Cancer genomics paves the way to targeted therapy.

RESUMEN: La lucha contra el cáncer es aún un desafío mayor, con cerca de 14 millones de nuevos casos de cáncer al año y más de 8 millones de muertes anuales atribuidas al cáncer. Con la ayuda de múltiples servicios clínicos del HUMV y otras Instituciones, trabajamos para demostrar la hipótesis de que análisis integrados de genómica y secuenciación dirigida de alta profundidad en especímenes quirúrgicos de rutina puede generar datos firmes y relevantes sobre la complejidad molecular, composición subclonal, índice mutacional, firmas mutacionales y mutaciones precisas en genes con implicaciones terapéuticas; así generando una herramienta diagnóstica robusta que permita predecir sensibilidad a terapias específicas. In este proyecto, hemos podido demostrar que los estudios genómicos del cáncer demuestran dianas útiles para la intervención terapéutica y que la combinación de múltiples terapias inactivando rutas oncogénicas convergentes representa una opción plausible para pacientes con cáncer avanzado.ABSTRACT: Cancer is still a mayor challenge with something more than 14M new cases per year in the world and more of 8M patients dying yearly because of cancer. With the collaboration of multiple clinical services at the HUMV and other clinical institutions, we are working to demonstrate the hypothesis that genomics integrative analysis and high-depth targeted mutational analysis in routine cancer specimens may generate consistent, relevant data informing about molecular complexity, subclonal composition, mutational rate, mutational signatures and precise mutations in genes with therapeutic implications; thus generating a robust, solid, diagnostic tool that may allow to predict the sensitivity to specific therapies. In this project we have been able to demonstrate that cancer genome studies do demonstrate actionable targets, and that the combination of multiple therapies targeting convergent pathways represent a plausible option for advanced cancer patients

Increased risk of MAFLD and liver fibrosis in inflammatory bowel disease independent of classic metabolic risk factors

ackground & Aims There is conflicting evidence regarding the prevalence of and risk factors for metabolic-associated fatty liver disease (MAFLD) in patients with inflammatory bowel disease (IBD). We aimed to determine MAFLD prevalence and risk factors in IBD patients. Methods Cross-sectional, case-control study included all consecutive IBD patients treated at 2 different university hospitals. Controls were subjects randomly selected from the general population and matched by age, sex, type 2 diabetes status, and body mass index in a 1:2 ratio. MAFLD was confirmed by controlled attenuation parameter. Liver biopsies were collected when MAFLD with significant liver fibrosis was suspected. In addition, age- and fibrosis stage-paired non-IBD patients with biopsy-proven MAFLD served as a secondary control group. Results Eight hundred thirty-one IBD patients and 1718 controls were included. The prevalence of MAFLD and advanced liver fibrosis (transient elastography ≥9.7 kPa) was 42.00% and 9.50%, respectively, in IBD patients and 32.77% and 2.31%, respectively, in the general population (P < .001). A diagnosis of IBD was an independent predictor of MAFLD (adjusted odds ratio, 1.99; P < .001) and an independent risk factor for advanced liver fibrosis (adjusted odds ratio, 5.55; P < .001). Liver biopsies were obtained from 40 IBD patients; MAFLD was confirmed in all cases, and fibrosis of any degree was confirmed in 25 of 40 cases (62.5%). Body mass index and type 2 diabetes prevalence were significantly lower in IBD-MAFLD patients than in severity-paired patients with biopsy-proven MAFLD. Conclusions MAFLD and liver fibrosis are particularly prevalent in IBD patients, regardless of the influence of classic metabolic risk factors.Acknowledgements: The authors report funding support from the Spanish Instituto de Salud Carlos III-FEDER Grant (FIS - PI18/01304) related to this manuscript

Applied diagnostics in liver cancer. Efficient combinations of sorafenib with targeted inhibitors blocking AKT/mTOR

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. There is increasing interest in developing specific markers to serve as predictors of response to sorafenib and to guide targeted therapy. Using a sequencing platform designed to study somatic mutations in a selection of 112 genes (HepatoExome), we aimed to characterize lesions from HCC patients and cell lines, and to use the data to study the biological and mechanistic effects of case-specific targeted therapies used alone or in combination with sorafenib. We characterized 331 HCC cases in silico and 32 paired samples obtained prospectively from primary tumors of HCC patients. Each case was analyzed in a time compatible with the requirements of the clinic (within 15 days). In 53% of the discovery cohort cases, we detected unique mutational signatures, with up to 34% of them carrying mutated genes with the potential to guide therapy. In a panel of HCC cell lines, each characterized by a specific mutational signature, sorafenib elicited heterogeneous mechanistic and biological responses, whereas targeted therapy provoked the robust inhibition of cell proliferation and DNA synthesis along with the blockage of AKT/mTOR signaling. The combination of sorafenib with targeted therapies exhibited synergistic anti-HCC biological activity concomitantly with highly effective inhibition of MAPK and AKT/mTOR signaling. Thus, somatic mutations may lead to identify case-specific mechanisms of disease in HCC lesions arising from multiple etiologies. Moreover, targeted therapies guided by molecular characterization, used alone or in combination with sorafenib, can effectively block important HCC disease mechanisms.FUNDING: Grants from ISCIII, co-financed by the European Union (FEDER) (PI16/00156), Ramón and Cajal research program from MINECO (RYC-2013-14097) and FUNDACIÓN LUCHAMOS POR LA VIDA to JPV. Grants from ISCIII (RD06/0020/0107-RD012/0036/0060) to MAP. Grant from ISCIII (Ref. PIE15/00079) to JC & JPV. NGD is a recipient of a UC-IDIVAL pre-doctoral fellow. I.V. was also supported by the Ramón and Cajal research program

Shared Oncogenic Pathways Implicated in Both Virus-Positive and UV-Induced Merkel Cell Carcinomas

Merkel cell carcinoma (MCC) is a highly malignant neuroendocrine tumor of the skin whose molecular pathogenesis is not completely understood, despite the role that Merkel cell polyomavirus can play in 55e90% of cases. To study potential mechanisms driving this disease in clinically characterized cases, we searched for somatic mutations using whole-exome sequencing, and extrapolated our findings to study functional biomarkers reporting on the activity of the mutated pathways. Confirming previous results, Merkel cell polyomavirus-negative tumors had higher mutational loads with UV signatures and more frequent mutations in TP53 and RB compared with their Merkel cell polyomavirus-positive counterparts. Despite important genetic differences, the two Merkel cell carcinoma etiologies both exhibited nuclear accumulation of oncogenic transcription factors such as NFAT or nuclear factor of activated T cells (NFAT), P-CREB, and P-STAT3, indicating commonly deregulated pathogenic mechanisms with the potential to serve as targets for therapy. A multivariable analysis identified phosphorylated CRE-binding protein as an independent survival factor with respect to clinical variables and Merkel cell polyomavirus status in our cohort of Merkel cell carcinoma patients.This work was supported by grants from Instituto de Salud-Carlos III (ISCIII); cofinanced by the European Union; (FEDER) (PI12/00357), and a Ramón and Cajal research program (MINECO; RYC-2013-14097) to JPV, Asociación Española Contra el Cáncer and ISCIII grants (RD06/0020/0107, RD012/0036/0060) to MAP, and Coordinated Project of Excellence inter-Institutos de investigación acreditados institutes (ISCIII; PIE15/00081) to MAP. The Ramón and Cajal research program also supports IV. SD was supported by the Torres Quevedo subprogram (MICINN; PTQ-12-05391)

COVID-19 outbreaks in a transmission control scenario: challenges posed by social and leisure activities, and for workers in vulnerable conditions, Spain, early summer 2020

Severe acute respiratory syndrome coronavirus 2 community-wide transmission declined in Spain by early May 2020, being replaced by outbreaks and sporadic cases. From mid-June to 2 August, excluding single household outbreaks, 673 outbreaks were notified nationally, 551 active (>6,200 cases) at the time. More than half of these outbreaks and cases coincided with: (i) social (family/friends’ gatherings or leisure venues) and (ii) occupational (mainly involving workers in vulnerable conditions) settings. Control measures were accordingly applied

Medicina de precisión en el carcinoma de células de Merkel y el melanoma cutáneo avanzado: implicaciones de la caracterización molecular en el diagnóstico, el pronóstico y la terapia dirigida

ABSTRACT: In this work, two types of aggressive skin cancer have been studied; Merkel cell carcinoma (MCC) and advanced cutaneous melanoma. Their study has been addressed starting with the molecular characterization of tumors by means of DNA sequencing techniques, whose results have been later used to develop potential translational applications to diagnosis and treatment. It has been designed a novel and original approach to molecularly characterize MCC tumors, consisting in a combination of mutational and immunohistochemical analysis. Such analysis has allowed the identification of new disease mechanisms, besides two adverse prognostic markers. Furthermore, it has been developed a targeted mutational analysis platform to characterize advanced cutaneous melanoma cases, in a time compatible with the clinic. As well, specific combinatorial therapies have been designed, which have shown a clear efficacy both ex vivo and in vivo.RESUMEN: En este trabajo se han estudiado dos tipos de cáncer de piel agresivos; el carcinoma de células de Merkel (MCC) y el melanoma cutáneo avanzado. Su estudio se ha abordado partiendo de la caracterización molecular de los tumores mediante el uso de técnicas de secuenciación de ADN, cuyos resultados se han utilizado posteriormente para desarrollar posibles aplicaciones traslacionales para el diagnóstico y el tratamiento. Se ha diseñado un abordaje novedoso y original para caracterizar molecularmente tumores de MCC, consistente en la combinación de análisis mutacionales e inmunohistoquímicos. Este análisis ha permitido identificar nuevos mecanismos de enfermedad, además de dos marcadores pronósticos adversos. También se ha desarrollado una plataforma de análisis mutacional dirigido para caracterizar casos de melanoma cutáneo avanzado al diagnóstico, en un tiempo compatible con la práctica clínica, y se han diseñado terapias de combinación específicas que han demostrado ser eficaces tanto ex vivo como in vivo.La financiación necesaria para la realización de esta tesis ha sido aportada por el Ministerio de Economía y Competitividad (RD12/0036/0060), el Ministerio de Educación y Ciencia (RD06/0020/0107), el Instituto de Investigación Carlos III (PIE15/00081 y PI12/00357) y la asociación Luchamos por la Vida (LPLV-2015, LPLV- 2016)

Comparison between the compound ETP-39010 and other pan-PIMi.

<p>(A) Selectivity profile showing the IC₅₀ values of each of the compounds for the kinase activity of the indicated enzymes. (B) Percentage of inhibition of a panel of unrelated kinases by ETP-39010 and ETP-47551. A similar profile was found for ETP-47551, ETP-47652 and ETP-46638 compounds. (C) Sensitivity of PTCL cell lines to all pan-PIMi. (D) The newly developed pan-PIMi ETP-47551 reduced cell viability in all studied PTCL cell lines (IC₅₀ values calculated after 72 h of treatment are shown). (E) The pan-PIMi ETP-47551 strongly induced apoptosis in a time-dependent manner in all studied PTCL cell lines (*, p<0.05, from comparison with DMSO-treated cells). The percentage of non-viable cells was calculated as Annexin V+/7AAD− plus Annexin V+/7AAD+ cells in the PIMi-treated condition minus the DMSO-treated control. (F) The combination of ALKi + ETP-39010 was highly synergistic only in ALK+ ALCL cell lines, as was (G) the combination of ALKi + ETP-47551 (Combination Index, CI, <1 indicates synergism between the two drugs; CI ≈1 indicates an additive effect; CI>1 indicates antagonism).</p

Synergism between ALK and PIM inhibition in ALCL.

<p>(A) ALK expression was explored by Western blot in 4 PTCL cell lines. (B) IC₅₀ values were measured upon 72 h treatment with the ALKi Crizotinib: ALK+ ALCL cell lines were around 10 times as sensitive to the ALKi as the ALK− cells. (C) Cells were treated for 24 h with IC₅₀ of ALKi and pan-PIMi, alone and combined. The combination of ALKi + PIMi was highly synergistic (Combination Index, CI<1) and strongly enhanced apoptosis in ALK+ ALCL cell lines after 24 h, while this combination was antagonistic in ALK− PTCL cell lines (CI>1) (*, p<0.05 in comparison with DMSO). Data represent Annexin V+/PI− and Annexin V+/PI+ cells in each treatment. Black columns highlight the combined treatment.</p

PIM kinases as potential therapeutic targets in PTCL.

<p>(A) Gene expression profiling of tumoral samples from 38 human PTCL patients compared with 6 reactive lymph nodes (LN) by microarrays revealed a significantly increased expression of <i>PIM1</i> and <i>PIM2</i> genes (FDR<0.05), but not <i>PIM3</i>. The heatmap is shown in the upper panel, and the relative quantification (Log₂ ratio) comparing PIM expression in PTCL <i>versus</i> LN is shown in the lower panel. (B) GSEA ranked all significantly altered genes between PTCL and LN according to its correlation with <i>PIM1</i> or <i>PIM2</i> expression and displayed them in the red-to-blue bar. Each gene belonging to every pathway was interrogated whether it appeared positively (in the red region of the bar) or negatively (in the blue side) correlated. Using this approach GSEA identified a positive and significant correlation between <i>PIM1</i> and <i>PIM2</i> expression and Jak/STAT, NF-κB and IL-2 signaling pathways in the PTCL molecular signature (FDR<0.25). (C) PIM family genes mRNA level was measured by RT-qPCR in eight PTCL cell lines and (D) primary tumoral T cells from 5 Sézary Syndrome patients (SS #1–5), and compared with normal T cells isolated from 3 healthy donors (Control #1–3). The relative RNA amount of PIM has been calculated as a relative quantification, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112148#s2" target="_blank">Methods</a> section (RQ = 2^−ΔCt), normalized with non-tumoral cells: RQ in PTCL/RQ in healthy #3. In both settings, <i>PIM1</i>, and especially <i>PIM2</i>, but not <i>PIM3</i> expression was found to be increased in PTCL. (E) PIM kinase protein basal levels in PTCL cell lines measured by Western blot. PIM1 and PIM2 isoforms are also shown. (F) Distribution of PIM2 protein in a series of tumoral samples from 136 PTCL patients measured by immunohistochemistry. Negative, weakly positive and strongly positive samples were defined by the presence of <5%, 5–20% and >20% positive cells. (G) Distribution of PIM2 protein in the most common PTCL subtypes measured by immunohistochemistry.</p