Dissecting the role of TP53 alterations in del(11q) chronic lymphocytic leukemia

© 2021 The Authors.[Background]: Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high-risk alterations such as del(11q)/ATM-mutations and del(17p)/TP53-mutations have not been established.[Methods]: We integrated next-generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response.[Results]: Targeted sequencing analysis of the co-occurrence of high-risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co-occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9-edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition.[Conclusions]: Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.Spanish Fondo de Investigaciones Sanitarias, Grant/Award Numbers: PI15/01471, PI18/01500); Fundación Memoria Don Samuel Solórzano Barruso, Grant/Award Number: RD12/0036/006

Gut microbiota composition and arterial stiffness measured by pulse wave velocity: Case-control study protocol (MIVAS study)

[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)

Lifestyles, arterial aging, and its relationship with the intestinal and oral microbiota (MIVAS III study): a research protocol for a cross-sectional multicenter study

The microbiota is increasingly recognized as a significant factor in the pathophysiology of many diseases, including cardiometabolic diseases, with lifestyles probably exerting the greatest influence on the composition of the human microbiome. The main objectives of the study are to analyze the association of lifestyles (diet, physical activity, tobacco, and alcohol) with the gut and oral microbiota, arterial aging, and cognitive function in subjects without cardiovascular disease in the Iberian Peninsula. In addition, the study will examine the mediating role of the microbiome in mediating the association between lifestyles and arterial aging as well as cognitive function.Methods and analysisMIVAS III is a multicenter cross-sectional study that will take place in the Iberian Peninsula. One thousand subjects aged between 45 and 74 years without cardiovascular disease will be selected. The main variables are demographic information, anthropometric measurements, and habits (tobacco and alcohol). Dietary patterns will be assessed using a frequency consumption questionnaire (FFQ) and the Mediterranean diet adherence questionnaire. Physical activity levels will be evaluated using the International Physical Activity Questionnaire (IPAQ), Marshall Questionnaire, and an Accelerometer (Actigraph). Body composition will be measured using the Inbody 230 impedance meter. Arterial aging will be assessed through various means, including measuring medium intimate carotid thickness using the Sonosite Micromax, conducting analysis with pulse wave velocity (PWA), and measuring pulse wave velocity (cf-PWV) using the Sphygmocor System. Additional cardiovascular indicators such as Cardio Ankle Vascular Index (CAVI), ba-PWV, and ankle-brachial index (Vasera VS-2000®) will also be examined. The study will analyze the intestinal microbiota using the OMNIgene GUT kit (OMR−200) and profile the microbiome through massive sequencing of the 16S rRNA gene. Linear discriminant analysis (LDA), effect size (LEfSe), and compositional analysis, such as ANCOM-BC, will be used to identify differentially abundant taxa between groups. After rarefying the samples, further analyses will be conducted using MicrobiomeAnalyst and R v.4.2.1 software. These analyses will include various aspects, such as assessing α and β diversity, conducting abundance profiling, and performing clustering analysis.DiscussionLifestyle acts as a modifier of microbiota composition. However, there are no conclusive results demonstrating the mediating effect of the microbiota in the relationship between lifestyles and cardiovascular diseases. Understanding this relationship may facilitate the implementation of strategies for improving population health by modifying the gut and oral microbiota

Clinical, biological, and prognostic implications of SF3B1 co-occurrence mutations in very low/low- and intermediate-risk MDS patients

SF3B1 is a highly mutated gene in myelodysplastic syndrome (MDS) patients, related to a specific subtype and parameters of good prognosis in MDS without excess blasts. More than 40% of MDS patients carry at least two myeloid-related gene mutations but little is known about the impact of concurrent mutations on the outcome of MDS patients. In applying next-generation sequencing (NGS) with a 117 myeloid gene custom panel, we analyzed the co-occurrence of SF3B1 with other mutations to reveal their clinical, biological, and prognostic implications in very low/low- and intermediate-risk MDS patients. Mutations in addition to those of SF3B1 were present in 80.4% of patients (median of 2 additional mutations/patient, range 0–5). The most frequently mutated genes were as follows: TET2 (39.2%), DNMT3A (25.5%), SRSF2 (10.8%), CDH23 (5.9%), and ASXL1, CUX1, and KMT2D (4.9% each). The presence of at least two mutations concomitant with that of SF3B1 had an adverse impact on survival compared with those with the SF3B1 mutation and fewer than two additional mutations (median of 54 vs. 87 months, respectively: p = 0.007). The co-occurrence of SF3B1 mutations with specific genes is also linked to a dismal prognosis: SRSF2 mutations were associated with shorter overall survival (OS) than SRSF2wt (median, 27 vs. 75 months, respectively; p = 0.001), concomitant IDH2 mutations (median OS, 11 [mut] vs. 75 [wt] months; p = 0.001), BCOR mutations (median OS, 11 [mut] vs. 71 [wt] months; p = 0.036), and NUP98 and STAG2 mutations (median OS, 27 and 11 vs. 71 months, respectively; p = 0.008 and p = 0.002). Mutations in CHIP genes (TET2, DNMT3A) did not significantly affect the clinical features or outcome. Our results suggest that a more comprehensive NGS study in low-risk MDS SF3B1mut patients is essential for a better prognostic evaluation.This work was supported by grants from the following: Contrato Rio Hortega, CM17/00171; Gerencia Regional de Salud (Castilla y León) para proyectos de investigación año 2018, 1850/A/18; 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 (SA271P18); Proyectos de Investigación del SACYL, Spain, GRS1847/A/18, GRS1653/A17; 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”, by grants from Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD12/0036/0069) and Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233). JMHS is supported by a research grant from Fundación Española de Hematología y Hemoterapia. MM is currently supported by an Ayuda predoctoral de la Junta de Castilla y León from the Fondo Social Europeo (JCYL- EDU/556/2019 PhD scholarship)

Co-occurrence of cohesin complex and Ras signaling mutations during progression from myelodysplastic syndromes to secondary acute myeloid leukemia

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

CRISPR/Cas9-generated models uncover therapeutic vulnerabilities of del(11q) CLL cells to dual BCR and PARP inhibition

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.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” (SA271P18), “Proyectos de Investigación del SACYL”, Spain GRS 1847/A/18, GRS1653/A17,“Fundación Memoria Don Samuel Solórzano Barruso” (FS/23-2018), 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 SYNtherapy “Synthetic Lethality for Personalized Therapy-based Stratification In Acute Leukemia” (ERAPERMED2018-275); ISCIII (AC18/00093). MQÁ is fully supported by an “Ayuda predoctoral de la Junta de Castilla y León” by the Fondo Social Europeo (JCYL-EDU/529/2017 PhD scholarship); MHS was supported by a grant from FEHH/Janssen (“Sociedad Española de Hematología y Hemoterapia”) and now holds a Sara Borrell postdoctoral contract (CD19/00222) from Instituto de Salud Carlos III (ISCIII), co-funded by Fondo Social Europeo (FSE) “El Fondo Social Europeo invierte en tu futuro”; AERV is supported with a research grant by FEHH (“Fundación Española de Hematología y Hemoterapia”); MG is supported by a Marie Curie Action International Outgoing Fellowship (PIOF-2013-624924); EtH is a Special Fellow of the Leukemia and Lymphoma Society (LLS) and a Scholar of the American Society of Hematology (ASH) and JLO is supported by a grant from the University of Salamanca (“Contrato postdoctoral programa II”).Peer reviewe

Search for the standard model Higgs boson in the H to ZZ to 2l 2nu channel in pp collisions at sqrt(s) = 7 TeV

A search for the standard model Higgs boson in the H to ZZ to 2l 2nu decay channel, where l = e or mu, in pp collisions at a center-of-mass energy of 7 TeV is presented. The data were collected at the LHC, with the CMS detector, and correspond to an integrated luminosity of 4.6 inverse femtobarns. No significant excess is observed above the background expectation, and upper limits are set on the Higgs boson production cross section. The presence of the standard model Higgs boson with a mass in the 270-440 GeV range is excluded at 95% confidence level.Comment: Submitted to JHE

Constraints on the χ_(c1) versus χ_(c2) polarizations in proton-proton collisions at √s = 8 TeV

The polarizations of promptly produced χ_(c1) and χ_(c2) mesons are studied using data collected by the CMS experiment at the LHC, in proton-proton collisions at √s=8  TeV. The χ_c states are reconstructed via their radiative decays χ_c → J/ψγ, with the photons being measured through conversions to e⁺e⁻, which allows the two states to be well resolved. The polarizations are measured in the helicity frame, through the analysis of the χ_(c2) to χ_(c1) yield ratio as a function of the polar or azimuthal angle of the positive muon emitted in the J/ψ → μ⁺μ⁻ decay, in three bins of J/ψ transverse momentum. While no differences are seen between the two states in terms of azimuthal decay angle distributions, they are observed to have significantly different polar anisotropies. The measurement favors a scenario where at least one of the two states is strongly polarized along the helicity quantization axis, in agreement with nonrelativistic quantum chromodynamics predictions. This is the first measurement of significantly polarized quarkonia produced at high transverse momentum