A gene expression assay based on chronic lymphocytic leukemia activation in the microenvironment to predict progression

Gene expression; Chronic lymphocytic leukemiaExpresión génica; Leucemia linfocítica crónicaExpressió gènica; Leucèmia limfocítica crònicaSeveral gene expression profiles with a strong correlation with patient outcomes have been previously described in chronic lymphocytic leukemia (CLL), although their applicability as biomarkers in clinical practice has been particularly limited. Here we describe the training and validation of a gene expression signature for predicting early progression in patients with CLL based on the analysis of 200 genes related to microenvironment signaling on the NanoString platform. In the training cohort (n = 154), the CLL15 assay containing a 15-gene signature was associated with the time to first treatment (TtFT) (hazard ratio [HR], 2.83; 95% CI, 2.17-3.68; P < .001). The prognostic value of the CLL15 score (HR, 1.71; 95% CI, 1.15-2.52; P = .007) was further confirmed in an external independent validation cohort (n = 112). Notably, the CLL15 score improved the prognostic capacity over IGHV mutational status and the International Prognostic Score for asymptomatic early-stage (IPS-E) CLL. In multivariate analysis, the CLL15 score (HR, 1.83; 95% CI, 1.32-2.56; P < .001) and the IPS-E CLL (HR, 2.23; 95% CI, 1.59-3.12; P < .001) were independently associated with TtFT. The newly developed and validated CLL15 assay successfully translated previous gene signatures such as the microenvironment signaling into a new gene expression–based assay with prognostic implications in CLL.This work was supported by research funding from the Asociación Española Contra el Cáncer grant [5U01CA157581-05, ECRIN-M3 - A29370] and in part by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias [PI17/00950, M.C., PI17/00943, F.B, PI18/01392, P.A.], and the Spanish Ministry of Economy and Competitiveness [CIBERONC-CB16/12/00233], the Education Council or Health Council of the Junta de Castilla y León [GRS 2036/A/19], and Gilead Sciences [GLD15/00348]. This work was supported by research funding from the Asociación Española Contra el Cáncer grant [5U01CA157581-05, ECRIN-M3 - A29370] and in part by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias [PI17/00950, M.C., PI17/00943, F.B, PI18/01392, P.A.], and the Spanish Ministry of Economy and Competitiveness [CIBERONC-CB16/12/00233], the Education Council or Health Council of the Junta de Castilla y León [GRS 2036/A/19], Gilead Sciences [GLD15/00348] and Gilead Fellowships [GLD16/00144, GLD18/00047, F.B.], and Fundació la Marató de TV3 [201905-30-31 F.B]. All Spanish funding was cosponsored by the European Union FEDER program “Una manera de hacer Europa”. M.C. holds a contract from Ministerio de Ciencia, Innovación y Universidades [RYC-2012-2018]

Immunological and genetic kinetics from diagnosis to clinical progression in chronic lymphocytic leukemia

Progressió clínica; Evasió immuneProgresión clínica; Evasión inmuneClinical progression; Immune evasionBackground Mechanisms driving the progression of chronic lymphocytic leukemia (CLL) from its early stages are not fully understood. The acquisition of molecular changes at the time of progression has been observed in a small fraction of patients, suggesting that CLL progression is not mainly driven by dynamic clonal evolution. In order to shed light on mechanisms that lead to CLL progression, we investigated longitudinal changes in both the genetic and immunological scenarios. Methods We performed genetic and immunological longitudinal analysis using paired primary samples from untreated CLL patients that underwent clinical progression (sampling at diagnosis and progression) and from patients with stable disease (sampling at diagnosis and at long-term asymptomatic follow-up). Results Molecular analysis showed limited and non-recurrent molecular changes at progression, indicating that clonal evolution is not the main driver of clinical progression. Our analysis of the immune kinetics found an increasingly dysfunctional CD8+ T cell compartment in progressing patients that was not observed in those patients that remained asymptomatic. Specifically, terminally exhausted effector CD8+ T cells (T-betdim/−EomeshiPD1hi) accumulated, while the the co-expression of inhibitory receptors (PD1, CD244 and CD160) increased, along with an altered gene expression profile in T cells only in those patients that progressed. In addition, malignant cells from patients at clinical progression showed enhanced capacity to induce exhaustion-related markers in CD8+ T cells ex vivo mainly through a mechanism dependent on soluble factors including IL-10. Conclusions Altogether, we demonstrate that the interaction with the immune microenvironment plays a key role in clinical progression in CLL, thereby providing a rationale for the use of early immunotherapeutic intervention.This work was supported by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI17/00950, M.C., PI18/01392, P.A. and PI17/00943, F.B.) and co-financed by the European Regional Development Fund (ERDF) and Fundación Asociación Española Contra el Cáncer (M.C. and P.A.), Gilead Fellowships (GLD16/00144, GLD18/00047, F.B.) and Fundació la Marató de TV3 (201905–30-31 F.B). S.B. is the recipient of a postdoctoral fellowship from Fundación Alfonso Martin Escudero. R.V-M. is supported by a Torres Quevedo fellowship from the Spanish Ministry of Science and Innovation (PTQ-16-08623). A.E-C. is funded by ISCIII/MINECO (PT17/0009/0019) which is co-funded by FEDER. M.C. holds a contract from Ministerio de Ciencia, Innovación y Universidades (RYC-2012-2018)

Cell free circulating tumor DNA in cerebrospinal fluid detects and monitors central nervous system involvement of B-cell lymphomas

Limfoma no Hodgkin agressiu; Limfoma del SNCLinfoma no Hodgkin agresivo; Linfoma del SNCAggressive Non-Hodgkin's Lymphoma; CNS lymphomaThe levels of cell free circulating tumor DNA (ctDNA) in plasma correlated with treatment response and outcome in systemic lymphomas. Notably, in brain tumors, the levels of ctDNA in the cerebrospinal fluid (CSF) are higher than in plasma. Nevertheless, their role in central nervous system (CNS) lymphomas remains elusive. We evaluated the CSF and plasma from 19 patients: 6 restricted CNS lymphomas, 1 systemic and CNS lymphoma, and 12 systemic lymphomas. We performed whole exome sequencing or targeted sequencing to identify somatic mutations of the primary tumor, then variant-specific droplet digital PCR was designed for each mutation. At time of enrolment, we found ctDNA in the CSF of all patients with restricted CNS lymphoma but not in patients with systemic lymphoma without CNS involvement. Conversely, plasma ctDNA was detected in only 2/6 patients with restricted CNS lymphoma with lower variant allele frequencies than CSF ctDNA. Moreover, we detected CSF ctDNA in 1 patient with CNS lymphoma in complete remission and in 1 patient with systemic lymphoma, 3 and 8 months before CNS relapse was confirmed; indicating CSF ctDNA might detect CNS relapse earlier than conventional methods. Finally, in 2 cases with CNS lymphoma, CSF ctDNA was still detected after treatment even though a complete decrease in CSF tumor cells was observed by flow cytometry (FC), indicating CSF ctDNA better detected residual disease than FC. In conclusion, CSF ctDNA can better detect CNS lesions than plasma ctDNA and FC. In addition, CSF ctDNA predicted CNS relapse in CNS and systemic lymphomas.This work was supported by research funding from Fundación Asociación Española contra el Cáncer (AECC) (to JS, MC and PA); FERO (to JS), laCaixa (to JS), BBVA (CAIMI) (to JS), the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI16/01278 to JS; PI17/00950 to MC; PI17/00943 to FB) cofinanced by the European Regional Development Fund (ERDF) and Gilead Fellowships (GLD16/00144, GLD18/00047, to FB). MC holds a contract from Ministerio de Ciencia, Innovación y Universidades (RYC-2012-12018). SB received funding from Fundación Alfonso Martin Escudero. LE received funding from the Juan de la Cierva fellowship. We thank CERCA Programme / Generalitat de Catalunya for institutional support

Expressió de ZAP-70 en linfòcits B normals i síndromes limfoproliferatives B

[cat] La Leucèmia Limfàtica Crònica (LLC), la més freqüent de les leucèmies en el món occidental, és una malaltia produïda per la proliferació i cúmul de limfòcits B madurs, actualment incurable. Aproximadament el 50 de les LLCs presenten hipermutacions al gen de les immunoglobulines (IgVH), el que indica que podrien representar un subgrup diferent de cèl·lules que passen pel centre germinal, el lloc fisiològic de les hipermutacions.6. A més a més, els pacients sense hipermutacions tenen un pitjor pronòstic. L'anàlisi de les immunoglobulines és de gran interès pel pronòstic dels malalts d'LLC. Malauradament aquest és costós i llarg. És per això que una de les prioritats a la recerca en LLC és identificar marcadors relacionats amb les hipermutacions de les immunoglobulines. Recentment s'ha descrit la presència de la proteïna ZAP-70 en cèl·lules de LLC sense mutacions a les immunoglobulines. ZAP-70 és una kinasa de la família de tirosines kinases Syk/ZAP-70 que s'expressa normalment en limfòcits T i NK, on participa en la senyalització a través del TCR. També es troba en limfòcits pro/pre-B de ratolí, però no s'ha descrit en limfòcits B humans.La hipòtesi de treball del present projecte consisteix en que la expressió de ZAP-70 podria estar relacionada amb l'estat mutacional de les immunoglobulines i, per tant, amb la progressió de la LLC cap a estadis més avançats. Aquesta proteïna podria trobar-se en limfòcits pro/pre-B humans i en les neoplàsies que en deriven, les leucèmies agudes limfoblàstiques B.RESULTATS I CONCLUSIONS:- El 57 % dels casos de LLC tenen una elevada expressió de ZAP-70. La determinació de ZAP-70 per citometria és un factor pronòstic ràpid i fiable i de gran utilitat clínica en la LLC.- L'expressió de ZAP-70 es troba en limfòcits pro/pre-B normals i desapareix en estadis maduratius posteriors. - El 56% de les leucèmies agudes limfoblàstiques B expressen ZAP-70, probablement com a reflex del seu orígen cel·lular- La proteïna ZAP-70 es troba expressada de manera aberrant en aproximadament un trenta per cent de les leucèmies/limfomes de Burkitt, ja que no es troba el limfòcits B de fenotip madur- la proteïna ZAP-70 i d'altres elements senyalitzadors del pre-TCR/TCR es troben expressats i fosforilats en les cèl·lules de leucèmia aguda limfoblàstica B, possiblement col·laborant amb altres proteïnes senyalitzadores en garantir la senyalització a través del pre-BCR.[eng] BACKGROUND: The mutational status of immunoglobulin variable region genes (IgVH) is an important prognostic parameter in chronic lymphocytic leukemia (CLL). Unfortunately, no good surrogates for IgVH mutations have as yet been identified. The ZAP-70 gene is overexpressed in a subset of CLL cases, this change correlating with the lack of mutations of IgVH.METHODS: To probe the suggestive link between ZAP-70 expression and IgVH mutations, ZAP-70 was analyzed in different cell lines and in 56 CLL pacients using flow cytometry, Western Blot, and/or immunohistochemistry methods. The results were correlated with the IgVH mutational status and patients outcome.RESULTS: Using flow cytometry ZAP-70 expression was higher in IgVH unmutated CLL (n=35) (48 percent of cells ± 21 percent) than in mutated cells (n=21) (5.7 percent of cells ± 3.8 percent). All CLL patients with ZAP-70 expression > 20 percent (n=32) lacked IgVH mutations, whereas 21/24 CLL with ZAP-70 < 20 percent exhibited IgVH mutations. Finally, ZAP-70 expression had prognostic significance for progression and survival in Binet A cases.CONCLUSIONS: ZAP-70 expression by flow cytometry correlated with the IgVH mutational status and had prognostic significance in CLL patients. Therefore, ZAP-70 analysis can be considered a reliable surrogate for the IgVH mutational status in CLL and a useful tool for assessing prognosis in this disease.PURPOSE: ZAP-70 gene is normally expressed in T and NK cells, where is required for the T-cell receptor signaling. It has been described that ZAP-70 contributes to the B-cell development at early stages of B-cell differentiation in mice. The purpose was to investigate the presence of ZAP-70 in normal pro/pre B-cells and mature B-cells, and in tumoral cells from B-acute lymphoblastic leukemias (B-ALLs).EXPERIMENTAL DESIGN: ZAP-70 expression was ascertained by flow cytometry, immunofluorescence, Western blot, and quantitative RT-PCR. Analysis of ZAP-70 and other signalling proteins of the pre-TCR/TCR was performed by Western Blot.RESULTS: ZAP-70 was expressed in pro/pre B-cells, but not in normal mature B-cells derived from bone marrow, peripheral blood or tonsil. Among tumoral cells, ZAP-70 was expressed in 56% of B-ALLs with pro/pre B-cell phenotype and in 4/6 Burkitt/ALL lymphomas. Moreover, other elements of the pre-TCR/TCR signaling pathway, like LAT and Lck, were also found in B-ALL cells.CONCLUSIONS: Amongst normal B-cell subsets, ZAP-70 was found expressed in normal pro/pre B-cells, but not in a significant proportion of normal B-cells with mature phenotype. Moreover, the presence of ZAP-70 in B-ALLs probably reflects their cellular origin. The lack of ZAP-70 expression in normal mature B-cells suggests that its expression in mature-derived neoplasms with different cellular origin, such as Burkitt's lymphoma and chronic lymphocytic leukemia, might be due to an aberrant phenomenon

Increased anandamide induced relaxation in mesenteric arteries of cirrhotic rats. Role of cannabinoid and vanilloid receptors

Background and aims: Anandamide is an endocannabinoid that evokes hypotension by interaction with peripheral cannabinoid CB1 receptors and with the perivascular transient receptor potential vanilloid type 1 protein (TRPV1). As anandamide has been implicated in the vasodilated state in advanced cirrhosis, the study investigated whether the mesenteric bed from cirrhotic rats has an altered and selective vasodilator response to anandamide. Methods: We assessed vascular sensitivity to anandamide, mRNA and protein expression of cannabinoid CB1 receptor and TRPV1 receptor, and the topographical distribution of cannabinoid CB1 receptors in resistance mesenteric arteries of cirrhotic and control rats. Results: Mesenteric vessels of cirrhotic animals displayed greater sensitivity to anandamide than control vessels. This vasodilator response was reverted by CB1 or TRPV1 receptor blockade, but not after endothelium denudation or nitric oxide inhibition. Anandamide had no effect on distal femoral arteries. CB1 and TRPV1 receptor protein was higher in cirrhotic than in control vessels. Neither CB1 mRNA nor protein was detected in femoral arteries. Immunochemistry showed that CB1 receptors were mainly in the adventitia and in the endothelial monolayer, with higher expression observed in vessels of cirrhotic rats than in controls. Conclusions: These results indicate that anandamide is a selective splanchnic vasodilator in cirrhosis which predominantly acts via interaction with two different types of receptors, CB1 and TRPV1 receptors, which are mainly located in perivascular sensory nerve terminals of the mesenteric resistance arteries of these animals

Regulación de la expresión del microRNA miR-21 por la proteína ZAP-70 en células de leucemia linfática crónica

La leucemia linfática crónica (LLC) está asociada a factores biológicos como la expresión de la proteína ZAP-70 y la expresión aberrante del miR-21. OBJETIVOS: Determinar la expresión de miR-21 en líneas celulares B y en células primarias de LLC y su asociación con la expresión de ZAP-70 en la LLC. MATERIAL Y MÉTODOS: Análisis de la expresión de miR-21 en la línea celular transfectada con ZAP-70 y en células de LLC. RESULTADOS: Se observó mayor expresión de miR-21 en las células con ZAP-70 tras estimulación del BCR y una correlación positiva entre la expresión de miR-21 y la de ZAP-70.La leucèmia linfàtica crònica (LLC) està associada a factors biològics com l'expressió de la proteïna ZAP-70 i l'expressió aberrant de miR-21. OBJECTIUS: Determinar l'expressió de miR-21 en les línees cel·lulars B i en les cèl·lules primàries de LLC i la seva associació amb l'expressió de ZAP-70 en la LLC. MATERIAL i MÈTODES: Anàlisi de l'expressió de miR-21 en la línia cel·lular transfectada amb ZAP-70 i en les cèl·lules de LLC. RESULTATS: S'observà una major expressió de miR-21 en les cèl·lules amb ZAP-70 després de l'estimulació del BCR i una correlació positiva entre l'expressió de miR-21 i ZAP-70

Regulación de la expresión del microRNA miR-21 por la proteína ZAP-70 en células de leucemia linfática crónica

La leucemia linfática crónica (LLC) está asociada a factores biológicos como la expresión de la proteína ZAP-70 y la expresión aberrante del miR-21. OBJETIVOS: Determinar la expresión de miR-21 en líneas celulares B y en células primarias de LLC y su asociación con la expresión de ZAP-70 en la LLC. MATERIAL Y MÉTODOS: Análisis de la expresión de miR-21 en la línea celular transfectada con ZAP-70 y en células de LLC. RESULTADOS: Se observó mayor expresión de miR-21 en las células con ZAP-70 tras estimulación del BCR y una correlación positiva entre la expresión de miR-21 y la de ZAP-70.La leucèmia linfàtica crònica (LLC) està associada a factors biològics com l'expressió de la proteïna ZAP-70 i l'expressió aberrant de miR-21. OBJECTIUS: Determinar l'expressió de miR-21 en les línees cel·lulars B i en les cèl·lules primàries de LLC i la seva associació amb l'expressió de ZAP-70 en la LLC. MATERIAL i MÈTODES: Anàlisi de l'expressió de miR-21 en la línia cel·lular transfectada amb ZAP-70 i en les cèl·lules de LLC. RESULTATS: S'observà una major expressió de miR-21 en les cèl·lules amb ZAP-70 després de l'estimulació del BCR i una correlació positiva entre l'expressió de miR-21 i ZAP-70

Clinical characteristics and outcome of SARS-CoV-2 infection in admitted patients with chronic lymphocytic leukemia from a single European country

Infección por SARS-CoV-2; Leucemia linfocítica crónicaInfecció per SARS-CoV-2; Leucèmia limfocítica crònicaSARS-CoV-2 infection; Chronic lymphocytic leukemi

Increased anandamide induced relaxation in mesenteric arteries of cirrhotic rats. Role of cannabinoid and vanilloid receptors

Background and aims: Anandamide is an endocannabinoid that evokes hypotension by interaction with peripheral cannabinoid CB1 receptors and with the perivascular transient receptor potential vanilloid type 1 protein (TRPV1). As anandamide has been implicated in the vasodilated state in advanced cirrhosis, the study investigated whether the mesenteric bed from cirrhotic rats has an altered and selective vasodilator response to anandamide. Methods: We assessed vascular sensitivity to anandamide, mRNA and protein expression of cannabinoid CB1 receptor and TRPV1 receptor, and the topographical distribution of cannabinoid CB1 receptors in resistance mesenteric arteries of cirrhotic and control rats. Results: Mesenteric vessels of cirrhotic animals displayed greater sensitivity to anandamide than control vessels. This vasodilator response was reverted by CB1 or TRPV1 receptor blockade, but not after endothelium denudation or nitric oxide inhibition. Anandamide had no effect on distal femoral arteries. CB1 and TRPV1 receptor protein was higher in cirrhotic than in control vessels. Neither CB1 mRNA nor protein was detected in femoral arteries. Immunochemistry showed that CB1 receptors were mainly in the adventitia and in the endothelial monolayer, with higher expression observed in vessels of cirrhotic rats than in controls. Conclusions: These results indicate that anandamide is a selective splanchnic vasodilator in cirrhosis which predominantly acts via interaction with two different types of receptors, CB1 and TRPV1 receptors, which are mainly located in perivascular sensory nerve terminals of the mesenteric resistance arteries of these animals

Repolarization of tumor infiltrating macrophages and increased survival in mouse primary CNS lymphomas after XPO1 and BTK inhibition

XPO1; BTK; Sistema immunitari innatXPO1; BTK; Sistema inmune innatoXPO1; BTK; Innate immune systemBackground Patients diagnosed with primary central nervous system lymphoma (PCNSL) often face dismal outcomes due to the limited availability of therapeutic options. PCNSL cells frequently have deregulated B-cell receptor (BCR) signaling, but clinical responses to its inhibition using ibrutinib have been brief. In this regard, blocking nuclear export by using selinexor, which covalently binds to XPO1, can also inhibit BCR signaling. Selinexor crosses the blood–brain barrier and was recently shown to have clinical activity in a patient with refractory diffuse large B-cell lymphoma in the CNS. We studied selinexor alone or in combination with ibrutinib in pre-clinical mouse models of PCNSL. Methods Orthotopic xenograft models were established by injecting lymphoma cells into the brain parenchyma of athymic mice. Tumor growth was monitored by bioluminescence. Malignant cells and macrophages were studied by immunohistochemistry and flow cytometry. Results Selinexor blocked tumor growth and prolonged survival in a bioluminescent mouse model, while its combination with ibrutinib further increased survival. CNS lymphoma in mice was infiltrated by tumor-promoting M2-like macrophages expressing PD-1 and SIRPα. Interestingly, treatment with selinexor and ibrutinib favored an anti-tumoral immune response by shifting polarization toward inflammatory M1-like and diminishing PD-1 and SIRPα expression in the remaining tumor-promoting M2-like macrophages. Conclusions These data highlight the pathogenic role of the innate immune microenvironment in PCNSL and provide pre-clinical evidence for the development of selinexor and ibrutinib as a new promising therapeutic option with cytotoxic and immunomodulatory potential.This work was supported by research funding from the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI17/00950, M.C., PI17/00943, F.B, PI18/01392, P.A., PI16/01278, J.S) cofinanced by the European Regional Development Fund (ERDF); Fundación Asociación Española Contra el Cáncer (M.C. and P.A.) and Gilead Fellowships (GLD16/00144, GLD18/00047, F.B). M.C. holds a contract from Ministerio de Ciencia, Innovación y Universidades (RYC-2012-12018). S.B. is the recipient of a postdoctoral fellowship from Fundación Alfonso Martin Escudero