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This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS09/01543, 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 nº 306242-NGS-PTL. ; Hernandez, JA; Hernandez-Sanchez, M; Rodriguez-Vicente, AE; Grossmann, V; Collado, R; Heras, C; Puiggros, A; Martin, AA; Puig, N; Benito, R; Robledo, C; Delgado, J; Gonzalez, T; Queizan, JA; Galende, J; de la Fuente, I; Martin-Nunez, G; Alonso, JM; Abrisqueta, P; Luno, E; Marugan, I; Gonzalez-Gason, I; Bosch, F; Kohlmann, A; Gonzalez, M; Espinet, B; Hernandez-Rivas, JM.

The rapid progress in next-generation sequencing technologies has significantly contributed to our knowledge of the genetic events associated with the development, progression and treatment resistance of chronic lymphocytic leukemia patients. Together with the discovery of new driver mutations, next-generation sequencing has revealed an immense degree of both intra- and inter-tumor heterogeneity and enabled us to describe marked clonal evolution. Advances in immunogenetics may be implemented to detect minimal residual disease more sensitively and to track clonal B cell populations, their dynamics and molecular characteristics. The interpretation of these aspects is indispensable to thoroughly examine the genetic background of chronic lymphocytic leukemia. We review and discuss the recent results provided by the different next-generation sequencing techniques used in studying the chronic lymphocytic leukemia genome, as well as future perspectives in the methodologies and applications. ; This work received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under Grant Agreement n°306242-NGS-PTL and MSMT CR (2013–2015/no.7E13008). It was also supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS 09/01543, PI12/00281 and PI15/01471, Proyectos de Investigación del SACYL 355/A/09, 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 the 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). AERV is supported by a grant from the Fundación Ramón Areces. MHS is fully supported by an Ayuda Predoctoral de la Junta de Castilla y León from the Fondo Social Europeo (JCYL-EDU/346/2013 PhD scholarship). SP, VB and JM were supported by AZV grants from the Ministry of Health of the Czech Republic No. 15-31834A, 15-30015A and 16-29447A, MH CR project 65269705 and MSMT CR project CEITEC2020 LQ1601 under the National Sustainability Programme II. ; Peer Reviewed

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 <40% del(11q) (11q-L) (P<0.0001). In the multivariate analysis, only the presence of 11q-L, mutated IGHV status, early Binet stage and absence of extended lymphadenopathy were associated with longer TFT. Patients with 11q-H had an OS of 90 months, while in the 11q-L group the OS was not reached (P = 0.008). The absence of splenomegaly (P = 0.02), low LDH (P = 0.018) or β2M (P = 0.006), and the presence of 11q-L (P = 0.003) were associated with a longer OS. In addition, to detect the presence of mutations in the ATM, TP53, NOTCH1, SF3B1, MYD88, FBXW7, XPO1 and BIRC3 genes, a select cohort of CLL patients with losses in 11q was sequenced by next-generation sequencing of amplicons. Eighty %of CLLs with 11qshowed mutations and fewer patients with low frequencies of 11q- had mutations among genes examined (50% vs 94.1%, P = 0.023). In summary, CLL patients with <40% of 11qhad a long TFT and OS that could be associated with the presence of fewer mutated genes. ; 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. ; Peer Reviewed

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al. ; [Background]: MicroRNAs are known to inhibit gene expression by binding to the 3'UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients. [Methods]: By applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3'UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status, HSP90B1 expression and clinico-biological data were assessed. [Results]: HSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome. [Conclusions]: HSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target. ; This work was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS 09/01543 and PI12/00281, Proyectos de Investigación del SACYL 355/A/09, COST Action EuGESMA (BM0801), Fundación Manuel Solórzano, Obra Social Banca Cívica (Caja Burgos), 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). 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. MHS is fully supported by an Ayuda predoctoral de la Junta de Castilla y Leon by the Fondo Social Europeo. ME Sarasquete is supported by Contrato Miguel Servet (CP13/00080). ; Peer Reviewed

[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). ; Peer Reviewed