Genomic determinants of chronic lymphocytic leukemia progression: from individual drivers to a heterogeneous genetic makeup

[eng] Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries. Although the disease might follow an indolent course, it rapidly progresses in a fraction of cases, become resistant to treatment, and eventually transform to a more aggressive B-cell lymphoma, known as Richter syndrome. The mechanisms underlying these distinct clinical courses are not fully understood. In this Thesis, we aimed to elucidate the genomic determinants of CLL progression, to provide tools to characterize these tumors from next-generation sequencing data, and to extract key biological findings that could improve the management of the patients. In the first chapter (Studies 1 and 2), we characterized a noncoding mutation effecting the small nuclear RNA U1, a component of the spliceosome involved in the 5’ splice site recognition via base paring. Mutations in this gene altered the splicing and expression of multiple genes, were found in CLL tumors lacking clinically relevant genomic alterations, and were independently associated with patients’ outcome. In the next chapter (Studies 3, 4, and 5), we aimed to deeper into the subclonal architecture of CLL. We identified mutations present in small subpopulations associated with disease progression, recognized common evolutionary trajectories, and showed that the integration of the whole tumor architecture into prognostic models could improve the stratification of the patients. In the third chapter (Study 6), we analyzed the whole genome of CLL patients undergoing Richter syndrome and observed that this transformation was accompanied by an increased mutational and genomic complexity. We identified a unifying mutational process that could orchestrate this genomic chaos. In the fourth chapter (Studies 7 and 8), we developed a bioinformatic algorithm aimed to reconstruct the immunoglobulin gene rearrangements in lymphoid neoplasms from whole-genome sequencing, which might facilitate the use of this methodology in the future clinical practice. By applying this algorithm, we studied a recurrent mutation in the IGLV3-21 gene associated with an aggressive disease with a strong influence on the current and future risk stratification of CLL patients. Altogether, this Thesis has contributed to understand the genomic determinants of CLL progression through the analysis of its dynamic and heterogeneous genetic makeup.[cat] La leucèmia limfocítica crònica (LLC) és la forma més freqüent de leucèmia en adults als països occidentals. Malgrat que la malaltia pot seguir un curs clínic indolent, aquesta pot progressar ràpidament en una fracció dels casos, tornant-se resistent al tractament i, fins i tot, transformar a un limfoma de cèl·lules B més agressiu, fenomen conegut com síndrome de Richter. Els mecanismes que condicionen aquests diferents cursos clínics no són del tot coneguts. Els objectius d’aquesta tesi doctoral van ser elucidar els determinants genòmics de la progressió de la LLC, proporcionar eines per caracteritzar aquests tumors a partir de dades de seqüenciació de nova generació i extreure aspectes biològics clau d’aquests tumors que permetin millorar el maneig dels pacients. Al primer capítol (estudis 1 i 2), vam caracteritzar una mutació no codificant que afectava l’ARN nuclear petit U1, un component de l’espliceosoma implicat en el reconeixement del lloc 5’ d’empalmament (o splicing) per complementarietat de seqüència. Les mutacions d’aquest gen van alterar l’splicing i l’expressió de múltiples gens, es van trobar en tumors de LLC mancats d’alteracions genòmiques clínicament rellevants i es van associar de forma independentment amb el pronòstic dels pacients. En el següent capítol (estudis 3, 4 i 5), vam intentar aprofundir en l’arquitectura subclonal de la LLC. Es van identificar mutacions presents en petites subpoblacions associades a la progressió de la malaltia, es van reconèixer trajectòries evolutives comunes i es va demostrar que la integració de l'arquitectura tumoral en models pronòstics podria millorar l'estratificació dels pacients. En el tercer capítol (estudi 6), vam analitzar el genoma complet de pacients amb LLC que desenvolupaven la síndrome de Richter i vam observar que aquesta transformació anava acompanyava d’un augment de la complexitat mutacional i genòmica. Vam identificar un procés mutacional unificador que podria orquestrar aquest caos genòmic. Al quart capítol (estudis 7 i 8), vam desenvolupar un algoritme bioinformàtic dirigit a reconstruir els reordenaments genètics de les immunoglobulines en les neoplàsies limfoides a partir de la seqüenciació del genoma complet, cosa que podria facilitar l’ús d’aquesta metodologia en la pràctica clínica. Mitjançant l’aplicació d’aquest algoritme, vam estudiar una mutació recurrent en el gen IGLV3-21 associada a una malaltia agressiva amb una forta influència en la present i futura estratificació dels pacients amb LLC. En conjunt, aquesta tesi ha contribuït a entendre els determinants genòmics de la progressió de la LLC mitjançant l’anàlisi de la seva dinàmica i heterogènia composició genètica

Circular consumption and production of electronic devices: an approach to measuring durability, upgradeability, reusability, obsolescence and premature recycling

What is circular consumption of electronics? Is it measurable? Can we set goals or compare with other consumers? A principle of circular economy is durability, prolonging the useful life of products. If we move the principle to the function of consumption, we would have that: circular consumption is to make the most of time resources by ensuring that in the end they are recycled, but never before time or prematurely, but only when they can no longer be used or reused by anyone else. In this article we propose a set of measures, metrics and progress indicators to measure the use of resources that consumers make in their use phase. With these metrics we can identify which consumers are the most circular; those who are able to use the same electronic devices for the longest time, either internally, or by collaborating with external agents so that these devices are reused and recycled properly. We have been able to validate usage performance metrics and premature recycling in the analysis of more than 3,000 desktop and laptop type electronic devices. These devices have been discarded by hundreds of organisations in 2018 and 2019. Finally, we propose the metrics of durability and obsolescence for models and brands of devices, which although it does not allow us to know the reasons for a low durability; absence or high cost of spare parts, difficulty of repair, etc., it does allow us to elaborate a ranking so that consumers can reward with their consumption choice, manufacturers who make products that reach high thresholds of durability.This work is partially supported by the Venture Builder program of the Ledger project of the European Commission #825268, the Agència de Residus de Catalunya, the Spanish government TIN2016-77836-C2-2R and the Catalan government AGAUR SGR 990. We thank the eReuse team, Xavier Bustamante, the developers of the smartmontools software, and the community of circular economy activists in eReuse, the Pangea circuit, and the open-source community in general, as metaphorically, we stand on the shoulders of giants that allow us to see beyond the horizon.Peer ReviewedPostprint (published version

Unraveling the genetics of transformed splenic marginal zone lymphoma

Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Large B-Cell, Diffuse; MutationLeucemia linfocítica crónica de células B; Linfoma de células B grandes difuso; MutaciónLeucèmia limfocítica crònica de cèl·lules B; Limfoma de cèl·lules B grans difús; MutacióThe genetic mechanisms associated with splenic marginal zone lymphoma (SMZL) transformation are not well defined. We studied 41 patients with SMZL that eventually underwent large B-cell lymphoma transformation. Tumor material was obtained either only at diagnosis (9 patients), at diagnosis and transformation (18 patients), and only at transformation (14 patients). Samples were categorized in 2 groups: (1) at diagnosis (SMZL, n = 27 samples), and (2) at transformation (SMZL-T, n = 32 samples). Using copy number arrays and a next-generation sequencing custom panel, we identified that the main genomic alterations in SMZL-T involved TNFAIP3, KMT2D, TP53, ARID1A, KLF2, 1q gains, and losses of 9p21.3 (CDKN2A/B) and 7q31-q32. Compared with SMZL, SMZL-T had higher genomic complexity, and higher incidence of TNFAIP3 and TP53 alterations, 9p21.3 (CDKN2A/B) losses, and 6p gains. SMZL and SMZL-T clones arose by divergent evolution from a common altered precursor cell that acquired different genetic alterations in virtually all evaluable cases (92%, 12 of 13 cases). Using whole-genome sequencing of diagnostic and transformation samples in 1 patient, we observed that the SMZL-T sample carried more genomic aberrations than the diagnostic sample, identified a translocation t(14;19)(q32;q13) present in both samples, and detected a focal B2M deletion due to chromothripsis acquired at transformation. Survival analysis showed that KLF2 mutations, complex karyotype, and International Prognostic Index score at transformation were predictive of a shorter survival from transformation (P = .001; P = .042; and P = .007; respectively). In summary, SMZL-T are characterized by higher genomic complexity than SMZL, and characteristic genomic alterations that could represent key players in the transformation event.This study was supported by Fundacion Asociación Española Contra el Cancer (AECC)/Centro de Investigación Biomédica en Red de Cancer (CIBERONC): PROYE18020BEA (S.B.), fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III “Cofinanciado por la Union Europea ´ and Fondos FEDER: European Regional Development Fund “Una manera de hacer Europa”: PI17/ 01061 (S.B.), PI19/00887 (A.L.-G. and E.G.), INT20/00050 (A.L.- G.), MaratO TV3-Cancer/201904-30 (S.B.), Generalitat de Catalunya Suport Grups de Recerca AGAUR (2021-SGR-01293 [S.B.] and 2021-SGR-01172 [E.C.]), and Ministerio de Ciencia e Innovacion (PID2021-123054OB-I00 [E.C.]). C.L. is supported by postdoctoral Beatriu de Pinos grant, Secretaria d´Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and by Marie Sklodowska-Curie COFUND program from H2020 (2018-BP-00055). E.C. is an Academia Researcher of the "Institucio Catalana de Recerca i Estudis Avançats" of the Generalitat de Catalunya. This work was mainly developed at the Centre Esther Koplowitz (CEK), Barcelona, Spain

From genetics to therapy: Unraveling the complexities of Richter transformation in chronic lymphocytic leukemia

Chronic lymphocytic leukemia; Clonal evolution; Richter's transformationLeucèmia limfocítica crònica; Evolució clonal; Transformació de RichterLeucemia linfocítica crónica; Evolución clonal; Transformación de RichterRichter transformation (RT) refers to the progression of chronic lymphocytic leukemia, the most prevalent leukemia among adults, into a highly aggressive lymphoproliferative disorder, primarily a diffuse large B-cell lymphoma. This is a severe complication that continues to be a therapeutic challenge and remains an unmet medical need. Over the last five years, significant advances have occurred in uncovering the biological processes leading to the RT, refining criteria for properly diagnose RT from other entities, and exploring new therapeutic options beyond the ineffective chemotherapy. This review summarizes current knowledge in RT, including recent advances in the understanding of the pathogenesis of RT, in the classification of RT, and in the development of novel therapeutic strategies for this grave complication.This work was supported in part by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias [PI17/00943, F.B, PI18/01392, P.A.], and Gilead Fellowships, United States [GLD18/00047, F.B.] and Fundació la Marató de TV3, Spain [201905-30-31 F.B]. F.N. acknowledges research support from the American Association for Cancer Research (2021 AACR-Amgen Fellowship in Clinical/Translational Cancer Research, 21-40-11-NADE), the European Hematology Association (EHA Junior Research Grant 2021, RG-202012-00245), the Lady Tata Memorial Trust, India (International Award for Research in Leukaemia 2021–2022, LADY_TATA_21_3223)

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high–B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.The authors thank the Hematopathology Collection registered at the Biobank of Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and the Biobank HUB-ICO-IDIBELL (PT20/00171) for sample procurement, S. Martín, F. Arenas, the Genomics Core Facility of the IDIBAPS, CNAG Sequencing Unit, Mission Bio, Omniscope and Barcelona Supercomputing Center for the technical support and the computer resources at MareNostrum4 (RES activity, BCV-2018-3-0001). This study was supported by the la Caixa Foundation (CLLEvolution-LCF/PR/HR17/52150017, Health Research 2017 Program HR17-00221, to E.C.), the European Research Council under the European Union’s Horizon 2020 Research and Innovation Program (810287, BCLLatlas, to E.C., J.I.M.-S., H.H. and I.G.), the Instituto de Salud Carlos III and the European Regional Development Fund Una Manera de Hacer Europa (PMP15/00007 to E.C. and RTI2018-094584-B-I00 to D.C.), the American Association for Cancer Research (2021 AACR-Amgen Fellowship in Clinical/Translational Cancer Research, 21-40-11-NADE to F.N.), the European Hematology Association (EHA Junior Research Grant 2021, RG-202012-00245 to F.N.), the Lady Tata Memorial Trust (International Award for Research in Leukaemia 2021-2022, LADY_TATA_21_3223 to F.N.), the Generalitat de Catalunya Suport Grups de Recerca AGAUR (2017-SGR-1142 to E.C., 2017-SGR-736 to J.I.M.-S. and 2017-SGR-1009 to D.C.), the Accelerator award CRUK/AIRC/AECC joint funder partnership (AECC_AA17_SUBERO to J.I.M.-S.), the Fundació La Marató de TV3 (201924-30 to J.I.M.-S.), the Centro de Investigación Biomédica en Red Cáncer (CIBERONC; CB16/12/00225, CB16/12/00334, CB16/12/00236), the Ministerio de Ciencia e Innovación (PID2020-117185RB-I00 to X.S.P.), the Fundación Asociación Española Contra el Cáncer (FUNCAR-PRYGN211258SUÁR to X.S.P.), the Associazione Italiana per la Ricerca sul Cancro Foundation (AIRC 5 × 1,000 no. 21198 to G.G.) and the CERCA Programme/Generalitat de Catalunya. H.P.-A. is a recipient of a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (FPU19/03110). A.D.-N. is supported by the Department of Education of the Basque Government (PRE_2017_1_0100). E.C. is an Academia Researcher of the Institució Catalana de Recerca i Estudis Avançats of the Generalitat de Catalunya. This work was partially developed at the Center Esther Koplowitz (Barcelona, Spain).Peer Reviewed"Article signat per 52 autors/es: Ferran Nadeu, Romina Royo, Ramon Massoni-Badosa, Heribert Playa-Albinyana, Beatriz Garcia-Torre, Martí Duran-Ferrer, Kevin J. Dawson, Marta Kulis, Ander Diaz-Navarro, Neus Villamor, Juan L. Melero, Vicente Chapaprieta, Ana Dueso-Barroso, Julio Delgado, Riccardo Moia, Sara Ruiz-Gil, Domenica Marchese, Ariadna Giró, Núria Verdaguer-Dot, Mónica Romo, Guillem Clot, Maria Rozman, Gerard Frigola, Alfredo Rivas-Delgado, Tycho Baumann, Miguel Alcoceba, Marcos González, Fina Climent, Pau Abrisqueta, Josep Castellví, Francesc Bosch, Marta Aymerich, Anna Enjuanes, Sílvia Ruiz-Gaspà, Armando López-Guillermo, Pedro Jares, Sílvia Beà, Salvador Capella-Gutierrez, Josep Ll. Gelpí, Núria López-Bigas, David Torrents, Peter J. Campbell, Ivo Gut, Davide Rossi, Gianluca Gaidano, Xose S. Puente, Pablo M. Garcia-Roves, Dolors Colomer, Holger Heyn, Francesco Maura, José I. Martín-Subero & Elías Campo "Postprint (published version

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia

B-cell lymphoma; Cancer epigenetics; Chronic lymphocytic leukaemiaLinfoma de células b; Epigenética del cáncer; Leucemia linfocítica crónicaLimfoma de cèl·lules b; Epigenètica del càncer; Leucèmia limfocítica crònicaRichter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high–B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.The authors thank the Hematopathology Collection registered at the Biobank of Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and the Biobank HUB-ICO-IDIBELL (PT20/00171) for sample procurement, S. Martín, F. Arenas, the Genomics Core Facility of the IDIBAPS, CNAG Sequencing Unit, Mission Bio, Omniscope and Barcelona Supercomputing Center for the technical support and the computer resources at MareNostrum4 (RES activity, BCV-2018-3-0001). This study was supported by the la Caixa Foundation (CLLEvolution-LCF/PR/HR17/52150017, Health Research 2017 Program HR17-00221, to E.C.), the European Research Council under the European Union’s Horizon 2020 Research and Innovation Program (810287, BCLLatlas, to E.C., J.I.M.-S., H.H. and I.G.), the Instituto de Salud Carlos III and the European Regional Development Fund Una Manera de Hacer Europa (PMP15/00007 to E.C. and RTI2018-094584-B-I00 to D.C.), the American Association for Cancer Research (2021 AACR-Amgen Fellowship in Clinical/Translational Cancer Research, 21-40-11-NADE to F.N.), the European Hematology Association (EHA Junior Research Grant 2021, RG-202012-00245 to F.N.), the Lady Tata Memorial Trust (International Award for Research in Leukaemia 2021-2022, LADY_TATA_21_3223 to F.N.), the Generalitat de Catalunya Suport Grups de Recerca AGAUR (2017-SGR-1142 to E.C., 2017-SGR-736 to J.I.M.-S. and 2017-SGR-1009 to D.C.), the Accelerator award CRUK/AIRC/AECC joint funder partnership (AECC_AA17_SUBERO to J.I.M.-S.), the Fundació La Marató de TV3 (201924-30 to J.I.M.-S.), the Centro de Investigación Biomédica en Red Cáncer (CIBERONC; CB16/12/00225, CB16/12/00334, CB16/12/00236), the Ministerio de Ciencia e Innovación (PID2020-117185RB-I00 to X.S.P.), the Fundación Asociación Española Contra el Cáncer (FUNCAR-PRYGN211258SUÁR to X.S.P.), the Associazione Italiana per la Ricerca sul Cancro Foundation (AIRC 5 × 1,000 no. 21198 to G.G.) and the CERCA Programme/Generalitat de Catalunya. H.P.-A. is a recipient of a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (FPU19/03110). A.D.-N. is supported by the Department of Education of the Basque Government (PRE_2017_1_0100). E.C. is an Academia Researcher of the Institució Catalana de Recerca i Estudis Avançats of the Generalitat de Catalunya. This work was partially developed at the Center Esther Koplowitz (Barcelona, Spain)

RNA editing contributes to epitranscriptome diversity in chronic lymphocytic leukemia

RNA editing—primarily conversion of adenosine to inosine (A > I)—is a widespread posttranscriptional mechanism, mediated by Adenosine Deaminases acting on RNA (ADAR) enzymes to alter the RNA sequence of primary transcripts. Hence, in addition to somatic mutations and alternative RNA splicing, RNA editing can be a further source for recoding events. Although RNA editing has been detected in many solid cancers and normal tissue, RNA editing in chronic lymphocytic leukemia (CLL) has not been addressed so far. We determined global RNA editing and recurrent, recoding RNA editing events from matched RNA-sequencing and whole exome sequencing data in CLL samples from 45 untreated patients. RNA editing was verified in a validation cohort of 98 CLL patients and revealed substantially altered RNA editing profiles in CLL compared with normal B cells. We further found that RNA editing patterns were prognostically relevant. Finally, we showed that ADAR knockout decreased steady state viability of MEC1 cells and made them more susceptible to treatment with fludarabine and ibrutinib in vitro. We propose that RNA editing contributes to the pathophysiology of CLL and targeting the RNA editing machinery could be a future strategy to maximize treatment efficacy

Minimal spatial heterogeneity in chronic lymphocytic leukemia at diagnosis

Acknowledgements This study was supported by the Instituto de Salud Carlos III and the European Regional Development Fund “Una manera de hacer Europa” (grant PMP15/00007), and the “la Caixa” Foundation (grant CLLEvolution-HR17-00221). EC is an Academia Researcher of the ‘Institució Catalana de Recerca i Estudis Avançats’ (ICREA) of the Generalitat de Catalunya. FN is supported by a predoctoral fellowship of the Ministerio de Economía y Competitividad (BES-2016-076372). FM is supported by the Memorial Sloan Kettering Cancer Center NCI Core Grant (P30 CA 008748).Peer ReviewedPostprint (author's final draft

Author Correction: A practical guide for mutational signature analysis in hematological malignancies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper