Nuevas aportaciones de la hibridación in situ fluorescente y el estado mutacional del gen de las cadenas pesadas de inmunoglobulinas para predecir el curso clínico de los pacientes con leucemia linfocítica crónica

La leucemia linfocítica crónica (LLC) es una enfermedad con un curso clínico extremadamente variable. Por este motivo, con el objetivo de predecir qué pacientes van a progresar en poco tiempo, en las últimas décadas se han identificado varios factores pronósticos de gran relevancia. Entre ellos, el estado mutacional de la cadena pesada de las inmunoglobulinas (IGHV) y las anomalías citogenéticas detectadas por hibridación in situ fluorescente (FISH) se han consolidado como dos de los más potentes. Además, en algunos casos indican la necesidad de un abordaje terapéutico diferente. En relación con el estado mutacional IGHV, el patrón no mutado se observa en aproximadamente la mitad de los pacientes con LLC y normalmente se caracteriza por presentar una enfermedad agresiva, al contrario que el patrón mutado. La FISH es capaz de detectar alteraciones genéticas clonales en aproximadamente el 80% de los sujetos con LLC. Estas alteraciones son, por orden de frecuencia: deleción de 13q (13q-), deleción de 11q (11q-), trisomía 12 (+12) y deleción de 17p (17p-), las cuales, junto a la ausencia de anormalidades citogenéticas, definen cinco grupos con pronóstico distinto. Además, el porcentaje de pérdidas en los casos de 11q-, 13qy 17p- también tiene valor pronóstico, de tal modo que los enfermos con un porcentaje elevado de células con deleción son los que presentan una evolución peor. Objetivos, material, métodos y resultados: El objetivo de esta Tesis Doctoral es analizar el impacto pronóstico de aspectos concretos y novedosos acerca del estado mutacional IGHV y de la FISH en pacientes con LLC. Se presentan a continuación los resúmenes de las publicaciones que han dado lugar a esta Tesis..

New Insights in Prognosis and Therapy of Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease with a very variable clinical outcome. New biological markers, such as cytogenetic abnormalities or mutation status, have become important prognostic factors. Whole-genome sequencing studies have revealed novel genomic mutations, NOTCH1, SF3B1, BIRC3, TP53 and MYD88 being the most important. All these mutations have also been associated with the disease outcome. The treatment of CLL has evolved favourably in recent years. However, adverse events or chemorefractoriness occurs in some cases. Luckily, an increasing number of compounds are under development with promising results. Some of these new targeted therapies include B-cell receptor inhibitors, new anti-CD20 antibodies, Bcl-2 inhibitors, immunomodulatory drugs or chimeric antigen receptors (CARs). In this chapter, we will conduct a review of the new prognostic markers of CLL, the relationship they have with each other to build prognostic scores, the role they have in guiding treatment decisions and the novel therapies that have emerged recently with immunologic, biochemical and genetic targets

Ocratoxina A e resíduos de produtos fitofarmacêuticos em vinhos elaborados nos distritos sanitários de La Roda e Villarrobledo (Albacete) 2008- 2015: avaliação

The presence of pesticide residues and/or ochratoxin A in wine can affect the health of consumers. Fewwineries have taken control measures or have verified the effectiveness of the control measures they have taken.It was of interest to evaluate the results for pesticide residues and ochratoxin A in wine samples taken by wineriesor by official inspection bodies.All analytical results for pesticides and/or ochratoxin A in wine samples taken between 2008 and 2015 and available at the wineries in both districts, or in the official control samples, were considered. All in all, 20 analytical results on pesticide residues and 64 Ochratoxin A determinations.No results exceeded the maximum limit of pesticide residue onwine grapes, since there is no legal limit onwine. The determination of pesticides in wine only makes sense when concentration or dilution factors during the winemaking process, with regard to the maximum limits of pesticide residueson wine grapes, are justified, but this did not happeninanycase. Two different pesticide residues were found in one sample, and a concentration of 5 mg kg-1 of iprodione (maximum limit of iprodione residue on wine grapes: 10 meaning kg-1) was found in an organic wine sample, which gives an idea of wrong agricultural practices.No ochratoxin A result exceeded the legal limit for wine of 2 μg L-1(Commission Regulation (EC) No.1881/2006). Concentrations of up to 0.44 μg L-1 were obtained, which shows that ochratoxin A can occur inwine.Wineries need to define and take effective control measures to deal with these two hazards.La presencia de residuos fitosanitarios o de ocratoxina A en vinos puede afectar a la salud de los consumidores. Pocas bodegas han implantando medidas de control, o han verificado la eficacia de las medidas adoptadas.Era interesante valorar los resultados sobre residuos fitosanitarios y ocratoxina A en muestras de vino tomadas por las bodegas o por el control oficial.Se consideraron todos los resultados analíticos sobre fitosanitarios o ocratoxina A en muestras de vino tomadas entre 2008 y 2015 disponibles en las bodegas de los distritos de salud de La Roda y Villarrobledo, o en muestras de control oficial. En total 20 analíticas de fitosanitarios y 64 determinaciones de ocratoxina A.Puesto que no hay límite legal en vino, ningún resultado sobrepasó el límite máximo de residuos fitosanitarios en uva de vinificación. La determinación de fitosanitarios en vino tiene sentido si se justifican factores de dilución o concentración durante el proceso de elaboración, respecto a los límites máximos de residuos fitosanitarios en uva, lo cual no sucedió en ningún caso. Una muestra presentó residuos de dos fitosanitarios distintos, y en una muestra de vino ecológico se obtuvo 5 mg kg-1 de iprodiona (límite máximo de residuo de iprodiona en uva: 10 mg kg-1), orientando sobre prácticas agrícolas incorrectas.Ningún resultado de ocratoxina A superó el límite legal en vino de 2 μg L-1 (Reglamento(CE)Nº 1881/2006). Se obtuvieron concentraciones de hasta 0,44 μg L-1, demostrando que es posible la ocurrencia de ocratoxina A en vino.Es necesario que las bodegas definan y apliquen medidas de control eficaces sobre estos dos peligros.A presença de resíduos fitofarmacêuticos e/ou de ocratoxina A em vinhos pode afetar a saúde dos consumidores. Poucas adegas têm estabelecido medidas de controlo, ou verificado a eficácia das medidas adotadas.Assim, mostrou-se interessante avaliar os resultados de resíduos fitofarmacêuticos e ocratoxina A em amostras de vinho recolhidas pelas adegas ou por Controlo Oficial.Foram considerados todos os resultados analíticos sobre fitofarmacêuticos e/ou ocratoxina A em amostras de vinho recolhidas entre 2008 e 2015 nas adegas situadas nos Distritos Sanitários de La Roda e Villarrobledo, ou em amostras de Controlo Oficial. No total, realizaram-se 20 análises de fitofarmacêuticos e 64 determinações de ocratoxina A.Considerando que não existe limite legal para o vinho, nenhum resultado ultrapassou o limite máximo de resíduos fitofarmacêuticos na uva de vinificação. A determinação de fitofarmacêuticos em vinho faz sentido, caso se justifiquem fatores de diluição ou de concentração no processo de elaboração, em relação ao limite máximo de resíduos em uva, situação que não aconteceu. Uma amostra revelou resíduos de dois fitofarmacêuticos distintos. Numa outra amostra de vinho biológico obtiveram-se 5 mg kg-1 de iprodiona (limite máximo de resíduo em uva: 10 mg kg-1), o que é sugere práticas agrícolas incorretas.Nenhum valor de ocratoxina A excedeu o limite legal em vinho de 2 μg L-1(Regulação (CE) Nº 1881/2006). Foram atingidas concentrações de 0,44 μg L-1, mostrando que é possível a presença de ocratoxina A em vinho.Deste modo, é necessário que as adegas definam e apliquem medidas eficazes para controlar estes dois perigos

Mutation status and immunoglobulin gene rearrangements in patients from northwest and central region of Spain with chronic lymphocytic leukemia

This is an open access article distributed under the Creative Commons Attribution License.-- et al.The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL.The study was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias 02/1041, FIS 09/01382, FIS 09/01543, and PI12/00281; RD12/0036/0069 from Red Tematica 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”; and Caja de Burgos-Banca Cívica. A. Rodrígues was fully supported by an Ayuda Predoctoral FIS de Formación en Investigacion by the Spanish Fondo de Investigaciones Sanitarias. M. Hernandez-Sanchez was partially supported by a grant from the “Fundacion Española de Hematología y Hemoterapia.” Partially supported by grants from “Proyectos de Investigacion Biomédica del SACYL” 106/A/06.Peer Reviewe

From Biomarkers to Models in the Changing Landscape of Chronic Lymphocytic Leukemia: Evolve or Become Extinct

hronic lymphocytic leukemia (CLL) is an extremely heterogeneous disease. With the advent of oral targeted agents (Tas) the treatment of CLL has undergone a revolution, which has been accompanied by an improvement in patient’s survival and quality of life. This paradigm shift also affects the value of prognostic and predictive biomarkers and prognostic models, most of them inherited from the chemoimmunotherapy era but with a different behavior with Tas. This review discusses: (i) the role of the most relevant prognostic and predictive biomarkers in the setting of Tas; and (ii) the validity of classic and new scoring systems in the context of Tas. In addition, a critical point of view about predictive biomarkers with special emphasis on 11q deletion, novel resistance mutations, TP53 abnormalities, IGHV mutational status, complex karyotype and NOTCH1 mutations is stated. We also go over prognostic models in early stage CLL such as IPS-E. Finally, we provide an overview of the applicability of the CLL-IPI for patients treated with Tas, as well as the emergence of new models, generated with data from patients treated with Tas

TRAF3 alterations are frequent in del-3′IGH chronic lymphocytic leukemia patients and define a specific subgroup with adverse clinical features

Interstitial 14q32 deletions involving IGH gene are infrequent events in chronic lymphocytic leukemia (CLL), affecting less than 5% of patients. To date, little is known about their clinical impact and molecular underpinnings, and its mutational landscape is currently unknown. In this work, a total of 871 CLLs were tested for the IGH break-apart probe, and 54 (6.2%) had a 300 kb deletion of 3′IGH (del-3′IGH CLLs), which contributed to a shorter time to first treatment (TFT). The mutational analysis by next-generation sequencing of 317 untreated CLLs (54 del-3′IGH and 263 as the control group) showed high mutational frequencies of NOTCH1 (30%), ATM (20%), genes involved in the RAS signaling pathway (BRAF, KRAS, NRAS, and MAP2K1) (15%), and TRAF3 (13%) within del-3′IGH CLLs. Notably, the incidence of TRAF3 mutations was significantly higher in del-3′IGH CLLs than in the control group (p < .001). Copy number analysis also revealed that TRAF3 loss was highly enriched in CLLs with 14q deletion (p < .001), indicating a complete biallelic inactivation of this gene through deletion and mutation. Interestingly, the presence of mutations in the aforementioned genes negatively refined the prognosis of del-3′IGH CLLs in terms of overall survival (NOTCH1, ATM, and RAS signaling pathway genes) and TFT (TRAF3). Furthermore, TRAF3 biallelic inactivation constituted an independent risk factor for TFT in the entire CLL cohort. Altogether, our work demonstrates the distinct genetic landscape of del-3′IGH CLL with multiple molecular pathways affected, characterized by a TRAF3 biallelic inactivation that contributes to a marked poor outcome in this subgroup of patients.Funding information: Universidad de Salamanca; Fundación Española de Hematología y Hemoterapia (FEHH); Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Grant/Award Number: CB16/12/00233; Red Temática de Investigación Cooperativa en Cáncer (RTICC); “Fundación Memoria Don Samuel Solórzano Barruso”: FS/33–2020, Grant/Award Number: RD12/0036/0069; “Gerencia Regional de Salud, SACYL”:, Grant/Award Numbers: GRS2385/A/21, GRS2140/A/20; Consejería de Educación, Junta de Castilla y León, Grant/Award Number: SA118P20; European Regional Development Fund and Instituto de Salud Carlos III, Grant/Award Numbers: CD19/00222, FI19/00191; Spanish Fondo de Investigaciones Sanitarias, Grant/Award Numbers: PI21/00983, PI18/0150

Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression

© The Author(s) 2021.BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation.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 2062/A/19, 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), Universidad de Salamanca (Programa XIII), 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), co-funded by ERDF/ESF, “Investing in your future”. M.Q.Á. and A.E.R.V. are supported with a research grant by FEHH (“Fundación Española de Hematología y Hemoterapia”); M.H.S. holds a Sara Borrell postdoctoral contract (CD19/00222) from the Instituto de Salud Carlos III (ISCIII). C.P.C. was supported by an “Ayuda predoctoral en Oncología” (AECC) and is a recipient of a PFIS grant (FI19/00191) from Instituto de Salud Carlos III; PFIS grant and Sara Borrell postdoctoral contrat are co-founded by Fondo Social Europeo (FSE) “El Fondo Social Europeo invierte en tu futuro”; J.L.O. and R.B.S. are supported by a grant from the University of Salamanca (“Contrato postdoctoral programa II”)

Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression

Article number: 127[EN]BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation

Prognosis assessment of early-stage chronic lymphocytic leukemia: Are we ready to predict clinical evolution without a crystal ball?

On behalf ofGrupo Español de Leucemia Linfática Crónica and Grupo Cooperativo Español de Citogenética Hematológica.[Background]: The discovery of new biologic variables with high prognostic effect has been accompanied by the emergence of different prognostic indexes (PIs) to assess the time to first treatment in patients with early-stage (Binet A) chronic lymphocytic leukemia (CLL). The present study compared the prognostic value of 5 PIs: CLL international prognostic index (CLL-IPI), Barcelona-Brno, international prognostic score-A (IPS-A), CLL-01, and a tailored approach.[Patients and Methods]: We applied the 5 PIs to a cohort of 428 unselected patients with Binet A CLL from a multicenter Spanish database with clinical and biologic information available. The predictive value of the scores was assessed using Harrell’s concordance index (C index) and area under the receiver operating characteristic curve (AUC).[Results]: We found a significant association between time to first treatment and risk subgroups for all 5 PIs used. The most accurate PI was the IPS-A (C-index, 0.72; AUC, 0.76), closely followed by CLL-01 (C-index, 0.69; AUC, 0.70), CLL-IPI (C-index, 0.69; AUC, 0.69), Barcelona-Brno (C-index, 0.67; AUC, 0.69), and the tailored approach (C-index, 0.61 and 0.58; AUC, 0.58 and 0.54).[Conclusions]: The concordance between the PIs was low (44%), suggesting that although all these PIs improve clinical staging and help physicians in routine clinical practice, it will be necessary to harmonize larger cohorts of patients to define the best PI for treatment decision-making in the real world.The present study was supported by the Spanish Fondo de Investigaciones Sanitarias (grants PI15/01471 and PI18/01500), Instituto de Salud Carlos III, European Regional Development Fund (Una manera de hacer Europa), Consejería de Educación, Junta de Castilla y León (grant SA271P18), Proyectos de Investigación del SACYL (grants GRS1847/A/18 and GRS1653/A17), Fundación Memoria Don Samuel Solórzano Barruso (grant FS/23-2018), Red Temática de Investigación Cooperativa en Cáncer (grant RD12/0036/0069), Centro de Investigación Biomédica en Red de Cáncer (grant CIBERONC CB16/12/00233), and Synthetic Lethality for Personalized Therapy-based Stratification in Acute Leukemia (grant ERAPERMED2018-275); and Instituto de Salud Carlos III (grant AC18/00093). M.H.S. holds a Sara Borrell postdoctoral contract (CD19/00222) from the Spanish Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, European Regional Development Fund (El Fondo Social Europeo invierte en tu future). A.E.R.V. is supported by a research grant from the Fundación Española de Hematología y Hemoterapia. M.Q.Á. is fully supported by the Ayuda predoctoral de la Junta de Castilla y León from the Fondo Social Europeo (PhD scholarship JCYL EDU/529/2017). C.P.C. is a recipient of a PFIS grant (FI19/00191) from Instituto de Salud Carlos III, cofounded by Fondo Social Europeo (El Fondo Social Europeo invierte en tu future).Peer reviewe

COVID-19 Severity and Survival over Time in Patients with Hematologic Malignancies: A Population-Based Registry Study

Mortality rates for COVID-19 have declined over time in the general population, but data in patients with hematologic malignancies are contradictory. We identified independent prognostic factors for COVID-19 severity and survival in unvaccinated patients with hematologic malignancies, compared mortality rates over time and versus non-cancer inpatients, and investigated post COVID-19 condition. Data were analyzed from 1166 consecutive, eligible patients with hematologic malignancies from the population-based HEMATO-MADRID registry, Spain, with COVID-19 prior to vaccination roll-out, stratified into early (February–June 2020; n = 769 (66%)) and later (July 2020–February 2021; n = 397 (34%)) cohorts. Propensity-score matched non-cancer patients were identified from the SEMI-COVID registry. A lower proportion of patients were hospitalized in the later waves (54.2%) compared to the earlier (88.6%), OR 0.15, 95%CI 0.11–0.20. The proportion of hospitalized patients admitted to the ICU was higher in the later cohort (103/215, 47.9%) compared with the early cohort (170/681, 25.0%, 2.77; 2.01–3.82). The reduced 30-day mortality between early and later cohorts of non-cancer inpatients (29.6% vs. 12.6%, OR 0.34; 0.22–0.53) was not paralleled in inpatients with hematologic malignancies (32.3% vs. 34.8%, OR 1.12; 0.81–1.5). Among evaluable patients, 27.3% had post COVID-19 condition. These findings will help inform evidence-based preventive and therapeutic strategies for patients with hematologic malignancies and COVID-19 diagnosis.Depto. de MedicinaFac. de MedicinaTRUEFundación Madrileña de Hematología y HemoterapiaFundación Leucemia y LinfomaAsociación Madrileña de Hematología y Hemoterapiapu