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Inherited antithrombin deficiency, the most severe form of thrombophilia, is predominantly caused by variants in SERPINC1. Few causal structural variants have been described, usually detected by multiplex ligation-dependent probe amplification or cytogenetic arrays, which only define the gain or loss and the approximate size and location. This study has done a complete dissection of the structural variants affecting SERPINC1 of 39 unrelated patients with antithrombin deficiency using multiplex ligation-dependent probe amplification, comparative genome hybridization array, long-range PCR, and whole genome nanopore sequencing. Structural variants, in all cases only affecting one allele, were deleterious and caused a severe type I deficiency. Most defects were deletions affecting exons of SERPINC1 (82.1%), but the whole cohort was heterogeneous, as tandem duplications, deletion of introns, or retrotransposon insertions were also detected. Their size was also variable, ranging from 193 bp to 8 Mb, and in 54% of the cases involved neighboring genes. All but two structural variants had repetitive elements and/or microhomologies in their breakpoints, suggesting a common mechanism of formation. This study also suggested regions recurrently involved in structural variants causing antithrombin deficiency and found three structural variants with a founder effect: the insertion of a retrotransposon, duplication of exon 6, and a 20-gene deletion. Finally, nanopore sequencing was determined to be the most appropriate method to identify and characterize all structural variants at nucleotide level, independently of their size or type., Supported by the National Institute for Health Research (NIHR) for the NIHR BioResource project (grant numbers RG65966 and RG94028), by the Instituto de Salud Carlos III grant; Fondo Europeo de Desarrollo Regional (FEDER) grant PI18/00598; and Fundación Séneca 19873/GERM/15. M.E.d.l.M.-B. has a postdoctoral contract from University of Murcia, Murcia, Spain. C.B.-P. has a Río Hortega fellowship. B.d.l.M.-B. has a postdoctoral fellowship from Fundación Séneca. J.C.-G. has a predoctoral fellowship from the Ministry of Universities FPU19/03662.

Purpose To assess whether subjects with Philadelphia negative myeloproliferative neoplasms (Ph-MPNs) show differences in the presence of vascular, cardiac or renal target organ damage (TOD) and other vascular function parameters as compared to individuals without this condition. Methods An observational study was conducted. Fifty-seven subjects diagnosed with Ph-MPNs used as cases and 114 subjects without Ph-MPNs as controls. We matched the subjects with and without Ph-MPNs using the propensity scores in a 1:2 ratio using the variables gender, type 2 diabetes mellitus, high blood pressure, hyperlipidaemia and smoking. Vascular, cardiac and renal TOD were established according to the criteria of the European Society of Hypertension and Cardiology guidelines. Arterial stiffness was also assessed using the cardio-ankle vascular index (CAVI). Results Mean age was 63.50 ± 11.70 and 62.90 ± 8.32 years in subjects with and without Ph-MPNs, 32 females (56%) in the first group and 62 (54%) in the second. Subjects with Ph-MPNs have a higher percentage of carotid injury than subjects without Ph-MPNs (35.1% vs. 21.1%) and higher albumin/creatinine ratio. In the logistic regression analysis, subjects with Ph-MPNs had an OR = 2.382 (IC95% 1.066–5.323) for carotid injury versus those without haematological disease. Conclusions Subjects with Ph-MPNs have twice the risk of by carotid injury than those without haematological disease.

Context: There is a debate regarding the impact of t(1;19) (q23;p13) in adult BCP ALL. While the MD Anderson group suggests it may be a low-risk subtype, the German, English, and French study groups have shown no differential outcome, and Italian and SWOG groups have reported poor outcomes. Objective: To analyze the frequency and clinical impact of t(1;19) in a series of adult BCP ALL patients (pts). Design & Patients: A review of 513 adult BCP ALL pts (15 to 60 years) diagnosed between 2003 and 2017 and treated with MRD-oriented protocols of the PETHEMA Group. Interventions: G-banding and FISH were performed on BM samples. Measurable residual disease (MRD) was centrally assessed by multi-parametric flow cytometry. Main Outcomes Measures: Complete response (CR), overall survival (OS) and cumulative incidence of relapse (CIR), assessed by competing risk analysis. Results: Total of 26 pts with t(1;19)/TCF3-PBX1 (representing 5% of all BCP ALL). 9/23 (39%) cases showed isolated t(1;19) while 14/23 (61%) had additional chromosomal aberrations (ACA). Pts with t(1;19) were more likely to be female (73% vs 45%, p=0.006) and pre-B phenotype (63% vs 17%, p Conclusions: Although showing favorable initial treatment response, pts with t(1;19) experience a higher rate of relapse (especially those with ACA to t(1;19)) than the remaining BCP ALL pts, without differences in OS. A deeper genetic analysis may identify markers of poor outcome enabling a more precise risk stratification of t(1;19) pts.

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.

The development of Next-Generation Sequencing (NGS) has provided useful diagnostic, prognostic, and therapeutic strategies for individualized management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Consequently, NGS is rapidly being established in clinical practice. However, the technology’s complexity, bioinformatics analysis, and the different available options difficult a broad consensus between different laboratories in its daily routine introduction. This collaborative study among Spanish centers was aimed to assess the feasibility, pros, and cons of our customized panel and other commercial alternatives of NGS-targeted approaches. The custom panel was tested in three different sequencing centers. We used the same samples to assess other commercial panels (OncomineTM Childhood Cancer Research Assay, Archer®FusionPlex® ALL, and Human Comprehensive Cancer Panel GeneRead Panel v2®). Overall, the panels showed a good performance in different centers and platforms, but each NGS approach presented some issues, as well as pros and cons. Moreover, a previous consensus on the analysis and reporting following international guidelines would be preferable to improve the concordance in results among centers. Our study shows the challenges posed by NGS methodology and the need to consider several aspects of the chosen NGS-targeted approach and reach a consensus before implementing it in daily practice.

© The Author(s)., [Background]: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has an unacceptably low cure rate. In recent years, a number of new treatment strategies and compounds were developed for the treatment of AML. There were several randomized controlled clinical trials with the objective to improve patients’ management and patients’ outcome in AML. Unfortunately, these trials are not always directly comparable since they do not measure the same outcomes, and currently there are no core outcome sets that can be used to guide outcome selection and harmonization in this disease area. The HARMONY (Healthcare Alliance for Resourceful Medicine Offensive against Neoplasms in Hematology) Alliance is a public-private European network established in 2017 and currently includes 53 partners and 32 associated members from 22 countries. Amongst many other goals of the HARMONY Alliance, Work Package 2 focuses on defining outcomes that are relevant to each hematological malignancy. Accordingly, this pilot study will be performed to define a core outcome set in AML., [Methods]: The pilot study will use a three-round Delphi survey and a final consensus meeting to define a core outcome set. Participants will be recruited from different stakeholder groups, including patients, clinicians, regulators and members of the European Federation of Pharmaceutical Industries and Associations. At the pre-Delphi stage, a literature research was conducted followed by several semi-structured interviews of clinical public and private key opinion leaders. Subsequently, the preliminary outcome list was discussed in several multi-stakeholder face-to-face meetings. The Delphi survey will reduce the preliminary outcome list to essential core outcomes. After completion of the last Delphi round, a final face-to-face meeting is planned to achieve consensus about the core outcome set in AML., [Discussion]: As part of the HARMONY Alliance, the pilot Delphi aims to define a core outcome set in AML on the basis of a multi-stakeholder consensus. Such a core outcome set will help to allow consistent comparison of future clinical trials and real-world evidence research and ensures that appropriate outcomes valued by a range of stakeholders are measured within future trials., This work was funded through the Innovative Medicines Initiative 2 joint undertaking and listed under grant agreement 116026. This joint undertaking receives support from the European Union’s Horizon 2020 research and innovation program and the EFPIA. An additional cash grant was obtained from Bayer AG for conducting the Delphi survey development

© 2020 by the authors., The clonal basis of relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is complex and not fully understood. Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and multiplex ligation-dependent probe amplification (MLPA) were carried out in matched diagnosis–relapse samples from 13 BCP-ALL patients to identify patterns of genetic evolution that could account for the phenotypic changes associated with disease relapse. The integrative genomic analysis of aCGH, MLPA and NGS revealed that 100% of the BCP-ALL patients showed at least one genetic alteration at diagnosis and relapse. In addition, there was a significant increase in the frequency of chromosomal lesions at the time of relapse (p = 0.019). MLPA and aCGH techniques showed that IKZF1 was the most frequently deleted gene. TP53 was the most frequently mutated gene at relapse. Two TP53 mutations were detected only at relapse, whereas the three others showed an increase in their mutational burden at relapse. Clonal evolution patterns were heterogeneous, involving the acquisition, loss and maintenance of lesions at relapse. Therefore, this study provides additional evidence that BCP-ALL is a genetically dynamic disease with distinct genetic profiles at diagnosis and relapse. Integrative NGS, aCGH and MLPA analysis enables better molecular characterization of the genetic profile in BCP-ALL patients during the evolution from diagnosis to relapse., This work was supported in part by a grant from the Consejería de Educación, Junta de Castilla y León, Fondos FEDER (SA085U16, SA271P18), Proyectos de Investigación de la Gerencia Regional de Sanidad, SACYL, (GRS 1847/A/18; GRS 2062/A/19), SYNtherapy. Synthetic Lethality for Personalized Therapy-based Stratification In Acute Leukemia (ERAPERMED2018-275); ISCIII (AC18/00093), co-funded by ERDF/ESF, “Investing in your future”, Fundación Castellano Leonesa de Hematología y Hemoterapia (FUCALHH 2017), and Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233). A grant to AM from the Junta Provincial de Salamanca of the Asociación Española Contra el Cáncer (AECC), a grant to MFC from the Universidad Pedagógica y Tecnológica de Colombia—Vicerrectoría de Investigación y Extensión (Grupo de Investigación en Ciencias Biomédicas UPTC—GICBUPTC, Escuela de Ciencias Biológicas). M. Hernández-Sánchez holds a Sara Borrell post-doctoral contract (CD19/00222) from the Instituto de Salud Carlos III (ISCIII). co-founded by Fondo Social Europeo (FSE) “El Fondo Social Europeo invierte en tu futuro”, MM is currently supported by an “Ayuda predoctoral de la Junta de Castilla y León” by the Fondo Social Europeo (JCYL- EDU/556/2019 PhD scholarship) and a grant to JR and JMR from Instituto Carlos III (PI14/01971).

Machine learning-inspired approaches allow for processing of multidimensional data and have shown to uncover clinical patterns in several human diseases. Recently it has been used to model cGVHD risk groups according to clinical data, but the role of this approach applied to gene-expression-profile (GEP) data has not been established yet. In this study, we aimed to unravel specific GEP patterns associated to cGVHD using machine-learning analyses. For this purpose, we isolated peripheral blood T-lymphocytes drawn from normal healthy donors and from patients who had underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) with and without cGVHD symptoms. Dataset obtained in HG-U133 Plus 2.0 Gene Chip oligonucleotide array were wrapped by Boruta method based on Random Forest. Thus, a limited set of 53 genes was retained and two-dimensional Principal Component Analysis plots projection was plotted showing a clear distinction of cGVHD with No-cGVHD and healthy Control groups with area under the curve (AUC) over 0.75 for each comparison. The highest scored genes were CDKN2A, SERPINB9, LYPLA1 y CKTM1A/B genes which are involved in positive regulation of cellular senescense with protection against perforin-dependent apoptosis. Altogether our novel findings, using machine-learning approach applied to GEP in cGVHD unravel a limited panel of five genes with a potential diagnostic and targeted therapy.

Background Turner syndrome is a genetic disorder that affects women. It is caused by an absent or incomplete X chromosome, which can be presented in mosaicism or not. There are 12 cases of Turner syndrome patients who present structural alterations in autosomal chromosomes. Case presentation The present case report describes a patient with a reciprocal, maternally inherited translocation between chromosomes 2 and 12 with a mosaicism of X monosomy 45,X,t(2;12)(p13;q24)[95]/46,XX,t(2;12)(p13;q24)[5]. Through genetic mapping arrays, altered genes in the patient were determined within the 23 chromosome pairs. These genes were associated with the patient’s clinical features using a bioinformatics tool. Conclusion To our knowledge, this is the first case in which a translocation (2;12) is reported in a patient with Turner syndrome and confirmed by conventional cytogenetics, FISH and molecular genetics. Clinical features of our patient are closely related with the loss of one X chromosome, however mild intellectual disability can be likely explained by autosomal genes. The presence of familial translocations was a common finding, thus emphasizing the need for familiar testing for further genetic counselling.

Treatment with azacitidine (AZA) has been suggested to be of benefit for higher-risk myelodysplastic syndrome (HR-MDS) patients with chromosome 7 abnormalities (Abn 7). This retrospective study of 235 HR-MDS patients with Abn 7 treated with AZA (n = 115) versus best supportive care (BSC; n = 120), assessed AZA treatment as a time-varying variable in multivariable analysis. A Cox Regression model with time-interaction terms of overall survival (OS) at different time points confirmed that, while chromosome 7 cytogenetic categories (complex karyotype [CK] versus non-CK) and International Prognostic Scoring System risk (high versus intermediate-2) retained poor prognosis over time, AZA treatment had a favourable impact on OS during the first 3 years of treatment compared to BSC (Hazard ratio [HR] 0·5 P

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders., This study was supported by research grants from the Gerencia Regional de Salud (GRS 1370/A/16), ISCIII & Feder (PI14/01956), CIBERER CB15/00055, Fundación Séneca (19873/GERM/15) and Sociedad Española de Trombosis y Hemostasia (SETH). SPW holds a British Heart Foundation chair.

Essentials Vitamin K-dependent coagulant factor deficiency (VKCFD) is a rare autosomal recessive disorder. We describe a case of inherited VKCFD due to uniparental disomy. The homozygous mutation caused the absence of GGCX isoform 1 and overexpression of Δ2GGCX. Hepatic and non-hepatic vitamin K-dependent proteins must be assayed to monitor VKCFD treatment. Summary: Background Inherited deficiency of all vitamin K-dependent coagulant factors (VKCFD) is a rare autosomal recessive disorder caused by mutations in the γ-glutamyl carboxylase gene (GGCX) or the vitamin K epoxide reductase gene (VKORC1), with great heterogeneity in terms of both clinical presentation and response to treatment. Objective To characterize the molecular basis of VKCFD in a Spanish family. Methods and Results Sequencing of candidate genes, comparative genomic hybridization and massive sequencing identified a new mechanism causing VKCFD in the proband. Uniparental disomy (UPD) of chromosome 2 caused homozygosity of a mutation (c.44-1G>A) resulting in aberrant GGCX splicing. This change contributed to absent expression of the mRNA coding for the full-length protein, and to four-fold overexpression of the smaller mRNA isoform lacking exon 2 (Δ2GGCX). Δ2GGCX might be responsible for two unexpected clinical observations in the patient: (i) increased plasma osteocalcin levels following vitamin K supplementation; and (ii) a mild non-bleeding phenotype. Conclusions Our study identifies a new autosomal disease, VKCFD1, caused by UPD. These data suggest that the Δ2GGCX isoform may retain enzymatic activity, and strongly encourage the evaluation of both hepatic and non-hepatic vitamin K-dependent proteins to assess differing responses to vitamin K supplementation in VKCFD patients., This work was supported by PI15/00079 and 19873/GERM/15 (Fundacion Seneca). M. E. de la Morena-Barrio holds a postdoctoral contract from MEC FPDI-2013-17273.

Altres ajuts: This work was supported by a grant from the Spanish Group of MDS (GESMD, 2017). LP, FF, PA and FS research is supported by a grant from (GRC) Generalitat de Catalunya, economical support from CERCA Programme/Generalitat de Catalunya, Fundacio Internacional Josep Carreras and from Celgene International. LP and JMHS are supported by a research grant from FEHH (Fundacion Española de Hematología y Hemoterapia, 2017). IV acknowledges support from Pethema. MC and LZ research is supported by a grant from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain. MFM and her research is supported by the Spanish Association against Cancer (AECC, AIO2014), and the Ministerio de Economía y Competitividad of Spanish Central Government. The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies.

[Introduction]: Intestinal microbiota is arising as a new element in the physiopathology of cardiovascular diseases. A healthy microbiota includes a balanced representation of bacteria with health promotion functions (symbiotes). The aim of this study is to analyse the relationship between intestinal microbiota composition and arterial stiffness. [Methods and analysis]: An observational case—control study will be developed. Cases will be defined by the presence of at least one of the following: carotid-femoral pulse wave velocity (cf-PWV), Cardio-Ankle Vascular Index (CAVI), brachial ankle pulse wave velocity (ba or ba-PWV) above the 90th percentile, for age and sex, of the reference population. Controls will be selected from the same population as cases. The study will be developed in Primary Healthcare Centres. We will select 500 subjects (250 cases and 250 controls), between 45 and 74 years of age. Cases will be selected from a database that combines data from EVA study (Spain) and Guimarães/Vizela study (Portugal). Measurements: cf-PWV will be measured using the SphygmoCor system, CAVI, ba-PWV and Ankle-Brachial Index will be determined using VaSera device. Gut microbiome composition in faecal samples will be determined by 16S ribosomal RNA sequencing. Lifestyle will be assessed by food frequency questionnaire, adherence to the Mediterranean diet and IPAQ (International Physical Activity Questionnaire). Body composition will be evaluated by bioimpedance. [Ethics and dissemination]: The study has been approved by ‘Committee of ethics of research with medicines of the health area of Salamanca’ on 14 December 2018 (cod. 2018-11-136) and the ’Ethics committee for health of Guimaraes’ (Portugal) on 15 October 2019 (ref: 67/2019).All study participants will sign an informed consent form agreeing to participate in the study, in compliance with the Declaration of Helsinki and the WHO standards for observational studies. The results of this study will allow a better description of gut microbiota in patients with arterial stiffness., The project has been funded by the Carlos III Health Institute (Spain) through the Network of preventive activities and health promotion (redIAPP, RD16/0007), co-financed with European funds for regional development (FEDER) and the Autonomous Government of Castilla y León (GRS 1820/B/18; GRS 1944/B/19 and intensification programme).

Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse., This work was partially supported by the Instituto de Salud Carlos III-Cofinanciación con fondos FEDER (PI080568, PS0901897 and PI13/00111), the Gerencia Regional de Salud, Junta de Castilla y León (GRS202/A08 and GRS 702/A/11), the Spanish Myeloma Network Program (RD06/0020/0006) and the Asociación Española Contra el Cáncer (AECC, GCB120981SAN).

Despite significant advances in molecular genetic approaches, fluorescence in situ hybridization (FISH) remains the gold standard for the diagnostic evaluation of genomic aberrations in patients with chronic lymphocytic leukemia (CLL). Efforts to improve the diagnostic utility of molecular cytogenetic testing have led to the expansion of the traditional 4-probe CLL FISH panel. Not only do these efforts increase the cost of testing, they remain hindered by the inherent limitations of FISH studies - namely the inability to evaluate genomic changes outside of the targeted loci. While array-based profiling and next generation sequencing (NGS) have critically expanded our understanding of the molecular pathogenesis of CLL, these methodologies are not routinely used by diagnostic laboratories to evaluate copy number changes or the mutational profile of this disease. Mitogenic stimulation of CLL specimens with CpG-oligonucleotide (CpG-ODN) has been identified as a reliable and reproducible means of obtaining a karyotype, facilitating a low-resolution genome-wide analysis. Across a cohort of 1255 CpG-ODN-stimulated CLL specimens, we describe the clinical utility associated with the combinatorial use of FISH and karyotyping. Our testing algorithm achieves a higher diagnostic yield (∼10%) through the detection of complex karyotypes, well-characterized chromosomal aberrations not covered by the traditional CLL FISH panel and through the detection of concurrent secondary malignancies. Moreover, the single cell nature of this approach permits the evaluation of emerging new clinical concepts including clonal dynamics and clonal evolution. This approach can be broadly applied by diagnostic laboratories to improve the utility of traditional and molecular cytogenetic studies of CLL. Am. J. Hematol. 91:978-983, 2016. © 2016 Wiley Periodicals, Inc.

The current consensus on the diagnosis, prognosis, and treatment of essential thrombocythemia (ET) is based on experts' recommendations. However, several aspects of the diagnosis of, prognosis of, and therapy for ET are still controversial. The Delphi method was employed with an expert panel of members of the Spanish Group of Ph-negative Myeloproliferative Neoplasms in order to identify the degree of agreement on the diagnosis, prognosis, and treatment of ET. Nine leading experts selected a total of 41 clinical hematologists with well-known expertise in ET. An electronic questionnaire was used to collect the questions rated in a four-step scale. The questions were grouped into four blocks: diagnosis, risk stratification, goals of therapy, and treatment strategy. After the first round consisting of 80 questions, a second round including 14 additional questions focused on the recommendations advocated by experts of the European LeukemiaNet in 2011 was analyzed. The median and mean values for the first and second rounds were calculated. A summary of the conclusions considered as the most representative of each block of questions is presented. The Delphi method is a powerful instrument to address the current approaches and controversies surrounding ET.

B-cell chronic lymphocytic leukaemia (B-CLL) is the most common haematological malignancy in Western countries. Trisomy 12 (12%), and deletions of 13q14 (50%), 11q23 (18%) and 17p13 (7%) are the most frequent chromosomal aberrations (Palamarchuk et al, 2010). 13q14 losses, associated with better prognosis, are primary changes resulting in loss of MIR15A/MIR16-1 and DLEU7 (Palamarchuk et al, 2010). Notably, the 10% of these deletions occur together with unbalanced translocations with multiple partners (Puiggros et al, 2014). Two cases with t(8;13)(q14;q24) translocations have been described, although not characterized at molecular level (Gardiner et al, 1997; Put et al, 2012). 8q24 rearrangements are generally rare in CLL and their prognostic significance is unclear., This work was supported by the AIRC (Associazione Italiana per la Ricerca sul Cancro).

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.

This is an open-access paper.-- et al., A multistep model has been proposed of disease progression starting in monoclonal gammopathy of undetermined significance continuing through multiple myeloma, sometimes with an intermediate entity called smoldering myeloma, and ending in extramedullary disease. To gain further insights into the role of the transcriptome deregulation in the transition from a normal plasma cell to a clonal plasma cell, and from an indolent clonal plasma cell to a malignant plasma cell, we performed gene expression profiling in 20 patients with monoclonal gammopathy of undetermined significance, 33 with high-risk smoldering myeloma and 41 with multiple myeloma. The analysis showed that 126 genes were differentially expressed in monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma as compared to normal plasma cell. Interestingly, 17 and 9 out of the 126 significant differentially expressed genes were small nucleolar RNA molecules and zinc finger proteins. Several proapoptotic genes (AKT1 and AKT2) were down-regulated and antiapoptotic genes (APAF1 and BCL2L1) were up-regulated in multiple myeloma, both symptomatic and asymptomatic, compared to monoclonal gammopathy of undetermined significance. When we looked for those genes progressively modulated through the evolving stages of monoclonal gammopathies, eight snoRNA showed a progressive increase while APAF1, VCAN and MEGF9 exhibited a progressive downregulation. In conclusion, our data show that although monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma are not clearly distinguishable groups according to their gene expression profiling, several signaling pathways and genes were significantly deregulated at different steps of the transformation process., This study was partially supported by Spanish FIS (PI080568, PS09/01450 and PS0901897), “Gerencia Regional de Salud, Junta de Castilla y León” (GRS 702/A/11) grant, and the Spanish Myeloma Network Program (RD06/0020/0006, RD12/0036/0058 and RD12/0036/0046).

Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet, and even within some cytogenetically well-defined subclasses there is considerable clinical heterogeneity. Recent advances in genomics technologies such as gene expression profiling (GEP) provide powerful tools to further characterize myeloid malignancies at the molecular level, with the goal to refine the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML with regard to diagnosis, prediction of clinical outcome, discovery of novel subclasses and identification of novel therapeutic targets and novel drugs. As many challenges remain ahead, we discuss the pitfalls of this technology and its potential including future integrative studies with other genomics technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients. © 2011 Macmillan Publishers Limited All rights reserved.

Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL) is still a challenge. Aims: To characterize the presence of additional DNA copy number alterations (CNAs) in children and adults with ALL by whole-genome oligonucleotide array (aCGH) analysis, and to identify their associations with clinical features and outcome. Array-CGH was carried out in 265 newly diagnosed ALLs (142 children and 123 adults). The NimbleGen CGH 12x135K array (Roche) was used to analyze genetic gains and losses. CNAs were analyzed with GISTIC and aCGHweb software. Clinical and biological variables were analyzed. Three of the patients showed chromothripsis (cth6, cth14q and cth15q). CNAs were associated with age, phenotype, genetic subtype and overall survival (OS). In the whole cohort of children, the losses on 14q32.33 (p = 0.019) and 15q13.2 (p = 0.04) were related to shorter OS. In the group of children without good- or poor-risk cytogenetics, the gain on 1p36.11 was a prognostic marker independently associated with shorter OS. In adults, the gains on 19q13.2 (p = 0.001) and Xp21.1 (p = 0.029), and the loss of 17p (p = 0.014) were independent markers of poor prognosis with respect to OS. In summary, CNAs are frequent in ALL and are associated with clinical parameters and survival. Genome-wide DNA copy number analysis allows the identification of genetic markers that predict clinical outcome, suggesting that detection of these genetic lesions will be useful in the management of patients newly diagnosed with ALL., The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n°306242-NGS-PTL, the Fundación Castellano Leonesa de Hematología y Hemoterapia (FUCALHH 2013), the Consejería de Educación, Junta de Castilla y León (HUS272U13), Proyectos de Investigación del SACYL, Spain: GRS 994/A/14, BIO/SA10/14, BIO/SA31/13. The work was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS 09/01543, PI12/00281, COST Action EuGESMA (BM0801), Fundación Española de Hematología y Hemoterapia (FEHH), and by grants (RD12/0036/0069) from the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) “Una manera de hacer Europa” (Innocampus; CEI-2010-1-0010). MFC was supported by study commission (no. 223-2011) granted by the Universidad Pedagógica y Tecnológica de Colombia, Colombia.

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

Gene expression profiling signatures may be used to classify the subtypes of Myelodysplastic syndrome (MDS) patients. However, there are few reports on the global methylation status in MDS. The integration of genome-wide epigenetic regulatory marks with gene expression levels would provide additional information regarding the biological differences between MDS and healthy controls. Gene expression and methylation status were measured using high-density microarrays. A total of 552 differentially methylated CpG loci were identified as being present in low-risk MDS; hypermethylated genes were more frequent than hypomethylated genes. In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and the control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hypermethylated genes were underexpressed in low-risk MDS cases. Gene network analysis revealed molecular mechanisms associated with the low-risk MDS group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and ETS1 were identified as silenced genes. In addition, the immune response and micro RNA biogenesis were affected by the hypermethylation and underexpression of IL27RA and DICER1. Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could have a central role in these diseases., This study was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS 09/01543, Proyectos de Investigación del SACYL 355/A/09, COST Action “EuGESMA” (BM0801), Northern Ireland Leukaemia Research Fund (NILRF), Northern Ireland Assembly Department of Education and Learning, Obra Social Banca Cívica (Caja Burgos), 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, the Cancer Research Foundation of Salamanca University and the Redes de Investigación RTIIC (FIS). MdR is fully upported by an >Ayuda Predoctoral” by the Fundación Española de Hematología y Hemoterapia.

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.

Genetic events mediating transformation from premalignant monoclonal gammopathies (MG) to multiple myeloma (MM) are unknown. To obtain a comprehensive genomic profile of MG from the early to late stages, we performed high-resolution analysis of purified plasma cells from 20 MGUS, 20 smoldering MM (SMM) and 34 MM by high-density 6.0 SNP array. A progressive increase in the incidence of copy number abnormalities (CNA) from MGUS to SMM and to MM (median 5, 7.5 and 12 per case, respectively) was observed (P=0.006). Gains on 1q, 3p, 6p, 9p, 11q, 19p, 19q and 21q along with 1p, 16q and 22q deletions were significantly less frequent in MGUS than in MM. Although 11q and 21q gains together with 16q and 22q deletions were apparently exclusive of MM status, we observed that these abnormalities were also present in minor subclones in MGUS. Overall, a total of 65 copy number-neutral LOH (CNN-LOH) were detected. Their frequency was higher in active MM than in the asymptomatic entities (P=0.047). A strong association between genetic lesions and fragile sites was also detected. In summary, our study shows an increasing genomic complexity from MGUS to MM and identifies new chromosomal regions involved in CNA and CNN-LOH.

Recently, an acute restriction of imiglucerase has occurred as a result of viral contamination and manufacturing problems. A position statement from the European Working Group for Gaucher Disease and European Gaucher Alliance established a set of key recommendations for identifying and monitoring at-risk patients. In Spain, a profile of the shortage situation was obtained through follow-up of patients with Gaucher disease (GD) and compliance with the therapy recommendations. Here we describe a group of patients, with modified doses of imiglucerase, during the shortage. Fifty adult GD1 patients (25 males/25 females), previously on ERT, were analysed before and after the 6-month shortage. The mean age was 45.3 ± 15.3 years (range: 18-84). The mean Severity Score Index at diagnosis was 8.7 ± 3.8 (range: 3-19); 20% of patients were splenectomized; and 78% had bone disease. During the shortage, 23 patients (46%) discontinued therapy; as complications in this group only one patient suffered a bone crisis and another anaemia (Hb

Background: Although there has been remarkable progress, there is a need to improve the molecular dissection of subtypes, identifying genetic alterations that predict the risk of treatment failure and developing novel and targeted therapies. B-ALL patients (pts) that do not have the most recurrent adult rearrangements (BCR-ABL1 t(9;22); TCF3-PBX1 t(1;19); MLL-AF4 t(4;11)) are collectively referred to as “triple negative” (Ph-/-/-) ALL. Aims: Biologic characterization of Ph-/-/- ALL considering CRLF2 overexpression event (that represents near to 57% of B-ALL; Roberts KG, J Clin Oncol 2016), in in order to define and assess biomarkers in this subgroup to test new drugs. Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) was performed on 51 Ph-/-/- ALL, 25 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized and analyzed with the Expression Console and the Transcriptome Analysis Console (TAC) Software (Affymetrix). Successively we cluster triple-negative GEP data with our validated pipeline, based in a top ten gene list. Results: Comparing GEP of Ph-/-/- and Ph+ to donors we found some shared top upreg genes to focus on (e.g., EBF1, CD19, BLNK, PDLIM1, PXDN, NAV1, CTGF, LEF1, CD200, CRLF2). In triple-negative ALL GEP top upreg gene analysis we identify a well-defined 2-clusters-subdivision (Gr1 and Gr2). Furthermore, a third group , in the Gr1, can be identified by the algorithm without ambiguous assignments. The Gr2 is characterized by CTGF, CRLF2 and CD200 overexpression and it represents 11.3% of all B-ALL. Two groups t-test has been performed between Ph+ and the isolated subgroups of Ph- to determine the similarity of these two groups to Ph+. The Gr2 GEP is similar to Ph+ one. Conclusions: We identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B-based on 10 genes. Gr2 represents 11.3% of all B-ALL and it is characterized by high expression of three main genes: CRLF2, CTGF and CD200. Gr1 represents 46% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identifies new potential therapeutic targets to test as a single agent or in combination. ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Citation Format: Anna Ferrari, Silvia Vitali, Valentina Robustelli, Andrea Ghelli Luserna Di Rorà, Simona Righi, Cristina Papayannidis, Giovanni Marconi, Enrica Imbrogno, Alessandra Santoro, J M. Hernández-Rivas, Carmen Baldazzi, Maria Chiara Abbenante, Stefania Paolini, Nicoletta Testoni, Gastone Castellani, Elena Sabattini, Michele Cavo, Daniel Remondini, Giovanni Martinelli. Gene expression profiling identifies new adult "triple-negative" acute lymphoblastic leukemia (ALL) subgroups [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2951.

Background Acute promyelocytic leukemia is a subtype of acute myeloid leukemia characterized by the t(15;17). The incidence and prognostic significance of additional chromosomal abnormalities in acute promyelocytic leukemia is still a controversial matter. Design and Methods Based on cytogenetic data available for 495 patients with acute promyelocytic leukemia enrolled in two consecutive PETHEMA trials (LPA96 and LPA99), we analyzed the incidence, characteristics, and outcome of patients with acute promyelocytic leukemia with and without additional chromosomal abnormalities who had been treated with all-trans retinoic acid plus anthracycline monochemotherapy for induction and consolidation. Results Additional chromosomal abnormalities were observed in 140 patients (28%). Trisomy 8 was the most frequent abnormality (36%), followed by abn(7q) (5%). Patients with additional chromosomal abnormalities more frequently had coagulopathy (P=0.03), lower platelet counts (P=0.02), and higher relapse-risk scores (P=0.02) than their counterparts without additional abnormalities. No significant association with FLT3/ITD or other clinicopathological characteristics was demonstrated. Patients with and without additional chromosomal abnormalities had similar complete remission rates (90% and 91%, respectively). Univariate analysis showed that additional chromosomal abnormalities were associated with a lower relapse-free survival in the LPA99 trial (P=0.04), but not in the LPA96 trial. However, neither additional chromosomal abnormalities overall nor any specific abnormality was identified as an independent risk factor for relapse in multivariate analysis. Conclusions The lack of independent prognostic value of additional chromosomal abnormalities in acute promyelocytic leukemia does not support the use of alternative therapeutic strategies when such abnormalities are found.

The presence of cytogenetic aberrations on mesenchymal stem cells (MSC) from myelodysplastic syndrome (MDS) patients is controversial. The aim of the study is to characterize bone marrow (BM) derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridization (FISH) and genetic changes by array-based comparative genomic hybridization (array-CGH). In all 36 cases of untreated MDS were studied. MDS-MSC achieved confluence at a significantly slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic. These results were confirmed by FISH. To confirm that genomic changes were also present in freshly obtained MSCs they were enriched by sorting BM cells with the following phenotype: CD45(-)/CD73(++)/CD34(-)/CD271(++). They also showed genomic changes that were confirmed by FISH. To analyze the relationship of these aberrations with clinical-biological data an unsupervised hierarchical cluster analysis was performed, two clusters were identified: the first one included the 5q- syndrome patients, whereas the other incorporated other MDS. Our results show, for the first time that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to a particular MDS subtype, the 5q- syndrome.

Part of this study has been reported as an oral presentation at the EHA Meeting in Vienna 2015., The introduction of Rituximab has improved the outcome and survival rates of Burkitt lymphoma (BL). However, early relapse and refractoriness are current limitations of BL treatment and new biological factors affecting the outcome of these patients have not been explored. This study aimed to identify the presence of genomic changes that could predict the response to new therapies in BL. Forty adolescent and adult BL patients treated with the Dose-Intensive Chemotherapy Including Rituximab (Burkimab) protocol (Spanish Programme for the Study and Treatment of Haematological Malignancies; PETHEMA) were analysed using array-based comparative genomic hybridization (CGH). In addition, the presence of TP53, TCF3 (E2A), ID3 and GNA13 mutations was assessed by next-generation sequencing (NGS). Ninety-seven per cent of the patients harboured genomic imbalances. Losses on 11q, 13q, 15q or 17p were associated with a poor response to Burkimab therapy (P = 0·038), shorter progression-free survival (PFS; P = 0·007) and overall survival (OS; P = 0·009). The integrative analysis of array-CGH and NGS showed that 26·3% (5/19) and 36·8% (7/19) of patients carried alterations in the TP53 and TCF3 genes, respectively. TP53 alterations were associated with shorter PFS (P = 0·011) while TCF3 alterations were associated with shorter OS (P = 0·032). Genetic studies could be used for risk stratification of BL patients treated with the Burkimab protocol., This work was supported in part by: grant no 306242-NGS-PTL from European Union’s Seventh Framework Programme (FP7/2007-2013), Fundacion-Castellano-Leonesa de Hematologia-y-Hemoterapia (FUCALHH 2013), Consejeria de Educacion, Junta de Castilla y León (HUS272U13), SACYL research projects: GRS1172/A/15, GRS 994/A/14, BIO/SA10/14, BIO/SA31/13. Fondo de Investigaciones Sanitarias FISPI09/01543, FIS-PI12/00281, PI14/01971, COST-ActionEuGESMA (BM0801), Fundacion Española de Hematologia y Hemoterapia (FEHH), and by grants RD12/0036/0069, RD12/ 0036/0029 and RD12/0036/0044 from the Red Tematica de Investigacion Cooperativa en Cancer (RTICC), Instituto de Salud Carlos III (ISCIII), the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’ (Innocampus). NGS was performed as part of the IRON-II collaborative network of haematological laboratories. MFC was supported by study commission no 223-2011 granted by the UPTC, Colombia. MHS was supported by Junta de Castilla y Leon from the ERDF.

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al., The presence of SF3B1 gene mutations is a hallmark of refractory anemia with ring sideroblasts (RARS). However, the mechanisms responsible for iron accumulation that characterize the Myelodysplastic Syndrome with ring sideroblasts (MDS-RS) are not completely understood. In order to gain insight in the molecular basis of MDS-RS, an integrative study of the expression and mutational status of genes related to iron and mitochondrial metabolism was carried out. A total of 231 low-risk MDS patients and 81 controls were studied. Gene expression analysis revealed that iron metabolism and mitochondrial function had the highest number of genes deregulated in RARS patients compared to controls and the refractory cytopenias with unilineage dysplasia (RCUD). Thus mitochondrial transporters SLC25 (SLC25A37 and SLC25A38) and ALAD genes were over-expressed in RARS. Moreover, significant differences were observed between patients with SF3B1 mutations and patients without the mutations. The deregulation of genes involved in iron and mitochondrial metabolism provides new insights in our knowledge of MDS-RS. New variants that could be involved in the pathogenesis of these diseases have been identified., This work was partially supported by grants from: the Spanish Fondo de Investigaciones Sanitarias (PI14/1971, PI12/00281); Proyectos de Investigación del SACYL (BIO/SA52/14, BIO/SA10/14, BIO/SA31/13, GRS 994/A/14); Consejería Educación, Junta Castilla y León (HUS272U13); COST Action "EuGESMA" (BM0801); 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) (RD12/0036/0069, RD12/0036/0029, RD12/0036/0044); European Union's Seventh Framework Programme (FP7/2007-2013 nº 306242- NGS-PTL). MDR is fully supported by a "Grant from Fundación Española de Hematología y Hemoterapia". MA was fully supported by a "Grant from the Spanish Consejo Superior de Investigaciones Científicas, Junta para la Ampliación de Estudios (JAE Predoctoral) [09-02402]". MH is fully suported by an "Ayuda predoctoral de la Junta de Castilla y León" from "European Regional Development Fund". DMZ was funded by the Spanish Ministry of Science and Innovation (PI082025) and Junta de Castilla y León (CSI224A11-2, SAN/103/2011).

Acute lymphoblastic leukemia (ALL) following solid organ or hematologic malignancy (secondary ALL, s-ALL) is not well characterized. We analyzed the characteristics and outcome of patients with s-ALL and compared them with those of patients with de novo- ALL. Of 448 patients, 24 (5%) had previous neoplasia. Sixteen patients had received previous cytotoxic therapy (therapy-associated ALL, t-ALL), and eight had not (antecedent-malignancy ALL, am-ALL). Except for more advanced age in patients with s-ALL, no statistically significant differences were observed in WBC count, CNS involvement, immunophenotype or cytogenetics between the groups, nor in complete remission (t-ALL: 94%; am-ALL: 75%; de novo-ALL: 85%), 3-year remission duration (58%; 50%; 72%), overall survival (71%; 38%; 60%) or event-free survival (53%, 38%; 53%). Our study did not show poor clinical or cytogenetic features or inferior outcome in ALL patients with antecedent neoplastic disease, irrespective of the type of treatment received for the neoplasia.