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Background: Tumor heterogeneity is a hurdle to effective therapy, as illustrated by the ‘mixed responses’ frequently seen in immunotherapy-treated patients. Previously, AXL+ tumor cells were identified to be highly resistant to targeted therapy, whereas more differentiated MITF+ tumor cells do respond to RAF and MEK inhibitors. Patients and methods: In this study, we analyzed tumor heterogeneity and explored the presence of the previously described AXL+ or MITF+ melanoma subpopulations in metastatic tissues by NanoString gene expression analysis, single-cell RNA sequencing and in situ multiplex immunofluorescence. Furthermore, we analyzed how these subpopulations correlate with immunological pressure and response to immunotherapy by immunomodulating antibodies or autologous tumor lysate-loaded dendritic cell vaccination. Results: Our data demonstrate large interpatient variability and variable therapy-induced changes independent of the type of therapy. We identify the presence of previously described AXL+ and MITF+ subpopulations in metastatic tissues both at the mRNA level and in situ at the protein level, and demonstrate that MITF+ melanoma cells are significantly decreased upon immunotherapy, while AXL+ melanoma cell numbers are stable. MITF+ tumor cells showed the most significant inverse correlation with CD8+ T cells. Our patient cohort also shows that immunotherapy-induced changes in the abundance of AXL+ or MITF+ tumor cells did not correlate with improved survival. Conclusions: Overall, this study suggests that more differentiated MITF+ tumors are efficiently targeted by immunotherapy, while AXL+ tumor cells may be more resistant, analogous to their response to targeted therapy.

RNA binding proteins (RBP) have a function in T cell activation and differentiation. Here the authors show that in the absence of two RBPs, ZFP36 and ZFP36L1, T cells acquire more rapidly superior effector capabilities, showing a limiting function for RBP in T cell activation and differentiation.

The present work represents the second and final part of a twofold study aiming at the definition and validation of an integrated methodology for the analysis and modeling of road tunnel ventilation systems. A numerical approach is presented, based on the Finite Volume integration of the 1D mechanical and thermal energy conservation equations on a network of ducts, representing the ventilation system of the 11.6 km long Mont Blanc Tunnel. The set of distributed and concentrated loss coefficients, representing dissipation of mechanical energy by friction in each part of the ventilation system, is calibrated against a rich experimental dataset, collected throughout a dedicated set of in situ tests and presented in the first part of the work. The calibration of the model is carried out by means of genetic optimization algorithms. Predictions of the flow field using the calibrated parameters are in remarkable agreement with the experimental data, with an overall RMS error of ± 0.27 m/s, i.e. of the same order of the accuracy of the measurement probes. Further validation against a selection of field data recorded by the tunnel monitoring and control system is brought forward, highlighting the robustness and potential general applicability of the proposed approach.

The knowledge of the flow field inside road tunnels under normal operation, let alone fire conditions, is only approximate and partial. The reason is that while the full three-dimensional, unsteady problem is out of reach of numerical methods, on the other hand accurate measurement of the airflow in road and railway tunnels constitutes an extremely demanding task. The present work, structured as a twofold study, takes up the challenge and proposes an original integrated experimental and numerical approach for the analysis and modeling of flow inside a road tunnel and its ventilation systems, aiming at defining a methodology for the creation of “digital twins” of the system itself, on which advanced ventilation and smoke control strategies can be tested and fine-tuned. In this first part, an innovative experimental facility for the continuous acquisition of the longitudinal velocity profile along the whole length of a road tunnel has been designed and built. The facility consists of a survey rake with five bidirectional vane anemometers, which is mounted on a small electric vehicle that can travel through the tunnel at constant speed. This paper reports the design procedure of the measurement facility, with particular focus on the conception and realization of the vehicle carrying the survey rake. Results of the first experimental campaign carried out under the 11611 meters long Mont Blanc road tunnel are presented to corroborate the validity of the approach adopted and the accuracy of the measurement chain.

Objective: Extensive stage Small-Cell Lung Cancer (ES-SCLC) is the most lethal lung cancer, and the addition of immunotherapy conferred a slight survival benefit for patients. Extensive molecular profiling of patients treated with chemotherapy (CT) or chemotherapy plus immunotherapy (CT+IO) would be able to identify molecular factors associated with patients' survival., Material and Methods: In this retrospective study, 99 ES-SCLC patients were considered. Of the 79 includible patients, 42 received CT (median age 71 y/o, I-IIIQ: 65-76), and 37 received CT+IO (median age 71 y/o, I-IIIQ 66-75). The FoundationOne CDx assay was performed on patients' tumor tissues., Results: The most mutated genes were TP53 (99%), RB1 (78%), PTEN (23%) and MLL2 (20%), with no significant differences between the treatment groups. As a continuous variable, Tumor Mutation Burden (TMB) had an effect on patients' progression-free survival (PFS) by type of treatment (HR 1.81 (95%, CI: 0.99-3.31) and HR 0.84 (95%, CI: 0.56-1.26) for patients treated with CT and CT+IO, respectively). TMB was also computed and dichotomized using two different cut-offs: considering cut-offs of 10 mut/Mb and >16 mut/Mb, 45 patients (57%) and 68 patients (86.1%) had a low TMB, respectively. A high TMB (cut-off 10 mut/Mb) predicted worse PFS in patients treated with CT ( p =0.046); even though not statistically significant, a high TMB (cut-off 16 mut/Mb) predicted a better survival in patients treated with CT+IO. Moreover, at univariate analysis, MLL2 mutations were associated with better prognosis in the overall case series (HR PFS = 0.51, 95% CI: 0.28-0.94), and overall survival (HR OS = 0.52, 95% CI: 0.28-0.97)., Conclusion: In ES-SCLC, TMB is associated with worse survival in patients treated with CT alone, and with better survival in patients treated with CT+IO, whether considered as a continuous or a dichotomized variable, at different cut-offs. Alterations in epigenetic factors are also associated to better patient prognosis., Competing Interests: The authors report no conflicts of interest in this work., (© 2025 Canale et al.)

COVID-19 remains a significant global health problem with uncertain long-term consequences for convalescents. We investigated the relationships between anti-N protein antibody levels, severe acute respiratory syndrome (SARS)-CoV-2-associated TCR repertoire parameters, HLA type and epidemiological information from three cohorts of 524 SARS-CoV-2-infected subjects subgrouped in acute phase, seronegative and seropositive convalescents from the Emilia Romagna region. Epidemiological information and anti-N antibody index were associated with TCR repertoire data. HLA type was inferred from TCR repertoire using the HLA3 tool and its association with clonal breadth (CB) and clonal depth (CD) was assessed. Age above 58 years, male and COVID-19 hospitalisation were significantly and independently associated with seropositivity (p = 0.004; p = 0.004; p = 0.04), suggesting an association between high antibody titres and symptoms' severity. As for the TCR repertoire analysis, we found no difference in CB among the cohorts, while CD was higher in seronegative than acute (p = 0.04). However, clustering analysis supported that seronegative patients are endowed with broader CB and deeper CD indicating a compensatory mechanism without effective seroconversion. The CD calculated on the TCRs associated with the single SARS-CoV-2 ORFs in convalescents is higher when compared to the acute. Lastly, we identified and reported on novel HLAs significantly associated with increased risk of hospitalisation such as HLA-C*07:02 carriers (OR = 3.9, CI = 1.1-13.4, p = 0.03) and on HLAs that associate significantly with lower or higher TCR repertoire parameters in a population exposed for the first time to SARS-CoV-2., (© 2025 The Author(s). HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

Background: Adjuvant trastuzumab is the standard of care for HER2+ breast cancer (BC) patients. However, >50% of patients become resistant. This study aimed at the identification of the molecular factors associated with disease relapse and their further investigation as therapeutically exploitable targets., Methods: Analyses were conducted on formalin-fixed paraffin-embedded tissues of the primary tumors of relapsed (cases) and not relapsed (controls) HER2+ BC patients treated with adjuvant trastuzumab. The nCounter Human Breast Cancer Panel 360 was used. Logistic regression and partitioning around medoids were employed to identify the genes associated with disease recurrence. Cytotoxicity experiments using trastuzumab-resistant cell lines and a network pharmacology approach were carried out to investigate drug efficacy., Results: A total of 52 patients (26 relapsed and 26 not relapsed) were analyzed. We found that a higher expression of HDAC6 was significantly associated with an increased risk of recurrence, with an adjusted OR of 3.20 (95% CI 1.38-9.91, p = 0.016). Then, we investigated the cytotoxic activity of the selective HDAC6 inhibitor Nexturastat A (NextA) on HER2+ cell lines, which were both sensitive and trastuzumab-resistant. A sub-cytotoxic concentration of NextA, combined with trastuzumab, showed a synergistic effect on BC cell lines. Finally, using a network pharmacology approach, we identified HSP90AA1 as the putative molecular candidate responsible for the synergism observed in vitro., Conclusions: Our findings encourage the exploration of the role of HDAC6 as a prognostic factor and the combinatorial use of HDAC6 selective inhibitors combined with trastuzumab in HER2+ BC, in particular for those patients experiencing drug resistance.

Background: The purpose of the study was to conduct a comprehensive genomic characterization of gene alterations, microsatellite instability (MSI), and tumor mutational burden (TMB) in submucosal-penetrating (Pen) early gastric cancers (EGCs) with varying prognoses., Methods: Samples from EGC patients undergoing surgery and with 10-year follow-up data available were collected. Tissue genomic alterations were characterized using Trusight Oncology panel (TSO500). Pathway instability (PI) scores for a selection of 218 GC-related pathways were calculated both for the present case series and EGCs from the TCGA cohort., Results: Higher age and tumor location in the upper-middle tract are significantly associated with an increased hazard of relapse or death from any cause (p = 0.006 and p = 0.032). Even if not reaching a statistical significance, Pen A tumors more frequently present higher TMB values, higher frequency of MSI-subtypes and an overall increase in PI scores, along with an enrichment in immune pathways. ARID1A gene was observed to be significantly more frequently mutated in Pen A tumors (p = 0.006), as well as in patients with high TMB (p = 0.027). Tumors harboring LRP1B alterations seem to have a higher hazard of relapse or death from any cause (p = 0.089), being mutated mainly in relapsed patients (p = 0.093)., Conclusions: We found that the most aggressive subtype Pen A is characterized by a higher frequency of ARID1A mutations and a higher genetic instability, while LRP1B alterations seem to be related to a lower disease-free survival. Further investigations are needed to provide a rationale for the use of these markers to stratify prognosis in EGC patients., (© 2024. The Author(s).)

Background: Hearing efficiency is known to influence and interact with communication and mental health. Hearing impairment may be hidden when co-occurring with neurological disorders., Purpose: We performed a systematic review and meta-analysis in order to address the following questions: 1) which specific tools of auditory processing show clear deficits, separating Temporal Lobe Epilepsy (TLE) patients from normal controls,2) How well is TLE evaluated in terms of hearing and auditory processing?, Methods: The study inclusion criteria were: 1) patients diagnosed with temporal lobe epilepsy, 2) presence of a normal control group, 3) auditory processing assessment using auditory stimuli with behavioral tests and/or P300 or Mitch Match Negativity (MMN) latency and/or amplitude, 4) publications written in English, 5) publication date after 2000. 132 articles were retrieved and based on PRISMA & PICO criteria 23 articles were analyzed., Results: Temporal resolution and processing as measured by the behavioral tests of Gaps-In-Noise (GIN) and Duration Pattern Test (DPT) document deficiencies in TLE patients and separate them from normal controls. Electrophysiology as measured by MMN & P300 shows statistically significant differences in TLE patients compared to controls with patients showing deficient auditory processing. A clear difference between studies with psychoacoustic assessment as opposed to electrophysiology ones may be due to lacking or incomplete evaluation of peripheral hearing by gold standard tools (76.9% in electrophysiology studies)., Conclusion: Auditory processing is deficient in patients with TLE. There is a clear need to evaluate hearing efficiency before proceeding to auditory processing evaluation with behavioral or electrophysiological tests., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background : Patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (mBC) generally receive hormonal therapy (HT) combined with CDK4/6 inhibitors (CDK4/6i). Despite this treatment, resistance mechanisms to CDK4/6i emerge and the majority of these patients experience disease progression (PD). This highlight the necessity to uncover the resistance mechanism to CDK4/6i through the identification of specific biomarkers. The primary objective is to assess the accuracy and feasibility of a novel multi-gene target panel NGS assay on circulating tumor DNA (ctDNA) to detect molecular alterations of AKT1 , ERBB2 , ESR1 , KRAS , PIK3CA , and TP53 genes in women with BC undergoing HT plus CDK4/6i treatment. Secondarily, the study aims to explore the relationship between genomic profiling and clinical outcomes. Materials and Methods : Plasma samples were collected from 16 patients diagnosed with advanced/locally advanced HR+/HER2- BC at 2 time points: T0 (baseline) and at T1 (3 months after CDK4/6i treatment). Starting from 2 mL of plasma, ctDNA was isolated and libraries were set up using the Plasma-SeqSensei (PQS) ® Breast Cancer IVD Kit, sequenced on Nextseq 550 and analyzed using the Plasma-SeqSensei™ IVD Software ® . Results : Among the five patients who presented PD, three had PIK3CA mutations and, of these, two showed a higher mutant allele frequency (MAF) at T1. In three patients with stable disease and in eight patients with partial response, the MAF of the detected alterations decreased dramatically or disappeared during CDK4/6i treatment. Conclusions : Based on our findings, the liquid biopsy analysis using the PQS panel seems to be both feasible and accurate, demonstrating a strong sensitivity in detecting mutations. This exploratory analysis of the clinical outcome associated to the mutational status of patients highlights the potential of molecular analysis on liquid biopsy for disease monitoring, although further validation with a larger patient cohort is necessary to confirm these preliminary observations.

Purpose: Plasma circulating tumor DNA (ctDNA) is a valuable resource for tumor characterization and for monitoring of residual disease during treatment; however, it is not yet introduced in metastatic colorectal cancer (mCRC) routine clinical practice. In this retrospective exploratory study, we evaluated the role of ctDNA in patients with mCRC treated with chemotherapy plus bevacizumab., Materials and Methods: Fifty-three patients were characterized for RAS and BRAF status on tumor tissue before the start of treatment. Plasma was collected at baseline, at first clinical evaluation, and at disease progression. ctDNA analysis was performed using Oncomine Colon cfDNA Assay on the Ion S5 XL instrument., Results: At baseline, from a plasma sample, RAS , BRAF , or PIK3CA mutations were detected in 44 patients. A high correspondence was observed between ctDNA and tumor tissue mutations ( KRAS 100%, NRAS 97.9%, BRAF 97.9%, PIK3CA 90%). Low baseline variant allele frequency (VAF) was found to be associated with longer median progression-free survival (PFS) compared with those with high VAF (15.9 v 12.2 months, P = .02). A higher PFS {12.29 months (95% CI, 9.03 to 17.9) v 8.15 months (95% CI, 2.76 to not available [NA]), P = .04} and overall survival (34.1 months [95% CI, 21.68 to NA] v 11.1 months [95% CI, 3.71 to NA], P = .003) were observed in patients with large decline in VAF at first evaluation., Conclusion: ctDNA analysis is useful for molecular characterization and tumor response monitoring in patients with mCRC. Quantitative variations of released ctDNA are associated with clinical outcomes.

Extracellular vesicles (EVs) act as molecular mediators in the tumor microenvironment, by shuttling information contained within malignant cells and functioning as regulators of the immune system. Circular (circ)RNAs are characterized by a closed loop-like structure that makes them more stable in the extracellular milieu and suitable to be packaged inside EVs. circPVT1 (hsa_circ_0001821) showed an oncogenic role in several cancer types and immunosuppressive properties in myeloid and lymphoid cell subsets. In this study, we characterized EVs from acute myeloid leukemia (AML) patients in terms of size, concentrations, surface markers and circPVT1 cargo. We showed that circPVT1 is overexpressed by primary blast cells from newly-diagnosed AML patients compared with hematopoietic stem-progenitor cells and is released as cell-free RNA in the plasma. We isolated EVs from the plasma of AML patients and healthy subjects by size exclusion chromatography and characterized them by nanoparticle tracking analysis. EVs from patients' plasma are larger compared with those from healthy subjects and their surface profile is characterized by higher levels of the leukemic cell markers CD133, CD105, CD49e and other immune-related epitopes, with differences according to AML molecular profile. Moreover, digital PCR analysis revealed that circPVT1 is more abundant inside EVs from the plasma of AML patients compared with healthy subjects. Our findings provide new insights on the features and content of AML EVs and suggest a role of circPVT1 in the crosstalk between AML cells and the tumor microenvironment., Competing Interests: Declaration of Competing Interest The other authors declare that they have no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Adenocarcinoma of the esophagus (EAC) and gastroesophageal junction (GEJ-AC) is associated with poor prognosis, treatment resistance and limited systemic therapeutic options. To deeply understand the genomic landscape of this cancer type, and potentially identify a therapeutic target in a neoadjuvant chemotherapy non-responder 48-year-old man, we adopted a multi-omic approach. We simultaneously evaluated gene rearrangements, mutations, copy number status, microsatellite instability and tumor mutation burden. The patient displayed pathogenic mutations of the TP53 and ATM genes and variants of uncertain significance of three kinases genes ( ERBB3 , CSNK1A1 and RPS6KB2 ), along with FGFR2 and KRAS high copy number amplification. Interestingly, transcriptomic analysis revealed the Musashi-2 ( MSI2)-C17orf64 fusion that has never been reported before. Rearrangements of the RNA-binding protein MSI2 with a number of partner genes have been described across solid and hematological tumors. MSI2 regulates several biological processes involved in cancer initiation, development and resistance to treatment, and deserves further investigation as a potential therapeutic target. In conclusion, our extensive genomic characterization of a gastroesophageal tumor refractory to all therapeutic approaches led to the discovery of the MSI2-C17orf64 fusion. The results underlie the importance of deep molecular analyses enabling the identification of novel patient-specific markers to be monitored during therapy or even targeted at disease evolution.

Objectives: Poorly cohesive carcinomas (PCCs) are neoplasms defined by a predominantly dyshesive growth pattern with single cell or cord-like stromal infiltration. The -distinctive clinicopathologic and prognostic features of small bowel PCCs (SB-PCCs) in comparison with conventional-type small intestinal adenocarcinomas have only recently been characterized. However, as SB-PCCs' genetic profile is still unknown, we aimed to analyze the molecular landscape of SB-PCCs., Methods: A next-generation sequencing analysis through Trusight Oncology 500 on a series of 15 nonampullary SB-PCCs was performed., Results: The most frequently found gene alterations were TP53 (53%) and RHOA (13%) mutations and KRAS amplification (13%), whereas KRAS, BRAF, and PIK3CA mutations were not identified. Most SB-PCCs (80%) were associated with Crohn disease, including both RHOA-mutated SB-PCCs, which featured a non-SRC-type histology, and showed a peculiar appendiceal-type, low-grade goblet cell adenocarcinoma (GCA)-like component. Rarely, SB-PCCs showed high microsatellite instability, mutations in IDH1 and ERBB2 genes, or FGFR2 amplification (one case each), which are established or promising therapeutic targets in such aggressive cancers., Conclusions: SB-PCCs may harbor RHOA mutations, which are reminiscent of the diffuse subtype of gastric cancers or appendiceal GCAs, while KRAS and PIK3CA mutations, commonly involved in colorectal and small bowel adenocarcinomas, are not typical of such cancers., (© American Society for Clinical Pathology, 2023.)

In the field of bone tissue engineering, particular interest is devoted to the development of 3D cultures to study bone cell proliferation under conditions similar to in vivo ones, e.g. by artificially producing mechanical stresses promoting a biological response (mechanotransduction). Of particular relevance in this context are the effects generated by the flow shear stress, which governs the nutrients delivery rate to the growing cells and which can be controlled in perfusion reactors. However, the introduction of 3D scaffolds complicates the direct measurement of the generated shear stress on the adhered cells inside the matrix, thus jeopardizing the potential of using multi-dimensional matrices. In this study, an anisotropic hydroxyapatite-based set of scaffolds is considered as a 3D biomimetic support for bone cells deposition and growth. Measurements of sample-specific flow resistance are carried out using a perfusion system, accompanied by a visual characterization of the material structure. From the obtained results, a subset of three samples is reproduced using 3D-Computational Fluid Dynamics (CFD) techniques and the models are validated by virtually replicating the flow resistance measurement. Once a good agreement is found, the analysis of flow-induced shear stress on the inner B-HA structure is carried out based on simulation results. Finally, a statistical analysis leads to a simplified expression to correlate the flow resistance with the entity and extensions of wall shear stress inside the scaffold. The study applies CFD to overcome the limitations of experiments, allowing for an advancement in multi-dimensional cell cultures by elucidating the flow conditions in 3D reactors., (© The Author(s) 2023. Published by Oxford University Press.)

Combining phenotypical and molecular characterization of rare cells is challenging due to their scarcity and difficult handling. In oncology, circulating tumor cells (CTCs) are considered among the most important rare cell populations. Their phenotypic and molecular characterization is necessary to define the molecular mechanisms underlying their metastatic potential. Several approaches that require cell fixation make difficult downstream molecular investigations on RNA. Conversely, the DEPArray technology allows phenotypic analysis and handling of both fixed and unfixed cells, enabling a wider range of applications. Here, we describe an experimental workflow that allows the transcriptomic investigation of single and pooled OE33 cells undergone to DEPArray analysis and recovery. In addition, cells were tested at different conditions (unfixed, CellSearch fixative (CSF)- and ethanol (EtOH)-fixed cells). In a forward-looking perspective, this workflow will pave the way for novel strategies to characterize gene expression profiles of rare cells, both single-cell and low-resolution input., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rossi, Angeli, Martinelli, Fabbri and Gallerani.)

Circulating tumor cells' (CTCs) heterogeneity contributes to counteract their introduction in clinical practice. Through single-cell sequencing we aim at exploring CTC heterogeneity in metastatic breast cancer (MBC) patients. Single CTCs were isolated using DEPArray NxT. After whole genome amplification, libraries were prepared for copy number aberration (CNA) and single nucleotide variant (SNV) analysis and sequenced using Ion GeneStudio S5 and Illumina MiSeq, respectively. CTCs demonstrate distinctive mutational signatures but retain molecular traces of their common origin. CNA profiling identifies frequent aberrations involving critical genes in pathogenesis: gains of 1q ( CCND1 ) and 11q ( WNT3A ), loss of 22q ( CHEK2 ). The longitudinal single-CTC analysis allows tracking of clonal selection and the emergence of resistance-associated aberrations, such as gain of a region in 12q ( CDK4 ). A group composed of CTCs from different patients sharing common traits emerges. Further analyses identify losses of 15q and enrichment of terms associated with pseudopodium formation as frequent and exclusive events. CTCs from MBC patients are heterogeneous, especially concerning their mutational status. The single-cell analysis allows the identification of aberrations associated with resistance, and is a candidate tool to better address treatment strategy. The translational significance of the group populated by similar CTCs should be elucidated.

This study investigates the biological effects on a 3D scaffold based on hydroxyapatite cultured with MC3T3 osteoblasts in response to flow-induced shear stress (FSS). The scaffold adopted here (B-HA) derives from the biomorphic transformation of natural wood and its peculiar channel geometry mimics the porous structure of the bone. From the point of view of fluid dynamics, B-HA can be considered a network of micro-channels, intrinsically offering the advantages of a microfluidic system. This work, for the first time, offers a description of the fluid dynamic properties of the B-HA scaffold, which are strongly connected to its morphology. These features are necessary to determine the FSS ranges to be applied during in vitro studies to get physiologically relevant conditions. The selected ranges of FSS promoted the elongation of the attached cells along the flow direction and early osteogenic cell differentiation. These data confirmed the ability of B-HA to promote the differentiation process along osteogenic lineage. Hence, such a bioactive and naturally derived scaffold can be considered as a promising tool for bone regeneration applications., (© The Author(s) 2022. Published by Oxford University Press.)

Aims: Next-generation sequencing (NGS) is becoming a new gold standard for determining molecular predictive biomarkers. This study aimed to evaluate the reliability of NGS in detecting gene fusions, focusing on comparing gene fusions with known and unknown partners., Methods: We collected all gene fusions from a consecutive case series using an amplicon-based DNA/RNA NGS platform and subdivided them into two groups: gene fusions with known partners and gene fusions with unknown partners. Gene fusions involving ALK, ROS1 and RET were also examined by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH)., Results: Overall, 1174 malignancies underwent NGS analysis. NGS detected gene fusions in 67 cases (5.7%), further subdivided into 43 (64.2%) with known partners and 24 (35.8%) with unknown partners. Gene fusions were predominantly found in non-small cell lung carcinomas (52/67, 77.6%). Gene fusions with known partners frequently involved ALK (20/43, 46.5%) and MET (9/43, 20.9%), while gene fusions with unknown partners mostly involved RET (18/24, 75.0%). FISH/IHC confirmed rearrangement status in most (89.3%) of the gene fusions with known partners, but in only one (4.8%) of the gene fusions with unknown partners, with a significant difference (p < 0.001). In 17 patients undergoing targeted therapy, the log-rank test revealed that the overall survival was higher in the known partner group than in the unknown partner group (p = 0.002)., Conclusions: NGS is a reliable method for detecting gene fusions with known partners, but it is less accurate in identifying gene fusions with unknown partners, for which further analyses (such as FISH) are required., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Interacting subsystems are commonly described by networks, where multimodal behaviour found in most natural or engineered systems found recent extension in form of multilayer networks. Since multimodal interaction is often not dictated by network topology alone and may manifest in form of cross-layer information exchange, multilayer network flow becomes of relevant further interest. Rationale can be found in most interacting subsystems, where a form of multimodal flow across layers can be observed in e.g., chemical processes, energy networks, logistics, finance, or any other form of conversion process relying on the laws of conservation. To this end, the formal notion of heterogeneous network flow is proposed, as a multilayer flow function aligned with the theory of network flow. Furthermore, dynamic equivalence is established with the framework of Petri nets, as the baseline model of concurrent event systems. Application of the resulting multilayer Laplacian flow and flow centrality is presented, along with graph learning based inference of multilayer relationships over multimodal data. On synthetic data the proposed framework demonstrates benefits of multimodal flow derivation in critical component identification. It also displays applicability in relationship inference (learning based function approximation) on multimodal time series. On real-world data the proposed framework provides, among others, multimodal flow interpretation of U.S. economic activity, uncovering underlying empirical steady state probability distribution, as well as inherent network (economic) robustness., (© 2022. The Author(s).)

Background: Here, we monitored the evolution of CTCs spread in 11 patients affected by locally advanced EC who were undergoing therapy., Methods: In this perspective study, we designed multiple blood biopsies from individual patients: before and after neoadjuvant chemo-radio therapy and after surgery. We developed a multi-target array, named Grab-all assay, to estimate CTCs for their epithelial (EpCAM/E-Cadherin/Cytokeratins) and mesenchymal/stem (N-Cadherin/CD44v6/ABCG2) phenotypes. Identified CTCs were isolated as single cells by DEPArray, subjected to whole genome amplification, and copy number aberration (CNA) profiles were determined. Through bioinformatic analysis, we assessed the genomic imbalance of single CTCs, investigated specific focal copy number changes previously reported in EC and aberrant pathways using enrichment analysis., Results: Longitudinal monitoring allowed the identification of CTCs in at least one time-point per patient. Through single cell CNA analysis, we revealed that CTCs showed significantly dynamic genomic imbalance during treatment. Individual CTCs from relapsed patients displayed a higher degree of genomic imbalance relative to disease-free patients' groups. Genomic aberrations previously reported in EC occurred mostly in post-neoadjuvant therapy CTCs. In-depth analysis showed that networks enrichment in all time-point CTCs were inherent to innate immune system. Transcription/gene regulation, post-transcriptional and epigenetic modifications were uniquely affected in CTCs of relapsed patients., Conclusions: Our data add clues to the comprehension of the role of CTCs in EC aggressiveness: chromosomal aberrations on genes related to innate immune system behave as relevant to the onset of CTC-status, whilst pathways of transcription/gene regulation, post-transcriptional and epigenetic modifications seem linked to patients' outcome.

Superficial scald is a post-harvest chilling storage injury leading to browning of the surface of the susceptible cv Granny Smith apples. Wounding of skins has been reported to play a preventive role on scald development however its underlying molecular factors are unknown. We have artificially wounded the epidermal and sub-epidermal layers of apple skins consistently obtaining the prevention of superficial scald in the surroundings of the wounds during two independent vintages. Time course RNA-Seq analyses of the transcriptional changes in wounded versus unwounded skins revealed that two transcriptional waves occurred. An early wave included genes up-regulated by wounding already after 6 h, highlighting a specific transcriptional rearrangement of genes connected to the biosynthesis and signalling of JA, ethylene and ABA. A later transcriptional wave, occurring after three months of cold storage, included genes up-regulated exclusively in unwounded skins and was prevented from its occurrence in wounded skins. A significant portion of these genes was related to decay of tissues and to the senescence hormones ABA, JA and ethylene. Such changes suggest a wound-inducible reversed hormonal balance during post-harvest storage which may explain the local inhibition of scald in wounded tissues, an aspect that will need further studies for its mechanistic explanation.