Leucemia linfática crónica B: una enfermedad heterogénea. Estudio del rol de las mutaciones de IgVH y BCL-6 en esta heterogeneidad: aproximaciones genómica y funcional.

RESUMEN La leucemia linfática crónica B (LLC-B) presenta un comportamiento clínico variable. Los pacientes sin mutaciones en la región variable del gen de la cadena pesada de las inmunoglobulinas (IgVH) sufren una enfermedad agresiva con menor tiempo libre de progresión y supervivencia global que los pacientes con IgVH mutado. Además, las mutaciones en el gen BCL6 proveen información clínica valiosa, permitiendo identificar un subgrupo de pacientes con alto riesgo de progresión, a pesar de tener mutaciones en IgVH. Sin embargo, el conocimiento de la biología subyacente en estos subgrupos moleculares de LLC-B aún no ha sido completamente dilucidado. El objetivo de este trabajo es el de valorar la implicación de las mutaciones en IgVH y BCL-6 como responsables de la heterogeneidad en el comportamiento biológico y expresión génica en la LLC-B en pacientes en estadío temprano de la enfermedad, para luego integrar la información obtenida con el fin de hallar genes y/o rutas metabólicas implicados e identificar posibles dianas diagnósticas/ pronósticas en LLC-B. Los estudios funcionales en células de LLC-B indican que, la inducción de apoptosis in vitro por Bortezomib (inhibidor de proteasoma) es estadísticamente diferente cuando las muestras se agrupan de acuerdo al estdo mutacional del gen BCL6, en el grupo de muestras mutadas en IgVH. Además, pudimos demostrar que esta inducción de apoptosis es dependiente del tiempo, de la dosis del fármaco utilizado, y de la activación de caspasas. Avanzando sobre los mecanismos que pudieran ocasionar un fracaso terapéutico, mediante modelos de co-cultivos, hemos comprobado que tanto células dendríticas como de estroma medular, son capaces de proteger a las células de LLC de la inducción de apoptsis por Bortezomib. En el estudio transcriptómico, encontramos unos 150 genes con expresión diferencial de acuerdo al estado mutacional de IgVH, mientras que no pudimos identificar ninguna diferencia significativa de acuerdo a las mutaciones en BCL6. Un grupo de 15 genes fueron escogidos para validar nuestros resultados, y cuantificamos su expresión por PCR cuantitativa a tiempo real, corroborándose los hallazgos obtenidos en el estudio de microarrays. Además, el uso de herramientas bioinformáticas para asignar perfiles funcionales, permitió distinguir cambios de expresión de manera coordinada en grupos de genes pertenecientes a la determinadas vías KEGG o agrupados dentro de un mismo término de ontología génica. La identificación de nuevos marcadores pronósticos en LLC fue el siguiente paso realizado en este estudio, para lo cual utilizamos un grupo muestral independiente para validar biológicamente los hallazgos provenientes del estudio de perfiles de expresión. Así, mediante el uso de PCR cuantitativa a tiempo real demostramos que la expresión de LPL, ZAP70, CRY1, BCL7A, DUSP22/BCL7A o CD82/BCL7A, determinados en linfocitos CD19+, correlacionan con el estado mutacional de IgVH y con el tiempo libre de tratamiento en pacientes con LLC en estadíos tempranos. Nuestros resultados mostraron además que CRY1 exhibía muy buena sensibilidad y especificidad al ser utilizado como marcador subrogado del estudio mutacional del gen IgVH . CRY1 también resultó ser un excelente biomarcador de necesidad de tratamiento, pudiendo utilizarse sobre muestras de LLC sin necesidad de seleccionar linfocitos B, por lo que este marcador podría ser introducido en la práctica clínica debido a lo simple de su metodología de cuantificación que es menos laboriosa de realizar que el estudio mutacional de IgVH. En conclusión, los estudios de expresión génica señalan que existen subgrupos moleculares bien definidos dentro de la LLC-B, sobre todo en base al estado mutacional de IgVH. Existen varios genes cuya expresión podría utilizarse como marcador de pronóstico en LLC-B, siendo CRY1, un componente esencial del reloj biológico, uno de los que proponemos para ser incorporado en la práctica clínica. __________________________________________________________________________________________________CLL is a heterogeneous disease with a variable clinical course. Patients with unmutated IgVH gene show a shorter progression free and overall survival than patients with IgVH mutated. In addition, BCL6 mutations identifies a subgroup of patients with high risk of progression. Anyway, the underlying biologic process involved in the different behaviour of the CLL molecular subgroups is not very well understood. The aim of this work is to evaluate the role of IgVH and BCL6 mutations as responsible of the different biologic behaviour and gene expression profiles encounter in subgroups of early-stage CLL patients and also, the identification of new diagnostic/prognostic markers for the disease. Functional in vitro studies reveal that IgVH/BCL6 subgroups have different behavior in response to apoptosis induction by Bortezomib, a farmacologic agent use for proteasome inhibition. Gene expression analysis was performed using a high density microarrays. Around 150 genes differentially expressed were found according to IgVH mutations, whereas no difference was found according to BCL6 mutations. Functional profiling methods allowed us to distinguish KEGG and GO terms showing coordinated gene expression changes across subgroups of CLL. Biologic validation of a set of differentially expressed genes by RTqPCR revealed that LPL, ZAP70, CRY1, BCL7A, DUSP22/BCL7A or CD82/BCL7A, quantified on CD19+cells are reliable biomarkers to assess IgVH mutational status and also prognostic markers in early-stage CLL patients. Among them, CRY1, a gene involved in circadian rhythm control, performed as good or better than other well established biomarkers in CLL. This observation, along with the easiness of CRY1 determination by RTqPCR on B lymphocytes or unselected PBMC, indicate that CRY1 may be used as a new marker of disease progression for the initial prognostic assessment of early-stage CLL. In addition, it would be interesting to introduce this marker in clinical trials for its evaluation in larger cohorts of patients

Ipilimumab

En definitiva, el desarrollo del ipilimumab, el primer anticuerpo contra un punto de control inmunológico, ha demostrado su eficacia en el control de diversos tumores malignos, ha iniciado el renacer de la inmunooncología que está revolucionando el tratamiento de estas enfermedades y está generando nuevas esperanzas a los pacientes. Esta revolución ha supuesto nuevas formas de evaluar la eficacia de los tratamientos y están apareciendo nuevos efectos adversos que los profesionales sanitarios están aprendiendo a diagnosticar y tratar con eficacia y seguridad. En conclusión, con el desarrollo del ipilimumab se ha iniciado una auténtica revolución en el campo de la oncología y nuevas esperanzas en el tratamiento del cáncer.Sirera Pérez, R.; Jantus-Lewintre, E. (2019). Ipilimumab. Inmunología. 38(4):25-27. http://hdl.handle.net/10251/157310S252738

Inmunoterapia del cáncer. Realidades y perspectivas.

Sirera Pérez, R.; Jantus-Lewintre, E. (2019). Inmunoterapia del cáncer. Realidades y perspectivas. Inmunología. 38(1):33-34. http://hdl.handle.net/10251/157287S333438

MicroRNAs: Promising New Antiangiogenic Targets in Cancer

[EN] MicroRNAs are one class of small, endogenous, non-coding RNAs that are approximately 22 nucleotides in length; they are very numerous, have been phylogenetically conserved, and involved in biological processes such as development, differentiation, cell proliferation, and apoptosis. MicroRNAs contribute to modulating the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules and so they play a key role in both health and disease. Angiogenesis is the process of new blood vessel formation from preexisting ones, which is particularly relevant to cancer and its progression. Over the last few years, microRNAs have emerged as critical regulators of signalling pathways in multiple cell types including endothelial and perivascular cells. This review summarises the role of miRNAs in tumour angiogenesis and their potential implications as therapeutic targets in cancer.This study was partially supported by a Grant from Ministerio de Ciencia e Inovacion de Espana (TRA09-0132), Beca Roche en Onco-Hematologia 2009, and Red Tematica de Investigacion Cooperativa en Cancer (RD12/0036/0025).Gallach, S.; Calabuig-Farinas, S.; Jantus Lewintre, E.; Camps, C. (2014). MicroRNAs: Promising New Antiangiogenic Targets in Cancer. BioMed Research International. 2014. https://doi.org/10.1155/2014/878450S201

Comprehensive cross-platform comparison of methods for non-invasive EGFR mutation testing: results of the RING observational trial

Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next-generation sequencing (NGS)-based methods, three high-sensitivity PCR-based platforms, and two FDA-approved methods were compared using 72 plasma samples, from EGFR-mutant non-small-cell lung cancer (NSCLC) patients progressing on a first-line tyrosine kinase inhibitor (TKI). NGS platforms as well as high-sensitivity PCR-based methodologies showed excellent agreement for EGFR-sensitizing mutations (K = 0.80–0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86–0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false-positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs

miRNA detection methods and clinical implications in lung cancer

[EN] Lung cancer is the leading cause of cancer death worldwide. Therefore, advances in the diagnosis and treatment of the disease are urgently needed. miRNAs are a family of small, noncoding RNAs that regulate gene expression at the transcriptional level. miRNAs have been reported to be deregulated and to play a critical role in different types of cancer, including lung cancer. Thus, miRNA profiling in lung cancer patients has become the core of several investigations. To this end, the development of a multitude of platforms for miRNA profiling analysis has been essential. This article focuses on the different technologies available for assessing miRNAs and the most important results obtained to date in lung cancer.This study was partially supported by a grant from the Ministerio de Ciencia e Inovacion de Espana (TRA09-0132), Beca Roche en Onco-Hematologia 2009 and Red Tematica de Investigacion Cooperativa en Cancer (RD12/0036/0025). 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Lung tumorspheres as a drug screening platform against cancer stem cells

Treatment resistance and metastasis are linked to cancer stem cells (CSCs). This population represents a promising target, but remains unexplored in lung cancer. The main objective of this study was to characterize lung CSCs and discover new therapeutic strategies

Analysis of the prognostic role of an immune checkpoint score in resected non-small cell lung cancer patients

This is an Accepted Manuscript of an article published by Taylor & Francis in Oncoimmunology on 2017, available online: http://www.tandfonline.com/10.1080/2162402X.2016.1260214[EN] Tumors develop mechanisms to recruit tolerogenic immune cells and to induce the expression of molecules that act as immune checkpoints. This regulation of the immune microenvironment favors immune tolerance to the neoplastic cells. In this study, we have investigated the prognostic role of immune-checkpoint expression markers in a cohort of resectable non-small cell lung cancer (NSCLC) patients. RNA was isolated from fresh-frozen lung specimens (tumor and normal lung) (n = 178). RTqPCR was performed to analyze the relative expression of 20 immune-related genes that were normalized by the use of endogenous genes selected by GeNorm algorithm. Patients with higher expression levels of IL23A and LGALS2 presented better outcomes. In the clustering expression patterns, we observed that patients with higher expression of immunoregulatory genes had better survival rates. Additionally, these data were used to develop a gene expression score. Since CTLA4 and PD1 were associated with prognosis based on Cox regression analysis (Z-score > 1.5), a multivariate model including these two genes was created. Absolute regression coefficients from this analysis were used in order to calculate the immunecheckpoint score: (PD1 x 0.116) + (CTLA4 x 0.059) for each case. Kaplan-Meier survival analysis showed that patients with high immune-checkpoint score have longer overall survival (OS) [NR vs. 40.4 mo, p = 0.008] and longer relapse-free survival (RFS) [82.6 vs. 23 mo, p = 0.009]. Multivariate analysis in the entire cohort indicated that the immune-checkpoint score was an independent biomarker of prognosis for OS [HR: 0.308; 95% CI, 0.156-0.609; p = 0.001] and RFS [HR: 0.527; 95% CI, 0.298-0.933; p = 0.028] in early-stage NSCLC patients. In conclusion, this score provides relevant prognostic information for a better characterization of early stage NSCLS patients with strikingly different outcomes and who may be candidates for immune-based therapies.This work was supported by the Red Tematica de Investigacion Cooperativa en Cancer (RD12/0036/0025) and the Fondo de Investigacion Sanitaria-Fondo Europeo de Desarrollo Regional (PI09/01147, PI09/01149 and PI12/02838)Usó, M.; Jantus-Lewintre, E.; Calabuig-Farinas, S.; Blasco, A.; Garcia Del Olmo, E.; Guijarro, R.; Martorell, M.... (2017). Analysis of the prognostic role of an immune checkpoint score in resected non-small cell lung cancer patients. Oncoimmunology (Online). 6(1):1-10. https://doi.org/10.1080/2162402X.2016.1260214S1106

CD5 and CD6 as immunoregulatory biomarkers in non-small cell lung cancer

Background: The study of immune surveillance in the tumour microenvironment is leading to the development of new biomarkers and therapies. The present research focuses on the expression of CD5 and CD6 - two lymphocyte surface markers involved in the fine tuning of TCR signaling - as potential prognostic biomarkers in resectable stages of non-small cell lung cancer (NSCLC). Methods: CD5 and CD6 gene expression was analysed by reverse transcription quantitative polymerase chain reaction (RTqPCR) in 186 paired fresh frozen tumour and normal tissue samples of resected NSCLC. Results: Patients with higher CD5 expression had significantly increased overall survival (OS, 49.63 vs. 99.90 months, p=0.013). CD5 expression levels were correlated to CD4 infiltration and expression levels, and survival analysis showed that patients with a higher CD5/CD4+ ratio had significantly improved prognosis. Multivariate analysis established CD5 expression as an independent prognostic biomarker for OS in early stages of NSCLC [HR=0.554; 95% CI, 0.360-0.853; p=0.007]. Further survival analysis of NSCLC cases (n=97) from The Cancer Genome Atlas (TCGA) database, confirmed the prognostic value of both CD5 and CD6 expression¸ although CD6 expression alone did not reach significant prognostic value in our NSCLC training cohort. Conclusions: Our data support further studies on CD5 and CD6 as novel prognostic markers in resectable NSCLC and other cancer types (i.e., melanoma), as well as a role for these receptors in immune surveillance

Soluble galectin-3 as a microenvironment-relevant immunoregulator with prognostic and predictive value in lung adenocarcinoma

Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin-3 (GAL-3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non-small cell lung cancer (NSCLC) patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early-stage patients and commercial cell lines were cultured, using tumorsphere-forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL-3 using reverse transcription-quantitative real-time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry and immunoassay analysis. Our results using three-dimensional (3D) models of lung tumor cells revealed that soluble GAL-3 (sGAL-3) is highly expressed and secreted. To more accurately mimic the TME, a co-culture of tumorspheres and fibroblasts was used, revealing that GAL-3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (TREGS). In the translational phase, we confirmed that patients with high expression levels of GAL-3 had more TREGS, which suggests that tumors may be recruiting this population through GAL-3. Next, we evaluated levels of sGAL-3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL-3 could be used as an independent prognostic biomarker for overall survival and relapse-free survival in early-stage LUAD patients. Additionally, levels of sGAL-3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first-line pembrolizumab were evaluated, further supporting that sGAL-3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve (AUC) of 0.801 (p=0.011). Moreover, high levels might predict decreased progression-free survival and overall survival to anti-PD-1 therapy, with sGAL-3 being a prognosis-independent biomarker for advanced LUAD