P = 0.07). For never-smokers with advanced NSCLC, chemotherapy, not ICIs, was significantly associated with improvement of PFS . In more than second-line setting, ICIs significantly prolonged OS over that with chemotherapy in ever-smokers . For never-smokers with NSCLC, however, ICIs failed to significantly improve OS . In conclusion, this meta-analysis indicates that ICIs can prolong survival over that with chemotherapy in ever-smokers with advanced NSCLC. However, ICIs failed to improve survival in never-smokers. These results suggest that smoking status may be a predictive marker for survival benefits to ICIs.Immune checkpoint inhibitors (ICIs) have emerged as a new treatment option for patients with advanced non-small-cell lung cancer (NSCLC). Some studies with ICIs in NSCLC suggested that smoking history was associated with improved survival outcomes. We conducted this meta-analysis to investigate if survival benefits of ICIs in patients with advanced NSCLC are different according to smoking status. Electronic databases were searched for eligible studies. We included randomized controlled trials with the data of survival outcomes and extracted progression-free survival (PFS) or overall survival (OS) stratified by smoking status. From 6 studies, 2,389 ever-smokers and 413 never-smokers were included in the meta-analysis. In first-line treatment setting, ICIs tended to improve PFS in patients with smoking history (HR = 0.85 ,  Lung cancer is the leading cause of cancer-related death all over the world , 2. TobaImmunotherapy has emerged as a new treatment option for patients with advanced non-small-cell lung cancer (NSCLC) . The proTherefore, it is unclear whether the efficacy of ICIs in patients with NSCLC is associated with smoking history. We conducted this meta-analysis of randomized controlled studies to investigate if survival benefits of ICIs in patients with advanced NSCLC are different according to smoking status.Figure Of the six studies, four were conducted in patients with previously treated NSCLC –14 and tTable P = 0.07) . For never-smokers with advanced NSCLC, chemotherapy, not ICIs, was significantly associated with improvement of PFS  .From 2 studies conducted in first-line setting, 756 current or former smokers with advanced NSCLC and 83 patients with no history of tobacco use were included in the meta-analysis of hazard ratios (HRs) and 95% confidence intervals (CIs) for progression-free survival (PFS) , 16. Com) Figure . The ran) Figure . There wP < 0.00001) . For never-smokers with advanced NSCLC, ICIs failed to significantly improve OS, compared with chemotherapy  .From 4 studies performed in patients with previously treated NSCLC –14, 1,63) Figure . There w) Figure . There wIn this meta-analysis, we investigated whether survival benefits of ICIs in advanced NSCLC were different between ever-smokers and never-smokers. We found that ICIs as a first-line or salvage therapy for advanced NSCLC could prolong PFS or OS over those with chemotherapy in ever-smokers, but not in never-smokers.et al. recently reported the results of an interesting study to assess the effects of smoking on the mutational landscape and pembrolizumab response in NSCLC [ICIs have proved survival benefit in patients with advanced NSCLC, but there is a critical need to identify predictive biomarkers associated with advantages from ICIs. Although some studies have showed the significant correlation between the efficacy of ICIs and PD-L1 expression level , 11, 13,in NSCLC . AccordiP = 0.07). In never-smokers with NSCLC, however, ICIs failed to significantly improve survival (PFS or OS) regardless of treatment setting. These results indicate that smoking status is a simple but useful clinical predictive marker for survival benefits to ICIS in patients with advanced NSCLC. Interestingly, smoking status has also shown a significant clinical impact in NSCLC patients with epidermal growth factor receptor (EGFR) mutations [et al. reported that smoking history was detrimental to patients with NSCLC harboring EGFR mutations [et al., PFS benefit of EGFR-TKIs over platinum doublet chemotherapy was significantly better in patients with no smoking history [In this meta-analysis, ever-smokers with advanced NSCLC derived significant OS benefit from ICIs over chemotherapy (docetaxel) as a salvage therapy. ICIs also tended to prolong PFS for patients with a history of smoking in first-line treatment setting ; providing HRs with their 95% CIs for PFS or OS.The following data were collected from the eligible studies: the first author's name, year of publication, study phase, number of patients, treatment setting and regimen, PD-L1 expression level, PFS and OS stratified by smoking status and HRs with their 95% CIs.Data extractions were carried out independently by two authors (BJK and HSK). If the two authors could not reach a consensus, the other (JHK) was consulted to resolve the dispute.Q statistic and the I2 statistic. The fixed-effect model (Mantel–Haenszel method) was selected for pooling the homogeneous outcomes when P ≥ 0.1 and 2I ≤ 50%, and the random-effects model (DerSimonian–Laird method) was applied for pooling heterogeneous outcomes when P < 0.1 and 2I > 50%.Statistical values used in the meta-analysis were obtained directly from the original articles or abstracts. The effect size of PFS and OS was pooled through HR and its 95% CI, whereas the effect size of the other outcomes was evaluated via the number of patients. The heterogeneity across studies was examined by P-values were from two-sided versions of the respective test and P < 0.05 was considered statistically significant. RevMan version 5.2 software was used to report outcomes.The plots show a summary estimate of the results from all the studies combined. The size of the squares shows the estimate from each study and reflects the statistical ‘weight’ of that study (the relative contribution of that study to the summary estimate). Results are graphically presented as forest plots with diamonds representing estimate of the pooled effect and the width of diamond representing its precision. The line of no effect is number one for binary outcomes, which, if not crossed by the diamond, indicates statistical significance. All


Media and World Health Organization (WHO) attention on Zika virus transmission at the 2016 Rio Olympic Games and the 2015 Ebola virus outbreak in West Africa diverted the attention of global public health authorities from other lethal infectious diseases with epidemic potential. Mass gatherings such as the annual Hajj pilgrimage hosted by Kingdom of Saudi Arabia attract huge crowds from all continents, creating high-risk conditions for the rapid global spread of infectious diseases. The highly lethal Middle Eastern respiratory syndrome coronavirus (MERS-CoV) remains in the WHO list of top emerging diseases likely to cause major epidemics. The 2015 MERS-CoV outbreak in South Korea, in which 184 MERS cases including 33 deaths occurred in 2 months, that was imported from the Middle East by a South Korean businessman was a wake-up call for the global community to refocus attention on MERS-CoV and other emerging and re-emerging infectious diseases with epidemic potential. The international donor community and Middle Eastern countries should make available resources for, and make a serious commitment to, taking forward a “One Health” global network for proactive surveillance, rapid detection, and prevention of MERS-CoV and other epidemic infectious diseases threats. Mass gatherings at sporting , 2 and rBetween 8 and 14 September 2016, the cities of Jeddah, Makkah, and Madinah, Kingdom of Saudi Arabia (KSA), hosted the annual Hajj pilgrimage . An estiSerological studies show that camels have been infected with MERS-CoV for 20 years. MERS-CoV is a common infection in dromedary camels, and there is accumulating evidence that the sporadic human outbreaks are seeded by zoonotic infection from camels . There hThe risk of sustained person-to-person transmission appears to be very low and MERS-CoV is considered to have a low epidemic potential , 22. NevIt remains an enigma that, while SARS-CoV spread rapidly globally and caused >8,000 cases and 775 deaths  before disappearing within 8 months of discovery , causingThe past three annual Hajj pilgrimages  have passed by without any increase in travel-related MERS-CoV cases . While KThe risk of returning pilgrims infected with MERS-CoV to their home countries remains. Thus, health professionals and healthcare systems should remain vigilant at all times , and espMERS-CoV remains in the WHO list of top “emerging diseases likely to cause major epidemics” . However


Spalax  endures extreme hypoxic conditions and fluctuations in oxygen levels that threaten DNA integrity. Nevertheless, Spalax is long-lived, does not develop spontaneous cancer, and exhibits an outstanding resistance to carcinogenesis in vivo, as well as anti-cancer capabilities in vitro. We hypothesized that adaptations to similar extreme environmental conditions involve common mechanisms for overcoming stress-induced DNA damage. Therefore, we aimed to identify shared features among species that are adapted to hypoxic stress in the sequence of the tumor-suppressor protein p53, a master regulator of the DNA-damage response (DDR).The subterranean blind mole rat, Spalax p53.We found that the sequences of p53 transactivation subdomain 2 (TAD2) and tetramerization and regulatory domains (TD and RD) are more similar among hypoxia-tolerant species than expected from phylogeny. Specific positions in these domains composed patterns that are more frequent in hypoxia-tolerant species and have proven to be good predictors of species’ classification into stress-related categories. Some of these positions, which are known to be involved in the interactions between p53 and critical DDR proteins, were identified as positively selected. By 3D modeling of p53 interactions with the coactivator p300 and the DNA repair protein RPA70, we demonstrated that, compared to humans, these substitutions potentially reduce the binding of these proteins to We conclude that extreme hypoxic conditions may have led to convergent evolutionary adaptations of the DDR via TAD2 and TD/RD domains of p53.The online version of this article (doi:10.1186/s12862-016-0743-8) contains supplementary material, which is available to authorized users. Nannospalax  is a solitary subterranean mammal , or  or , have disrupted the binding of p53 to RPA70. Each of these combinations includes residues that participate in the pattern identified in the current study as stress-related in TAD2 (highlighted in bold). Other studies . Habitat and behavioral information were obtained from the Animal Diversity Web (ADW) online database . This server implements an empirical Bayesian method for dN/dS calculation and allows to compare the M8 model (that assumes selection) vs. the M8a null model (that assumes no selection) by LRT as described above, and to identify site-specific selection, accordingly. It is important to clarify that site-specific positive selection models do not provide information regarding the lineage in which the positive selection might occur. Methods that provide information regarding the site and the branch  were not used since these methods are not reliable for regions containing alignment gaps, which were tested here [Positive-selection analysis was conducted using: (1) The HyPhy module MEME . This mesion 4.8 , 97. Thies (BEB) . (3) Thees (BEB)  [http://ted here .


The objective of this study was to evaluate the feasibility of using paired-associative stimulation (PAS) to study excitatory and inhibitory plasticity in adolescents while examining variables that may moderate plasticity (such as sex and environment).We recruited 34 healthy adolescents . To evaluate excitatory plasticity, we compared mean motor-evoked potentials (MEPs) elicited by single-pulse transcranial magnetic stimulation (TMS) before and after PAS at 0, 15, and 30 min. To evaluate inhibitory plasticity, we evaluated the cortical silent period (CSP) elicited by single-pulse TMS in the contracted hand before and after PAS at 0, 15, and 30 min.p < 0.01) at all post-PAS intervals. Neither socioeconomic status nor sex was associated with post-PAS MEP changes. PAS induced significant CSP lengthening in males but not females.All participants completed PAS procedures. No adverse events occurred. PAS was well tolerated. PAS-induced significant increases in the ratio of post-PAS MEP to pre-PAS MEP amplitudes (PAS is a feasible, safe, and well-tolerated index of adolescent motor cortical plasticity. Gender may influence PAS-induced changes in cortical inhibition. PAS is safe and well tolerated by healthy adolescents and may be a novel tool with which to study adolescent neuroplasticity. Adolescence is a period of tremendous neuronal plasticity characterized by synaptic pruning, and axonal myelination as the brain matures into its adult state , 2. TherPsychiatric disorders such as depression and schizophrenia typically arise in late adolescence –8 and arIn the original PAS experiment, Stefan et al.  administin vivo in humans  early in development, it induces hyperpolarization (inhibition) in the mature brain . PediatrB cortical inhibition is cortical silent period (CSP) which PAS lengthens . For example, previous work has demonstrated sex differences in synaptic connectivity in the frontal cortex in response to chronic  and prenTherefore, the aim of this study was twofold: (1) to determine whether PAS was feasible in adolescents and (2) to characterize potential external factors influencing PAS outcomes. To determine the feasibility of PAS in adolescents, we evaluated PAS-induced excitatory (motor facilitation) and inhibitory (CSP) plasticity in healthy adolescents, reporting on these measures as well as participant dropout. We also evaluated whether external factors such as sex, age, SES, and academic achievement influenced PAS-induced plasticity. We hypothesized that PAS would induce motor facilitation in healthy adolescents. Moreover, we hypothesized PAS would lengthen CSP in adolescents, as in adults, in the absence of evidence to the contrary.This study was approved by the Research Ethics Board of the Centre for Addiction and Mental Health (CAMH) in Toronto. In accordance with the recommendations of the Declaration of Helsinki, all participants provided their written informed consent prior to participation. The protocol was approved by the Research Ethics Board of the CAMH in Toronto, ON, Canada.To evaluate the feasibility of using PAS in healthy adolescents, we delivered PAS according to the methods described by Stefan et al. . All datWe recruited adolescents aged 13–19 from community agencies, schools, and the Internet. Research associates contacted interested respondents who called the Temerty Centre for Therapeutic Brain Intervention, provided a non-standardized introduction of the study objectives, and described the potential risks associated with TMS. Respondents completed a standardized TMS safety screen and had the opportunity to ask questions about the study to which research associates provided non-standardized responses.Eligible participants were English speakers, had parents who spoke conversational English, were right handed [according to the Oldfield handedness interview ], and weK 1.00), and test–retest  reliability  6.0 to rule out psychiatric illness . The MINiability . We usedThe Wide Range Achievement Test 4 (WRAT-4) was used to assess academic achievement . The WRAAll participants provided their parent’s level of education and occupation. We calculated the Hollingshead Index , which mEach neurophysiology testing session lasted 1.5 h. Participants sat in a comfortable chair, with a cushion in their lap, and right forearm exposed. We prepped the thumb and volar surface of the forearm with an alcohol wipe for recording. We collected EMG data from disposable disks in a tendon-belly arrangement. Participants were directed to relax their right hand for the entirety of the study. EMG recording and speakers at high gain served as another measure of muscle activity throughout testing. The EMG signal was amplified , filtered (band pass 2 Hz–2.5 kHz), digitized at 5 kHz  and stored in a laboratory computer for offline analysis.We delivered single-pulse TMS to the left motor cortex with a 7-cm figure-of-eight coil and a Magstim 200 stimulator . We used a moderately suprathreshold stimulus to identify the optimal APB stimulation location, marking it with a felt-tipped marker to ensure consistent coil placement across trials. The handle of the coil pointed backward at 45° to the mid-sagittal line and perpendicular to the central sulcus.mV) was then determined. Stefan and colleagues found the SI1mV to be approximately 120% of the RMT  at the optimal stimulation position. We defined the RMT as the minimum stimulus intensity needed to produce a response of at least 50 µV in the relaxed APB in 5 of 10 consecutive trials. The stimulus intensity required to evoke a 1-mV peak-to-peak response  with an a priori significance level of 0.0167. To evaluate PAS-induced changes in CSP, we used repeated measures ANOVA with “PAS session” as a within-subject factor and “sex” as a between-subject factor. We chose parametric testing for this measure since, unlike ratio data, CSP duration was normally distributed. The CSP duration was defined as the time between onset of MEP and return of voluntary contraction on EMG and determined by visual inspection as previously described .We calculated descriptive statistics of sample demographics and WRAT-4 scores. We used a non-parametric ANOVA test (the Friedman test) to compare mean MEPs evoked at baseline, 0-, 15-, and 30-min post-PAS since ratio data were not normally distributed. We then completed escribed . For CSPn = 17), high >54 (=17)].To evaluate the impact of various independent variables on MEP potentiation, we evaluated Spearman’s correlation between the average PAS ratio and ordinal independent variables . We used the Wilcoxon rank-sum test to compare group differences for MEP potentiation due to categorical variables, which include gender and Hollingshead high vs. low using a median split  with a mean age of 17 years. Our mean sample age was, therefore, situated in a continuum between Damji et al. , respectively. These higher stimulation intensities are similar to those used in previous pediatric TMS studies. Croarkin et al.  with a minority of male participants i.e., 13). It is possible that this smaller number of adolescent males showed less variability than females. We did not synchronize menstrual cycles or account for menarche in the female participants, potentially introducing variability in the female sample on account of varying brain GABA levels. Still, the variability in male and female CSP duration was comparable across groups as shown in Figure 1 with a . It is pOur results demonstrate significant PAS-induced motor cortical plasticity in healthy adolescents. Applying PAS to adolescents was safe and well tolerated. We also found significantly greater inhibitory neuroplasticity in young males compared to young females. It is possible that differences in testosterone-induced maturation of GABA neurons or fluctuating GABA levels with female menstrual cycles account for this finding. PAS may eventually serve as an investigational tool in at-risk adolescents, elucidating mechanisms of psychiatric illness.All authors  contributed to the conception, structure, and literature review for the manuscript. JL prepared the initial draft, and ZD and PC critically revised the draft. All the authors  prepared the final draft, approved the final draft for publication, and agreed to assume accountability for the accuracy and composition of the manuscript.JL, SA, TR, and YS report no competing interests. In the last 5 years, PC has received research support from Pfizer for an investigator-initiated study and equipment in-kind support from Neuronetics. DB receives research support and in-kind equipment support for an investigator-initiated study from Brainsway Ltd., and he is the site principal investigator for three sponsor-initiated studies for Brainsway Ltd. He also receives in-kind equipment support from Magventure for an investigator-initiated study. He receives medication supplies for an investigator-initiated trial from Invidior. In the last 5 years, ZD received research and equipment in-kind support for an investigator-initiated study through Brainsway Inc. and a travel allowance through Merck. ZD has also received speaker funding through Sepracor Inc., AstraZeneca and served on the advisory board for Hoffmann-La Roche Limited and Merck and received speaker support from Eli Lilly.


Recently a second endogenous ligand for APJ, named apela, has been discovered. We confirm that apela activates signal transduction pathways (ERK activation) in cells expressing the cloned rat APJ. Previous studies suggest that exogenous apela is diuretic, attributable wholly or in part to an action on renal APJ. Thus far the cellular distribution of apela in the kidney has not been reported. We have utilized in situ hybridization histochemistry to reveal strong apela labelling in the inner medulla (IM), with lower levels observed in the inner stripe of the outer medulla (ISOM), of rat and mouse kidneys. This contrasts with renal aplnr expression where the converse is apparent, with intense labelling in the ISOM (consistent with vasa recta labelling) and low-moderate hybridization in the IM, in addition to labelling of glomeruli. Apelin is found in sparsely distributed cells amongst more prevalent aplnr-labelled cells in extra-tubular regions of the medulla. This expression profile is supported by RNA-Seq data that shows that apela, but not apelin or aplnr, is highly expressed in microdissected rat kidney tubules. If endogenous tubular apela promotes diuresis in the kidney it could conceivably do this by interacting with APJ in vasculature, or via an unknown receptor in the tubules. The comparative distribution of apela, apelin and aplnr in the rodent kidney lays the foundation for future work on how the renal apelinergic system interacts.Apelin binds to the G protein-coupled apelin receptor (APJ; gene name  Upon binding APJ, apelin activates a myriad of signal transduction pathways that are often G protein-dependent, including inhibition of adenylate cyclase, stimulation of MAP kinase (ERK) phosphorylation, Akt phosphorylation that could contribute to neuroprotection, and enhancing nitric oxide synthase activity [Apelin exists as a number of proteoforms  gene is also translated [apela mRNA [apela null mutants invariably exhibit cardiac dysplasia [aplnr knockout mice [apelin knockout mice [apela or apelin are over-expressed in zebrafish [A second endogenous ligand for APJ has recently been discovered ,9. OrigiEnde ) expressanslated ,9. It enanslated ,9. The aanslated ,9. Apelaanslated ,12. Apelysplasia ,9,13, reout mice . Since cout mice ,15, it iebrafish , indicataplnr [apela binding heterogeneous nuclear ribonucleoprotein L (hnRNPL), an inhibitory binding partner of p53 in these cells. Whether apela acts as a protein in a non-APJ-dependent manner  or as a regulatory RNA in vivo is not known.Exogenously administered apela is anorexigenic , and proaplnr , while aaplnr . This deApela mRNA transcripts appear to be more prevalent during development [apela gene expression in the kidney has not been reported. Evidence for renal apelin expression is less convincing—it appears to be sparsely distributed in tissue endothelial cells and perhaps vascular epithelial cells and glomeruli [aplnr is mainly expressed in vascular elements (vasa recta) of the outer medulla and glomeruli [elopment ,9,10. Thelopment ,9, and aelopment , and in elopment  by RT-PClomeruli . In the lomeruli ,22,23—lolomeruli .in vitro and to characterize the anatomical relationship between apela and aplnr expression in the rat and mouse kidney using in situ hybridization histochemistry (ISHH) with gene-specific oligonucleotides. In an attempt to gain further insights into the possible functions of the renal apelinergic system we have also utilized branched-chain ISHH to determine whether the apelin, apela and/or aplnr genes are co-expressed in the same regions in the rat kidney. This distribution is supported by RNA-Seq studies on isolated renal tubule segments. Our studies reveal that while apela and aplnr expression overlap, apela is mainly expressed in the medullary collecting ducts and loops of Henle whereas aplnr is highly expressed in non-tubular structures in the outer medulla. Apelin expression is largely restricted to isolated cells mainly in the vicinity of aplnr cells in the medulla. This distribution raises questions about how the two-ligand apelinergic system operates in regulating renal function.The aims of this study are to confirm apela bioactivity via APJ aplnr knockout colony [ad libitum. Animal care and maintenance were performed in accordance with the Animal Scientific Procedures Act (1986) United Kingdom and approved by the Bristol University Animal Welfare and Ethical Review body.Adult (8–12 weeks old) male wildtype (n = 3) or null mutants (n = 2) mice from our t colony   was used for branched-chain in situ hybridization histochemistry and ERK signal transduction assays (see below).Chinese hamster ovary (CHO) cells were transfected with the rat DNA (B78 ) as prevDNA  or rat apela-32 , or vehicle (dH2O), the cells were immunostained for dual phosphorylated(pp) ERK1/2 and total(t) ERK1/2—cell images were acquired using the IN Cell Analyzer 1000 and analysis performed by In Cell Analyzer Workstation 3.5 software  as previously described [CHO-B78 cells were grown in 96-well plates at a concentration of approx 10,000 cells/well and serum-starved  O/N. Dose-response curves were performed for 10min, where [Pyrescribed ,28. The 35S-end-labelled 48bp-oligonucleotides targeting apela, as described in detail for oligonucleotide probes (http://www.wsyacy.com/SNGE/Protocols/ISHH/ISHH.html). The antisense probes used in this study were rAP-1 and rAP-2 specific for rat apela ), and mAP-1 and mAP-2 specific for mouse apela . ISHH with rat aplnr probes  were thaw-mounted onto polylysine-coated slides and ISHH performed with two ectively ) was useCorresponding sense probes used as negative controls gave little, or low, levels of uniform background labelling.Sections were exposed to X-ray film  for 1–3 months at RT or apposed to emulsion (Ilford K5) for up to a year at 4°C . Film images were scanned and pseudocoloured in Image J by inverting LUX of ‘16 colours’ in Lookup Tables. Emulsion-coated sections were developed manually with D-19 developer (Agar Scientific Ltd. U.K.) according to the manufacturer’s instructions, counterstained with toluidine blue and viewed on a Leica DM2000 microscope equipped with a Lecia DFC70000T camera and Leica Application Suit X (LAS X) workstation.apela, apelin and aplnr in the same rat heart or kidney sections RNAscope  with the Multiple Fluorescence Assay kit was used. In this technique, target-specific Z-shaped probes with overhangs create a ‘bridge’ that binds a preamplifier that can then bind multiple amplifiers. The technique provides single RNA copy detection [To compare the expression of etection , typicalapela (GenBank Accession XM_008772035 (LOC100912649); bp147-1053; 12 Z probe pairs), apelin  and aplnr . RNAscope was performed on 16μm frozen sections that were post-fixed in 4% PFA, according to the ACD user protocols (sheets#320513 and 320293). RNAscope detection of aplnr expression in CHO-APJ (B78) and nontransfected CHO cells  for 2 days) followed the tissue section protocol with the exception that pre-treatment was performed as in ACD sheet#320528. With cells, only the aplnr probe(s)  were used, with a blank probe in the other two channels.Three fluorescent colour combinations were available, Alexa 488 (green), Atto 550 (orange) and Atto 647 (Far red), the colour for each target probe set dependant on which fluorescent colour modules (Amp4 AltA-C) and channels (C1-3) are used. The proprietary probes were designed by ACD for the following rat sequences: POLR2A, PPIB and ubiquitin (UBC); 3-plex probe set; ACD#407301) and negative ) probes obtained from ACD were included in all experiments. Sections (and cells) were counterstained with DAPI as per ACD instructions.Positive . Z stacks were taken of all images with a Z step size of 0.5 μm. Images were exported to Image J (where Far-red labelling was sometimes converted to white) or Adobe Photoshop. No attempt was made to quantitate the amount of ‘dot’ labelling.apela is not annotated in the rat reference DNA databases in the original mapping of RNA-Seq reads we looked specifically in the mouse apela sequence to find the corresponding rat homologue; the distribution of aplnr and apelin in the same kidney structures was similarly extracted. Details of the methods and analysis have been outlined [Previously unpublished high-throughput, ‘deep’ sequencing (RNA-Seq) data was retrieved from a dataset on microdissected rat kidney tubules from adult male Sprague-Dawley rats . The preoutlined .post hoc Dunnett’s test with GraphPad Prism software (version 4.0b). p<0.05 was considered as statistically significant.IN Cell Analyser 1000 experiments were performed in triplicate wells, with experiments performed at least 2 times. Statistical analysis was with a one-way ANOVA and aplnr cDNA (B78) were used as a positive control for the aplnr probe, confirming expression of APJ, and functional activity of apela-32 at APJ, in this cell line. Aplnr mRNA is highly expressed as punctate dots in CHO-B78 cells  in CHO-B78 cells in a dose-responsive manner between 10−10–10-6M  with weaker expression in the inner stripe of the outer medulla (ISOM)  . Serial apela pattern of labelling is similar to that observed in the rat. In the cortex of both mouse and rat kidneys there was some light labelling of isolated structures. There is no apparent difference in the distribution of apela between aplnr wildtype and knockout mice  cells in the medulla  of glomeruli express aplnr (only 1–5 dots/cell) in a diffuse pattern in glomerular cells , whereas apelin is absent from the tubules. Low levels of aplnr are present in the distal convoluted tubule (DCT) and cortical thick ascending loop of Henle (cTAL)—aplnr is not detected in collecting ducts. The RNA-Seq data does not include extra-tubular elements such as vasa recta where ISHH shows aplnr to be highly expressed. It confirms and extends the tubular expression of apela obtained by ISHH.e tubule . Notablyin vitro [apela mRNA is not abundantly expressed in the adult rat heart, and that there are relatively more apela-expressing cells in the inner, compared to the outer, medulla of the rat and mouse kidney, whereas the converse is true for aplnr in rats. In addition, we show apela to be mainly found in tubular elements in contrast to aplnr (and apelin); findings that are supported by RNA-Seq data on microdissected rat tubular segments. In the renal medulla, apelin expression is aligned with aplnr expression, where some cells appear to express both genes.APJ was an orphan GPCR , 31 untiin vitro . Using Iapela is particularly low, with only sparse cells expressing 1–3 mRNA transcripts/cell, which is in contrast to reports of a more widespread apela expression as demonstrated by RT-PCR in endothelial cells, fibroblasts and cardiomyocytes isolated from adult rat heart [apela is expressed primarily in the embryonic heart [In vivo both apelin and apela increase cardiac contractility in adult rodents and humans [in vitro [apela RNA acts in vivo as an APJ-independent regulatory ncRNA, although this has been shown to be functionally relevant in only one system  to dateAplnr and apelin appear to be co-localized in endothelial cells in and around microvascular proliferations in brain tumour specimens [aplnr and apelin co-expression in the kidney is likely to be in vascular elements. Similarly, while aplnr-apelin-apela co-expression is rare in the kidney, the co-localization of multiple receptor-ligand combinations  knockdown alters the expression of the GPCR CXCR4 in cultured human umbilical vein endothelial cells, apela stimulation or knockdown has no effect [in vitro [apela over apelin in the kidney may suggest that renal apela  is the more important APJ ligand in regulating renal function—this of course does not take into account possible effects of circulating levels of apela and/or apelin. As measured by enzyme immunoassay, apela has been detected in human plasma at slightly higher levels than apelin [aplnr expression have been reported by others in microdissected collecting ducts [aplnr. We propose that renal apela in the collecting duct and other tubular elements may access extra-tubular APJ in structures like the vasa recta to regulate the osmotic gradient and blood flow throughout this region. There is close proximity of tubular and vascular elements in the medulla, and cross-talk involving these structures has been implicated in the function of other peptide-receptor systems involving vasoactive agents, including endogenous peptides such as endothelin-1 [The expression of genes for one or both APJ-ligands with their receptor in the same cell, or cells expressing either ligand gene in the close proximity of APJ-expressing cells, raises the possibility of autocrine and/or paracrine actions for the apelinergic system within the heart and kidney. The demonstration of co-localization of receptor/ligand pairs in individual cells is not without precedent, e.g., in the brain a GPCR may regulate the release of its own endogenous ligand co-expressed in the same (or neighbouring) cell . Aplnr apecimens  and we sd family ) in otheo effect . In the o effect ) are medin vitro . The reln apelin . We noteng ducts , an obvithelin-1 ,43 that thelin-1 . We cannthelin-1 .apela mRNA and/or apela levels are reduced in pulmonary arterial hypertension in human patients and rat models, and apela-32 attenuates the remodelling of pulmonary vasculature and hypertrophy in right ventricular cardiomyocytes [apela expression is regulated (in concert with apelin and/or aplnr expression where the genes co-exist) may also be informative as to which APJ ligand may be active in target tissues. From a functional viewpoint, based on studies in zebrafish [apela knockouts in rodents may not be viable and so genetic manipulation of apela will likely require the use of inducible, conditional knockouts or RNA-edited models, and/or RNA interference with apela-specific knockdown constructs, to unravel its relative importance compared to apelin in regulating renal function.Many questions remain to be explored. For example, does apela in its ncRNA or peptide form(s) contribute to the pathological dispositions of some cardiovascular and metabolic conditions? Apelin and apela have anti-apoptotic activity in a number of tissues and cells ,19 and bmyocytes . In addimyocytes . Studiesebrafish ,9, the mapela mRNA transcripts have been reported to be detected ‘exclusively’ in adult rat kidney [apela mRNA expression in isolated cells in other tissues such as the heart. The full spectrum of apela-expressing tissues and the phenotype of the cells therein, and the functional relevance of apela as a separate entity or in unison with apelin, awaits determination. These and other questions will benefit from a thorough characterization of the sites of apela expression in a cellular context.Despite the fact that t kidney , our stu


Host-level influenza virus–respiratory pathogen interactions are often reported. Although the exact biological mechanisms involved remain unelucidated, secondary bacterial infections are known to account for a large part of the influenza-associated burden, during seasonal and pandemic outbreaks. Those interactions probably impact the microorganisms’ transmission dynamics and the influenza public health toll. Mathematical models have been widely used to examine influenza epidemics and the public health impact of control measures. However, most influenza models overlooked interaction phenomena and ignored other co-circulating pathogens.Herein, we describe a novel agent-based model (ABM) of influenza transmission during interaction with another respiratory pathogen. The interacting microorganism can persist in the population year round  or cause short-term annual outbreaks . The agent-based framework enables precise formalization of the pathogens’ natural histories and complex within-host phenomena. As a case study, this ABM is applied to the well-known influenza virus–pneumococcus interaction, for which several biological mechanisms have been proposed. Different mechanistic hypotheses of interaction are simulated and the resulting virus-induced pneumococcal infection (PI) burden is assessed.This ABM generates realistic data for both pathogens in terms of weekly incidences of PI cases, carriage rates, epidemic size and epidemic timing. Notably, distinct interaction hypotheses resulted in different transmission patterns and led to wide variations of the associated PI burden. Interaction strength was also of paramount importance: when influenza increased pneumococcus acquisition, 4–27% of the PI burden during the influenza season was attributable to influenza depending on the interaction strength.This open-source ABM provides new opportunities to investigate influenza interactions from a theoretical point of view and could easily be extended to other pathogens. It provides a unique framework to generate in silico data for different scenarios and thereby test mechanistic hypotheses.The online version of this article (doi:10.1186/s12879-017-2464-z) contains supplementary material, which is available to authorized users. Streptococcus pneumoniae [Haemophilus influenza [Staphylococcus aureus [Influenza is a respiratory virus that imposes a heavy burden on human populations by causing millions of severe illness cases and deaths worldwide every year , despiteeumoniae , 7, 8, Hnfluenza , Staphyls aureus  etc. Eves aureus , 12. If Streptococcus pneumoniae (pneumococcus) [A long-studied interaction among respiratory pathogens is that between influenza viruses and ococcus) , 8, 13. ococcus) , 15. Pneococcus) , that asococcus) , 18. Wheococcus) . A synerococcus) , 8, 20. Mathematical modeling is a powerful tool to explore the epidemiology and spread of infectious diseases –23. HistHerein, we describe a novel individual-based simulator of the co-circulation of influenza virus (henceforth referred to as influenza) in interaction with another respiratory pathogen in a virtual population of humans, the Simulator of Flu in Interaction (SimFI) framework. We used this framework to explore the particular context of influenza and pneumococcus co-circulation. Based on simulated data, we assessed the potential burden of PI cases attributable to influenza, under different interaction hypotheses.We developed a stochastic ABM, in which each human individual is specifically modeled and his/her successive infectious states recorded. The model is applied to simulate influenza and a second pathogen, which can be either epidemic  or endemic (present in the population throughout the year). The between-pathogen interactions and their consequences can vary according to the chosen pathogen.http://b2phi.inserm.fr/#/resources/71/NetLogo-SimFI-model.We used the NetLogo multi-agent programmable modeling environment for the implementation . The modIndividuals are characterized by several variables monitoring their location, age and infectious state concerning the two pathogens. The simulation time step is the day. On each day of the simulation individuals move in the simulated world and come into contact with other people. From those contacts, the transmission of one or both pathogens is possible and will result in reevaluations of the variables describing infectious status. Here years define epidemic years, starting on October 1st and ending on September 30th.The model is artificially spatially explicit so that the individuals can move, in what the NetLogo software refers to as a “world”, which is a torus divided into patches. Two persons are considered to be in contact if they coincide on the same patch at a given time. The dimension of the patches – and thus of the world – is set in order to have, on average, 13 contacts per person and per day as reported by the Polymod study and its French equivalent , 32. TheFigure Once an individual is infected with an influenza virus, the incubation period is assumed to last an average of 2 days , 34, folOn the first day of each epidemiological year, the characteristics of the coming influenza outbreak are set. The influenza-associated infectiousness probability is drawn from a normal probability distribution with a mean value set by the user (3.3% per contact-day by default). A portion of randomly chosen individuals is immunized against the coming influenza virus (default at 23%) to take into account natural or acquired immunity in the population. The importation date of the first cases is randomly drawn from a gamma distribution of mean 71 and standard deviation 28, and the number of imported cases is randomly chosen between 20 and 30. Every individual has a 20% probability of being reported when they enter the symptomatic phase. Default values were chosen to obtain incidence series comparable to French surveillance data from the Sentinel Network .Individuals can acquire the second pathogen in a similar way as they acquire influenza, with user-defined characteristics of natural history . The generic timeline of the second pathogen is represented on Fig. If the second pathogen is epidemic, the outbreak set-up is similar to that of influenza, with a mean infectiousness rate, initial immunity proportion, importation period, and number of imported cases all to be defined by the user. If the second pathogen is endemic, a fraction of randomly chosen individuals (20% of the population by default) acquires the pathogen, thus entering the asymptomatic phase at the beginning of the simulation.Different interaction mechanisms influencing the pathogens’ infection dynamics are embedded in our model. They are based on the main biological mechanisms found in the literature on influenza interactions , 8, 13, β) or infection (p) probabilities  provided by the IT Department at Institut Pasteur, Paris. Graphs and statistical analyses were performed with R version 3.3.3.The pneumococcus parameters default values are given in Table PIs have a seasonal trend, with more cases during the winter than summer , 43. We Several studies have investigated a potential interaction between influenza virus and pneumococcus , 8, 20. ij), −transmission (θij), −cross-immunity (μij), and -pathogenicity (πij), many interaction scenarios can be simulated. We modified independently the parameters corresponding to the influenza-pneumococcus interaction for our simulations: when one interaction mechanism is activated, the corresponding parameter is set to a non-1 value, while the other interaction parameters are fixed to 1. For each of the three parameters considered here , the following values were independently tested: 2, 5, 10, 15, 25, 50, 75, 100, corresponding to a total of 24 scenarios. A baseline scenario with no interaction was also simulated. For each scenario, 1000 independent epidemiological years were simulated from the model.By varying the values of the different interaction parameters modulating pneumococcal-acquisition  and the mean total number of PIs for the baseline scenario in which no influenza–pneumococcus interaction mechanism was activated. The burden was assessed for three different periods of the simulated epidemiological year: during influenza season only, post-influenza season only, and during the entire year (therefore including the two previously cited periods). The influenza season is defined as weeks with >150 new influenza cases reported, the post-influenza season is the period starting immediately after the end of the influenza season, as we defined it, and lasting until the end of the simulated year (September 30th).In the following, all average numbers of cases are reported per 100,000 inhabitants.Influenza dynamics are well-reproduced by the model, as displayed in Fig. Weekly incidence series for PIs are presented in Fig. Figure In our simulations, the average influenza season lasted 11.6 weeks, and the post-influenza period lasted 26.6 weeks.As expected, an average annual number of 242 PIs (mean 4.6 per week) was estimated when no between-pathogen interaction was considered [see the table in Additional file The burden of influenza-attributable PIs increased slightly with the acquisition parameter: above the threshold value of 15 it remained stable, representing on average 34 PIs out of 276 annual cases  for the whole year, even for very high interaction-parameter values Fig. . During The burden of influenza-attributable PI cases increased with the transmission-related parameter, regardless of the time period , reaching an influenza-induced burden of 68% for the highest parameter value during the influenza season . The transmission- and pathogenicity-interaction mechanisms behaved differently. The PI burden increased with the transmission-interaction–parameter value for the three considered periods , with respective values in the ranges 1–52%, 3–72%, and 2–46% of total PIs. Same was observed for the pathogenicity mechanism, with respective average PI burdens in the ranges of 1–56%, 1–79%, and 0–24%.Herein, we tested a wide range of strengths for the influenza-virus–pneumococcus interaction  with interaction-parameter values ranging from 2 to 100, in accordance with the estimations of previous modeling studies for different types of pneumococcal infections , 45. BasAn advantage of the SimFI framework is the possibility to exactly count the number of infections with each pathogen, as well as the exact number of cases attributable to the between-pathogen interaction. The authors of several studies attempted to estimate the PI burden. Classically, burden estimations have been based on regression models, including influenza as a covariate, and comparison to a baseline deduced by removing influenza. For example, Walter et al.  estimateHerein, we aimed to formalize the simplest model incorporating the main features of influenza interactions with another pathogen. Our model is therefore a simplification of reality. A sensitivity analysis was carried out on several key parameters: pneumococcus carriage rate, mixing patterns in the simulated world, and size of the influenza epidemic. The movement patterns of the individuals did not affect the influenza-induced PI burden, however both the carriage rate of pneumococcus in the population and the size of the influenza epidemic had an impact on the PI burden .In conclusion, a novel individual-based simulator of influenza in interaction with another pathogen, including several ready-to-use interaction mechanisms, was described. As an application, we used this SimFI framework to assess what the influenza-associated PI burden could be for different hypothetical interactions. This open-access model will provide new opportunities to test hypotheses concerning pathogen  interactions, generate in silico data for different scenarios, and assess the statistical methods generally used to study interactions.Additional file 1:Natural histories of influenza and pneumococcus. Clinical status (upper rectangles) and infectious status  are shown for influenza (top timeline) and pneumococcus (bottom timeline). (JPEG 72 kb)Additional file 2:Sp of pneumococcus (Streptococcus pneumoniae) can be modulated by the different interaction mechanisms, depending on the infectious status of the two individuals in contact. α12 is the parameter for the acquisition-interaction directed from influenza on pneumococcus, and θ12 is the transmission-interaction parameter. (PNG 67 kb)Calculation of pneumococcus’ acquisition probability depending on the two in-contact individuals’ infectious statuses. The transmission probability βAdditional file 3:Weekly incidence of simulated cases per 100,000 for the three possible pathogens. (A–D) For influenza (red), the following parameter values were used for the simulations: transmission probability 3.3% per contact-day; 23% of the population initially immunized; 20% case-reporting probability; no interaction mechanism activated between influenza and the second pathogen. For PI cases (black), the following parameter values were used: carriage rate 22% per contact-day; pathogenicity probability 0.0042% per day; no immunity; 100% case-reporting probability; no interaction mechanism activated (A), acquisition-interaction strength 50 (B), transmission-interaction strength 50 (C), and pathogenicity-interaction strength 50 (D). (E) Pneumococcal carriage prevalence for the baseline scenario (orange), the acquisition-interaction strength 50 (green), the transmission-interaction strength 50 (blue), and the pathogenicity-interaction strength 50 (purple); (F–H) For influenza (red) and a second epidemic pathogen (black) cases, the following parameter values were used for the latter: transmission probability 2.8% per contact-day; 25% of the population initially immunized; 20% case-reporting probability; no interaction mechanism activated (F), acquisition-interaction strength 25 (G), and cross-immunity–interaction strength 0.8 (H). The represented data were chosen for five among the 1000 simulated years for each scenario for their explicit representation of each interaction-mechanism effect on infection dynamics. (PNG 424 kb)Additional file 4:Burden of influenza-related pneumococcal infections for all simulated scenarios. Eight different values of interaction parameters were tested for each interaction mechanism . Values in the table represent the influenza-induced excess numbers of pneumococcal infections and the associated percentages, presented with their 95% confidence intervals, per 100,000. Values are aggregated over the three different periods considered: whole epidemiological year, influenza season, and post-influenza season. (XLSX 16 kb)Additional file 5:Weekly influenza-induced PI burden after the introduction of influenza in the population. The average weekly burdens for different scenarios are represented if they are statistically significant . The x-axis represents the number of weeks after the introduction of influenza in the population. (PNG 213 kb)Additional file 6:Sensitivity analysis of pneumococcus carriage rate, movement patterns in the population, and influenza size. (PDF 181 kb)


Very few studies have examined the potential spill-over effect of a trial intervention in general practice. We investigated whether training and support of general practitioners in the intensive treatment of people with screen-detected diabetes improved rates of redeemed medication, morbidity and mortality in people with clinically-diagnosed diabetes.ADDITION-Denmark trial, 175 general practices were cluster randomised (i) to routine care, or (ii) to receive training and support in intensive multifactorial treatment of individuals with screen-detected diabetes (2001 to 2009). Using national registers we identified all individuals who were diagnosed with clinically incident diabetes in the same practices over the same time period. . We compared rates of redeemed medication, a cardiovascular composite endpoint, and all-cause mortality between the routine care and intensive treatment groups.This is a secondary, post-hoc, register-based analysis linked to a cluster randomised trial. In the ADDITION-Denmark practices between 2001 and 2009 . There were large and significant increases in the proportion of patients redeeming cardio-protective medication in both treatment groups during follow-up. After a median of seven years of follow-up, there was no difference in the incidence of a composite cardiovascular endpoint  or all-cause mortality between the two groups .In total, 4,107 individuals were diagnosed with clinically incident diabetes in There was no evidence of a spill-over effect from an intervention promoting intensive treatment of people with screen-detected diabetes to those with clinically-diagnosed diabetes. Overall, the proportion of patients redeeming cardio-protective medication during follow-up was similar in both groups.NCT00237549ClinicalTrials.gov  The benefit of managing cardiovascular risk factors among people with type 2 diabetes is well established . HoweverADDITION trial, 175 general practices were cluster randomised to routine care or to receive training and support in the implementation of an intensive diabetes treatment program for individuals with screen-detected diabetes [In the Danish arm of the diabetes . Given tdiabetes .ADDITION-Denmark, we used national registry data to compare rates of redeemed cardio-protective medication, morbidity and mortality among people with clinically-diagnosed diabetes in the routine care and intensive treatment trial groups.In order to assess a potential spill-over effect of the trial intervention among practices taking part in ADDITION-Denmark consists of two phases: (i) a pragmatic screening programme, and (ii) a cluster-randomised parallel-group trial comparing the effects of intensive multifactorial treatment with routine care among individuals with screen-detected type 2 diabetes [diabetes ,6. In brdiabetes . EthicalGPs and nurses received training and support in delivering intensive treatment via small group or practice-based educational meetings where treatment targets / algorithms, lifestyle advice, and supporting evidence were discussed. Patient audit and feedback was completed in face-to-face meetings held up to twice a year or was coordinated by post. Intensive treatment practices received additional funding to support the delivery of care, which included target-driven management of hyperglycaemia, blood pressure, and cholesterol levels by medical treatment and promotion of healthy lifestyles, based on the stepwise regimen used in the Steno-2 study and other trial results,9.In the routine care group, general practitioners were advised to follow Danish national recommendations on diabetes treatment ,11 and rADDITION-Denmark over the same time period as the intervention of the trial (2001 to 2009), using the target population from the ADDITION-Denmark . Our definition of clinically diagnosed diabetes was a proxy measure based on two different modes of identification. Participants were defined as having diabetes if they fulfilled at least one of the following criteria: (i) included on the Danish National Patient Register with a diagnosis of diabetes ; or (ii) redeemed glucose-lowering medication at least twice within six months during the study period (2001 to 2008). Patients with screen-detected diabetes who participated in the ADDITION trial were excluded. The practice and participants flow are shown in This is a secondary, post-hoc, register-based analysis linked to the ADDITION-Denmark trial. In order to assess the potential spill-over effect of the intervention, we identified individuals who were diagnosed with clinically incident diabetes in the same 175 GP practices taking part in 19. We calculated the score for the Charlson Comorbidity Index[We linked information about individuals with clinically incident diabetes to other Danish registers using unique civil registration numbers. We retrieved information on age, sex, education, marital status and redeemed cardio-protective medication . Educatity Index using inst December 2013, when national registers were searched for information on vital status and incident CVD events. For death, the primary outcome was all-cause mortality (based on underlying cause of death). We used a composite of first event of cardiovascular death, non-fatal MI  or non-fatal stroke  to delineate an incident CVD event. Data on incident CVD events was gathered from the National Patient Registry, which records all in-patient and out-patient hospitalisations in Denmark.Participants were followed for a median of 7.3 years to 31Baseline characteristics were summarised separately in the intensive treatment and routine care trial groups. Patient groups were compared using the χ2 test for categorical variables and the t test for continuous variables. P-values <0.05 were considered statistically significant. Date of entry to the study was date of diagnosis of clinically-incident diabetes. Individuals were censored on the date of first event, death, emigration or 31/12/2013, whichever was earliest. Hazard ratios comparing mortality and incident CVD events between the groups were estimated with a Cox proportional hazards regression model. We used a Nelson-Aalen hazard function to generate a cumulative incidenc curve based on this model. Since allocation to the treatment groups was at the practice level, robust standard errors were calculated that take into account the two-level structure of the data, using the “cluster” option in Stata. We adjusted for age, sex and the Charlson Comorbidity Index Score. In a sensitivity analysis, we further adjusted for education level among those with complete data for this variable (118 people had missing data for education). We calculated the proportion of participants who redeemed prescriptions for six cardio-protective medication classes in both groups during the following times periods: 0 to 12 months before clinical diagnosis of diabetes, from 01/07/2008 to 30/06/2009, and from 01/01/2012 to 31/12/2012. All analyses were completed using Stata Version 14.1 .Table 1 shows the characteristics of individuals diagnosed with clinically-incident diabetes from ADDITION-Denmark practices between 2001 and 2009. The mean age (SD) of the cohort was 59.6 (8.2) years and 41% were female. Levels of education, marital status and the Charlson Comorbidity Index score were similar between the trial groups.Patterns of redeemed medication during follow-up are shown in Table 3). Following adjustment, there was no significant difference in incident CVD between the intensive treatment and routine care groups  .The median (IQR) duration of follow-up was 7.3 years . During follow-up, there were 289 cardiovascular events (14.1%) in the intensive treatment group (incidence (95% CI): 20.7  and 258 events (12.6%) in the routine care group (incidence (95% CI): 18.4  per 1,000 person-years) . There was no difference in the overall result when adjusting for education level.During follow-up, there were 447 deaths (21.7%) in the intensive treatment group : 30.0  per 1,000 person-years) and 419 deaths (20.4%) in the routine care group : 29.8  per 1,000 person-years). Following adjustment, there was no significant difference in all-cause mortality between the two groups (HR 1.08 (95% CI: 0.94 to 1.23)) . The trial also took place against a background of increasing national interest in screening and early treatment for diabetes, which might have encouraged practitioners to improve detection and management of CVD risk factors. Furthermore, we observed a large and significant increase in redeemed cardio-protective treatment in both trial groups during follow-up, suggesting that both intensive treatment and routine care GPs changed their prescribing behaviour among clinically-diagnosed patients. Both trial groups had room for improvement in GP prescribing e.g. only 78% of clinically-diagnosed individuals redeemed glucose-lowering medication at the end of follow-up. This finding mirrors the main trial results among screen-detected individuals, where both treatment groups received higher levels of cardio-protective medication by the end of follow-up and there were only modest differences in levels of treatment and cardiovascular risk factors at the five-year health examination[There are a further explanations for the lack of a spill-over effect observed in this trial. Firstly, we did not aim to affect change in health practitioner behaviour with regards to treatment of clinically incident diabetes patients. As such, training and support of general practitioners in the intensive treatment of people with screen-detected diabetes may not be associated with any benefits for individuals found with clinically-diagnosed diabetes in the same practices. An alternative explanation is that general practitioners in both trial groups improved their management of screen-detected and clinically diagnosed diabetes patients. GPs taking part in amination.ADDITION-Denmark practices between 2001 and 2012. Trial groups were well balanced for patient level characteristics at baseline. Outcome ascertainment was very robust. The Danish National Death Registry estimates 100% coverage of mortality based on death certificates, while the National Patient Registry includes 99.4% of discharges from Danish hospitals. Deaths and incident CVD events were coded blind to study group.The Danish registry system allows a unique investigation of the long-term experience of individuals diagnosed with clinically-incident diabetes in ADDITION-Denmark were ≥40 years of age at the time of inclusion of GP practices, we assume that the number of type 1 diabetes cases is likely to be low and similar in both groups.Our definition of clinically diagnosed diabetes was a proxy measure based on date of inclusion in the Danish National Patient Register and redemption of glucose-lowering medication at least twice within six months during the study period (2001 to 2009). While we did not have formal clinical diagnosis of diabetes for each person included on the Register, a recent validation of the algorithm for including patients in the Register suggests that it has a sensitivity of ≥95% and a positive predictive value of around 80% . A furthADDITION-Denmark practices.While we were able to compare trends in redeemed medication to explore a potential effect of the intervention on GP prescribing patterns, it would also have been useful to examine diet, physical activity and smoking behavior to examine potential effects on motivation to change lifestyle behaviour. However, these data are not available for individuals with clinically-diagnosed diabetes in In conclusion, there was no evidence of a spill-over effect from the intensive treatment of people with screen-detected diabetes to those with clinically-diagnosed diabetes. The risk of incident CVD and mortality did not differ between trial groups.


Hypoplastic left heart syndrome (HLHS) covers a spectrum of rare congenital anomalies characterised by a non-apex forming left ventricle and stenosis/atresia of the mitral and aortic valves. Despite many studies, the causes of HLHS remain unclear and there are conflicting views regarding the role of flow, valvar or myocardial abnormalities in its pathogenesis, all of which were proposed prior to the description of the second heart field. Our aim was to re-evaluate the patterns of malformation in HLHS in relation to recognised cardiac progenitor populations, with a view to providing aetiologically useful sub-groupings for genomic studies.Nkx2–5-Cre labelled myocardial and endothelial cells within the left and right ventricles, Mef2c-AHF-Cre, which labels second heart field-derived cells only, was largely restricted to the endocardium and myocardium of the right ventricle. However, like Nkx2–5-Cre, Mef2c-AHF-Cre lineage cells made a significant contribution to the aortic and mitral valves. In contrast, Wnt1-Cre made a major contribution only to the aortic valve. This suggests that discrete cardiac progenitors might be responsible for the patterns of defects observed in the distinct ventricular sub-groups.We examined 78 hearts previously classified as HLHS, with subtypes based on valve patency, and re-categorised them based on their objective ventricular phenotype. Three distinct subgroups could be identified: slit-like left ventricle (24%); miniaturised left ventricle (6%); and thickened left ventricle with endocardial fibroelastosis . Slit-like ventricles were always found in combination with aortic atresia and mitral atresia. Miniaturised left ventricles all had normally formed, though smaller aortic and mitral valves. The remaining group were found to have a range of aortic valve malformations associated with thickened left ventricular walls despite being described as either atresia or stenosis. The degree of myocardial thickening was not correlated to the degree of valvar stenosis. Lineage tracing in mice to investigate the progenitor populations that form the parts of the heart disrupted by HLHS showed that whereas Only the slit-like ventricle grouping was found to map to the current nomenclature: the combination of mitral atresia with aortic atresia. It appears that slit-like and miniature ventricles also form discrete sub-groups. Thus, reclassification of HLHS into subgroups based on ventricular phenotype, might be useful in genetic and developmental studies in investigating the aetiology of this severe malformation syndrome. GJA1 [HAND1 [NOTCH1 [NKX2–5 [GATA4 [Hypoplastic left heart syndrome (HLHS) is the term applied by Noonan and Nadas  to a ranGJA1 , 13, HAN1 [HAND1 , 15, NOT [NOTCH1 , NKX2–5  [NKX2–5  and GATAIn contrast to the limited progress in finding the causes of HLHS, there has been a revolution in our understanding of cardiac morphogenesis since the description of the second heart field (SHF) in 2001. The previous theory proposing segmental patterning within the primitive heart tube was an attractive one, not least because it apparently explained many heart defects including HLHS. We now know it to be incorrect. Instead, it is recognised that a single atrial and ventricular chamber are initially formed from the first heart field (FHF), but that consequent addition of cells from the surrounding SHF produce parts of the atria, the right ventricle and the outflow tract (reviewed in ). StudieCre-lox mice, we ask if the features of HLHS subtypes can be adequately explained by abnormalities attributable to specific progenitor populations. We demonstrate three discrete ventricular phenotypes, but note that these are associated with a broad range of abnormalities affecting the aortic and mitral valves suggesting that HLHS may be the result of distinct developmental sequences. In some situations, valvar abnormalities might be the initiating abnormality, whilst in others the primary issue most likely lies in abnormal endocardial or myocardial growth or signalling. This study indicates how the heterogeneity of phenotype in HLHS can be grouped in studies aiming to explain the pathogenesis of HLHS.In this study, we take a novel approach to understanding HLHS, initially by reviewing the intra-cardiac morphology of hearts appropriately coded as HLHS, looking specifically for patterns of abnormalities that would correlate with disturbance of known developmental processes. Then, using genetic lineage tracing with The congenitally malformed heart archive at Birmingham Children’s Hospital contains approximately 2000 specimens collected since 1939. Permission to study the collection was granted by the R&D department at Birmingham Children’s Hospital NHS Foundation Trust and the Custodian Committee of the collection. Seventy-eight un-operated hearts, in good condition and fulfilling the accepted criteria for HLHS [Mef2c-AHF-Cre, Wnt1-Cre, Nkx2–5-Cre, and Tie2-Cre [ROSAeYFP and mTmG reporter lines were used in these experiments. Specificity of expression of these Cre drivers in our hands has been confirmed in previous studies [Tie2-Cre –23 mice, studies , 25. TheMouse embryos were collected at embryonic (E) days 10.5 and E15.5. Immunohistochemistry was carried out as described previously , 26. AleComparisons were made using ANOVA  assuming non-parametric distributions. Post Hoc testing with Dunn’s test allowed for unequal group size.Based on macroscopic examination and probe patency, we sub-classified 78 hearts with a previous diagnosis of HLHS Fig. , focussiExamination of the left ventricles in HLHS specimens revealed three different appearances Fig. . A distiWe next examined the aortic and mitral valves in more detail, relating them with the ventricular phenotypes as described above. Of the 24 hearts with aortic stenosis, all five with miniaturised ventricles contained remarkably large and well-formed aortic valves with three thin leaflets Fig. . The remAlthough a range of abnormalities was seen in the mitral valves, there was a general relationship between the size of papillary muscles, length of tendinous chords and the degree of leaflet formation. The largest valves with well-formed thin leaflets, fine separate tendinous cords, and prominent papillary muscles, were found in the five hearts with miniaturised ventricles (MS/AS) Fig. . In 15/1The ascending aorta was narrow in comparison to the pulmonary trunk in all the hearts Fig. . The ratCre-lox based lineage tracing in transgenic mice to determine the progenitor contributions and developmental processes common to the left-sided structures of the heart, focussing on stages prior to and following cardiac septation (embryonic day (E) 10.5 and E15.5 respectively).To investigate the developmental mechanisms underpinning the miniaturised, slit-like, and thickened with EFE categories of HLHS described above, we used Nkx2–5-Cre that identifies all cardiomyocytes [Mef2c-AHF-Cre, which specifically labels SHF-derived cardiomyocytes [Nkx2–5-Cre identified cardiomyocytes in the right and left atria and ventricles .Discrimination of FHF cells from SHF cells was performed by comparing the expression patterns of GFP reporter constructs in transgenic mice containing myocytes , with thmyocytes . At E10.les Fig. , c, Mef2ium Fig. , d. The cardium , second heart field-derived cells (Mef2c-AHF-Cre), endocardial cells (Tie2-Cre) and neural crest cells (Wnt1-Cre) [To explore these concepts of embryonic origins further, we carried out a developmental analysis using ructures . Recentlructures  althoughructures , all preelopment , 19. Thent1-Cre) –23 and wnt1-Cre) , 25). Alnt1-Cre) . Thus, tnt1-Cre) . Thus, int1-Cre) .The new classification proposed in this study is not intended to replace conventional descriptions that are based on the valvar pathology. The conventional classification of HLHS has important clinical implications. For example, hearts with mitral stenosis and aortic atresia are associated with adverse clinical outcome due to sinusoidal connections between the coronary circulation and the left ventricular cavity , 40. How


PGD for HLA typing is a procedure that can be performed when an affected childrequires a transplant to treat a non-hereditary disorder related to thehematopoietic and/or immune system. Hematopoietic stem cell transplantation froman HLA-identical donor provides the best treatment option. Three conventionalovarian stimulation procedures for IVF were performed in a couple with a10-year-old child diagnosed with T-cell acute lymphoblastic leukemia of highrisk. Trophectoderm biopsy and aCGH examination were performed on 15blastocysts, three on the first IVF procedure, four on the second cycle, andeight on the third. Three euploid blastocysts HLA-compatible with the genome ofthe affected child were identified. One euploid blastocyst HLA-compatible withthe affected child was warmed and transferred, resulting in an HLA-matched livebirth. In conclusion, combined aCGH for aneuploidy screening and Karyomappingmay be performed in a single biopsy procedure. T-cell acute lymphoblastic leukemia is an immature lymphoid tumor characterized bydiffuse infiltration of the bone marrow by malignant hematopoietic cells expressingimmature T-cell markers. Clinically, patients with T-cell acute lymphoblasticleukemia have elevated white blood cell counts and hematopoietic insufficiency,neutropenia, anemia, and thrombocytopenia. In addition, they often show thymicmediastinal masses and meningeal infiltration of the central nervous system at thetime of diagnosis. Immature T-cell tumors presenting as thymic masses with limitedbone marrow infiltration are diagnosed instead as T-lymphoblastic lymphomas .Preimplantation genetic diagnosis (PGD) has been applied as a selection tool to avoidinheritable diseases  method. Karyomapping provides anintegral diagnostic method based on the ligation of single-gene defects. Genotypingparents and close relatives with known diseases eliminates the need for custom testdevelopment, and since Karyomapping defines four sets of SNP markers for each of theparental chromosomes, it allows the simultaneous execution of molecular cytogeneticanalysis  and allow pregnancy to develop with an unaffectedHLA-compatible embryo .Their 10-year-old boy had been diagnosed with T-cell acute lymphoblastic leukemia,but tests for bone marrow transplantation performed at the National Institute forNeoplastic Diseases in Peru revealed he and his sister were not histocompatible.After having searched the Bone Marrow Donors Worldwide database, they were offeredthe possibility of undergoing IVF with PGD. They were informed of the scope andlimitations of PGD in HLA typing.Following our clinical protocol, the 36-year-old woman was instructed to perform ahormone profile test and undergo pelvic ultrasound examination. The tests revealedshe had polycystic ovaries, and treatment with Metformin was prescribed (Metformin -1500 mg/day). Though her hormone profile was normal, prolactin was found to beelevated (39 ng/mL) and she was treated with Cabergoline (Dostinex - Pfizer).The 42-year-old man was instructed to undergo conventional sperm analysis. Accordingto the WHO 2010 manual , he was Three controlled ovarian stimulation (COS) cycles were performed. The patientswere properly advised about the procedures and signed informed consentterms.The first COS cycle was performed in December of 2014. Ovarian induction was donewith 150 IU of FSH/LH (Bravelle - Ferring) and 150 IU of hp-HMG(Menopur-Ferring) from day 2 to day 10 of the cycle. Ant-GnRH with follicle ≥14 , and ovulation time was synchronizedwith r-hCG (Ovidrel-Merck). After oocyte retrieval, 34 oocytes were obtained; 17were mature, eight were fertilized and three blastocysts were vitrified forbanking. Vitrification was performed according to cryotech instructions The second COS cycle was performed in February of 2015. Follicular growth wasinduced with 150 IU of FSH/LH (Bravelle-Ferring) and 75 IU of hp-HMG(Menopur-Ferring) from day 3 to day 10 of the cycle. Ant-GnRH with follicle ≥14  and ovulation was triggered withAna-GnRH (Gonapeptil-Ferring). Twenty-eight oocytes were aspirated; 16 weremature, 10 were fertilized and four blastocysts were vitrified.The last COS cycle was performed in April of 2015. Patient was administrated 150IU of FSH/LH (Bravelle - Ferring) and 75 IU of hp-HMG (Menopur-Ferring) from day2 to day 11 of the cycle. Ant-GnRH (Cetrotide-Merck Serono) with follicle≥14  and ovulation was synchronized with Ana-GnRH(Gonapeptil-Ferring). In this cycle, 33 oocytes were aspirated; 26 were mature,18 were fertilized and eight blastocysts were biopsied on days 5/6. On the sameday, all blastocyst generated in previous cycles were warmed and biopsied. Allblastocysts were vitrified for genetics analysis. .Trophectoderm biopsy and aCGH examination were performed on 15 blastocysts, threeon the first IVF procedure, four on the second cycle, and eight on the third.PGS with aCGH results showed that eight blastocysts were euploid .The same DNA isolated and amplified for aCGH was used to perform PGD withKaryomapping. In this case, the HLA region has 128 informative SNPs in themother and 78 in the father. SNPs were classified as compatible ornon-compatible with respect to the analysis of the same SNPs in the affectedchild's cells.The SNPs reported as compatible were grouped as P1  and M1 . In orde® -Abbott), a GnRH agonist, 3.75 mg intra muscular. On the first day of subsequentmenses, transvaginal ultrasound was performed and hormone therapy with EstradiolValerate (Progynova® - Shering) was initiated in a variabledose protocol, with an oral dose of 4 mg/day from day 1 to day 7, and 6 mg/dayfrom day 8 until an endometrial thickness of 10 mm and trilaminar pattern wasreached, on day 18. At that time, Micronized Progesterone 600 mg vaginal per daywas initiated and the dose of Estradiol Valerate was increased to 8 mg/day.In the middle of the luteal phase (day 21) of the previous menstrual cycle thepatient was administered Leuprolide Acetate  or genetic diseases (Karyomapping). This case report described two clinical challenges. The first was the PCO-likeovaries. An antagonist GnRH protocol was used and modified after the first cycle,because of the low quality of oocytes determined by low blastocyst formation. In thefollowing cycles the dose of hp-HMG was decreased on account of the polycysticovaries. A GnRH agonist was also used as a final trigger for oocyte maturation tominimize the risk of ovarian hyperstimulation syndrome . In womeThe second challenge was the combination of PGD techniques (aCGH and Karyomapping).There are few reports describing this type of protocol. One of the studies wasperformed between 2007 and 2014 and enrolled 12 couples interested in taking part ina PGD-HLA-typing program. All couples had children affected by either a genetic oran acquired disease affecting their hematopoietic and/or immune system. Sevencouples were treated in 26 cycles and two healthy HLA-matched babies were born,leading to a live birth rate of 28.6% per transfer and 7.7% per initiated cycle wereeuploid on aCGH; and three (20%) were HLA-compatible with the affected child.Similar results have been reported in a French study, in which 19% (3 out of 16) ofthe embryos were unaffected and HLA-compatible  transplantation from an HLA-identical donor is the besttherapeutic option for genetic diseases affecting the hematopoietic and/or immunesystem in children, and may be an option for individuals with acquired diseases aswell (Regarding legislation, there is no regulation for ART procedures in Peru. Allclinical and laboratory practices were performed following the highest ethicalstandards and in close compliance with European legislation (In conclusion, combined aCGH for aneuploidy screening and Karyomapping was performedin a single biopsy procedure, resulting in an HLA-matched live birth. This type ofprocedure may be extended to other single gene disorders.


Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children. In recent Total Therapy studies conducted at St. Jude Children’s Research Hospital, children with ALL had a 5-year overall survival of around 94%. This is the result of a combination of risk stratification based on the biological features of the leukemic cells and the response to treatment , treatment modification based on pharmacodynamic and pharmacogenomic data, and improved supportive care. However, innovative approaches are required to further improve survival to as close to 100% as possible and to reduce the adverse effects of treatment. Next-generation sequencing of leukemic cell DNA and RNA, as well as of germline DNA, can identify submicroscopic genetic structural changes and sequence alterations that contribute to leukemogenesis. Next-generation sequencing data can be used to define new ALL subtypes, to help improve treatment response and reduce adverse effects, and to identify novel prognostic markers and therapeutic targets to facilitate personalized precision medicine. In this article, we describe our approach to detecting targetable lesions in patients with ALL by next-generation sequencing and explain how we integrate the sequencing data into the treatment of these patients. BCR-ABL1-positive ALL] or T-cell ALL (T-ALL) who were aged 1–18 years at diagnosis, 5-year event-free survival was increased to 87% and overall survival to 94%. In this protocol, prophylactic cranial irradiation was completely replaced by risk-adapted intrathecal chemotherapy and the incidence of isolated CNS relapse was reduced to 2.7%  have improved considerably over time . In the  to 2.7% , 4. HoweAlthough the outcomes for patients with St. Jude low-risk ALL [equivalent to National Cancer Institute (NCI) standard-risk ALL] are now excellent, there is still a need for substantial improvement in the cure rates for patients with St. Jude standard-risk and high-risk ALL . Our latest frontline ALL treatment protocol, Total XVII (NCT03117751), incorporates novel precision-medicine strategies based on inherited and leukemia/lymphoma-specific genomic features and targeted treatment approaches.BCR-ABL1–like ALL), a recently described subtype of B-ALL, is characterized by a gene-expression profile similar to that of Ph-positive ALL; a variety of genetic alterations that activate tyrosine kinase signaling; the mutation of lymphoid transcription factor genes such as IKZF1 (in 70%–80% of cases); and a poor outcome  or kappa (IGK) loci that result in their overexpression and the activation of receptor signaling ; activating mutations in the Janus kinase genes themselves (JAK1 or JAK3); and mutations that impair the function of negative regulators of JAK–STAT signaling (SH2B3)  .To capture these relevant alterations in the Total XVII protocol, diagnostic testing involves not only established morphologic, immunophenotyping, and molecular genetic approaches but also next-generation sequencing diagnostics that are performed as the clinical standard of care for all consented patients.TCF3-PBX1, ETV6-RUNX1, MLL rearrangement, and BCR-ABL1) by RT-PCR and fluorescence in situ hybridization (FISH) , which may be especially relevant to Ph-like ALL and actionable with TKIs. Comprehensive sequencing data are reported by days 28–42 of remission induction. After diagnostic testing is complete, patients are reapproached for germline testing. If consent is obtained for constitutional testing, data from the previously sequenced germline sample are reanalyzed to detect alterations in cancer predisposition genes.At study initiation, patients are also offered integrative whole-genome, whole-exome, and whole-transcriptome sequencing, performed in a College of American Pathologists (CAP)/Clinical Laboratory Improvement Amendments-accredited laboratory Figure . The anaCRLF2 rearrangement and Sanger sequencing for JAK1/JAK2 mutations for cases with high CRLF2 expression, and RT-PCR and/or transcriptome sequencing for cases with low CRLF2 expression)  uses tiered screening algorithms, employing TaqMan low-density array cards for NCI high-risk B-ALL, followed by sequential genomic profiling  . HoweverOf the patients with B-ALL treated on the St. Jude Total XV protocol, 12% had Ph-like ALL, which was associated with high levels of minimal residual disease (MRD) during induction . PossiblABL-class fusions , and the JAK inhibitor ruxolitinib is administered to those with CRLF2 rearrangements and/or other JAK–STAT pathway alterations   .ABL-class chimeric fusions  that are identified by RNA-Seq. Because of our previous experience with dasatinib and its safety profile  that results in the activation of JAK–STAT signaling and who also have MRD of 5% or more on day 15 of induction or 1% or more at the end of induction. Ruxolitinib will be administered with discontinuous dosing (2 weeks on and 2 weeks off), and a dose-finding phase is included in the study. In patients with MRD (≥0.01%) at the end of induction, subsequent MRD will be monitored closely, and immunotherapy  or a hematopoietic stem cell transplant will be considered for those with persistently MRD-positive disease.Clinical experience of administering ruxolitinib in combination with conventional chemotherapy in children is limited, as is data on its safety profile, and a comprehensive identification of the genetic alterations associated with activation of the JAK–STAT pathway will take 28–42 days. Thus, ruxolitinib will be administered after remission-induction therapy  to patients with a rearrangement, mutation, or genomic deletion  have been seen in approximately 25% of patients with T-ALL and are prevalent in the groups with LX1/TLX3 or HOXA mutations (The Total XVII protocol enrolls patients with T-ALL, and targetable genetic alterations and activated signaling pathways also represent important opportunities for precision-medicine approaches to treating T-ALL. th T-ALL , 20, andutations . Thus, rOur next-generation sequencing approach identifies therapeutic targets to facilitate personalized precision medicine while providing the flexibility to interrogate emerging prognostic and predictive markers in real time. This approach could lead to improved cure rates and reduced toxicities, especially in higher risk patients.Conception and design: HI. Manuscript writing and final approval: all authors.HI obtained drug support from Bristol-Myers Squibb and Incyte Corporation. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YP and handling Editor declared their shared affiliation.


Recent improvements in next-generation sequencing (NGS) technology have enabled detection of biomarkers in cell-free DNA in blood and may ultimately replace invasive tissue biopsies. However, a better understanding of the performance of blood-based NGS assays is needed prior to routine clinical use. As part of an IRB-approved molecular profiling registry trial of pancreatic ductal adenocarcinoma (PDA) patients, we facilitated blood-based NGS testing of 34 patients from multiple community-based and high-volume academic oncology practices. 23 of these patients also underwent traditional tumor tissue-based NGS testing. cfDNA was not detected in 9/34 (26%) patients. Overall concordance between blood and tumor tissue NGS assays was low, with only 25% sensitivity of blood-based NGS for tumor tissue NGS. Mutations in KRAS, the major PDA oncogene, were only detected in 10/34 (29%) blood samples, compared to 20/23 (87%) tumor tissue biopsies. The presence of mutations in circulating DNA was associated with reduced overall survival (54% in mutation-positive versus 90% in mutation-negative). Our results suggest that in the setting of previously treated, advanced PDA, liquid biopsies are not yet an adequate substitute for tissue biopsies. Further refinement in defining the optimal patient population and timing of blood sampling may improve the value of a blood-based test. Pancreatic ductal adenocarcinoma (PDA) is an aggressive cancer that is projected to become the second leading cause of cancer-related death by 2025 . This isWith the advent of new commercially available CLIA/CAP accredited lab testing for next-generation sequencing (NGS) panels, detection of actionable mutations from tissue biopsies no longer requires that the patient be seen at a specialized high-volume tertiary care academic medical center. Still, potential obstacles in detecting mutations from patient samples include the tumor not being accessible via biopsy and/or not enough tumor cells being extracted for DNA analysis. A potential solution for these problems is the development of “liquid biopsy” techniques that use the same NGS technologies for molecular profiling. Detection of circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA), circulating tumor cells (ctcDNA), and tumor exosome-containing genomic material has created the possibility of a non-invasive method for diagnosing and monitoring cancer .We previously launched an initiative , which includes multi-omic molecular profiling of PDA patients’ tumors and matches patients with appropriate clinical trials and therapies based on actionable molecular anomalies, treatment history and geographical locations. However, to effectively implement this precision medicine strategy, biopsy samples with relatively high levels of tumor cells are needed, forcing us to exclude PDA patients with locally advanced disease, or those with small volume, non-biopsiable disease. As a promising solution, we evaluated two CLIA/CAP accredited blood-based NGS assays as a potential substitute for gold standard tissue biopsy procedures. Here we describe our experience with a pilot study of 34 PDA patients that represent a “real world” setting of consecutive patients with metastatic, disseminated disease in community and academic settings from locations across the United States.n = 26) or the Cynvenio ClearID test (n = 8). Hereafter, these assays will be referred to as the cfDNA-based NGS assay and the ctcDNA-based NGS assay, respectively. The majority of patients had extensive disease burden that had metastasized to the liver, lung, or peritoneal cavity. However, several patients had either localized disease or no detectable disease due to distal pancreatectomy or Whipple procedure . The blood-based NGS assays did not detect 14 KRAS variants (61%) that were present in the tumor tissue. We noted that in patients for whom KRAS mutations were detected in both tumor tissue and blood, all tumor samples were biopsied from liver metastases  ranged from 1.2 to 10 ng. Of the 4 patients with tumor tissue KRAS mutations but no cfDNA KRAS mutations, one of them had a low cell-free DNA yield (1.7 ng), while the other three had high yields (>8 ng) that were similar to the two patients with KRAS mutations detected in both plasma and tumor tissue. This indicates that both technological (low cfDNA yields) and biological  limitations likely play a role.To determine whether technical limitations played a part in the low rate of detection of Medicine . QualityMedicine . For theTP53 status (3 mutants and 4 wild-type). The blood-based NGS assays did not detect 15 TP53 variants (65.2%) that were present in the tumor tissue. The three concordant TP53 variants were G325*, V272L, and R273C . Three patients had differing TP53 mutations in cfDNA-based and tumor tissue NGS: pan-545 , pan-594 , and pan-598 .In the 23 patients with both blood-based and tumor tissue NGS, six (26.1%) were concordant for CDKN2A or SMAD4 were detected in the blood-based assays  and SMAD4  variants detected by tissue-based NGS were deletions, indels, or splice site mutations and thus would not be expected to be detected by the blood-based NGS assays.No mutations in s Figure . MutatioTo systematically assess the performance of blood-based NGS assays across all genes, we determined the sensitivity and specificity of the cfDNA-based assay using the tumor tissue NGS data as the reference. For this analysis, we only considered the patients for whom both cfDNA and tumor tissue NGS data were available. Also, to be consistent with methodologies described previously , 11, we KRAS and MYC  between the level of CA19-9 marker and the maximum variant allele fraction . While a larger sample size is needed to draw definitive conclusions, these results indicate that clinical covariates may lack strong associations with the presence or absence of mutations in cfDNA and therefore may not be sufficient indicators of whether a blood-based NGS assay will reliably detect tumor mutations.Mutations in circulating DNA were only detected in blood-based NGS analysis of 25 (74%) of the patients: 19 out of 26 (73%) by the cfDNA-based assay and 6 out of 8 (75%) by the ctcDNA-based assay. Since one of our goals is to detect actionable mutations that can influence treatment decisions, it is critical to know when a blood-based assay is most likely to detect specific tumor DNA mutations. Therefore we explored the clinical factors related to the presence or absence of detectable mutations in the blood. We used the maximum variant allele fraction in the blood-based assays as a surrogate for level of tumor DNA in the blood . The patn = 24), with 11 deaths occurring in this subgroup compared to one in the subgroup with no detectable tumor DNA (n = 10) . The prognostic significance of the presence of mutations in the circulation highlights a possible role for blood-based NGS in clinical care of pancreatic cancer patients, in keeping with the recent role for CTC analysis in monitoring disease burden . A. AKRAS msurvival . The levsurvival .The low concordance between tumor tissue and cfDNA-based NGS that we observed here indicates that further technical development is needed in this specific setting of late-stage pancreatic cancer. Nonetheless, despite the limitations we observed, we have demonstrated that it is feasible to utilize blood-based NGS profiling across many different hospitals and community-based practices; this is in contrast to other similar studies that were conducted at large academic institutions , 26. ThePancreatic cancer patients were recruited under an IRB-approved registry into a molecular profiling program after obtaining informed consent. Patients from 18 different hospitals/high volume cancer centers and 7 different community oncology practices were enrolled and data collected in the registry. Patients were enrolled sequentially, during the time frame of this Pilot program.2EDTA tubes and then stabilized in a proprietary fixative, which allows for a window of four days between sample collection and analysis. The Guardant360 assay generates read depths above 8, 000x and has a detection limit for frequencies of 0.25% [Whole blood samples were sent to one of two commercial laboratories for targeted NGS analysis. The Guardant360 test  involves targeted sequencing of a 68-gene panel  in cfDNAof 0.25% . At an oof 0.25% . In all Formalin-fixed, paraffin-embedded tumor tissue samples were sent to a commercial laboratory for NGS testing , which targets a 321-gene panel  for sequsurvival package [All statistical analysis was performed in R. Survival differences between patient groups were determined using the log-rank test on Kaplan-Meier curves with the R  package . When peThe concordance between NGS assays was evaluated by calculating the gene-level sensitivity and precision of blood-based NGS assays in detecting mutations present in the tumor tissue, as identified by NGS. A gene variant was considered concordant if the exact nucleotide change was present in the same gene and patient in both blood-based and tumor tissue biopsy.