall survival and time to first therapy as well as more proliferation and less apoptosis. A quantification of the number of cells showing a genetic abnormality should, therefore, be included in the study of the prognostic factors of B-cell chronic lymphoid leukemia.

Clonal plasma cells (PC) from different types of monoclonal gammopathies (MG) display distinct phenotypes consistent with an increased antigen-presentation and T-cell costimulation in MG of undetermined significance that deteriorates in malignant conditions. Expression of other cell surface and soluble molecules (e.g. adhesion/proliferation molecules) involved in the interaction between clonal PC and the bone marrow (BM) microenvironment has also been related to malignant PC, although the exact clinical significance of their expression remains largely unknown. Analysis of cell surface levels of several of these molecules in multiple myeloma (MM) patients shows an association between lower expression on BMPC of the HLA-I and β2-microglobulin antigen-presenting molecules, the CD126 and CD130 IL6 receptor (IL6R) chains, and CD38, and adverse prognostic features of the disease. Likewise, patients showing higher soluble levels of antigen-presenting molecules (HLA-I and β2-microglobulin), IL6R and CD95 tended to be associated with more aggressive disease behavior. In contrast, CD40, CD86, CD56, CD19, and CD45 were not associated with patients' outcome. Interestingly, upon considering the ratio between the soluble and PC membrane expression of each molecule, an increased adverse prognostic impact was observed for both HLA-I and β2-microglobulin, but not for the other molecules. Multivariate analysis confirmed the independent prognostic value of cell surface expression of CD126 on BMPC together with serum β2-microglobulin and LDH. In summary, our results show an abnormal distribution of the cellular and soluble compartments of the HLA-I, IL6R, and to a lower extent, CD95 molecules, in MM, associated with the clinical characteristics and behavior of the disease., Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo (Madrid, Spain); Grant numbers: Spanish Network on Multiple Myeloma G03/136, RETICC RD06/0020/0035-FEDER.

[Objectives]: The information currently available about dendritic cells (DCs) in patients with different types of monoclonal gammopathy (MG) is limited and frequently controversial. In the present study, we analyzed the ex vivo distribution as well as the phenotypic and functional characteristics of peripheral blood (PB) DCs from different types of MG. [Methods]: For this purpose, 61 untreated patients in total with MG were analyzed - MG of undetermined significance (MGUS), 29 cases; multiple myeloma (MM), 28 cases; and plasma cell leukemia (PCL), 4 cases - in comparison with a group of 10 healthy controls. [Results]: Our results show an absolute overall higher number of all subsets of PB DCs in PCL, together with lower numbers of myeloid DCs in MM patients. From a phenotypic point of view, PB DC subsets from all types of MG expressed significantly higher levels of HLA molecules and altered patterns of expression of the CD2, CD11c, CD16, CD22, CD62L, and CD86 molecules, in association with altered patterns of secretion of inflammatory cytokines. [Conclusion]: In summary, we show the existence of significant abnormalities in the distribution, phenotype, and pattern of secretion of inflammatory cytokines by different subsets of PB DCs from patients with MGs, which could reflect a potentially altered homing of DCs, together with a greater in vivo activation and lower responsiveness of PB DCs, which are already detectable in MGUS patients. ©AlphaMed Press., This work was partially supported by the Spanish Red de mieloma multiple y otras gammapatías (Contract Grant RTIC G03/136), the Spanish Red de Centros de Cáncer

To explore novel genetic abnormalities occurring in myelodysplastic syndromes (MDS) through an integrative study combining array-based comparative genomic hybridization (aCGH) and next-generation sequencing (NGS) in a series of MDS and MDS/myeloproliferative neoplasms (MPN) patients. 301 patients diagnosed with MDS (n = 240) or MDS/MPN (n = 61) were studied at the time of diagnosis. A genome-wide analysis of DNA copy number abnormalities was performed. In addition, a mutational analysis of DNMT3A, TET2, RUNX1, TP53 and BCOR genes was performed by NGS in selected cases. 285 abnormalities were identified in 71 patients (23.6%). Three high-risk MDS cases (1.2%) displayed chromothripsis involving exclusively chromosome 13 and affecting some cancer genes: FLT3, BRCA2 and RB1. All three cases carried TP53 mutations as revealed by NGS. Moreover, in the whole series, the integrative analysis of aCGH and NGS enabled the identification of cryptic recurrent deletions in 2p23.3 (DNMT3A; n = 2.8%), 4q24 (TET2; n = 10%) 17p13 (TP53; n = 8.5%), 21q22 (RUNX1; n = 7%), and Xp11.4 (BCOR; n = 2.8%), while mutations in the non-deleted allele where found only in DNMT3A (n = 1), TET2 (n = 3), and TP53 (n = 4). These cryptic abnormalities were detected mainly in patients with normal (45%) or non-informative (15%) karyotype by conventional cytogenetics, except for those with TP53 deletion and mutation (15%), which had a complex karyotype. In addition to wellknown copy number defects, the presence of chromothripsis involving chromosome 13 was a novel recurrent change in high-risk MDS patients. Array CGH analysis revealed thepresence of cryptic abnormalities in genomic regions where MDS-related genes, such as TET2, DNMT3A, RUNX1 and BCOR, are located., This work was partially supported by Grants from the Spanish Fondo de Investigaciones Sanitarias FIS (PI12/00281); Proyectos de Investigación del SACYL (BIO/SA47/13; BIO/SA52/14; GRS/874/A13; GRS 994/A/14); COST Action “EuGESMA”(BM0801); Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanis Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) “Una manera de hacer Europa”(Innocampus, CEI2010-1-0010) (RD12/0036/0069; RD12/0036/0029; RD12/0036/0044); and the European Union Seventh Framework Programme [FP7/2007-2013] under Grant Agreement n˚306242-NGS-PTL. MA was supported by a “Junta para Ampliación de Estudios”fellowship [09-02402] of the Spanish National Research Council (Consejo Superior de Investigaciones Científicas, CSIC), cofunded by the European Social Fund, and by a “Grant from Fundación Española de Hematología y Hemoterapia”. AK is employed by AstraZeneca

Leukemic B-chronic lymphoproliferative disorders (B-CLPDs) are generally believed to derive from a monoclonal B cell; biclonality has only occasionally been reported. In this study, we have explored the incidence of B-CLPD cases with 2 or more B-cell clones and established both the phenotypic differences between the coexisting clones and the clinicobiologic features of these patients. In total, 53 B-CLPD cases with 2 or more B-cell clones were studied. Presence of 2 or more B-cell clones was suspected by immunophenotype and confirmed by molecular/genetic techniques in leukemic samples (n = 42) and purified B-cell subpopulations (n = 10). Overall, 4.8% of 477 consecutive B-CLPDs had 2 or more B-cell clones, their incidence being especially higher among hairy cell leukemia (3 of 13), large cell lymphoma (2 of 10), and atypical chronic lymphocytic leukemia (CLL) (4 of 29). In most cases the 2 B-cell subsets displayed either different surface immunoglobulin (sIg) light chain (n = 37 of 53) or different levels of the same sIg (n = 9 of 53), usually associated with other phenotypic differences. Compared with monoclonal cases, B-CLL patients with 2 or more clones had lower white blood cell (WBC) and lymphocyte counts, more frequently displayed splenomegaly, and required early treatment. Among these, the cases in which a CLL clone coexisted with a non-CLL clone were older and more often displayed B symptoms, a monoclonal component, and diffuse infiltration of bone marrow and required early treatment more frequently than cases with monoclonal CLL or 2 CLL clones.

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al., To analyze the impact of the 11q deleted (11q-) cells in CLL patients on the time to first therapy (TFT) and overall survival (OS), 2,493 patients with CLL were studied. 242 patients (9.7%) had 11q-. Fluorescence in situ hybridization (FISH) studies showed a threshold of 40% of deleted cells to be optimal for showing that clinical differences in terms of TFT and OS within 11q- CLLs. In patients with ≥40% of losses in 11q (11q-H) (74%), the median TFT was 19 months compared with 44 months in CLL patients with, This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS 09/01543, PI12/00281 and PI15/01471, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) "Una manera de hacer Europa", Proyectos de Investigación del SACYL 355/A/09, GRS/1172/A15, COST Action EuGESMA (BM0801), Fundación Manuel Solórzano, Obra Social Banca Cívica (Caja Burgos), Fundación Española de Hematología y Hemoterapia (FEHH), and by grants (RD12/0036/0069 and RD12/0036/0044) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness and European Regional Development Fund (ERDF) "Una manera de hacer Europa" (CEI 2010-1-0010). The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under Grant Agreement n°306242-NGS-PTL. María Hernández-Sánchez is fully supported by an Ayuda Predoctoral de la Junta de Castilla y León from the Fondo Social Europeo (JCYL-EDU/346/2013 Ph.D. scholarship). Vera Grossmann was supported by MLL Munich and Alexander Kohlmann was supported by MLL Munich and AstraZeneca in terms of salary.

This is an open access article distributed under the Creative Commons Attribution License.-- et al., The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL., The study was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias 02/1041, FIS 09/01382, FIS 09/01543, and PI12/00281; RD12/0036/0069 from Red Tematica de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) “Una manera de hacer Europa”; and Caja de Burgos-Banca Cívica. A. Rodrígues was fully supported by an Ayuda Predoctoral FIS de Formación en Investigacion by the Spanish Fondo de Investigaciones Sanitarias. M. Hernandez-Sanchez was partially supported by a grant from the “Fundacion Española de Hematología y Hemoterapia.” Partially supported by grants from “Proyectos de Investigacion Biomédica del SACYL” 106/A/06.

Fundamento y objetivo En los pacientes con sindromes mielodisplasicos (SMD) la acumulacion progresiva de hierro en el organismo representa uno de los problemas mas importantes de orbilidad y mortalidad. Presentamos un protocolo de diseno observacional, abierto, multicentrico y no aleatorizado, en pacientes diagnosticados de SMD en regimen transfusional con sobrecarga de hierro. En este estudio se pretende comprobar la eficacia del tratamiento parenteral con desferrioxamina, utilizando dispositivos de perfusion continua subcutanea, y valorar la evolucion de la sobrecarga de hierro y los requerimientos tranfusionales. Pacientes y metodo Se incluyeron 28 pacientes (12 varones, 16 mujeres), 12 con anemia refractaria (AR), 15 con anemia sideroblastica (AS) y 1 con un SMD no especificado. El seguimiento medio fue de 13,5 meses con perdidas paulatinas de pacientes, 11 de ellas durante el primer ano. Resultados A los 12 meses, la media (desviacion estandar [DE]) de ferritina disminuyo en 258,51 (1.208,04) ng/ml (p = 0,4) y a los 24 meses, en 979,6 (810,31) ng/dl (p = 0,1). A los 12 meses la media de transfundidos aumento en 60,57 (183,7) g Hb/mes (p = 0,029) y a los 24 meses, en 167,3 (406,5) g Hb/mes (p = 0,36). Conclusiones La administracion de desferrioxamina es eficaz en prevenir el aumento de la sobrecarga ferrica en los pacientes con sindromes mielodisplasicos y deberia incorporarse en su tratamiento.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: Patients with chronic lymphocytic leukemia and 13q deletion as their only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus, cases with a high number of 13q- cells (13q-H) had both shorter overall survival and time to first therapy. The goal of the study was to analyze the genetic profile of 13q-H patients. [Design and Methods]: A total of 102 samples were studied, 32 of which served as a validation cohort and five were healthy donors. [Results]: Chronic lymphocytic leukemia patients with higher percentages of 13q- cells (>80%) showed a different level of gene expression as compared to patients with lower percentages (, The study was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias 02/1041 and FIS 09/01543; Caja de Burgos-Banca Cívica, Proyectos de Investigación del SACYL 106/A/06 and by the Acción Transversal del Cáncer project, through an agreement between the Instituto de Salud Carlos III (ISCIII), the Spanish Ministry of Science and Innovation, the Cancer Research Foundation of Salamanca University and the Redes de Investigación RTIIC (FIS). AR is fully supported by an Ayuda Predoctoral FIS de Formación en Investigación by the Spanish Fondo de Investigaciones Sanitarias.

The progressive accumulation of iron in the organism contribute to one of the most important problems of morbidity and mortality in patients with myelodysplastic syndrome (MDS). We present an observational protocol, multicentre, open and non-aleatheorised, in patients diagnosed with MDS on transfusional regime with iron overload. The study was meant to prove the effectiveness of the parenteral treatment with desferrioxamine using continuous devices of subcutaneous profusion and evaluate the evolution of iron overload as well as transfusional requirements.There were 28 patients (12 men and 16 women), 12 AR, 15 AS and 1 unknown. Average monitoring lasted 13.5 months and there was a gradual loss of patients. 11 of them had gone during the first year.After 12 months the average of ferritin decreased by 258.51 ng/dl (DE 1208.04; p = 9.4) and after 24 months, it decreased by 979.6 ng/dl (DE 810.31; p = 0.1). After 12 months the average of requirements increased by 60.57 gHb/month (DE 183.7; p = 0.029) and after 24 months, it increased by 167.3 g/Hb/month (DE 406.5; p = 0.36).Desferroxiamine treatment is effective at least to prevent an iron overload in these patients, and therefore should be incorporated in the clinical practice.

The immunological bone marrow (BM) microenvironment plays a major role in controlling growth and survival of clonal plasma cells (PC); this might translate into different patterns of expression of molecules involved in immune responses on PC from different types of monoclonal gammopathies (MG). We have studied the expression of a group of nine such molecules on both BMPC and the plasma of 61 newly diagnosed MG patients (30 MG of undetermined significance (MGUS), 27 multiple myeloma (MM) and four plasma cell leukemia (PCL)) and five normal individuals. Clonal PC from all MG displayed significantly increased levels of CD56, CD86 and CD126, and decreased amounts of CD38 (P

Abstract 3827 Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological disorders in which diagnosis, risk stratification, and treatment selection are based on morphological and cytogenetic studies in bone marrow (BM) samples. MDS are characterized by several recurrent chromosomal abnormalities, most of them unbalanced, with a widely variable prognosis. The assessment of these genomic defects is essential for a correct risk stratification of these patients. However, conventional cytogenetic (CC) techniques are not sufficient for the study of all MDS patients, because of the high proportion of normal karyotypes (40–50%) and unsuccessful cytogenetics (10%) (defined as the absence of mitosis). Array-based comparative genomic hybridization (aCGH) technology allows the screening of copy number changes among the whole genome in one single experiment and offers a higher resolution than conventional cytogenetics. Aims: To assess the potential application of aCGH in the clinical diagnosis of MDS as complementary tool to conventional cytogenetics. Patients and Methods: The study cohort comprises a total of 263 patients: MDS (203) and MDS/MPN (60) patients that have been previously studied by CC and FISH. Among the whole series, 33 (12.5%) patients had no successful cytogenetic results due to the absence of mitosis. In the remaining 230 patients with evaluable metaphases, 42 (16%) had an aberrant, while 188 (71.5%) presented a normal karyotype. Within this last group, 141 had ≥20 good-quality metaphases evaluated, 37 had 10–20 metaphases studied, and 10 patients had ≤10 successful metaphases. Copy number changes were analysed in all patients included in the study using NimbleGen Human CGH 12×135K Whole-Genome Tiling Array (Roche NimbleGen). Sex-matched human commercial DNA samples were used as reference. Data were analysed using the segMNT algorithm in NimbleScanv2.6 Software. Subsequently all genomic abnormalities found by aCGH analysis were confirmed by FISH. Results: Using aCGH methodology, copy number changes (greater than 600 bp) were detected in 54 patients of the global series: 4.3% of the normal karyotype patients, 88.1% of cases with abnormal cytogenetics, and 27.3% of patients with unsuccessful cytogenetics. Overall a high correlation (94.3%) between the cytogenetic changes observed by CC and CGH arrays was observed. Thus aCGH analysis revealed the same genomic abnormalities showed by CC in 88.1% of patients. In the remaining 11.9% genomic results were discordant between aCGH and CC, because of the presence of balanced translocations, not assessable by aCGH, and clonal cell populations below 30%. Furthermore, additional genomic abnormalities (n=36) not detected by CC were found by aCGH. The most frequent aberrations were losses affecting chromosomes 5 (33%), 7/7q (17%), 20q (14%), and Y (14%), as well as gains involving chromosome 8 (14%). Interestingly, other abnormalities, mainly losses, were found in chromosomes 4, 12, and 17. Focusing on the 188 patients with normal karyotype by CC, the aCGH profiling results were concordant with cytogenetics in 98% of those patients with ≥20 metaphases studied and in 92% of those with 10–20 metaphases. However, only 80% of those patients with ≤10 successful metaphases and no changes by CC displayed no copy number changes by aCGH. The most frequent abnormality found by aCGH among these normal karyotype cases was the presence of 5q deletion (2%), while other chromosomes affected were 7, 8, 11, 12 and 20. All these abnormalities were confirmed by FISH. Regarding the patients with unsuccessful cytogenetics, 72.7% of cases displayed a normal aCGH profile, while 27.3% showed at least one genomic imbalance The most frequent genomic aberrations were losses in 4q (6%), 5q (12%) and 7q (9%), and gain of chromosome 8 (6%). In addition, three of these cases showed a complex karyotype, showing more than 5 abnormalities. Conclusion: The use of aCGH karyotyping in the diagnosis of MDS could be used as a complementary technique to conventional karyotyping in the evaluation of MDS patients. Mainly in patients with unsuccessful cytogenetics and those with normal karyotype and Disclosures: Hernández: Celgene: Research Funding.

Abstract 4725 In the last twenty years enzyme replacement therapy (ERT) with imiglucerase has been a clinically effective for Gaucher disease (GD). The recombinant glucocerebrosidase administered intravenously - usually at biweekly intervals by lifelong has improved the quality of life of patients, avoided spleen removal and bone complications. In the last months an acute shortage of imiglucerase manufactured by the Genzyme Corporation (MA, USA) has occurred as a result of viral contamination firstly and other deficiencies in the production facility. In September 2009 a position statement based on the findings of the European Working Group for Gaucher Disease and European Gaucher Alliance, established a set of key recommendations about identification and monitoring of at-risk patients threatened. In Spain the follow-up of patients and the strict complementation of rules of therapy have permitted to obtain a profile of the situation in a group of patients with restricted ERT. Patients and Methods: A total of 50 GD1 patients have been analyzed before and after 6 and 12 months of imiglucerase shortage. Have been excluded for analysis children in order to dose reduction has been minimal as well as patients who have switched to another ERT or miglustat therapy. Results: Gender: 25 males/25 females. Mean age of group: 45.3±15.3 (range:18-84) SSI at diagnosis(Dx): 8.7±3.8 (range:3-19) Chitotriosidase (CT) activity at Dx:13,383±12,783 nM/mL.h; CCL18/PARC at Dx: 767±1,198 ng/mL. 20% of patients were splenectomized and 78% had bone disease at Dx. During shortage 23 patients (46%) discontinued therapy, in this period only one patient suffered a bone crisis and other anaemia as complications. Mean reduction of haemoglobin level: 2.7% (NS), platelet counts: 5.4% (NS). CT activity was increased 135% (p Disclosures: Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma.

Background and objectives: Several reports describe the use of miglustat (Zavesca®), an oral inhibitor of glucosylceramide synthase, for type 1 Gaucher disease (GD1) in clinical trials. There are few data on the use of miglustat for GD1 in the real-world clinical experience. Here, we assess the long-term efficacy, tolerability and safety of miglustat in a prospective, open-label, observational study of GD1 patients attending routine clinic visits. Design and Methods: 48 patients with mild-to-moderate GD1 received miglustat 100 mg t.i.d. over 36 months. Patients were analysed in two groups: therapy-naïve patients (n = 11) and those switched from previous treatment with enzyme replacement therapy (ERT; n = 37). Assessments of clinical status, haematological parameters, and organomegaly were performed before treatment and after 6, 12, 24 and 36 months of therapy. Data were compared with a historical reference cohort 29 GD1 patients treated for 36 months with ERT. Results: Nine treatment-naïve patients completed 36 months of miglustat therapy. Sustained improvements in haemoglobin were seen in all patients who had anaemia before treatment. Platelet counts improved in patients with initial thrombocytopenia, and were maintained in patients with normal counts at baseline. Plasma chitotriosidase activity decreased in treatment-naïve patients during the first year on therapy and remained stable thereafter. S-MRI findings indicated improved bone status at 36 months. In patients switched from ERT, 15 completed 36 months of miglustat therapy, and displayed maintained or improved clinical and haematological parameters, and surrogate disease severity markers. No statistically significant differences were observed at 36 months between treatmentnaïve patients with similar baseline characteristics treated with either miglustat or ERT. Overall, miglustat was well tolerated. Interpretation and conclusions: Miglustat provided appreciable therapeutic responses on clinical, haematological, biomarker and S-MRI parameters at 36 months in patients with GD1, and was well tolerated. Therapeutic responses with miglustat were comparable with those seen with ERT.

Monoclonal Gammopathy of Uncertain Significance (MGUS) is a monoclonal disorder defined by the presence of a serum monoclonal protein

Abstract 3593 Azacitidine (AZA) is currently being used in AML patients (Pts) not deemed candidates for intensive chemotherapy. Most of this usage follows approved drug label by EMA, but off-label usage is not uncommon in Pts with >30% bone marrow blast (BMB) cells. Clinical trials are now comparing AZA vs. conventional care in selected populations, and data from daily practice may shed light on the generalizability of their results. A retrospective nationwide study was set up in Spain in order to evaluate the population of AML Pts that is receiving AZA as front-line therapy (Rx) in Spain, its patterns of usage, effectiveness and safety in daily practice conditions, as well as the factors linked to overall response rate (ORR) and overall survival (OS). Only Pts treated before Dec/2010 could be included in this study, that was approved by the Spanish Medicines Agency (AEMPS, code ACL-AZA-2011–01). Data were collected from Oct/2011 to Jan/2012 and analyzed as of Jun/2012. ORR was evaluated according to both ELN-2010 criteria for AML as well as IWG-2006 criteria for MDS (in order to assess the impact of PB changes), OS measured from 1st cycle of AZA to death or last follow-up, and toxicity coded according to NCI CTCAEv3.0. One-hundred and ten untreated Pts (79 M/31 F, median age 75, range 56–89) were recruited from 22 academic and community sites. Comorbidity was present in 96 Pts (cardiac 43, hepatic 10, renal 3, diabetes 26, other neoplasms 18, etc.), ECOG being ≥2 in 33. Thirty Pts had an antecedent hematological disorder. Five cases had recurrent genetic abnormalities (NPM1-mutated AML in 4), 61 had MDS-related AML, 16 therapy-related AML and 28 AML not otherwise specified. Cytogenetics (Cyto) was available in 95 (86.4%): 48 diploid and 47 abnormal. MRC-2010 cyto category was favourable in 1, intermediate in 64 and adverse in 30. Median WBC at Dx was 3.3 x10E9/L (0.8–172.4), WBC before 1st AZA cycle 2.8 (1.0–175.0), platelet count 56 (7–467), PB blast 4.0% (0–100) and hemoglobin 91 g/L (48–142). Sixty-four Pts (58.2%) had BMB>30% (median 35.0%, range 15.0–98.0%). Median time from Dx to Rx was 19.5 days (0–411). Pts received 745 AZA cycles (median 4, range 1–29; ≥6 cycles 45.4%, ≥12 cycles 18.8%), but 30.9% received ≤2 because of disease progression or toxicity. 7.2% received concomitantly hydroxiurea for WBC control. Route of administration was EV in 5.8%, home administration took place in only 1.5%, and AZA was given as inpatients in 27.3% of the cycles. The no. of days of AZA Rx was 7 in 63.6% of the cycles, week-end off Rx being common place (5–2–2 schedule in 71.9%). Median AZA daily dose was 73.6 mg/sqm ( During follow-up (median 8.6 months, 0.1–48.7), 56 Pts progressed (39 on and 17 off-Rx). In 18, response was not evaluable (lack of BM assessment or death before 8 weeks). Best ORR according to IWG-2006 was 44.5% and 53.3% in the ITT and evaluable populations, respectively, while ORR according to ELN-2010 was 17.3% and 20.7%, respectively. Complete response rate (including CRm/CRi) was 15.5% and 18.5%, respectively, with both criteria. Platelet count duplication after 1st cycle predicted ORR with IWG-2006 (82.4% vs. 33.8%, p=0.001, Fisher) but this was less evident with ELN-2010 (41.2% vs., 18.3%, p=0.057, Fisher). Median OS from 1st AZA cycle was 8.1 months (CI95% 5.3–10.9, range 0.1–47.9; OS at 12 months 36.7%, OS at 24 months 7%), OS from Dx 9.5 (CI 6.0–10.0, 0.1–48.7) and PFS 7.2 (CI 4.7–9.8; 0.1–29.5). Multivariate analysis showed that the best predictors of OS in our series were: ECOG ≤1, BMB ≤30%, a diploid cyto and WBC before 1st AZA cycle CONCLUSION: OS of AML Pts treated with AZA seems promising, although it depends on ECOG, BM blast proportion, cytogenetics and WBC before 1st AZA cycle. After adjusting for cytogenetics, multilineage dysplasia does not result informative for OS in this population. Disclosures: Ramos: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Off Label Use: Azacitidine in AML (even without multilineage dysplasia or with more than 30% bone marrow blast cells). Deben:Celgene: Honoraria. Colado:Celgene: Honoraria.