Acute care readmission risk is an increasingly recognized problem that has garnered significant attention, yet the reasons for acute care readmission in the inpatient rehabilitation population are complex and likely multifactorial. Information on both medical comorbidities and functional status is routinely collected for stroke patients participating in inpatient rehabilitation. We sought to determine whether functional status is a more robust predictor of acute care readmissions in the inpatient rehabilitation stroke population compared with medical comorbidities using a large, administrative data set.A retrospective analysis of data from the Uniform Data System for Medical Rehabilitation from the years 2002 to 2011 was performed examining stroke patients admitted to inpatient rehabilitation facilities. A Basic Model for predicting acute care readmission risk based on age and functional status was compared with models incorporating functional status and medical comorbidities (Basic-Plus) or models including age and medical comorbidities alone (Age-Comorbidity). C-statistics were compared to evaluate model performance.There were a total of 803,124 patients: 88,187 (11%) patients were transferred back to an acute hospital: 22,247 (2.8%) within 3 days, 43,481 (5.4%) within 7 days, and 85,431 (10.6%) within 30 days. The C-statistics for the Basic Model were 0.701, 0.672, and 0.682 at days 3, 7, and 30 respectively. As compared to the Basic Model, the best-performing Basic-Plus model was the Basic+Elixhauser model with C-statistics differences of +0.011, +0.011, and + 0.012, and the best-performing Age-Comorbidity model was the Age+Elixhauser model with C-statistic differences of -0.124, -0.098, and -0.098 at days 3, 7, and 30 respectively.Readmission models for the inpatient rehabilitation stroke population based on functional status and age showed better predictive ability than models based on medical comorbidities. Nearly one fifth of Medicare patients discharged from a hospital are rehospitalized within 30 days due to an acute medical condition . RecentlRelatively stable rates of reported all-cause readmissions, despite the identification of a growing number of possible risk factors, suggest that the reasons for acute care readmissions are complex and multifactorial . While vSeveral studies in relatively small populations found a significant relationship between functional status and acute care readmission from the post-acute inpatient rehabilitation setting, although these did not examine 30-day readmission risk specifically –9. Risk This study examines the role of functional status compared to that of medical comorbidities in risk prediction models for acute care readmissions in the inpatient rehabilitation stroke population at different time points following acute care discharge.This study uses a retrospective cross-sectional study design. We hypothesized that functional status would predict acute care readmissions and that its inclusion in risk prediction models would yield better predictive ability than models based on the presence of comorbidities alone. We analyzed data from the Uniform Data System for Medical Rehabilitation (UDSMR), a repository for inpatient rehabilitation facility (IRF) functional outcome data. CMS requires IRFs to complete the Inpatient Rehabilitation Facility Patient Assessment Instrument (IRF-PAI), which contains demographic, social, medical, and functional data. UDSMR serves approximately 70% of all IRFs in the United States data in . Data weInformation collected in the IRF-PAI includes up to 10 comorbidities, coded according to the International Classification of Diseases 9th edition Clinical Modification (ICD-9-CM). Transfer to an acute care hospital is designated as a disposition category within the IRF-PAI. The IRF-PAI also includes the FIM™ instrument, a standardized evaluation tool that assesses function and serves as a proxy for the intensity of resources required for care –15. The Medical comorbidities, designated by ICD-9-CM code in the IRF-PAI, were further analyzed according to three different comorbidity scoring systems: the Elixhauser comorbidity method  ,19, DeyoFirst, we created a pre-specified ‘Basic Model’ that included 3 variables related to age and functional status: age, FIM™ motor score, and FIM™ cognitive score. Next, we compared the performance of the Basic Model to models that added comorbidity data to the Basic Model according to each of the three comorbidity scoring systems (Basic-Plus models), and models that included only age and comorbidities from each scoring system (Age-Comorbidity models). Consequently, three Basic-Plus models and three Age-Comorbidity models were developed . For eacThe area under the receiver operator curve (C-statistic) was used to test model performance. The C-statistic is defined as the proportion of time a model correctly discriminates between a pair of high- and low-risk individuals and has been used to describe discriminative ability of models in prior readmission studies and in a systematic review of readmission risk prediction models . A C-staThe UDSMR database of adult IRF discharges comprised 4,467,307 total cases between 2002 and 2011. We excluded 19,177 patients who left the IRF setting against medical advice, leaving 4,448,130 eligible cases. Of these, 901,652 had an Impairment Group code of stroke. We excluded 26 patients with ages recorded as greater than 108 as presumed documentation errors, 30,135 patients with a delay of >90 days between stroke onset and IRF admission, 38,799 patients who were not admitted to inpatient rehabilitation directly from an acute hospital, and 1,865 patients who died while admitted to inpatient rehabilitation. The final sample size was 803,124 patients from 1,157 IRFs . Of thesLogistic regression coefficients for the Basic Model at each time point are shown in While functional status has been suggested as an important predictor of acute care readmissions, it remains comparatively understudied in studies of larger post-acute populations in the United States and larger, population-based or multi-center studies of readmission risk have generally demonstrated modest discriminative ability (C-statistic range 0.55–0.65) . SeveralThis study is the first to develop readmissions models using data obtained from a large, national data set and showed that age and functional status on admission predict the risk of acute care readmissions well and with good model calibration at 3, 7, and 30 days from IRF admission. Models incorporating age and functional status (Basic Models) on admission alone consistently outperformed models based on age and comorbidities (Age-Comorbidity Models). The addition of comorbidities to these basic models (Basic Plus Models) increased complexity but did not significantly enhance predictive ability at 3, 7, or 30 days. Risk prediction for the Basic Model at 3 days in particular showed substantially better predictive ability compared with large, population-based models incorporating comorbidities in prior literature , while rThere are several potential reasons for the improved predictive ability of models incorporating functional status as compared with models that rely on medical comorbidities for readmissions in the inpatient rehabilitation stroke population. While ICD-9-CM codes indicating the presence of medical comorbidities may be easily obtained from large, administrative data sets, information regarding disease severity and clinical instability is typically sparse and has been proposed as a key to improving models’ predictive ability . By contInterventions aimed at promoting successful recovery after hospitalization and reducing acute care readmissions often focus on improving care for specific medical conditions  and transitions of care at nodal time points (e.g. direct communication between inpatient and outpatient providers prior to acute care discharge) ,3. The rThe results of this study must be interpreted within the context of their limitations. This was an observational study and we are unable to demonstrate a direct, causative relationship between functional status and readmissions using our data set. Comorbidity data was obtained from ICD-9-CM codes, of which a maximum of 10 per patient are recorded, rather than all potential comorbidities. Furthermore, the documented presence of medical comorbidities in isolation is not a reliable indication of illness severity or clinical stability. We addressed this limitation by using three different and validated comorbidity scoring systems in the development of our risk prediction models to account for medical comorbidities using a standardized measure. Demographic and social information is collected within the IRF-PAI but was not included in our models because of the hypothesis-driven design of our study. However, prior studies have suggested that the relationship between functional status and readmissions in the inpatient rehabilitation stroke population are minimally confounded by demographic factors ,7,9. WhiFunctional status effectively predicts acute care readmissions after stroke in the inpatient rehabilitation setting. Models using functional status as indicated by FIM™ instrument scoring and age showed improved predictive ability compared with medical comorbidities and age alone in the inpatient rehabilitation stroke population using data from a large, administrative data set. Our data adds to increasing evidence that functional status is an important measure of health and predictor of risk for adverse health events following acute hospitalization in the stroke population ,31–36. MS1 Fig(PDF)Click here for additional data file.S2 Fig(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Secondary endpoints included repeated clinical assessments with DAS28(CRP), and assessments of osteitis and synovitis by the RAMRIS method. Infliximab showed greater decrease from baseline in DCE-MRI Ktrans of wrist and MCP at all visits compared with placebo (P<0.001). Statistical analysis was performed to identify genes associated with treatment-specific 14-week change in Ktrans. The 256 genes identified were used to derive a gene signature score by averaging their log expression within each patient. The resulting score correlated with improvement of Ktrans in infliximab-treated patients and with deterioration of Ktrans in placebo-treated subjects. Poor responders showed high expression of activated B-cell genes whereas good responders exhibited a gene expression pattern consistent with mobilization of neutrophils and monocytes and high levels of reticulated platelets. This gene signature was significantly associated with clinical response in two previously published whole blood gene expression studies using anti-TNF therapies. These data provide support for the hypothesis that anti-TNF inadequate responders comprise a distinct molecular subtype of RA characterized by differences in pre-treatment blood mRNA expression. They also highlight the importance of placebo controls and robust, objective endpoints in biomarker discovery.Approximately 30% of rheumatoid arthritis patients achieve inadequate response to anti-TNF biologics. Attempts to identify molecular biomarkers predicting response have met with mixed success. This may be attributable, in part, to the variable and subjective disease assessment endpoints with large placebo effects typically used to classify patient response. Sixty-one patients with active RA despite methotrexate treatment, and with MRI-documented synovitis, were randomized to receive infliximab or placebo. Blood was collected at baseline and genome-wide transcription in whole blood was measured using microarrays. The primary endpoint in this study was determined by measuring the transfer rate constant (KTrial Registration: ClinicalTrials.gov NCT01313520 Anti-TNF biologics are an important class of therapeutics in the treatment of rheumatoid arthritis, but unfortunately approximately 30% of patients achieve inadequate response. Variability in response is incompletely understood. It has been linked to smoking status, concomitant treatment with methotrexate (MTX) and other DMARDs, disease severity, and patient disability trans, the volume transfer constant of GBCA between blood plasma and the synovium. This endpoint is related to capillary permeability and vascularity in the synovium, and correlates strongly with histological measures of inflammation Given the limitations of the DAS28 and similar measures, biomarker discovery using objective disease assessments, like magnetic resonance imaging (MRI), is attractive. Uniquely, MRI is able to evaluate the inflammation of synovium and bone, which are thought to ultimately result in articular cartilage loss and bone erosion, respectively. It is most frequently monitored using the RAMRIS method trans.In the present study, we measured pre-treatment gene expression in whole blood and employed DCE-MRI of the wrist to monitor disease progression in a randomized, controlled, multi-site trial of infliximab plus MTX versus placebo plus MTX. Analysis of these data identified a 256 gene signature associated with disease activity measured by KThis analysis was conducted as part of a 14-week, randomized, double-blind, placebo-controlled, methodology study  conducted from April 6, 2011 to March 29, 2012 in 4 clinical centers in Europe. The study was conducted in accordance with principles of Good Clinical Practice and was approved by the National Ethics Committee in Romania and the National Ethics Committee, Clinical Research of Drugs and Methods of Treatment in Moldova. All subjects provided informed written consent. Details of the study methods are provided as a supplement and have been submitted elsewhere for publication.Participants had a diagnosis of RA for at least 6 months (based on the American College of Rheumatology (ACR) 1987 criteria), at least 6 tender and 6 swollen joints (using the 28 joint set), C-reactive protein (CRP) ≥1.0 mg/L, and were on a stable dose of methotrexate. Subjects who were on stable doses (<10mg/day) of prednisone or non-steroidal anti-inflammatory drugs (NSAIDs) maintained those treatments through the study. All subjects were naïve to anti-TNF biologics, and were required to have RAMRIS synovitis score ≥1 in the radio-carpal or intercarpal joints of one hand based on centralized expert assessment.At weeks 0, 2, 6, and 14, participants received infliximab 3 mg/kg in 0.9% NaCl or 0.9% NaCl alone. Patients continued to receive their standard dose and regimen of disease modifying antirheumatic drugs (DMARDS).DAS28(CRP) is a composite score of the number of tender joints (28 joint count), the number of swollen joints (28 joint count), patient global assessment of disease (GADP) on a 100 mm visual analog scale (VAS), and CRP (mg/dL) trans. Additionally, synovitis, osteitis and bone erosion were scored using the Outcome Measures in Rheumatology (OMERACT) RA MRI Score (RAMRIS) method MRI of the most clinically involved hand and wrist was acquired at baseline and weeks 2, 4, and 14 in order to measure Ktrans of wrist synovium was the primary endpoint of the study. Secondary endpoints included Ktrans of metacarpophalangeal joints (MCP), DAS28(CRP), and RAMRIS scores. The DCE-MRI endpoints and the continuous clinical endpoints were compared between treatment groups using constrained Longitudinal Data Analysis (cLDA) proposed by Liang and Zeger KRNA was isolated from PAXgene blood samples (2.5ml) according to the manufacturer's instructions. Isolated total RNA samples were assayed for quality  and yield (Ribogreen) metrics prior to amplification. Fifty-nine samples passing quality control (QC) were then amplified using the NuGEN Ovation WB protocol and hybridized to Rosetta/Merck Human RSTA Custom Affymetrix 2.0 microarrays. Microarray data have been deposited in the Gene Expression Omnibus archive (GEO accession GSE58795).QC analysis was performed in the absence of treatment allocation information. QC metrics, including average background signal, scale factor, beta-actin and GAPDH 3' to 5' ratios, and the number of genes called 'present' using the MAS5 algorithm, were examined to assess potential technical quality issues related to amplification and RNA degradation. No problematic samples were identified. Array probe intensity normalization was performed using the RMA algorithm in R/Bioconductor. Gene expression patterns and their principal components of variation were checked against the principal components of sample quality and process parameters (QC). The first two QC principal components were observed to correlate significantly with several principal components of variation in the expression data. Therefore, prior to further analysis, the RMA-normalized expression data was detrended by regressing the log2 intensity of each probe set against the first two QC principal components.Detectably expressed probe sets were defined as those with a MAS5 ‘present’ call in at least 25% of all samples. Log expression values of probe sets mapping to the same gene were averaged. Signature scores were used to summarize the expression of the genes comprising pre-specified signatures and were calculated by averaging the log expression intensity values for each gene within a subject. Since signatures may comprise genes both correlated and anti-correlated with treatment response, the mean log expression values for anti-correlated genes was multiplied by -1 to preserve the effect of directionality.trans to identify deviation from random expectation. For Ktrans, we defined a 256 gene signature associated with 14-week treatment response by identifying genes with a nominally significant gene expression by treatment interaction term (p<0.01). The Pearson correlation matrix for all 256 genes in the predictive signature was calculated and used to cluster the genes into four groups. Details on clustering are provided in the supplemental methods.To identify genes whose baseline expression correlated with treatment effect, we modeled the 14-week change in each disease activity endpoint as a function of treatment and gene expression at baseline. For each detectably expressed gene, we performed ordinary least squares regression with terms for log expression intensity, treatment allocation, their interaction, and baseline disease activity. Q-Q plots of interaction term p-values vs. the uniform distribution were generated for DAS28, RAMRIS osteitis, RAMRIS synovitis, and KThe value of gene expression for predicting 14-week change in disease activity was assessed using a random subsampling strategy. In each round of sampling a training set of 40 subjects was randomly selected, and genes with a significant (p<0.01) expression by treatment interaction term were identified in this training set. Expression of these genes was averaged into a score for each subject. Using only the subjects in the training set, we then fit two linear models of disease activity: the first included terms for baseline disease activity, treatment, signature score, and signature score by treatment interaction, the second included only terms for treatment and baseline disease activity. Each of these models was then used to predict 14-week change in disease activity for the held-out test subjects, and the resulting mean squared prediction errors over ten rounds of sampling were compared.To evaluate the statistical association between signature score and other disease activity endpoints, we fit a linear model of change from baseline data including terms for baseline disease activity, treatment allocation, signature score and the signature by treatment interaction, and examined the effect size and p-value associated with the gene signature main effect and interaction term.P = 0.058).Sixty-one adults with moderate to severe RA were enrolled into the study. The cohort was 92% female with a mean age (SD) of 50 (10) years. Ninety-one percent were positive for Rheumatoid Factor. Baseline characteristics are described in One participant meeting clinical criteria did not have sufficient synovitis at baseline to qualify, but was randomized to treatment in error. Since the patient was eligible on clinical grounds, the patient was not discontinued, and was included in all analyses. All subjects completed the study.trans of synovium in the wrist derived from DCE-MRI analysis showed a significant treatment effect as early as 2 weeks following initiation of infliximab. This treatment effect was observed at each subsequent time point as well  in the wrist and MCPs (data not shown).Mean K as well . PlaceboP<0.001, Infliximab significantly reduced the RAMRIS scores for both synovitis and osteitis in the wrist and MCPs as early as 2 weeks, and maintained reduction through 14 weeks P<0.001, .Infliximab reduced DAS28(CRP) disease activity at weeks 2, 4 and 14 compared with placebo . As expetrans by first applying linear regression to identify genes associated with response. The Q-Q plot of expected and observed p-values showed deviation from random expectation  with baseline-adjusted 14-week change in the infliximab-treatment arm and negative correlation (R2 = 0.62) in the placebo arm . Analogous coefficient estimates from a model fit to the 2-week response data were not significant, although the signature score main effect approached significance (p = 0.059). Although the signature was derived using Ktrans of wrist, the signature was also significantly associated with change in Ktrans in the MCP joints at both 14 weeks  and 4 weeks . Next, by repeated random sampling of training and test sets, we estimated whether inclusion of baseline gene expression in a regression model improved prediction of 14-week change in Ktrans. For Ktrans, gene expression improved mean-squared prediction error of held out data  (We assessed the ability of baseline whole blood gene expression to predict 14-week change in Kof wrist . The sigcebo arm . Given t t-test) .trans signature score and change in DAS28(CRP), RAMRIS synovitis, or RAMRIS osteitis at any time point. However, at 14 weeks and only in the infliximab treatment arm, we identified a weak positive correlation between signature score and change in DAS28(CRP)  despite the poor correlation  between 14-week change in DAS28 and change in log Ktrans. In the infliximab treatment arm we also examined the distribution of signature scores across EULAR response categories (an assessment of response based on pre-treatment DAS28 and post-treatment change in DAS28) Similar analyses attempting to derive a gene signature using disease activity endpoints RAMRIS osteitis, RAMRIS synovitis, and DAS28(CRP) revealed no deviation from random expectation in Q-Q plots and no improvement in prediction of 14-week change in disease activity. There was no significant association between the KWe examined the predictive ability of our signature to categorize clinical response using previously reported data from studies where baseline whole blood gene expression was measured in the context of anti-TNF biologic treatment, and where gene expression data passed quality control trans improvement showed greater median down-regulation  and 27/72 were significantly down-regulated by infliximab treatment (p = 3.2e-7 Fisher's exact test). Conversely, genes negatively correlated with 14-week Ktrans improvement had higher median fold changes compared to all detected genes , and were underrepresented in significantly down-regulated genes .To examine whether infliximab treatment affects the expression level of genes in the predictive signature, we examined the effect of 1 month of infliximab treatment on expression of the 256 genes using the whole blood gene expression data set of Van Baarsen et al. We assessed the ability of previously reported blood gene expression signatures to predict anti-TNF biologic response by evaluating their correlation with change in disease activity as measured by DCE-MRI, RAMRIS or DAS28(CRP). None of the previously reported gene signatures in A number of genes in the signature have been genetically associated with RA. Ten genes  are located within RA genetic risk loci, and of these AFF3, ANKRD55, FCGR3B, FCRL1, FCRLA, and IKZF3 were reported to have additional supporting biological evidence linking them to the disease in a recent genetic and bioinformatic analysis Expression of genes in two clusters correlated positively with infliximab response. Cluster 1 is highly concordant with the Chaussabel platelet module and is enriched for platelet markers and genes involved in platelet aggregation like calmodulin-3, CD36, platelet factor 4, prostacyclin receptor, and thromboxane A2 receptor. Cluster 2 is concordant with the Chaussabel myeloid/monocyte blood module and contains genes expressed specifically in myeloid lineage cells including the low affinity Fcγ receptor IIIb, CD14, CD300LB, and NQO2.In contrast, clusters 3 and 4 are comprised of genes whose expression levels correlate with infliximab non-response. Cluster 3 is enriched for markers of activated B-cells and plasma cells including B-cell linker, IGLJ3, CD79b, CD72, early B-cell factor 1, and IKZF3 and consequently shows high correlation with the B-cell blood module. Cluster 4 is enriched for genes down-regulated in human blood after administration of recombinant G-CSF, a cytokine that mobilizes myeloid cells from the bone marrow. This cluster shows the highest correlation with a metabolism-related Chaussabel blood module.2 = 0.137, P = 0.004) suggesting that its reported correlation with response may be explained, in part, by enriching for patients with flaring disease. More objective measures of RA disease activity and progression could aid in the identification of predictive biomarkers Clinical characteristics explain only a small portion of the total variability in patient response to anti-TNF treatment in RA trans of synovium in the wrist. In contrast to the standard clinical endpoint DAS28, DCE-MRI showed no improvement with placebo. Using DCE-MRI we derived a pre-treatment whole blood gene expression signature correlating with change in the imaging biomarker Ktrans. The signature was associated with Ktrans improvement in infliximab-treated patients but with worsening Ktrans in the placebo arm. A similar association was seen with Ktrans summed from the metacarpophalangeal joints of the hand in each treatment arm.In this study, we propose a path forward by profiling gene expression in both placebo and treatment arms and by employing a sensitive, quantitative, and objective DCE-MRI endpoint: Ktrans both represent gadolinium contrast transfer to synovium, and both have been validated as inflammation biomarkers through clinical association with joint swelling and with inflammatory pathology on biopsy trans and the rate of early enhancement) as opposed to static images obtained at the peak of contrast enhancement used for RAMRIS methodology We observed no correlation between the gene expression signature and 14-week change in RAMRIS synovitis or osteitis. This observation is potentially unexpected, because RAMRIS synovitis scores and Ktrans improvement after infliximab treatment were highly enriched for genes reported to be down-regulated by treatment. This observation provides confidence that the genes in our predictive signature are related to the biology of anti-TNF treatment in rheumatoid arthritis.Interestingly, evaluation of the effect of 1 month of infliximab treatment on the genes in the signature revealed evidence of infliximab-induced gene expression changes for many genes. In particular, genes whose expression correlated with Ktrans response in other patients will need to await the availability of other datasets. However, in both studies we examined EULAR responders had significantly higher signature scores than non-responders, consistent with expectation. Although the higher signature score observed in EULAR responders is interesting, the poor discrimination achieved using the signature to distinguish responders and non-responders indicates it would be of limited use in clinical practice. Nevertheless, to the extent that the signature genuinely defines molecular subtypes in RA it could serve as an interesting endpoint in future biomarker or genetic studies of the disease. One potentially interesting avenue would be to examine the association of genetic variants with gene signature score to identify genetic drivers of the putative patient subtypes reported here. In addition, by combining all genes into a single score representing an average log expression level, we have ignored the possibility that individual genes identified in this study may have greater predictive value than others. It would be interesting to explore predictive modeling approaches that take this into account.Unfortunately, prior gene expression studies have not employed DCI-MRI as an endpoint. DCE-MRI is infrequently used in studies of RA patients, and so independent validation of our signature and Kin vitro TNF inhibits hematopoiesis, chronic inflammation in RA can be associated with monocytosis and granulocytosis Unsupervised clustering of the genes making up the predictive signature described here suggests that they broadly reflect three sources: 1) myeloid lineage cells 2) B-cells, and 3) reticulated platelets. High expression of myeloid cell markers and low expression of genes down-regulated by recombinant G-CSF in peripheral blood is associated with response to infliximab in RA. Although Taken together, these results are consistent with the hypothesis that infliximab responders have higher levels of inflammatory processes driven by TNF than non-responders. In this model, anti-TNF responders may show increased induction of pro-myelopoietic factors which mobilize granulocytes and monocytes from the bone marrow, and collaborate with TNF to inhibit B-cell lymphopoiesis and increase megakaryocytopoiesis, platelet turnover, and hence the fraction of reticulated platelets in whole blood. These effects would be reflected in the myeloid, B-cell, and platelet components of the baseline whole blood gene expression signature we report here.This study provides evidence that a whole blood gene expression signature correlates with response to infliximab as assessed by DCE-MRI. High signature score associated with improvement in infliximab-treated patients but not in placebo-treated patients, however the ability of the signature to stratify EULAR responders and non-responders was poor. Additional studies should attempt to validate this finding and explore the molecular underpinnings of the gene signature and its relationship to anti-TNF response, in particular the hypothesis that anti-TNF inadequate responders comprise a distinct molecular subtype in RA.S1 FileSupplemental figures and methods. Figure S1, Q-Q plots for primary and secondary response endpoints. Linear regression was applied to identify genes associated with treatment-specific response to infliximab. Observed p-values for the gene expression by treatment interaction coefficients are plotted vs. random expectation based on a uniform null distribution for each response endpoint. Figure S2, Histograms of gene expression log fold changes observed after one month of infliximab treatment in the study of Van Baarsen et al. From top to bottom: all genes with detectable expression in whole blood, predictive signature genes whose expression is correlated with 14-week log Ktrans improvement in infliximab-treated patients, and signature genes whose expression is anti-correlated with 14-week change in log Ktrans. Figure S3, Heat map of clustered signature genes. Predictive signature genes were clustered into four groups based on their correlation in expression. Heat map color indicates the correlation in expression between genes across all patients' baseline whole blood samples.(DOCX)Click here for additional data file.S1 TableGenes associated with 14-week change in RA disease activity measured by DCE-MRI.(DOCX)Click here for additional data file.


Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells while maintaining their free-floating character. We use polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membranes. This coating remains optically clear, allowing capture of high-resolution images and videos of McTNs on viable free-floating cells. In addition, we show that tethering allows for the real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells can vastly enhance our understanding of CTCs under conditions that better recapitulate the microenvironments they encounter during metastasis.Free-floating tumor cells located in the blood of cancer patients, known as circulating tumor cells (CTCs), have become key targets for studying metastasis. However, effective strategies to study the free-floating behavior of tumor cells  Cancer metastasis occurs when epithelial tumor cells travel through non-adherent microenvironments, like the bloodstream or lymphatics, to a distant organ. The presence of tumor cells in the non-adherent microenvironment of the bloodstream, known as circulating tumor cells (CTCs), has been detected in numerous epithelial cancers including breast, prostate, colon, and lung . CTCs arMicroscopy analysis of CTCs has focused almost exclusively on cells adhered to surfaces ) owing to the ease of imaging and characterization of cells in these static positions. However, the functional and molecular characteristics of adherent and non-adherent tumor cells are dramatically different –11. ThusBiomaterials offer many attractive features – stability, biocompatibility, versatile chemistries – for controlling cell adhesion. Common approaches include chemically functionalizing surfaces, incorporating cell adhesion peptides, and micropatterning using polymer-based soft lithography or electrospinning techniques. Of particular note, several recent studies have exploited biomaterials to identify CTCs –21 or usTo enable this new ability, we identified three design features that would allow prolonged, real-time imaging and drug screening of McTNs on live tumor cells in a free-floating state: 1) optically-clear coatings to support imaging, 2) ability to control microfluidic flow over cells and 3) simple, low-energy manufacturing process. Past studies have demonstrated the utility of PEMs for tuning cell adhesion by varying polymer composition or through addition of lipids, RGD sequences, or other binding moieties –34. ThusThe responses of cancer cells detached from ECM  are highly important in survival, apoptosis, metastasis, and even in the expression of stem cell characteristics , 37. HowWe formed PEMs from two common polymers, poly(methacrylic acid) (PMA) and polyacrylamide (PAAm) that have previously been shown to limit cell adhesion of numerous cell types , 38. Subsn-glycero-3-phosphocholine (DOPC) as cationic and zwitterionic lipids, respectively, owing to the ability of these molecules to interact with the PEMs electrostatically , or with either terminal lipid layer . During successive wash steps, DOTAP retained tumor cells more efficiently compared to non-tethered cells seeded on microfluidic slides coated with PEM only  and 1,2-dioleoyl-y Figure . Followiy Figure . We nexty Figure . MCF-7 cy Figure . RepreseWe next determined if lipid tethering with DOTAP maintained free-floating tumor cell characteristics. As a first indicator, McTN frequency was assessed on PEM and PEM-DOTAP surfaces. Blinded McTN counts revealed no differences in McTN frequency on PEM-no tether and PEM-DOTAP surfaces compared to previously published counts on low-attach multi-well plates Figure . These rOne of the greatest challenges in studying free-floating cells is the difficulty in measuring their functional properties or behavior in real time. This is especially apparent when trying to image free-floating cells over time and in three dimensions. Epifluorescence is unable to capture McTNs in high resolution Figure . ConfocaThe major advantage in imaging McTNs over time is being able to study their responses to drugs not only by McTN frequency, but also McTN dynamics. To demonstrate this potential, we recorded three dimensional z-stacks of untreated MDA-MB-436 cells, cells treated with the microtubule destabilizing agent, colchicine, or the microtubule stabilizing agent, paclitaxel. Addition of colchicine decreased McTNs while paclitaxel enhanced McTNs Figure . Over tiThe majority of cancer-related deaths are due to the spread of tumor cells through the circulation from the primary site to a secondary organ . While iIn this proof-of-concept study we show that tethering is an effective way to retain the free-floating behavior of cancer cells and provides new opportunities to study their functional properties with high-resolution microscopy through spatial localization. This strategy offers the ability to coat a variety of surfaces, including microfluidics. We show that incorporating a lipid moiety on PEMs can passively immobilize tumor cells in a manner that preserves McTN formation and does not affect cell viability. In this method, no cellular adhesive properties are necessary because the interaction of the cell membrane with the lipid results in cell tethering. Lipids have previously been used to immobilize cells , 34; herIn our studies, each PEM lipid formulation tethered cancer cells, but the lipid moiety itself altered cell retention, with DOTAP – a cationic lipid – driving the best tethering. DOPC – a zwitterionic lipid – contains a phosphate group which may cause less favorable interactions with cell membranes compared to DOTAP. While cells were immobilized on the substrate, they remained free-floating owing to the cytophobic nature of the initial PEM coating. There was some loss of tethered cells as a function of the number of washes, but it is likely these interactions can be strengthened by incorporation of cross-linkable lipids , 42, 43.We demonstrate the utility of tethering for tumor cell analysis by assessing the response of McTNs to microtubule-targeting drugs. Unlike our previous studies, it is now possible to add drugs via microfluidic exchange without displacing cells, allowing measurements of colchicine-dependent McTN disruption  and paclEx vivo culture of CTCs in non-adherent conditions has provided one method to analyze CTCs from patients [High numbers of CTCs correlate with increased metastasis and decreased survival of patients with metastatic cancer , 45, 46.Studying the biology of CTCs has suggested important consequences for both metastatic efficiency and the sensitivity of these structures to candidate cancer drugs. Of note, patterns of drug sensitivities have been linked to the genetic mutations present in individual CTC samples from breast cancer and lung cancer patients, indicating that a change in tumor genotypes during the course of treatment can lead to drug resistance , 41, 47.sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids. Colchicine was purchased from Sigma and paclitaxel was purchased from Enzo Life Sciences.MDA-MB-436 and MCF-7 cell lines were purchased from ATCC and cultured with Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Poly(methacrylic acid)  and polyacrylamide (PAAm)  were purchased from Polysciences. Poly (PAH)  was purchased from Alfa Aesar. 1,2-dioleoyl-3-trimethylammonium-propane  (DOTAP) and 1,2-dioleoyl-For multilayer film deposition, similar to methods previously reported , PMA andUncoated microfluidic slides (1μ-Slide VI 0.4) were obtained from Ibidi and tissue culture treated 96-well plates were obtained from Corning. To coat the microfluidic slides, 120μL of each polyelectrolyte solution was added to the microchannels and 75μl of solution was added to each well of the multi-well plate. After incubation, solution was removed via aspiration and rinsed twice for 1 min using 120μL of pH 3 water. Bilayers of PMA and PAAm were assembled and terminated with either DOPC or DOTAP as described above. Following deposition, slides were allowed to air dry for 1 hr at room temperature then stored at room temperature.MDA-MB-436 and MCF-7 breast cancer cells were seeded on PEM coated microfluidic slides  ranging from 0 to 8 bilayers. An Olympus CKX4 microscope was used for all experiments to capture images at 4x magnification. Three pictures per channel were taken after cell seeding for each condition to quantify initial cell number (t0). At 6 and 24 hrs, media was removed from the channel and the channel was washed once before addition of new media. Three images per channel were taken for each condition. The area of the image occupied with cells (as a percent) was quantified using CellProfiler (Broad Institute) and the average from three images was calculated. The average percentage for each condition was then normalized to the area occupied at t0.MDA-MB-436 and MCF-7 breast cancer cells were seeded on PEM coated 96-well plates  ranging from 0 to 8 bilayers. At each time point , media was removed from the well and the well was washed once before addition of new media. After the 24hr time point an additional wash was done on all wells. Cell number was determined using CellTiter reagent according to manufacturer's instructions. Each time point was normalized to initial cell number from a reading done immediately after cell seeding.4/PAAm4 bilayers . At 0, 6, and 24 hrs Live/Dead (Life Technologies) reagent was added according to manufacturer's instructions. Corresponding phase contrast, live  green fluorescence, and dead (ethidium homodimer-1) red fluorescence images were taken in triplicate at 4x magnification with an Olympus CKX41 fluorescence microscope. The number of cells in each image was quantified using CellProfiler. The percent of live and dead cells were calculated by quantifying the number of green fluorescence positive and red fluorescence positive cells, respectively, and dividing by total number of cells in the phase contrast image. GFP and Texas Red filters were used to for imaging. MDA-MB-436 and MCF-7 breast cancer cells were plated on 96-well black plates with PMA4/PAAm4 bilayers . At time 0, 1, 3, 6, and 24 hours Live/Dead reagent was added and read on a plate reader according to manufacturer's instructions. Relative fluorescence units (RFU) were normalized to time 0.MDA-MB-436 and MCF-7 breast cancer cells were seeded on PEM coated microfluidic slides with PMA4/PAAm4 coated microfluidic slides with DOPC or DOTAP . Cells were incubated for 1 hr to allow for tethering. To quantify initial cell number, three images per channel were taken for each condition at time 0. After 1 hr, existing media was gently removed from the bottom port of each channel and fresh media was added to the top port. Following a wash, three images were taken per channel for each condition using an Olympus CKX41 microscope at 4x magnification. This process was repeated for each wash. The area of the image occupied with cells (as a percent) was quantified using CellProfiler and the average from three images was calculated. The average percentage for each condition was then normalized to the area occupied at time 0.MDA-MB-436 and MCF-7 cells were seeded on PMA4/PAAm4 coated microfluidic slides with DOPC or DOTAP . Cells were incubated for 1 hr to allow for tethering. After 1 hr, one wash was done where the existing media was gently removed from the bottom port of each channel and fresh media was added to the top port. This wash was to ensure only tethered cells were analyzed. At 0 and 6 hrs after washing, Live/Dead reagent was added according to manufacturer's instructions. Corresponding phase contrast, live  green fluorescence, and dead (ethidium homodimer-1) red fluorescence images were taken in triplicate. The number of cells in each image was quantified using CellProfiler. The percent of live and dead cells were calculated by quantifying the number of green fluorescence positive and red fluorescence positive cells, respectively, and dividing by total number of cells in the phase contrast image. GFP and Texas Red filters were used for imaging.MDA-MB-436 and MCF-7 cells were seeded on PMA4/PAAm4 coated microfluidic slides, PMA4/PAAm4 coated microfluidic slides with DOTAP, or a low attach 24-well plate . Cells were incubated for 1 hr to allow for tethering. After 1 hr, one wash was done where the existing media was gently removed from the bottom port of each channel and fresh media was added to the top port on the DOTAP slides. This wash was to ensure only tethered cells were analyzed. After this wash, CellMask Orange (Life Technologies) cell membrane dye was added to each channel to a final concentration of 1:10,000. McTNs were scored blindly in a population of 100 cells/well as previously described [MDA-MB-436 cells were trypsinized, spun down, and resuspended in phenol red-free and serum-free DMEM. Cells were seeded on PMAescribed . Represe4/PAAm4 coated microfluidic slides and PMA4/PAAm4 coated microfluidic slides with DOTAP . Cells were incubated for 1 hr to allow for tethering. After 1 hr, one wash was done where the existing media was gently removed from the bottom port of each channel and fresh media was added to top port on the DOTAP slides. This wash was to ensure only tethered cells were analyzed. After this wash, CellMask orange cell membrane dye was added to each channel to a final concentration of 1:10,000. Cells were treated with 5μM colchicine for 15 mins and 1μg/ml paclitaxel for 120 mins. McTN imaging was done on an Olympus FV100 confocal laser scanning microscope at 60x magnification. Five 1μm slices and 20 frames at a 10 sec frame rate were taken for at least five image sets for each condition. The number of McTNs on each cell was manually counted on five cells per condition using the maximum intensity z-projection at the last frame.MDA-MB-436 cells were trypsinized, spun down, and resuspended in phenol red-free and serum-free DMEM. Cells were seeded on PMAGraphpad Prism (version 6.02) was used to determine all statistic comparisons. Student's t-test and one-way ANOVA tests were performed with a Tukey multiple comparisons post-test as indicated. A p-value of 0.05 or less was considered statistically significant.


Availability of sophisticated statistical modelling for developing robust reference equations has improved interpretation of lung function results. In 2012, the Global Lung function Initiative(GLI) published the first global all-age, multi-ethnic reference equations for spirometry but these lacked equations for those originating from the Indian subcontinent (South-Asians). The aims of this study were to assess the extent to which existing GLI-ethnic adjustments might fit South-Asian paediatric spirometry data, assess any similarities and discrepancies between South-Asian datasets and explore the feasibility of deriving a suitable South-Asian GLI-adjustment.Spirometry datasets from South-Asian children were collated from four centres in India and five within the UK. Records with transcription errors, missing values for height or spirometry, and implausible values were excluded(n = 110).1) and forced vital capacity (FVC) in South-Asian children were on average 15% lower, ranging from 4–19% between centres. By contrast, proportional reductions in FEV1 and FVC within all but two datasets meant that the FEV1/FVC ratio remained independent of ethnicity. The ‘GLI-Other’ equation fitted data from North India reasonably well while ‘GLI-Black’ equations provided a better approximation for South-Asian data than the ‘GLI-White’ equation. However, marked discrepancies in the mean lung function z-scores between centres especially when examined according to socio-economic conditions precluded derivation of a single South-Asian GLI-adjustment.Following exclusions, cross-sectional data were available from 8,124 children . When compared with GLI-predicted values from White Europeans, forced expired volume in 1s (FEVUntil improved and more robust prediction equations can be derived, we recommend the use of ‘GLI-Black’ equations for interpreting most South-Asian data, although ‘GLI-Other’ may be more appropriate for North Indian data. Prospective data collection using standardised protocols to explore potential sources of variation due to socio-economic circumstances, secular changes in growth/predictors of lung function and ethnicities within the South-Asian classification are urgently required. Lung function tests are an integral part of clinical management of respiratory disease but reliable interpretation of results relies on availability of suitable reference data to help distinguish the effects of disease from those of growth and development. Appropriate reference equations are therefore crucial both in clinical management and for interpretation of clinical trials in which lung function is a primary outcome,2. In adSince publication of the most recent ERS/ATS guidelines for spirometry in 2005  there haDevelopment of sophisticated statistical modelling techniques for deriving more robust reference equations has provided opportunities to improve interpretation of lung function results across the age span, with the GLI publishing the first global all-age, multi-ethnic reference equations for spirometry in 2012. These e1) and forced vital capacity (FVC) were very similar in Indian urban children residing in Bangalore city to those living in the UK; both being approximately 11% (~0.9z-score) lower than that predicted for White European children[1/FVC ratio. The similarity of results from these two studies, suggested that it should be possible to derive an additional GLI-adjustment (coefficient) suitable for use in South-Asian children. The primary aim of this study was to assess the extent to which existing GLI-ethnic adjustments might fit South-Asian paediatric spirometry data. The secondary aim was to assess any similarities and discrepancies between South-Asian datasets and the feasibility of deriving a suitable GLI-adjustment if needed for interpreting such data. Some results from this study have been reported in abstract form[Two recent studies of 5–12 year old South-Asian school children in London, UK and in Bangalore, India using identical equipment, protocol and quality control(QC),23 showe children, with noract form.UK centres who had recently (last 15 years) published or collected spirometry data from healthy South-Asian subjects aged 5–18 years or Indian centres who had either submitted such data to the GLI team or recently published their findings were invited to collaborate. See supporting information in 1 or FVC, or where lung function values were deemed implausible  were discarded (n = 110).Among the 11 centres (six UK and five Indian centres) contacted, ten centres responded positively although only nine were able to submit the requested data within the available time frame. Datasets  were available from four centres in India  ,18,21,251, FVC, and FEV1/FVC were initially expressed as z-scores using the GLI-2012 equations for White subjects as a baseline[Results are presented as mean(SD) for continuous variables or as frequencies(%) for categorical variables. Anthropometry was expressed as sex-specific z-scores based on Indian growth charts derived from well-nourished children. Only th baseline.1 and FVC relative to the ‘GLI-White’ equation were proportional, as had been observed in over 60 datasets previously submitted to the GLI-2012[th centile  ≤ -1.645 z-scores).Within each centre, lung function results were then inspected to ascertain i) the relationship between the lung function z-scores against age and height to ensure that there were no trends in residuals . One-way ANOVA was performed to compare the lung function indices (z-scores based on GLI-White) between centres. Post-hoc pairwise comparisons were subsequently performed using the Tukey’s honestly significant difference test which takes into account sample size when calculating the 95% Confidence Intervals (CI).Data for analysis were available from 8,124 children , the majority of whom were of Indian origin  according to centre, with an excessive proportion of healthy children (i.e. >>5%) falling below the lower limit of normal (LLN) as defined by the 5th centile. Similar results were found for FVC , with Centre B (Delhi) and Centre G (Leicester) having significantly larger FEV1 and FVC than those from all other centres (p<0.01) except Centre I, in whom zFVC was similar to that in Centre G (p = 0.93). However, proportional reductions in FEV1 and FVC within all but two datasets (D and G) meant that FEV1/FVC generally remained independent of ethnicity[Lung function results are summarised in Among the UK studies, lung function differences were not explained by socio-economic factors–11. By c1 and zFVC were smaller than when compared with GLI-White equations, the GLI-South-East Asian equations provided a poorer fit for zFEV1/FVC, and an excessive proportion of healthy children had results that fell below the 5th centile for all outcomes. While the entire South-Asian dataset fitted the predicted values for Black-African origin subjects (15% lower than White subjects) reasonably well  fitted the South-Asian data is summarised in bly well , marked 1 and FVC approximating 0 (1), albeit with a slight negative offset for FEV1/FVC  relatively well, with mean (SD) for both FEVFEV1/FVC . Under ss centre .This exercise was not repeated for the GLI-North-East Asian prediction equations since on average these are only 3% lower than GLI-White, and would therefore under-estimate lung function for all South-Asian data.1 and FVC (1 and FVC (Model 3).The marked discrepancies in the mean lung function z-scores between centres  especial and FVC –6, these and FVC  and spirAfter extensive analyses, preliminary GLI-adjustments were derived for the three models . Details1 was, on average, 15% lower in South-Asian children, with similar results for FVC emphasising the need for an ethnic adjustment when interpreting spirometry results from such children. By contrast, proportional reductions in FEV1 and FVC in the majority of datasets meant that the FEV1/FVC ratio, which is commonly used in the diagnosis of airway obstruction, remained independent of ethnic group. Despite the smaller lung volumes in relation to height observed across all datasets, there was marked variability between centres in the magnitude of both the mean offset from predicted values in White subjects (which ranged from 4–19%) and the between-subject variability (coefficient of variation). Although the existing ‘GLI-Other’ equation fitted data from North India reasonably well while the ‘Black’ ethnic adjustment provided a better approximation for most of the other South-Asian data than the ‘White equation’, the marked disparities between centres persisted, irrespective of which equation was applied.In this study we collated spirometry data from 8,124 subjects from four Indian-based and five UK-based studies of South-Asian children. After adjustment for age, sex and height using the GLI equations for White Europeans, FEVThese findings confirm previous observations by Aggarwal and colleagues who compared spirometry equations from different subsets of the Indian adult population and found that the three sets of equations  did not yield interchangeable results, suggesting that there may be considerable heterogeneity in predicted lung function in various Indian subgroups. While sResults from this study represent the largest collation of spirometry data from South-Asian children. Data were subjected to sophisticated statistical modelling which has been shown to be highly effective for developing all-age, multi-ethnic reference ranges. HoweverThe issue of which subjects to include in a reference population remains debateable,35. We uAll data collated were community based, with most studies specifically designed to recruit from a random sample of schools which catered to a wide range of socio-economic circumstances to ensure a representative sample. Although the Hyderabad data were collected before publication of modern standards for paediatric spirometry which coTo assess how representative the data were, we also compared anthropometric data to the current Indian growth charts, derived from 18,666 children attending 10 affluent schools from five major geographical regions of India. After a1 and FVC were on average 15% lower than that in White children of similar age and height, this difference ranged from 4–19% across the datasets. While mean differences of up to 0.5 z-scores (~5%) in lung function can occur by chance in small samples[One of the most striking findings in this study was the variation in mean lung function between different centres. While FEVl samples, this col samples. Review l samples,39, reas1 and FVC between South-Asian and White children in this study were similar to other reports. While all centres strived to achieve international ATS/ERS standards, the quality of data will inevitably be influenced by the training, expertise and experience of the technical staff. Unfortunately, the lack of stored flow-volume curves with which to assess quality of spirometry data from six of nine centres. We have not reported detailed anthropometry regarding sit/stand ratio or chest dimensions in this report, as so few studies have included such measures, and those that did found that they made a relatively small contribution to observed differences between White and South-Asian children,26. Neve GLI-2012, resultibody size,23. Data1 and FVC observed do not reflect intrinsic differences in the functionality of the respiratory system, as reflected by the consistency of the FEV1/FVC ratio. While derivation of an ethnic GLI-adjustment would potentially be the most efficient and cost-effective way of accommodating those of South-Asian origin as has been done for those of Black-African origin, results from this study suggest that no single adjustment will currently fit all data collected from this group, since there appear to be complex factors contributing to the heterogeneity observed within the South-Asian population. While it may be possible to address some of these factors in future, for example by greater standardisation of equipment, methodology and quality control, whereas the contribution of others, such as extreme poverty, may diminish over time with improved living standards, there may be some intrinsic differences related to genetic ancestry, which need further investigation. Meanwhile, the clinical dilemma of which equations to choose when interpreting spirometry results from South-Asian children remains. Personal communication with the authors from the Indian based studies included in this initiative, revealed that within most clinical centres in India, technicians frequently rely on the unspecified and disparate reference values presented as default by the manufacturers, a practice not unknown even in the UK[http://www.ers-education.org/guidelines/global-lung-function-initiative.aspx) would be far preferable, even if this did entail a small offset. Results from the current study suggest that until an improved solution can be found, use of the GLI-Black equations for the majority of South-Asian children, or the GLI-other equation for those originating from North India, could provide an interim solution. For any clinical centre with access to a sizeable population  of healGiven that such an approach will need updating in future, we have included three alternative equations in the Variations in both the mean level of lung function and the degree of within-subject variability meant that none of the existing GLI-ethnic adjustments provided an ideal fit for all the datasets examined in this study and also precluded derivation of a single new South-Asian paediatric GLI adjustment. Given the current wide variation in equations used to interpret lung function from South-Asian children, an alternative approach would be to express results from South-Asian children using either the ‘GLI-Black’ or ‘GLI -Other’ equations, depending on region of origin, both of which are widely available in current spirometers.Further prospective data collection among South-Asians residing within the Indian sub-continent as well as those who have migrated, using standardised protocols over a wider age range, is required to explore the potential sources of variation observed, with regular updating if there is evidence of significant secular changes. When undertaking such studies, age should be recorded in decimal years and standardised anthropometry include assessments of sitting height. All lung function assessments need to be undertaken according to ATS/ERS guidelines, adaptedIn conclusion, despite some progress being made, the challenges we faced in attempting to derive spirometry reference equations for South-Asian children are perhaps best summarised by the following quote from Earl C. Kelley, Professor of Secondary Education at Wayne University (1951):"We have not succeeded in answering all our problems—indeed we sometimes feel we have not completely answered any of them. The answers we have found have only served to raise a whole set of new questions. In some ways we feel that we are as confused as ever, but we think we are confused on a higher level and about more important things. So this report does not purport to give final answers, or to claim that we now "know how to do it". We see more need for revision than ever. But we are doing better than we did. And this is a progress report, rendered with humility because of the unsolved problems we see now which we could not see before."S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.


M = 75.94, SD = 18.09) after Roux-en-Y gastric bypass  were compared with 50 age and gender matched bariatric surgery candidates . To measure cognitive function the following computer-assisted behavioral tasks were utilized: Iowa Gambling Task, Tower of Hanoi, Stroop Test, Trail Making Test-Part B, and Corsi Block Tapping Test. Impulsive symptoms and behaviors were assessed using impulsivity questionnaires and a structured interview for impulse control disorders (ICDs). No group differences were found with regard to performance-based cognitive control, self-reported impulsive symptoms, and ICDs. The results indicate that the general tendency to react impulsively does not differ between pre-surgery and post-surgery patients. The question of whether nonfood-related impulsivity in morbidly obese patients changes post-surgery should be addressed in longitudinal studies given that impulsive symptoms can be considered potential targets for pre- as well post-surgery interventions.Initial evidence that cognitive function improves after bariatric surgery exists. The post-surgery increase in cognitive control might correspond with a decrease of impulsive symptoms after surgery. The present study investigated cognitive function and nonfood-related impulsivity in patients with substantial weight loss due to bariatric surgery by using a comparative cross-sectional design. Fifty post-bariatric surgery patients (postBS group) who had significant percent weight loss ( Impulsivity is considered a predisposition toward rapid, unplanned reactions to internal or external cues and by the tendency to think, plan, control, and behave insufficiently . This co2; There is a growing body of research demonstrating the important role of impulsivity in overeating and obesity  or for those with obesity grade 2 (BMI: 35-39.9 kg/m2) who suffer from chronic obesity-related somatic disorders  by using a combination of behavioral tasks, self-ratings and clinical interview. In addition to tasks focusing on attention, mental flexibility, and response inhibition, we decided to apply tasks on decision-making and problem solving. Poor performance in such tasks is considered to be associated with disadvantageous decisions and an impulsive responding style . To the The postBS group was compared with an age and gender matched group of pre-bariatric surgery patients (preBS group). Based on previous research on cognitive functioning in bariatric surgery samples our hypotheses were twofold. First, we expected to find a better performance in neuropsychological tasks in the postBS group compared to the preBS group. Second, we hypothesized that the postBS group would have less impulsive symptoms given the aforementioned expected better cognitive abilities after bariatric surgery. In addition, we aimed to examine the relationship between the measures that were thought to tap different aspects of impulsivity, in particular performance-based tasks, questionnaires, and clinical interview. Based on earlier findings we expected a rather weak correspondence.2) from another study that had examined the role of somatic comorbidity, executive functions, and temperament in obesity (N = 19) or the University Hospital Erlangen (N = 31) and had completed the same assessments as the postBS group plus two additional cognitive tasks  was used to measure problem solving abilities . The patCognitive control including selective attention and response inhibition was assessed using a computerized version of the Stroop Test . The parThe Trail Making Test-Part B TMT-B;  was usedThe Corsi Block Tapping Test (Corsi) was administered to assess the capacity of visuospatial short-term memory . ParticiThe Behavioral Activation System (BAS) scale of the German version  of the BData on the German version of the Conners Adult ADHD Rating Short-Scale-self-report CAARS;  were avaImpulse control disorders were measured using the ICD module of the research version of the Structured Clinical Interview for DSM-IV . The intTo describe the sample, information on weight, height, and obesity-related somatic comorbidity  was taken from patient charts. Age, nationality, and information on school education were self-reported.The presence or absence of BED was assessed by interview using the BED module of the German version of the Eating Disorder Examination . PatientPercent excess weight loss (%EWL) was defined based on BMI changes by using the following formula: (preoperative BMI-postoperative BMI)/25 × 100.t-tests or Mann–Whitney’s-U tests when the variables were not normally distributed. Two-tailed Spearman’s rank-order correlations were run to determine the relationship between the different measures. The significance level for all tests was set at α = 0.05.Statistical analyzes were performed using IBM SPSS Statistics version 22. Chi-squared tests were used to compare the two groups with regard to categorical variables. To calculate differences between the groups in continuous variables, we performed independent sample Mpre = 48.01 kg/m2, SDpre = 6.56 vs. Mpost = 49.52 kg/m2, SDpost = 5.79, t(98) = -1.22, p = 0.224). Both groups were matched by age and gender; thus, no group differences were found on these variables. The mean age of the preBS group was 42.04 years (SD = 11.17) and the postBS group was 42.30 years (SD = 10.64). Each group consisted of 43 women (86%) and 7 men (14%).The postBS group reported an average time after surgery of 14.44 months  and a mean %EWL of 75.94% . The current BMI of the pre-surgery group did not differ significantly from the pre-surgery BMI of the post-surgery group  = 14.82, p < 0.001). Patients in the preBS group were more likely to have a comorbid somatic condition.As can be seen in Table 1).While about one third of the preBS group was diagnosed with BED, none of the patients in the postBS group met the criteria for BED. Also, at least one LOC eating episode per week during the last month occurred more often in the pre-operative group. No group differences were found in nationality or school education  = 2.08, one-tailed Fisher’s exact test p = 0.122, respectively).Table 3 displays the two-tailed Spearman’s rank-order correlations between variables.More correct answers in the Corsi Block Tapping Test were related to shorter completion time in the TMT-B and to better performance in the Stroop Test. Outcomes in the ToH and the IGT were positively correlated. Moreover, we found a significant relationship between scores on the CAARS subscale ‘Impulsivity’ and the number of impulse control disorders.The main result of the present study is that we did not find significant differences between patients after significant weight loss due to bariatric surgery and patients seeking surgical treatment for obesity with regard to cognitive function or nonfood-related impulsivity.In contrast to our first hypothesis, the postBS group did not show a better performance in neuropsychological tasks than the preBS group. Given initial evidence from a few longitudinal studies demonstrating increased cognitive control after surgery  we expecIn addition to variances in measurement, another important difference between our study and previous studies is the fact that we compared two different cross-sectional samples, whereas earlier studies used longitudinal designs. The intra-individual variability in test performance in longitudinal studies is probably lower than possible inter-individual differences between cross-sectional samples that may have contributed to the lack of group differences in our study. Nonetheless, the present age and gender matched postBS and preBS groups did not differ in pre-surgery BMI and seemed quite typical for post- and pre-surgery samples in terms of BMI, somatic comorbidity, and the absence/presence of BED and LOC eating . TherefoTo date, information on general impulsivity pre and post-surgery remains scarce. In the present sample, impulsive symptoms as measured by questionnaires or diagnosed by clinical interview did not vary between pre and post-surgery patients. These results support the findings of An additional aim of the present study was to examine the correspondence between different cognitive and impulsivity measures, which we expected to be weak or absent based on the existing literature . Indeed,Our results should be interpreted in the context of certain limitations. The most noteworthy of these limitations is our cross-sectional comparison of two independent patient groups, which may restrict the conclusions that can be drawn from our results. Additional studies addressing cognitive function and different facets of impulsivity after bariatric surgery should use adequately powered longitudinal designs with long-term observations. Another shortcoming of this study is that we relied on the BAS scale and the CAARS ‘Impulsivity’ subscale to measure self-reported impulsivity. The CAARS subscale might not have reflected the whole impulsivity spectrum. According to Overall, the present findings confirm earlier results with regard to significant weight loss, and include decreased somatic comorbidity, and less BED in post-bariatric surgery patients compared to patients with morbid obesity who did not undergo surgical treatment. With regard to cognitive function and nonfood-related impulsive symptoms, however, our results indicate a lack of group differences.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Individuals with peripheral arterial disease are at higher risk for cardiovascular events than the general population. While supplementation with omega-3 polyunsaturated fatty acids (PUFA) has been shown to improve vascular function, it remains unclear if supplementation decreases serious clinical outcomes. We conducted a systematic review and meta-analysis to determine whether omega-3 PUFA supplementation reduces the incidence of cardiovascular events and complications in adults with peripheral arterial disease.We searched five electronic databases  from inception to 6 December 2013 to identify randomized trials of omega-3 PUFA supplementation (from fish or plant oils) that lasted ≥12 weeks in adults with peripheral arterial disease. No language filters were applied. Data on trial design, population characteristics, and health outcomes were extracted. The primary outcome was major adverse cardiac events; secondary outcomes included myocardial infarction, cardiovascular death, stroke, angina, amputation, revascularization procedures, maximum and pain-free walking distance, adverse effects of the intervention, and quality of life. Trial quality was assessed using the Cochrane Risk of Bias tool.I2 75%, 2 trials, 288 individuals) or other serious clinical outcomes. Adverse events and compliance were poorly reported.Of 741 citations reviewed, we included five trials enrolling 396 individuals. All included trials were of unclear or high risk of bias. There was no evidence of a protective association of omega-3 PUFA supplementation against major adverse cardiac events (pooled risk ratio 0.73, 95% CI 0.22 to 2.41, Our results showed that insufficient evidence exists to suggest a beneficial effect of omega-3 PUFA supplementation in adults with peripheral arterial disease with regard to cardiovascular events and other serious clinical outcomes. Peripheral arterial disease is an atherosclerosis-induced blockage of non-coronary and non-cerebral arteries. Individuals diagnosed with peripheral arterial disease most often present with ischemic pain in the lower leg following exercise, and as the disease progresses, pain may also occur at rest . The manOmega-3 polyunsaturated fatty acids (PUFA) are a group of dietary fats obtained from fish and plant oils that are reported to have a protective role in coronary heart disease and other cardiovascular complications . Omega-3Given the high incidence of cardiovascular events in individuals with peripheral arterial disease  and the a priori published protocol .,38.34,38I2 89%). None of the trials reported on quality of life. The remaining secondary outcomes  were each reported by only one trial and were not found to be statistically different with omega-3 PUFA intervention  precluded most subgroup analyses. Comparisons of the effects of omega-3 PUFA intervention length, dose and type of omega-3 PUFA on pain-free walking distance did not reach significance, and statistical heterogeneity remained high Table .In this systematic review and meta-analysis of omega-3 PUFA in adults with peripheral arterial disease, we found no evidence to suggest a protective association between omega-3 PUFA supplementation and clinical cardiovascular outcomes, including myocardial infarction, cardiovascular death, angina, stroke, amputation, revascularization, pain-free walking distance or quality of life. One trial indicated that omega-3 PUFA intake may be associated with increased gastrointestinal side effects.The effect of omega-3 PUFA supplementation in cardiovascular disease is controversial. Some reviews and meta-analyses have demonstrated reductions in adverse events with both plant-based ALA and marine-sourced EPA and DHA supplementation in cardiovascular -14,39 anIn this review, the small number of trials available and the lack of uniformity in population and design of the included trials contributed to the relatively high statistical heterogeneity for some outcomes, and thus caution must be exercised in interpreting these results. Some of the key differences among the included trials included geographic differences , varOnly two of the included trials ,38 supplThe internal validity of the trials was often unclear due to underreporting of methods, which potentially could have biased the results. All of the trials rated poorly in the risk of bias assessment, mostly due to a lack of detailed reporting and uncertainty around the true effect of omega-3 PUFA alone  scholarship through the National Sciences and Engineering Research Council’s Collaborative Research and Training Experience (CREATE) program. The authors declare no conflicts of interest.An expert in vascular and metabolic disease (JE) led the review team, which included a panel of content experts from multiple fields: vascular physiology (PZ), lipid nutrition (CGT), and research methodology . All authors assisted in formulating the review question, CF assisted with developing the search strategies, RZ and AMAS guided the review methods, and AY assisted with screening and data extraction throughout the review process. JE drafted the manuscript with input from all other authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/14/70/prepub


This study was conducted to establish how well PREDICT performs in estimating survival in a large cohort of younger women (aged ≤40 years) recruited to the POSH study.Breast cancer is the most common cancer women in the UK, with approximately 50,000 new cases each year. PREDICT  survival estimates for younger women. However, short term (5-year) estimates were less accurate, with the tool overestimating survival by 25%, and by 56% for patients with ER positive tumours. PREDICT also underestimated survival at 5-years for patients with ER negative tumours.PREDICT is a user-friendly and reliable tool for providing accurate long-term survival estimates for younger women with breast cancer. However, the model requires further calibration for more accurate short-term estimates. Prediction in the short-term may be most relevant for the increasing number of women considering risk-reducing bilateral mastectomy.


Optimal applicator insertion is a precondition for the success of cervix cancer brachytherapy (BT). We aimed to assess feasibility and efficacy of MRI-assisted pre-planning, based on applicator insertion in para-cervical anaesthesia (PCA).Five days prior to BT, the pre-planning procedure was performed in 18 cervix cancer patients: tandem-ring applicator was inserted under PCA, pelvic MRI obtained and applicator removed. Procedure tolerability was assessed. High risk clinical target volume (HR CTV) and organs at risk were delineated on the pre-planning MRI, virtual needles placed at optimal positions, and dose planning performed. At BT, insertion was carried out in subarachnoidal anaesthesia according to pre-planned geometry. Pre-planned and actual treatment parameters were compared.Pre-planning procedure was well tolerated. Median difference between the pre-planned and actual needle insertion depth and position were 2 (0–10) mm and 4 (0–30) degrees, respectively. The differences between the pre-planned and actual geometric and dosimetric parameters were statistically non-significant. All actual needles were positioned inside the HR CTV and outside the organs at risk (OAR).Our pre-planning approach is well tolerated and effective. Pre-planned geometry and dose distribution can be reproduced at BT. MRI is the recommended modality for image guided adaptive brachytherapy (IGABT) of cervix cancer.25Accurate applicator insertion with optimal geometric distribution of eventual IS channels is a precondition for the tight control and fine-tuning of the dose distribution. The inadequacies of a suboptimal application cannot always be compensated by treatment plan optimization. Applicator insertion geometry is typically based on clinical and MRI findings at diagnosis and clinical findings at IGABT.Attempting to increase the likelihood of optimal implant geometry already at the first application, we developed an MRI-assisted pre-planning protocol, based on applicator insertion in paracervical anaesthesia (PCA). Our preliminary results on the efficacy of this technique were published recently.The study protocol was approved by the institutional and national ethics committees, multidisciplinary tumour board and the cost analysis office of the Institute of Oncology Ljubljana.Twenty consecutive patients with histologically verified inoperable cervix cancer, treated with curative intent, were enrolled after signing an informed consent. One patient was excluded from analysis due to non-compliance to the study protocol. In the second patient, pre-planning and inclusion in the dosimetric analysis was precluded by vasovagal syncope during PCA injection. Eighteen patients were eligible for the dosimetric analysis.Preplanning insertion was performed in sterile conditions immediately after or in the last week of EBRT. On the morning of the insertion, a laxative suppository and an anxiolytic were applied and one hour intravenous analgesic/antiemetic infusion of tramadol, metamizole and metoclopramide was administered. With the patient in lithotomy position, 10% lidocaine spray was applied topically on the vaginal mucosa and 3 mL of 2% lidocaine injected bilaterally in the paracervical region to achieve PCA . After 5Pre-planning MRI was performed after applicator insertion at a 1.5 Tesla scanner , using a pelvic surface phased-array coil. 2D T2w fast spin echo images , were obtained in paratransverse  plane. In addition, a 3D T2 weighted sequence with high sampling efficiency was performed . Both data sets were transferred to the treatment planning system  and co-registered, using shared DICOM coordinates. Registration was corrected manually when indicated. From the 3D data-set, para-transverse, para-coronal and para-sagittal reconstructions were resampled within the treatment planning system to match the slice thickness and acquisition planes of the 2D images.3 of the rectum, sigmoid colon and bladder below 12.5 Gy, 12.5 Gy and 15 Gy, respectively). When HR CTV was adequately covered following this modifications , no further action was taken and the preplan was considered optimized. When indicated, virtual interstitial channels were placed at optimal radial angles and optimal insertion depths in the parametria to improve the HR CTV coverage and/or OAR avoidance  were delineated and the applicator reconstructed. The preplanning process started by using the standard IC loading pattern. A nominal dose of 18.5 Gy in 25 hourly pulses was specified at point A. For treatment plan optimization and reporting, biologically equivalent doses , were used. After evaluating the dose distribution and DVH parameters, the standard IC pre-plan was modified, aiming to meet our dose constraints for the OAR  into account. T2 weighted FSE MR images in para-transverse, para-coronal and para–sagittal orientation were obtained in addition to the 3D isotropic MRI with high sampling efficiency. Contouring, applicator reconstruction and treatment planning was carried out. The actual radial angles of needle insertion positions and depths were recorded and compared with the pre-planned values . The StaThe non-parametric Wilcoxon signed-rank test for matched pairs was used to compare the treatment plan parameters. The p-value of 0.05 was used as the limit of statistical significance. Statistical program SPSS was used for statistical analysis.3 (range: 15.1 – 68.3 cm3).The patient who developed syncope following PCA injection recovered spontaneously, rapidly and without complications. However, the pre-planning procedure was terminated and the patient was ineligible for dosimetric analysis. FIGO stage distribution of the included patients was as follows: IB1: 2, IIB: 13, IIIB: 2 and IVA: 1 patient. The median HR CTV size was 26 cmSubjective pain sensation during the pre-planning insertion in PCA was reported by all 18 patients included in the dosimetric study. Eight (44.5%) patients reported a peak VAS score of 0 (no pain); 6 (33.5%) a peak VAS score of 1; and 4 (22%) patients a peak VAS score of 3. The median duration of the pre-planning procedure was 69 minutes (range 55 to 90 minutes).At first BT application, a total of 55 IS needles were inserted in 18 patients  through the ring template, respecting the pre-planned specifications. The differences between the pre-planned and actual geometric radial angle of insertion and insertion depth were statistically non-significant . All actAll patients benefited from 3D MRI-guided treatment plan optimization . In stanSeveral reports have demonstrated favourable results of cervix cancer IGABT when compared to conventional x-ray based method.27US-guidance has proven helpful in achieving good position of the intrauterine tandem and is a promising method in interstitial gynaecological BT.PCA is used for cervical dilatation in various obstetric and gynaecological procedures. Nevertheless, the data on its effectiveness and safety are conflicting. A recent review concluded that PCA is ineffective in achieving pain control for women undergoing uterine interventions.With exception of one case of syncope after PCA injection , we observed no adverse events of the pre-planning procedure. About 20% of women undergoing out-patient dilatation of cervical canal are reported to experience vasovagal reactions.3, range: 15.1 – 26.1 cm3), the IS needles were not used. In these cases, the DVH constraints could be met by adapting the standard IC plan and the dosimetric benefit of pre-panning was small. In the remaining 14 patients , the IS needles were used in addition to the IC applicator and the dosimetric benefit of pre-planning was demonstrated. To summarize, the pre-planning procedure, as described here, may be advantageous in cases with bad tumour response to EBRT and/or where unfavourable topography of residual pathological tissues at time of BT can be expected. In small and/or well responding tumours pre-planning was not beneficial.In our study, an excellent reproduction of the pre-planned implant geometry was achieved at first actual BT application, resulting in clinically and statistically non-significant dosimetric differences between the pre-plan and actual BT plan . At actuLimitations of our pre-planning approach include the need for an additional MRI study, staff requirements and administration of additional medications. Therefore, it may be limited to centres with high resources. However, according to our experience, the pre-planning reduced the time needed for the actual BT application and improved the operator confidence and patient compliance during the procedure. In addition, the need for iterative imaging and implant corrections or even removing the applicator due to a potentially suboptimal geometry could be avoided in all cases. By omitting multi-planar MRI and employing 3D MRI for pre-planning, total image acquisition time was shortened for approximately 50%, when compared to our standard MRI study at time of actual BT.44MRI-assisted cervix cancer BT pre-planning, based on applicator insertion in PCA is well tolerated, safe and fast. In cases with bad tumour response to EBRT and/or unfavourable target volume topography at time of BT it enables determination of an optimal distribution of the interstitial applicator channels. At actual BT implantation, an excellent reproduction of the pre-planned insertion geometry and DVH outcome can be achieved. MR assisted IGABT improves dosimetric outcome when compared with conventional dose prescription methods. PCA may be an effective method of pain control in a setting with limited resources and in patients with medical contraindications for general or spinal anaesthesia, requiring cervico-uterine BT.