In recent years, social media websites have been suggested as a novel, vast source of data which may be useful for deriving drug safety information. Despite this, there are few published reports of drug safety profiles derived in this way. The aims of this study were to detect and quantify glucocorticoid-related adverse events using a computerised system for automated detection of suspected adverse drug reactions (ADR) from narrative text in Twitter, and to compare the frequency of specific ADR mentions within Twitter to the frequency and patterns of spontaneous ADR reporting to a national drug regulatory body. Of 159,297 tweets mentioning either prednisolone or prednisone between 1st October 2012 and 30th June 2015, 20,206 tweets were deemed to contain information resembling an ADR. The top AE MedDRA® Preferred Terms were ‘insomnia’ and ‘weight increased’, both recognised non-serious but common side effects. These were proportionally over-reported in Twitter when compared to spontaneous reports in the UK regulator’s ADR reporting scheme. Serious glucocorticoid related AEs were reported less frequently. Pharmacovigilance using Twitter data has the potential to be a valuable, supplementary source of drug safety information. In particular, it can illustrate which drug side effects patients discuss most commonly, potentially because of important impacts on quality of life. This information could help clinicians to inform patients about frequent and relevant non-serious side effects as well as more serious side effects. Patients on steroid drugs often complain of insomnia and weight gain on Twitter, offering a window into commonly experienced side effects. William Dixon from the University of Manchester, UK, and colleagues searched for mentions of either prednisolone or prednisone on Twitter between October 2012 and June 2015. They documented around 20,000 tweets on the social media platform that contained discussion of an adverse drug reaction. The top side effects cited were “insomnia” and “weight increased”, both of which constituted a larger proportion of total steroid-related complaints on Twitter than in the official UK regulator’s reporting scheme. Serious side effects were mentioned less frequently on Twitter. The findings show the power of social media data to identify the side effects impacting quality of life among patients. Clinicians have long been aware of the many side effects of GC therapy, including osteoporosis, serious infection, diabetes mellitus and cardiovascular disease.2 Despite widespread use since the 1950s and an understanding of their mechanism of action,3 evidence on the frequency and impact of long-term glucocorticoid toxicity are still lacking.6Glucocorticoid (GC) therapy is used widely in patients with inflammatory diseases. It is estimated that the prevalence of oral GC use in the UK population is around 1%.7 For example, when groups of patients and rheumatologists were asked to rank the ten “most worrisome” GC-related adverse events, patients ranked renal function, fatigue and palpitations much higher than rheumatologists.8 Similarly, a study of patient-reported side effects of GC therapy in patients with asthma demonstrated patients often report adverse events in differing priorities and patterns to clinicians.5 Given that clinician–patient disconnect and treatment concerns are two key factors in influencing medication adherence,10 further work which focuses on patient experience and opinion is needed to develop a more complete understanding of GC safety.Clinicians and patients are known to have differing views on what the important side effects of GC therapy are. This matters because decision-making is influenced by the value judgement of the outcome under consideration, be it a benefit or a risk, alongside its probability and its impact.11In the last two decades, increasing numbers of patients have become active on the World Wide Web; seeking information about symptoms, disease and medications, as well as sharing their health related experiences online in forums, or social media platforms such as Twitter and Facebook. A reported 72% of internet users say they looked online for health information within the past year.13 This corpus of publically-facing data could generate new knowledge regarding drugs and their benefits and harms. In recent years, text mining techniques; which aim to automatically transform free text into structured data using Natural Language Processing (NLP) techniques, have proven useful in identifying potential drug safety concerns from text within Facebook and Twitter.14 Possible opportunities for drug safety arising from automated analysis of social media data include routine pharmacovigilance, the study of drug safety in particular populations of interest, or studying the impact of side effects on patients’ lives. Traditional methods of pharmacovigilance such as the MHRA’s Yellow Card scheme in the UK and the Food and Drug Administration Adverse Event Reporting System (FDA-FAERS) in the USA15 are prone to underreporting with an estimated 94% of adverse drug reactions (ADRs) unreported.17In the 3rd quarter of 2015, Twitter and Facebook had 320 million and 1.6 billion active monthly users, respectively.The aim of this study was to detect and quantify glucocorticoid-related adverse events using a computerised system for automated suspected ADR detection from narrative text in Twitter. A secondary aim was to compare the frequency of specific ADR mentions within Twitter to the frequency and patterns of spontaneous ADR reporting to a national drug regulatory body.18 Figure 19 After filtering out 17 non-medical event PTs and 41 treatment indications , ‘weight increased’ (8.2%), ‘non-specific reaction’ (7.8%), ‘increased appetite’ (7.5%) and ‘malaise’ (4.4%) Fig. . Non-spe23% of proto-AE tweets had more than one PT tag Fig. . The topWithin MHRA DAPs, there were 7457 suspected ADRs reported from 1963 to 2016 for both prednisolone and prednisone. 3022 were reported between 2012 and 2015. For MHRA DAPs, the five most frequently reported adverse event PTs were ‘gastrointestinal haemorrhage’, ‘drug ineffective’, ‘dyspnoea’, ‘malaise’ and ‘pyrexia’.1 (Twitter:MHRA) and PRR2 (MHRA: Twitter) for the top 25 AE PTs by source are reported in Table The PRRIn this study of nearly 100,000 tweets mentioning prednisolone or prednisone, the two most common adverse events mentioned were ‘insomnia’ and ‘weight increased’. These two outcomes were disproportionately detected in Twitter when compared to reports in the Yellow Card Scheme.20 Surprisingly, there is neither significant research previously undertaken to understand the frequency, progression and impact of these outcomes, nor on how best to manage them. In the electronic Medicines Compendium (eMC) patient information leaflet for prednisolone, there is no explicit mention of insomnia as a side effect , nor is there a reference to the predominance of any individual side effects.21 Within the scientific literature, there are very few publications specifically studying the effect of (high-dose) glucocorticoids on insomnia,23 despite it being one of the most commonly discussed side effects on social media.Clinicians may not be surprised by these findings as both insomnia and weight gain are commonly seen in clinical practice. Indeed, patients may opt not to take glucocorticoids on the basis of concerns about side effects, including weight gain, underlining its importance to patients.24 Whilst not identifying new safety signals, the analysis tells us what is happening commonly, and potentially where this has impact on patients’ lives. The importance of the side effects most commonly discussed in this Twitter analysis is supported by a recent survey that has found patients using glucocorticoid therapy are most concerned by weight gain and insomnia.25Within our study of Tweets during the 3 year interval 2012–2015, there were 20,210 glucocorticoid-related AE PTs compared to 3022 AE PTs spontaneously reported to the UK MHRA over the same period. The frequent discussion of common but non-serious side effects may complement traditional methods that more commonly collect unexpected events.26 We did note that a minority of tweets were written in the third person, for example parents tweeting about their children.There are clear limitations in this analysis, including the representativeness of the population and thus generalisability, accurate identification of AEs and appropriate attribution of causality. We did not have access to any demographic information on the study subjects who generated the tweets as such data are not routinely collected within Twitter. It is likely that most tweets were representative of the population of interest (adult users of GC therapy), although younger people may be over-represented.The type of PT detected in Twitter was different to that reported in Yellow Card. Only ‘insomnia,’ ‘drug ineffective’, ‘malaise’, ‘weight increased’, ‘abdominal pain’ and ‘depression’ were present in the top 25 AE list for both Twitter and the Yellow Card database. ‘Non-specific reaction’, the third most commonly detected PT in Twitter, was never coded in Yellow Card, perhaps reflecting the low likelihood of reporting a vague symptom to the regulators. Some of the discrepancies may be accounted for by different terminologies and coding methods: it is possible, for example, that the PT ‘altered state of consciousness’ seen only in the Twitter top 25 may overlap with the PT ‘confusional state’ seen only in the MHRA top 25. Within Twitter, asymptomatic adverse events such as biochemical disturbances were understandably not reported. It was noted that a higher number of less severe AE PTs were reported in Twitter compared to that in Yellow Card. This may be due to differences in the motivation for patients to discuss their medications in Twitter vs. the motivation to report to SRSs. Furthermore, most reports within the Yellow Card database are derived from clinical staff with patients only able to submit suspected ADRs from 2005. Interestingly, within Twitter, nine of the top 25 (40%) AE PTs fell under the System Organ Class (SOC) pertaining to psychiatric disorders, in contrast to just one in the top 25 PTs in Yellow Card. This may reflect willingness for patients to discuss these matters on social media and reluctance for to report such AEs to Yellow Card. It may also represent the greater challenge in attributing drug causality within this type of disorder, something required for the Yellow Card scheme but not for tweets.The upside of prednisone, I am rash free”), negative modifiers  and mapping temporal relationships .27 Others have proposed semantic approaches for identifying adverse events in social media, instead of the lexical approach used here.28Current NLP methods are not perfect in correctly identifying all ADRs. This includes problems of sensitivity (i.e. correctly identifying true ADRs) and specificity (correctly not detecting non-ADRs). Opportunities for improving the automated identification of ADRs include analysis of sentiment, for example using context to identify benefits rather than harms  dictionary. Tweets were automatically categorised based on their likelihood of containing text relating to a GC-related ADR. Tweets with high probability of containing an ADR were further analysed to determine which AE symptoms appeared most frequently. An AE refers to a negative symptom/term , whilst an ADR refers to a causal association between drug and AE. All work was approved by the University of Manchester Computer Science ethics board (ref: CS225).Tweets mentioning prednisolone and prednisone during the specified timeframe were acquired from an unknown denominator of all tweets (irrespective of content) using the search terms ‘prednisolone’ and ‘prednisone’, and misspellings, for example ‘predniolone’ and ‘prdnisone’. Data were processed by Epidemico, Inc. to identify possible ADRs and AEs as described below and detailed elsewhere.29 All data were anonymised, then transferred to the University of Manchester where tweets were de-duplicated  and underwent further analysis as below: user mentions were replaced by @user, all URLs were replaced by [link] and all named entities were replaced by ‘Someone’ or ‘Someone-or-other’ for consecutive entities.Publically-facing tweets were accessed by Epidemico using Twitter’s general-use streaming application programming interface (API) from 1st October 2012 to 30th June 2015. All tweets mentioning the glucocorticoid agents 19 Where tweets mentioned more than one possible AE, each AE was mapped to its respective PT. There were thus more PTs than there were tweets.A subset of all Tweets (28%) mentioning prednisolone or prednisone were manually analysed by human MedDRA® trained curators at Epidemico to train their proprietary NLP automated classifier. The remaining tweets then went through this classifier, which identified possible symptoms and mapped them to the MedDRA® dictionary. Figure 30 tweets tagged with an indicator score of ≥0.7 were categorised as a ‘proto-AE’, meaning the tweet was considered to represent a likely ADR. If a tweet was tagged with multiple PTs, it was assigned a single indicator score.After possible AEs had been identified and mapped to MedDRA®, individual tweets were given an ‘indicator score’ representing the probability that the tweet contained an ADR. In brief, the indicator score uses a Bayesian probabilistic model developed through statistical machine learning computation following manual coding by human curators on a training set. The model generates a score ranging between 0 and 1. The closer the assigned score is to 1, the higher the probability that the tweet contained an ADR. Based on previous work,It was noted during early exploratory analysis that the MedDRA-coded PT in Proto-AE tweets would sometimes represent the indication for treatment (e.g. rheumatoid arthritis), or a non-medical event . A clinical researcher (RP) thus manually assessed each unique PT and identified PTs deemed not to represent a medical event or which referred to a therapeutic indication  established in 1953 and which includes patient-reported data since 2005. Data held within Yellow Card is publically available in the MHRA’s Drug Analysis Prints (DAPs).A proportional reporting ratio (PRR) for each of the top 25 Twitter-reported AE PTs was generated by comparing the proportional frequency of each AE reported through Twitter to the proportional frequency of that same PT in the Yellow Card database for both prednisolone and prednisone. For example, suppose ‘cough’ accounted for 10% of all AE PTs in Twitter, and accounted for 5% of reported AEs in the Yellow Card database, the PRR would be 10/5 = 2.Supplemental Material


Glioblastoma is the most common and aggressive adult brain malignancy against which conventional surgery and chemoradiation provide limited benefit. Even when a good treatment response is obtained, recurrence inevitably occurs either locally (∼80%) or distally (∼20%), driven by cancer clones that are often genomically distinct from those in the primary tumour. Glioblastoma cells display a characteristic infiltrative phenotype, invading the surrounding tissue and often spreading across the whole brain. Cancer cells responsible for relapse can reside in two compartments of residual disease that are left behind after treatment: the infiltrated normal brain parenchyma and the sub-ventricular zone. However, these two sources of residual disease in glioblastoma are understudied because of the difficulty in sampling these regions during surgery.Here, we present the results of whole-exome sequencing of 69 multi-region samples collected using fluorescence-guided resection from 11 patients, including the infiltrating tumour margin and the sub-ventricular zone for each patient, as well as matched blood. We used a phylogenomic approach to dissect the spatio-temporal evolution of each tumour and unveil the relation between residual disease and the main tumour mass. We also analysed two patients with paired primary-recurrence samples with matched residual disease. Our results suggest that infiltrative subclones can arise early during tumour growth in a subset of patients. After treatment, the infiltrative subclones may seed the growth of a recurrent tumour, thus representing the ‘missing link’ between the primary tumour and recurrent disease.These results are consistent with recognised clinical phenotypic behaviour and suggest that more specific therapeutic targeting of cells in the infiltrated brain parenchyma may improve patient’s outcome. Key MessageIn this study, we investigate the evolution of residual disease in glioblastoma that is left behind after surgery, in particular cancer cells in the infiltrative tumour margin and in the sub-ventricular zone. We found that residual disease samples contained early precursor cancer clones that were involved in the development of the disease and may contribute to recurrence.Glioblastoma (GB) is a lethal brain cancer against which effective therapeutic options are lacking . The disThe accumulating clinical and genomic evidence suggests that infiltration may be a very early event in GB development, so that no matter how early a cancer is detected, it has already spread to distal regions of the brain, including the normal brain parenchyma and the SVZ Figure A. After Annals of Oncology online for clinical information). Between five and nine multiple samples from the tumour mass , SVZ and infiltrative margin (M) areas were collected from each patient . Copy number alterations per sample are reported in Figure Annals of Oncology online for details about bioinformatics analysis.Between 100 and 300 ng of DNA from each of the 69 tumour specimens and 11 blood samples were used for whole-exome sequencing using the Agilent SureSelectXT Human All Exon V5 Kit . Median coverage was ×157 . A custom targeted sequencing panel for 891 single nucleotide variants (SNVs)  identified from the exome sequencing data was designed using Agilent’s Haloplex technology (TES1). In addition, we designed a separate Agilent SureSelect XT2 capture panel to specifically validate 1054 SNVs found in the M and SVZ samples across all patients (TES2). Both amplicon (TES1) and targeted capture (TES2) libraries were sequenced on an Illumina HiSeq2500 obtaining a median coverage of ×4050 and ×1128, respectively, in reported variants . Samples from the tumour mass and SVZ from 7/11 patients were common to our previous studies, for which we had carried out microarray copy number profiling and gene expression alone .A key to understanding cancer is not just exposing ITH, the natural process that underlines clonal evolution, but also to understand such heterogeneity in a way that is clinically relevant and therapeutically tractable. An important aspect of genomic ITH is that it embeds the evolutionary history of the tumour, a fundamental biological element that cannot be directly measured in humans. Nevertheless, inferring and understanding that history may be critical in developing a rationale for combinatorial therapeutics .Specifically, in this study, we leveraged the spatio-temporal decomposition of the clonal architecture of the tumour to understand the link between subclones in the main tumour mass and in residual disease left behind in the surrounding brain parenchyma and SVZ following surgery. This residual disease is a key factor contributing to GB treatment failure because of resistance to radiation and alkylating chemotherapy coupled with an inherent ability to seed re-growth. Therefore, the main message of this study is that residual disease and not just the main tumour mass , 21, 22 This observation presents a challenge to the GB research community to develop the tools and strategies needed to collect and robustly analyse difficult samples from residual disease areas in prospective cohorts. Due to the limited number of patients in this study, additional analyses on larger cohorts are necessary to validate these findings. We acknowledge that analysing sparse cancer cells in the margin remains challenging, even with the most advanced sequencing and bioinformatics approaches currently available. Further work is also needed to improve purification of margin samples, which is not currently possible due to lack of reliable markers to sort GB cells. Therefore, new efforts of collecting infiltrative cells that lay distant from the main tumour mass will be needed to study residual disease with more accuracy. To do this, post-mortem efforts such as the PEACE study  are likely to play a key role in revealing the biology of infiltrative disease in GB. Supplementary DataClick here for additional data file.


Here, we investigated whether phospholipase C (PLC)-related catalytically inactive protein (PRIP), a metabolic modulator of PIP2, regulates PIP2-mediated cytokinesis. We found that PRIP localised to the cleavage furrow during cytokinesis. Moreover, HeLa cells with silenced PRIP displayed abnormal cytokinesis. Importantly, PIP2 accumulation at the cleavage furrow, as well as the localisation of RhoA and phospho-myosin II regulatory light chain to the cleavage furrow, were reduced in PRIP-silenced cells. The overexpression of oculocerebrorenal syndrome of Lowe-1 (OCRL1), a phosphatidylinositol-5-phosphatase, in cells decreased PIP2 levels during early cytokinesis and resulted in cytokinesis abnormalities. However, these abnormal cytokinesis phenotypes were ameliorated by the co-expression of PRIP but not by co-expression of a PIP2-unbound PRIP mutant. Collectively, our results indicate that PRIP is a component at the cleavage furrow that maintains PIP2 metabolism and regulates RhoA-dependent progression of cytokinesis. Thus, we propose that PRIP regulates phosphoinositide metabolism correctively and mediates normal cytokinesis progression.Cytokinesis is initiated by the formation and ingression of the cleavage furrow. Phosphatidylinositol 4,5-bisphosphate [PIP One major phosphoinositide involved is phosphatidylinositol 4,5-bisphosphate [PIP2]. During cytokinesis, PIP2 accumulates and is maintained at adequate levels at the cleavage furrow from the initiation of furrowing until the completion of constriction in mammalian cells3. PIP2 levels in the plasma membrane are regulated by a variety of metabolic enzymes such as phospholipase C (PLC), phosphoinositide kinases, and phosphoinositide phosphatases including oculocerebrorenal syndrome of Lowe-1 (OCRL1), an inositol polyphosphate 5-phosphatase4.Cytokinesis is the final step of cell division, in which a parent cell divides into two daughter cells by the formation and ingression of a cleavage furrow at the plasma membrane after chromosome segregation. A number of phosphoinositides and phosphoinositide-modifying enzymes are involved in cytokinesis, and phosphoinositide production and hydrolysis, which are required for specific steps of cytokinesis, are regulated spatially and temporally2 levels at the cleavage furrow causes cytokinesis abnormalities. For example, the overexpression of a kinase-deficient phosphatidylinositol 4-phosphate 5-kinase (PIP5K) mutant and of PIP2-phosphatase synaptojanin or the microinjection of anti-PIP2 antibodies impairs cytokinesis in mammalian cells5. Therefore, properly maintaining PIP2 levels at the cleavage furrow is crucial for normal cytokinesis progression6. However, the mechanism by which PIP2 metabolism is regulated during furrow ingression is not fully understood.Disruption of PIP7. An important role of PIP2 is the recruitment of RhoA to the target membrane, as the depletion of PIP2 inhibits the recruitment of RhoA to the cleavage furrow in cytokinesis9. RhoA has a positively charged amino acid cluster at the carboxy-terminus, which can directly interact with negatively charged phosphoinositides, including PIP2, in vitro10. ECT2 also interacts with phosphoinositides, including PIP2, in vitro11. Therefore, the direct binding of Rho-GEF and RhoA to PIP2 in the cleavage furrow could be an important process in cytokinesis progression.Cleavage furrow progression requires the activation of the small GTPase RhoA, which requires epithelial cell transforming sequence 2 (ECT2), a Rho GDP/GTP exchange factor (RhoGEF)12, which enhances the ATPase activity of myosin II and promotes the assembly of monomers of myosin II into bipolar filaments, followed by the generation of a contractile force, which then cleaves the parent cell into two daughter cells14.Actin filaments and myosin-II are evolutionarily conserved force-generating components of the contractile ring during cytokinesis. Contractile force is generated by non-muscle myosin II, which is regulated by RhoA/Rho-associated protein kinase (ROCK) signalling. Activated ROCK phosphorylates myosin II regulatory light chain (MRLC) directly and also phosphorylates myosin phosphatase-targeting subunit 1, inactivating it. Consequently, phospho-MRLC (Ser19) is augmented17. There are two isoforms of PRIP in mammals, PRIP1 and PRIP2: the former is expressed mainly in the brain and the lung, and the latter is expressed ubiquitously19. PRIP1 was originally isolated as a cytosolic protein containing a pleckstrin homology (PH) domain, which binds to inositol-1,4,5-triphosphate [InsP3]21. Subsequently, PRIP1 and PRIP2 were reported to bind to PIP2 via its PH domain and localise to the plasma membrane24. Although the sequence of the PRIP PH domain is similar to that of PLCδ1, PRIPs exhibit distinct functional characteristics with respect to InsP3 and PIP2 binding25. Amino acid R40 in PLCδ PH is a critical amino acid residue for InsP3 binding, and PRIP1 PH(R134Q) mutant (R134 corresponds to R40 in PLCδ PH) fails to bind with InsP3 or PIP226.PLC-related catalytically inactive protein (PRIP) is a unique protein with high homology to the PLCδ1 isozyme but lacking PLC activity2 into phosphatidylinositol 3,4,5-trisphosphate by phosphoinositide 3-kinase (PI3K), a process that suppresses the migration of cancer cells27. However, little is known regarding the involvement of PRIP in PIP2-dependent cytokinetic events. Therefore, we examined the potential role and regulatory mechanism of PRIP in cytokinesis.We recently reported that PRIP negatively regulates the conversion of PIPWe first investigated the localisation of PRIP during cytokinesis using HeLa and HEK293 cells transiently transfected with PRIP1 or PRIP2. Both enhanced green fluorescent protein (EGFP)-PRIP1 and EGFP-PRIP2 signals in HeLa cells clearly localised to the cleavage furrow, where phosphorylated MRLC signal  was observed Fig. . Similar24. Therefore, we next examined the localisation of PRIP1 and PRIP2 during interphase. EGFP-PRIP1 and EGFP-PIRP2 mainly localised to the cytoplasm, with some signal being observed on the plasma membrane in both HEK293 and HeLa cells , HEK293 (a human embryonic kidney cell line), and MCF-7 (a human breast cancer cell line). PRIP1 is not expressed, resulting in levels of about 40% and 10% that in control cells following transient transfection with PRIP2-specific siRNAs PRIP2-si1 and PRIP2-si2, respectively  and a non-phosphorylatable form of MRLC . EGFP-AA-MRLC localised to the cleavage furrow in HeLa cells -mediated actin nucleation and polymerisation, as well as ROCK/pMRLC-dependent myosin II activation, consequently leading to the assembly and contraction of the actomyosin ring at the cleavage furrow32. PRIP2-knockdown HeLa cells exhibited a decrease in the thickness of F-actin as well as a reduced signal intensity of pMRLC in the cleavage furrow P2 directly, which is required to complete cytokinesis9. In addition, PIP2 regulates the accumulation of RhoA at the cleavage furrow in HeLa cells3. We previously reported a protective effect of PRIP on PIP2 metabolism in migrating cells27. Therefore, we predicted that PIP2 accumulation at the cleavage furrow would be regulated by PRIP. To directly monitor plasma membrane PIP2 levels during cytokinesis, we successfully established a HEK293 cell line that stably expressed the EGFP-tagged PH domain of PLCδ1 (EGFP-PLCδPH), a probe for PIP2. The isolated cell line had a single peak distribution of EGFP intensity, as determined by flow cytometry Ptry Fig. , and EGFtry Fig. . Endogentry Fig. . PRIP2 sasm Fig. . Howevernal Fig. .2, BODIPY FL-PIP2 was exogenously added to HeLa cell culture before examination of PRIP localisation. BODIPY FL-PIP2 signal was observed at the cleavage furrow and the plasma membrane, and in intracellular components , in which the PH domain fails to bind PIP233, was observed in the cytoplasm and failed to accumulate at the cleavage furrow P2 levels in the cleavage furrow P2 levels at the cleavage furrow is either via the upregulation of new PIP2 synthesis or the downregulation of PIP2 metabolism into other phospholipids. To address this issue, we examined the turnover of PIP2 at the cleavage furrow using stably EGFP-PLCδPH-expressing HEK293 cells. The synthesis and metabolism rates of PIP2 were assessed by measuring the GFP intensity of PLCδPH via fluorescence recovery after photobleaching (FRAP) analysis. We selected a cell starting cytokinesis and bleached the region of the cleavage furrow with a laser line. At the non-bleached side of the cleavage furrow, the GFP intensity did not differ between control siRNA- and PRIP2-si1-transfected cells P2 metabolism.Although PRIP is engaged in maintaining PIProw Fig. , it remalls Fig. , suggestlls Fig. , suggestOCRL1 (an inositol polyphosphate 5-phosphatase) in HeLa cells results in the accumulation of PIP2 at the intercellular bridge in late cytokinesis34. We ensured that Halo-OCRL1 overexpression efficiently reduced PIP2 levels at the intercellular bridge compared with that in control cells in late cytokinesis P2 metabolism by OCRL1 at the cleavage furrow. We found that, during cleavage furrow formation, exogenously expressed Halo-OCRL1 was diffusely distributed in HEK293 cells, and PIP2 levels  were obviously reduced at the plasma membrane, including the cleavage furrow, compared with that in Halo vector-transfected control cells -mutant and Halo-OCRL1-co-transfected HEK293 cells exhibited blurred signals of PLCδPH and DsRed-PRIP1(R134Q) P2 levels to those in non-Halo-OCRL1-transfected cells, but Prip1(R134Q) overexpression did not , or EGFP vector27 in which Halo vector or Halo-OCRL1 was transiently transfected. Approximately 80% of OCRL1-transfected EGFP vector-expressing MCF-7 cells showed abnormal cytokinesis -expressing cells. These results suggest that PRIP protects PIP2 from OCRL1-mediated hydrolysis, which requires the binding of PRIP and PIP2 via the PRIP PH domain.To investigate the effects of PRIP in OCRL1-expressed cells on cytokinesis progression, we performed live-cell imaging. We used MCF-7 cells stably expressing sis Fig. . Importa2 recognition of OCRL1 and PLCδ1, PIP2-metabolising enzymes. An in vitro co-sedimentation assay was performed using liposomes composed of 100% phosphatidylcholine (PC) or 5% PIP2 and 95% PC following the procedure described in Fig. OCRL1-expressing or PLCδ1-expressing HeLa cells by centrifugation and used in the precipitation assays. Halo-OCRL1 precipitated with liposomes composed of 5% PIP2 and 95% PC but did not precipitate with 100% PC liposomes P2 was inhibited by the addition of PRIP1 P2 metabolism.We next examined whether PRIP affects the PIPted Fig. . Similar2 and modulates PIP2-mediated cytokinesis9. Therefore, to determine whether PRIP affects the association between RhoA and PIP2, we performed the PIP2-liposome sedimentation assay in the presence of OCRL1, RhoA, and PRIP1 P2 was not affected, even at a high dose of PRIP PIP1 Fig. . The assRIP Fig. . These r2, which is an integral signalling molecule despite being a minor component of cellular membranes, is generated by metabolic enzymes at the cleavage furrow and functions there during cytokinesis35. Therefore, adequate PIP2 metabolic regulation is crucial for correct cytokinesis progression. The findings in this study demonstrated that the binding of PRIP to PIP2 maintains normal PIP2 levels at the cleavage furrow, which leads to the proper completion of cytokinesis. We thus propose that PRIP is a new candidate for protecting PIP2 from metabolism by associated enzymes during cytokinesis. Elucidation of this novel PRIP-mediated cytokinesis regulation pathway may aid our understanding of fundamental cellular mechanisms.Cytokinesis is a highly ordered process requiring an intricate interplay among cytoskeletal, chromosomal, and cell cycle regulatory pathways. Additional cellular processes, including phosphoinositide metabolism on the plasma membrane, are also important for cytokinesis. PIP2 interacts with Rho, regulates Rho GTPase activity at the cleavage furrow, and facilitates the recruitment of contractile ring constituents to the cleavage furrow as a scaffold lipid9. We previously reported that PRIP negatively regulates PIP2 metabolism in migrating cells27. In this study, a deficiency of PRIP2 in HeLa cells was found to cause cytokinesis defects and delay by suppressing the accumulation of PIP2 and RhoA at the cleavage furrow. The demonstrated PRIP role here is that PRIP maintains PIP2 levels by protecting PIP2 from its metabolising enzymes at the cleavage furrow and regulates the RhoA/pMRLC-dependent progression of cytokinesis. Rho also stimulates PIP5K activity36 and ROCK-mediated phosphatase and tensin homologue (PTEN) signalling37, which results in the upregulation of PIP2 synthesis. Thus, PIP2, under the guardianship of PRIP, may accelerate the accumulation of RhoA at the cleavage furrow, followed by further PIP2 production by PI5K and/or PTEN. This positive feedback loop contributes to the initiation of the cleavage furrow and the promotion of cleavage furrow ingression to complete cytokinesis.PIP31. RhoA activates ROCK and citron kinase, which phosphorylate the MRLC of non-muscle myosin II31. RhoA also binds and activates formins that elicit actin nucleation and polymerisation. Thus, RhoA coordinates the RhoA/ROCK/pMRLC and RhoA/formin signalling pathways and facilitates actomyosin ring assembly and contraction. In this study, PRIP2-silenced cells exhibited reduced pMRLC levels and the accumulation of F-actin and RhoA at the cleavage furrow. Therefore, these results indicate that PRIP is the most upstream molecule in the RhoA-mediated signalling pathways, profoundly modulating F-actin formation and MRLC phosphorylation. Although PRIP2-silenced cells displayed reduced accumulation of F-actin at the cleavage furrow, the transfection of a phospho-mimic MRLC mutant (AD-MRLC) alone efficiently rescued the cytokinesis delay in PRIP2-silenced cells. It was previously reported that actomyosin contractile ring assembly is regulated by actin-related protein 2 and 3 (ARP2/3), which enhance actin nucleation39. Myosin II heavy chain accumulates at the cleavage furrow and promotes the elongation of actin bundles40. These are independently modulated by the RhoA pathway during cytokinesis. Therefore, regulation of F-actin assembly at basal levels during cytokinesis is most likely modulated by RhoA-independent signalling.Anillin, a highly concentrated protein in the cleavage furrow, is a scaffolding protein that links RhoA, F-actin, and myosin II and regulates the contractile ring2 is a substrate of PLC, OCRL1, and PI3K. These enzymes are involved in PIP2 turnover. Several PLC isoforms are localised to the cleavage furrow, and PLC activity is required for cytokinesis42. The overexpression of synaptojanin failed to lead to PIP2 and RhoA accumulation in the cleavage furrow3. In addition, a PIP2 phosphatase, OCRL, which is mutated in patients with oculocerebrorenal syndrome of Lowe, participates in cell abscission during cytokinesis34. Inhibition of the PIP2 kinase PI3K by a PI3K inhibitor such as LY294002 or wortmannin induces a delay in cytokinesis when the inhibitor is added at during furrow cleavage formation43. These reports indicate that the dynamic regulation of PIP2 metabolism and PIP2 maintenance at the cleavage furrow are crucial for correct cytokinesis progression. Here, we showed that the overexpression of OCRL1 resulted in reduced PIP2 accumulation at the cleavage furrow and abnormal and delayed cytokinesis. This phenotype was rescued by the additional expression of PRIP1 but not of a PIP2-unbound PRIP mutant. Importantly, FRAP analysis confirmed the inhibitory effect of PRIP on PIP2 metabolism at the cleavage furrow. Collectively, our data demonstrate that PRIP protects PIP2 via the PRIP PH domain from metabolism by its enzymes and maintains sufficient levels of PIP2 for cytokinesis.PIP2, in vitro. This binding activity requires the recruitment or stabilisation of RhoA at the cleavage furrow44. In our experiments, PRIP competitively inhibited substrate recognition by OCRL1 or PLCδ1 but did not affect the binding of RhoA to PIP2. These data indicate that the binding mode of RhoA to PIP2 differs from those of PRIP, OCRL1, and PLCδ1. These results support the positive role of PRIP in regulating the PIP2/RhoA signalling pathway.RhoA has a carboxy-terminal polybasic tail, which can, through electrostatic interactions, directly bind to several phosphatidylinositols, including PIP227. PRIP1(R134Q), a PIP2-unbound mutant with a mutation in the PH domain33, slightly accumulated at the cleavage furrow and slightly rescued cytokinesis abnormalities observed in OCRL1-overexpressing cells. The binding of the X-Y domain to PIP2 in the PRIP(R134Q) mutant may inhibit the association between PIP2 and OCRL1. Consequently, the PRIP(R134Q) mutant partially restored cytokinesis.PRIP contains an X-Y domain, which is similar to the PLC catalytic core subunit in PLCδ1. Although PRIP1 and PRIP2 do not have activity, the X-Y domain of PRIP can associate with PIP2 metabolism in the cleavage furrow during cytokinesis. Our findings conclusively demonstrate that, even if PIP2 is exposed to a number of its metabolic enzymes, PRIP can maintain the high levels of PIP2 required at the cleavage furrow to promote correct cytokinesis. The continuous phosphoinositide cycling required at the cleavage furrow is regulated by a positive feedback mechanism involving PIP2 accumulation via PRIP-mediated RhoA signalling, which is necessary for normal cytokinesis progression.This is the first study to elucidate the existence of a rheostat for PIPPLCδPH, Prip1, Prip1(R134Q), and PRIP2; DsRed2-tagged Prip1 and Prip1(R134Q); GST-tagged PRIP2 and PLCδ1; and Halo-PRIP1 were described previously45. EGFP-tagged AD-MRLC and AA-MRLC were described previously46. Halo-tagged OCRL1 (FHC01740) and RHOA (FHC01522) were purchased from Promega . Human PRIP2-siRNAs  were purchased from Sigma Genosys . A negative control siRNA (S10C-0600) was purchased from Cosmo Bio .EGFP-tagged 49. Anti-β-actin antibody (IMG-5142A) was obtained from Imgenex . Anti-β-tubulin (D66) and anti-total MRLC (MY-21) antibodies were purchased from Sigma-Aldrich . Anti-GST antibody (M209-3) was obtained from Medical & Biological Laboratories . Anti-RhoA (sc-418) and anti-PLCδ1 (D-7) antibodies were purchased from Santa Cruz Biotechnology . Anti-HaloTag antibody (G928A) was purchased from Promega. Alexa Fluor 488-conjugated antibodies , Alexa Fluor 594-conjugated anti-mouse IgG (A11005), and Alexa Fluor 405-conjugated antibodies [anti-rabbit IgG (A31556) and anti-mouse IgG (A31553)] were obtained from Invitrogen . Alexa 594-conjugated anti-mouse IgM (21044) antibody was purchased from Thermo Fisher Scientific .Anti-phospho-MRLC , anti-GAPDH (#2118), anti-OCRL1 (#8797), HRP-conjugated anti-rabbit IgG (#7074), and HRP-conjugated anti-mouse IgG (#7076) antibodies were purchased from Cell Signaling Technology . Anti-PRIP1 and anti-PRIP2 polyclonal antibodies were developed previously27. HEK293 cells were transfected with an expression vector encoding EGFP-PLCδPH or a control EGFP vector and cultured in the presence of 1 mg/ml G418  for 14 days. Stably EGFP-expressing colonies were selected. Cells were cultured under conventional growth conditions and transfected with plasmids or siRNA using the Lonza 4D-Nucleofector X Unit  or Lipofectamine 3000 (Invitrogen).MCF-7 cells stably expressing EGFP-PRIP1 or EGFP-PRIP1(R134Q) were developed previouslyPRIP1, PRIP2, and GAPDH were amplified using specific primer pairs whose sequences were previously described27. The PCR products were distinguished by agarose gel electrophoresis.A 5-μg sample of total RNA from HeLa cells, isolated using an RNeasy minikit , was used for reverse transcription reactions using a cDNA synthesis kit . 29.Cells were seeded on μ-dishes  and cultured until attached. Transfection was performed as described above. Cells were synchronised with 50 μM monastrol  for 12 h, and then monastrol was washed out. For detection of HaloTag, cells were incubated with 1 μM of HaloTag TMR ligand or HaloTag Coumarin ligand (Promega) for 1 h before washout of the ligand. The cells were recorded every minute for 3 h by live-cell imaging on a BZ-9000 microscope . During FRAP analysis, EGFP images were scanned before and after bleaching at low laser power on a confocal laser scanning microscope . To destroy EGFP fluorescence, maximal laser power was applied to a furrowing region for 30 s. Images were taken before and after bleaching at intervals of 1 min until completion of cytokinesis. Cytokinesis analysis and measurement of fluorescent intensity were performed using ImageJ 1.45 s  following previously described methods51. F-actin was visualised by ActinRedTM 555 ReadyProbes (R37112) (Thermo Fisher Scientific) or Alexa Fluor 350-labelled phalloidin (Invitrogen). RhoA staining was carried out following the previously described method52. Fluoview FV10i was used for observations. The acquired images were analysed by ImageJ 1.45 s.Immunofluorescence and western blotting were carried out following previously described methods2 was carried out using the method described previously27, with modifications, using PIP2  and/or phosphatidylcholine . Cell lysates were obtained from cells transiently expressing Halo-OCRL1 and/or Halo-RhoA. Recombinant EGFP-PRIP1 and GST-PRIP2 were purified from MCF-7 cells stably expressing EGFP-Prip1 and HEK293 cells transiently expressed GST-PRIP2. The detailed method is described in Fig. A binding assay of OCRL1 and RhoA to liposomes containing PIPp-value less than 0.05 was considered statistically significant.GraphPad Prism  was used for statistical analyses. The non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test was used. A Supplementary Information


While intratumour genetic heterogeneity of primary clear cell renal cell carcinoma (ccRCC) is well characterized, the genomic profiles of metastatic ccRCCs are seldom studied. We profiled the genomes and transcriptomes of a primary tumour and matched metastases to better understand the evolutionary processes that lead to metastasis. In one ccRCC patient, four regions of the primary tumour, one region of the thrombus in the inferior vena cava, and four lung metastases (including one taken after pegylated (PEG)-interferon therapy) were analysed separately. Each sample was analysed for copy number alterations and somatic mutations by whole exome sequencing. We also evaluated gene expression profiles for this patient and 15 primary tumour and 15 metastasis samples from four additional patients. Copy number profiles of the index patient showed two distinct subgroups: one consisted of three primary tumours with relatively minor copy number changes, the other of a primary tumour, the thrombus, and the lung metastases, all with a similar copy number pattern and tetraploid-like characteristics. Somatic mutation profiles indicated parallel clonal evolution with similar numbers of private mutations in each primary tumour and metastatic sample. Expression profiling of the five patients revealed significantly changed expression levels of 57 genes between primary tumours and metastases, with enrichment in the extracellular matrix cluster. The copy number profiles suggest a punctuated evolution from a subregion of the primary tumour. This process, which differentiated the metastases from the primary tumours, most likely occurred rapidly, possibly even before metastasis formation. The evolutionary patterns we deduced from the genomic alterations were also reflected in the gene expression profiles. Kidney cancer is a usually lethal urologic malignancy with an annual mortality rate of 50% and an annual incidence of 337,000 new cases worldwide . About 3Although somatic mutation and RNA expression profiles of primary ccRCC have been extensively described in the Cancer Genome Atlas project , the genUnderstanding how molecular (genomic) alterations accumulate during tumour evolution is important to gain insight into the development of metastasis and might provide clues to more optimal treatment strategies. As the majority of metastases are thought to establish through hematogenous dissemination , studyinIn this study, we extensively analysed samples from a unique ccRCC patient for whom nine samples were available taken from multiple regions of a primary ccRCC tumour, tumour thrombus reaching inferior vena cava, and four pulmonary metastases from different sites in the lung. We analysed the pattern of CNAs, SNVs, and gene expression in these samples to interrogate the process of tumour evolution in this patient. To explore the differences in gene expression among primary ccRCCs and metastases, we then analysed samples from four additional patients.Array comparative genomic hybridization (CGH)-based copy number profiles were generated for three primary tumour samples , the inferior vena cava tumour thrombus (VT), and four lung metastases (M1–M4) of the index patient . All samA phylogenetic tree based on the CNA data  shows a We generated B-allele frequency (BAF) plots for all samples, including Pr2, for which we had no array CGH data. The BAF plot of Pr2 resembled that of Pr1 and Pr3, indicating a similar CNA pattern . We thenWhole exome sequencing (WES) was conducted for all tumour samples of RC1 with matched normal kidney cortex used as control. The mean target coverage for all samples was 57×, with a coverage >10× for 90% of the target region . A total of 146 non-synonymous somatic mutations identified in 138 genes were defined as a major clone mutation in at least one tumour sample, adding up to 390 events of major clone mutations in nine tumour samples . In an aVHL and PBRM1) and in EPHA4, a lung adenocarcinoma driver gene [ARID1A. Twenty mutations were unique for the metastases. We did not observe a clear increase in mutational load consistent with the evolutionary path reflected by CNA patterns depicted in BLOC1S4, WDFY4, and CUBN) were shared with VT and all lung metastases.Eighteen of the 146 non-synonymous somatic mutations (12.3%) were shared by all tumour samples. Among these were mutations in two well-known ccRCC cancer driver genes , 32 of which were upregulated and 25 that were downregulated in metastases versus the primary tumours (p < 0.009)  and extracellular matrix organization (p < 0.03) . All differentially expressed genes that clustered in the blood micro-particle and extracellular matrix organization groups were upregulated in metastases, except for ALB and ACAN. Although not indicated as an enriched pathway, seven genes  in this list are related to the epithelial–mesenchymal transition pathway [We next identified genes consistently differentially expressed between primary tumours and metastases of all five patients. As the VT sample of the index patient (RC1) could not be clearly classified as a primary tumour or a metastasis, this sample was not included. The analysis identified 57 genes with an absolute fold change > 3 , and all lung metastases, had a complex, largely similar, copy number profile with tetraploid characteristics. We assumed that conversion of a near diploid tumour cell (Pr1–Pr3) to a near tetraploid state (Pr4) resulted in a tumour–cell lineage with metastatic potential in this patient.Theoretically, the VT could have acted as a “distribution station” between the primary tumour subclone with metastatic potential and all other metastases, as has been observed by Turajlic et al . AlternaVHL and PBRM1, which were, therefore, most likely involved in the initiation of tumour development in this patient. A low number of shared mutations between primary tumours and their metastasis was also reported in a study where single primary tumour samples were compared with a single metastatic sample per patient [Compared to the analysis of the copy number changes, the somatic mutation profiles of the tumours were less informative for reconstructing metastatic origin but did reveal early evolutionary steps. We did not observe a clear difference in the mutation load between Pr1, Pr2, and Pr3, on the one hand, and Pr4, VT, and the lung metastasis, on the other. Only 12% of the somatic mutations were shared between all primary tumour subregions and the metastases. These included mutations in the well-known ccRCC-specific cancer driver genes  patient . We did  patient . It probWe identified several somatic mutations restricted to the different metastatic sites in the lungs, but none of these mutations were shared between all lung metastasis sites. This, again, indicates that metastasis-to-metastasis dissemination of cancer cells, e.g., as seen in prostate cancer , did notARHGAP12 and a frameshift deletion of CENPN in the VT and all lung metastatic samples. Gene ARHGAP12 encodes a junctional complex protein that affects tumour cell adhesion, scattering, and migration driven by hepatocyte growth factor [ARHGAP12 has been related to the increased invasive growth of human cancer cell lines from lung epithelial cells, prostate, thyroid, and breast [CENPN is important in kinetochore assembly prior to mitosis [CUBN. Being expressed in several normal epithelial cell types, including those in the kidney, CUBN encodes a receptor for intrinsic factor-vitamin B12 complexes [CUBN expression has been found by others in venous tumour thrombus and lung metastases, as compared to primary tumours of ccRCC [CUBN expression was associated with poor overall and cancer-specific survival in ccRCC patients. In our patient’s tumours, we did not observe low CUBN expression. In fact, none of the genes that showed differential expression between primary tumour and metastases carried a mutation in any of the tumour samples. Conversely, the mutations that occurred in these tumours, apparently did not influence mRNA levels of the mutated genes.With respect to the mutations that may have facilitated the process of metastasis, we found it interesting that we observed a missense mutation of h factor ,24. Thesh factor . Downreg mitosis . Althougomplexes . Low CUBof ccRCC . The samTo characterize tumour subclones in primary tumours and metastases based on gene expression, we analysed mRNA profiles of the index patient and four additional ccRCC patients. Through gene expression profiling, we were able to confirm the putative metastatic seeding subclone in the index patient. In the additional patients, we succeeded in one case (RC3). Our inability to identify the putative metastasis-seeding subclone in the other three patients was most likely due to the incomplete sampling and did not exclude the presence of such a subclone in the primary tumour. Among the 57 differentially expressed genes in the metastatic versus primary tumour samples, six extracellular matrix genes were upregulated, and one was downregulated in the metastases. We also observed upregulation of seven genes related to the epithelial–mesenchymal transition in the metastases, three of which are part of the extracellular matrix pathway as well. Together with intratumoural hypoxia, the extracellular matrix has been suggested to play a crucial role in metastasis development  through Formalin-fixed paraffin-embedded (FFPE) tissues were collected from four different regions of the primary tumour, from the venous tumour thrombus and from four metastatic lesions of one ccRCC patient—the index patient . With thFor four additional ccRCC patients, multiple regions of a primary ccRCC tumour and metastases were available . HaematoFor all of the index patient’s tumour samples, array CGH was carried out using 500 ng genomic DNA from FFPE tumour samples using the Complete Genomic SureTag DNA Enzymatic Labelling Kit protocol and an OligoaCGH/ChIP-on-Chip Hybridization kit , according to the manufacturer’s instructions. Normal kidney cortex DNA isolated from FFPE material from the index patient was used as reference. Labelled DNA samples were hybridized on the Agilent Microarray, Custom HD-CGH, 4 × 180 K  following the manufacturer’s protocol. After scanning of the arrays, data were analysed with Nexus 7.5 software .https://github.com/mmterpstra/molgenis-c5-TumorNormal/tree/459417cc9553fae8c3040953970938860dafdfea), as described previously. The GATK HaplotypeCaller (downloaded from https://software.broadinstitute.org/gatk/) was used as variant caller [All tumour samples from the index patient were subjected to whole exome sequencing (WES) . Exome capturing and subsequent library preparation were conducted using 100 ng of DNA isolated from FFPE material using SureSelect-All exon V2™ . The final library was quantified using an Agilent 2100 Bioanalyzer . Paired-end sequencing with 100 bp reads was performed on the Illumina HiSEQ 4000™ . Sequencing data were processed using our in-house bioinformatics pipeline , according to the protocol for degraded (FFPE) RNA. The enriched libraries were sequenced on the Illumina NextSeq 500 System . Processing of raw sequence reads and subsequent gene expression analysis are described in the Copy number alteration and somatic mutation profiling from multi-region sampling of the primary tumour and metastases facilitates the identification of somatic alterations that underlie early events in tumour evolution and subsequent events in metastatic development. Gene expression profiling can reveal additional alterations at the transcriptome level. Together, these techniques may help to further identify the genomic profiles in primary renal cell cancer and their metastases, with the ultimate goal of finding patterns that can improve diagnostics and guide clinical management of this severe condition.


ETV6–NTRK3 fusion. Treatment with larotrectinib led to rapid clinical improvement with near total resolution of primary and metastatic lesions on MRI imaging. This is the first report of a TRK fusion glioma successfully treated with a TRK inhibitor.Genes encoding TRK are oncogenic drivers in multiple tumour types including infantile fibrosarcoma, papillary thyroid cancer and high-grade gliomas (HGG). TRK fusions have a critical role in tumourigenesis in 40% of infant HGG. Here we report the first case of a TRK fusion-driven HGG treated with larotrectinib—the first selective pan-TRK inhibitor in clinical development. This 3-year-old girl had failed multiple therapies including chemotherapy and radiotherapy. Tumour profiling confirmed an  Progression following RT leads to a dismal outcome.5High-grade gliomas (HGG) are highly aggressive brain tumours that affect both adults and children.7 Three members of the TRK proto-oncogene family have been described: TRKA, TRKB and TRKC, coded by the NTRK1, NTRK2 and NTRK3 genes, respectively.8 TRK fusions have been identified at varying frequency in paediatric gliomas, including in 3 out of 7 cases of infant HGG in one study, and have a critical role in tumourigenesis in mouse models.9Genes encoding neurotrophin receptor kinases (TRK) have recently been implicated as oncogenic drivers in multiple tumour types including infantile fibrosarcoma, papillary thyroid cancer and, rarely, adult HGG.10 Conversely, solid tumours and HGG without TRK fusions were unresponsive to the inhibitor.11 Here we report the first case of a patient with a TRK fusion-driven HGG treated with a TRK inhibitor.Larotrectinib is the first selective pan-TRK inhibitor in clinical development. Recent Phase 1 results showed that larotrectinib is well tolerated in children, with very high response rates in solid tumours that harbour TRK fusions.5The patient is a 3-year-old girl who was diagnosed with a brain tumour at 5 months of age. She presented initially with vomiting and seizures and an MRI showed a heterogeneous mass measuring 6 × 3 × 2 cm in the right lateral ventricle. Following gross total resection pathology showed predominance of large epithelioid and spindle-shaped cells with mild pleomorphism, mitotic index of 14 per 10 high power fields and a Ki67 proliferative index of 40%. The tumour showed patchy positivity for GFAP, strong nuclear staining for p53, and was negative for synaptophysin, chromogranin, NeuN, BRAF V600E, H3K27M and ATRX. She was diagnosed with a HGG and was treated with an infant brain tumour protocol with 13 cycles of chemotherapy.Four months after completing treatment, she had disease progression in the tumour bed with multiple nodules in the lateral and third ventricles. Further tumour debulking confirmed recurrent HGG. After 6 months, a new mass in the tumour bed was subtotally resected and she received focal radiotherapy of 54 Gy to the tumour bed. The resected tumour was profiled on a pilot personalised medicine study. Three months following completion of radiation therapy, she represented with difficulty walking, drowsiness, vomiting and irritability. MRI showed widespread progressive disease with increased enhancement at the resection site, and enlarging suprasellar and subependymal nodules in the lateral and third ventricles. Dexamethasone was continued at 1.5 mg daily. The parents were told that she was incurable, and she was referred to palliative care for symptom management.ETV6–NTRK3 fusion  domain, as well as the NTRK3 protein tyrosine kinase (PTK) domain , as well as somatic copy neutral loss of heterozygosity on chromosome 17, resulting in clonal biallelic loss of TP53, plus hemizygous loss of 9p and 18q, three copies of chrX, and biallelic focal deletion of CDKN2A/B. No germline mutations were identified.Whole-genome sequencing of fresh-frozen tumour DNA (116× average depth) and matched germline DNA (43× average depth) revealed a t translocation, resulting in an ion Fig. . The resain Fig. . RNA-Seqsed Fig. . Immunoh2 bd. After 4 weeks, she had no further lethargy, drowsiness, headaches or vomiting, was eating well, started talking clearly and had been weaned off dexamethasone. After 6 weeks, she was able to walk independently, was speaking in 2–3 word sentences, and had normal energy levels. By week 8, she was running, dancing, and continued to gain new words and language. No adverse events attributable to larotrectinib were observed.Compassionate access to larotrectinib was obtained and commenced at a dose of 100 mg/mAn MRI performed after 8 weeks of therapy showed resolution of the enhancing suprasellar mass, with improvement or resolution of all metastatic ventricular nodules and significantly less enhancement in the surgical bed. MRI at 5 months confirmed the response, with resolution of enhancement in the tumour bed and almost all metastatic lesions Fig. . As of t5 The response to larotrectinib following failure of multiagent chemotherapy and radiotherapy suggests that TRK inhibitor therapy should be tested in the treatment of other TRK fusion-driven gliomas.To our knowledge, this is the first reported case of treatment with, and response to, a TRK inhibitor, in a patient with a TRK fusion-driven HGG. The marked response opens a new paradigm in the management of patients with this aggressive brain tumour. While 50% of infants with HGG respond to systemic chemotherapy, progression following chemotherapy and radiotherapy remains incurable, with dismal outcomes.13 In the recent report on larotrectinib efficacy in paediatric and adult clinical trials, 75% of patients with a TRK-driven tumour had an objective response to therapy.14 However, no patients were reported who had brain tumours harbouring TRK fusions, and the utility of this inhibitor for these HGG patients has, until now, been unknown. This report indicates that larotrectinib penetrates the BBB and may have potent activity in TRK-driven HGG.There are several TRK inhibitors currently in development, but no clinical results have till now been published in patients with TRK-driven brain tumours. Entrectinib is a selective tyrosine kinase inhibitor of the TRK kinases, C-Ros oncogene 1 and anaplastic lymphoma kinase. It has been shown to be active in a patient with a TRK-driven non-small cell lung cancer with cerebral metastases, suggesting the potential for this class of drugs to target primary intracranial tumours.6 however, given the paucity of effective treatment options for these patients, the results here suggest that biomarker-driven trials of TRK inhibitors are warranted in adult HGG patients.It is unclear whether the activity seen in this case can be translated to adults with HGGs that harbour TRK fusions. TRK fusions in adult HGG are rare,15 Further assessment of larotrectinib in clinical trials of HGG patients is needed to determine whether genomic profiling should be considered early in patient work-up. More evidence is also required to integrate TRK inhibitors into standard treatment, especially as some infants have durable responses to first-line chemotherapy. TRK inhibitors could be trialled in infants with relapsed or progressive disease, and as a strategy to avoid the damaging effects of high-dose radiation therapy, or combined with chemotherapy at diagnosis. The potential for integration of this molecularly driven, targeted therapy into the treatment for patients with HGG warrants further testing in clinical trials.How can TRK inhibitors be incorporated into the treatment of TRK fusion-driven HGG? Most important will be the detection of fusions by immunohistochemistry, FISH or sequenced-based tumour profiling . The latter techniques provide an unbiased approach, and may identify fusions in atypical tumour types, or novel rearrangements which may not be detected with standard tests. Notably, the pan-TRK antibody used here failed to recognise the ETV6–TRK3 fusion in this case, similar to a recent analysis.


Sci., 2016, 7, 5893–5899.Correction for ‘Face and edge directed self-assembly of Pd An incorrect grant number was quoted for this paper and the corrected Acknowledgement section is as shown below:P. S. M. thanks the DST (New Delhi) for the research grant [DST/SJF/CSA-01/2011-2012(G)]. P. H. is grateful to Mr Sayantan Chatterjee for the rheology experiments and also thankful to Mr Shaunak Chakraborty for fruitful discussion on the single crystal structure analysis.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


B and Hartmann number H. Skin friction coefficient decreases for large values of Hartmann number H and fluid parameter B. Also, heat transfer rate monotonically enhances with conjugate parameter of Newtonian heating γ and Prandtl number Pr. This article addresses the effects of homogenous–heterogeneous reactions on electrically conducting boundary layer fluid flow and heat transfer characteristics over a stretching sheet with Newtonian heating are examined. Using similarity transformations, the governing equations are transformed into nonlinear ordinary differential equations. The constricted ordinary differential equations are solved computationally by shooting technique. The impact of pertinent physical parameters on the velocity, concentration and temperature profiles is discussed and explored via figures and tables. It is clear from figures that the velocity profile reduces for large values of fluid parameter  Although the considered model is very intricate and has favorite over the other fluid models, power law model is induced rather than the empirical relation from kinetic theory of liquid. It condenses to Newtonian relevance at high and low shear rates. Recently, the Powell–Eyring fluid was numerically studied by Akbar et al. [ Fluids used in industries maximum are non-Newtonian in nature which do not have a direct relationship between stress and deformation rate, e.g., molten polymers, biological fluids, lubricants, mud and some fluids occurring naturally such as animal blood. These fluids have been distributed into different classes. Among these, one of the important models is Powellr et al. . They rer et al.  analyzedr et al.  illustrar et al.  observedr et al.  examinedr et al.  used Kelr et al. –10, authThe heterogeneous and homogenous reactions are natural processes of chemical reactions. Several reactions progress very slowly, without a catalyst. The natural processes of heterogeneous and homogenous reactions are actually very complex including the consumption and production of reactant species at various rates on both the catalyst surface and fluid, i.e., reactions occurring in biochemical system, combustion and catalysis. Merkin  investig Flow generated by a shrinking/stretching surface produces a great interest due to several applications in industries and engineering, e.g., two-dimensional steady flow is used for contracting and expanding the surface such as stretching/shrinking wrapping bundle, aluminum bottle industrial procedures, wrapping and hot rolling. These flows with heat transfer have a great importance in paper production, enhanced petroleum resources, food processing, etc. Nadeem et al. , 20 analThe main motivations of this analysis are to analyze the effects of MHD flow of electrically conducting Powell–Eyring fluid over a stretching plate and also consider Newtonian heating with heterogeneous and homogenous reactions. The nonlinear expression concentration is transformed into ordinary differential expressions, and then, shooting method is employed in computing the result in MATLAB package. Numerical results are explained with tables and graphs.uw = cx, where c is a nonnegative constant. Consider two-dimensional coordinates system such that x is taken along with the plate, while the y-axis is taken at right angle to the sheet. We assume interaction between homogenous and heterogeneous reaction for a small model. Mathematical form of these reactions is given as:Consider incompressible two-dimensional steady flow of non-Newtonian fluid over a stretching surface with homogenous and heterogeneous reactions. The sheet is suddenly stretched such that the fluid flow with velocity b and a are the concentration of chemical species B and A and Ki  are the rate constant. The stream function ψ such that In the above equations, v and u are the components of velocity in the y- and x-directions, respectively. μ is the dynamic viscosity, ρ is the density, β is the fluid parameter, σ is the electrical conductivity of the fluid, H is the strength of magnetic field, α is the thermal diffusivity, for Newtonian heating hs is the heat transfer parameter and τ is the ratio of heat capacity and effective heat capacity of the fluid. After applying the boundary layer approximation in Eqs. , we , we v anΨ is the stream function, and by using these similarity transformations, Eqs.  bec bec14\doη. T∞ is the free stream and T is the fluid temperature, respectively. The quantity of practical interest such as skin friction coefficient is defined as:In the above mathematical equations, prime denotes the differentiation w.r.t. Equations –18) alo alo18) aWe get the system of ODEsh3 (0), h5 (0) and h7 (0) as 1, −1 and 1, respectively. Then, the system of first-order ODEs is solved using Runge–Kutta fifth-order technique. The convergence criterion for shooting method is 10−6 which absolute value of given and computed values of h2, h4 and h6. Thus, if the error tolerance, i.e., 10−6, is less than boundary residuals, then re-adjust initial guesses by Newton’s method. This loop is recycled again until the required criteria are satisfied.Choose a suitable initial approximations for H on velocity profile. It is examined that by enhancing the Hartmann number H the velocity profile decreases. This is due to the retarding nature of the Lorentz forces which reduces the motion of the fluid in the boundary layer and enhances its temperature. Figure M on velocity profile. It is evident that by hiking the values of fluid parameter M the velocity profile enhances. Because, it is illustrated that by enhancing the M the density of the fluid particles reduces which causes increase in velocity. Figure B on velocity. From this figure, decrease in velocity profile is noticed for increase values of fluid parameter B. Because, for increment in fluid parameter B, the viscosity of fluid particle enhances, and due to this reason, the velocity profile reduces. Figure γ on dimensionless temperature profile. As for Newtonian heating the value of conjugate parameter γ increase the thermal boundary layer thickness reduces eventually the temperature profile rise up. Figure Pr on dimensionless temperature profile. This figure reveals the reverse result for thermal boundary layer with increase in Prandtl number Pr when the thermal boundary layer falls down. The fluid has high conductivity for insignificant values of Prandtl number Pr. So by increasing the values of Prandtl number Pr, the thermal diffusivity reduces, which causes the decrease in temperature and thermal boundary layer of the fluid. Figures K and Ks on concentration profile. It is clear from this figure that thickness of concentration boundary layer enhances with η and after certain values of η it has no effect of concentration profile in both cases. Figure Sc on mass transfer rate. As the Schmidt number Sc is the diffusivity ratio, i.e., mass and momentum diffusivity, the mass diffusivity is small for large number of Schmidt number Sc due to this fact that the concentration profile enhances. Figure B and strength of magnetic parameter H. It is manifested from figure that skin friction enhances by increasing the fluid parameter B and Hartmann number H. Figure Pr and γ on Nusselt number. It is analyzed that for Newtonian heating the conjugate parameter γ reduces but Prandtl number Pr increase the Nusselt number. Table B and Hartmann number H skin friction increases, while it shows opposite behavior in the case of fluid parameter M. Table Pr and conjugate parameter of Newtonian heating γ. Concentration profile increases for large values of Schmidt number Sc and falls down for homogenous reaction parameter K. Tables θ′(0) and f′′(0) with previous results of the literature against variation of Hartmann number H and Prandtl number Pr.The impact of certain flow parameters on concentration, temperature and velocity profiles is visualized in this section. Figure B and Hartmann number H was noticed in velocity profile.A substantial reduction in the fluid parameters γ and Prandtl number Pr.A qualitatively opposite behavior was analyzed in the velocity profile for increasing values of for Newtonian heating parameter K and Ks, but showed opposite behavior for Schmidt number Sc.Concentration profile reduced by increasing homogenous and heterogeneous reactions parameter H and fluids parameters B and M increased.Skin friction coefficient enhanced when Hartmann number K, heterogeneous reaction parameter Ks and Schmidt number Sc.Concentration profile enhanced for large values of homogenous reaction parameter The study of two-dimensional homogenous and heterogeneous reactions in Powell–Eyring fluid model is considered numerically under the influence of MHD and Newtonian heating. The main results of this problem are:


Hypoxia is one of the most common phenotypes of malignant tumours. Hypoxia leads to the increased activity of hypoxia-inducible factors (HIFs), which regulate the expression of genes controlling a raft of pro-tumour phenotypes. These include maintenance of the cancer stem cell compartment, epithelial-mesenchymal transition (EMT), angiogenesis, immunosuppression, and metabolic reprogramming. Hypoxia can also contribute to the tumour progression in a HIF-independent manner via the activation of a complex signalling network pathway, including JAK-STAT, RhoA/ROCK, NF-κB and PI3/AKT. Recent studies suggest that nanotherapeutics offer a unique opportunity to target the hypoxic microenvironment, enhancing the therapeutic window of conventional therapeutics. In this review, we summarise recent advances in understanding the impact of hypoxia on tumour progression, while outlining possible nanotherapeutic approaches for overcoming hypoxia-mediated resistance. The hostile microenvironment within a solid tumour is increasingly recognized as a major impediment to effective cancer therapy . Hypoxia2 (15 mmHg), while pathological hypoxia defined as ≤1% O2 and radiobiological hypoxia as ≤0.4% . Si. Si69]. Exosome liberated miR-310a-30p is understood to polarize macrophages into an M2 phenotype via the activation of PTEN/P13Kγ signalling, favouring the malignant properties of tumour cells, due in part to the expression of the anti-inflammatory cytokine (IL-10) and arginase-1 (Arg1) . AdditioAs outlined above, the ability of cancer cells to invade local tissue and spread to distant sites is a critical step in disease progression, often accompanied by a poorer clinical prognosis. EMT is the proposed mechanism by which cells acquire the properties necessary for invasion and migration. Intrinsic properties of NPs have the potential to inhibit cancer progression through the regulation of EMT. For example, Arvizao et al. (2013) reported that gold NP (AuNP) treatment can delay tumour metastases through the inhibition of MAPK signalling and EMT reversal. They found that unmodified AuNPs not only downregulated the phosphorylation of MAPK but also reversed EMT by downregulating Snail, N-Cadherin, Vimentin . Polyeth2 NPs) significantly inhibited the proliferation, migration and invasion of HCC cell lines, attenuating in vivo tumour growth by 2.2-fold in comparison to NP-only control, mediated through the upregulation of SH2-containing protein tyrosine phosphatase 1 (SHP-1) and the corresponding suppression of JAK2/STAT3 signalling . In. In99]. Hypoxia also serves as an important driver of MDSCs recruitment. In a model of hepatocellular carcinoma (HCC), HIF-1α upregulated the expression of chemokine (C-C motif) ligand 26 (CCL-26) in cancer cells, recruiting chemokine (C-X3-C motif) receptor 1 (CX3CR1) expressing-MDSCs to primary tumours and promoting HCC tumour growth. Furthermore, inhibition of CCL-26 by the HIF inhibitor digoxin or through the blockade of CX3CR1 using a neutralizing antibody suppressed MDSC recruitment and tumour growth . HypoxiaTAMs are one of the most abundant forms of immune cell populations within the TME, emerging as an important regulator in fostering malignancy, cancer progression and therapeutic resistance . Tumour Macrophages can also undergo phenotypic changes and different forms of activation depending on the signal. Typically, classically activated macrophages are stimulated by T helper 1 (Th1) cytokines  as well as microbial cell wall components. Indeed, the classical M1 macrophage phenotype is known to possess potential antibacterial and anti-inflammatory activity through the secretion of reactive oxygen species and nitrogen intermediates, thereby counteracting cancer progression. An alternative M2 macrophage immunosuppressive phenotype is stimulated by Th2 cytokines  and other cytokines, which are responsible for blocking Th1 response and promote angiogenesis and cell proliferating. Tumour hypoxia has been indicated to play a pivotal role in the phenotypical control of TAMs .Hypoxia-induced extracellular vesicle (EV) miR-103a from lung cancer cells increased M2-type polarization, mediated by suppressed PTEN activity and the subsequent activation of the PI3/AKT and STAT pathway. Inhibition of miR-103a led to a decrease in hypoxia-induced M2-type polarization, while macrophages treated with EV miR-103a further enhanced cancer progression and tumour angiogenesis . In addiTumour derived exosomes enriched in immunosuppressive proteins including the chemokines/chemoattractant (CSF-1), monocyte chemoattractant protein-1/C-C chemokine 2 (MCP-1/CCL-2), and TGF-β, are also enhanced during hypoxia, leading to the macrophage recruitment and M2-like polarization both in vitro and in vivo . FurtherUpregulation of negative immune checkpoint molecule of programmed death ligand (PD-L1) in tumour cells, macrophages and dendritic cells under hypoxia, can initiate the interactions between PD-L1 and cell surface checkpoint receptor programmed cell death-1 (PD-1) expressed on effector T cells, resulting in the increased apoptosis of cytotoxic T lymphocytes (CTLs) and subsequent downregulation of T-cell antitumour reactivity. Increased PD-L1 and HIF target genes (CAIX and GLUT1) expression have been observed in a renal cell carcinoma (RCC) model possessing a VHL mutation. Furthermore, there is almost no PD-L1 expression in the presence of pVHL or absence of HIF-2α, indicating that PD-L1 expression is specifically regulated by the pVHL/HIF-2α axis in RCC . HypoxiaImmunotherapy has revolutionised cancer treatment. Immune checkpoint inhibitors, including those to PD-1, PD-L1 and CTL antigen 4 (CTLA-4), have now been approved for a number of cancers . HoweverGiven their high plasticity, reprogramming or repolarisation of TAMs from an immunosuppressive M2-like phenotype to an anti-tumourigenic M1-like phenotype is an attractive approach. Furthermore, as these cells readily internalise particles, nanoparticles may be used to deliver agents directly to these cells. β-cyclodextrin nanoparticles loaded with the toll-like receptor (TLR) 7 and 8 agonist R848 were selectively delivered to TAMs in vivo, altering the TME phenotype to that of an M1 . FurtherTargeting the MDSC compartment may be another approach for improving immune tolerance within the TME. As mentioned above, PI3K signalling is crucial in the functioning of myeloid suppressor cells in response to chemokines, particularly the PI3K-γ isoform. PLGA NPs have been developed to incorporate the PI3K-γ inhibitor IPI-549, and co-functionalised to target the TME with aminoethyl anisamide (AEAA), a ligand for sigma-1 receptor which is overexpressed in a number of tumour types including pancreatic adenocarcinoma. IPI-549 loaded NPs significantly reduced both MDSC and tumour associated B cell proportions in KPC pancreatic tumour-bearing mice while also inhibiting tumour growth in comparison to free-IPI-549 . An alteLyp-1, a Nrp-1-binding peptide, homes to lymphatics, TAMs and tumour cells, particularly those within hypoxic regions of the tumour . Lyp-1 cMolecular oxygen is a critical component of mitochondrial ATP production. However, tumour cells often forgo oxidative phosphorylation (OXPHOS) in the mitochondria in favour of increased glycolysis, even in the presence of oxygen. This aerobic glycolysis phenomenon in tumour cells is often known as the “Warburg effect”, following observations of Otto Warburg in the early twentieth century ,121. ThiIn addition to increasing the glycolytic activity, hypoxia can also suppress the production of mitochondrial ROS by uncoupling glycolysis and OXPHOS via the upregulation of pyruvate dehydrogenase kinase-1 (PDK-1). Hypoxia-induced PDK-1 expression can block the conversion of pyruvate to acetyl-CoA, hereby preventing ATP production via the TCA cycle, attenuating ROS production, which in turn protects cancer cells from hypoxia-induced apoptosis . HypoxiaHypoxia attenuation of metabolism may also occur through modulating the function of the electron transport chain via the downregulation of cytochrome-c oxidase . HIF-1α have been shown to activate the transcription of genes encoding COX4-2 and the mitochondrial protease LON, leading to the degradation of the COX4-1 subunit, aiding in better adaption to hypoxia with reduced ROS production . Hypoxia+ across the cell membrane and the induction of an acidic tumour microenvironment [The metabolic transformation of tumour cells to favour glycolysis over OXPHOS represents a promising therapeutic target that may be exploited by nanoparticles. Among the hexokinase family of proteins, HK-2, which catalyses the phosphorylation of glucose, is frequently overexpressed in tumour cells . As a reironment . Targetiironment ; the autAn alternative approach to overcome hypoxia-driven metabolic reprogramming within the TME is to alleviate tumour hypoxia by reducing the oxygen consumption (OC) within cells. To this end, a limited number of therapeutics have been identified with the potential to reduce the OC in tumour cells including the anti-malarial atovaquone and the anti-diabetic metformin. Atovaquone has been shown to reduce the OC by more than 80% in a number of tumour cells in vitro, and could abolish hypoxia in xenograft models of head and neck cancer and colon cancer after seven days of treatment, by inhibiting mitochondrial complex III. This was also associated with an improved radiation tumour growth response in vivo (growth delay of 13.2 days between control and atovaquone irradiation groups) . NPs for18O49 have been used as effective PDT and PPT agents due to their ability to generate ROS and produce heat when combined with NIR laser irradiation [18O49 have been shown to improve responses to PDT and PPT in vitro by reducing the OC in tumour cells, and significantly inhibiting tumour growth and increasing TUNEL staining (a measure of tumour apoptosis) in Raji-lymphoma xenografts [Conversely, the anti-diabetic metformin has been shown to reduce OC by inhibiting complex I in the mitochondrial electron transport chain, leading to improvements in tumour oxygenation and radiation responses in models of colon cancer in vivo . PEG-PCLadiation . HoweverTumour hypoxia is a critical feature of the TME, contributing to disease progression and resistance to chemo and radiotherapy. The response to tumour hypoxia is mainly driven by oxygen-dependent HIFs that enable tumour progression. However, a complete understanding of the mechanisms driving the response to hypoxia within the TME remains elusive. Hypoxia has effects not only on tumour cells and on the maintenance of CSCs, but also on those cells of the surrounding stroma, driving angiogenesis, malignant progression, immunosuppression and aiding in metabolic reprogramming . DespiteSignificant barriers to the translation of NPs include sufficient tumour penetration, stability and potential systemic toxicity. However, meaningful achievements have been made to improve nanomedicine delivery and retention in solid tumours through smart nanoparticle design and TME modification. Development of self-recognition biomimetic nanodelivery systems have shown great potential in increasing the circulation and biocompatibility of nanoparticles within the host organism; this includes coating NPs with an erythrocyte or cancer cell membrane. Alternatively, enhanced tumour penetration could be achieved through the use of circulating monocytes or macrophages . Tumour Future research efforts and clinical translation of nanotherapeutics will require a detailed understanding of the molecular nature of the hypoxic TME to optimise treatment combinations. This approach will undoubtedly facilitate the development of more promising nanotherapeutic platforms for the future treatment of hypoxic tumours that are not only targeted towards tumour cells, but have dual-targeting effects on cells of the TME in addition to augmenting intratumoural oxygenation.


We were recently alerted by an anonymous tip that at least two paragraphs in the title review  appeared


The indication, composition and timing of administration of non-resuscitation fluid in septic shock have so far received little attention and accordingly the potential to reduce this source of fluid is unknown. The objective of the study was to quantify and characterize non-resuscitation fluid administered to patients with septic shock.This prospective observational study was performed in eight intensive care units in Sweden and Canada during 4 months in 2018. Adult patients with septic shock within 24 h of admission to the intensive care unit were eligible for inclusion. Non-resuscitation fluids were defined as fluids other than colloids, blood products and crystalloids at a rate ≥ 5 ml/kg/h. Indication, volume and type of fluid were recorded during the first 5 days after admission. A maximum of 30 patients could be included per centre. To estimate the potential to reduce administration of non-resuscitation fluid, a pragmatic “restrictive” protocol for administration of non-resuscitation fluids was devised based on the most restrictive practice already in place for non-resuscitation fluids at any of the participating centres. Data are presented as median (interquartile range [IQR]).A total of 200 patients were included in the study and the 30-day mortality was 35%. Patients received a total of 7870  ml of non-resuscitation fluids and 2820 (1430–4580) of resuscitation fluids during the observation period. Median volumes of non-resuscitation and resuscitation fluids were similar at day 1 (1620 [710–2320] and 1590 [520–3000]) ml, respectively) and non-resuscitation fluids represented the largest source of fluid from day 2 and onwards after admission to the ICU. Vehicles for drugs such as vasoactive drugs and antibiotics constituted the largest fraction of non-resuscitation fluids (2400 [1270–4030] ml) during the 5-day observation period. Modelling suggested that volume of non-resuscitation fluids could be reduced by 2840 (1270–4900) ml during the first 5 days of admission to the ICU, mainly through reducing maintenance fluids.Non-resuscitation fluids constitute the major fraction of fluids administered in the ICU to patients suffering from septic shock and may represent the largest modifiable target to reduce fluid overload. Administration of resuscitation fluids intravenously to ensure adequate tissue perfusion is a cornerstone in the treatment of sepsis and septic shock . HoweverThere are many indications for fluid administration in the critically ill. Observational studies have shown that the majority of fluid administered to patients admitted to an intensive care unit (ICU) is for indications other than maintaining intravascular volume, such as nutrition or as a vehicle for intravenous medications , 8, 9. TThe indication, composition and timing of administration of non-resuscitation fluid in septic shock have so far received little attention and accordingly the potential to reduce this source of fluid is unknown. Based on these considerations, the objective of the present study was to quantify and characterize the fluid given to patients with septic shock during the first 5 days of admission to the ICU and to assess the potential to reduce the non-resuscitation fluid, by modelling a restrictive fluid protocol. For this purpose, we conducted a prospective observational study in eight intensive care units in Sweden and Canada.We conducted this multicenter observational study on eight different sites: six intensive care units in southern Sweden , and two in Vancouver, British Columbia, Canada . Patients were included between March 1st 2018 and June 30th 2018. Follow-up time was 30 days from inclusion. The regional ethical boards in Lund, Sweden (application # 2017/565) and Vancouver, Canada (#H17-03504) approved the study. The ethical board in Sweden required informed consent from patients or their legal surrogates before enrollment, whereas the ethical board in Vancouver waived the need for informed consent. We prepared the manuscript according to the STROBE guidelines for observational studies and registered the study at Clinicaltrials.gov NCT03438097 prior to inclusion of the first patient.We included consecutive adult patients (≥ 18 years) with septic shock per SEPSIS-3 criteria  within 2During the first 5 days of admission to the ICU, we registered fluid input, output and fluid balance. Nurses registered fluid input, output and fluid balance manually, in all sites but one. At this site, computer software collected fluid input and fluid balance electronically, while a nurse registered fluid output manually. We collected data on patient characteristics from the electronic medical records/paper records and the electronic records differed across the study sites. A designated researcher at each site transferred the data to the CRF. This researcher was carefully informed on how to register the data onto the CRF and was not blinded to the study objectives.We specified fluids as either non-resuscitation fluids or resuscitation fluids. The non-resuscitation fluids were subdivided into six groups: vehicle for drugs, enteral and parenteral nutrition, glucose solutions, enteral water and crystalloid administered at a rate of < 5 ml/kg/h . The groDaily fluid balance was determined by subtracting total fluid output, except for perspiration from total fluid intake. Bowel movements were included in the fluid balance according to local protocol. Demographic data were collected from the patient charts. Simplified Acute Physiology Score (SAPS-3) and Sequential Organ Failure Assessment (SOFA) were calculated on the day of admission. Mechanical ventilation or renal replacement therapy (RRT) during any of the first 5 days was registered, as well as source of sepsis and surgery as a source of infection. Length of stay in the ICU, ICU mortality and 30-day mortality were also recorded.In an attempt to estimate the potential to reduce administration of non-resuscitation fluid, we devised a pragmatic “restrictive” protocol for administration of non-resuscitation fluids based on the most restrictive practice already in place for non-resuscitation fluids at any of the participating centres. In this protocol, we assumed the following: no maintenance fluid was given to patients with a positive cumulative fluid balance, no intravenous glucose was given for nutritional purposes, and enteral nutrition was changed to a concentration of 2 kcal/ml in centres using less concentrated formulas.No sample size calculation was performed. Based on historical admission rates in the smaller participating ICUs during a typical 4-month period, a maximum inclusion of 30 patients per ICU was set in an attempt to balance the cohort. We performed statistical analyses using GraphPad Prism 8.1.1  and R v. 3.5.2 . We did not use imputation for missing data. Data are presented as medians and interquartile ranges.n = 3, death before consent could be obtained n = 2) leaving 200 patients for inclusion in the analysis. Each centre included a median of 24 patients (min–max 16–30). The 30-day mortality in the cohort was 71/200 (35%) and the most common source of sepsis was abdominal infection. Length of ICU stay was 78 (47–169) h. A detailed description of characteristics is presented in Table We screened a total of 1946 consecutive patients for eligibility during the study period. Of these, 208 patients were diagnosed with septic shock within 24 h of admission. A total of 8 patients were not included , respectively. From day 2 and onwards, daily volume of non-resuscitation fluids was larger than daily volume of resuscitation fluids. A daily breakdown of the different subgroups of non-resuscitation fluids is presented in Fig. Patients received a median of 7870 4060–12,340) ml of non-resuscitation fluids and 2820 (1430–4580) ml of resuscitation fluids during the observation period. Daily intake of fluids and daily fluid balance are presented in Fig. 2,340 ml By modelling a restrictive protocol of non-resuscitation fluids in the respective centre, we obtained a theoretical reduction of 2840 (1270–4900) ml per patient of non-resuscitation fluids for the whole cohort, during the observation period . *Kruskal–Wallis test.Additional file 2. Volume and type of fluid during day 1–5 in “standard care” vs “restrictive” protocol. Volumes are presented in millilitres (median [IQR]).Additional file 3. Potential non-resuscitation fluid reduction during day 1–5, per site, in the “restrictive” protocol. Volumes are presented in millilitres (median [IQR]). Please note that the sum of the medians does not equal median of the sum because of the skewed distribution of data.


Perioperative fluid and hemodynamic management have been much-debated topics over the last few years. Recently, a number of large trials have been published to help inform this debate. The Restrictive versus Liberal Fluid Therapy for Major Abdominal Surgery (RELIEF) study is the largest trial to date of perioperative fluid management. The 3000-patient trial comparing 2 different fluid regimes showed that a restrictive fluid regimen during and up to 24 h after surgery was associated with an increase in acute kidney injury (AKI). This result is at odds with a recent trend to a more restrictive fluid approach during major surgery and suggests that practice may have become too restrictive. A moderately liberal (aiming for 1–2 l positive) or goal-directed approach is therefore recommended. Optimal perioperative fluid and hemodynamic management remain topics of significant debate in perioperative medicine. One reason for this is that perioperative care has changed significantly over the last 10 years as approaches such as laparoscopic surgery and enhanced recovery after surgery (ERAS) pathways have become routine. Much of the older clinical research in this field has lost relevance and new questions need to be answered. Another reason is that although we have had many trials of fluid and hemodynamic management over the years, most have been small single-center studies rather than large pragmatic multicenter trials. Fortunately, and thanks to the hard work of leading investigators worldwide, this has changed significantly over the last few years with the publication of a number of large multicenter studies on these topics  study published in the New England Journal of Medicine in May 2018 is the largest trial published to date on perioperative fluid management  rather than using a standardized fluid algorithm. RELIEF was a pragmatic trial and clinicians were allowed to use ERAS y and goal-directed therapy pathways according to local practices. The fact that many centers did not use ERAS pathways in many ways reflects current practice with real-world implementation of ERAS being much lower in practice than in the literature, with many centers also overrating their ERAS compliance. Subgroup analyses show that the use of GDT and ERAS did not affect the primary results, suggesting that the findings are generalizable.We believe that the findings of the RELIEF trial will change practice for patients undergoing major surgery. There are times during any major surgical procedure when there is uncertainty regarding the risks and benefits of further fluid administration. This is true both for low-risk patients without advanced hemodynamic monitors and higher risk patients undergoing goal-directed therapy. In recent years, clinicians may have become less likely to give a fluid bolus when they are uncertain of the best course of action. RELIEF may have redressed our equipoise. The risks and benefits of intravenous fluid seem likely to remain a topic of discussion for some time to come.