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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). ; Peer Reviewed

The research leading to these results has mainly received funding from the European Union Seventh Framework Programme [FP7/2007–2013] under Grant Agreement no 306242-NGS-PTL. In addition, this work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI15/01471, PI18/01500, 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 994/A/14, BIO/SA10/14, BIO/SA31/13, GRS 1172/A/15,"Fundación Memoria Don Samuel Solórzano Barruso", by grants (RD12/0036/0069) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) CB16/12/00233 and USAL "Programa XIII". M. Hernández-Sánchez is supported by FEHH-Janssen ("Sociedad Española de Hematología y Hemoterapia"). M Quijada-Álamo is supported by an "Ayuda Predoctoral de la Junta de Castilla y León" (JCYL-EDU/529/2017). We are grateful to I. Rodríguez, S. González, T.Prieto, M. Á. Ramos, A. Martín, A. Díaz, A. Simón, M.del Pozo, V. Gutiérrez and S. Pujante from Centro de Investigación del Cáncer, Salamanca, for their technical assistance. D. Tamborero is supported by project SAF2015–74072-JUN, which is funded by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Dearrollo Regional (FEDER). This work was supported by Seventh Framework Programme (NGS-PTL/2012–2015/no.306242) and Ministry of Education, Youth and Sports (2013–2015, no. 7E13008); by the Ministry of Education, Youth and Sports of the Czech Republic under the CEITEC 2020 project (LQ1601); by the Ministry of Health, Czech Republic - conceptual development of research organization (FNBr, 65269705); by the Specific University Research (nr. MUNI/A/0968/2017) provided by MEYS; and by the project CZ.02.1.01/0.0/0.0/16_013/0001634 National Center for Medical Genomic - modernization of infrastructure and research of genetic variation in the population, funded by OP RDE. We acknowledge the CF Genomics CEITEC MU supported by the NCMG research infrastructure (LM2015091 funded by MEYS CR) for their support with obtaining the scientific data presented in this paper. We acknowledge S. Takacova from CEITEC MU for her help with the sample selection and processing.

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 ; Peer Reviewed

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.

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.

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. ; Peer Reviewed

[Background]: In B-cell precursor acute lymphoblastic leukaemia (B-ALL), the identification of additional genetic alterations associated with poor prognosis is still of importance. We determined the frequency and prognostic impact of somatic mutations in children and adult cases with B-ALL treated with Spanish PETHEMA and SEHOP protocols. [Methods]: Mutational status of hotspot regions of TP53, JAK2, PAX5, LEF1, CRLF2 and IL7R genes was determined by next-generation deep sequencing in 340 B-ALL patients (211 children and 129 adults). The associations between mutation status and clinicopathological features at the time of diagnosis, treatment outcome and survival were assessed. Univariate and multivariate survival analyses were performed to identify independent prognostic factors associated with overall survival (OS), event-free survival (EFS) and relapse rate (RR). [Results]: A mutation rate of 12.4% was identified. The frequency of adult mutations was higher (20.2% vs 7.6%, P=0.001). TP53 was the most frequently mutated gene (4.1%), followed by JAK2 (3.8%), CRLF2 (2.9%), PAX5 (2.4%), LEF1 (0.6%) and IL7R (0.3%). All mutations were observed in B-ALL without ETV6-RUNX1 (P=0.047) or BCR-ABL1 fusions (P<0.0001). In children, TP53mut was associated with lower OS (5-year OS: 50% vs 86%, P=0.002) and EFS rates (5-year EFS: 50% vs 78.3%, P=0.009) and higher RR (5-year RR: 33.3% vs 18.6% P=0.037), and was independently associated with higher RR (hazard ratio (HR)=4.5; P=0.04). In adults, TP53mut was associated with a lower OS (5-year OS: 0% vs 43.3%, P=0.019) and a higher RR (5-year RR: 100% vs 61.4%, P=0.029), whereas JAK2mut was associated with a lower EFS (5-year EFS: 0% vs 30.6%, P=0.035) and a higher RR (5-year RR: 100% vs 60.4%, P=0.002). TP53mut was an independent risk factor for shorter OS (HR=2.3; P=0.035) and, together with JAK2mut, also were independent markers of poor prognosis for RR (TP53mut: HR=5.9; P=0.027 and JAK2mut: HR=5.6; P=0.036). [Conclusions]: TP53mut and JAK2mut are potential biomarkers associated with poor prognosis in B-ALL patients. ; This work was supported in part by a grant from the European Union's Seventh Framework Programme (FP7/2007–2013) under Grant Agreement 306242-NGS-PTL, the Consejeria de Educacion, Junta de Castilla y León (HUS272U13, SA085U16 to JMHR and JCYL-EDU/346/2013 PhD scholarship to MHS), Fundación Castellano Leonesa de Hematología y Hemoterapia (FUCALHH 2013) to JMHR, Proyectos de Investigacion del SACYL, Spain (BIO/SA31/13 and BIO/SA10/14) to RB; The Instituto de Salud Carlos III from Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ISCIII-FEDER) ''Una manera de hacer Europa'' Spanish Cancer Network (RD12/0036/0069 and RD12/0036/0061 to JM) and FIS grants PI15/01471 to JMHR and PI15/00032 to EFR. Universidad Pedagogica y Tecnologica de Colombia—Vicerrectoría de Investigacion y Extension (Grupo de Investigacion en Ciencias Biomedicas UPTC—GICBUPTC, Escuela de Ciencias Biologicas) to MFC. ; Peer Reviewed