Influenza B virus (IBV) is an acute, respiratory RNA virus that has been assumed to induce the eventual death of all infected cells. We and others have shown however, that infection with apparently cytopathic viruses does not necessarily lead to cell death; some cells can intrinsically clear the virus and persist in the host long-term. To determine if any cells can survive direct IBV infection, we here generate a recombinant IBV capable of activating a host-cell reporter to permanently label all infected cells. Using this system, we demonstrate that IBV infection leads to the formation of a survivor cell population in the proximal airways that are ciliated-like, but transcriptionally and phenotypically distinct from both actively infected and bystander ciliated cells. We also show that survivor cells are critical to maintain respiratory barrier function. These results highlight a host response pathway that preserves the epithelium to limit the severity of IBV disease. Infection of a cell with influenza B virus (IBV) often results in cell death and the role of surviving cells in pathogenesis is unclear. Here, Dumm et al. generate a recombinant IBV that activates a host-cell reporter to permanently label infected cells, and show that surviving cells are important to preserve epithelial barrier function. Influenza A virus (IAV) and influenza B virus (IBV) are the two genera of this family that cause the majority of disease in humans. Despite causing up to 45% of annual influenza-induced mortality2, IBV has been relatively understudied compared to IAV. Although highly related, IAV and IBV are molecularly distinct in their protein products4, tropisms6, and have been shown to induce different antiviral responses8. Clinically, it has traditionally been assumed that IBV induces a milder form of disease. However, several recent epidemiological studies suggest that IBV disease can be just as severe as that induced by IAV in terms of clinical symptoms and outcomes12. Thus, a more complete understanding of the mechanisms of IBV disease is highly relevant.Influenza viruses cause acute respiratory disease in up to 20% of the global population annually14. This tissue damage, combined with the rapid influx of immune cells and inflammatory cytokines, underlies the clinical symptoms of influenza disease. Because the lung epithelium is the first line of defense against incoming debris and pathogens, an ineffective epithelial barrier leaves the host susceptible to respiratory deficits, decreased mucociliary clearance and secondary infections. Previously, it has been thought that virus and immune-induced cell death account for all of the epithelial disruption observed during and after influenza virus infection. There is emerging evidence, however, that the mechanisms of epithelial barrier maintenance during infection may be more nuanced than previously appreciated.In the lung, influenza viruses cause widespread cell death and changes to the structure and composition of the epithelium18. Interestingly, these “survivor” cells appear to persist in the host long-term; however, for the most part, their effects on host physiology are unclear19. A number of reports have shown striking changes to respiratory epithelium during and after influenza virus infection;14 in particular, a significant reduction in the number of ciliated cells has been reported20. However, there has not been a previous examination of whether cellular survival occurs after direct IBV infection. The mechanisms for how respiratory barrier function is maintained in the face of significant cellular damage are incompletely understood, and the potential contributions of cells that can survive direct infection have not been evaluated.While acute viral infections have been thought to uniformly lead to the lysis of infected cells, we and others have demonstrated that cells can non-lytically clear viral replication and survive direct infection with orthomyxo-, corona-, and rhabdovirusesIn this report, we first test if cellular survival can occur after IBV infection. To accomplish this, we generate a Cre-expressing reporter virus in the B/Malaysia/2506/2004 background. We use this tool to demonstrate that epithelial cells are capable of surviving IBV infection in mice. We report that the majority of the cells that survive IBV infection are ciliated-like cells that display significant transcriptional alterations relative to bystander ciliated cells. These transcriptional changes correlate with a number of unique cellular morphology changes such as the absence of apical cilia. Upon depletion of the survivor cell population, we observe increased epithelial permeability, decreased pulmonary compliance, and delayed recovery from infection. Based on these data, we propose a model in which non-lytic clearance of IBV and subsequent cellular survival is a host-adaptive process to preserve critical respiratory barrier function during an acute viral infection.21. When this reporter virus infects a cell with a Cre-responsive cassette, it removes the STOP cassette flanked by loxP sites to allow constitutive expression of the reporter protein and permanently labels any cell that has been infected  Mal/04 or Mal/04-Cre reporter virus, expression of zsGreen was observed exclusively with the Cre-expressing virus  background. We accomplished this by encoding Cre recombinase in the polymerase (PB1) segment of the viral genome Fig. , an apprted Fig. . After gted Fig. . To confrus Fig. .Fig. 1De24.To determine the stability of the Cre insert in the viral genome, we performed serial passaging of the virus in eggs and found that both the insert length and Cre recombinase activity were maintained over four passages Fig. . When we3. We then infected mice and analyzed mNeon positive cells for EpCam and CD45, cell surface markers specific for epithelial and hematopoietic lineages, respectively. We collected cells from infected mice at 2 days post-infection (DPI) and found that ~47% of mNeon + cells were epithelial while ~23% were hematopoietic in origin  as a surface-expressed marker for flow cytometry identification of ciliated cells39, suggesting that this marker may be useful for further study of surviving FOXJ1 + cells. We first verified that CD24 did indeed specifically label ciliated cells by co-staining tracheal tissue with canonical cell markers , during active infection (2 DPI), and matched populations at 14 days post-infection that had either experienced direct viral infection (14 DPI tdTomato + ) or those that had never been infected (14 DPI tdTomato−) Fig. . In orde41 to classify pathways dysregulated between uninfected and survivor cells at 14DPI based on the top 500 significant differentially expressed genes. Some pathways, which are not typically highly active in ciliated cells, such as surfactant homeostasis42 were upregulated uniquely in the survivor cell population  for some of the genes most dysregulated in survivor cells. For the upregulated genes, we tested expression of the surfactant components lls Fig. . To ensulls Fig. .Fig. 5Ci14. However, our data suggest that respiratory epithelium cilia loss is not solely the result of the lytic elimination of ciliated cells.We next wanted to define if the altered gene expression profiles in survivor cells translated into phenotypic differences in this cell population relative to uninfected ciliated cells. Since genes related to cilia structure and function were highly downregulated in survivor cells, we began by determining the presence of apical cilia on survivor and bystander ciliated cells in murine tracheal sections. Using acetylated tubulin as a marker for motile cilia, we found that cilia were readily detected on bona fide FOXJ1 + ciliated cells from uninfected mice, where ~91% of ciliated cells displayed mature surface cilia Fig. . In cont46 and also for the frequency of these ciliated-like survivor cells to increase over time. A time course analysis of survivor CD24 + cell frequency within the total survivor cell population revealed stability in the frequency of survivor ciliated-like cells -labeled dextran intranasally then collected the tracheas. After washing residual dextran from the lumen of the trachea, we analyzed the surrounding tracheal tissue for fluorescence. This analysis revealed that the leakage of particulate into tracheal tissue was significantly increased when survivor cells were absent from the large airways . This line allows for the depletion of entire population of survivor cells via diphtheria toxin (DT) administration. For DT depletion, we chose a timepoint after the resolution of viral infection in vivo  up to the point of viral clearance at 9 days post-infection. After toxin administration at days 9 and 10 post-infection, however, the recovery rates of the two groups diverged. The group with intact survivor cell populations continued to recover while mice without survivor cells had significantly delayed recovery from infection Fig. . DT treaGas-exchange in the lung requires respiratory epithelial cells to maintain constant contact with the air and as a result, there is frequent contact with airborne pathogens. When the lung epithelium encounters a cytopathic pathogen such as influenza virus, a large number of epithelial cells can be damaged and eliminated. When this happens, pulmonary barrier function can be compromised, leading to fluid leaking into the airways and decreased gaseous exchange, potentially resulting in the host succumbing to the infection. To maintain lung function, there are a number of characteristic alterations to the epithelium that occur during and after viral infection. While epithelial lung damage has been studied extensively after influenza virus infection, models have traditionally assumed that changes to epithelium composition are solely the results of cellular death and regenerative potential of the epithelium.Although there were no previous reports of any cell surviving IBV infection, we hypothesized that some epithelial cells would have the ability to survive direct viral infection, and that this population of survivor cells could play a role in viral pathogenesis. To test this hypothesis, we engineered an IBV strain to express Cre recombinase and found that epithelial cells can survive IBV infection and that the majority of survivor cells were ciliated-like cells located in the proximal airways. In addition to their survival, the unique gene expression profiles of these cells allowed significant morphological and physiological flexibility. Finally, we demonstrated that the presence of these cells, and potentially their unique transcriptional state, contributed to the maintenance of epithelial barrier function and benefited the host during recovery from viral infection.57 and refuted in the literature58. Regardless of the exact mechanism of their formation, it is clear that viral infection induces a population of ciliated-like cells that uniquely express specific  genes and acquire a phenotype not normally associated with ciliated cells.It is currently unclear how IBV infection can induce long-term transcriptional changes in a surviving cell. It is likely that at least some of the long-term changes to these cells are the results of virally induced stress and/or host shutoff experienced during infection. Our survivor ciliated-like cells could be derived from either the loss of full ciliated cell identity after infection of a mature ciliated cell, or the infection of a progenitor cell that prevents complete differentiation. The stability of the ciliated cell component of the population of survivor cells and lack of BrdU labeling of ciliated survivor cells  argues that the processes reported here are a specific response to surviving direct IBV infection. Further, we found that cytoskeletal changes to survivor cells were conserved across both in vivo and in vitro studies, implying that this response is not due to factors outside of the infected cell, such as the immune system. While the tissue-level loss of cilia of respiratory cilia during and after viral infection has been previously reported by a number of groups20, our work reveals that cilia loss is not solely the result of lytic killing of ciliated cells, but also potentially the virally induced elimination of functional cilia from ciliated cells. Whether or not these cells ever regain functionality or if they maintain their non-ciliated phenotype indefinitely is currently unknown. An important future area of research will be to define how these cells affect epithelial regeneration and if their dysfunction contributes to the impaired mucociliary clearance that is associated with bacterial secondary infection.Ciliated cells are crucial for the innate defense of the lung airways, using mechanical cilia beating to clear debris and pathogens from the airways of the lungs. In both mouse and human airways, ciliated cells are present in the large airways and orchestrate mucociliary clearance thereby preventing damage and infection. Our data suggest that some ciliated cells survive direct infection with an acute, cytopathic virus and display phenotypic alterations. While dynamic changes in ciliated cell populations are consistent with other lung injury models53. It is likely that the most critical roles for survivor cells are during these early infection timepoints, and the phenotypes observed after DT ablation of survivor cells are consistent with a role for survivor cells limiting the severity of the primary viral disease.In order to assess the functional relevance of survivor cell populations, we used toxin depletion experiments to show that these cells contribute to the preservation of pulmonary barrier integrity during and after viral infection. While intuitive that depletion of survivor cells would increase epithelial permeability, we argue that toxin depletion simply reveals the total amount of epithelial damage, which would naturally result from IBV infection if no cells had the ability to survive and persist in the epithelium. Since we experimentally depleted survivor cells after the resolution of active viral infection  into the PB1 segment of the Mal/04 genome, where the two genes are separated by PTV-1 self-cleavage site for co-translational separation. Our B/Malaysia/2506/2004 reverse genetic system is derived from a laboratory passaged virus and is no longer a perfect match to the reference sequences, however, they are still highly homologous; the exact sequences of each viral segment are available upon request. Mal/04-mNeon was generated by inserting the mNeon fluorescent gene59 (cloned from PR/8-mNeon24) into the HA segment of the Mal/04 genome. All viruses were grown in a humidified incubator at 33 °C. Similarly, all viral growth curves and serial passaging was done in embryonated chicken eggs at 33 °C. The sequences for primers used in cloning are provided in Supplementary Table Influenza B virus  expressing Cre recombinase was generated by inserting Cre . After applying an agar overlay, the MDCKs were incubated for 72 h at 33 °C then fixed and stained with polyclonal serum to recognize viral proteins. MDCK cells were not monitored for mycoplasma infection.Mal/04-Cre was rescued using the pDZ plasmid systemlox-STOP-lox-zsGreen transgenic insert. These cultures were then infected with WT Mal/04 or Mal/04-Cre at a range of MOI’s. The expression of zsGreen was monitored using the ZOE Fluorescent Cell Imager (BioRad). A549 cells were not monitored for mycoplasma infection.To test the efficiency of Cre recombination by Mal/04-Cre, A549 (ATCC), a lung epithelial cell line, was transduced with a Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (tdTomato) and C57BL/6-Gt(ROSA) 26Sortm1(HBEGG)Awai/J (DTR) mice were purchased from The Jackson Laboratory. Foxj1CreERt2 transgenic mice61 were developed by Dr. Chay T. Kuo. Colonies of tdTomato homogenic, DTR homogenic and tdTomato;DTR heterogenic were maintained by the Duke Breeding Facility. Mice from 6–10weeksold were anesthetized with ketamine/xylazine and infected intranasally with 40 μL of virus diluted in pharmaceutical grade phosphate-buffered saline (PBS) at the indicated doses. Body weight was measured over the course of infection with 80% body weight designated as the humane endpoint. All experiments included 3–5 mice per sample, as indicated in the figure legend. No blinding was performed in animal experiments. Both male and female animals were used and randomly assigned to mock and infected treatment groups. All experiments involving animals were conducted in accordance with Duke University Animal Care and Use Committee.Wild-type C57BL/6, B6.Cg-(Supplementary Table 4). Cells were incubated in primary antibodies for 1 h on ice and Live/Dead fixable blue dye (Life Technologies). Cells were analyzed on a FACSCanto II or a Fortessa X-20 (BD) machine with standard laser and filter combinations. Data was visualized and processed with FlowJo software.Mouse lung epithelial cells were isolated from WT, tdTomato and DTR mice for differentiated cell culture. Lungs were perfused with 5 mL PBS then inflated with 1 mL Dispase (Corning) and 1 mL 1% low-melt NuSieve agarose (Lonza) in water. Lungs were then digested at room temperature in dispase for 45 min, further homogenized using a razor blade in dulbecco's modified eagle media (DMEM) containing DNase I (Sigma-Aldrich) and filtered through a 70 μm filter (Corning). Red blood cells were removing using 1x Red blood cell lysis buffer (BD Biosciences) then washed in PBS 1% Bovine serum albumin (BSA) before staining. Antibodies used for staining include: CD326/EpCam , CD45 , CD31 , CD24 , SSEA-1 , Isolectin B4  Mice were infected and monitored until 10 days post-infection. At this point, mice were treated with BrdU (Sigma) intraperitoneally injected with 1 mg/200 μL once, and through their water at 0.8 mg/mL on days 10–14 post-infection. At 14 days, post-infection mice were collected and lung cells were analyzed using flow cytometry with an antibody staining for intracellular BrdU antibody .62. Fibroblasts were eliminated using differential adherence on Primaria plates (Corning) and cells were seeded on 4 μm pore transwell membranes (Corning) in mTec Plus media with Y27632 (Tocris) for 48 h before differentiation in mTec Serum-free media. Differentiation was followed by visualizing beating cilia in real time after 8 days in air–liquid interface culture. Membranes were infected between 14–21 days post-differentiation. At these timepoints, there were approximately equal numbers of apical ciliated and secretory cells.Basal stem cells were isolated from the tracheas of WT, tdTomato, DTR, and tdTomato;DTR mice. Tracheas were dissected, minced and digested in Pronase (Sigma) according to published protocolsMouse lung tissues Fig.  were fixFor membranes, whole-mount tissue sections and sectioned trachea microscopy Figs. –6 mice wDifferentiated membranes from tdTomato mice were infected with Mal/04-mNeon. At 2 DPI, membranes were fixed and stained FoxJ1 . Samples were imaged at the same time and using consistent microscope settings on a Leica SP5 inverted confocal microscope and processed identically. The brightness of FOXJ1 in the nucleus was quantified for all cells within five images. Cells were then marked as infected or uninfected based on virally expressed mNeon and the data was normalized to uninfected cells.The surface area of survivor cells was quantified using ALI culture membranes and in vivo. tdTomato transgenic mice Fig.  or diffetdTomato transgenic mice were infected with Mal/04-Cre and collected at 14 DPI. Tracheas were then fixed and stained with Actub  and FoxJ1 . Survivor cells were identified based on tdTomato expression and cells were manually scored for the presence of FOXJ1 and ACTUB in both survivor cells and uninfected cells.63. Libraries were then sequenced over two lanes of Illumina HiSeq 4000 with 150 bp paired end reads. The raw and normalized data is accessible at NCBI GEO under the accession number GSE116032. The reads were then processed according to 10x Genomics Cell Ranger pipeline. Samples were demultiplexed and converted to FASTQ files, then mapped against the mouse transcriptome (mm10) and the B/Mal/04 viral genome. Finally, samples were aggregated to normalize samples to the same sequencing depth. After the cell ranger pipeline, there were 7852 cells with an average of 41,405 reads per cell, a median 7165 UMI counts per cell and a median 1986 genes per cell. This count matrix was then analyzed using R and the R package Seurat64.Epithelial cells were sorted (BD DiVa) using EpCam and CD45 using expression of tdTomato as the marker for infection in the 2 DPI timepoint sample. Cells were washed and libraries were prepped from 5000 cells using Chromium Single-Cell 3′ Reagent Kit (10x Genomics) by the Duke University Molecular Genomics Core. Based on the 10x Genomics Chromium literature, there is a multiplet rate of 1.6% cells/sampleUsing the basic filtering functions, genes expressed in fewer than three cells, cells that express fewer than 200 genes, and cells expressing >5000 genes were removed from the analysis to minimize doublets. This resulted in a total of 7833 cells for further analysis. The data matrix was then scaled to normalize gene expression counts for each cell by the total expression. These values were then multiplied by the default scale factor: 10,000 and log-normalized. Finally, variable genes were calculated using the average expression and dispersion for each gene across cells, which resulted in 1238 variable genes being identified. Common sources of variation can be regressed out based on Seurat’s built-in function to construct linear models predicting gene expression based on noise-contributing variables. Using this function, variation caused by the number of detected molecules per cell was accounted for in the data. Next, principal component analysis was performed on the scaled and normalized data. Mock and 2 DPI samples were then combined using a canonical correlation analysis and the first 9CCs were used align the two samples. All samples were then grouped using the aligned CCA subspaces to cluster cells. Finally, the top 25 variably expressed genes for each cluster were calculated using the “bimod” likelihood-ratio test for single-cell gene expression.65.Cells were sorted (BD DiVa) based on tdTomato, CD24, EpCam, and CD45 as indicated in the figure legend then RNA was prepped from at least  cells using RNeasy Plus Mini kit (74104). Samples were then prepped using NEBNext Poly(A) messenger RNA (mRNA) Magnetic Isolation Module (NEB #E74905). NEBNext Ultra II RNA Library Prep Kit (E7770S) was used to prep libraries with NEBNext Multiplex Oligos for Illumina  for adaptor ligation and enrichment of libraries. Samples were analyzed on two lanes of Illumina HiSeq 2500 in rapid run mode. The raw and normalized data is accessible at NCBI GEO under the accession number GSE116032. The reads were mapped to the mouse transcriptome (mm10) and the B/Mal/04 genome using Bowtie, and samples were run on Basespace (Illumina). Finally reads were imported into DeSeq2 for analysisp-values were calculated and corrected using the Benjamini-Hochberg procedure for each gene between the 14 DPI uninfected and the 14 DPI survivor cells. The top 500 genes ranked by p-value were sorted and the upregulated and downregulated genes were categorized by gene ontology using DAVID Functional Annotation Bioinformatics Microarray Analysis41. Finally, the normalized read counts were used to generate bar plots for genes of interest.First, technical duplicates across lanes were collapsed by summing them together. Then the library was scaled using the default DESeq function, which smooths over outlier read counts by log transforming read counts and puts emphasis on moderately expressed genes for normalization. Variance estimates for genes across samples were calculated and ordered and the top 200 genes were selected to plot in heatmaps. Finally, the fold change and Wald test SftpA Mm00499170_m1, SftpD Mm00517321_m1, Cldn18 Mm00517321_m1, Itga7 Mm00434400_m1, and Foxj1 Mm0126729_m1. RNA was normalized to the endogenous 18S primer probe set (Thermo 4319413E).Cells were sorted (BD DiVa) based on tdTomato, CD24 and EpCam as indicated in the figure legend then RNA was prepped using an Rneasy Plus Mini kit (74104). One-step qRT-PCR was done using the EXPRESS One-Step Superscript qRT-PCR Kit and following commercial TaqMan probes: The effect of survivor cells on epithelial permeability was tested using ALI culture membranes  and intraperitoneally (100 ng). Lastly, mice were treated intranasally with 10 mg/mL FITC-4kDa Dextran. One hour later, mice were sacrificed and tracheas were collected, rinsed thoroughly with PBS and incubated in formamide before quantifying fluorescence intensity on a plate reader in duplicate. Fluorescence values were normalized to the weight of the trachea digested.Leakage of albumin into the lung was measured using bronchoalveolar lavage fluid from WT mice that were mock infected with PBS, WT mice that were infected with Mal/04-Cre and DTR-expressing transgenic mice that were infected with Mal/04-Cre. Both infected groups were treated with diphtheria toxin on 9 and 10 DPI both intranasally (10 ng) and intraperitoneally (100 ng) and bronchoalveolar lavage fluid was collected on day 11 post-infection. Albumin was quantified by ELISA (Bethyl E90–134) and absorbance was measured on a plate reader and compared to a standard curve.2O and ventilation was initiated at 150 breaths/min, tidal volume 10 mL/kg). Mice were paralyzed with i.p. pancuronium bromide (0.8 mg/kg). An electrocardiogram (ECG) monitor was connected via copper electrodes inserted subcutaneously into the limbs of the mice to determine heart rate as a monitor of depth of anesthesia. The animals were stabilized for about 5 min of regular ventilation and a standard lung volume history established by delivering two total lung capacity (TLC) maneuvers to a pressure limit of 27 cmH2O and holding for three seconds. Next, two sets of baseline measurements were collected with Snapshot and Quickprime perturbations in addition to PV-loops. Data were fit to the single-compartment model to provide values for compliance.To assess lung physiology, mice were anesthetized by i.p. injection of urethane (1 g/kg), and the trachea dissected free of surrounding tissue and cannulated with a blunted 19-gauge needle. The mice were then connected to a SciReq flexiVent FX small rodent ventilator. The ventilator was programmed to maintain a positive end expiratory pressure (PEEP) of 3 cm HMouse morbidity was measured in both tdTomato and DTR-transgenic mice to measure the effect of survivor cells. At 9 and 10 DPI, mice were treated with diphtheria toxin both intranasally (10 ng) and intraperitoneally (100 ng). Mice that were not recovering at 9 DPI, prior to when DT was administered, were excluded from analysis.T-test for tests involving one comparison between two samples. For comparisons between > 2 samples, a one-way or two-way ANOVA were used to account for numbers of independent variables. These ANOVAs were paired for different timepoints when appropriate. Fisher’s least significant difference (LSD) test was used for multiple comparisons testing and S.E.M was plotted to indicate variability around the mean for tested samples66. All experiments included at least three biological replicates as indicated in the figure legend and were replicated at least twice. Sample sizes were chosen based on the expected magnitude and variance of the phenotype and most experiments included 3–6 samples per treatment group. No samples were excluded unless specifically stated in the figure legend and methods. Analysis was performed using Prism 7 software (Graphpad). For all figures, *p ≤ 0.05, **p ≤ 0.001, ns not significant.Unless otherwise noted, all statistical analysis was performed using either a two-tailed Student’s Further information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Early detectionof autonomic changes, either provoked for therapeutic purposes2 one of the mostcommon and overlooked complications and a significant cardiovascular risk factor.Classic (linear) analysis of heart rate variability (HRV) has been routinely used toassess autonomic behavior in diabetic patients in order to promptly detectneuropathy,3 the authorscompared linear and nonlinear indexes and studied their correlation. While symbolicanalysis presented partial correlation with linear methods, Shannon entropy index wassimilar in DM individuals and controls, and these findings raise two importantissues:As the linear analysis provides important data, non-linear indexes of HRV have beenproposed as well, emerging as potential ancillary tools to investigate dysautonomia intype 1 and 2 DM. In the paper “Nonlinear Dynamics in young people withdiabetes”,What could be the clinical value of determining the complexity and randomnessof HRV by nonlinear methods? Are they sensitive and efficient?7 The observed lack of correlation ofnonlinear methods with standard measures may imply low sensitivity, and it stands indisagreement with Javorka et al.,4which claim that “The complexity of HRV appears to be even more affected (in DMpatients) than the magnitude of HRV that is commonly assessed by cardiac autonomictests.”Several authors agreed that linear indexes (time and frequency domains) are simple andreproducible methods to evaluate the cardiac autonomic system and are consistentlyreduced in diabetic patients.4On the other hand, a perfect correlation between nonlinear techniques and standard HRVmeasures would provide only limited additional diagnostic information. In fact, previousauthors verified that linear HRV indexes performed even better than most complexitymeasures in discriminating DM patients from controls.So, where do we stand on the noninvasive diagnosis of dysautonomia?8 medium-term(~1h),7 ultra short-term (<5 min)9 and short-term (from 5-10 min)10 data recordingindexes, all of them dealing with noninterchangeable information.To the best of our knowledge, time and frequency domain indexes remain the most acceptedand used methods to assess HRV. Nonlinear measures are potential tools, but to reachoptimal HRV assessment, the methods must be standardized: it is possible to find studieswith 24-hour,Nonlinear methods’ contribution to evaluate diabetic autonomic system dysfunction is yetto be demonstrated by large-scale comparison studies. Once complexity evaluation provesits value, however, one last question will remain: to what extent would it help patientsprevent diabetic neuropathy progression?


C-reactive protein (CRP) is a member of the pentraxin family of proteins. These proteins are highly conserved over the course of evolution being present as far back as 250 million years ago. Mammalian pentraxins are characterized by the presence of five identical non-covalently linked subunits. Each subunit has a structurally conserved site for calcium-dependent ligand binding. The biological activities of the pentraxins established over many years include the ability to mediate opsonization for phagocytosis and complement activation. Pentraxins have an important role in protection from infection from pathogenic bacteria, and regulation of the inflammatory response. It was recognized early on that some of these functions are mediated by activation of the classical complement pathway through C1q. However, experimental evidence suggested that cellular receptors for pentraxins also play a role in phagocytosis. More recent experimental evidence indicates a direct link between pentraxins and Fc receptors. The Fc receptors were first identified as the major receptors for immunoglobulins. The avidity of the interaction between IgG complexes and Fc receptors is greatly enhanced when multivalent ligands interact with the IgG binding sites and activation of signaling pathways requires Fc receptor crosslinking. Human pentraxins bind and activate human and mouse IgG receptors, FcγRI and FcγRII, and the human IgA receptor, FcαRI. The affinities of the interactions between Fc receptors and pentraxins in solution and on cell surfaces are similar to antibody binding to low affinity Fc receptors. Crystallographic and mutagenesis studies have defined the structural features of these interactions and determined the stoichiometry of binding as one-to-one. Pentraxin aggregation or binding to multivalent ligands increases the avidity of binding and results in activation of these receptors for phagocytosis and cytokine synthesis. This review will discuss the structural and functional characteristics of pentraxin Fc receptor interactions and their implications for host defense and inflammation. Pentraxins are an ancient family of serum proteins that are part of the innate immune system. Pentraxins are defined by a homologous pentraxin (PTX) domain of ~200 amino acids that contains a calcium-dependent ligand-binding site. The two classical pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP) are composed of non-covalently linked subunits arranged in a planar cyclic pentamer or hexamer Figure  1). Bot. Bot1). Streptococcus pneumoniae. SAP was initially identified as the precursor for a shared component of amyloid. Each subunit has a single ligand binding site allowing multivalent binding on one face of the pentamer. Multivalent binding by pentraxins initiates complement activation through the classical pathway  whereas CRP is an acute phase protein that increases dramatically in concentration from < 1 μg/ml to several hundred μg/ml during acute inflammation. In the mouse CRP is only found at low concentrations and SAP is an acute phase reactant , 12 (TabImmunoglobulin Fc receptors (FcRs) expressed primarily on hematopoietic cells provide essential links between antibody and cellular responses , 14. FcROpsonization of bacteria and complement activation were the earliest recognized activities of CRP , 20. UsiUsing a solution BIAcore-based binding assay, the binding affinities between CRP, SAP, and the long pentraxin PTX3 and FcγR were examined systematically Table . The resStructural characteristics of the interaction between pentraxins and FcγRs were determined from the crystal structure of the complex between human SAP and FcγRIIa Figure  25). Fc. Fc25). The pentraxin binding partially overlap with the IgG binding sites on the receptor. This is consistent with cellular studies showing the inhibition of CRP and SAP function by IgG.Pentraxin binding to peripheral blood cells leads to signaling, opsonization and cytokine production. The opsonic activity of CRP was recognized soon after its discovery as a pneumococcal binding protein. CRP increased phagocytosis of C-polysaccharide coated erythrocytes by both complement-dependent and complement-independent mechanisms . AggregaS. pneumonia , and CRP enhanced proinflammatory cytokine production by human peripheral blood mononuclear cells (PBMC) responding to neumonia . Cytokinneumonia .in vitro activities of pentraxins mediated through FcγR are under further investigation. A limiting role for CRP in dendritic cell maturation and T cell activation in a mouse model of experimental autoimmune encephalomyelitis (EAE) was shown to require the inhibitory receptor FcγRIIb  cells. CRP not only bound FcαRI on transfected cells, crosslinking by CRP induced ERK  phosphorylation, degranulation, and cytokine production in transfected RBL cells. In addition, CRP induced surface expression of FcαRI on neutrophils, resulting in phagocytosis and TNF-α production . Th. Thin vioduction  33)..in vivo In vivo functions of pentraxins have primarily been studied in mouse models using human injected or transgenic human CRP. Mice genetically deficient in individual FcγR or complement components have been used to delineate the role of FcγR in these functional settings. Human CRP and human SAP were shown to mediate phagocytosis through mouse FcγRs  have been used extensively to study IgG-FcγR interactions in immune complex and autoimmune disease. Injection of anti-platelet antibodies  induces platelet clearance over a period of 24 h with recovery by 48 h. Injection of either human CRP or human IgG (IVIG) prevented platelet depletion in this model and protection was also seen following transfer of CRP-treated spleen cells . The traCRP injection was also protective in immune complex mediated-nephritis . NephrotS. pneumoniae infection is primarily mediated by complement activation.Together these studies demonstrate that CRP can activate macrophages through FcγR to regulate inflammatory responses. In contrast CRP protection against Effects of either injected or transgenic CRP have also been seen in more complex autoimmune mouse models , 44, 45.H1 to TH2  is a model for multiple sclerosis and has been studied extensively as a T cell-mediated autoimmune disease. CRP expressed from a transgene is protective in mouse EAE by shifting the phenotype of autoimmune T cells from T1 to TH2 . More re FcγRIIb .In vivo SAP attenuated fibrosis in both renal and pulmonary models by regulating macrophage polarization (SAP also regulates the inflammatory response by binding to FcγR. SAP promotes phagocytosis and enhances cytokine production by monocytes and macrophages through activating FcγR as described above. In addition, SAP was identified as a serum factor responsible for inhibiting the differentiation PBMC into fibrocytes, and this effect was inhibited by aggregated IgG . In vivorization , 50. Recrization .Pentraxins are serum acute phase proteins conserved throughout the animal kingdom. While they are known to activate complement to provide innate immunity against infections, recent work established their role in activating cellular immune functions. Members of the pentraxin family can crosslink and activate a subgroup of Fc receptors upon opsonization, similar to Fc receptor activation by immune-complexes. Because of their ability to activate both soluble and cellular immune responses and their inflammatory nature, pentraxins interface innate, and adaptive immunity. These features are essentially shared with antibodies. However, while antibodies are highly restricted to individual antigenic epitopes, pentraxins are pattern recognition receptors with broad specificity for microbial glycans. They are therefore not redundant but complementary to each other in providing host immune surveillance.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Intradural spinal varices are rare lesions, with only three cases being previously reported in the literature. Previously described patients underwent MRI for non-specific low back pain and radiculopathy and were found to have an intradural lesion adjacent to the cauda equina, mimicking a nerve sheath tumour or ependymoma. Consideration of an intradural varix in the differential diagnosis of an intradural extramedullary spinal lesion is necessary to guide appropriate management. We report a case of an intradural spinal varix diagnosed with first-pass arterial and blood pool phase gadolinium-enhanced auto-triggered elliptic centric-ordered MR angiography. Digital subtraction angiography confirmed that there was no shunt but failed to demonstrate the varix. We reviewed the existing literature to look for common clinical and imaging features. A 69-year-old female presented with a 9-year history of low back pain progressing over 2 years, radiating to the left hip and leg. There was no history of weakness, sensory loss, and bowel or bladder dysfunction. Past medical history was unremarkable and there was no prior surgical history. Neurological examination demonstrated normal strength and sensation in the lower extremities. Deep tendon reflexes and plantar responses were normal.T1 weighted sequences and had peripheral hypointensity with central hyperintensity on T2 weighted sequences. The lesion enhanced intensely on delayed post-contrast T1 weighted images. On axial images, the lesion was serpiginous and extended from the T12 to L3 level. There was no spinal cord oedema and no perimedullary flow voids. A 6-month follow-up MRI with arterial and blood pool phase auto-triggered elliptic centric-ordered MR angiography (MRA) sequences  definitively excluded an arteriovenous shunt . The DSA3 They must be differentiated from intradural tumours or spinal arteriovenous fistulas (AVFs). The differential diagnoses for intradural tumours include nerve sheath tumours, haemangioblastomas, ependymomas, meningiomas, paragangliomas, lymphoma and drop metastases.1 Spinal AVFs could include both dural and spinal cord AVFs. In children, perimedullary AVFs often have high flow with large feeding arteries and marked dilatation of the veins.4 Auto-triggered elliptic centric-ordered MRA and DSA in our case confirmed the lack of arterial–venous shunting.5 Avid enhancement of the lesion on blood pool phase MRA and post-contrast sequences, in addition to the elongated serpiginous morphology with intermediate signal on T1 weighted imaging, peripheral hypointensity and central hyperintensity on T2 weighted imaging were characteristic of an intradural venous varix described by Moonis et al,1 which was previously confirmed by surgical intradural exploration. A similar pattern of enhancement on delayed post-contrast imaging is encountered in varices found in more common locations such as the orbit.6 The DSA failed to demonstrate the large varix owing to its poor contrast compared to the excellent contrast achieved by MRA.We report a case of a spinal intradural venous varix characterized with first-pass arterial and blood pool phase MRA in a patient with longstanding low back pain and radiculopathy. Spinal intradural varices are rare lesions with only three cases being previously reported .1–3 They3 In the case of a thrombosed intradural venous varix, symptoms may include severe pain and urinary retention.2 Patient ages have ranged from 51 to 87 years, with a 1 : 1 male to female ratio after inclusion of our patient in the series. Previously reported levels of varices have spanned from L2 to L4 over one to two vertebral body segments. As demonstrated in our case, and in the case by Moonis et al,1 the appearance of intermediate signal on T1 weighted imaging with lack of expected flow void on conventional T1 sequences most likely relates to MR artefacts from slow venous flow.7 The peripheral hypointensity and central hyperintensity on T2 weighted imaging, which we have been referred to as the “doughnut” sign , and serpiginous morphology are signs that can be recognized to consider this rare diagnosis.This is the first case of an intradural venous varix evaluated with gadolinium-enhanced arterial and blood pool MRA. The Intradural venous varices are rare intradural vascular lesions that may mimic an intradural extramedullary tumour or a spinal AVF.T2 “doughnut sign” and evaluation with MRA with arterial and blood pool phases may be helpful in establishing the diagnosis of an intradural venous varix.The Written informed consent was obtained from the patient for publication of this case report, including accompanying images.


Abnormal Ca2+ levels can activate toxic cascades leading to characteristic death of Purkinje cells, cerebellar atrophy, and ataxia that occur in many SCAs. The output of the cerebellar cortex is conveyed to the deep cerebellar nuclei (DCN) by Purkinje cells via inhibitory signals; thus, Purkinje cell dysfunction or degeneration would partially or completely impair the cerebellar output in SCAs. In the absence of the inhibitory signal emanating from Purkinje cells, DCN will become more excitable, thereby affecting the motor areas receiving DCN input and resulting in uncoordinated movements. An outstanding advantage in studying the pathogenesis of SCAs is represented by the availability of a large number of animal models which mimic the phenotype observed in humans. By mainly focusing on mouse models displaying mutations or deletions in genes which encode for Ca2+ signaling-related proteins, in this review we will discuss the several pathogenic mechanisms related to deranged Ca2+ homeostasis that leads to significant Purkinje cell degeneration and dysfunction.Spinocerebellar ataxias (SCAs) constitute a heterogeneous group of more than 40 autosomal-dominant genetic and neurodegenerative diseases characterized by loss of balance and motor coordination due to dysfunction of the cerebellum and its efferent connections. Despite a well-described clinical and pathological phenotype, the molecular and cellular events that underlie neurodegeneration are still poorly undaerstood. Emerging research suggests that mutations in SCA genes cause disruptions in multiple cellular pathways but the characteristic SCA pathogenesis does not begin until calcium signaling pathways are disrupted in cerebellar Purkinje cells. Ca The cerebellum, the second largest area exceeded only by the cerebral cortex, contains more neurons than the rest of the brain. For most of the 20th century, it has long been thought that the cerebellum was involved in motor learning and coordination ,2. More 2+) signaling in Purkinje cells is thought to be a key mechanism in the pathogenesis of SCAs i) is required to stimulate the cytosolic PKCγ to translocate towards the plasma membrane, subsequent DAG binding is mandatory for enzyme activation. PKCγ is expressed solely in the brain and spinal cord and its localization is restricted to neurons i is remarkably increased in the presence of mutant PKCγ, thereby leading to aberrant intracellular signaling. These results indicate that an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may be responsible for the neurodegeneration characteristic of SCA14. However, another report showed that SCA14 mutant PKCγ activity is reduced in living cells causing an inefficient activation of downstream mitogen-activated protein kinase (MAPK) signaling. These events might therefore lead to reduced expression of target genes and affect neural development and survival . m. mGRM1 gdination ,232,233.dination . On otheg levels ,235,236.ing SCAs .2+ signaling has been identified as a common underlying mechanism in most of SCAs . Since disrupted Purkinje cell Ca of SCAs , one pha2+ stabilizer) resulted in improved motor performance and decreased Purkinje cell loss in mouse models of SCA2 and SCA3 [Atxn1 inclusions in Purkinje cells. Finally, AONs are relatively short, single-stranded DNA/RNA sequences that can bind the target mRNA through Watson–Crick base pairing. This binding arrests translation and decreases synthesis of the encoded protein. SCA2 mice treated with intrathecal AONs injections displayed a reduced cerebellar ATXN2 expression associated with improvements in motor functions. Interestingly, Purkinje cell firing frequency returned to normal levels [Atxn3 and to rescue motor impairment. A correlation was also found with a recovery of defects in Purkinje cell firing frequency [Long-term feeding with intracellular dantrolene 2.1 is involved in development, functional differentiation and maturation of climbing fiber synapses. Studies on ataxic mutant mice have also resulted in the identification of other molecules involved in the phase of climbing fiber synapse elimination, such as PKCγ and its downstream signal transduction pathway, as well as Atxn1. These mechanisms mentioned above could act independently or, more likely, interact and synergize with each other, triggering the accumulation of Purkinje cell damage that eventually leads to cerebellar dysfunction. This suggests that simultaneous targeting of several pathways might be therapeutically necessary to prevent neurodegeneration and preserve neuronal function [A wide variety of mutations causing ataxia reveals the central role of cerebellar Purkinje cells in the pathogenesis of motor dysfunctions in SCAs. Almost all ionic channels or receptors expressed by Purkinje cells involved in Caje cells , but thefunction .


Heterogeneity and low incidence comprise the biggest challenge in sarcoma diagnosis and treatment. Chemotherapy, although efficient for some sarcoma subtypes, generally results in poor clinical responses and is mostly recommended for advanced disease. Specific genomic aberrations have been identified in some sarcoma subtypes but few of them can be targeted with approved drugs.We cultured and characterised patient-derived sarcoma cells and evaluated their sensitivity to 525 anti-cancer agents including both approved and non-approved drugs. In total, 14 sarcomas and 5 healthy mesenchymal primary cell cultures were studied. The sarcoma biopsies and derived cells were characterised by gene panel sequencing, cancer driver gene expression and by detecting specific fusion oncoproteins in situ in sarcomas with translocations.Soft tissue sarcoma cultures were established from patient biopsies with a success rate of 58%. The genomic profile and drug sensitivity testing on these samples helped to identify targeted inhibitors active on sarcomas. The cSrc inhibitor Dasatinib was identified as an active drug in sarcomas carrying chromosomal translocations. The drug sensitivity of the patient sarcoma cells ex vivo correlated with the response to the former treatment of the patient.Our results show that patient-derived sarcoma cells cultured in vitro are relevant and practical models for genotypic and phenotypic screens aiming to identify efficient drugs to treat sarcoma patients with poor treatment options. In spite the fact that some sarcoma subtypes such as osteosarcomas and Ewing sarcomas are sensitive to cytostatic drugs, conventional chemotherapy has limited benefit for most sarcoma subtypes and is usually recommended for advanced disease.4 Heterogeneity and low incidence have been a great challenge for sarcoma diagnosis, treatment, drug development and clinical trials.Sarcomas are aggressive tumours originating from connective tissues with an incidence of 1% of all adult cancers. They comprise approximately 130 distinct histological subtypes according to the World Health Organization (WHO) classification of bone and soft tissue tumours.5Genetically, sarcomas divide into two groups: those with complex genomes comprising of approximately 60% of all cases and those with characteristic genetic alterations, such as chromosomal translocations, that are often drivers of sarcomagenesis. This group accounts for 40% of the sarcoma cases.6 It has shown that cancer cells gradually acquire genetic aberrations such as mutations, changes in gene copy numbers, and gene fusions, but only a few hundred of these alterations are essential for tumourigenesis or for cancer cell survival.7New generation sequencing technology has been used to elucidate the genomes and transcriptomes of several human cancers, including sarcomas.8 osteosarcoma,9 undifferentiated pleomorphic sarcoma9 and dedifferentiated liposarcoma.10 Sarcomas with chromosomal translocations express specific fusion genes that are drivers of sarcomagenesis but seldom acquire secondary genetic aberrations.11 This has been confirmed in a comprehensive study with 206 sarcomas representing six different subtypes.12 The most common genetic aberrations found in this study were amplifications of the MDM2, CDK4, HMG2 genes, and deletions in the RB1, TP53, PTEN,CDKN2A and NF1 genes. Sarcomas acquire less gene mutations compared to tumours of epithelial origin. These are most common in the TP53, RB and ATRX genes.12Sarcomas with high rate of mutations include subtypes such as leiomyosarcoma,13 However, to date, only a few of these alterations are matched to approved drugs and less than 5% of the information generated is used to guide patient treatment. The therapeutic efficacy of large-scale genetic profiling on patient tumours is still challenging.This information has been useful to identify genetic aberrations in tumours that can be targeted by newly developed drugs.15 The use of PDCs and PDXs in drug screens is gaining considerable interest since these models represent a closer surrogate to the patient tumour. Furthermore, the use of approved drugs for other treatment indications can be accelerated since the preclinical and safety studies in humans have already been performed.Phenotypic or functional screening can be an alternative to overcome this gap. It refers to the identification of molecules with particular biological effects using cell, tissue-based assays or animal models. Phenotypic screening using cancer models such as patient-derived tumour cells (PDCs), organoids and patient-derived xenografts (PDX) is an efficient approach to identify treatments or new therapeutic indications for approved drugs.In the present study, we cultured patient-derived sarcoma cell and characterised these cells and the biopsy of origin by analysing gene mutations, cancer driver gene and fusion protein expression. We further determined the sensitivity of the patient-derived sarcoma cells to a library of approved and investigational drugs to evaluate if this approach can be useful to identify targeted inhibitors with potential therapeutic effects for individual sarcoma patients.Patient-derived cells were dissociated from solid biopsies through mechanic grinding followed by enzymatic digestion using a 0.1% collagenase mixture for 1−2 h at 37 °C under gentle agitation. The cell mixture was then passed through a 100 μm mesh, and the collagenase solution was replaced by fresh culturing medium.Most sarcoma cells were grown in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12 (DMEM/F12) supplemented with 15% fetal bovine serum (FBS), non-essential amino acids, penicillin and streptomycin. Angiosarcoma cells were cultured in endothelial cell media containing vascular endothelial growth factor (VEGF), insulin growth factor (IGF) and basic fibroblast growth factor (FGF). Muscle cells were grown in skeletal muscle cell media containing 5% fetal calf serum, basic FGF, insulin, and dexamethasone .Ewing’s sarcoma (ES), alveolar rhabdomyosarcoma (aRMS) and angiosarcoma samples were obtained by fine needle aspiration (FNA) from palpable tumours. The content of tumour cells in the FNA biopsy was assessed on site by microscopic evaluation of Grunewald−Giemsa-stained FNA smears prior to in vitro cultivation.Logarithmic growing patient-derived cells (PDC) were established within a range from 2 days to 18 weeks depending on the biopsy size, cell viability and histological subtype.Primary cells were cultured in chamber slides for 24 h; thereafter the medium was removed, and the cells were washed twice with phospate buffered saline (PBS) and fixed for 15 min in 4% paraformaldehyde, following permeabilisation with 0.1% TritonX100 for 15 min. The PLA was performed according to the in situ Fluorescence PLA protocol (Sigma-Aldrich). For detection of fusion proteins, we used two antibodies raised in different species, one recognising the N-terminus and the second recognising the C-terminus of each chimeric protein. Species-specific antibodies conjugated with DNA oligonucleotides were then used to recognise the primary antibody. These DNA probes can be ligated to form a circular probe only in the case that the two antibodies are in the proximity. The DNA probe is subsequently amplified using DNA polymerase and a fluorescent-labelled deoxynucleotide in the amplification reaction allowing the visualisation of the fusion protein in the UV-light range using a confocal microscope.TM Green Fast qPCR Mix green master mix . Gene expression analysis was calculated using the Livak method, in which the ΔCT values were calculated by normalising values for each gene to the housekeeping genes, ΔΔCT was then calculated by normalising the ΔCT values of either biopsy of patient-derived cells to that of the normal muscle cells and used to determine the fold change (2−ΔΔCT). Log2 (fold change) values were then plotted in a HeatMap using the GENE-E software (Broad Institute).RNA was isolated using AllPrep  according to the manufacturer’s instructions and the quality of the RNA was evaluated using a bioanalyser . The expression of 180 cancer driver genes .18We used the drug sensitivity and resistance testing (DSRT) developed at the Institute for Molecular Medicine Finland (FIMM).The compounds were dispensed in 384-well plates  at five different concentrations covering a range between 1 and 10,000 nM using acoustic liquid handling .Early passaged (passages 2−5) PDC growing as monolayers were collected by tripsinisation. Twenty-five microliters of single-cell suspensions containing 500–2000 cells were dispensed to each well using a Multidrop Combi Reagent Dispenser . Plates were subsequently centrifuged, put on an orbital shaker for 10 min followed by additional 10 min incubation at room temperature to allow the cells to plate to the bottom.2, and a humidified environment. Cell viability was assessed by determining the total adenosine tri-phosphate (ATP) levels in living cells using CellTiter-Glo (CTG) luminescent cell viability assay . Luminescence was measured using a multimode multiplate reader .The plates were incubated for 72 h at 37 °C, 5% COThe selective drug sensitivity score (sDSS) was calculated by subtracting the DSS of normal cell controls  from the DSS of sarcoma cells.Target enrichment was performed with the HaloPlex target enrichment system (Agilent Technologies), according to the manufacturer’s instructions. A custom gene panel was designed, covering the coding regions of the 16 target genes listed in Supplementary Table 20 and the trimmed reads were subsequently aligned to the reference genome  using BWA mem version 0.7.12,21 allowing for a maximum of 5% mismatches in a read. Single-nucleotide variation detection was performed using SNPmania version 0.0.7  . Insertion and deletion detection was performed using Pindel version 0.2.5a8 with parameters -x 2, allowing the detection of indels up to 512 bp, and -B 100 allowing the detection of structural variants of size ~100 bp. Only positions having a read depth ≥ 30 and variant allele frequency > 1% were considered. The variants were further filtered to remove all synonymous mutations, as well as variants having a frequency > 1% in the 1000Genome project21 and the trimmed reads were subsequently aligned to the reference genome  using BWA mem version 0.7.12, allowing for a maximum of 5% mismatches in a read. Single-nucleotide variation detection was performed using SNPmania version 0.0.7   only considering aligned reads with an alignment quality of at least 5 and read bases with a minimum base quality of 20. These thresholds were set based on optimal sequence concordance with HapMap variant data. Insertion and deletion detection was performed using Pindel version 0.2.5a8 with parameters -x 2, allowing the detection of indels up to 512 bp, and -B 100 allowing the detection of structural variants of size ~100 bp.Illumina sequencing adaptors were removed by cutAdapt version 1.8.0 Soft tissue sarcomas were obtained by surgical excision, or by FNA and were used to establish patient-derived sarcoma cell cultures, hereby denoted as PDC. Normal healthy control cells were also isolated and cultured from three healthy muscle biopsies, one epithelial bladder biopsy, from umbilical cord-derived mesenchymal stem cells  and from the bone marrow of two healthy donors. Representative samples from the biopsies were dissected for (a) deep freezing, (b) formalin-fixed and paraffin embedding (FFPE), (c) genotypic and transcription profiling and (d) establishment of the PDC Fig. . FourteeThe comparison of the DSS values among our sarcoma cohort (14 cases) showed that drug classes such as histone deacetylase (HDAC), cyclin-dependent kinase (CDK), proteasome, mitosis, and mTOR inhibitors were active in most of the sarcoma subtypes tested. However, when normalising the DSS of the sarcoma PDCs to that of healthy cells , to obtain the sDSS, we identified selective inhibitors such as Dasatinib , one synovial sarcoma (SS) and two Ewing sarcoma (ES).A 19-year-old male developed a primary tumour in the prostate that was diagnosed as a PAX3-FOXO1-positive aRMS. He underwent treatment according to the Italian Sarcoma Group/Scandinavian Sarcoma Group protocol III (ISG/SSGIII) consisting of doxorubicin, vincristine and cisplatin  by RT-PCR confirming the presence of aRMS cells Fig. . Among tTCH Fig. .Figure Two cases were investigated; one expressing the fusion protein ASPS1-TFE3 (K-ASPS2) and a case where the fusion gene transcript was not detected in either biopsy nor the PDC (K-ASPS3).K-ASPS2 originated from the tumour of a 20-year-old female, with a mass in the soleus muscle with a tumour morphology characteristic of alveolar soft part sarcoma consisting of tumour cells with eosinophilic cytoplasm and prominent nucleoli arranged in small alveoli Fig. . Lung meAt the time of biopsy, the patient had received treatment with Sunitinib (Sutent®) Crizotinib , Trabectedin (Yondelis®), Denosumab (Xgeva®) and Pazopanib (Votrient®) with poor responses and developed progressive disease , the dexamethasone-induced Ras-related (RASD1), the Wilms Tumour 1 (WT1) and mTOR (FRAP1) kinase genes were among the most expressed genes in relation to normal muscle cells  kinase inhibitor BMS-863233, and the topoisomerase inhibitor Topotecan. Similar to K-ASPS2 and K-RMS1, Dasatinib was an active drug in K-ASPS3 , the mTOR kinase gene FRAP1, the anti-apoptotic gene BCL2 and several autophagy-related genes of the ATG and SEC family, and downregulation of the TP53 gene among others  protocol consisting of cisplatin, doxorubicin, etoposide and vincristine with no clinical response  and radiotherapy , followed by autologous stem cell transplantation , ex vivo culturing of tissue slices,26Sarcoma is a highly heterogeneous and aggressive disease in which some histological subgroups lack efficient treatments. Following the breakthrough of the approval of Imatinib™/Gleevec® for the treatment of gastrointestinal stromal tumours, other specific targets have been identified for sarcomas such as PDGFRα and β, c-Met, PI3K-AKT, HDAC, MDM2 and IGF1R, and inhibitors targeting these pathways have been developed and evaluated in preclinical and clinical trials.In this study, we cultured and characterised patient-derived sarcoma cells to test their sensitivity towards a comprehensive library of cancer drugs as an approach to identify selective inhibitors for individual cases. Twenty-four sarcoma biopsies were taken but only 14 of them could be grown in vitro. Most of the biopsy donors were affected by progressive disease and had been treated with systemic chemotherapy or radiation at the time of biopsy. Although it is difficult to draw conclusions from a small cohort we observed that poor cell viability in the sample, and adjuvant treatment prior to biopsy, had negative effects on the establishment of PDCs.27Several sarcoma groups are already using functional screens to identify targeted inhibitors for sarcoma. In a recent study, 63 human adult and paediatric sarcoma cell lines were screened against 445 cancer agents including 100 approved drugs. The cell panel consisted of 15 sarcoma subtypes with an overrepresentation of osteosarcoma and Ewing sarcoma cell lines. The drug response results showed that sarcomas are in general poor responders. Ewing sarcoma however was the most responsive group in this series. The drug responses varied within the groups and was associated with specific gene or microRNA expression. Normal healthy controls were not assessed, and several cell lines used in this study have been cultured in vitro for several years.15 In this study, whole exome sequencing was performed in a large panel of tumours and demonstrated that patients with advanced cancers carry limited number of targetable genomic aberrations. Similar to our results they show that in vitro drug testing on patient-derived cells can potentially predict patient response to therapy, and it is advantageous when genomic profiling fails to identify targetable genomic alterations.Two recent studies have shown that patient-derived tumour xenografts (PDX) and low passaged s PDCs preserve the intra-tumour heterogeneity and the genomic fingerprint of the tumours of origin.28 and this represents a limitation if the patient has progressive disease. These results however demonstrate that patient-derived sarcoma cells or xenografts are relevant models that can be used to identify effective treatments for sarcoma patients.The power of using patient-derived sarcoma cells to predict patient response to drug treatment has not been evaluated in big cohorts. Recently, patient tumours engrafted in mice were used as a predictive model for response to therapy. In this study, 29 human sarcomas were transplanted in immunodeficient mice (TumorGraft) and 22 (76%) of them engrafted and were subsequently passed in mice for drug sensitivity testing. The most relevant finding was that TumorGrafts could predict response to treatment in 13 of 16 cases of sarcoma patients undergoing treatment; however, time from tumour engraftment to drug sensitivity assay was several months,In our study we found several indications that support the use of sarcoma PDC in drug screens as a predictive model of patient treatment response. First, we could confirm that the genetic characteristics of the sarcoma biopsies such as gene mutations and fusion gene expression were preserved in early passaged PDCs. Secondly, there was an association between drug sensitivity and the response to the patient’s actual treatment. Poor DSS in drugs used in routine treatment were observed in patients with refractory disease, contrary to a case of a taxane responsive Ewing sarcoma in which taxanes showed activity in vitro.Thirdly, c-Met inhibitors were active in an ASPS1-TFE3-positive alveolar soft part sarcoma consistent with the targeting of the cMET promoter by ASPS1-TFE3.It is also possible that the lack of response of cultured sarcoma cells from patients with recurrent disease is the result of the clonal evolution of their tumour clones resistant to treatment that are preserved in the PDC. The comparison of drug responses between primary and metastatic tumours could shed information and is an issue of interest for further investigations.In summary, our pilot study shows that patient-derived sarcoma cells can be isolated from surgical and FNA biopsies and expanded in vitro to be used in comprehensive drug sensitivity testing. This is a rapid approach that does not require animal facilities or the reprogramming of cells to establish the PDCs. It can identify selective agents with activity in sarcomas from individual patients and can predict the patient response to standard-of-care treatment. This method can be used as a guide to select treatments for sarcoma patients with otherwise few therapeutic options; however, trials with larger cohorts need to be performed to confirm its clinical value.Gene expression in sarcoma biopsies and derived PDCDrug sensitivity scores (DSS) and selective DSS in all samplesSarcoma Gene PanelS Table 2Translocation-associated soft tissue sarcomas- Clinical data at time of biopsyCancer Driver Gene Q-RT-PCR library


ALK tyrosine kinase receptor is oncogenically activated in neuroblastoma. Whereas numerous ALK fusion genes have been reported in different malignancies, in neuroblastoma ALK is mainly activated through point mutations. Three hotspot residues  account for 85% of mutant ALK seen in neuroblastoma. In a cohort of 105 Swedish neuroblastoma cases of all stages, these hotspot regions were re-sequenced (>5000X). ALK mutations were detected in 16 of 105 patients . Mutations at the F1174 and F1245 hotspot were observed in eleven and three cases respectively. ALK mutations were also detected at the I1171 and L1240 codons in one tumor each. No mutations were detected at R1275. Sanger sequencing could confirm ALK status for all mutated samples with variant allele fraction above 15%. Four of the samples with subclonal ALK mutation fraction below this would have gone undetected relying on Sanger sequencing only. No distinct mutation spectrum in relation to neuroblastoma tumours genomic subtypes could be detected although there was a paucity of ALK mutations among 11q-deleted tumors. As ALK mutations status opens up an excellent opportunity for application of small molecule inhibitors targeting ALK, early and sensitive detection of ALK alterations is clinically important considering its potential role in tumour progression.The  Although there is treatment available for high-risk NB cases, long-term survival for this patient group is less than 50% despite aggressive treatment. The adverse outcome of high-risk NB constitutes a major clinical problem, mainly attributed to insufficient means to treat refractory or relapsed disease2. Consequently, one of the most important practical utilities of studying NB tumour heterogeneity lies in its implications for improving therapeutic strategy and ultimately, increased survival.Neuroblastoma (NB), the most common extracranial solid cancer in childhood, displays unique heterogeneity in terms of both genomic and clinical behavior3. Importantly, not all mutations detected in a cancer cell are helpful for increasing knowledge regarding the mechanism of transformation. Whereas some genetic defects provide selective advantage for cancer development and/or progression, others are mere passengers without functional relevance and thus, not valuable as targetable candidates4. In NB, only a limited number of recurrent somatic mutations have been reported, these include mutations in ALK (Anaplastic Lymphoma Kinase), and a set of genes involved in chromatin-remodelling and neuritogenesis. Apart from the hotspot mutations in ALK most variants identified are private6. Activating germline mutations in ALK are the major cause of hereditary NB although somatically acquired ALK alterations are observed in 6–12% of sporadic cases9. Activating point mutations are mainly seen at three “hot spot” residues; F1174 , F1245 , and R1275 (mutated to Q or L), all localized within the kinase domain of ALK and together accounting for 85% of all mutant ALK in NB10. Less frequent observed are substitutions at I1170 (to N or S) and I1171 (to N)11. Besides activating missense mutations, ALK can also be activated by amplification or rare translocation events, further supporting the necessity of this tyrosine kinase receptor for NB tumourigenesis13. The recent discovery of the ALK ligand, ALKAL2 (FAM150B/AUGα),15 with a genetic locus in the genomic proximity of ALK, makes it tempting to speculate a role for ligand-dependent activation of the wildtype ALK receptor in NB pathogenesis. This makes ALK an attractive therapeutic target in NB. A number of small molecule ALK inhibitors are currently under clinical or preclinical investigation for treatment of ALK-positive malignancies. Single drug treatment with the small molecule ALK/MET inhibitor crizotinib has shown very promising results in adult non-small cell lung cancer and in large cell anaplastic lymphoma that harbour ALK translocations16. However, inhibition of ALK mutation in the context of the full-length receptor is complex, and crizotinib has proven to be less clinically effective in treatment of NB17. Biochemical studies of ALK mutations have shown that the different variants display both variable kinase activity and variable inhibitor susceptibility10 but this could possibly be surmountable through more high affinity inhibitors recently developed18. Second and third generation ALK specific inhibitors such as ceritinib (LDK378)19, brigatinib (AP26113)21, alectinib (CH-5424802)22, and lorlatinib (PF-06463922)24 might significantly improve NB treatment options. We recently reported on a child with underlying Fanconi anemia (FA) and ALK mutant high-risk NB responding strongly to precision therapy with the ALK TKI ceritinib25.In-depth molecular characterization of cancer specimens can provide prognostic or diagnostic information and identify molecular therapeutic targetsALK mutations, either already present at time of diagnosis or acquired during disease progression, is of major clinical importance for NB patients as this may lead to new therapeutic possibilities27. However, only a few comprehensive studies have explored the genomic landscape of relapsing NB30. We recently showed that ALK alterations are enriched at relapse and that these mutations can be detected as minor subclones in the primary tumour that subsequently emerge as the major relapse clone31. It is highly likely that these initial subclones present at diagnosis confer a selective advantage after first line treatment with chemotherapy, ultimately leading to clonal expansion and tumour relapse. These data strongly suggest that precise molecular characterization of ALK mutations should be included in clinical diagnostics of NB tumours not only at diagnosis but also continuously throughout the clinical management of the patient.More sensitive detection of ALK copy number gain as well as the frequency and type of ALK mutations that may remain undetected using Sanger-based sequencing methodology. To do this we have employed deep parallel DNA sequencing techniques that allow detection of very low frequency of mutations and we compare this method with previously used Sanger sequencing methodology. We further intend to define the subclonal heterogeneity of the tumour and their potential role in clonal evolution and progression of NB.To investigate this rigorously we have now undertaken a study of a series of 105 NB tumours to explore NB tumour samples were obtained through surgery/biopsying after informed consent from parents/guardians. The tumours were graded according the international NB staging system (INSS) and international NB risk group (INRG). All samples belong to a Swedish cohort of patients with NB tumours of all stages, Table MYCN status and tumour copy number aberrations has been determined earlier, using SNP arrays and/or FISH32. Clinical data and genomic profiles of included patients are summarized in Table ALK gene were analyzed by two different deep parallel sequencing methods (i) the HaloPlex™ target enrichment system and (ii) an amplicon-based assay for sequencing of three hotspot regions in ALK  using Illumina MiSeq sequencing platform . In order to ensure the potential of our method in detection and discrimination between ALK three hotspot residues F1174, F1245 and R1275, three serial dilutions  from each of the three hotspot amplicons  were generated using 20 ng DNA of three patients each carrying the relevant mutation and dilute it with 20 ng of unmatched germline DNA. Subsequently, eight germline genomic DNAs from healthy donors and three tumours with known ALK mutation served as controls to quantify background abundances.Following DNA extraction using standard procedures, mutations of the receptor tyrosine kinase (RTK) domain of the ALK gene. Custom probe design for the ALK region of interest (exon 21–25) was created with SureDesign (Agilent). In brief, 225 ng of genomic DNA from each sample were fragmented using eight seperate restriction enzyme digestion reactions and denatured prior hybridization to the HaloPlex probe library. The HaloPlex Target Enrichment System Protocol (Version D.5) was followed without modification to perform the library preparation and target capture using the HaloPlex Exome capture baits. We assessed library quality using Agilent’s 2200 TapeStation system. Enriched and indexed library were prepared for 150-bp paired-end sequencing (2 × 150 bp) on an Illumina MiSeq sequencing system .HaloPlex  is a custom in-solution capture hybridization method that was used for deep sequencing of ALK regions of interest containing hotspots F1174 (exon 23), F1245 (exon 24), and R1275 (exon 25). During the PCR, using KAPA HiFi HotStart DNA Polymerase , the region-specific primers including Illumina sequencing adapters overhang were used. After amplicon purification with Ampure magnetic beads , dual indices are attached using Nextera XT Index Kit (Illumina). Following another clean-up and quality control, the final libraries were pooled equimolarly, denatured prior pair-end sequencing (2 × 150 bp) using MiSeq v.3 reagent kit (Illumina). The number of libraries sequenced per flow cell was adjusted to achieve a depth of coverage of at least 5000x for the amplicons in each sample.For sequencing library construction, 12 ng of genomic DNA were used to amplify the 33, quality of reads was assessed through Fastqc showing that majority of reads never dropped below a phred score of 30 at any position  were mapped against Human genome build hg19  with BWA-MEM default settings35. We did variant calling with the tool UnifiedGenotyper with the option to EMIT_ALL_CONFIDENT_SITES offered in the Genome Analysis Toolkit (GATK)36. This option was used to be able to predict low frequency alleles, since it gives the allele count for each allele at each position (Supplementary Table ALK tyrosine kinase domain. The most frequently observed sporadic ALK substitutions at amino acids positions F1174 (exon 23), F1245 (exon 24) and R1275 (exon 25) were studied in detail . The percentages of bases at coordinates that correspond to positions of ALK hotspots mutations were studied in detail. The background variation frequency at the relevant coordinates was determined in the controls and compared to cases followed by analysis of the mutational status of ALK exon 21–25 in the hybridization-based assay and exon 23–25 in the amplicon-based assay. ALK mutations with a variant allele frequency less than 20% were detected in six tumours while ten other cases showed ALK mutations with higher variant allele frequency (between 20–60%). At residue I1171 in the ALK locus (chr2:29445213), an ALK variant was detected in one case with the frequency of mutated allele as low as 2.7%. This was the lowest variant frequency level detected by deep-targeted sequencing in our cohort. At the F1174 hotspot (chr2:29443695-29443697), alterations were observed in eleven cases: seven cases harbored a mutation leading to the amino acid change F1174L, two cases with F1174I, one each of the F1174C, and F1174S substitutions, with the mutated allele fractions ranging from 14% to 60%. Interestingly the patient harbouring F1174S mutation with 58% frequency also exhibited a subclone with an F1174I mutation detected at 8% frequency of the mutated allele  could be identified .ALK variants with mutated allele fraction above 20% might be interpreted as clonal, whereas the six ALK mutations observed with a fraction below 20% might be interpreted as subclonal events that might be unrecognized through Sanger sequencing. All ALK mutated samples were also tested by Sanger sequencing which confirmed all ALK mutations occurring with a mutated allele fraction above 15% leaving four samples with a mutated allele fraction below 15% undetected by Sanger sequencing in our hands.In this study, tumour samples with a mix of up to 50% normal cells were included which may underestimate the mutation frequencies of heterogeneous tumour samples. Thus it would be expected that subclonal mutation events would represent a greater fraction of the tumour cell population in the actual tumour mass as compared to analyzed DNA fragments. In this regard, our detection of ten ALK gene amplification detected from previously performed SNP microarrays. All four cases also had co-amplification of MYCN and no ALK mutation was detected in any of these samples. Focal low copy number gain for ALK was also detected in one MYCN-amplified patient who also had a chromothripsis pattern for chromosome 2 (case #57). Partial chromosome 2p gain that includes the ALK locus was observed in 26 patients while gain of whole chromosome 2 was seen in 11 cases. Interestingly, all patients (n = 4) with partial 2p gain that also carry an ALK mutation are dead of disease (Table ALK and MYCN except in 6 patients (data available on request). In these six cases, the MYCN amplification segment was present with a breakpoint proximal to the start genomic position of gained region. The partial 2p gains encompassing the ALK locus were present in only 4 of 16 mutated tumours indicating that 2p gain is not a common mechanism for increasing mutated ALK copy number.In addition, four of the 105 tumours included also had 38. This type of heterogeneity may thereby hinder therapeutic strategies that depend on results from single tumour-biopsy samples and provides a risk to render clinically important variants undetected if using less sensitive detection methods.With the emergence of targeted therapeutic strategies, full characterization of genetic alterations becomes crucial to improve treatment and patient outcome. Intratumour genetic heterogeneity has been reported in number of human malignancies and provides insights into clonal evolution and pathogenesisALK mutations in exon 23, 24 and 25 including those also present at subclonal level, 105 diagnostic NB samples were analyzed through ultra-deep sequencing that provides increased sensitivity to that of conventional Sanger sequencing . Variant allele fraction of observed ALK point mutations in this study ranged from 2.7% to 60% of all NB cases, with a subclonal  observed in 6 of 16 ALK-mutated samples. This is in concordance with the work of Bellini et al. who performed deep sequence of exons 23 and 25 of ALK in a series of 276 NB samples and showed that more than half of the identified point mutations were subclonal events that might have gone undetected by Sanger sequencing. This highlights the importance of deep sequencing techniques for the identification of mutations in NB, especially when targeted therapy is a clinical consideration39. All of the NB -associated ALK mutations, observed in our dataset are known to confer strong gain of function effect to ALK in vivo assays, and are therefore known as drivers in the progression of NB tumours40. The potent ligand-independent F1174L mutation was present in 7 cases of which 4 had MYCN-amplification whereas the other three displayed other genomic profiles. Three of four patients with MYCN amplification and ALK F1174L co-occurrence showed adverse outcome (Table MYCN-amplified NBs42. We observed that the majority of MYCN-amplified tumours (4 of 6) contained subclonal  ALK aberrations while in MYCN-nonamplified tumours the majority of ALK mutations (8 of 10) were clonal. This implies that MYCN-nonamplified tumours are dependent on the presence of this constitutive active kinase in a higher percentage of the cell population. In addition to mutations, ALK activation can also result from high-level gene amplification. In keeping with previous studies, amplification of ALK almost exclusively occurs in MYCN-amplified tumours43. Interestingly, we do not detect any mutations among the 11q-deleted genomic subgroup of NB tumours 15 with a genetic locus in the genomic proximity of ALK and MYCN, may aid in further development of therapeutic tools for intervention in NB development in the near future.Despite the encouraging development of next generation ALK inhibitors, both intrinsic and acquired resistance may occur when using single drug treatment for receptor tyrosine kinase inhibitors. One mechanism of resistance is acquirement of secondary genetic changes that reduce the accessibility of the hydrophobic inhibitor-binding pocket for small molecule inhibitor by increasing the affinity for ATP instead. A number of these resistant promoting gatekeeper mutations have been demonstrated in non-small cell lung cancer patients treated with crizotinib or alectinibALK is the most frequently mutated gene in NB and has provided new hope for NB treatment using ALK-specific inhibitors. As ALK mutations may contribute to disease progression and relapse, early detection and eradication of these subclones is of uttermost importance as they may improve patient prognosis. We show here that through ultra-deep sequencing, ALK point mutations were detected in 16 samples whereof 6 were subclonal that may remain undetected through conventional sequencing methods. This proves that ultra-deep sequencing could become crucial in clinical analysis as it allows sensitive detection of activating ALK mutations as well as of resistance promoting mutations present at subclonal level.Supplementary dataset