Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations

Estudo de Usabilidade de Assinaturas Digitais em Objetos Metrológicos

INMETR

Targeting CD34(+) cells of the inflamed synovial endothelium by guided nanoparticles for the treatment of rheumatoid arthritis

Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34 + endothelial precursors were addressed in inflamed synovium using targeted biodegradable nanoparticles (tBNPs). These nanostructures were made of poly-lactic acid, poly-caprolactone, and PEG and then coated with a synovial homing peptide. Immunofluorescence analysis clearly demonstrated their capacity to selectively address CD34 + endothelial cells in synovial tissue obtained from human, mouse, and rat. Biodistribution studies in two different animal models of rheumatoid arthritis (antigen-induced arthritis/AIA and collagen-induced arthritis/CIA) confirmed the selective accumulation in inflamed joints but also evidenced the capacity of tBNP to detect early phases of the disease and the preferential liver elimination. The therapeutic effect of methotrexate (MTX)-loaded tBNPs were studied in comparison with conventional MTX doses. MTX-loaded tBNPs prevented and treated CIA and AIA at a lower dose and reduced administration frequency than MTX. Moreover, MTX-loaded tBNP showed a novel mechanism of action, in which the particles target and kill CD34 + endothelial progenitors, preventing neo-angiogenesis and, consequently, synovial inflammation. tBNPs represent a stable and safe platform to develop highly-sensitive imaging and therapeutic approaches in RA targeting specifically synovial neo-angiogenesis to reduce local inflammation

Integrative methylome-transcriptome analysis unravels cancer cell vulnerabilities in infant MLL-rearranged B cell acute lymphoblastic leukemia

B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. As predicted by its prenatal origin, infant B-ALL (iB-ALL) shows an exceptionally silent DNA mutational landscape, suggesting that alternative epigenetic mechanisms may substantially contribute to its leukemogenesis. Here, we have integrated genome-wide DNA methylome and transcriptome data from 69 patients with de novo MLL-rearranged leukemia (MLLr) and non-MLLr iB-ALL leukemia uniformly treated according to the Interfant-99/06 protocol. iB-ALL methylome signatures display a plethora of common and specific alterations associated with chromatin states related to enhancer and transcriptional control in normal hematopoietic cells. DNA methylation, gene expression, and gene coexpression network analyses segregated MLLr away from non-MLLr iB-ALL and identified a coordinated and enriched expression of the AP-1 complex members FOS and JUN and RUNX factors in MLLr iB-ALL, consistent with the significant enrichment of hypomethylated CpGs in these genes. Integrative methylome-transcriptome analysis identified consistent cancer cell vulnerabilities, revealed a robust iB-ALL–specific gene expression–correlating dmCpG signature, and confirmed an epigenetic control of AP-1 and RUNX members in reshaping the molecular network of MLLr iB-ALL. Finally, pharmacological inhibition or functional ablation of AP-1 dramatically impaired MLLr-leukemic growth in vitro and in vivo using MLLr-iB-ALL patient–derived xenografts, providing rationale for new therapeutic avenues in MLLr-iB-ALL.We thank CERCA/Generalitat de Catalunya (SGR180) and Fundació Josep Carreras-Obra Social la Caixa for their institutional support. Financial support for this work was obtained from the European Research Council (CoG-2014-646903 and PoC-2018-811220 to PM), the Spanish Ministry of Economy and Competitiveness (SAF-2019-108160-R and SAF2016-76758-R to PM and IV, respectively), the Spanish Association against cancer (AECC-CI-2015 and PROYE18061FERN to CB and MFF), the Fundación Uno entre Cienmil (to PM), the Health Institute Carlos III (ISCIII/FEDER, PI17/01028, PI15/00892, PI18/01527 to CB and AFF/MFF, respectively). We also acknowledge the Plan de Ciencia, Tecnología e Innovación from the Asturias Government cofunding 2018–2022/FEDER (IDI/2018/146to MFF). MFF also acknowledges funding from Fundación General CSIC (0348_CIE_6_E). PM also acknowledges financial support from Fundación Leo Messi. JRT and MV are supported by Juan de la Cierva fellowships by the Spanish Ministry of Science and Innovation (FJCI-2015-26965, IJC2018-36825-I, IJCI-2017-3317) and IUOPA-ISPA-FINBA (The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain). RTR is supported by a fellowship from the AECC scientific foundation. RFP and PSO are supported by the Severo Ochoa program (BP17-114 and BP17-165, respectively).Peer reviewe

Diolein based nanostructures as targeted theranostics

Diolein based non-targeted theranostic nanoparticles (DO-NPs) containing 10%wt of the amphiphilic Gadolinium complex (C18)2DTPA(Gd), and targeted NPs, obtained by introducing growing amounts (3%wt, 6%wt or 10%wt) of (C18)2-Peg3000-FA in the sample composition, have been studied for their in vitro and in vivo properties. Cellular binding was studied by ICP-MS analysis of the Gadolinium content and by Surface Plasmon Resonance (SPR) assays. The best formulation in terms of selectivity towards IGROV-1 cells with respect to non-targeted DO-NPs, was that containing 3% (C18)2Peg3000-FA (P<0.01). Cytotoxic studies and confocal microscopy analysis of IGROV-1 cells indicate high selective properties of the targeted doxorubicin (DOX) loaded NPs. Nanoparticles described here represent the first example in which a targeted carrier characterized by a stable foamy mesophase, provided by the Diolein component, combine the therapeutic effect due to the anticancer drug doxorubicin, with the imaging properties provided by paramagnetic gadolinium complexes for MRI. As evidenced by T1w and T2w MRI images and by the in vivo antitumor effect in IGROV-1 tumor-bearing mice, DO-NP3-FA/DOX provides very high therapeutic efficacy with a tumor growth regression of 80% and 50% higher as compared to the mice treated with saline solution and with Doxil, respectively

In Vitro and In Vivo Assessment of Nonionic Iodinated Radiographic Molecules as Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Tumor Perfusion Agents

The aim of this study was to evaluate 4 nonionic x-ray iodinated contrast agents (CAs), commonly used in radiographic procedures, as novel chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) agents by assessing their in vitro exchange properties and preliminary in vivo use as tumor enhancing agents.The CEST properties, as function of pH (range, 5.5-7.9) and of radio frequency conditions (irradiation field strength range of 1-9 \u3bcT and time of 1-9 seconds), have been determined at 7 T and 310 K for 4 x-ray CAs commonly used in clinical settings, namely, iomeprol, iohexol, ioversol, and iodixanol. Their in vivo properties have been investigated upon intravenous injection in a murine HER2+ breast tumor model (n = 4 mice for each CA) using both computed tomography (CT) and MRI modalities.The prototropic exchange rates measured for the 4 investigated iodinated molecules showed strong pH dependence with base catalyzed exchange rate that was faster for monomeric compounds (20-4000 Hz in the pH range of 5.5-7.9). Computed tomography quantification showed marked (up to 2 mg I/mL concentration) and prolonged accumulation (up to 30 minutes postinjection) inside tumor regions. Among the 4 agents we tested, iohexol and ioversol display good CEST contrast properties at 7 T, and in vivo results confirmed strong and prolonged contrast enhancement of the tumors, with elevated extravasation fractions (74%-91%). A strong and significant correlation was found between CT and CEST-MRI tumor-enhanced images (R = 0.70, P < 0.01).The obtained results demonstrate that iohexol and ioversol, 2 commonly used radiographic compounds, can be used as MRI perfusion agents, particularly useful when serial images acquisitions are needed to complement CT information

Innate Immune Environment in Ileal Pouch Mucosa: \u3b1-5 Defensin Up-Regulation as Predictor of Chronic/Relapsing Pouchitis

Background Defensins are small cationic peptides with antibacterial activity expressed only in Paneth cells (\u3b1-defensins) or generally in intestinal epithelial cells (\u3b2-defensins) that have a profound effect on gut microbiota. We have recently shown that chronic pouchitis, that occurs in 5% of patients after restorative proctocolectomy and can cause pouch failure, is associated to a significant increase Clostridiaceae spp. The aim of this study was to gain further insight in the pathogenesis of chronic/relapsing pouchitis by exploring the interplay between the microbiota adherent to the ileal pouch mucosa and defensin expression. Patients and methods Thirty-two consecutive patients coming for follow up endoscopy, were recruited. On pouch biopsies we determined bacteria adherent to the mucosa, by culture method, and mRNA for \u3b1-and \u3b2-defensins and Toll Like Receptor-4 and -2, by quantitative Real Time RT-PCR. Serum and mucosal levels of IL-1\u3b2, IL-6 and TNF-\u3b1 were measured with immunometric assays. Faecal lactoferrin was analyzed by quantitative ELISA. After a median follow up of 23 (IQR 20-24) months the patients were contacted for a reassessment of current and past disease activity. Results During the follow up chronic/relapsing pouchitis was diagnosed in 6 patients. Mucosal level of \u3b1-5 and \u3b1-6 defensins correlated with chronic/ relapsing pouchitis onset (tau=0.30, p=0.034 and tau=0.28, p=0.053, respectively). High levels of \u3b1-5 defensin resulted predictive of chronic/relapsing pouchitis [AUC=74%(95% CI=53-89%), p=0.052]. Patients with high level of \u3b1-5 and \u3b1-6 defensins had earlier pouchitis relapses (p=0.009 and p=0.034, respectively). High levels of \u3b1-5 defensin were associated to a significant risk of chronic/relapsing pouchitis [OR=10.6(95% CI=1.2-97.6), p=0.027]. At multivariate analysis mucosal levels of \u3b1-5 defensin and the number of CFU of mucosa- associated Clostridiaceae spp resulted to be independent predictors of chronic/relapsing pouchitis [tau=0.46(0.18), p=0.024 and tau=0.44(0.18), p=0.027, respectively]. Conclusion Chronic/relapsing pouchitis is associated to increased expression of mucosal \u3b1-5 defensin. High levels of \u3b1-5 defensin may be the response to challenge of Clostridiaceae spp antigens or, alternatively they may favour Clostridiaceae spp decreasing other competing bacteria familiae

Mucosal Immune Environment in Colonic Carcinogenesis: CD80 Expression on Epithelial Cell Peaks in Dysplasia and Its Inhibition Leads to Carcinogenesis Progression

Background A significantly higher expression of the co-stimulatory molecule CD80 in ulcerat- ive colitis (UC) and dysplasia suggesting the presence of an immunosurveillance against cancerogenesis was recently observed. The aims of this study were to verify this hypothesis exploring the interplay between epithelium and CD8 T cell in human colonic carcinogenesis and evaluating the effect of CD80 signalling inhibition. Patients and methods Twenty nine patients diagnosed with UC, UC with colonic dysplasia, colonic adenoma, or colonic cancer were recruited at the moment of colonic resection. Mucosal samples were obtained during the colectomy and included specimens from healthy, dysplastic and neoplastic colon. Single cell suspensions were obtained by enzymatic digestion of mucosal specimens and subjected to Fluorescence-Activated Cell Sorting (FACS) to determine the proportion of epithelial cells (Cytokeratine 20, Cyt-20+) acting as antigen presenting cells (expressing CD80, CD40, HLA I or HLA II) and the proportion of CD8+ T cells activated (positive for CD28, CD38 or CD69). The second part of the study involved a mouse model of inflammation-related colon carcinogenesis with azoxymethane (AOM) and dextran sodium sulfate-induced (DSS). After the injection of AOM mice received 3 or 5 cycles of 3% DSS in their drinking water, each cycle consisted of 5 days of treatment followed by 14 days of rest. Animals were randomized to receive monoclonal anti-CD80 antibodies (10 ug/mouse) or isotype control, and sacrificed after 28 days. Results The proportion of Cyt-20+ CD80+ and Cyt-20+ HLA II+ cells was higher in dysplasia than in non dysplastic colonic tissue (p=0.03 and p=0.07, respectively). The proportion of Cyt-20+ CD80+ and Cyt-20+ CD40+ cells was higher in dysplasia than in invasive adenocarcinoma (p=0.07 and p=0.04, respectively). The proportion of CD8+ T cell expressing CD28 and CD38 was higher in dysplastic mucosa than in the mucosa with no neoplastic changes (p=0.01 and p=0.01, respectively). In mice that received anti-CD80 antibodies after AOM and 3 cycles of DSS inflammation was significantly inhibited (p=0.01) while high grade dysplasia extension was significantly higher than in mice that received a control antibody (p=0.01). The extension of low grade dysplasia was similar in the two groups. Conclusion This study confirmed that the proportion of epithelial cells acting as antigen presenting cells peaks in the dysplastic colonic mucosa and it is associated to activation of mucosal CD8+ T cell. CD80 inhibition increased high grade dysplasia extension without influencing the low grade dysplasia one. All together these data confirm the hypo- thesis that an active immunosurveillance mechanism centred on CD80 signalling is implicated in controlling the colonic carcinogenesis progression at the passage from low to high grade dysplasia