Explore the vast range of titles available.

In this report we offer the widest comparison of links removal (attack) strategies efficacy in impairing the robustness of six real-world complex weighted networks. We test eleven different link removal strategies by computing their impact on network robustness by means of using three different measures, i.e. the largest connected cluster ( LCC ), the efficiency ( Eff ) and the total flow ( TF ). We find that, in most of cases, the removal strategy based on the binary betweenness centrality of the links is the most efficient to disrupt the LCC . The link removal strategies based on binary-topological network features are less efficient in decreasing the weighted measures of the network robustness (e.g. Eff and TF ). Removing highest weight links first is the best strategy to decrease the efficiency ( Eff ) in most of the networks. Last, we found that the removal of a very small fraction of links connecting higher strength nodes or of highest weight does not affect the LCC but it determines a rapid collapse of the network efficiency Eff and the total flow TF . This last outcome raises the importance of both to adopt weighted measures of network robustness and to focus the analyses on network response to few link removals.

Background Resistance to osimertinib in advanced EGFR- mutated non-small cell lung cancer (NSCLC) constitutes a significant challenge for clinicians either in terms of molecular diagnosis and subsequent therapeutic implications. Methods This is a prospective single-centre study with the primary objective of characterising resistance mechanisms to osimertinib in advanced EGFR- mutated NSCLC patients treated both in first- and in second-line. Next-Generation Sequencing analysis was conducted on paired tissue biopsies and plasma samples. A concordance analysis between tissue and plasma was performed. Results Sixty-five advanced EGFR -mutated NSCLC patients treated with osimertinib in first- ( n  = 56) or in second-line ( n  = 9) were included. We managed to perform tissue and liquid biopsies in 65.5% and 89.7% of patients who experienced osimertinib progression, respectively. Acquired resistance mechanisms were identified in 80% of 25 patients with post-progression samples, with MET amplification ( n  = 8), EGFR C797S ( n  = 3), and SCLC transformation ( n  = 2) the most frequently identified. The mean concordance rates between tissue and plasma for the EGFR activating mutation and for the molecular resistance mechanisms were 87.5% and 22.7%, respectively. Conclusions Resistance to osimertinib demonstrated to be highly heterogeneous, with MET amplification the main mechanism. Plasma genotyping is a relevant complementary tool which might integrate tissue analysis for the study of resistance mechanisms.

BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

We compared seven node vaccination strategies in twelve real-world complex networks. The node vaccination strategies are modeled as node removal on networks. We performed node vaccination strategies both removing nodes according to the initial network structure, i.e., non-adaptive approach, and performing partial node rank recalculation after node removal, i.e., semi-adaptive approach. To quantify the efficacy of each vaccination strategy, we used three epidemic spread indicators: the size of the largest connected component, the total number of infected at the end of the epidemic, and the maximum number of simultaneously infected individuals. We show that the best vaccination strategies in the non-adaptive and semi-adaptive approaches are different and that the best strategy also depends on the number of available vaccines. Furthermore, a partial recalculation of the node centrality increases the efficacy of the vaccination strategies by up to 80%.

In this study, we investigated the effects and the underlying molecular mechanisms of the multi-kinase inhibitor sorafenib in a panel of breast cancer cell lines. Sorafenib inhibited cell proliferation and induced apoptosis through the mitochondrial pathway. These effects were neither correlated with modulation of MAPK and AKT pathways nor dependent on the ERα status. Sorafenib promoted an early perturbation of mitochondrial function, inducing a deep depolarization of mitochondrial membrane, associated with drop of intracellular ATP levels and increase of ROS generation. As a response to this stress condition, the energy sensor AMPK was rapidly activated in all the cell lines analyzed. In MCF-7 and SKBR3 cells, AMPK enhanced glucose uptake by up-regulating the expression of GLUT-1 glucose transporter, as also demonstrated by AMPKα1 RNA interference, and stimulated aerobic glycolysis thus increasing lactate production. Moreover, the GLUT-1 inhibitor fasentin blocked sorafenib-induced glucose uptake and potentiated its cytotoxic activity in SKBR3 cells. Persistent activation of AMPK by sorafenib finally led to the impairment of glucose metabolism both in MCF-7 and SKBR3 cells as well as in the highly glycolytic MDA-MB-231 cells, resulting in cell death. This previously unrecognized long-term effect of sorafenib was mediated by AMPK-dependent inhibition of the mTORC1 pathway. Suppression of mTORC1 activity was sufficient for sorafenib to hinder glucose utilization in breast cancer cells, as demonstrated by the observation that the mTORC1 inhibitor rapamycin induced a comparable down-regulation of GLUT-1 expression and glucose uptake. The key role of AMPK-dependent inhibition of mTORC1 in sorafenib mechanisms of action was confirmed by AMPKα1 silencing, which restored mTORC1 activity conferring a significant protection from cell death. This study provides insights into the molecular mechanisms driving sorafenib anti-tumoral activity in breast cancer, and supports the need for going on with clinical trials aimed at proving the efficacy of sorafenib for breast cancer treatment.

Purpose To investigate whether eosinophils and other white blood cell subtypes could be used as response and prognostic markers to anti-Programmed cell Death-1 or anti-PD-Ligand-1 treatments in non-small cell lung cancer patients. Methods We retrospectively analyzed data from the NSCLC patients consecutively treated at our hospital with a PD-1/PD-L1 inhibitor in monotherapy for advanced disease. A total of 191 patients were evaluated at three time-points to investigate any relation between tumor response and WBC counts. Results Baseline WBC and subtypes did not differ according to the type of response seen under treatment. A higher relative eosinophil count (REC) correlated with more objective responses ( p  = 0.019 at t1 and p  = 0.014 at t2; OR for progression = 0.54 and 0.53, respectively) independently of the smoking status, PD-L1 status, and immune-related toxicity (IRT). Higher REC was also associated with a longer duration of treatment ( p  = 0.0096). Baseline absolute neutrophil count was prognostic ( p  = 0.049). At t1 relative lymphocytes, absolute and relative neutrophils, and neutrophil-to-lymphocyte ratio were prognostic ( p  = 0.044, p  = 0.014, p  = 0.0033, and p  = 0.029, respectively). Conclusion Our results show that in NSCLC patients anti-PD-1/PD-L1 therapy induces an early increase only in blood eosinophils, more prominent in responding patients and independent of the smoking status, PD-L1 status, and IRT. Eosinophils are also associated with a longer duration of treatment. Furthermore, our data support a prognostic role of neutrophils, lymphocytes, and their ratio for NSCLC patients with advanced disease treated with PD(L)-1 blockade.

Background:VEXAS syndrome is an adult-onset, X-linked, life-threatening, autoinflammatory disease with predominant hematological involvement caused by somatic mutation in UBA1 gene whose pathophysiology is still unknown.Objectives:To achieve a molecular and phenotypic characterization of hematopoiesis of VEXAS patients and to develop cellular and humanized mouse models by gene editing.Methods:Six VEXAS patients (p.Met41>Thr; p.Met41>Val; p.Met41>Leu; c.118-1 G>C) were recruited from our Unit. Variant allele frequency (VAF) of UBA1 mutant cells was quantified by targeted sequencing in isolated hematopoietic lineages and hematopoietic stem/progenitor cells (HSPCs). Multiparametric immunophenotypic analysis and single cell RNA seq were performed on peripheral blood and bone marrow (BM), focusing on HSPCs. Circulating monocytes were analyzed by whole RNA-seq and metabolome analysis. Healthy age and sex-matched controls were included. To introduce UBA1 mutations and develop VEXAS models, cutting-edge gene editing technologies were adopted in healthy human HSPCs.Results:Targeted sequencing in VEXAS patients showed >0.8 VAF in HSPCs. Conversely, VAF largely differed across mature cells, averaging 0.81 in neutrophils, 0.64 in monocytes, 0.42 in NK, 0.07 in T cells, and 0.09 in B cells, supporting a myeloid skewing of mutant HSPCs. Multiparametric immunophenotypic analyses showed unbalanced composition of HSPCs in the BM, with 2-to-3-fold reduction of primitive stem cells, multipotent and lymphoid progenitors, and 2-fold increase of myeloid progenitors, compared to matched healthy individuals. HSPCs, myeloid-biased HSPCs and immature myeloid cells were increased by 3-to-4 fold in the circulation (p<0.03). Gene expression analysis of circulating monocytes displayed upregulation of inflammatory pathways and metabolic rewiring (Figure. 1, panels A-B). Metabolomic analyses confirmed hyperactivation of the glycolytic pathway and specific lipid metabolism (Figure. 1, C-D). Single-cell RNA-seq of bone marrow mononuclear cells identified a subpopulation of CD34+ cell specific of VEXAS patients and revealed upregulated stress response and immune activation pathways across VEXAS cell clusters compared with healthy controls. Models of VEXAS generated by gene editing recapitulated patients’ hematopoiesis and pathophysiology in vitro and in vivo. UBA1 mutations were installed at VAF>0.9 in HSPCs and generated a myeloid bias in vitro. Transplantation of edited HSPCs in immunodeficient mice resulted in a 100-fold reduction in circulating B cells, while NK and myeloid compartments were preserved. Human BM HSPCs were 5-fold lower than control mice, largely myeloid-biased and presented abnormal vacuolar morphology. Concordantly, VAF was >0.8 in myeloid cells and HSPCs and <0.3 in B cells. Figure 1. Panel A. Gene expression analysis of peripheral monocytes in VEXAS patients compared to controls. Panel B. Gene-enrichmed pathway analysis in VEXAS patients compared to controls. Panel C. Metabolic analysis in VEXAS patients compared to controls. Panel D. Metabolic-enriched pathway analysis in VEXAS patients compared to controls.Figure 1.Conclusion:Mutations in UBA1 drive expansion of HSPCs and enhance myelopoiesis-guided accumulation of myeloid precursors. Mutant lymphoid cells are negatively selected and their myeloid counterpart in peripheral blood displays upregulation of transcriptomic signatures and metabolic pathways indicative of inflammatory activation. Gene editing-based models hold promise to enable preclinical testing and validation of novel therapeutics to treat VEXAS syndrome.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:Corrado Campochiaro: None declared, Raffaella Molteni: None declared, Guido Pacini: None declared, Martina Fiumara: None declared, Alessandro Tomelleri: None declared, Elisa Diral: None declared, Davide Stefanoni: None declared, Angelica Varesi: None declared, Alessandra Weber: None declared, Roberta Alfieri: None declared, Luisa Albano: None declared, Maddalena Panigada: None declared, Eleonora Cantoni: None declared, Daniele Canarutto: None declared, Luca Bassoricci: None declared, Pamela Quaranta: None declared, Angelo D’Alessandro: None declared, Marco Matucci-Cerinic: None declared, Raffaella Di Micco: None declared, Alessandro Aiuti: None declared, Fabio Ciceri: None declared, Ivan Merelli: None declared, Lorenzo Dagna: None declared, Serena Scala: None declared, Simone Cenci: None declared, Luigi Naldini: None declared, Samuele Ferrari: None declared, Giulio Cavalli Novartis, Novartis.

Background HER-2 represents a relatively new therapeutic target for non small cell lung cancer (NSCLC) patients. The incidence for reported HER-2 overexpression/amplification/mutations ranges from 2 to 20% in NSCLC. Moreover, HER-2 amplification is a potential mechanism of resistance to tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKI) (about 10% of cases). T-DM1, trastuzumab emtansine is an antibody-drug conjugate composed by the monoclonal antibody trastuzumab and the microtubule polymerization inhibitor DM1. The activity of T-DM1 has been studied in breast cancer but the role of T-DM1 in lung cancer remains unexplored. Methods Antiproliferative and proapoptotic effects of T-DM1 have been investigated in different NSCLC cell lines by MTT, crystal violet staining, morphological study and Western blotting. HER-2 expression and cell cycle were evaluated by flow cytometry and Western blotting. Antibody dependent cell cytotoxicity (ADCC) was measured with a CytoTox assay. Xenografted mice model has been generated using a NSCLC cell line to evaluate the effect of T-DM1 on tumor growth. Moreover, a morphometric and immunohistochemical analysis of tumor xenografts was conducted. Results In this study we investigated the effect of T-DM1 in a panel of NSCLC cell lines with different HER-2 expression levels, in H1781 cell line carrying HER-2 mutation and in gefitinib resistant HER-2 overexpressing PC9/HER2cl1 cell clone. T-DM1 efficiently inhibited proliferation with arrest in G2-M phase and induced cell death by apoptosis in cells with a significant level of surface expression of HER-2. Antibody-dependent cytotoxicity assay documented that T-DM1 maintained the same activity of trastuzumab. Our data also suggest that targeting HER-2 with T-DM1 potentially overcomes gefitinib resistance. In addition a correlation between cell density/tumor size with both HER-2 expression and T-DM1 activity was established in vitro and in an in vivo xenograft model. Conclusions Our results indicate that targeting HER-2 with T-DM1 may offer a new therapeutic approach in HER-2 over-expressing lung cancers including those resistant to EGFR TKIs.

Despite the initial response, all patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). The EGFR-T790M secondary mutation is responsible for half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5-15% of NSCLCs. Clinical findings indicate the retained addiction of resistant tumors on EGFR signaling. Therefore, we evaluated the molecular mechanisms supporting the therapeutic potential of gefitinib maintenance in the HCC827 GR5 NSCLC cell line harbouring MET amplification as acquired resistance mechanism. We demonstrated that resistant cells can proliferate and survive regardless of the presence of gefitinib, whereas the absence of the drug significantly enhanced cell migration and invasion. Moreover, the continuous exposure to gefitinib prevented the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and down-regulation of vimentin and N-cadherin. Importantly, the inhibition of cellular migration was correlated with the suppression of EGFR-dependent Src, STAT5 and p38 signaling as assessed by a specific kinase array, western blot analysis and silencing functional studies. On the contrary, the lack of effect of gefitinib on EGFR phosphorylation in the H1975 cells (EGFR-T790M) correlated with the absence of effects on cell migration and invasion. In conclusion, our findings suggest that certain EGFR-mutated patients may still benefit from a second-line therapy including gefitinib based on the specific mechanism underlying tumor cell resistance.

Background The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro . Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.